Definition of the immunogenic forms of modified human LDL recognized by human autoantibodies and by rabbit hyperimmune antibodies

Humans and laboratory animals recognize human modified LDL as immunogenic. Immune complexes (ICs) isolated from human sera contain malondialdehyde-modified LDL (MDA-LDL) and N (epsilon)(carboxymethyl)lysine-modified LDL (CML-LDL) as well as antibodies reacting with MDA-LDL, copper-oxidized LDL (OxLDL), CML-LDL, and advanced glycosylation end product (AGE)-modified LDL. OxLDL and AGE-LDL antibodies isolated from human sera recognize the same LDL modifications and do not react with modified non-LDL proteins. Rabbit antibodies have different reactivity patterns: MDA-LDL antibodies react strongly with MDA-LDL and MDA-BSA but weakly with OxLDL; OxLDL antibodies react strongly with OxLDL and weakly with MDA-LDL; CML-LDL antibodies react with CML-LDL > CML-BSA > AGE-LDL > OxLDL; AGE-LDL antibodies react strongly with AGE-LDL, react weakly with OxLDL, and do not react with CML-LDL. Thus, human and rabbit antibodies seem to recognize different epitopes. Capture assays carried out with all rabbit antibodies showed binding of apolipoprotein B-rich lipoproteins isolated from ICs, suggesting that laboratory-generated epitopes are expressed by in vivo-modified LDL, although they are not necessarily recognized by the human immune system. Thus, the definition of immunogenic forms of modified LDL eliciting human autoimmune responses requires the isolation and characterization of autoantibodies and modified LDL from human samples, whereas rabbit antibodies can be used to detect in vivo-modified human LDL.     

Unique structural properties of apolipoprotein B in low-density lipoproteins produced by several human hepatoma-derived cell lines

Previous work has shown that low-density lipoproteins (LDL) secreted by hepatoma-derived cell lines have an unusual composition compared to plasma LDL; rather than cholesteryl ester, the hepatoma cell-secreted LDL have a triacylglycerol core. We have found that they also have an increased negative charge, as judged by agarose electrophoresis. Since apolipoprotein B is a glycoprotein containing carbohydrate chains terminated with negatively charged sialic acid residues, we examined whether increased glycosylation of the apolipoprotein B from three hepatoma cell lines (Hep G2, Hep 3B and Huh 7) might account for the differences in LDL charge. The weight percent carbohydrate for Hep G2, Hep 3B and Huh 7 LDL-protein (1.1 +/- 0.2; 1.7 +/- 0.8; 0.4 +/- 0.1) was found to be extremely low compared with the 2.8-9% range we found for plasma LDL-protein, while the amount of LDL-lipid associated carbohydrate from hepatoma LDL was similar to that we found in plasma LDL. Furthermore, desialation of hepatoma cell-secreted LDL with neuraminidase did not normalize the negative charge to that of neuraminidase-treated plasma LDL. Western blots of thrombin proteolytic fragments indicated that, in addition to the T1-T4 fragments seen in plasma apolipoprotein B, apolipoprotein B of hepatoma-derived LDL produced four to five new fragments (T5-T9), suggesting increased exposure of proteolytic sites. Western blotting of the new fragments with antibodies specific for known apolipoprotein B sequences suggests that many of the new cleavage sites cluster in or near the putative LDL receptor recognition site.     

Stoichiometric binding of apolipoprotein B-specific monoclonal antibodies to low density lipoproteins

The structure of apolipoprotein B and its stoichiometry on plasma lipoproteins has been a major issue and one refractory to a variety of analyses. Immunochemical analyses represent an independent approach. Examinations of apolipoprotein B (apo-B) epitopes on human plasma low density lipoproteins (LDL) using monoclonal antibodies have consistently revealed the existence of extensive apo-B heterogeneity. In the present study, we have addressed the solution of the stoichiometry problem using quantitative analysis of the maximum number of identical antibodies that can be bound per LDL particle in which we take into account this ligand heterogeneity. We have estimated the molecular weight of apo-B by quantifying the number of times a given apo-B epitope is expressed on the surface of LDL. The quantitative binding of eight previously characterized monoclonal antibodies was measured in a fluid phase radioimmunoassay. The results were analyzed by Scatchard analysis and expressed on the basis of independent measurements of the maximum amount of LDL that could be bound by each antibody. Affinity constants for each of the eight antibodies varied between 8.5 X 10(7) and 80 X 10(7) M-1. For these same antibodies, the concentration of maximally bound antibody at a normalized LDL concentration of 1000 ng/ml was estimated to be 0.9-1.8 nM with a mean of 1.23 nM. Adopting a molecular mass from physicochemical analysis for LDL apo-B of 550,000 daltons, the molar ratio between bound antibody and LDL varied between 0.5 and 1.2 (mean 0.75 +/- 0.15). The results supported the hypothesis that apo-B is present as a single large molecular weight polypeptide in LDL.     

Apolipoproteins and their electrophoretic pattern throughout the reproductive cycle in the green frog Rana esculenta

Lipoprotein fractions in Rana esculenta were separated using the same salt intervals currently applied for human lipoproteins. Very low density lipoproteins (VLDL), low density lipoproteins (LDL) and high density lipoproteins (HDL) were analyzed with reference to the electrophoretic pattern. The lipoprotein electrophoretic pattern in males and females throughout the reproductive cycle showed minor differences. In general, each fraction was characterized by a specific apolipoprotein content. VLDL and LDL fractions were dominated by a high molecular weight (MW) band, most likely the counterpart of human Apolipoprotein B (apo B). The apo B in R. esculenta cross reacted, although weakly, with antibodies raised against chicken apo B. The HDL fraction showed a band with an apparent MW of 29 kDa. The electrophoretic mobility of the protein moiety of HDL was similar to human apolipoprotein A-I (apo A-I). However, HDL apolipoprotein of R. esculenta did not cross react with antibodies against chicken apo A-I under either denaturing or native conditions. The HDL apolipoprotein of R. esculenta was purified by DEAE-Sephacel chromatography followed by HPLC. Its amino acid composition showed a moderate correlation with trout, salmon, chicken and human apo A-I.     

ELISA quantitation of apolipoproteins in plasma lipoprotein fractions: ApoE in apoB-containing lipoproteins (Lp B:E) and apoB in apoE-containing lipoproteins (Lp E:B)

Growing clinical evidence suggests that metabolic behavior and atherogenic potential vary within lipoprotein subclasses that can be defined by apolipoprotein variation. Variant constituency of apolipoproteins B and E (apoB and apoE) may be particularly important because of the central roles of these apolipoproteins in the endogeneous lipid delivery cascade. ApoB is the sole protein of low-density lipoprotein (LDL), and like LDL cholesterol, the plasma apoB level has been positively correlated with risk for atherosclerotic disease. ApoE is a major functional lipoprotein in the triglyceride-rich lipoproteins, and may be crucial in the conversion of very low density lipoprotein (VLDL) to LDL. Based on work by others that enabled the quantititation of apoB-containing particles by content of up to two other types of apolipoprotein, we have developed a method for determining the amount of apoE in apoB-containing lipoproteins (Lp B:E) and the amount of apoB in apoE-containing lipoproteins (Lp E:B). From the Lp B:E and Lp E:B concentrations, the molar ratio of apoE to apoB in lipoproteins containing apoB and/or apoE in plasma can be determined. The methodology is fast, specific, and sensitive and should prove extremely useful in further categorizing lipoproteins and characterizing their behavior. In applying this method to clinical groupings of normo- and hyperlipidemia, we found that the plasma triglyceride level correlated with the apoE and Lp B:E concentrations in plasma, while the total cholesterol level correlated with the apoB and Lp E:B levels.     

Provision of cholesterol to lymphocytes by high density and low density lipoproteins. Requirement for low density lipoprotein receptors

The capacity of lipoprotein fractions to provide cholesterol necessary for human lymphocyte proliferation was examined. When endogenous synthesis of cholesterol was blocked, proliferation of mitogen-stimulated normal human lymphocytes was markedly inhibited unless an exogenous source of sterol was supplied. All lipoprotein fractions with the exception of high density lipoprotein subclass 3 were able to provide cholesterol for lymphocyte proliferation. Each of the lipoprotein subfractions capable of providing cholesterol was also able to regulate endogenous sterol synthesis in cultured human lymphocytes. Provision of cholesterol by lipoproteins required the interaction of apolipoprotein B or apolipoprotein E with specific receptors on normal lymphocytes. Apolipoprotein modification by acetylation or methylation, which markedly reduced the ability to regulate sterol biosynthesis, also diminished the capacity of lipoproteins to provide cholesterol. In addition, depletion of apolipoprotein B- and apolipoprotein E-containing particles from high density lipoprotein decreased its ability to suppress cholesterol synthesis and prevented it from providing cholesterol to proliferating lymphocytes. Monoclonal antibodies directed against the receptor-recognition sites on apolipoprotein B and apolipoprotein E were used to define the specific apolipoproteins required for the provision of cholesterol to lymphocytes by the various lipoprotein fractions. The antibody to apolipoprotein B inhibited cholesterol provision by both low density lipoprotein (LDL) and other lipoprotein fractions. The antibody to apolipoprotein E did not decrease provision of cholesterol by LDL but did inhibit the capacity of other fractions to provide cholesterol. In addition, a monoclonal antibody against the ligand binding site on the LDL receptor inhibited provision of cholesterol to normal lymphocytes by all lipoproteins. Finally, lymphocytes lacking LDL receptors were unable to obtain cholesterol from any lipoprotein fraction. These studies demonstrate that LDL receptor-mediated interaction with apolipoprotein B or apolipoprotein E is essential for the provision of cholesterol to normal human lymphocytes from all lipoprotein sources.     

Characterization of a monoclonal antibody that binds equally to all apolipoprotein and lipoprotein forms of human plasma apolipoprotein B. I. Specificity and binding studies

A stable mouse hybridoma cell line has been developed that produces monoclonal antibody to human plasma apolipoprotein B. This antibody was proven to be specific for apolipoprotein B immunoblotting and an enzyme immunoassay using apolipoprotein B and other apolipoproteins. The antibody bound with comparable affinities to soluble apolipoprotein B, chylomicrons, very-low-density (VLDL) and low-density lipoproteins (LDL). Coupled to agarose, this antibody allowed complete removal of apolipoprotein B-containing lipoproteins from normolipidemic, hypertriglyceridemic and hypercholesterolemic plasma. Desialyzation and deglycosylation had no effect on its binding to LDL. The described antibody had no effect on the receptor-mediated binding of radiolabeled LDL to the human hepatoma cells (HepG2) in culture. Analysis of 25 different samples of human plasma indicated identical expression of the corresponding epitope in these individuals. The described monoclonal antibody, most likely, binds to a rather stable domain of apolipoprotein B that is not altered by the interaction with lipids or polymorphism of the apolipoprotein B. We propose that this antibody be called 'Pan B' antibody.     

Characterization of a monoclonal antibody that binds equally to all apolipoprotein and lipoprotein forms of human plasma apolipoprotein B. II. Isolation of apolipoprotein B-containing lipoproteins from human plasma

Monoclonal antibody ('Pan B' antibody) that binds equally to all major forms of human plasma apolipoprotein B was used in an immunoaffinity chromatography procedure to isolate apolipoprotein B-containing lipoproteins from hyperlipidemic human plasma. These lipoproteins were compared with lipoproteins in native plasma, with lipoproteins isolated by polyclonal antibodies and with lipoproteins isolated by the conventional ultracentrifugational method. Judged by the apolipoprotein and lipid composition, lipoproteins isolated with 'Pan B' antibody were virtually identical to those isolated by ultracentrifugation or polyclonal antibodies. Lipoproteins isolated by 'Pan B' antibody were comparable in size and shape to the lipoproteins in native plasma and to the lipoproteins isolated by polyclonal antibodies or ultracentrifugation. The immunoaffinity column with monoclonal 'Pan B' antibody retained all apolipoprotein B-containing lipoproteins and showed significantly higher capacity than polyclonal immunoaffinity column. The column with the highest capacity allowed the isolation from whole plasma of 0.144 mg of apolipoprotein B per ml of gel in less than 2 h.     

Interactions of low density lipoprotein2 and other apolipoprotein B-containing lipoproteins with lipoprotein(a)

Studies were undertaken to investigate potential interactions among plasma lipoproteins. Techniques used were low density lipoprotein2 (LDL2)-ligand blotting of plasma lipoproteins separated by nondenaturing 2.5-15% gradient gel electrophoresis, ligand binding of plasma lipoproteins by affinity chromatography with either LDL2 or lipoprotein(a) (Lp(a)) as ligands, and agarose lipoprotein electrophoresis. Ligand blotting showed that LDL2 can bind to Lp(a). When apolipoprotein(a) was removed from Lp(a) by reduction and ultracentrifugation, no interaction between LDL2 and reduced Lp(a) was detected by ligand blotting. Ligand binding showed that LDL2-Sepharose 4B columns bound plasma lipoproteins containing apolipoproteins(a), B, and other apolipoproteins. The Lp(a)-Sepharose column bound lipoproteins containing apolipoprotein B and other apolipoproteins. Furthermore, the Lp(a) ligand column bound more lipoprotein lipid than the LDL2 ligand column, with the Lp(a) ligand column having a greater affinity for triglyceride-rich lipoproteins. Lipoprotein electrophoresis of a mixture of LDL2 and Lp(a) demonstrated a single band with a mobility intermediate between that of LDL2 and Lp(a). Chemical modification of the lysine residues of apolipoprotein B (apoB) by either acetylation or acetoacetylation prevented or diminished the interaction of LDL2 with Lp(a), as shown by both agarose electrophoresis and ligand blotting using modified LDL2. Moreover, removal of the acetoacetyl group from the lysine residues of apoB by hydroxylamine reestablished the interaction of LDL2 with Lp(a). On the other hand, blocking of--SH groups of apoB by iodoacetamide failed to show any effect on the interaction between LDL2 and Lp(a). Based on these observations, it was concluded that Lp(a) interacts with LDL2 and other apoB-containing lipoproteins which are enriched in triglyceride; this interaction is due to the presence of apolipoprotein(a) and involves lysine residues of apoB interacting with the plasminogen-like domains (kringle 4) of apolipoprotein(a). Such results suggest that Lp(a) may be involved in triglyceride-rich lipoprotein metabolism, could form transient associations with apoB-containing lipoproteins in the vascular compartment, and alter the intake by the high affinity apoB, E receptor pathway.     

Metabolism of human plasma triacylglycerol-rich lipoproteins in rodent macrophages: capacity for interaction at beta-VLDL receptor

The capacity of human plasma triacylglycerol-rich lipoproteins to be metabolized by rat macrophages was studied with plasma triacylglycerol-rich lipoproteins obtained from subjects with fasting chylomicronemia or from normal subjects after a fat meal. Triacylglycerol-rich lipoproteins were separated by chromatography into two fractions designated TRL1 and TRL2; from their composition and changing concentration during alimentary lipemia, TRL1 contained a higher proportion of chylomicron remnants than TRL2. Degradation of 125I-labeled TRL1 was greater than that of 125I-labeled TRL2. In competition studies with 125I-labeled beta-VLDL from cholesterol-fed rabbits, unlabeled TRL1 displaced beta-VLDL as completely as did unlabeled beta-VLDL, being slightly more potent than TRL2, which contained less apolipoprotein E than TRL1. This reflected common interaction at receptors that probably included both beta-VLDL and B/E receptors, since: (1) in fresh macrophages, VLDL from hypertriglyceridemic subjects partially displaced beta-VLDL; (2) in B/E receptor-repressed macrophages, TRL1 maintained capacity to totally displace beta-VLDL. This was confirmed in experiments with J774 murine macrophages in which triacylglycerol-rich lipoproteins and beta-VLDL displaced each other equally, whereas LDL was ineffective in displacing beta-VLDL. Furthermore, monoclonal antibodies raised against apolipoprotein B48 and reacting strongly with LDL, failed to inhibit the binding of triacylglycerol-rich lipoprotein to the macrophages. This indicates an interaction through apolipoprotein E which is present in high concentration in triacylglycerol-rich lipoprotein as well as in beta-VLDL. It applies to triacylglycerol-rich particles derived from either the intestine (chylomicron remnants) or the liver (VLDL remnants from hypertriglyceridemic subjects).     

Immunological heterogeneity of rat apolipoprotein B epitope expression. A study using monoclonal antibodies to rat apolipoprotein B

Epitope expression of rat apolipoprotein B on lipoproteins was investigated with the help of six monoclonal antibodies produced from mice. Through a variety of techniques, which include cotitrations, ELISAs and quantitative immunoadsorption precipitation, we concluded that the six monoclonal antibodies recognize five different epitopes. LRB 110 and LRB 260 recognize epitopes that may be overlapping. LRB 240 and LRB 250 recognize epitopes that are preferentially expressed in triacylglycerol-rich particles. LRB 220 recognizes an epitope that is expressed by all apolipoprotein-B-containing lipoproteins. We have also determined that apolipoprotein B epitope expression in rat lipoproteins is very similar to its human counterpart. Both rat and human apolipoprotein B epitope expression on lipoproteins showed heterogeneities even in homologous lipoprotein preparations. We concluded that a variety of techniques are necessary to fully characterize monoclonal antibodies to apolipoproteins. The possible implications of epitope expression in pathophysiology are also discussed.     

Allotypes associated with B apolipoproteins in rabbits

Western blot analysis of the alloantisera (i.e., anti-Lpq1, anti-Lpq2, anti-Lpq3, and anti-Lpq4) which defined the three lpq genes of rabbit linkage group VIII showed that they reacted strongly with an apolipoprotein of molecular weight 320,000. They also cross-reacted with an apolipoprotein of molecular weight 220,000. The two apolipoproteins that reacted with the alloantisera were found by SDS-polyacrylamide gel electrophoresis to be present in very low density (VLDL), intermediate density (IDL), and low density (LDL) lipoprotein fractions and by Western blot analysis to react with an anti-apolipoprotein B antiserum. These results support the conclusion that the alloantisera react with allotypes associated with the B apolipoproteins. The distribution of the four allotypes among different lipoprotein fractions, however, differed. The quantitative competitive Enzyme Linked Immunosorbant Assay (ELISA) showed that the Lpq1, Lpq2, and Lpq4 allotypes were found in the highest concentration in VLDL, IDL, and LDL, and in significantly lower concentrations in plasma chylomicrons. The concentrations of these allotypes in high density lipoproteins (HDL) as measured in the ELISA were about 1% of the concentrations found in LDL. The Lpq3 allotype, on the other hand, was present in the highest concentrations only in IDL and LDL and in significantly lower concentrations in VLDL and plasma chylomicrons. Surprisingly, the concentration of the Lpq3 allotype in HDL was 20% of the level found in LDL.     

Interaction of LDL, Lp[a], and reduced Lp[a] with monoclonal antibodies against apoB

Five monoclonal antibodies (2A, 9A, 6B, L3, L7) produced in mice against human apolipoprotein B were investigated by competitive and inhibitive electroimmunoassay (EIA) for their reactivity with low density lipoprotein (LDL), lipoprotein[a] (Lp[a]), and reduced Lp[a]. All of the antibodies reacted with apoB of the different lipoproteins indicated by very similar slopes of the binding curves. None of them gave a positive reaction with apolipoprotein[a]. The amount of apoB required for 50% inhibition of antibody binding varied for the different antibodies and lipoproteins. Antibody 9A showed almost the same affinity for LDL, Lp[a], and reduced Lp[a]. Antibodies 2A and 6B bound about twofold better to LDL and reduced Lp[a] than to untreated Lp[a]. Antibodies L3 and L7 needed nearly threefold higher amounts of Lp[a]-apoB for 50% inhibition of antibody binding than of apoB of LDL and reduced Lp[a]. The amount of apoB required for 50% inhibition of antibody binding was somewhat higher in inhibitive assay than in competitive assay. We suggest that apo[a] covers certain epitopes of apoB in native Lp[a] leading to a reduced reaction with the monoclonal antibodies. However, it could also be that the binding of the [a]antigen to apoB via disulfide bridges causes profound conformational changes of the apoB region exposed to the surface.     

Immunochemical properties of human low density lipoproteins as explored by monoclonal antibodies. Binding characteristics distinct from those of conventional serum antibodies

Spleen cells obtained from mice immunized with human plasma low-density lipoproteins (LDL) were fused with mouse myeloma cells. The resulting hybridoma cells secreting immunoglobulin specific for LDL were screened and scored by radioimmunoassay and cloned by multiple limiting dilutions. Immunochemical properties of the monoclonal antibodies were compared with convential mouse serum antibodies. It was found that conventional antibodies precipitated LDL and bound more than 95% of 125I-labeled LDL and the maximal binding was independent of temperature. The monoclonal antibodies were incapable of precipitating LDL and bound a maximum of only 20% of the total 125I-labeled LDL. The maximal binding between monoclonal antibodies and LDL was extremely temperature-dependent. An optimal degree of binding was observed at 4 degrees C, whereas binding at 37 degrees C was only 30% of that achieved at 4 degrees C. Although the binding at 37 degrees C was low, the maximal binding could be re-established following a subsequent incubation at 4 degrees C, suggesting that the antigenic structure of LDL is reversibly modulated at temperatures between 4 and 37 degrees C. Since the orientation of apolipoprotein B in LDL is known to be dynamic at different temperatures, this result suggests that monoclonal antibodies, but not conventional antibodies, are capable of detecting subtle conformational changes in LDL. In addition, we have determined the binding affinity of LDL to monoclonal antibodies and to conventional antibodies. Only monoclonal antibodies showed a linear Scatchard plot, suggesting that the binding was to a single site with a single affinity. The monoclonal antibodies also possessed high specificity and failed to react with porcine LDL, while serum antibodies could recognize both human and porcine LDL.     

Metabolism of very low density lipoproteins in the pig. An in vivo study.

1. The metabolism of apolipoprotein B (apoB) was investigated in pigs injected with [125I]very low density lipoproteins (VLDL) to determine to which extent the two distinct low density lipoprotein subclasses (LDL1 and LDL2) derive from VLDL. 2. The lipoproteins were isolated by density gradient ultracentrifugation and the transfer of radioactivity from VLDL into LDL1 and LDL2 apoB was measured. 3. Only a minor portion of VLDL apoB was converted to LDL1 (7.7 +/- 3.2%) and LDL2 (3.6 +/- 1.5%), respectively. Thus, we conclude that the major portion of LDL, especially LDL2, is synthesized independently from VLDL catabolism.     

Monoclonal antibodies to low density lipoprotein used for the study of low- and very-low-density lipoproteins, in "ELISA" and immunoprecipitation technics

Seven monoclonal antibodies to low-density lipoprotein were studied by the ELISA for their reactivity with LDL or VLDL. Cotitration experiments showed that five of them are addressed to different antigenic epitopes. Two of the monoclonal antibodies were temperature independent whereas the others had a decreased binding activity at 37 degrees C compared to that obtained at 25 degrees C or 4 degrees C, suggesting the presence of antibodies directed to sequence or conformation epitopes, respectively. All antibodies reacted with both LDL and VLDL; four of them had a higher affinity for LDL and two others for VLDL. Immunoprecipitation of LDL and/or VLDL was observed upon immunodiffusion with certain pairs of antibodies. This may allow the use of pairs of monoclonal antibodies to LDL for the quantitative determination of apolipoprotein B in serum LDL and VLDL.     

Immunoelectron microscopic visualization of the transcytosis of low density lipoproteins in perfused rat arteries

A postembedding labeling technique was employed to visualize human native low density lipoproteins (LDL) during transcytosis in rat arterial endothelium. For this purpose human LDL was perfused through rat vasculature before fixation and processing for immunoelectron microscopy. The LDL particles were located on sections by anti-human apolipoprotein B-100 (LDL) antibodies and secondary antibodies or protein-A conjugated to 10-nm colloidal gold. LDL molecules were seen in plasmalemmal vesicles as well as in the subendothelial space. No colloidal gold was found in the intercellular junctions. Perfusion with reductively methylated LDL, which cannot bind to the LDL receptor, gave a similar labeling pattern, indicating that transcytosis of LDL via plasmalemmal vesicles is most likely receptor independent. Furthermore, the passage of LDL through intact vascular endothelium is a vesicular transport rather than an intercellular diffusion process.     

Lipolytic degradation of human very low density lipoproteins by human milk lipoprotein lipase: the identification of lipoprotein B as the main lipoprotein degradation product

Although the direct conversion of very low density lipoproteins (VLDL) into low density (LDL) and high density (HDL) lipoproteins only requires lipoprotein lipase (LPL) as a catalyst and albumin as the fatty acid acceptor, the in vitro-formed LDL and HDL differ chemically from their native counterparts. To investigate the reason(s) for these differences, VLDL were treated with human milk LPL in the presence of albumin, and the LPL-generated LDL1-, LDL2-, and HDL-like particles were characterized by lipid and apolipoprotein composition. Results showed that the removal of apolipoproteins B, C, and E from VLDL was proportional to the degree of triglyceride hydrolysis with LDL2 particles as the major and LDL1 and HDL + VHDL particles as the minor products of a complete in vitro lipolysis of VLDL. In comparison with native counterparts, the in vitro-formed LDL2 and HDL + VHDL were characterized by lower levels of triglyceride and cholesterol ester and higher levels of free cholesterol and lipid phosphorus. The characterization of lipoprotein particles present in the in vitro-produced LDL2 showed that, as in plasma LDL2, lipoprotein B (LP-B) was the major apolipoprotein B-containing lipoprotein accounting for over 90% of the total apolipoprotein B. Other, minor species of apolipoprotein B-containing lipoproteins included LP-B:C-I:E and LP-B:C-I:C-II:C-III. The lipid composition of in vitro-formed LP-B closely resembled that of plasma LP-B. The major parts of apolipoproteins C and E present in VLDL were released to HDL + VHDL as simple, cholesterol/phospholipid-rich lipoproteins including LP-C-I, LP-C-II, LP-C-III, and LP-E. However, some of these same simple lipoprotein particles were present after ultracentrifugation in the LDL2 density segment because of their hydrated density and/or because they formed, in the absence of naturally occurring acceptors (LP-A-I:A-II), weak associations with LP-B. Thus, the presence of varying amounts of these cholesterol/phospholipid-rich lipoproteins in the in vitro-formed LDL2 appears to be the main reason for their compositional difference from native LDL2. These results demonstrate that the formation of LP-B as the major apolipoprotein B-containing product of VLDL lipolysis only requires LPL as a catalyst and albumin as the fatty acid acceptor. However, under physiological circumstances, other modulating agents are necessary to prevent the accumulation and interaction of phospholipid/cholesterol-rich apolipoprotein C- and E-containing particles.     

Studies on the binding and degradation of human very-low-density lipoproteins by human hepatoma cell line HepG2

The regulation of the hepatic catabolism of normal human very-low-density lipoproteins (VLDL) was studied in human-derived hepatoma cell line HepG2. Concentration-dependent binding, uptake and degradation of 125I-labeled VLDL demonstrated that the hepatic removal of these particles proceeds through both the saturable and non-saturable processes. In the presence of excess unlabeled VLDL, the specific binding of 125-labeled VLDL accounted for 72% of the total binding. The preincubation of cells with unlabeled VLDL had little effect on the expression of receptors, but reductive methylation of VLDL particles reduced their binding capacity. Chloroquine and colchicine inhibited the degradation of 125I-labeled VLDL and increased their accumulation in the cell, indicating the involvement of lysosomes and microtubuli in this process. Receptor-mediated degradation was associated with a slight (13%) reduction in de novo sterol synthesis and had no significant effect on the cellular cholesterol esterification. Competition studies demonstrated the ability of unlabeled VLDL, low-density lipoproteins (LDL) and high-density lipoproteins (HDL) to effectively compete with 125I-labeled VLDL for binding to cells. No correlation was observed between the concentrations of apolipoproteins A-I, A-II, C-I, C-II and C-III of unlabeled lipoproteins and their inhibitory effect on 125I-labeled VLDL binding. When unlabeled VLDL, LDL and HDL were added at equal contents of either apolipoprotein B or apolipoprotein E, their inhibitory effect on the binding and uptake of 125I-labeled VLDL only correlated with apolipoprotein E. Under similar conditions, the ability of unlabeled VLDL, LDL and HDL to compete with 125I-labeled LDL for binding was a direct function of only their apolipoprotein B. These results demonstrate that in HepG2 cells, apolipoprotein E is the main recognition signal for receptor-mediated binding and degradation of VLDL particles, while apolipoprotein B functions as the sole recognition signal for the catabolism of LDL. Furthermore, the lack of any substantial regulation of beta-hydroxy-beta-methylglutaryl-CoA reductase and acyl-CoA:cholesterol acyltransferase activities subsequent to VLDL degradation, in contrast to that observed for LDL catabolism, suggests that, in HepG2 cells, the receptor-mediated removal of VLDL proceeds through processes independent of those involved in LDL catabolism.     

Catabolism of very low density lipoproteins in the rabbit. Effect of changing composition and pool size.

To determine the metabolic mechanism of hypercholesterolemia in rabbits produced by feeding cholesterol-rich diets, control and hypercholesterolemic rabbits were injected with I-labelled very low density lipoproteins (VLDL, d 1.006 g/ml) from control and/or hypercholesterolemic donors. Apolipoprotein B in VLDL decayed biphasically. The first phase occurred much more rapid than the second. 95% of the VLDL apolipoprotein B was catabolized via the first phase (t1/2 = 0.55 +/- 0.19 h) in normal rabbit with the immediate appearance of this radioactivity in intermediate density lipoproteins (IDL, d 1.006-1.025 g/ml) and low density lipoproteins (LDL, d 1.025-1.063 g/ml). The apolipoproteins C and E at the same time were transferred to high density lipoproteins where they decayed biphasically. The apolipoprotein B from hypercholesterolemic VLDL in the normal recipient disappeared at a similar rate as from normal VLDL via phase I; however, it was incompletely converted to IDL and LDL. Apolipoprotein B from normal VLDL in cholesterol-fed rabbits disappeared at a normal rate via phase I, but only 82% was catabolized by this phase. Hypercholesterolemic VLDL injected into the hypercholesterolemic recipient was less rapidly catabolized via phase I (T1/2 = 2.5 +/- 0.89 H) and only a small fraction was converted to IDL and LDL.