Distinct patterns of lipoproteins with apoB defined by presence of apoE or apoC-III in hypercholesterolemia and hypertriglyceridemia

Apolipoprotein (apo) E and apoC-III concentrations in VLDL and LDL are associated with coronary heart disease. We studied the relationship between apoE and apoC-III and the abnormal concentrations and distribution of apoB lipoproteins in 10 hypercholesterolemic and 13 hypertriglyceridemic patients compared with 12 normolipidemic subjects (mean age, 45 years). Sixteen distinct types of apoB lipoprotein particles were separated by first using anti-apoE and anti-apoC-III immunoaffinity chromatography in sequence and then ultracentrifugation [light VLDL, dense VLDL, IDL, and LDL, with apoE with or without apoC-III (E(+)C-III(+), E(+)C-III(-)) or without apoE with or without apoC-III (E(-)C-III(+), E(-)C-III(-))]. The concentrations of VLDL particles with apoC-III (E(+)C-III(+), E(-)C-III(+)) were increased in the hypertriglyceridemic group compared with the hypercholesterolemic and normolipidemic groups. These particles were the most triglyceride rich of the particle types, and their triglyceride content was twice as high in hypertriglyceridemics compared with the other two groups. Hypertriglyceridemics had a similar concentration of total E(-)C-III(-) particles compared with normolipidemics, but the E(-)C-III(-) particles were distributed more to VLDL and IDL than to LDL. Hypercholesterolemics, in contrast, were distinguished from the normolipidemic group by 2-fold higher concentrations of apoB lipoproteins without apoE or apoC-III (E(-)C-III(-)), mainly LDL, which had high cholesterol content. Nonetheless, both normolipidemics and hypercholesterolemics had apoC-III-containing VLDL, which comprised 68% and 43% of their total VLDL particles. E(+)C-III(-) particles were a minor type, comprising <10% of particles in all lipoproteins and patient groups. Therefore, VLDL particles with apoC-III may play a central role in identifying the high risk of coronary heart disease in hypertriglyceridemia, but their substantial prevalence in normolipidemics may be of clinical significance as well.     

Apolipoprotein C-III deficiency accelerates triglyceride hydrolysis by lipoprotein lipase in wild-type and apoE knockout mice

Previous studies with hypertriglyceridemic APOC3 transgenic mice have suggested that apolipoprotein C-III (apoC-III) may inhibit either the apoE-mediated hepatic uptake of TG-rich lipoproteins and/or the lipoprotein lipase (LPL)-mediated hydrolysis of TG. Accordingly, apoC3 knockout (apoC3(-/-)) mice are hypotriglyceridemic. In the present study, we attempted to elucidate the mechanism(s) underlying these phenomena by intercrossing apoC3(-/-) mice with apoE(-/-) mice to study the effects of apoC-III deficiency against a hyperlipidemic background. Similar to apoE(+/+) apoC3(-/-) mice, apoE(-/-)apoC3(-/-) mice exhibited a marked reduction in VLDL cholesterol and TG, indicating that the mechanism(s) by which apoC-III deficiency exerts its lipid-lowering effect act independent of apoE. On both backgrounds, apoC3(-/-) mice showed normal intestinal lipid absorption and hepatic VLDL TG secretion. However, turnover studies showed that TG-labeled emulsion particles were cleared much more rapidly in apoC3(-/-) mice, whereas the clearance of VLDL apoB, as a marker for whole particle uptake by the liver, was not affected. Furthermore, it was shown that cholesteryl oleate-labeled particles were also cleared faster in apoC3(-/-) mice. Thus the mechanisms underlying the hypolipidemia in apoC3(-/-) mice involve both a more efficient hydrolysis of VLDL TG as well as an enhanced selective clearance of VLDL cholesteryl esters from plasma. In summary, our studies of apoC3(-/-) mice support the concept that apoC-III is an effective inhibitor of VLDL TG hydrolysis and reveal a potential regulating role for apoC-III with respect to the selective uptake of cholesteryl esters.     

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.     

Structural peculiarities of the binding of very low density lipoproteins and low density lipoproteins to the LDL receptor in hypertriglyceridemia: role of apolipoprotein E

Very low (VLDL) and low density lipoproteins (LDL) were isolated from plasma of patients with the E3/3 phenotype which were divided into three groups based on their plasma triglyceride content: low (TG<200 mg/dl, TG(l)), intermediate (200<300 mg/dl, TG(i)300 mg/dl, TG(h)). The protein density (PD) on the VLDL and LDL surface was calculated from lipoprotein composition and protein location was studied by tryptophan fluorescence quenching by I(-) anions at 25 degrees C and 40 degrees C. A comparison of the TG(h) with the TG(l) group revealed a significant (<0.05) increase of the PD parameter as much as 21% for VLDL, but not for LDL where this parameter did not change for any group; generally, PD(LDL) values were 3.2-3.8-fold lower than PD(VLDL). In accordance with this difference, the tryptophan accessibility f in VLDL vs. LDL was lower at both temperatures. There were temperature-induced changes of the f parameter in opposite directions for these lipoproteins. The difference in f value gradually decreased for VLDL in the direction TG(l)TG(i)TG(h) while for LDL there was a U-shaped dependence for these groups. The Stern-Volmer quenching constant K(S-V) which is sensitive to both temperature and viscosity, did not change for VLDL, but K(S-V)(LDL) was 2-3-fold higher for the TG(i) group compared to the other two. The efficiencies of VLDL and LDL binding to the LDL receptor (LDLr) in vitro were compared by solid-phase assay free of steric hindrance observed in cell binding. The maximal number of binding sites did not change for either type of particles and between groups. The association constant K(a) and apolipoprotein (apo) E/apoB mole ratio values all increased significantly for VLDL, but not for LDL, in comparison of the TG(i+h) with the TG(l) group. Based on VLDL and LDL concentrations in serum and on the affinity constant values obtained in an in vitro assay, VLDL concentrations corresponding to 50% inhibition of LDL binding (IC(50)) were calculated in an assumption of the competition of both ligands for LDLr in vivo; the mean values of IC(50) decreased 2-fold when plasma TG exceeded 200 mg/dl. The functional dependences of K(a)(VLDL), IC(50) and apoE content in VLDL (both fractional and absolute) and in serum on TG content in the whole concentration range studied were fitted to a saturation model. For all five parameters, the mean half-maximum values TG(1/2) were in the range 52-103 mg/dl. The efficiency of protein-protein interactions is suggested to differ in normolipidemic vs. HTG-VLDL and apoE content and/or protein density on VLDL surface may be the primary determinant(s) of the increased binding of HTG-VLDL to the LDL receptor. ApoCs may compete with apoE for the binding to the VLDL lipid surface as plasma triglyceride content increases. The possible competition of VLDL with LDL for the catabolism site(s) in vivo, when plasma TG increases, could explain the atherogenic action of TG-rich lipoproteins. Moreover, the 'dual action' hypothesis on anti-atherogenic action of apoE-containing high density lipoproteins (HDL) in vivo is suggested: besides the well-known effect of HDL as cholesteryl ester catabolic outway, the formation of a transient complex of apoE-containing discs appearing at the site of VLDL TG hydrolysis by lipoprotein lipase with VLDL particles proposed in our preceding paper promotes the efficient uptake of TG-rich particles; in hypertriglyceridemia due to the diminished HDL content this uptake seems to be impaired which results in the increased accumulation of the remnants of TG-rich particles. This explains the observed increase in cholesterol and triglyceride content in VLDL and LDL, respectively, due to the CETP-mediated exchange of cholesteryl ester and triglyceride molecules between these particles.     

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.     

Apolipoprotein E Structure and Substrate and Receptor-Binding Activities of Triglyceride-Rich Human Plasma Lipoproteins in Normo- and Hypertriglyceridemia

Cysteine-arginine interchanges along the primary sequence of human plasma apolipoprotein E (apoE) play an important role in determining its biological functions due to a high mutation frequency of cytosine in CGX triplet that codes 33 of 34 apolipoprotein arginine residues. The contribution of apoE secondary structure to apolipoprotein-lipid interaction is described. The significance of apolipoprotein in triglyceride synthesis, lipoprotein lipolysis, and receptor-mediated clearance of lipolytic remnants of triglyceride-rich lipoproteins is discussed as well. The metabolic flow of lipoproteins in normo- and hypertriglyceridemia can be described by separate compartments that contribute to lipoprotein interaction with at least six different receptors: 1) low density lipoprotein (LDL) receptor; 2) LDL receptor-related protein (LRP); 3) apoB(48) macrophage receptor for hypertriglyceridemic very low density lipoproteins (VLDL); 4) scavenger receptors; 5) VLDL receptor; 6) lipolysis-stimulated receptor. The contribution of the exposure of apoE molecules on the surface of triglyceride-rich particles sensitive both to lipolysis and plasma triglyceride content to the interaction with LDL receptor and LRP is emphasized.     

Apolipoprotein E mediates binding of normal very low density lipoprotein to heparin but is not required for high affinity receptor binding

The relationship between the cholesteryl ester content of normal human very low density lipoprotein (VLDL) and its ability to bind to apolipoprotein E (apoE), heparin, and the low density lipoprotein (LDL) receptor have been compared. Plasma VLDL were separated by heparin affinity chromatography into two fractions: one with apoE and one without. Both fractions had the same cholesteryl ester content relative to apolipoprotein B (apoB). LDL, on the other hand, had a greater cholesteryl ester content. VLDL were modified by lipolysis to express the ability to bind apoE (Ishikawa, Y., Fielding, C. J., and Fielding, P. E. (1988) J. Biol. Chem. 263, 2744-2749). Lipolyzed VLDL with or without apoE were compared for their ability to bind to heparin or the up-regulated fibroblast LDL receptor. Lipolyzed VLDL bound with the same affinity to the receptor whether or not the particles contained apoE. ApoB, not apoE, appears then to be the important ligand for normal VLDL. On the other hand, modified VLDL without apoE, even though binding to the LDL receptor, did not bind to heparin. These data suggest that apoE mediates heparin binding in normal VLDL, that apoB mediates receptor binding, and that the cholesteryl ester content of VLDL is not a factor in the induction of the ability to bind apoE.     

ApoC-III content of apoB-containing lipoproteins is associated with binding to the vascular proteoglycan biglycan

Retention of apolipoprotein (apo)B and apoE-containing lipoproteins by extracellular vascular proteoglycans is critical in atherogenesis. Moreover, high circulating apoC-III levels are associated with increased atherosclerosis risk. To test whether apoC-III content of apoB-containing lipoproteins affects their ability to bind to the vascular proteoglycan biglycan, we evaluated the impact of apoC-III on the interaction of [(35)S]SO(4)-biglycan derived from cultured arterial smooth muscle cells with lipoproteins obtained from individuals across a spectrum of lipid concentrations. The extent of biglycan binding correlated positively with apoC-III levels within VLDL (r = 0.78, P < 0.01), IDL (r = 0.67, P < 0.01), and LDL (r = 0.52, P < 0.05). Moreover, the biglycan binding of VLDL, IDL, and LDL was reduced after depletion of apoC-III-containing lipoprotein particles in plasma by anti-apoC-III immunoaffinity chromatography. Since apoC-III does not bind biglycan directly, enhanced biglycan binding may result from a conformational change associated with increased apo C-III content by which apoB and/or apoE become more accessible to proteoglycans. This may be an intrinsic property of lipoproteins, since exogenous apoC-III enrichment of LDL and VLDL did not increase binding. ApoC-III content may thus be a marker for lipoproteins characterized as having an increased ability to bind proteoglycans.     

Heparin binding triggers human VLDL remodeling by circulating lipoprotein lipase: Relevance to VLDL functionality in health and disease

Hydrolysis of VLDL triacylglycerol (TG) by lipoprotein lipase (LpL) is a major step in energy metabolism and VLDL-to-LDL maturation. Most functional LpL is anchored to the vascular endothelium, yet a small amount circulates on TG-rich lipoproteins. As circulating LpL has low catalytic activity, its role in VLDL remodeling is unclear. We use pre-heparin plasma and heparin-sepharose affinity chromatography to isolate VLDL fractions from normolipidemic, hypertriglyceridemic, or type-2 diabetic subjects. LpL is detected only in the heparin-bound fraction. Transient binding to heparin activates this VLDL-associated LpL, which hydrolyses TG, leading to gradual VLDL remodeling into IDL/LDL and HDL-size particles. The products and the timeframe of this remodeling closely resemble VLDL-to-LDL maturation in vivo. Importantly, the VLDL fraction that does not bind heparin is not remodeled. This relatively inert LpL-free VLDL is rich in TG and apoC-III, poor in apoE and apoC-II, shows impaired functionality as a substrate for the exogenous LpL or CETP, and likely has prolonged residence time in blood, which is expected to promote atherogenesis. This non-bound VLDL fraction increases in hypertriglyceridemia and in type-2 diabetes but decreases upon diabetes treatment that restores the glycemic control. In stark contrast, heparin binding by LDL increases in type-2 diabetes triggering pro-atherogenic LDL modifications. Therefore, the effects of heparin binding are associated negatively with atherogenesis for VLDL but positively for LDL. Collectively, the results reveal that binding to glycosaminoglycans initiates VLDL remodeling by circulating LpL, and suggest heparin binding as a marker of VLDL functionality and a readout for treatment of metabolic disorders.     

Capillary isotachophoresis study of lipoprotein network sensitive to apolipoprotein E phenotype. 2. ApoE and apoC-III relations in triglyceride clearance

The plasma (P), VLDL (V) triglyceride and apoB (B) clearance rates were measured both as ‘mass’ clearance (k ₁) and ‘within the particle’ clearance in three patient groups (E33, E23 and E34 phenotypes) at heparin-induced lipolysis in vivo. The lipid (C)- and apoE (E)-specific lipoprotein profiles both before and after heparin were followed by capillary isotachophoresis. The displacement of apoE by exogenous apoC-III at plasma titration in vitro was measured as well. The phenotype-sensitive lipoprotein networks were constructed based on an established set of metabolic rules. The k ₁(V) values did not differ between the three groups, but the lower k ₁(P) values showed significant differences. The k ₁(P) values for E33 and E23 groups were twofold higher compared to E34. A twofold increase in the rate constant for VLDL triglyceride clearance within the particle in E34 group compared to E23 reflected the inhibition of lipolysis by apoE2. For E33 group, (i) the k ₁(V) value was negatively correlated to the size of non-displaceable apoE pool in 2E lipoprotein and to the maximal apoE sorbtion capacity for 2E and 3E lipoproteins; (ii) the k ₁(P) value was not associated to the apoE binding parameters; (iii) the k ₁(V) value was positively correlated to the 4C level and the magnitude of apoC-III removal from VLDL particle; (iv) the k ₁(P) value was positively correlated to the content of apoE, while negatively with apoC-III, in VLDL remnants. For E34 group, the k ₁(V) value was positively correlated to 11C and 1–7C pool levels. Lipolysis- and receptor-mediated TG runways seem to be mostly balanced in E33 group, and VLDL TG clearance may be controlled by HDL through apoE dissociation from VLDLs and apolipoprotein accumulation within ‘fast’ HDLs at lipolysis.     

Capillary isotachophoresis study of lipoprotein network sensitive to apolipoprotein E phenotype. 1. ApoE distribution between lipoproteins

Sixteen patients differing widely in plasma triglyceride content were divided into three groups by their apolipoprotein E (apoE) phenotype—E33 homozygotes, E23, and E34 heterozygotes. The plasma lipid and apoE distribution between individual lipoproteins was followed by capillary isotachophoresis (CITP) of plasma samples pre-stained with lipid fluorescent probe NBD-C6-ceramide and by fluorescein-labeled apoE, respectively. Among 12 peaks visualized by ceramide staining, an individual peak with very low density lipoproteins (VLDL) was identified. The VLDL cholesterol and apoE content determined by CITP directly in whole plasma were significantly related to their content as determined by conventional analysis with isolated VLDL. The ceramide distribution among lipoprotein pools was insensitive to apoE phenotype (49–53 : 7–11 : 39–43% for HDL, VLDL, and IDL/LDL, respectively) while the preferential binding of apoE to VLDL was observed in E34 patients compared to E33 (62 : 19 : 20 vs. 70 : 9 : 22%). In a study of apoE/F displacement from lipoproteins at plasma titration by apoC-III in vitro, apoE was found to bind more tightly to VLDL from E34 compared to E33 patients as evidenced by both the increased non-displaceable apoE pool, the increased VLDL sorbtion capacity for apoE, and the decreased displacement parameter in a “container” model of lipoprotein binding. Two different types of apoE package in a whole lipoprotein profile were observed. ApoE structure in a particular lipoprotein may underlie the phenotype-sensitive apoE distribution and apoC-III interference in hypertriglyceridemia.     

Effect of apolipoprotein E genotype on apolipoprotein B-100 metabolism in normolipidemic and hyperlipidemic subjects

The effect of apolipoprotein (apo) E genotype on apoB-100 metabolism was examined in three normolipidemic apoE2/E2, five type III hyperlipidemic apoE2/E2, and five hyperlipidemic apoE3/E2 subjects using simultaneous administration of ¹³¹I-VLDL and ¹²⁵I-LDL, and multi-compartmental modeling. Compared with normolipidemic apoE2/E2 subjects, type III hyperlipidemic E2/E2 subjects had increased plasma and VLDL cholesterol, plasma and VLDL triglycerides, and VLDL and intermediate density lipoprotein (IDL) apoB concentrations (P < 0.05). These abnormalities were chiefly a consequence of decreased VLDL and IDL apoB fractional catabolic rate (FCR). Compared with hyperlipidemic E3/E2 subjects, type III hyperlipidemic E2/E2 subjects had increased IDL apoB concentration and decreased conversion of IDL to LDL particles (P < 0.05). In a pooled analysis, VLDL cholesterol was positively associated with VLDL and IDL apoB concentrations and the proportion of VLDL apoB in the slowly turning over VLDL pool, and was negatively associated with VLDL apoB FCR after adjusting for subject group. VLDL triglyceride was positively associated with VLDL apoB concentration and VLDL and IDL apoB production rates after adjusting for subject group. A defective apoE contributes to altered lipoprotein metabolism but is not sufficient to cause overt hyperlipidemia. Additional genetic mutations and environmental factors, including insulin resistance and obesity, may contribute to the development of type III hyperlipidemia.     

Increased production of apolipoprotein B and its lipoproteins by oleic acid in Caco-2 cells

The production of lipids, apolipoproteins (apo), and lipoproteins induced by oleic acid has been examined in Caco-2 cells. The rates of accumulation in the control medium of 15-day-old Caco-2 cells of triglycerides, unesterified cholesterol, and cholesteryl esters were 102 +/- 8, 73 +/- 5, and 11 +/- 1 ng/mg cell protein/h, respectively; the accumulation rates for apolipoproteins A-I, B, C-III, and E were 111 +/- 9, 53 +/- 4, 13 +/- 1, and 63 +/- 4 ng/mg cell protein/h, respectively. Whereas apolipoproteins A-IV and C-II were detected by immunoblotting, apoA-II was absent in most culture media. In contrast to an early production of apolipoproteins A-I and E occurring 2 days after plating, the apoB expression appeared to be differentiation-dependent and was not measurable in the medium until the sixth day post-confluency. In the control medium, very low density lipoproteins (VLDL), low density lipoproteins (LDL), high density lipoproteins (HDL), and lipid-poor very high density lipoproteins (VHDL) accounted for 12%, 46%, 18%, and 24% of the total lipid and apolipoprotein contents, respectively. The triglyceride-rich VLDL contained mainly apoE (75%) and apoB (23%), while the protein moiety of LDL was composed of apoB (59%), apoE (20%), apoA-I (15%), and apoC-III (6%). The cholesterol-rich HDL contained mainly apoA-I (69%) and apoE (27%). In the control medium, major portions of apolipoproteins B and C-III (93-97%) were present in LDL, whereas the main parts of apoA-I (92%) and apoE (76%) were associated with HDL and VHDL. Oleate increased the production of triglycerides 10-fold, cholesteryl esters 7-fold, and apoB 2- to 4-fold. There was also a moderate increase (39%) in the production of apoC-III but no significant changes in those of apolipoproteins A-I and E. These increases were reflected mainly in a 55-fold elevation in the concentration of VLDL, and a 2-fold increase in the level of LDL; there were no significant changes in HDL and VHDL. VLDL contained the major parts of total neutral lipids (74-86%), apoB (65%), apoC-III (81%) and apoE (58%). In the presence of oleate, the VLDL, LDL, HDL, and VHDL accounted for 76%, 15%, 3%, and 6% of the total lipoproteins, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)     

Conformation of apolipoprotein E both in free and in lipid-bound form may determine the avidity of triglyceride-rich lipoproteins to the LDL receptor: structural and kinetic study

Slow refolding of human apolipoprotein E (apoE) in solution after guanidine- or cholate-induced denaturation followed by dialysis under controlled conditions was investigated using various spectroscopic properties of fluorescein- and dansyl-labeled apolipoprotein molecules. The results suggest that the last phase(s) of apoE refolding in solution include a slow (several hours at 24 degrees C) interconversion of a self-associated 'open' conformer into a more dense 'closed' conformer. The hydrophobic interactions are primarily responsible for the formation of this more compact apoE structure. To visualize the contribution of apolipoprotein conformation and/or the number of 'active' lipid-bound apoE molecules in the reaction of binding to the low density lipoprotein receptor (LDLr) by solid-phase binding assay, the complexes of human plasma apolipoprotein or recombinant (rec) apoE3 with dipalmitoylphosphatidylcholine (DPPC) or palmitoyloleoylphosphatidylcholine (POPC) varying in size were used. For seven complexes with plasma protein (four DPPC and three POPC complexes), the final phosphatidylcholine (PC)/protein mole ratio ranged from 117 to 279; affinity constant K(a) averaged for both PCs and plotted against this ratio abruptly increased from 3.8 x 10(7) to 3.8 x 10(8) M(-1) with a transition midpoint of 150-180 PC/apoE, mole ratio. Two DPPC complexes with rec protein bind much more efficiently. Complexes with both plasma and rec apoE were able to compete with very low density lipoproteins (VLDL) or low density lipoproteins (LDL) isolated from patients with E3/3 phenotype, for binding to the LDLr. Again, the competition efficiency abruptly increased at the increase in PC content with a transition midpoint of 130 PC/apoE, mole ratio. The transitions observed both in direct and competitive binding assay probably correspond to the abrupt increase in the number of 'active' apoE molecules on the complex surface accompanying the change in the size and/or in the shape of the complexes. The efficiency of apoE and apoB as the corresponding major ligands in the binding reaction of VLDL and LDL to the LDL receptor was compared. VLDL bind to LDLr following a simple encounter complex model, while LDL binding was characterized by a more complex two-step model with an additional isomerization step. The analysis of the binding data led us to suggest the existence of the continuum from several (2-3) apoE molecules on the surface of TG-rich particles that resulted in the increased binding affinity, on average 3.5-fold higher, compared to LDL. The existence of a complex equilibrium between aqueous and different lipid-bound forms of apoE is proposed, in particular, the formation of a transient disc-lipoprotein particle structure during the interaction with LDLr in vivo as well as in LPL-stimulated lipolysis of the lipid phase of the particle.     

Apolipoprotein C-III displacement of apolipoprotein E from VLDL: effect of particle size.

ApoC-III and apoE are important determinants of intravascular lipolysis and clearance of triglyceride-rich chylomicrons and VLDL from the blood plasma. Interactions of these two apolipoproteins were studied by adding purified human apoC-III to human plasma at levels observed in hypertriglyceridemic subjects and incubating under specific conditions (2 h, 37 degrees C). As plasma concentrations of apoC-III protein were increased, the contents in both VLDL and HDL were also increased. Addition of apoC-III at concentrations up to four times the intrinsic concentration resulted in the decreasing incremental binding of apoC-III to VLDL while HDL bound increasing amounts without evidence of saturation. No changes were found in lipid content or in particle size of any lipoprotein in these experiments. However, distribution of the intrinsic apoE in different lipoprotein particles changed markedly with displacement of apoE from VLDL to HDL. The fraction of VLDL apoE that was displaced from VLDL to HDL at these high apoC-III concentrations varied among individuals from 20% to 100% its intrinsic level. The proportion of VLDL apoE that was tightly bound (0% to 80%) was found to be reproducible and to correlate with several indices of VLDL particle size. In the group of subjects studied, strongly adherent apoE was essentially absent from VLDL particles having an average content of less than 50,000 molecules of triglyceride.Addition of apoC-III to plasma almost completely displaces apoE from small VLDL particles. Larger VLDL contain tightly bound apoE which are not displaced by increasing concentration of apoC-III.     

Antisense inhibition of apoB synthesis with mipomersen reduces plasma apoC-III and apoC-III-containing lipoproteins

Mipomersen, an antisense oligonucleotide that reduces hepatic production of apoB, has been shown in phase 2 studies to decrease plasma apoB, LDL cholesterol (LDL-C), and triglycerides. ApoC-III inhibits VLDL and LDL clearance, and it stimulates inflammatory responses in vascular cells. Concentrations of VLDL or LDL with apoC-III independently predict cardiovascular disease. We performed an exploratory posthoc analysis on a subset of hypercholesterolemic subjects obtained from a randomized controlled dose-ranging phase 2 study of mipomersen receiving 100, 200, or 300 mg/wk, or placebo for 13 wk (n = 8 each). ApoC-III-containing lipoproteins were isolated by immuno-affinity chromatography and ultracentrifugation. Mipomersen 200 and 300 mg/wk reduced total apoC-III from baseline by 6 mg/dl (38-42%) compared with placebo group (P < 0.01), and it reduced apoC-III in both apoB lipoproteins and HDL. Mipomersen 100, 200, and 300 mg doses reduced apoB concentration of LDL with apoC-III (27%, 38%, and 46%; P < 0.05). Mipomersen reduced apoC-III concentration in HDL. The drug had no effect on apoE concentration in total plasma and in apoB lipoproteins. In summary, antisense inhibition of apoB synthesis reduced plasma concentrations of apoC-III and apoC-III-containing lipoproteins. Lower concentrations of apoC-III and LDL with apoC-III are associated with reduced risk of coronary heart disease (CHD) in epidemiologic studies independent of traditional risk factors.     

Increased concentrations of circulating vitamin E in carriers of the apolipoprotein A5 gene - 1131T>C variant and associations with plasma lipids and lipid peroxidation

The aim of this study was to investigate the effects of the apolipoprotein A5 (APOA5) 1131T>C gene variant on vitamin E status and lipid profile. The gene variant was determined in 297 healthy nonsmoking men aged 20-75 years and recruited in the VITAGE Project. Effects of the genotype on vitamin E in plasma, LDL, and buccal mucosa cells (BMC) as well as on cholesterol and triglyceride (TG) concentrations in plasma and apolipoprotein A-I (apoA-I), apoB, apoE, apoC-III, and plasma fatty acids were determined. Plasma malondialdehyde concentrations as a marker of in vivo lipid peroxidation were determined. C allele carriers showed significantly higher TG, VLDL, and LDL in plasma, higher cholesterol in VLDL and intermediate density lipoprotein, and higher plasma fatty acids. Plasma alpha-tocopherol (but not gamma-tocopherol, LDL alpha- and gamma-tocopherol, or BMC total vitamin E) was increased significantly in C allele carriers compared with homozygote T allele carriers (P = 0.02), but not after adjustment for cholesterol or TG. Plasma malondialdehyde concentrations did not differ between genotypes. In conclusion, higher plasma lipids in the TC+CC genotype are efficiently protected against lipid peroxidation by higher alpha-tocopherol concentrations. Lipid-standardized vitamin E should be used to reliably assess vitamin E status in genetic association studies.     

The beta very low density lipoprotein present in hepatic lipase deficiency competitively inhibits low density lipoprotein binding to fibroblasts and stimulates fibroblast acyl-CoA:cholesterol acyltransferase

Beta very low density lipoprotein (VLDL) was isolated from a patient with hepatic lipase deficiency. The particles were found to contain apolipoprotein B-100 (apoB) and apolipoprotein E (apoE) and were rich in cholesterol and cholesteryl ester relative to VLDL with pre beta electrophoretic mobility. These particles were active in displacing human low density lipoprotein (LDL) from the fibroblast apoB,E receptor and produced a marked stimulation of acyl-CoA:cholesterol acyltransferase. Treatment of intact beta-VLDL with trypsin abolished its ability to displace LDL from fibroblasts. Incubation of trypsin treated beta-VLDL with fibroblasts resulted in a significant stimulation of acyl-CoA:cholesterol acyltransferase activity. beta-VLDL isolated from a patient with Type III hyperlipoproteinemia and an apoE2/E2 phenotype had a higher cholesteryl ester/triglyceride ratio than the beta-VLDL of hepatic lipase deficiency and contained apoB48. It displaced LDL from fibroblasts to a small but significant extent. The Type III beta-VLDL stimulated acyl-CoA:cholesterol acyltransferase to a level similar to that of trypsin-treated beta-VLDL isolated from the hepatic lipase-deficient patient. These results demonstrate that the cholesterol-rich beta-VLDL particles present in patients with hepatic lipase deficiency are capable of interacting with fibroblasts via the apoB,E receptor and that this interaction is completely due to trypsin-sensitive components of the beta-VLDL. These particles were very effective in stimulating fibroblast acyl-CoA:cholesterol acyltransferase. This stimulation was due to both trypsin-sensitive and trypsin-insensitive components.     

Plasma kinetics of apoC-III and apoE in normolipidemic and hypertriglyceridemic subjects

Apolipoprotein (apo) C-III and apoE play a central role in controlling the plasma metabolism of triglyceride-rich lipoproteins (TRL). We have investigated the plasma kinetics of total, very low density lipoprotein (VLDL) and high density lipoprotein (HDL) apoC-III and apoE in normolipidemic (NL) (n = 5), hypertriglyceridemic (HTG, n = 5), and Type III hyperlipoproteinemic (n = 2) individuals. Apolipoprotein kinetics were investigated using a primed constant (12 h) infusion of deuterium-labeled leucine. HTG and Type III patients had reduced rates of VLDL apoB-100 catabolism and no evidence of VLDL apoB-100 overproduction. Elevated (3- to 12-fold) total plasma and VLDL apoC-III levels in HTG and Type III patients, although associated with reduced apoC-III catabolism (i.e., increased residence times (RTs)), were mainly due to increased apoC-III production (plasma apoC-III transport rates (TRs, mean +/- SEM): (NL) 2.05 +/- 0.22 (HTG) 4.90 +/- 0.81 (P < 0.01), and (Type III) 8.78 mg. kg(-)(1). d(-)(1); VLDL apoC-III TRs: (NL) 1.35 +/- 0. 23 (HTG) 5.35 +/- 0.85 (P < 0.01), and (Type III) 7.40 mg. kg(-)(1). d(-)(1)). Elevated total plasma and VLDL apoE levels in HTG (2- and 6-fold, respectively) and in Type III (9- and 43-fold) patients were associated with increased VLDL apoE RTs (0.21 +/- 0.02, 0.46 +/- 0. 05 (P < 0.01), and 1.21 days, NL vs. HTG vs. Type III, respectively), as well as significantly increased apoE TRs (plasma: (NL) 2.94 +/- 0.78 (HTG) 5.80 +/- 0.59 (P < 0.01) and (Type III) 11.80 mg. kg(-)(1). d(-)(1); VLDL: (NL) 1.59 +/- 0.18 (HTG) 4.52 +/- 0.61 (P < 0.01) and (Type III) 11.95 mg. kg(-)(1). d(-)(1)).These results demonstrate that hypertriglyceridemic patients, having reduced VLDL apoB-100 catabolism (including patients with type III hyperlipoproteinemia) are characterized by overproduction of plasma and VLDL apoC-III and apoE.     

Lipolysis is a prerequisite for lipid accumulation in HepG2 cells induced by large hypertriglyceridemic very low density lipoproteins.

Lipoprotein kinetic studies have demonstrated that a large proportion of Sf 60-400 very low density lipoprotein (VLDL) is cleared directly from the circulation in Type IV hypertriglyceridemic subjects, at an unknown tissue site. The present studies were designed to investigate the role of hepatocytes in this process and to define the conditions, whereby Type IV Sf 60-400 VLDL would induce lipid accumulation in HepG2 cells. Type IV VLDL (Sf 60-400) failed to augment the total cholesterol, esterified cholesterol, or triglyceride content of HepG2 cells following 24-h incubations. Coincubation of bovine milk lipoprotein lipase (LPL) and Type IV VLDL with HepG2 cells induced a 3-fold increment in cellular esterified cholesterol mass (p less than 0.005) and a 7-fold increase in cellular triglyceride mass (p less than 0.005), compared to VLDL alone. The increased cellular lipid mass was associated with increased oleate incorporation into cellular cholesterol esters and triglycerides. Exogenous LPL hydrolyzed 76% of the VLDL triglyceride over 24 h. LPL action on Type IV VLDL was sufficient to promote cellular uptake of these lipoproteins, while elevated media-free fatty acid levels were not. Although HepG2 cells secrete apolipoprotein (apo) E, we assessed the role of VLDL-associated apoE in the lipid accumulation induced by VLDL plus LPL. ApoE-rich and apoE-poor Type IV VLDL subfractions induced similar increments in cellular esterified cholesterol in the presence of LPL, despite a 4-fold difference in apoE content. Sf 60-400 VLDL, from subjects homozygous for the defective apoE2, plus LPL, behaved identically to Type IV VLDL plus LPL. Type IV VLDL plus LPL, preincubated with anti-apoE (1D7) and apoB (5E11) monoclonal antibodies, known to block the binding of apoE and -B, respectively, to the LDL receptor failed to block lipid accumulation. In contrast, apoE-poor Type IV VLDL, apoE2 VLDL, and VLDL plus 1D7 were taken up poorly by J774 cells, cells that secrete LPL, but not apoE. These studies suggest that lipolytic remodeling of large Type IV VLDL by LPL is a prerequisite for their uptake by HepG2 cells and that HepG2 cell-secreted apoE rather than VLDL-associated apoE is the ligand involved in uptake.