Suppression of 3-hydroxy-3-methylglutaryl-CoA reductase by low density lipoproteins produced in vitro by lipoprotein lipase action on nonsuppressive very low density lipoproteins.

Very low density lipoproteins (VLDL), Sf60 to 400, from normolipemic individuals do not suppress 3-hydroxy-3-methylglutaryl-CoA reductase activity in cultured normal human fibroblasts at concentrations 20-fold higher than those of low density lipoproteins (LDL) that give total suppression. To determine if these VLDL contain all of the structural elements necessary for receptor-mediated suppression, they were converted in vitro with bovine milk lipoprotein lipase to low density lipoproteins. These LDL-like lipoproteins were as effective in suppression as LDL isolated directly from plasma, with half-maximal and complete suppression at 1 and 4 microgram of cholesterol ml-1. Neither native LDL nor LDL produced in vitro suppressed receptor-negative fibroblasts. We conclude that action of lipoprotein lipase on VLDL leads to a rearrangement of lipoprotein components that permits interaction of LDL produced in vitro with the LDL-specific cell surface receptor of fibroblasts and subsequent suppression of 3-hydroxy-3-methylglutaryl-CoA reductase.     

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.     

Interactions of high density lipoproteins with very low and low density lipoproteins during lipolysis

Interactions of high density lipoproteins (HDL) with very low (VLDL) and low (LDL) density lipoproteins were investigated during in vitro lipolysis in the presence of limited free fatty acid acceptor. Previous studies had shown that lipid products accumulating on lipoproteins under these conditions promote the formation of physical complexes between apolipoprotein B-containing particles (Biochim. Biophys. Acta, 1987. 919: 97-110). The presence of increasing concentrations of HDL or delipidated HDL progressively diminished VLDL-LDL complex formation. At the same time, association of HDL-derived apolipoprotein (apo) A-I with both VLDL and LDL could be demonstrated by autoradiography of gradient gel electrophoretic blots, immunoblotting, and apolipoprotein analyses of reisolated lipoproteins. The LDL increased in buoyancy and particle diameter, and became enriched in glycerides relative to cholesterol. Both HDL2 and HDL3 increased in particle diameter, buoyancy, and relative glyceride content, and small amounts of apoA-I appeared in newly formed particles of less than 75 A diameter. Association of apoA-I with VLDL or LDL could be reproduced by addition of lipid extracts of lipolyzed VLDL or purified free fatty acids in the absence of lipolysis, and was progressively inhibited by the presence of increasing amounts of albumin. We conclude that lipolysis products promote multiple interactions at the surface of triglyceride-rich lipoproteins undergoing lipolysis, including physical complex formation with other lipoprotein particles and transfers of lipids and apolipoproteins. These processes may facilitate remodeling of lipoproteins in the course of their intravascular metabolism.     

Secondary structure in very low density and intermediate density lipoproteins of human serum.

We have studies the secondary structures of the protein moieties of very low density lipoproteins (VLDL) and intermediate density lipoproteins (IDL) of human serum by circular dichroism (CD). Two potential complications in the application of this technique to lipoproteins have been evaluated. First, using chronographic potentiometry in CD measurements of VLDL fractions of different mean particle diameters, we have analyzed statistically the CD signals in order to define the limits imposed by light scattering with respect to both particle diameter and wavelength. We found that CD measurements can be made to as low as 210 nm on particles of 520 A or smaller, and to 194 nm on particles of 450 A and below. Second, we have evaluated the CD contribution of lipid chromophores. Despite the high ratio of lipid to protein, the relative CD effect of the lipids is smaller than for low density lipoproteins (LDL). due to the extremely small ellipticity of natural VLDL triglycerides. Thus, CD measurements can be obtained with confidence on the preponderant bulk of normal VLDL. For the first time we report the CD spectra of human VLDL and IDL. In contrast with human LDL and the lipoproteins of the hypercholesterolemic rabbit, the entire CD SPECTRUM OF HUMAN VLDL shows increased ellipticity with decreasing temperature, which is completely reversible. We have found that the protein moieties of human VLDL and IDL contain substantially more helix (approximately 50%) than does that of human LDL.     

Dissociation of high density lipoprotein precursors from apolipoprotein B-containing lipoproteins in the presence of unesterified fatty acids and a source of apolipoprotein A-I

Incubation of low (LDL), intermediate (IDL), or very low density lipoproteins (VLDL) with palmitic acid and either high density lipoproteins (HDL), delipidated HDL, or purified apolipoprotein (apo) A-I resulted in the formation of lipoprotein particles with discoidal structure and mean particle diameters ranging from 146 to 254 A by electron microscopy. Discs produced from IDL or LDL averaged 26% protein, 42% phospholipid, 5% cholesteryl esters, 24% free cholesterol, and 3% triglycerides; preparations derived from VLDL contained up to 21% triglycerides. ApoA-I was the predominant protein present, with smaller amounts of apoA-II. Crosslinking studies of discs derived from LDL or IDL indicated the presence of four apoA-I molecules per particle, while those derived from large VLDL varied more in size and contained as many as six apoA-I molecules per particle. Incubation of discs derived from IDL or LDL with purified lecithin:cholesterol acyltransferase (LCAT), albumin, and a source of free cholesterol produced core-containing particles with size and composition similar to HDL2b. VLDL-derived discs behaved similarly, although the HDL products were somewhat larger and more variable in size. When discs were incubated with plasma d greater than 1.21 g/ml fraction rather than LCAT, core-containing particles in the size range of normal HDL2a and HDL3a were also produced. A variety of other purified free fatty acids were shown to promote disc formation. In addition, some mono and polyunsaturated fatty acids facilitated the formation of smaller, spherical particles in the size range of HDL3c. Both discoidal and small spherical apoA-I-containing lipoproteins were generated when native VLDL was incubated with lipoprotein lipase in the presence of delipidated HDL. We conclude that lipolysis product-mediated dissociation of lipid-apoA-I complexes from VLDL, IDL, or LDL may be a mechanism for formation of HDL subclasses during lipolysis, and that the availability of different lipids may influence the type of HDL-precursors formed by this mechanism.     

Metabolism of apoprotein B in selectively bred baboons with low and high levels of low density lipoproteins

Very low density lipoprotein (VLDL) and low density lipoprotein (LDL) apoprotein (apo)-B turnover rates were measured simultaneously by injecting 131I-labeled VLDL and 125I-labeled LDL into fasting baboons (Papio sp.) selectively bred for high serum cholesterol levels and having either low or high LDL levels. The radioactivities in VLDL, intermediate density lipoprotein (IDL), LDL apoB, and urine were measured at intervals between 5 min and 6 days. Kinetic parameters for apoB were calculated in each baboon fed a chow diet or a high cholesterol, high fat diet (HCHF). VLDL apoB residence times were similar in the two groups of animals fed chow; they were increased by HCHF feeding in high LDL animals, but not in low LDL animals. Production rates of VLDL apoB were decreased by the HCHF diet in both high and low LDL animals. Most of the radioactivity from VLDL apoB was transferred to IDL. However, a greater proportion of radioactivity was removed directly from IDL apoB in low LDL animals than in high LDL animals, and only about one-third appeared in LDL. In high LDL animals, a greater proportion of this radioactivity was converted to LDL (61.4 +/- 7.2% in chow-fed animals and 49.2 +/- 10.9% in animals fed the HCHF diet; mean +/- SEM, n = 5). Production rates for LDL apoB were higher in high LDL animals than those in low LDL animals on both diets. The HCHF diet increased residence times of LDL apoB without changing production rates in both groups. VLDL apoB production was not sufficient to account for LDL apoB production in high LDL animals, a finding that suggested that a large amount of LDL apoB was derived from a source other than VLDL apoB in these animals.     

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.     

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.     

Size transformations of intermediate and low density lipoproteins induced by unesterified fatty acids

Plasma from individual human subjects is known to contain multiple discrete subpopulations of low (LDL) and intermediate (IDL) density lipoproteins that differ in particle size and density. The metabolic origins of these subpopulations are unknown. Transformation of IDL and larger LDL to smaller, denser LDL particles had been postulated to occur as a result of the combined effects of triglyceride hydrolysis and lipid transfer. However, the presence of multiple small LDL subspecies has been described in patients lacking cholesteryl ester transfer protein. We have characterized an alternative pathway in which size decrements in IDL or LDL are produced in the presence of unesterified fatty acids and a source of apolipoprotein (apo) A-I. Incubation of IDL or LDL subfractions with palmitic acid and either high density lipoproteins (HDL), apoHDL, or purified apoA-I gives rise to apoA-I, apoB-containing complexes that can dissociate into two particles, an apoB-containing lipoprotein with particle diameter 10-30 A smaller than the starting material, and a still smaller species (apparent peak particle diameter 140-190 A) containing lipid and apoA-I but no apoB. The newly formed IDL or LDL are depleted in phospholipid and free cholesterol with no change in apoB-100 as assessed by SDS gel electrophoresis. We hypothesize that this reaction may contribute to the formation of discrete IDL and LDL subpopulations of varying size during the course of hydrolysis of triglyceride-rich lipoproteins in plasma.     

Metabolism of apoB-100 in lipoproteins separated by density gradient ultracentrifugation in normal and Watanabe heritable hyperlipidemic rabbits

We have examined the capability of a previously developed compartmental model to explain the kinetics of radioiodinated apolipoprotein (apo) B-100 in very low density lipoproteins (VLDL), intermediate density lipoproteins (IDL), and low density lipoproteins (LDL) separated by density gradient ultracentrifugation after intravenous injection of radioiodinated VLDL into New Zealand white (NZW) and Watanabe heritable hyperlipidemic (WHHL) rabbits. Our model was developed primarily from kinetics in whole blood plasma of apoB-100 in particles with and without apoE after intravenous injection of large VLDL, total VLDL, IDL, and LDL. When the initial conditions for this model were assumed to be an intravenous injection of radiolabeled VLDL, the plasma VLDL and LDL simulations for NZW rabbits and the VLDL, IDL, and LDL simulations for WHHL rabbits were found to be inconsistent with the observed density gradient data. By adding a new pathway in the VLDL portion of the model for NZW rabbits and a new compartment in VLDL for WHHL rabbits, and by assuming some cross-contamination in the density gradient ultracentrifugal separations, it was possible to bring our model, which was based upon measurements of 125I-labeled apoB-100 in whole plasma, into conformity with the data obtained by density gradient ultracentrifugation. The relatively modest changes required in the model to fit the gradient ultracentrifugation data support the suitability of our approach to the kinetic analysis of the metabolism of apoB-100 in VLDL and its conversion to IDL and LDL based upon measurements of 125I-labeled apoB-100 in whole plasma after injection of radiolabeled VLDL, IDL, and LDL. Furthermore, the differences in kinetics observed by us between data from whole plasma and data from plasma submitted to ultracentrifugal separation from the same or similar animals highlight the fact that small variations that can occur in the separation of lipoprotein classes by buoyant density can lead to confusing results.     

Resistance of a very low density lipoprotein subpopulation from familial dysbetalipoproteinemia to in vitro lipolytic conversion to the low density lipoprotein density fraction

In vitro lipolysis of very low density lipoprotein (VLDL) from normolipidemic and familial dysbetalipoproteinemic plasma by purified bovine milk lipoprotein lipase was studied using the combined single vertical spin and vertical autoprofile method of lipoprotein analysis. Lipolysis of normolipidemic plasma supplemented with autologous VLDL resulted in the progressive transformation of VLDL to low density lipoprotein (LDL) via intermediate density lipoprotein (IDL) with the transfer of the excess cholesterol to high density lipoprotein (HDL). At the end of 60 min lipolysis, 92-96% of VLDL triglyceride was hydrolyzed, and, with this process, greater than 95% of the VLDL cholesterol and 125-I-labeled VLDL protein was transferred from the VLDL to the LDL and HDL density region. When VLDL from the plasma of an individual with familial dysbetalipoproteinemia was substituted for VLDL from normolipidemic plasma, less than 50% of the VLDL cholesterol and 65% of 125I-labeled protein was removed from the VLDL density region, although 84-86% of VLDL triglyceride was lipolyzed. Analysis of familial dysbetalipoproteinemic VLDL fractions from pre- and post-lipolyzed plasma showed that the VLDL remaining in the postlipolyzed plasma (lipoprotein lipase-resistant VLDL) was richer in cholesteryl ester and tetramethylurea-insoluble proteins than that from prelipolysis plasma; the major apolipoproteins in the lipoprotein lipase-resistant VLDL were apoB and apoE. During lipolysis of normolipidemic VLDL containing trace amounts of 125I-labeled familial dysbetalipoproteinemic VLDL, removal of VLDL cholesterol was nearly complete from the VLDL density region, while removal of 125I-labeled protein was only partial. A competition study for lipoprotein lipase, comparing normolipidemic and familial dysbetalipoproteinemic VLDL to an artificial substrate ([3H]triolein), revealed that normolipidemic VLDL is clearly better than familial dysbetalipoproteinemic VLDL in competing for the release of 3H-labeled free fatty acids. The results of this study suggest that, in familial dysbetalipoproteinemic individuals, a subpopulation of VLDL rich in cholesteryl ester, apoB, and apoE is resistant to in vitro conversion by lipoprotein lipase to particles having LDL-like density. The presence of this lipoprotein lipase-resistant VLDL in familial dysbetalipoproteinemic subjects likely contributes to the increased level of cholesteryl ester-rich VLDL and IDL in the plasma of these subjects.     

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.     

Mechanisms of inhibition by apolipoprotein C of apolipoprotein E-dependent cellular metabolism of human triglyceride-rich lipoproteins through the low density lipoprotein receptor pathway

The mechanism of inhibition by apolipoprotein C of the uptake and degradation of triglyceride-rich lipoproteins from human plasma via the low density lipoprotein (LDL) receptor pathway was investigated in cultured human skin fibroblasts. Very low density lipoprotein (VLDL) density subfractions and intermediate density lipoprotein (IDL) with or without added exogenous recombinant apolipoprotein E-3 were used. Total and individual (C-I, C-II, C-III-1, and C-III-2) apoC molecules effectively inhibited apoE-3-mediated cell metabolism of the lipoproteins through the LDL receptor, with apoC-I being most effective. When the incubation was carried out with different amounts of exogenous apoE-3 and exogenous apoC, it was shown that the ratio of apoE-3 to apoC determined the uptake and degradation of VLDL. Excess apoE-3 overcame, at least in part, the inhibition by apoC. ApoC, in contrast, did not affect LDL metabolism. Neither apoA-I nor apoA-II, two apoproteins that do not readily associate with VLDL, had any effect on VLDL cell metabolism. The inhibition of VLDL and IDL metabolism cannot be fully explained by interference of association of exogenous apoE-3 with or displacement of endogenous apoE from the lipoproteins. IDL is a lipoprotein that contains both apoB-100 and apoE. By using monoclonal antibodies 4G3 and 1D7, which specifically block cell interaction by apoB-100 and apoE, respectively, it was possible to assess the effects of apoC on either apoprotein. ApoC dramatically depressed the interaction of IDL with the fibroblast receptor through apoE, but had only a moderate effect on apoB-100. The study thus demonstrates that apoC inhibits predominantly the apoE-3-dependent interaction of triglyceride-rich lipoproteins with the LDL receptor in cultured fibroblasts and that the mechanism of inhibition reflects association of apoC with the lipoproteins and specific concentration-dependent effects on apoE-3 at the lipoprotein surface.     

Binding of intermediate density lipoproteins rich or poor in high molecular weight apolipoprotein B to rat liver membranes

Very low density lipoproteins rich or poor in high molecular weight apolipoprotein B (Bh-rich or Bh-poor VLDL, respectively) were prepared from rats fasted for 2 days and animals fasted and then refed for 2 days, respectively. Bh-rich or Bh-poor VLDL remnants (IDL) were also prepared by in vitro lipolysis of the corresponding VLDL preparations, and their apolipoprotein (apo) profile and lipid composition determined. Bh-rich IDL are richer in esterified cholesterol than Bh-poor IDL, but poorer in apoC and triglycerides. The binding of 125I-labeled Bh-rich IDL and 125I-labeled Bh-poor IDL to rat liver membranes was assessed by saturation-curve studies. Both types of IDL bound to high- and low-affinity sites on rat liver membranes. There were no significant differences between the binding of IDL produced from Bh-rich or Bh-poor VLDL to either the high- or low-affinity sites. However, by masking the low-affinity binding sites with saturating amounts of human high density lipoproteins 3 (HDL3), we were able to demonstrate that Bh-rich IDL bound to high-affinity binding sites with five times less affinity than Bh-poor IDL. These results show that saturating the low-affinity binding sites of rat liver membranes reveals differences in the binding abilities of lipoproteins to the high-affinity sites. Also, an analysis of apo and lipid compositions of the two types of IDL reveals that the apoBh contribution is likely to be responsible for differences in affinities of IDL for the high-affinity binding sites of rat liver membranes.     

Lovastatin therapy reduces low density lipoprotein apoB levels in subjects with combined hyperlipidemia by reducing the production of apoB-containing lipoproteins: implications for the pathophysiology of apoB production

We investigated the metabolism of very low density lipoprotein (VLDL), intermediate density lipoprotein (IDL), and low density lipoprotein (LDL) apolipoprotein B (apoB) in seven patients with combined hyperlipidemia (CHL), using 125I-labeled VLDL and 131I-labeled LDL and compartmental modeling, before and during lovastatin treatment. Lovastatin therapy significantly reduced plasma levels of LDL cholesterol (142 vs 93 mg/dl, P less than 0.0005) and apoB (1328 vs 797 micrograms/ml, P less than 0.001). Before treatment, CHL patients had high production rates (PR) of LDL apoB. Three-fourths of this LDL apoB flux was derived from sources other than circulating VLDL and was, therefore, defined as "cold" LDL apoB flux. Compared to baseline, treatment with lovastatin was associated with a significant reduction in the total rate of entry of apoB-containing lipoproteins into plasma in all seven CHL subjects (40.7 vs. 25.7 mg/kg.day, P less than 0.003). This reduction was associated with a fall in total LDL apoB PR and in "cold" LDL apoB PR in six out of seven CHL subjects. VLDL apoB PR fell in five out of seven CHL subjects. Treatment with lovastatin did not significantly alter VLDL apoB conversion to LDL apoB or LDL apoB fractional catabolic rate (FCR) in CHL patients. In three patients with familial hypercholesterolemia who were studied for comparison, lovastatin treatment increased LDL apoB FCR but did not consistently alter LDL apoB PR. We conclude that lovastatin lowers LDL cholesterol and apoB concentrations in CHL patients by reducing the rate of entry of apoB-containing lipoproteins into plasma, either as VLDL or as directly secreted LDL.     

The peroxisome proliferator-activated receptor alpha (PPARalpha) agonist ciprofibrate inhibits apolipoprotein B mRNA editing in low density lipoprotein receptor-deficient mice: effects on plasma lipoproteins and the development of atherosclerotic lesions

Low density lipoprotein receptor (LDLR)-deficient mice fed a chow diet have a mild hypercholesterolemia caused by the abnormal accumulation in the plasma of apolipoprotein B (apoB)-100- and apoB-48-carrying intermediate density lipoproteins (IDL) and low density lipoproteins (LDL). Treatment of LDLR-deficient mice with ciprofibrate caused a marked decrease in plasma apoB-48-carrying IDL and LDL but at the same time caused a large accumulation of triglyceride-depleted apoB-100-carrying IDL and LDL, resulting in a significant increase in plasma cholesterol levels. These plasma lipoprotein changes were associated with an increase in the hepatic secretion of apoB-100-carrying very low density lipoproteins (VLDL) and a decrease in the secretion of apoB-48-carrying VLDL, accompanied by a significant decrease in hepatic apoB mRNA editing. Hepatic apobec-1 complementation factor mRNA and protein abundance were significantly decreased, whereas apobec-1 mRNA and protein abundance remained unchanged. No changes in apoB mRNA editing occurred in the intestine of the treated animals. After 150 days of treatment with ciprofibrate, consistent with the increased plasma accumulation of apoB-100-carrying IDL and LDL, the LDLR-deficient mice displayed severe atherosclerotic lesions in the aorta. These findings demonstrate that ciprofibrate treatment decreases hepatic apoB mRNA editing and alters the pattern of hepatic lipoprotein secretion toward apoB-100-associated VLDL, changes that in turn lead to increased atherosclerosis.     

The catabolism of human and rat very low density lipoproteins by perfused rat hearts.

The catabolism of human and rat 125I-labelled very low density lipoproteins (VLDL) was compared by perfusing the lipoproteins through beating rat hearts. Triacylglycerol was removed from the VLDL to a greater extent than the protein moiety, leaving remnants containing relatively more apo-B and less apo-C. The change in apo-C content of the remnants correlated with the loss of triacylglycerol. The extent of removal of triacylglycerol from the rat and human VLDL was similar and in most cases appeared to saturate the heart lipoprotein lipase. The remnants were slightly smaller in size than the VLDL, and included particles which appeared to be partially emptied. In addition to remnants of d less than 1.019 g/ml, iodinated lipoproteins derived from rat and human VLDL were recovered at d 1.019-1.063 and 1.063-1.21 g/ml. The former contained largely cholesterol and cholesteryl esters, while phospholipids were the dominant lipid in the latter. An average of 40% of the 125I-labelled apoprotein lost from the VLDL was associated with the perfused hearts. Very little d 1.019-1.063 g/ml lipoprotein was produced from low (physiological) concentrations of rat VLDL, most of the lipoprotein being removed by the heart. However, lipoproteins of density 1.019-1.063 g/ml were formed from human VLDL at all concentrations in the perfusate, as well as from higher concentrations of the rat VLDL. Agarose gel filtration of lipoproteins following heart perfusion with human VLDL revealed large aggregates containing particles which resemble low density lipoproteins (LDL) in electron microscopic appearance and apoprotein composition, since they contain largely apo-B. These data suggest that at normal concentrations rat VLDL are almost completely catabolised and taken up by the heart without the formation of LDL, while LDL is produced from human VLDL at all concentrations.     

Structural heterogeneity of apoB-containing serum lipoproteins visualized using cryo-electron microscopy.

Cryo-electron microscopy was used to analyze the structure of lipoprotein particles in density gradient subfractions of human very low density lipoprotein (VLDL), intermediate density lipoprotein (IDL), and low density lipoprotein (LDL). Lipoproteins from a normolipidemic subject with relatively large and buoyant LDL (pattern A) and from a subject with a predominance of small dense LDL (pattern B) were compared. Projections of VLDL in vitreous ice were heterogeneous in size, but all were circular with a relatively even distribution of contrast. Selected projections of LDL, on the other hand, were circular with a high density ring or rectangular with two high density bands. Both circular and rectangular LDL projections decreased in average size with increasing subfraction density, but were found in all of 10 density gradient subfractions, both in pattern A and in pattern B profiles. Preparations of total IDL contained particles with the structural features of VLDL as well as particles resembling LDL. IDL particles resembling LDL were observed in specific density gradient subfractions in the denser region of the VLDL;-IDL density range. Within the group of IDL particles resembling LDL considerable heterogeneity was observed, but no structural features specific for the pattern A or pattern B lipoprotein profile were recognized.The observed structural heterogeneity of the apolipoprotein B-containing serum lipoproteins may reflect differences in the composition of these particles that may also influence their metabolic and pathologic properties.     

Dynamic structure of the lower density lipoproteins. II. Deuterium NMR studies of the monolayer of very low and low density lipoproteins

The order of phosphatidylcholine (PC) acyl chains in the surface monolayer of very low density lipoproteins (VLDL) and low density lipoproteins (LDL) has been determined from 2H nuclear magnetic resonance order parameters, SCD, using selectively deuterated PC or palmitic acids. From the computer simulated line shapes, we find two distinct phospholipid domains within the amphiphilic monolayer of both VLDL and LDL. In the more ordered domain of LDL, SCD was approximately 0.3 for the "plateau" chain region. The SCD values of VLDL particles are similar to those of LDL for the 5,6- and 11,12-positions, hence we suggest the organization of the more ordered region of VLDL and LDL are similar. The domain of low order in LDL comprises less than 10% of the phospholipid molecules (we do not distinguish between PC and sphingomyelin), having approximately the same order (SCD less than 0.1) as egg PC - sphingomyelin unilamellar vesicles. In VLDL, the domain of low order comprises between approximately 10 and approximately 20% of the phospholipid molecules and the entire acyl chain is in an essentially isotropic environment (SCD less than 0.02). We prepared VLDL-sized microemulsions composed of egg PC, deuterated PC, and triolein to test whether the apoproteins were responsible for creating the two differently organized domains in VLDL and LDL. Surprisingly, these protein-free particles also showed two domains of different order at two temperatures. The high order region, however, is less ordered than in VLDL and LDL. We explain two surface domains of PC in terms of lipid organization and the unique interactions of lipids in the various lipoprotein particles.     

Studies on the metabolism of apolipoprotein B in hypertriglyceridemic subjects using simultaneous administration of tritiated leucine and radioiodinated very low density lipoprotein

To study the metabolic pathways of apolipoprotein B (apoB), a series of studies were carried out in which both radioiodinated very low density lipoproteins (VLDL) and tritiated leucine were simultaneously injected into three hypertriglyceridemic subjects. The appearance and disappearance of tritium activity in VLDL apoB, intermediate density lipoprotein (IDL) apoB, and low density lipoprotein (LDL) apoB were followed as was the disappearance of iodine activity from VLDL and the appearance and disappearance of iodine activity in IDL apoB and LDL apoB. It was found that a delipidation chain could describe the kinetics of both endogenously and exogenously labeled VLDL. A slow component of VLDL was necessary to fit the VLDL 131I-labeled apoB data and was consistent with the observed VLDL [3H]apoB kinetics. In addition, the estimated rate of conversion of VLDL apoB to LDL exceeded that which appeared to pass through the measured IDL pools, suggesting that a fraction of the IDL was not directly observed. It was also found that a higher percentage of VLDL 131I-labeled apoB was converted to LDL apoB than was VLDL [3H]apoB. To evaluate possible causes of this apparent anomaly, simultaneous examination of all kinetic data was performed. This exercise resulted in the resolution of removal pathways from multiple compartments in the VLDL delipidation chain and the conversion of slowly metabolized VLDL to IDL and LDL. The wide spectrum of this loss pathway indicates that previous estimates of VLDL apoB production rate using the radioiodinated methodology probably represent lower bounds for the true physiologic variable. It is important to note that these direct losses were apparent only when the combination of endogenous and exogenous labeling was used.