Metabolism of high density lipoprotein subfractions.

Over the past few years, new experimental approaches have reinforced the awareness among investigators that the heterogeneity of HDL particles indicates significant differences in production and catabolism of HDL particles. Recent kinetic studies have suggested that small HDL, containing two apolipoprotein A-I molecules per particle, are converted in a unidirectional manner to medium HDL or large HDL, containing three or four apolipoprotein A-I molecules per particle, respectively. Conversion appears to occur in close physical proximity with cells and not while HDL particles circulate in plasma. The medium and large HDL are terminal particles in HDL metabolism with large HDL, and perhaps medium HDL, being catabolized primarily by the liver. These novel kinetic studies of HDL subfraction metabolism are compelling in-vivo data that are consistent with the proposed role of HDL in reverse cholesterol transport.     

Effect of alcohol on lipoprotein metabolism. I. High density lipoprotein binding

The effects of ethanol upon the binding of [125I]-labelled human high density lipoprotein 3 (HDL3) was examined in rat liver microsomes and monolayer cultures of human hepatoma (Hep G2) cells. Alcohol feeding to rats (35% caloric content) caused a significant (p less than 0.05) increase in serum cholesterol concentrations relative to pair-fed controls, but HDL3 binding to rat liver microsomes was unaffected by alcohol consumption. By contrast, addition of 10 mM ethanol to Hep G2 cells increased HDL3 binding, and this increase was observed after 14, 28 and 40 days of exposure. This alcohol-dependent rise in HDL3 binding was associated with a 2.3- to 5-fold rise in receptor number (Bmax), and a 2- to 6-fold increase in the dissociation constant (Kd). The data suggest that the net effect of increased receptor number and lower receptor affinity is to increase the capacity of hepatocytes to metabolize circulating high density lipoproteins, and that this increase in the face of elevated plasma high density lipoprotein cholesterol consequent upon alcohol consumption would facilitate greater mobilization of cholesterol from peripheral tissues to the liver.     

Immunological relationship between bile lipoprotein complex and high density lipoprotein.

Apo bile lipoprotein complex was isolated from human gallbladder bile and an antiserum was prepared. Double immunodiffusion studies show that apo bile lipoprotein complex is present in human plasma and more precisely one of its proteic component is common to bile lipoprotein complex and HDL fraction. This proteic component is different from apo AI, AII, apo B, apo CI, CII, CIII, apo D, apo E and albumin.     

High density lipoprotein metabolism in low density lipoprotein receptor-deficient mice

The LDL receptor (LDLR) and scavenger receptor class B type I (SR-BI) play physiological roles in LDL and HDL metabolism in vivo. In this study, we explored HDL metabolism in LDLR-deficient mice in comparison with WT littermates. Murine HDL was radiolabeled in the protein (¹²⁵I) and in the cholesteryl ester (CE) moiety ([³H]). The metabolism of ¹²⁵I-/[³H]HDL was investigated in plasma and in tissues of mice and in murine hepatocytes. In WT mice, liver and adrenals selectively take up HDL-associated CE ([³H]). In contrast, in LDLR^−/− mice, selective HDL CE uptake is significantly reduced in liver and adrenals. In hepatocytes isolated from LDLR^−/− mice, selective HDL CE uptake is substantially diminished compared with WT liver cells. Hepatic and adrenal protein expression of lipoprotein receptors SR-BI, cluster of differentiation 36 (CD36), and LDL receptor-related protein 1 (LRP1) was analyzed by immunoblots. The respective protein levels were identical both in hepatic and adrenal membranes prepared from WT or from LDLR^−/− mice. In summary, an LDLR deficiency substantially decreases selective HDL CE uptake by liver and adrenals. This decrease is independent from regulation of receptor proteins like SR-BI, CD36, and LRP1. Thus, LDLR expression has a substantial impact on both HDL and LDL metabolism in mice.     

Low density lipoprotein turnover in swine.

The catabolism of intravenously injected 125I-labelled low density lipoproteins (LDL) was followed in normal miniature swine for 2 weeks. When compared with the two-exponential model, the decay curve of the plasma radioactivity associated with the LDL fraction was best described by a three-exponential model. In this system, the half-lives were 4.5 +/- 3.7, 19.7 +/- 6.6, and 127 +/- 70 h (mean of four studies). Assuming a kinetic model with metabolism of LDL in the rapidly equilibrating compartment and two slower equilibrating compartments (a model requiring three exponentials), the mean fractional catabolic rate for apo-LDL was calculated to be 0.015 h-1. Therefore, if at steady state, the synthetic rate for apo-LDL in the same pigs would be 5.6 +/- 4.1 mg/h. Different kinetic models using two or three exponentials would provide different values for the synthetic rate of apo-LDL. However, in view of the known existence of at least three major equilibrating pools for LDL in plasma, liver, and lymph, and in view of the present results, the kinetic model for LDL metabolism should be better represented by a three-exponential system.     

High density lipoprotein modulates platelet function.

BACKGROUND: Platelet activation by atherogenic lipoproteins can be antagonized by high density lipoprotein (HDL), probably via interaction with the ATP-binding cassette transporter A1 (ABCA1). METHODS: ABCA1 expression and its association with cholesterol rich membrane domains was analyzed by mRNA and Western blot analysis. HDL effects on platelet receptor clustering were analyzed by flow cytometric analysis of fluorescence resonance energy transfer between fluorochrome-labeled antibodies. RESULTS: ABCA1 expression increased upon megakaryocytic differentiation of human stem cells and ABCA1 protein partially associated to LubroIWX-resistant membrane domains. Plasma HDL-cholesterol in healthy donors negatively correlated to the platelet membrane cholesterol content. Receptor cluster analysis revealed a decrease in the association of Gplb and FcgammaRII upon incubation of platelets with HDL3. CONCLUSION: Our results suggest that HDL modulates platelet reactivity by altering lipid raft associated receptor clustering.     

Low density lipoprotein binding induces asymmetric redistribution of the low density lipoprotein receptors in endothelial cells.

The uptake and transport of cholesterol-carrying low density lipoprotein (LDL) by the arterial wall is a continuous dynamic process, contributing to the cholesterol homeostasis in the plasma and in the cellular components of the vessel wall. Upon exposure to endothelial cells (EC), LDL interacts in part, with specific surface receptors (LDL-R). In this study we questioned: (i) the distribution of LDL receptors on the apical and basal cell membranes in endothelial cells; (ii) the role of LDL receptors in the control of cholesterol homeostasis and (iii) the translocation of LDL receptor across the EC. To this purpose bovine aortic EC were cultured on filters in a double-chamber system, in Dulbecco's medium supplemented either with 10% fetal calf serum (FCS) or with 10% lipoprotein-deficient serum (LPDS). The cells were exposed for 3h to 13H]acetate (40 microCi) added to both compartments of the cell culture inserts. The newly synthesized [3H]cholesterol was detected by thin layer chromatography and quantified by liquid scintillation counting. The LDL-R were detected in EC protein homogenates by immunoblotting using a monoclonal antibody against LDL-R (IgG-C7); the intracellular pathway of LDL-R was examined by electron microscopy using a complex made of protein A 5 nm or 20 nm colloidal gold particles and an anti-LDL receptor antibody (Au-PA-C7). To evaluate the distribution and the transport of LDL-R from one cell surface to the other, EC grown in LPDS were radioiodinated either on the apical or on the basolateral surface, incubated on the same surface with LDL, and subsequently biotinylated on the opposite non-radiolabeled surface. The EC were further solubilized and the protein extract immunoprecipitated with anti-LDL-R antibody or with mouse IgG (as control). The eluted antigen-antibody complexes were precipitated with streptavidin-agarose beads, solubilized, and subjected to SDS-PAGE. The results showed that: (a) the LDL-R were present on both endothelial cell fronts; (b) using the complex Au-PA-C7, the LDL-R were localized in endothelial plasmalemmal vesicles as well as coated pits and coated vesicles in multivesicular bodies and lysosomes, irrespective of the cell surface exposed to the complex; (c) biochemical assays indicated that upon ligand binding, the LDL-R were translocated preferentially from the apical to the basal plasma membrane.     

Heterogeneity of human plasma low density lipoprotein.

Low density lipoprotein (LDL) was fractionated into subspecies by the use of DEAE-agarose column chromatography and the peptide compositions of the LDL subspecies which eluted at different NaCl concentrations were determined. LDL which elutes at low NaCl concentration has relatively less non-B apoprotein than does LDL which elutes at high salt concentration. The LDL subspecies which elute at high NaCl concentration contain more apo A-1 than do those which elute at the lower NaCl molarity. These results indicate that LDL consists of subfractions which differ in their peptide compositions.     

Induction of low density lipoprotein receptor synthesis by high density lipoprotein in cultures of human skin fibroblasts.

Further studies have been made of the effects of high density lipoprotein (HDL) on the surface binding, internalization and degradation of 125I-labeled low density lipoprotein (125I-labeled LDL) by cultured normal human fibroblasts. In agreement with earlier studies, during short incubations HDL inhibited the surface binding of 125I-labeled LDL. In contrast, following prolonged incubations 125I-labeled LDL binding was consistently greater in the presence of HDL. The increment in 125I-labeled LDL binding induced by HDL was: (a) associated with a decrease in cell cholesterol content; (b) inhibited by the addition of cholesterol or cycloheximide to the incubation medium; and (c) accompanied by similar increments in 125I-labeled LDL internalization and degradation. It is concluded that HDL induces the synthesis of high affinity LDL receptors in human fibroblasts by promoting the efflux of cholesterol from the cells.     

Human platelets exclusively bind oxidized low density lipoprotein showing no specificity for acetylated low density lipoprotein.

The widely studied macrophage scavenger receptor system is known to bind both acetylated low density lipoprotein and oxidized low density lipoprotein. Although only the latter ligand has been shown to occur in vivo, acetylated low density lipoprotein is often used to evaluate the contribution of scavenger receptors to different (patho)physiologic processes, assuming that all existing subtypes of scavenger receptors recognise both lipoproteins. In the present work, we identify human platelets as the first natural cell type to bind oxidized low density lipoprotein without showing specificity for acetylated low density lipoprotein. Consequently, platelets possess exclusive receptor(s) for oxidized low density lipoprotein distinct from the 'classical' scavenger receptor AI/AII. From the data presented in this work, we conclude that the class B scavenger receptor CD36 (GPIV) is responsible for this exclusive oxidized low density lipoprotein binding.     

Very low density lipoprotein. Dissociation of apolipoprotein C during lipoprotein lipase induced lipolysis.

The fate of apo C in rat plasma very low density lipoprotein (VLDL) during lipolysis was studied using VLDL labeled specifically with 125I-labeled apo C and purified bovine milk lipoprotein lipase. Incubations were carried out in vitro and included serum-containing systems and albumin containing systems. Free fatty acids generation proceeded with time of incubation in the two systems. It, however, was enhanced 1.5--2 fold by the presence of serum. 125I-labeled apo C equilibrated between very low and high density lipoprotein (HDL) in both systems even when enzyme was not present in the incubation medium, or when the incubation was carried out at 0 degrees C. Upon initiation of lipolysis, more 125I-labeled apo C was transferred to HDL and the transfer was proportional to the magnitude of free fatty acids release. 125I-labeled apo C was also progressively removed from VLDL in the albumin-containing system, although no known lipoprotein acceptor to apo C was present in the medium. The 125I-labeled apo C was recovered predominantly with the medium fraction of d greater than 1.21 g/ml (60--70%), and to a lesser degree with that of d= 1.019--1.21 g/ml. However, the relationship between lipolysis (measured as free fatty acids release) and removal of 125I-labeled apo C from VLDL were indistinguinshable in the albumin containing system and the serum containing system. On the basis of these observations, it is postulated that the removal of apo C during lipolysis of VLDL reflects the nature of the partially degraded VLDL particles, and is independent of the presence of a lipoprotein acceptor to apo C.     

Comparison of plasma clearance of low density lipoprotein with beta-very low density lipoprotein or acetoacetylated low density lipoprotein in cholesterol-fed rabbits

The plasma clearance and tissue distribution of radioiodinated low-density lipoprotein (LDL), beta-very low density lipoprotein (beta-VLDL), and acetoacetylated LDL were studied in cholesterol-fed rabbits. Radioiodinated LDL ([125I]LDL) was cleared more slowly than either [125I]beta-VLDL or acetoacetylated-[125I]LDL and its fractional catabolic rate was one-half that of [125I]beta-VLDL and one-ninth that of acetoacetylated-[125I]LDL. Forty-eight hours after the injection of the labeled lipoproteins, the hepatic uptake was the greatest among the organs evaluated with the uptake of [125I]LDL being one-third that of either [125I]beta-VLDL or acetoacetylated-[125I]LDL. The reduction in the hepatic uptake of LDL due to a down-regulation of the receptors would account for this retarded plasma clearance.     

Low density lipoprotein receptor regulation. Kinetic models

The macromolecular species distribution in a receptor-mediated endocytotic pathway was computer simulated based on kinetic data reported in the literature. In the proposed model, the rapidity with which the recycled receptor is shuttled to the cell surface is indicated by the magnitude of k-3, the shuttling constant. The magnitude of k-3 will vary with the experimental conditions, but when this value is large, the internalized receptor is shuttled back to the cell surface with a traverse time of 14 min. Under steady-state conditions, after the cells have been incubated in the presence of LDL for 5 h (M.S. Brown and J.L. Goldstein, Cell 9 (1976) 663), the time required for a receptor to traverse the entire endocytotic pathway is 52 min. Our simulation suggests that normal LDL binding in such a short-term experiment may be independent of receptor synthesis. Thus, the degradation of LDL and resultant build-up of cholesterol would have no apparent inhibitory effect on the down-regulation of receptor synthesis.     

Incorporation of cholesterol into high density lipoprotein recombinants.

Apo-A-1, the principal apoprotein of high density lipoprotein, was incubated with cholesterol containing liposomes of dimyristoyl lecithin, lecithin from high density lipoprotein or sphingomyelin. Conditions were chosen to give 100% conversion of cholesterol-free liposomes into recombinants which were isolated by density gradient ultracentrifugation. For all phospholipids, there was a progressive decrease in incorporation of lipid into recombinants with increasing cholesterol/phospholipid ratio. The cholesterol/phospholipid ratio of recombinants was ～ 45% of unreacted liposomes, for all initial cholesterol/phospholipid ratios. The reduced cholesterol content suggests exclusion of cholesterol from a fraction of recombinant phospholipid, probably a boundary layer in contact with apo A-1.     

Prevention of low density lipoprotein aggregation by high density lipoprotein or apolipoprotein A-I

We have shown previously that low density lipoprotein (LDL) subjected to vortexing forms self-aggregates that are avidly phagocytosed by macrophages. That phagocytic uptake is mediated by the LDL receptor. We now show that LDL self-aggregation is strongly inhibited (80-95%) by the presence of high density lipoprotein (HDL) or apolipoprotein (apo) A-I. Another type of LDL aggregation, namely that induced by incubation of LDL with phospholipase C, was also markedly inhibited by HDL or apoA-I. The aggregation of LDL induced by vortexing was not inhibited by 2.5 M NaCl, and apoA-I was still able to block LDL aggregation at this high salt concentration, strongly suggesting hydrophobic interactions as the basis for the effect of apoA-I. The fact that apoA-I protected against LDL aggregation induced by two apparently quite different procedures suggests that the aggregation in these two cases has common features. We propose that these forms of LDL aggregation result from the exposure of hydrophobic domains normally masked in LDL and that the LDL-LDL association occurs when these domains interact. ApoA-I, because of its amphipathic character, is able to interact with the exposed hydrophobic domains of LDL and thus block the intermolecular interactions that cause aggregation.     

Diffusion of nitric oxide into low density lipoprotein.

A key early event in the development of atherosclerosis is the oxidation of low density lipoprotein (LDL) via different mechanisms including free radical reactions with both protein and lipid components. Nitric oxide (( small middle dot)NO) is capable of inhibiting LDL oxidation by scavenging radical species involved in oxidative chain propagation reactions. Herein, the diffusion of ( small middle dot)NO into LDL is studied by fluorescence quenching of pyrene derivatives. Selected probes 1-(pyrenyl)methyltrimethylammonium (PMTMA) and 1-(pyrenyl)-methyl-3-(9-octadecenoyloxy)-22,23-bisnor-5-cholenate (PMChO) were chosen so that they could be incorporated at different depths of the LDL particle. Indeed, PMTMA and PMChO were located in the surface and core of LDL, respectively, as indicated by changes in fluorescence spectra, fluorescence quenching studies with water-soluble quenchers and the lifetime values (tau(o)) of the excited probes. The apparent second order rate quenching constants of ( small middle dot)NO (k(NO)) for both probes were 2.6-3.8 x 10(10) m(-1) s(-1) and 1.2 x 10(10) m(-1) s(-1) in solution and native LDL, respectively, indicating that there is no significant barrier to the diffusion of ( small middle dot)NO to the surface and core of LDL. Nitric oxide was also capable of diffusing through oxidized LDL. Considering the preferential partitioning of ( small middle dot)NO in apolar milieu (6-8 for n-octanol:water) and therefore a larger ( small middle dot)NO concentration in LDL with respect to the aqueous phase, a corrected k(NO) value of approximately 0.2 x 10(10) m(-1) s(-1) can be determined, which still is sufficiently large and consistent with a facile diffusion of ( small middle dot)NO through LDL. Applying the Einstein-Smoluchowsky treatment, the apparent diffusion coefficient (D(')NO) of ( small middle dot)NO in native LDL is on average 2 x 10(-5) cm(2) s(-1), six times larger than that previously reported for erythrocyte plasma membrane. Thus, our observations support that ( small middle dot)NO readily traverses the LDL surface accessing the hydrophobic lipid core of the particle and affirm a role for ( small middle dot)NO as a major lipophilic antioxidant in LDL.