125I-labeled HDL Research Articles

Niacin inhibits surface expression of ATP synthase β chain in HepG2 cells: implications for raising HDL

Niacin is an effective agent for raising HDL, but its cellular target sites are largely unknown. We examined effects of niacin on the surface expression of ATP synthase beta chain, a newly described HDL/apolipoprotein A-I (apoA-I) receptor for HDL endocytosis, in HepG2 cells. A significant amount of immunodetectable beta chain was observed on the surface of HepG2 cells, which was competitively displaced by apoA-I. Niacin treatment reduced the surface expression of beta chain in HepG2 cells by approximately 27%, and decreased (125)I-labeled HDL uptake up to approximately 35%. However, nicotinamide, a niacin metabolite that does not have clinical lipid effects, exhibited weaker inhibition on the beta chain cell surface expression, and failed to show inhibitory action on (125)I-labeled HDL uptake. Furthermore, anti-beta chain antibody significantly reduced (125)I-labeled HDL uptake and abolished the inhibitory effect of niacin. Niacin did not change beta chain mRNA expression. These data suggest that niacin inhibits cell surface expression of the ATP synthase beta chain, leading to reduced hepatic removal of HDL protein, thus implicating a potential cellular target for niacin action to raise HDL.

Endothelial lipase is inactivated upon cleavage by the members of the proprotein convertase family

Mature endothelial lipase (EL) is a 68 kDa glycoprotein. In HepG2 cells infected with adenovirus encoding human EL, the mature EL was detectable in the cell lysates and heparin-releasable fractions. In contrast, cell media of these cells contained two EL fragments: an N-terminal 40 kDa fragment and a C-terminal 28 kDa fragment. N-terminal protein sequencing of the His-tagged 28 kDa fragment revealed that EL is cleaved on the C terminus of the sequence RNKR330, the consensus cleavage sequence for mammalian proprotein convertases (pPCs). Replacement of Arg-330 with Ser by site-directed mutagenesis totally abolished EL processing. EL processing could efficiently be attenuated by specific inhibitors of pPCs, alpha1-antitrypsin Portland (alpha1-PDX) and alpha1-antitrypsin variant AVRR. Coexpression of the pPCs furin, PC6A, and PACE4 with EL resulted in a complete conversion of the full-length EL to a truncated 40 kDa fragment. Exogenously added EL was also processed by cells, and the processing could be attenuated by alpha1-PDX. The expressed N-terminal 40 kDa fragment of EL (EL-40) harboring the catalytic site failed to hydrolyze [14C]NEFA from [14C]dipalmitoyl-PC-labeled HDL. EL-40 was incapable of bridging 125I-labeled HDL to the cells and had no impact on plasma lipid concentration when overexpressed in mice. Thus, our results demonstrate that pPCs are involved in the inactivation process of EL.

Administration of tyrosyl radical-oxidized HDL inhibits the development of atherosclerosis in apolipoprotein E-deficient mice.

Tyrosyl radical-oxidized HDL (tyrHDL) increases the ability of cells to donate cholesterol to apolipoprotein (apo) A-I for HDL particle formation. We tested whether treatment with tyrHDL raises endogenous HDL cholesterol levels and decreases atherosclerosis development in apoE-deficient mice. Tyrosyl radical oxidation of mouse HDL induced formation of apoAI-AII heterodimers and enhanced the ability of mouse HDL to deplete cultured fibroblasts of their regulatory pool of cholesterol. 125I-labeled HDL and tyrHDL delivered intraperitoneally were cleared at similar rates from plasma of chow-fed apoE-deficient mice. ApoE-deficient mice injected intraperitoneally twice weekly with 150 microg tyrHDL from age 10 to 18 weeks showed a maximum 2.3-fold increase in endogenous HDL cholesterol levels, which fell toward the end of the treatment period. tyrHDL treatment resulted in 37% less aortic lesion development than in control HDL-treated mice (P<0.001) and 67% less than in saline-injected animals (P<0.001). Administration of tyrHDL for 8 weeks resulted in significantly less atherosclerosis development in apoE-deficient mice than injection of HDL or saline. Molecules increasing mobilization of cellular cholesterol to apoAI for HDL particle formation would be expected to decrease atherosclerosis without necessarily causing sustained increases in circulating HDL cholesterol levels.

High density lipoprotein3 binding sites are related to DNA biosynthesis in the adenocarcinoma cell line A549

The effect of high density (apoE-depleted HDL3) on cell growth of a human tumor cell line (A549) was studied and related to its binding on the plasma membrane. HDL3 were shown to stimulate the incorporation of [3H]thymidine into DNA and cell proliferation; these effects were dose-dependent. As HDL3, apoA-I and apoA-I-liposomes complexes (but not apoA-II) were able to stimulate DNA synthesis in serum-free conditions. This effect was maximum for 15-30 micrograms HDL3 protein/ml concentration. Binding of HDL3 on whole cells occurred by two mechanisms: the first was specific for HDL3; the second, of lower affinity, was phospholipid-dependent and was inhibited by low density lipoprotein or by phospholipid particles. Internalization and degradation of bound HDL3 were not observed. The specific sites (27.9 +/- 2.2 ng HDL3 protein/ng cell protein) accounted for only 2.5% of total (specific+phospholipid) binding sites and they bound HDL3 with a dissociation constant (KD) of 2.47 +/- 0.46 microgram HDL3 protein/ml (2.6 +/- 0.5 x 10(-8) M). The apparent KD value of total binding sites (specific+phospholipid) was eightfold higher (20.4 +/- 6.1 micrograms HDL3 protein/ml). Analysis of the membrane specific binding sites by ligand blotting with 125I-labeled HDL3 showed a single protein with an apparent molecular mass of 110 kDa. When HDL3 binding on phospholipid sites was inhibited by rigid phospholipid particles, the stimulation of [3H]thymidine incorporation related to HDL3 concentration did not show a maximum peak as previously observed but reached a plateau at a concentration as low as 5 micrograms HDL3 protein/ml. This low concentration also nearly saturated the specific binding sites with HDL3. When binding on specific protein sites was suppressed by tetranitromethane, DNA synthesis was not stimulated but, in contrast, inhibited. The stimulating effect of HDL3 on DNA biosynthesis is therefore likely dependent on HDL3 occupying specific binding sites.

Putative mechanisms of action of probucol on high-density lipoprotein apolipoprotein A-I and its isoproteins kinetics in rabbits

Probucol is a widely prescribed lipid-lowering agent, the major effects of which are to lower cholesterol in both low- and high-density lipoproteins (LDL and HDL, respectively). The mechanism of action of probucol on HDL apolipoprotein (apo) A-I kinetics was investigated in rabbits, with or without cholesterol feeding. 125I-labeled HDL was injected intravenously, and blood samples were taken periodically for 6 days. Kinetic parameters were calculated from the apo A-I-specific radioactivity decay curves. Fractional catabolic rate (FCR) and synthetic rate (SR) of apo A-I in rabbits fed a normal chow and normal chow with 1% probucol were similar. Apo A-I FCR of the rabbits fed 0.5% cholesterol was significantly increased but there were no changes in SR, compared to findings in the normal chow-fed group. Apo A-I FCR of the rabbits fed 1% probucol with 0.5% cholesterol (both 1 month and 2 months) was significantly increased compared to findings in rabbits fed the normal chow as well as 0.5% cholesterol diet group, while SR of apo A-I was significantly reduced in the former groups. Kinetics at 1 month after discontinuation of 1% probucol (under cholesterol feeding) showed a similar FCR of HDL-apo A-I to that of the rabbits fed 0.5% cholesterol, but the SR of apo A-I remained lower. Apo A-I isoproteins kinetics assessed by autoradiography of isoelectric focusing slab gels showed that the synthesis of proapo A-I was significantly reduced in the 1% probucol with 0.5% cholesterol administered, compared to, the 0.5% cholesterol group. Thus, the action of probucol on HDL apo A-I kinetics was only prominent in case of higher serum cholesterol levels. The decreased HDL or apo A-I seen with probucol was apparently the result of an increase in FCR and a decrease in SR of HDL-apo A-I. A decreased synthesis of apo A-I remained evident even 1 month after discontinuing probucol. The action of probucol on the intracellular synthetic processes of apo A-I was revealed by the reduced synthesis of proapo A-I.

Metabolism of apoE-free high density lipoproteins in rat hepatoma cells: evidence for a retroendocytic pathway.

The cellular metabolism of apoE-free HDL (HDL) was studied in rat hepatoma cells (FU5AH). Cells incubated with HDL showed a dose-dependent decreased incorporation of [14C]acetate into cell sterol, indicating a net cholesterol delivery to the cells. HDL was localized both at the cell surface and inside the cell. This conclusion was drawn from both the association of 125I-labeled HDL with the cells under different experimental conditions and morphological evidence based on the association of colloidal gold-labeled HDL with the cells. Up to 63% of the 125I-labeled HDL protein initially inside the cell was subsequently recovered in the media as trichloroacetic acid precipitable (TCA-ppt) protein after a 30-min, 37 degrees C chase with a 100-fold concentration of unlabeled HDL. About 27% of the TCA-ppt apoprotein originally inside the cell was recovered as TCA-soluble material. Thus, we conclude that of the HDL apoprotein taken up by the cells, the majority is resecreted by a retroendocytosis pathway. The quantity of HDL apoprotein reappearing in the media was stimulated by the presence of unlabeled HDL in the media, while the amount of TCA-soluble material produced was not. Retroendocytosis of HDL was inhibited at 0 degree C and by the presence of 10 mM NaCN, 20 mM 2-deoxy-D-glucose in the media. Thus, the pathway appears to be both temperature- and energy-sensitive. HDL resecreted by the cell were depleted of cholesteryl ester and showed an altered size distribution, indicative of lipoprotein catabolism and remodeling. This study provides evidence for the existence of an endocytosis-retroendocytosis pathway for HDL apoproteins in a rat hepatoma cell and for the possibility that the endocytosis-retroendocytosis pathway may be involved in lipid delivery to the cell.

Cross-linking of apoproteins in high density lipoprotein by dimethylsuberimidate inhibits specific lipoprotein binding to membranes.

Apoprotein E-free high density lipoproteins (HDL) bind to various cells and cell membrane preparations with properties typical of ligand-receptor interactions. This specific binding can be inhibited by treatment of HDL with tetranitromethane (TNM). During treatment of HDL with TNM, in addition to the expected nitration of tyrosine residues, cross-linking of lipids to apoproteins and of apoproteins to each other occurs. We have recently shown that cross-linking of phospholipids to apoproteins is not responsible for the inhibition of binding (1987. Chacko, G. K., et al. J. Lipid Res. 28: 332-337). To determine the role of cross-linking of apoproteins to each other in the inhibition, we used the bifunctional reagent dimethylsuberimidate (DMS) to cross-link the apoproteins in HDL3. Over 80% of apoproteins in DMS-HDL3 were cross-linked, as analyzed by SDS-polyacrylamide gel electrophoresis. DMS-HDL3 was similar to control HDL3 in its lipid composition. Gel filtration chromatography did not reveal any significant difference in size between DMS-HDL3 and control HDL3. As determined by competitive binding with 125I-labeled HDL3, DMS-HDL3 was almost completely unable to bind specifically to rat liver plasma membranes and human skin fibroblasts. It is concluded from these results that TNM inhibits the specific binding of HDL3 to membranes by a mechanism that involves cross-linking of apoproteins to each other in HDL3 particles. This observation implies that the specific binding of HDL3 to cells may depend on the native quaternary structure of apoproteins in the HDL particle. Because of its reduced ability to bind to the specific binding sites, DMS-HDL3 may be useful for studies related to the functional aspects of HDL binding sites.

Immunochemical characterization of two antigenic sites on human apolipoprotein A-I; localization and lipid modulation of these epitopes

Two monoclonal antibodies, A17 and A30, were raised against human apolipoprotein A-I (apo A-I). They were studied by competitive inhibition of 125I-labeled HDL 3 with HDL subfractions, delipidated apo A-I, and complexes of dimyristoylphosphatidylcholine (DMPC) containing apo A-I and apo A-II. Immunoblotting located the A17 antibody on CNBr fragment 4 of apo A-I and the A30 antibody on CNBr fragment 1. The A17 antigenic determinant was expressed identically in all HDL subclasses, on delipidated apo A-I as well as all on the DMPC-apo A-I and DMPC-apo A-I/apo A-II complexes. In contrast, the apparent affinity constant of the A30 antibody for delipidated apo A-I was about 30-times less than for HDL 3 or for apo A-I/apo A-II-phospholipid complexes. These data suggest that the association of apo A-I with phospholipids improves the reactivity of the A30 monoclonal antibody towards apo A-I, and that this antigenic determinant has a different conformation in delipidated apo A-I compared to apo A-I complexed with phospholipids. Turbidimetric and fluorescence experiments monitoring the phospholipid-apo A-I association in the presence and in the absence of the A17 and A30 antibodies were consistent with the competition experiments carried out by solid phase radioimmunoassay (RIA). After reaction of apo A-I with the A30 antibody, we observed an enhancement of the degradation kinetics of large multilamellar vesicles (LMV), while the A17 antibody did not have a significant effect. Calcein leakage experiments carried out below the transition temperature of DPPC showed an enhancement of the degradation kinetics with both monoclonal antibodies, while the phase-transition release was independent of the reaction of apo A-I with the monoclonal antibodies. These data therefore suggest the existence of at least two different types of epitope on apo A-I, which might account for the differences in immunological reactivity of apo A-I that is either delipidated or present on HDL.

Effects of monocyte macrophages stimulation on hepatic lipoprotein receptors

The first series of in vivo experiments were designed to investigate the effects of monocytic macrophages (MMφ) stimulation by zymosan in cholesterol-fed rats. We found that the MMφ stimulation significantly decreased plasma very-low-density lipoprotein-cholesterol, low-density lipoprotein-cholesterol but not high-density lipoprotein-cholesterol. The hepatic and aortic cholesterol levels were also significantly decreased; meanwhile, the biliary total bile acid and fecal sterol excretion were significantly enhanced. These results were beneficial to the prevention and regression of atherosclerosis. The second series of in vitro experiments led to the discovery that zymosan did not have effect on HDL and LDL binding, uptake and degradation of hepatic parenchymal and nonparenchymal cells isolated from normal rats, but did have significant effects on those isolated from cholesterol-fed rats. The experiments of Kupffer cells modulating hepatocytes also demonstrated that hepatocyte HDL receptor activity was significantly enhanced by conditioned medium from acetylated LDL plus zymosan added to Kupffer cells, B max of 125I-labeled HDL specific binding was increased from 237.8 to 295.2 ng/mg cell protein. The K a value was not affected, indicating that there might be an increment in receptor number, but not receptor affinity. Cholesterol-loaded zymosan-stimulated Kupffer cells might secrete a soluble mediator affecting hepatocyte HDL receptor activity. Zymosan and other MMφ-stimulating reagents are promising in the exploration of a new approach for prevention and treatment of hypercholesterolemia and atherosclerosis.

Regulation of apolipoprotein A-I synthesis in lymph-drained rats

The effect of lymph diversion on plasma apolipoprotein A-I levels was studied. In lymph fistula rats apolipoprotein A-I levels in plasma stayed constant in spite of a loss of an equivalent of one-half plasma pool of apolipoprotein A-I per day through the lymph fistula. This indicates that synthesis of apolipoprotein A-I increases or that catabolism of apolipoprotein A-I decreases in a compensatory manner as intestinal apolipoprotein A-I is diverted. By using incorporation of [ 3H]leucine into newly synthesized apolipoprotein A-I it was shown that 2.6-times as much [ 3H]leucine was incorporated into apolipoprotein A-I in thoracic duct drained animals compared to controls. In experiments in which 125I-labeled HDL was injected intravenously into rats, it was shown that catabolism of HDL and apolipoprotein A-I was not decreased in lymph-drained rats. These data thus suggest that an increased synthesis of apolipoprotein A-I occurs when the intestinal contribution of apolipoprotein A-I diminishes. This is probably due to an increase in liver protein synthesis.

Tetranitromethane modification of human high density lipoprotein (HDL3): inactivation of high density lipoprotein binding is not related to cross-linking of phospholipids to apoproteins.

Treatment of human high density lipoprotein (HDL) with tetranitromethane (TNM) inhibits its binding to HDL-specific binding sites of cells and isolated membranes. The mechanism of this inhibition, however, is not known; during treatment of HDL with TNM, in addition to the expected nitration of tyrosine residues, cross-linking of lipids to apoproteins and of apoproteins to one another occurs. In order to determine whether the cross-linking of lipids to apoproteins occurs through the carbon-carbon double bonds in the acyl chains, and to determine whether the cross-linking of phospholipids to apoproteins is a possible mechanism of inhibition of binding, we have prepared a reconstituted HDL3 in which the native phospholipids were replaced with dimyristoyl phosphatidylcholine (DMPC). As a control, a reconstituted HDL3 (C-r-HDL3) was also prepared using the total apoproteins and the total lipid constituents of native HDL3. The reconstituted DMPC-containing HDL3 (DMPC-r-HDL3) was similar to native HDL3 and to C-r-HDL3 in its agarose gel electrophoretic mobility, in its chemical composition, and in its binding to rat liver plasma membranes. When treated with TNM, DMPC-r-HDL3, like the native HDL3 and C-r-HDL3, lost its ability to bind to the HDL binding sites of rat liver plasma membranes, as determined by competitive binding assays with 125I-labeled human HDL3 as the tracer. Nitrated DMPC-r-HDL3 contained only traces of phospholipids covalently linked to apoproteins, whereas 21-26% of the total phospholipids were cross-linked to apoproteins of nitrated C-r-HDL3 and nitrated native HDL3.(ABSTRACT TRUNCATED AT 250 WORDS)

The role of apolipoproteins of HDL in the selective uptake of cholesteryl linoleyl ether by cultured rat and bovine adrenal cells

Rat adrenal cells in culture were used to study the uptake of cholesteryl linoleyl ether ([ 3H]cholesteryl linoleyl ether), a nonhydrolyzable analog of cholesteryl ester. When ([ 3H]cholesteryl linoleyl ether was added in the form of liposomes, its uptake was enhanced by adrenocorticotropin (ACTH) and by addition of milk lipoprotein lipase and interfered by heparin. When the adrenal cells were incubated with homologous [ 3H]cholesteryl linoleyl ether-HDL, ACTH treatment also resulted in an increase in ([ 3H]cholesteryl linoleyl ether uptake. The uptake of [ 3H]cholesteryl linoleyl ether was in excess of the uptake and metabolism of 125I-labeled HDL protein and was not sensitive to heparin. Unlabeled HDL or delipidated HDL reduced very markedly the uptake of [ 3H]cholesteryl linoleyl ether, while addition of phosphatidylcholine liposomes had little effect. Attempts were made to deplete and enrich the adrenal cells in cholesterol and, while depletion resulted in a decrease in [ 3H]cholesteryl linoleyl ether-HDL uptake, enrichment of cells with cholesterol had no effect. Among the individual apolipoproteins tested, apolipoprotein A-I and the C apolipoproteins reduced [ 3H]cholesteryl linoleyl ether uptake, while apolipoprotein E was not effective. Since the labeled ligand studied was a lipid, these effects could not be due to an exchange of apolipoproteins, but indicated competition for binding sites. Preferential uptake of human [ 3H]cholesteryl linoleyl ether-HDL 3 by bovine adrenal cells was found when compared to the uptake and metabolism of 125I-labeled HDL. The present results suggest that the preferential uptake of HDL cholesteryl ester (as studied with [ 3H]cholesteryl linoleyl ether) requires an interaction between the apolipoproteins of HDL and cell surface components.

Discrimination between subclasses of human high-density lipoproteins by the HDL binding sites of bovine liver

The binding of human 125I-labeled HDL 3 (high-density lipoproteins, ρ 1.125–1.210 g/ cm 3) to a crude membrane fraction prepared from bovine liver closely fit the paradigm expected of a ligand binding to a single class of identical and independent sites, as demonstrated by computer-assisted binding analysis. The dissociation constant ( K d ), at both 37 and 4°C, was 2.9 μg protein/ml (approx. 2.9 · 10 −8 M); the capacity of the binding sites was 490 ng HDL 3 (approx. 4.9 pmol) per mg membrane protein at 37°C and 115 at 4°C. Human low-density lipoproteins (LDL) and very-low-density lipoproteins (VLDL) also bound to these sites ( K d = 41 μg protein/ml, approx. 6.7 · 10 −8 M for LDL, and K d = 5.7 μg protein/ml, approx. 7.0 · 10 −9 M for VLDL), but this observation must be considered in light of the fact that the normal circulating concentrations of these lipoproteins are much lower than those of HDL. The binding of 125I-labeled HDL 3 to these sites was inhibited only slightly by 1 M NaCl, suggesting the presence of primarily hydrophobic interactions at the recognition site. The binding was not dependent on divalent cations and was not displaceable by heparin; the binding sites were sensitive to both trypsin and pronase. Of exceptional note was the finding that various subclasses of human HDL (including subclasses of immunoaffinity-isolated HDL) displaced 125I-Iabeled HDL 3 from the hepatic HDL binding sites with different apparent affinities, indicating that these sites are capable of recognizing highly specific structural features of ligands. In particular, apolipoprotein A-I-containing lipoproteins with prebeta electrophoretic mobility bound to these sites with a strikingly lower affinity ( K d = 130 μg protein/ml) than did the other subclasses of HDL.

Evidence for non-equilibrating pools of apolipoprotein C-III in plasma lipoproteins.

Using immunoaffinity chromatography to isolate apoC-III from radiolabeled lipoproteins for direct determination of specific radioactivity, we have studied the metabolism of human apoC-III in VLDL and in HDL following the bolus injection of 125I-labeled VLDL. Transfer of apoC-III radioactivity from VLDL to HDL was detected in the plasma sample drawn 5 min after injection of the tracer. However, the specific radioactivity of apoC-III in VLDL was found to be higher than that in HDL, with this difference being maintained throughout the sampling period (48-72 hr). The ratios of the respective specific activities ranged from 1.2 to 1.9 in six subjects studied (two normolipidemics and four hypertriglyceridemics). When 125I-labeled HDL was injected as the tracer, however, the higher apoC-III specific radioactivity was associated with the HDL fraction. This lack of complete equilibration of apoC-III between VLDL and HDL in vivo was further characterized by in vitro studies using either 125I-labeled VLDL or 125I-labeled HDL. All incubations were carried out for 3 hr at 37 degrees C followed by 16 hr at 4 degrees C and the apoC-III specific activity in each lipoprotein fraction was directly determined after immunoaffinity chromatography. In a study of plasma from a mildly hypertriglyceridemic subject in which 125I-labeled VLDL was incubated with unlabeled HDL, apoC-III specific activities in VLDL remained 30% greater than that in HDL. When 125I-labeled HDL (from the same subject) was incubated with unlabeled VLDL of apoC-III, final specific activity in VLDL was less than 10% of that of HDL apoC-III. Differences in specific activities were also demonstrated when radiolabeled purified apoC-III was exchanged onto VLDL prior to its incubation with HDL. A consistent difference in apoC-III specific activities in VLDL and HDL was observed after isolation of the particles either by molecular sieve chromatography or by ultracentrifugation. These studies demonstrated that, while the exchange of apoC-III between VLDL and HDL may be very rapid, this equilibration is not complete. Pools of apoC-III that do not participate in the equilibration process are present in both the VLDL and HDL fractions and could account for 30-60% of the total apoC-III mass in each lipoprotein fraction.

Aortic accumulation and plasma clearance of beta-VLDL and HDL: effects of diet-induced hypercholesterolemia in rabbits.

To assess the role of beta-VLDL in diet-induced atherogenesis, the in vivo metabolism and aortic accumulation of 125I-labeled beta-VLDL were investigated in cholesterol-fed rabbits and chow-fed controls. 125I-labeled HDL and 125I-labeled albumin were studied for comparison. The fractional catabolic rate of 125I-labeled beta-VLDL was reduced in cholesterol-fed rabbits (0.011 vs 0.139 hr-1), but due to the high endogenous pool, the total beta-VLDL flux was very high (13.1 vs less than 1.1 mg/kg per 24 hr). These results suggest that elevated levels of beta-VLDL during cholesterol feeding were due to an enhanced rate of synthesis, a finding confirmed in hypercholesterolemic rabbits subjected to plasmapheresis. Following acute reduction of plasma cholesterol by plasmapheresis, the quantitative increases in beta-VLDL cholesterol concentrations (210 to 364 mg/dl) over the subsequent 24 hr were in agreement with the rise calculated from the plasma clearance kinetics of 125I-labeled beta-VLDL (378 mg/dl per 24 hr). Aortic accumulation of beta-VLDL in hypercholesterolemic rabbits was increased greater than 15-fold over controls. Accumulation was predominantly in the intimal atheromatous lesions. The fractional catabolic rate of 125I-labeled HDL was increased during cholesterol feeding (0.037 vs 0.021 hr-1). A decreased rate of synthesis appeared to be responsible for the markedly depleted plasma HDL. HDL accumulation within the aorta was attenuated greater than 9-fold in cholesterol-fed rabbits compared to those fed normal chow. Plasma kinetics and aortic accumulation of 125I-labeled albumin were similar in hypercholesterolemic and control rabbits.(ABSTRACT TRUNCATED AT 250 WORDS)

Modification of human high density lipoprotein (HDL3) with tetranitromethane and the effect on its binding to isolated rat liver plasma membranes.

Apolipoprotein E-free high density lipoproteins (HDL) bind to various cells and cell membrane preparations, with properties typical of ligand-receptor interactions. In order to further characterize the binding sites and to investigate the functional role of binding, a chemically modified HDL without the specific binding properties would be highly desirable. We have reacted human HDL3 with tetranitromethane, a relatively specific nitrating reagent for tyrosine residues, in 50 mM Tris HCL buffer, pH 8.0, and at a reagent concentration 10 times the molar excess of tyrosine residues. The resulting nitrated HDL3 completely lost its ability to bind to high affinity saturable binding sites of rat liver plasma membranes, as determined by competitive binding with 125I-labeled HDL3, and also by direct binding assays using 125I-labeled nitrated HDL3. Although nitrated HDL3 did not bind to the high affinity saturable binding sites, it bound to the membranes, but the binding was not saturable, and was not competed for by unlabeled nitrated HDL3. On agarose gel electrophoresis, pH 8.6, the nitrated HDL3 moved ahead of the control HDL3, indicating an increase in negative charges in the molecule. No difference in size was noted in the nitrated HDL3 when analyzed either by negative stain electron microscopy or by gel filtration chromatography. Spectroscopic analysis of the nitrated HDL3 at pH 8.0 revealed a prominent absorption with maximum at around 360 nm, but none in the region expected for nitrotyrosine residues. At pH 10.0, however, the nitrated HDL3 showed an absorption band with a maximum at around 440 nm, possibly related to nitrotyrosine residues. Nitrotyrosine was detected in the nitrated HDL3 on amino acid analysis. Comparison of the amino acid analysis of the nitrated HDL3 and control HDL3 showed no difference in composition of any of the amino acids except tyrosine; tyrosine content was reduced more than 90% in the nitrated HDL3. SDS-polyacrylamide gel electrophoresis analysis of apoproteins of nitrated HDL3 revealed changes in apolipoprotein profile. Bands corresponding to the apolipoproteins of the starting HDL3 almost disappeared and a series of new bands appeared at the high molecular weight region of the gel, indicating extensive cross-linking of apolipoproteins during the reaction. In addition, a substantial amount of phospholipids and cholesteryl esters, but not unesterified cholesterol, was found covalently linked, possibly through the unsaturated centers of the fatty acid chains, to apolipoproteins.(ABSTRACT TRUNCATED AT 400 WORDS)

Specific binding of high density lipoprotein (HDL3) is not related to sterol synthesis in rat intestinal mucosa.

There is good evidence that high density lipoprotein (HDL) is involved in the flux of cholesterol into the cells of some organs and out of the cells of other tissues. Because we have previously found that HDL is bound specifically by mucosal cells of the small intestine, we have examined the possibility that this was associated with regulation of cholesterol flux. We have, therefore, compared the specific binding of 125I-labeled HDL3 with cholesterol synthesis in mucosal cells obtained from rats that had been treated to alter intestinal cholesterol metabolism. The rate of sterol synthesis measured in tissue slices, by the incorporation of [3H]water into sterols, was altered up to fivefold by treatment with cholestyramine (to induce bile salt loss), by surformer treatment (to reduce absorption of cholesterol), and by biliary diversion. Yet the capacity of mucosal cells to bind, internalize, and degrade 125I-labeled HDL3 was unchanged. Cholesterol feeding influenced neither the interaction of 125I-labeled HDL3 with cells nor the rate of sterol synthesis. Furthermore, the interactions of 125I-labeled HDL3 with mucosal cells isolated from the proximal and distal halves of the intestine or between the upper and lower villus cells were similar, despite differences in sterol synthesis. These data suggest that, in rat intestine, the specific binding of HDL is not related to sterol synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding and degradation of human high-density lipoproteins by human hepatoma cell line HepG2

The catabolism of human HDL was studied in human hepatoma cell line HepG2. The binding of 125I-labeled HDL at 4°C was time-dependent and reached completion within 2 h. The observed rates of binding of 125I-labeled HDL at 4°C and uptake and degradation at 37°C indicated the presence of both high-affinity and low-affinity binding sites for this lipoprotein density class. The specific binding of 125I-labeled HDL accounted for 55% of the total binding capacity. The lysosomal degradation of 125I-labeled HDL was inhibited 25 and 60% by chloroquine at 50 and 100 μM, respectively. Depolymerization of microtubules by colchicine (1 μM) inhibited the degradation of 125I-labeled HDL by 36%. Incubation of cells with HDL caused no significant change in the cellular cholesterol content or in the de novo sterol synthesis and cholesterol esterification. Binding and degradation of 125I-labeled HDL was not affected by prior incubation of cells with HDL. When added at the same protein concentration, unlabeled VLDL, LDL and HDL had similar inhibitory effects on the degradation of 125I-labeled HDL, irrespective of a short or prolonged incubation time. Reductive methylation of unlabeled HDL had no significant effect on its capacity to inhibit the 125I-labeled HDL degradation. The competition study indicated no correlation between the concentrations of apolipoproteins A-I, A-II, B, C-II, C-III, E and F in VLDL, LDL and HDL and the inhibitory effect of these lipoprotein density classes on the degradation of 125I-labeled HDL. There was, however, some association between the inhibitory effect and the levels of apolipoprotein D and C-I.

Direct determination of human and rabbit apolipoprotein B selectively precipitated with butanol-isopropyl ether.

A method is described for the rapid, selective, and quantitative precipitation of apolipoprotein B from isolated hypercholesterolemic rabbit and human very low density lipoproteins (VLDL), intermediate density lipoproteins (IDL), and low density lipoproteins (LDL). Lipoprotein samples are heat-treated at 100 degrees C in 1% SDS. The denatured apoprotein solutions are then mixed briefly with two volumes of butanol-isopropyl ether 45:55 (v/v) to precipitate the apoB. The supernatant solutions, containing the non-apoB proteins and lipids, are removed and the apoB pellet is washed once with water. To determine apoB specific activity, the apoB pellet is resolubilized in 0.5 M NaOH by heating for 30 min at 120 degrees C. The hydrolyzed apoB protein is quantitated by fluorescence of a fluorescamine derivative. The precipitation of apoB is quantitative and selective: 99.5% of rabbit 125I-labeled LDL-apoB and 97.5% of human 125I-labeled LDL-apoB is precipitated and less than 5% of 125I-labeled HDL added to unlabeled VLDL, IDL, or LDL is precipitated. Triglyceride and cholesteryl ester contamination of the apoB pellet is less than 2% of their original radioactivities.

Characterization of high density lipoprotein binding activity in rat adrenocortical cells.

Rat adrenocortical cells take up high density lipoprotein cholesterol for use as steroidogenic substrate. To better understand this unique uptake process, we have first characterized HDL binding. Infusion of human 125I-labeled HDL into rats pretreated with 4-APP demonstrated that the adrenal and ovary accumulate HDL in a saturable fashion in vivo. Subsequent studies using isolated rat adrenocortical cells demonstrated that cellular uptake of HDL is comprised of two events. One event is characterized by reversible membrane binding and is complete by 60 min (t1/2 = 20 min). The second event is marked by irreversible apoprotein accumulation which continues for at least 3 hr. Reversibly bound material exhibits the same apoprotein distribution as unincubated HDL. Irreversible accumulation could not be attributed to internalization or lysosomal accumulation inasmuch as it also occurred with partially purified plasma membranes and was not enhanced by addition of chloroquine. Reversible binding of human HDL3 exhibited a saturable dependence on concentration (Kd = 27 micrograms protein/ml; N = 3.0 X 10(6) sites/cell) similar to that previously reported for rat liver, ovary, and testis. Cell accumulation of HDL decreased by over 80% at 4 degrees C compared to 37 degrees C, did not require calcium, and was not diminished by prior cell treatment with trypsin or pronase. These results indicate that rat adrenocortical cells possess plasma membrane recognition sites for HDL with different properties than those of the LDL receptor. Moreover, adrenal accumulation of HDL apoproteins does not lead to secondary lysosome formation.