Selective uptake of cholesteryl ester from high density lipoproteins by plasma membranes of adipose tissue

1990 ◽  
Vol 68 (5) ◽  
pp. 870-879 ◽  
Author(s):  
Joel G. Parkes ◽  
Aubie Angel
Keyword(s):  
Adipose Tissue ◽  
Cholesteryl Ester ◽  
Plasma Membranes ◽  
Divalent Cations ◽  
High Density ◽  
Metabolic Energy ◽  
High Density Lipoproteins ◽  
High Affinity ◽  
Lipoprotein Binding ◽  
Adipocyte Plasma Membranes

The interaction between high density lipoproteins (HDL) and adipose tissue is an important pathway for cholesterol and cholesteryl ester flux. In intact fat cells, a disproportionately greater net uptake of cholesteryl ester occurs subsequent to lipoprotein binding than would have been predicted from a consideration of holoparticle uptake alone. To characterize the early events in this process, cholesteryl hexadecyl ether, a nonmetabolizable, accumulative marker of cholesteryl ester, was incorporated into canine HDL2, and its uptake by omental adipocyte plasma membranes was measured in relation to the binding of HDL2, which in this animal species is enriched in apolipoprotein A-I and free of apolipoprotein E. The dose–response profile for HDL2 binding was consistent with a single lipoprotein binding site at all concentrations of HDL2, whereas uptake of cholesteryl ester from HDL2 was biphasic, suggesting a high affinity site at low HDL2 concentrations and a low affinity site at high lipoprotein concentrations. Pronase treatment stimulated binding twofold and this was accompanied by a parallel twofold stimulation of cholesteryl ester uptake. EDTA, on the other hand, reduced binding and uptake of cholesteryl ester by 20%, indicating partial dependence upon divalent cations. The proportion of HDL2 cholesteryl ester accumulated by plasma membranes relative to HDL2 protein bound was not altered by either pronase or EDTA, despite the fact that these agents had opposite effects upon binding. In dissociation studies, a portion of membrane-associated HDL2 did not equilibrate with exogenous HDL2 and a greater proportion of the cholesteryl ester failed to dissociate. A stepwise mechanism for cholesteryl ester uptake, involving (i) saturable, high affinity HDL2 binding to cell surface sites, (ii) vectoral, HDL2 concentration-dependent delivery of cholesteryl ester to the membrane, and (iii) cholesteryl ester sequestration into a nonexchangeable membrane compartment, appears to be independent of metabolic energy or cell processing.Key words: cholesteryl ester transport, high density lipoprotein receptor, cholesterol storage.

Characterization of high-density lipoprotein binding to rat adipocytes and adipocyte plasma membranes

1988 ◽  
Vol 66 (9) ◽  
pp. 986-997 ◽  
Author(s):  
Eva Zsigmond ◽  
Bessie Fong ◽  
Aubie Angel
Keyword(s):  
Adipose Tissue ◽  
Plasma Membranes ◽  
Density Lipoprotein ◽  
Membrane System ◽  
Rat Plasma ◽  
High Density ◽  
High Density Lipoproteins ◽  
Binding Characteristics ◽  
Isolated Adipocytes ◽  
Adipocyte Plasma Membranes

The interaction of high-density lipoproteins (HDL) with adipocytes is important in the regulation of cellular cholesterol flux. To study the mechanisms of HDL binding and cellular processing, we incubated adipocytes isolated from epididymal and perirenal adipose tissue of male Wistar rats (300 g) with HDL1 (1.07–1.10 g/mL) and HDL2 (1.10–1.14 g/mL) fractions separated from rat plasma by gradient ultracentrifugation. Freshly isolated adipocytes were incubated with 125I-labeled HDL for 2 h at 37 °C to determine cell-associated uptake and degradation. Adipocytes from both fat regions showed significant cell-associated HDL1 and HDL2 uptake and very high medium degradation (2- to 6-fold higher than uptake). To assess 125I-labeled HDL binding independent of cellular metabolism, we purified adipocyte plasma membranes from isolated adipocytes and used them in binding assays. Binding of HDL1 and HDL2 in the membrane system was 85–95% specific, sensitive to high NaCl concentrations, and abolished by pronase treatment. In contrast to HDL2 binding, the maximum HDL1 binding to perirenal plasma membranes was significantly higher than its binding to epididymal membranes (7.2 ± 1.3 vs. 4.4 ± 0.2 μg/mg, n = 6, p < 0.05). This increment in HDL1 binding to perirenal membranes represented an EDTA- sensitive, calcium-dependent component. These results indicate that HDL binding to adipocyte plasma membranes depends on both adipose tissue region and HDL subtype. The membrane binding characteristics, taken together with the cellular uptake results, suggest that adipocytes bind and metabolize HDL and that this interaction may involve a protein receptor.

scholarly journals Characterization and isolation of a high-density-lipoprotein-binding protein from bovine corpus luteum plasma membrane

1992 ◽  
Vol 287 (3) ◽  
pp. 841-848 ◽  
Author(s):  
K Ferreri ◽  
K M J Menon
Keyword(s):  
Plasma Membrane ◽  
Membrane Fraction ◽  
Plasma Membranes ◽  
Binding Protein ◽  
High Density ◽  
High Density Lipoproteins ◽  
Plasma Membrane Fraction ◽  
High Affinity ◽  
High Concentrations ◽  
Single Polypeptide

The ovary uses the cholesterol from high-density lipoproteins (HDL) as a substrate source for steroid hormone production. It is not clear, however, how ovarian cells acquire the lipoprotein cholesterol. This study describes the characterization and isolation of a high-affinity-binding protein for apolipoprotein E-free HDL from the plasma-membrane fraction of bovine corpora lutea. Plasma membranes were prepared by differential centrifugation with 5-6-fold enrichment of 5′-nucleotidase activity. The binding of 125I-HDL to the plasma membranes was time-dependent, and there appeared to be a single high-affinity site with a Kd of 6.7 micrograms of HDL/ml of assay buffer. The binding was not affected by high concentrations of low-density lipoproteins or the Ca2+ chelator EDTA, nor by changes in pH in the range 6.5-9.0. The binding was affected by the salt concentration in the buffer, with a dose-dependent increase that reached a maximum at 150-250 mM-NaCl. Binding was increased in the presence of high concentrations of KCl and KBr, and most significantly increased by high concentrations of bivalent metal ions. Ligand-blot analysis under reducing conditions revealed that the binding protein was a single polypeptide of about 108 kDa that was associated with the plasma-membrane fraction. This HDL-binding protein was purified to homogeneity by solubilization with Triton X-100, poly(ethylene glycol) precipitation, DEAE-Sephadex chromatography, and preparative SDS/PAGE. The purified binding protein is a single polypeptide of 108 kDa that retains high affinity and specificity for HDL as assayed by ligand blotting.

Enhanced binding of low and high density lipoproteins to human adipocyte plasma membranes: effects of temperature and proteases

1986 ◽  
Vol 64 (12) ◽  
pp. 1378-1382
Author(s):  
Bessie S. Fong ◽  
Pedro O. Rodrigues ◽  
Aubie Angel
Keyword(s):  
Plasma Membranes ◽  
Specific Binding ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
High Density ◽  
High Density Lipoproteins ◽  
Dependent Manner ◽  
Human Adipocyte ◽  
Ldl Binding ◽  
Adipocyte Plasma Membranes

The specific binding of 125I-labelled low density lipoprotein ([125I]LDL to human adipocyte plasma membranes was higher at 37 than at 0 °C. Prior treatment of membranes with pronase had no effect on LDL binding measured at 0 °C but consistently stimulated binding at 37 °C. Plasmin was similar to pronase in enhancing LDL-specific binding, but thrombin was not as effective. 125I-labelled high density lipoprotein ([125I]HDL2) specific binding to human adipocyte plasma membranes was similarly sensitive to temperature and pronase treatment. Addition of the protease inhibitor aprotinin in the adipocyte membrane binding assay significantly reduced [125I]LDL binding at 37 °C (p < 0.05), suggesting the involvement of a protease activity intrinsic to the lipoproteins and (or) membranes. These data demonstrate that both LDL and HDL binding in human adipocyte plasma membranes can be "up-regulated" by specific proteolytic perturbations in a temperature-dependent manner.

scholarly journals Characterization of high density lipoprotein binding to human adipocyte plasma membranes.

1985 ◽  
Vol 75 (6) ◽  
pp. 1804-1812 ◽  
Author(s):  
B S Fong ◽  
P O Rodrigues ◽  
A M Salter ◽  
B P Yip ◽  
J P Despres ◽  
...  
Keyword(s):  
High Density Lipoprotein ◽  
Plasma Membranes ◽  
Density Lipoprotein ◽  
High Density ◽  
Human Adipocyte ◽  
Lipoprotein Binding ◽  
Adipocyte Plasma Membranes

Identification of high density lipoprotein binding proteins in mature adipocyte plasma membranes

1993 ◽  
Vol 71 (7-8) ◽  
pp. 348-354 ◽  
Author(s):  
Xin-Yi Shen ◽  
Aubie Angel
Keyword(s):  
High Density Lipoprotein ◽  
Plasma Membranes ◽  
Binding Proteins ◽  
Density Lipoprotein ◽  
High Density ◽  
Rat Adipocytes ◽  
Apolipoprotein A ◽  
Molecular Masses ◽  
Lipoprotein Binding ◽  
Adipocyte Plasma Membranes

High density lipoprotein (HDL) binding proteins were identified in nonreduced detergent extracts of plasma membranes or crude membrane fractions of rat adipocytes by ligand blotting. Using 125I-labelled human apolipoprotein-E-free HDL ([125I]HDL3), two binding proteins in adipocyte membranes were detected with apparent molecular masses of 122 and 88 kilodaltons (kDa), respectively. The binding of HDL3 to both binding proteins was abolished by pronase treatment and was inhibited by excess unlabelled HDL3. Excessive unlabelled low density lipoprotein reduced the binding of [125I]HDL3 to the 122-kDa binding protein relatively less than that to the 88-kDa binding protein. Polyclonal antisera against purified rat apolipoprotein A-I (apoA-I) effectively inhibited the binding of HDL3 to adipocyte membranes. Affinity-purified antibodies against rat apoA-I also revealed two HDL-binding proteins in rat adipocyte and liver plasma membranes preincubated with rat HDL. The sizes of the HDL-binding proteins in adipocyte plasma membranes detected by anti-apoA-I were similar to those detected by radiolabelled ligand blotting and their counterparts in rat liver plasma membranes. The study demonstrates two HDL-binding proteins, distinguishable by apparent molecular masses and ligand binding affinity, in plasma membrane proteins of mature rat adipocytes using radiolabelled ligand and immunoligand blotting techniques. The results suggest that apoA-I is involved in the interactions between HDL and both variants of HDL-binding proteins.Key words: high density lipoprotein binding proteins, rat adipocytes, apolipoprotein A-I.

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