Human high density lipoprotein (HDL3) binding to rat liver plasma membranes

The binding of human 125I-labeled HDL3 to purified rat liver plasma membranes was studied. 125I-labeled HDL3 bound to the membranes with a dissociation constant of 10.5 micrograms protein/ml and a maximum binding of 3.45 micrograms protein/mg membrane protein. The 125I-labeled HDL3-binding activity was primarily associated with the plasma membrane fraction of the rat liver membranes. The amount of 125I-labeled HDL3 bound to the membranes was dependent on the temperature of incubation. The binding of 125I-labeled HDL3 to the rat liver plasma membranes was competitively inhibited by unlabeled human HDL3, rat HDL, HDL from nephrotic rats enriched in apolipoprotein A-I and phosphatidylcholine complexes of human apolipoprotein A-I, but not by human or rat LDL, free human apolipoprotein A-I or phosphatidylcholine vesicles. Human 125I-labeled apolipoprotein A-I complexed with egg phosphatidylcholine bound to rat liver plasma membranes with high affinity and saturability, and the binding constants were similar to those of human 125I-labeled HDL3. The 125I-labeled HDL3-binding activity of the membranes was not sensitive to pronase or phospholipase A2; however, prior treatment of the membranes with phospholipase A2 followed by pronase digestion resulted in loss of the binding activity. Heating the membranes at 100 degrees C for 30 min also resulted in an almost complete loss of the 125I-labeled HDL3-binding activity.     

Characterization of the specific binding of rat apolipoprotein E-deficient HDL to rat hepatic plasma membranes

We have used a preparation of rat liver plasma membranes to study the binding of rat apolipoprotein E-deficient HDL to rat liver. The membranes were found to bind HDL by a saturable process that was competed for by excess unlabeled HDL. The binding was temperature-dependent and was 85% receptor-mediated when incubated at 4, 22 and 37 degrees C. The affinity of the binding site for the HDL was consistent at all temperatures, while the maximum binding capacity increased at higher temperatures. The specific binding of HDL to the membranes did not require calcium and was independent of the concentration of NaCl in the media. The effect of varying the pH of the media on HDL binding was small, being 30% higher at pH 6.5 than at pH 9.0. Both rat HDL and human HDL3 were found to compete for the binding of rat HDL to the membranes, whereas rat VLDL remnants and human LDL did not compete. At 4 degrees C, complexes of dimyristoylphosphatidylcholine (DMPC) and apolipoproteins A-I, A-IV and the C apolipoproteins, but not apolipoprotein E, competed for HDL binding to the membranes. At 22 and 37 degrees C, all DMPC-apolipoprotein complexes competed to a similar extent, DMPC vesicles that contained no protein did not compete for the binding of HDL. These results suggest that the rat liver possesses a specific receptor for apolipoprotein E-deficient HDL that recognizes apolipoproteins A-I, A-IV and the C apolipoproteins as ligands.     

Detection and characterization of anionic polypeptide fraction binding sites in rat liver plasma membranes and cultured hepatocytes

The binding of human 125I-labeled 'anionic polypeptidic fraction' (APF) to purified rat liver plasma membranes was studied. The dissociation constant for this binding was 3.0 micrograms protein/mg membrane protein. Binding was competitively inhibited by unlabeled human APF, but not by human LDL (low density lipoproteins). When unlabeled HDL3 was added, binding of labeled APF was competitively reduced to a level between that of unlabeled APF and unlabeled LDL. Experiments with cultured rat hepatocytes confirmed those obtained with liver membranes and suggested the presence in rat liver of saturable APF-binding sites which seem to be specific for APF. The physiologic significance of these APF binding sites is discussed in relation to the fate of cholesterol in the liver.     

Comparison of binding and degradation of high density lipoprotein by intestinal mucosal cells, fibroblasts and adrenal cortical cells in culture

In this study we have compared the binding and degradation of human high density lipoprotein (HDL3), devoid of apolipoprotein E, by rat intestinal (mucosal) and adrenal cells and by human fibroblasts in culture. Binding of HDL3 to adrenal and intestinal cells was characterised by saturable, specific processes whereas skin fibroblasts from normal humans did not convincingly demonstrate saturability and had a lower affinity and capacity compared with adrenal and intestinal cells. Post-receptor events also appeared to differ. Cells from the adrenal cortex and gut showed similar binding affinities for HDL3 but the capacity for binding and for degrading HDL3 was much higher with intestinal cells. The large amounts of HDL degraded by intestinal cells suggest a specific role for the gut in HDL catabolism, and that, in the rat, intestinal cholesterol may be derived from circulating HDL. Finally, it is suggested that rat adrenal cortical and intestinal mucosal cells possess surface receptors for HDL3 which differ from the LDL receptor.     

Binding properties of high-density lipoprotein subfractions and low-density lipoproteins to rabbit hepatocytes

Primary cultures of rabbit hepatocytes which were preincubated for 20 h in a medium containing lipoprotein-deficient serum subsequently bound, internalized and degraded 125I-labeled high-density lipoproteins2 (HDL2). The rate of degradation of HDL2 was constant in incubations from 3 to 25 h. As the concentration of HDL2 in the incubation medium was increased, binding reached saturation. At 37 degrees C, half-maximal binding (Km) was achieved at a concentration of 7.3 micrograms of HDL2 protein/ml (4.06 X 10(-8)M) and the maximum amount bound was 476 ng of HDL2 protein/mg of cell protein. At 4 degrees C, HDL2 had a Km of 18.6 micrograms protein/ml (1.03 X 10(-7)M). Unlabeled low-density lipoproteins (LDL) inhibited only at low concentrations of 125I-labeled HDL2. Quantification of 125I-labeled HDL2 binding to a specific receptor (based on incubation of cells at 4 degrees C with and without a 50-fold excess of unlabeled HDL) yielded a dissociation constant of 1.45 X 10(-7)M. Excess HDL2 inhibited the binding of both 125I-labeled HDL2 and 125I-labeled HDL3, but excess HDL3 did not affect the binding of 125I-labeled HDL3. Preincubation of hepatocytes in the presence of HDL resulted in only a 40% reduction in specific HDL2 receptors, whereas preincubation with LDL largely suppressed LDL receptors. HDL2 and LDL from control and hypercholesterolemic rabbits inhibited the degradation of 125I-labeled HDL2, but HDL3 did not. Treatment of HDL2 and LDL with cyclohexanedione eliminated their capacity to inhibit 125I-labeled HDL2 degradation, suggesting that apolipoprotein E plays a critical role in triggering the degradative process. The effect of incubation with HDL on subsequent 125I-labeled LDL binding was time-dependent: a 20 h preincubation with HDL reduced the amount of 125I-labeled LDL binding by 40%; there was a similar effect on LDL bound in 6 h but not on LDL bound in 3 h. The binding of 125I-labeled LDL to isolated liver cellular membranes demonstrated saturation kinetics at 4 degrees C and was inhibited by EDTA or excess LDL. The binding of 125I-labeled HDL2 was much lower than that of 125I-labeled LDL and was less inhibited by unlabeled lipoproteins. The binding of 125I-labeled HDL3 was not inhibited by any unlabeled lipoproteins. EDTA did not affect the binding of either HDL2 or HDL3 to isolated liver membranes. Hepatocytes incubated with [2-14C]acetate in the absence of lipoproteins incorporated more label into cellular cholesterol, nonsaponifiable lipids and total cellular lipid than hepatocytes incubated with [2-14C]acetate in the presence of any lipoprotein fraction. However, the level of 14C-labeled lipids released into the medium was higher in the presence of medium lipoproteins, indicating that the effect of those lipoproteins was on the rate of release of cellular lipids rather than on the rate of synthesis.     

Determination of the structural domain of ApoAI recognized by high density lipoprotein receptors.

There is good evidence that high density lipoprotein (HDL) interacts with high affinity sites present on hepatocytes. The precise nature of the ligand recognized by putative HDL receptors remains controversial, although there is a consensus that apolipoprotein AI (apoAI) is involved. This suggestion would be strengthened if a biologically active site demonstrating a high affinity for the receptor could be isolated. Cyanogen bromide fragments (CF) of apoAI (CF1-CF4) were complexed with phospholipid, and their ability to associate with the receptor was compared in various binding studies. Careful analysis of the concentration-dependent association of 125I-labeled dimyristoyl phosphatidylcholine (DMPC) recombinants to rat liver plasma membranes revealed high and low affinity binding components. As all DMPC recombinants displayed the low affinity binding component, it was postulated that this interaction was independent of the protein present in the particle and may well represent a lipid-lipid or lipid-protein association with the membranes. Only 125I-labeled CF4.DMPC displayed a high affinity binding component with similar Kd and Bmax (8 x 10(-9) M, 1.6 x 10(-12) mol/mg plasma membrane protein) to that of 125I-labeled AI.DMPC (7 x 10(-9), 1.4 x 10(-12) mol/mg plasma membrane protein). Similarly, egg yolk phosphatidylcholine complexes containing CF4 (CF4.egg PC) showed higher affinity binding than CF1-egg yolk phosphatidylcholine complexes confirming the results obtained with DMPC complexes. Furthermore, ligand blotting studies showed that only 125I-labeled CF4.DMPC associated specifically with HB1 and HB2, two HDL binding proteins recently identified in rat liver plasma membranes. We conclude that a region within the carboxyl-terminus of apoAI is responsible for the interaction with putative HDL receptors present in rat liver plasma membranes.     

Binding of apolipoprotein A-I and A-II after recombination with phospholipid vesicles to the high density lipoprotein receptor of luteinized rat ovary

To determine the apolipoprotein specificity of high density lipoprotein (HDL) receptor, apolipoprotein A-I (apo-AI) and apolipoprotein A-II (apo-AII) purified from high density lipoprotein3 (HDL3) were reconstituted into dimyristoyl phosphatidylcholine vesicles (DMPC) and their ability to bind to luteinized rat ovarian membranes was examined. Both 125I-apo-A-I.DMPC and 125I-apo-A-II.DMPC were shown to bind to ovarian membranes with Kd = 2.87 and 5.70 micrograms of protein/ml, respectively. The binding of both 125I-apo-A-I.DMPC and 125I-apo-A-II.DMPC was inhibited by unlabeled HDL3, apo-A-I.DMPC, apo-A-II.DMPC, apo-C-I.DMPC, apo-C-II.DMPC, apo-C-III1.DMPC, and apo-C-III2.DMPC, but not by DMPC vesicles, bovine serum albumin.DMPC or low density lipoprotein. Since the binding labeled apo-A-I.DMPC and apo-A-II.DMPC was inhibited by the DMPC complexes of apo-C groups, the direct binding of 125I-apo-C-III1.DMPC was also demonstrated with Kd = 9.6 micrograms of protein/ml. In addition, unlabeled apo-A-I.DMPC, and apo-A-II.DMPC, as well as apo-C.DMPC, inhibited 125I-HDL3 binding. 125I-apo-A-I, 125I-apo-A-II, and 125I-apo-C-III1 in the absence of DMPC also bind to the membranes. These results suggest that HDL receptor recognizes apolipoprotein AI, AII, and the C group and that the binding specificity of the reconstituted lipoproteins is conferred by their apolipoprotein moiety rather than the lipid environment. In vivo pretreatment of rats with human chorionic gonadotropin resulted in an increase of 125I-apo-A-I.DMPC, 125I-apo-A-II.DMPC, and 125I-apo-C-III1.DMPC binding activities. However, no induction of binding activity was observed when the apolipoprotein was not included in DMPC vesicles. An examination of the equilibrium dissociation constant and binding capacity for 125I-apo-A-I.DMPC and 125I-apo-A-II.DMPC after human chorionic gonadotropin treatment revealed that the increase in binding activity was due to an increase in the number of binding sites rather than a change in the binding affinity. These results further support our contention that apo-A-I, apo-A-II, and the apo-C group bind to HDL receptor. In conclusion, the HDL receptor of luteinized rat ovary recognizes apolipoproteins A-I, A-II, and the C group but not low density lipoprotein, and the binding is induced by human chorionic gonadotropin in vivo.     

Catabolism of human low density lipoproteins by human hepatoma cell line HepG2

The mechanism of hepatic catabolism of human low density lipoproteins (LDL) by human-derived hepatoma cell line HepG2 was studied. The binding of 125I-labeled LDL to HepG2 cells at 4 degrees C was time dependent and inhibited by excess unlabeled LDL. The specific binding was predominant at low concentrations of 125I-labeled LDL (less than 50 micrograms protein/ml), whereas the nonsaturable binding prevailed at higher concentrations of substrate. The cellular uptake and degradation of 125I-labeled LDL were curvilinear functions of substrate concentration. Preincubation of HepG2 cells with unlabeled LDL caused a 56% inhibition in the degradation of 125I-labeled LDL. Reductive methylation of unlabeled LDL abolished its ability to compete with 125I-labeled LDL for uptake and degradation. Chloroquine (50 microM) and colchicine (1 microM) inhibited the degradation of 125I-labeled LDL by 64% and 30%, respectively. The LDL catabolism by HepG2 cells suppressed de novo synthesis of cholesterol and enhanced cholesterol esterification; this stimulation was abolished by chloroquine. When tested at a similar content of apolipoprotein B, very low density lipoproteins (VLDL), LDL and high density lipoproteins (HDL) inhibited the catabolism of 125I-labeled LDL to the same degree, indicating that in HepG2 cells normal LDL are most probably recognized by the receptor via apolipoprotein B. The current study thus demonstrates that the catabolism of human LDL by HepG2 cells proceeds in part through a receptor-mediated mechanism.     

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 degrees C was time-dependent and reached completion within 2 h. The observed rates of binding of 125I-labeled HDL at 4 degrees C and uptake and degradation at 37 degrees 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 microM, respectively. Depolymerization of microtubules by colchicine (1 microM) 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.     

The uptake of the apoprotein and cholesteryl ester of high-density lipoproteins by the perfused rat liver

The uptake of the 125I-labeled apolipoprotein and 3H-labeled cholesteryl ester components of rat apolipoprotein E-deficient HDL by the perfused liver was studied. The uptake of the cholesteryl ester moiety was 4-fold higher than that of apolipoprotein. The concentration-dependent uptake of labeled protein was saturable and competed for by an excess of unlabeled HDL. The uptake of cholesteryl ester was not saturable over the concentration range studied. In the presence of a 50-fold excess of unlabeled HDL, the uptake of both radiolabeled components was decreased by over 75%, indicating that three-quarters of the hepatic uptake of HDL is by a receptor-mediated process. After 15 min of perfusion, 37% of the apolipoprotein radioactivity that was initially bound at 5 min was released into the perfusate as a more dense particle. After 5, 15, 30 and 60 min of perfusion the subcellular distribution of the apolipoprotein and cholesteryl ester components was analyzed by Percoll density gradient centrifugation. Over the 60 min period, there appeared to be transfer of radioactivity from the plasma membrane fraction to the lysosomal fraction. However, the internalization and degradation of cholesteryl ester was more rapid than that of the apolipoprotein. Our findings indicate that there is preferential uptake of HDL cholesteryl ester relative to protein by the liver and that the internalization of these components may occur independently.     

Studies on the binding and degradation of human very-low-density lipoproteins by human hepatoma cell line HepG2

The regulation of the hepatic catabolism of normal human very-low-density lipoproteins (VLDL) was studied in human-derived hepatoma cell line HepG2. Concentration-dependent binding, uptake and degradation of 125I-labeled VLDL demonstrated that the hepatic removal of these particles proceeds through both the saturable and non-saturable processes. In the presence of excess unlabeled VLDL, the specific binding of 125-labeled VLDL accounted for 72% of the total binding. The preincubation of cells with unlabeled VLDL had little effect on the expression of receptors, but reductive methylation of VLDL particles reduced their binding capacity. Chloroquine and colchicine inhibited the degradation of 125I-labeled VLDL and increased their accumulation in the cell, indicating the involvement of lysosomes and microtubuli in this process. Receptor-mediated degradation was associated with a slight (13%) reduction in de novo sterol synthesis and had no significant effect on the cellular cholesterol esterification. Competition studies demonstrated the ability of unlabeled VLDL, low-density lipoproteins (LDL) and high-density lipoproteins (HDL) to effectively compete with 125I-labeled VLDL for binding to cells. No correlation was observed between the concentrations of apolipoproteins A-I, A-II, C-I, C-II and C-III of unlabeled lipoproteins and their inhibitory effect on 125I-labeled VLDL binding. When unlabeled VLDL, LDL and HDL were added at equal contents of either apolipoprotein B or apolipoprotein E, their inhibitory effect on the binding and uptake of 125I-labeled VLDL only correlated with apolipoprotein E. Under similar conditions, the ability of unlabeled VLDL, LDL and HDL to compete with 125I-labeled LDL for binding was a direct function of only their apolipoprotein B. These results demonstrate that in HepG2 cells, apolipoprotein E is the main recognition signal for receptor-mediated binding and degradation of VLDL particles, while apolipoprotein B functions as the sole recognition signal for the catabolism of LDL. Furthermore, the lack of any substantial regulation of beta-hydroxy-beta-methylglutaryl-CoA reductase and acyl-CoA:cholesterol acyltransferase activities subsequent to VLDL degradation, in contrast to that observed for LDL catabolism, suggests that, in HepG2 cells, the receptor-mediated removal of VLDL proceeds through processes independent of those involved in LDL catabolism.     

Specific saturable binding of rat high-density lipoproteins to rat kidney membranes

The binding of rat 125I-labelled high-density lipoprotein (HDL) to rat kidney membranes was studied using HDL fractions varying in their apolipoprotein E content. The apolipoprotein E/apolipoprotein A-I ratio (g/g) in the HDL fractions ranged from essentially 0 to 1.5. All these HDL preparations showed the same binding characteristics. The saturation curves, measured at 0 degrees C in the presence of 2% bovine serum albumin, consisted of two components: low-affinity non-saturable binding and high-affinity binding (Kd about 40 micrograms of HDL protein/ml). Scatchard analyses of the high-affinity binding suggest a single class of non-interacting binding sites. These sites could be purified together with the plasma membrane marker enzyme 5'-nucleotidase. The binding of rat HDL to rat kidney membranes was not sensitive to high concentrations of EDTA, relatively insensitive to pronase treatment and influenced by temperature. The specific binding of rat HDL was highest at acid pH and showed an additional optimum at pH 7.5. On a total protein basis unlabelled rat VLDL competed as effectively as unlabelled rat HDL for binding of 125I-labelled rat HDL to partially purified kidney membranes. Rat LDL, purified by chromatography on concanavalin A columns and human LDL did not compete. Unlabelled human HDL was a much weaker competitor than unlabelled rat HDL and the maximal specific binding of 125I-labelled human HDL was only 10% of the value for 125I-labelled rat HDL.     

Characterization of low density lipoprotein binding to human adipocytes and adipocyte membranes

125I-labeled low density lipoprotein (LDL) binding to purified plasma membranes prepared from freshly isolated human adipocytes was saturable, specific, and displaceable by unlabeled ligand. The maximum specific binding capacity measured at saturating concentrations of 125I-LDL was 1.95 +/- 1.17 micrograms of LDL bound/mg of membrane protein (mean +/- S.D., n = 16). In contrast to cultured fibroblasts, specific binding of LDL to adipocyte membranes was calcium-independent, was not affected by EDTA or NaCl, and was not destroyed by pronase. Plasma membranes purified directly from homogenized adipose tissue also showed calcium-independent LDL specific binding (0.58 +/- 0.33 micrograms of LDL bound/mg of membrane protein, mean +/- S.D. n = 11). Specific binding, internalization, and degradation of 125I-methylated LDL was demonstrated in isolated adipocytes and competition experiments showed that native and methylated LDL interacted with adipocytes through some common recognition mechanism(s). Compared to native LDL, specific binding of methylated LDL to adipocyte membranes was significantly reduced (43%), indicating that interaction of LDL with adipocyte was dependent in part on the lysine residues of apolipoprotein B. LDL binding to adipocyte plasma membranes was also competitively inhibited by human high density lipoprotein subfractions HDL2 and HDL3. Thus, LDL metabolism in mature adipocytes appears to be regulated by mechanisms distinctly different from a variety of cultured mesenchymal cells. In addition, the ability of adipocytes to bind, internalize, and degrade significant amounts of methylated LDL supports the view that adipose tissue is involved in the metabolism of modified lipoproteins in vivo.     

Solubilization of the 17 alpha-ethinyl estradiol-stimulated low density lipoprotein receptor of male rat liver

Pharmacological doses of 17 alpha-ethinyl estradiol induce a low density lipoprotein (LDL) receptor in the liver of male rats. Our aim was to solubilize this receptor. Isolated liver membranes (8,000-100,000 g fraction) from male rats treated with 17 alpha-ethinyl estradiol and from control rats were solubilized in 1% (w/v) Triton X-100. Using Amberlite XAD-2, more than 90% of the detergent was then removed. Liposomes were prepared by precipitating the solubilized proteins with acetone in the presence of phosphatidylcholine. The receptor activity of these liposomes was assayed using human 125I-labeled LDL. Filtration was used to separate bound from free 125I-labeled LDL. The assay was optimized; 0.25 mM CaCl2, 25 mM NaCl, pH 8.0, were chosen as the standard conditions. Binding of 125I-labeled LDL was dependent on Ca2+. Liposomes containing solubilized membrane proteins from treated rats displayed Ca2+-dependent binding which was 11 times higher than for control rats. The specific binding of 125I-labeled LDL was saturable with a Kd = 18 micrograms/ml. 125I-Labeled LDL was displaced by unlabeled lipoproteins containing apolipoproteins B and E and by dimyristoylphosphatidylcholine liposomes containing purified apolipoprotein E, but not by HDL3. The binding was abolished by pronase and was inhibited by suramin. Ligand blotting with 125I-labeled LDL revealed one band of protein with an apparent molecular weight of 133,000 daltons. These properties are characteristic of the low density lipoprotein receptor.     

Uptake of rat plasma HDL subfractions labeled with [3H]cholesteryl linoleyl ether or with 125I by cultured rat hepatocytes and adrenal cells

Rat plasma low- and high-density lipoproteins were labeled with [3H]cholesteryl linoleyl ether and isolated by rate-zonal ultracentrifugation into apolipoprotein B-containing LDL, apolipoprotein E-containing HDL1 and apolipoprotein E-poor HDL2. These fractions were incubated with cultured rat hepatocytes and comparable amounts of all lipoproteins were taken up by the cells. Rat HDL was isolated at d 1.085-1.21 g/ml and apolipoprotein E-free HDL was prepared by heparin Sepharose chromatography. The original HDL and the apolipoprotein E-free HDL were labeled with 125I or with [3H]cholesteryl linoleyl ether and incubated with rat hepatocytes or adrenal cells in culture. The uptake of apolipoprotein E-free [3H]cholesterol linoleyl ether HDL by the cultured hepatocytes was 20-40% more than that of the original HDL. Comparison of uptake of cholesteryl ester moiety (represented by uptake of [3H]cholesteryl linoleyl ether) and of protein moiety (represented by metabolism of 125I-labeled protein) was carried out using both original and apolipoprotein E-free HDL. In experiments in which low concentrations of HDL were used, the ratio of 3H/125I exceeded 1.0. In cultured adrenal cells, the uptake of [3H]cholesteryl linoleyl ether-labeled HDL was stimulated 3-6-fold by 1 X 10(-7) M ACTH, while the uptake of 125I-labeled HDL increased about 2-fold. The ratio of 3H/125I representing cellular uptake was 2-3 and increased to 5 in ACTH-treated cells. The present results indicate that in cultured rat hepatocytes the uptake of homologous HDL does not depend on the presence of apolipoprotein E. Evidence was also presented for an uptake of cholesteryl ester independent of protein uptake in cultured rat adrenal cells and to a lesser extent in rat hepatocytes.     

Rat high density lipoprotein subfraction (HDL3) uptake and catabolism by isolated rat liver parenchymal cells.

Rat liver parenchymal cell binding, uptake, and proteolytic degradation of rat 125I-labeled high density lipoprotein (HDL) subfraction, HDL3 (1.10 less than d less than 1.210 g/ml), in which apo-A-I is the major polypeptide, were investigated. Structural and metabolic integrity of the isolated cells was verified by trypan blue exclusion, low lactic dehydrogenase leakage, expected morphology, and gluconeogenesis from lactate and pyruvate. 125I-labeled HDL3 was incubated with 10 X 10(6) cells at 37 degrees and 4 degrees in albumin and Krebs-Henseleit bicarbonate buffer, pH 7.4. Binding and uptake were determined by radioactivity in washed cells. Proteolytic degradation was determined by trichloroacetic acid-soluble radioactivity in the incubation medium. At 37 degrees, maximum HDL3 binding (Bmax) and uptake occurred at 30 min with a Bmax of 31 ng/mg dry weight of cells. The apparent dissociation constant of the HDL3 receptor system (Kd) was 60 X 10(-8) M, based on Mr = 28,000 of apo-A-I, the predominant rat HDL3 protein. Proteolytic degradation showed a 15-min lag and then constant proteolysis. After 2 hours 5.8% of incubated 125I-labeled HDL3 was degraded. Sixty per cent of cell radioactivity at 37 degrees was trypsin-releasable. At 37 degrees, 125I-labeled HDL3 was incubated with cells in the presence of varying concentrations of native (cold) HDL3, very low density lipoproteins, and low density lipoproteins. Incubation with native HDL3 resulted in greatest inhibition of 125I-labeled HDL3 binding, uptake, and proteolytic degradation. When 125I-labeled HDL3 was preincubated with increasing amounts of HDL3 antiserum, binding and uptake by cells were decreased to complete inhibition. Cell binding, uptake, and proteolytic degradation of 125I-labeled HDL3 were markedly diminished at 4 degrees. Less than 1 mM chloroquine enhanced 125I-labeled HDL3 proteolysis but at 5 mM or greater, chloroquine inhibited proteolysis with 125I-labeled HDL3 accumulation in cells. L-[U-14C]Lysine-labeled HDL3 was bound, taken up, and degraded by cells as effectively as 125I-labeled HDL3. These data suggest that liver cell binding, uptake, and proteolytic degradation of rat HDL3 are actively performed and linked in the sequence:binding, then uptake, and finally proteolytic degradation. Furthermore, there may be a specific HDL3 (lipoprotein A) receptor of recognition site(s) on the plasma membrane. Finally, our data further support our previous reports of the important role of liver lysosomes in proteolytic degradation of HDL3.     

The role of apolipoprotein A-I and apolipoprotein A-II in high-density lipoprotein binding to human adipocyte plasma membranes

Adipocyte plasma membranes purified from omental fat tissue biopsies of massively obese subjects possess specific binding sites for high-density lipoprotein (HDL3). This binding was independent of apolipoprotein E as HDL3 isolated from plasma of an apolipoprotein E-deficient individual was bound to a level comparable to that of normal HDL3. To examine the importance of apolipoprotein A-I, the major HDL3 apolipoprotein, in the specific binding of HDL3 to human adipocytes, HDL3 modified to contain varying proportions of apolipoproteins A-I and A-II was prepared by incubating normal HDL3 particles with different amounts of purified apolipoprotein A-II. As the apolipoproteins A-I-to-A-II ratio in HDL3 decreased, the binding of these particles to adipocyte plasma membranes was reduced. Compared to control HDL3, a 92 +/- 3.1% reduction (mean +/- S.E., n = 3) in maximum binding capacity was observed along with an increased binding affinity for HDL3 particles in which almost all of the apolipoprotein A-I had been replaced by A-II. The uptake of HDL cholesteryl ester by intact adipocytes as monitored by [3H]cholesteryl ether labeled HDL3, was also significantly reduced (about 35% reduction, P less than 0.005) by substituting apolipoprotein A-II for A-I in HDL3. These data suggest that HDL binding to human adipocyte membranes is mediated primarily by apolipoprotein A-I and that optimal delivery of cholesteryl ester from HDL to human adipocytes is also dependent on apolipoprotein A-I.     

The effects of human low and high density lipoproteins on the binding of rat intermediate density lipoproteins to rat liver membranes

Upon incubation with rat liver membranes, radioiodinated rat intermediate density lipoproteins (IDL) interacted with at least two binding sites having a low and a high affinity as demonstrated by the curvilinear Scatchard plots obtained from the specific binding data. The purpose of our work was to identify the nature of these binding sites. Human low density lipoproteins (LDL), contain apolipoprotein B only, and human high density lipoproteins (HDL3), containing neither apolipoprotein B nor E, were both capable of decreasing the specific binding of rat 125I-IDL. The Scatchard analysis clearly revealed that only the low affinity component was affected by the addition of these human lipoproteins. In fact, the low affinity binding component gradually decreased as the amount of human LDL or HDL3 increased in the binding assay. At a 200-fold excess of human LDL or HDL3, the low affinity binding was totally masked, and the Scatchard plot of the specific 125I-IDL binding became linear. Only the high affinity binding component was left, enabling a precise measurement of its binding parameters. In a series of competitive displacement experiments in which the binding assay contained a 200-fold excess of human LDL or HDL3, only unlabeled rat IDL effectively displaced the binding of rat 125I-IDL. We conclude that the low affinity binding of rat IDL to rat liver membranes is due to weak interactions with unspecified lipoprotein binding sites. The camouflage of these sites by human lipoproteins makes possible the study of IDL binding to the high affinity component which likely represents the combined effect of IDL binding to both the remnant and the LDL receptors.     

Characterization of high-density lipoprotein binding and cholesterol efflux in cultured mouse adipose cells

Binding of high-density lipoproteins to cultured mouse Ob1771 adipose cells was studied, using labeled human HDL3, mouse HDL and apolipoprotein AI- or AII-containing liposomes. In each case, saturation curves were obtained, yielding linear Scatchard plots. The Kd values were found to be respectively 18, 42, 30 and 3.4 micrograms/ml, whereas the maximal binding capacities were found to be 160, 100, 90 and 21 ng/mg of cell protein. Apoprotein AI not inserted into liposomes did not bind. The binding of 125I-HDL3 was competitively inhibited by apolipoprotein AI-containing liposomes greater than mouse HDL greater than HDL3. The binding of 125I-labeled apolipoprotein AI- and 125I-labeled apolipoprotein AII-containing liposomes was competitively inhibited by HDL3, apolipoprotein AI- and apolipoprotein AII-containing liposomes. Dimyristoylphosphatidylcholine liposomes containing or not cholesterol did not interfere with the binding of labeled HDL3 or apolipoprotein-containing liposomes. Binding studies on crude membranes of Ob1771 adipose cells revealed the presence of intracellular binding sites for LDL and HDL3. Thus, adipose cells have specific binding sites for apolipoprotein E-free HDL and apolipoprotein AI (or AII) is the ligand for these binding sites. Long-term exposure of adipose cells to LDL cholesterol as a function of LDL concentration led to an accumulation of cellular unesterified cholesterol. This process was saturable and reversible as a function of time and concentration by exposure to HDL3 or apolipoprotein AI-containing liposomes, whereas apolipoprotein AII-containing liposomes did not promote any cholesterol efflux. Since long-term exposure of adipose cells to LDL and HDL3 did not affect the number of apolipoprotein B,E receptors and apolipoprotein E-free binding sites, respectively, it appears that adipose cells do not show efficient cholesterol homeostasis and thus could accumulate or mobilize unesterified cholesterol.     

Binding characteristics of high density lipoprotein subclasses to porcine liver, adrenal and skeletal muscle plasma membranes

1. We compared binding characteristics of 125I-labeled high density lipoprotein (HDL) subclasses to porcine liver, adrenal and skeletal muscle plasma membranes. 2. HDL subclasses were discriminated by their buoyant densities (HDL2 and HDL3) or by their apolipoprotein (apo) content (Lp-AI (particles containing apoA-I but no apoA-II) and LpA-I/A-II (particles containing both apoA-I and apoA-II)). 3. HDL2 and HDL3 showed saturable binding to the three types of membrane preparations. 4. No differences were found in the Kds within one HDL subclass. 5. Kds and maximal binding of HDL2 were lower than these of HDL3. Unlabeled HDL2 and HDL3, but not LDL, effectively displaced 125I-HDL2 and 125I-HDL3. 6. Binding of HDL was independent of the concentration of NaCl and did not require calcium. 7. These results suggest a process mediated by a single specific receptor in porcine liver, adrenal and skeletal muscle plasma membranes. 8. We also studied binding characteristics of HDL subclasses Lp-AI and LpA-I/A-II to porcine liver membranes. LpA-I showed the highest Kd and maximal binding. 9. All types of HDL subclasses studied (i.e. HDL2, HDL3, LpA-I and LpA-I/A-II) effectively competed for binding of both Lp-AI and LpA-I/A-II, suggesting that the HDL subclasses studied bind to the same receptor by their apoA-I moiety.