Low density lipoprotein binding, internalization, and degradation in human adipose cells.

Human adipose tissue derives its cholesterol primarily from circulating lipoproteins. To study fat cell-lipoprotein interactions, low density lipoprotein (LDL) uptake and metabolism were examined using isolated human adipocytes. The 125I-labelled LDL (d = 1.025-1.045) was bound and incorporated by human fat cells in a dose-dependent manner with an apparent Km of 6.9 + 0.9 microgram LDL protein/mL and a Vmax of 15-80 microgram LDL protein/mg lipid per 2 h. In time-course studies, LDL uptake was characterized by rapid initial binding followed by a linear accumulation for at least 4 h. The 125I-labelled LDL degradation products (trichloroacetic acid soluble iodopeptides) accumulated in the incubation medium in a progressive manner with time. Azide and F- inhibited LDL internalization and degradation, suggesting that these processes are energy dependent. Binding and cellular internalization of 125I-labelled LDL lacked lipoprotein class specificity in that excess (25-fold) unlabelled very low density lipoprotein (VLDL) (d less than 1.006) and high density lipoprotein (HDL) (d = 1.075-1.21) inhibited binding and internalization of 125I-labelled LDL. On an equivalent protein basis HDL was the most potent. The 125I-labelled LDL binding to an adipocyte plasma membrane preparation was a saturable process and almost completely abolished by a three- to four-fold greater concentration of HDL. The binding, internalization, and degradation of LDL by human adipocytes resembled that reported by other mesenchymal cells and could account for a significant proportion of in vivo LDL catabolism. It is further suggested that adipose tissue is an important site of LDL and HDL interactions.     

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.     

Differences in the processing of chylomicron remnants and beta-VLDL by macrophages

To gain a detailed understanding of those factors that govern the processing of dietary-derived lipoprotein remnants by macrophages we examined the uptake and degradation of rat triacylglycerol-rich chylomicron remnants and rat cholesterol-rich beta-very low density lipoprotein (beta-VLDL) by J774 cells and primary cultures of mouse peritoneal macrophages. The level of cell associated 125I-labeled beta-VLDL and 125I-labeled chylomicron remnants reached a similar equilibrium level within 2 h of incubation at 37 degrees C. However, the degradation of 125I-labeled beta-VLDL was two to three times greater than the degradation of 125I-labeled chylomicron remnants at each time point examined, with rates of degradation of 161.0 +/- 36.0 and 60.1 +/- 6.6 ng degraded/h per mg cell protein, respectively. At similar extracellular concentrations of protein or cholesterol, the relative rate of cholesteryl ester hydrolysis from [3H]cholesteryl oleate/cholesteryl [14C]oleate-labeled chylomicron remnants was one-third to one-half that of similarly labeled beta-VLDL. The reduction in the relative rate of chylomicron remnant degradation by macrophages occurred in the absence of chylomicron remnant-induced alterations in low density lipoprotein (LDL) receptor recycling or in retroendocytosis of either 125I-labeled lipoprotein. The rate of internalization of 125I-labeled beta-VLDL by J774 cells was greater than that of 125I-labeled chylomicron remnants, with initial rates of internalization of 0.21 ng/min per mg cell protein for 125I-labeled chylomicron remnants and 0.39 ng/min per mg cell protein for 125I-labeled beta-VLDL. The degradation of 125I-labeled chylomicron remnants and 125I-labeled beta-VLDL was dependent on lysosomal enzyme activity: preincubation of macrophages with the lysosomotropic agent monensin reduced the degradation of both lipoproteins by greater than 90%. However, the pH-dependent rate of degradation of 125I-labeled chylomicron remnants by lysosomal enzymes isolated from J774 cells was 50% that of 125I-labeled beta-VLDL. The difference in degradation rates was dependent on the ratio of lipoprotein to lysosomal protein used and was greatest at ratios greater than 50. The degradation of 125I-labeled beta-VLDL by isolated lysosomes was reduced 30-40% by preincubation of beta-VLDL with 25-50 micrograms oleic acid/ml, suggesting that released free fatty acids could cause the slower degradation of chylomicron remnants. Thus, differences in the rate of uptake and degradation of remnant lipoproteins of different compositions by macrophages are determined by at least two factors: 1) differences in the rates of lipoprotein internalization and 2) differences in the rate of lysosomal degradation.     

Processing of follitropin and its receptor by cultured pig Sertoli cells. Effect of monensin

Immature pig Sertoli cells, cultured in a chemically defined medium, are able to maintain many of their functional characteristics for at least two weeks. This model was used to investigate the binding, internalization and degradation of 125I-labelled human follitropin (hFSH) and the effects of pig FSH (pFSH) on its own receptors. The binding of 125I-labelled hFSH was dependent on time, temperature and concentration. At 4 degrees C, the apparent steady state was reached in 8-12 h and remained constant for at least 24 h, whereas at 33 degrees C the apparent equilibrium was reached in 4-6 h. Thereafter the total binding declined and by 24 h it was less than 50% of the maximum binding. At 33 degrees C the binding for the hormone to its surface receptor was followed by internalization of the hormone (half-life approximately equal to 1 h) and its degradation (half-life approximately equal to 3 h). The receptor-mediated internalization of hFSH was blocked by phenylarsine oxide. In the presence of the ionophore monensin (20 microM) the rates of binding and internalization were not modified but the degradation rate was much lower (half-life approximately equal to 18 h). Thus, in the presence of monensin, maximum binding increased twofold to threefold, and remained constant for 24 h. This increase was mainly due to an increase of the internalized hormone. When Sertoli cells were exposed to pFSH there was a loss of its own receptor, which was both dose-dependent (ED50 = 250 ng/ml) and time-dependent (t 1/2 = 14 h). Cycloheximide did not modify the FSH-induced down-regulation, whereas monensin enhanced the down-regulation process. These results show that FSH, like other ligands, is internalized and degraded by its target cells and indicate that the hormone-mediated down-regulation is related to the internalization process. However, the discrepancy between the rate of internalization and of hormone-induced down-regulation, suggests that some of the internalized receptors are recycled.     

Metabolism of low-density lipoproteins by cultured hepatocytes from normal and homozygous familial hypercholesterolemic subjects

The profoundly elevated concentrations of low-density lipoproteins (LDL) present in homozygous familial hypercholesterolemia lead to symptomatic cardiovascular disease and death by early adulthood. Studies conducted in nonhepatic tissues demonstrated defective cellular recognition and metabolism of LDL in these patients. Since mammalian liver removes at least half of the LDL in the circulation, the metabolism of LDL by cultured hepatocytes isolated from familial hypercholesterolemic homozygotes was compared to hepatocytes from normal individuals. Fibroblast studies demonstrated that the familial hypercholesterolemic subjects studied were LDL receptor-negative (less than 1% normal receptor activity) and LDL receptor-defective (18% normal receptor activity). Cholesterol-depleted hepatocytes from normal subjects bound and internalized 125I-labeled LDL (Bmax = 2.2 micrograms LDL/mg cell protein). Preincubation of normal hepatocytes with 200 micrograms/ml LDL reduced binding and internalization by approx. 40%. In contrast, 125I-labeled LDL binding and internalization by receptor-negative familial hypercholesterolemic hepatocytes was unaffected by cholesterol loading and considerably lower than normal. This residual LDL uptake could not be ascribed to fluid phase endocytosis as determined by [14C]sucrose uptake. The residual LDL binding by familial hypercholesterolemia hepatocytes led to a small increase in hepatocyte cholesterol content which was relatively ineffective in reducing hepatocyte 3-hydroxy-3-methylglutaryl-CoA reductase activity. Receptor-defective familial hypercholesterolemia hepatocytes retained some degree of regulatable 125I-labeled LDL uptake, but LDL uptake did not lead to normal hepatocyte cholesterol content or 3-hydroxy-3-methylglutaryl-CoA reductase activity. These combined results indicate that the LDL receptor abnormality present in familial hypercholesterolemia fibroblasts reflects deranged hepatocyte LDL recognition and metabolism. In addition, a low-affinity, nonsaturable uptake process for LDL is present in human liver which does not efficiently modulate hepatocyte cholesterol content or synthesis.     

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

The specific binding of 125I-labelled low density lipoprotein ([125I]LDL to human adipocyte plasma membranes was higher at 37 than at 0 degree C. Prior treatment of membranes with pronase had no effect on LDL binding measured at 0 degree C but consistently stimulated binding at 37 degrees 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 degrees C (p less than 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.     

A macrophage receptor that recognizes oxidized low density lipoprotein but not acetylated low density lipoprotein

The formation of cholesterol-loaded macrophage foam cells in arterial tissue may occur by the uptake of modified lipoproteins via the scavenger receptor pathway. The macrophage scavenger receptor, also called the acetylated low density lipoprotein (Ac-LDL) receptor, has been reported to recognize Ac-LDL as well as oxidized LDL species such as endothelial cell-modified LDL (EC-LDL). We now report that there is another class of macrophage receptors that recognizes EC-LDL but not Ac-LDL. We performed assays of 0 degrees C binding and 37 degrees C degradation of 125I-Ac-LDL and 125I-EC-LDL by mouse peritoneal macrophages. Competition studies showed that unlabeled Ac-LDL could compete for only 25% of the binding and only 50% of the degradation of 125I-EC-LDL. Unlabeled EC-LDL, however, competed for greater than 90% of 125I-EC-LDL binding and degradation. Unlabeled Ac-LDL was greater than 90% effective against 125I-Ac-LDL; EC-LDL competed for about 80% of 125I-Ac-LDL binding and degradation. Copper-oxidized LDL behaved the same as EC-LDL in all the competition studies. Copper-mediated oxidation of Ac-LDL produced a superior competitor which could now displace 90% of 125I-EC-LDL binding. After 5 h at 37 degrees C in the presence of ligand, macrophages accumulated six times more cell-associated radioactivity from 125I-EC-LDL than from 125I-Ac-LDL, despite approximately equal amounts of degradation to trichloroacetic acid-soluble products, which may imply different intracellular processing of the two lipoproteins. Our results suggest that 1) there is more than one macrophage "scavenger receptor" for modified lipoproteins; and 2) oxidized LDL and Ac-LDL are not identical ligands with respect to macrophage recognition and uptake.     

Biochemical and morphological effects of gentamicin in human proximal tubular cells: effects on lipid and lipoprotein metabolism

The effects of gentamicin, an antibiotic used extensively for antimicrobial therapy on the ultrastructure, binding, internalization, degradation, and cholesterol esterification of low-density lipoproteins, were investigated in cultured human proximal tubular cells. Cells were incubated with 0.3 mM gentamicin for 21 days with the following observations. Cells treated with gentamicin contained numerous "myeloid bodies." The binding, internalization, and degradation of 125I-labeled low-density lipoproteins ([125I]LDL) in cells treated with gentamicin was twofold lower than control cells. Pulse-chase experiments demonstrated that gentamicin did not impair the internalization of receptor-bound LDL and their subsequent transport to the lysosome. The relative amounts of [125I]LDL displaced by increasing concentrations of unlabeled LDL were the same in both gentamicin-treated and control cells. This pattern was reflected in the cell surface binding, internalization, and degradation of [125I]LDL. Gentamicin did not alter the degradation of [125I]LDL in cell homogenates at 4.0. The data suggest that gentamicin decreases the receptor-mediated endocytosis of LDL and subsequent lipid metabolism.     

Uptake and degradation of high density lipoprotein: comparison of fibroblasts from normal subjects and from homozygous familial hypercholesterolemic subjects.

Fibroblasts cultured from the skin of subjects with homozygous familial hyperlipoproteinemia (HFH) internalize and degrade low density lipoproteins at a much lower rate than do fibroblasts from normal subjects. Evidence has been presented that this reflects the absence from such mutant cells of specialized binding sites with high affinity for low density lipoproteins. The specificity of this membrane defect in familial hypercholesterolemia is further supported by the present studies comparing the metabolism of low density lipoproteins (LDL) and high density lipoproteins (HDL) in normal fibroblasts and in fibroblasts from HFH patients. The surface binding (trypsin-releasable (125)I) of (125)I-labeled LDL by HFH cells was approximately 30% of that by normal cells at a concentration of 5 micro g LDL protein per ml. At the same concentration the internalization (cell-associated (125)I after trypsinization) and degradation (trichloroacetic acid-soluble non-iodide (125)I) of (125)I-labeled LDL were less than 10% of the values obtained with normal cells. In contrast, the binding of (125)I-labeled HDL to HFH cells was actually somewhat greater than that to normal cells. Despite this, the internalization and degradation of (125)I-labeled HDL by HFH cells averaged only 70% of that by normal cells. [(3)H]- or [(14)C]Sucrose uptake, a measure of fluid uptake by pinocytosis, was similar in normal and HFH fibroblasts. These findings are consistent with the proposal that fibroblasts from subjects with HFH lack high-affinity receptors for LDL. These receptors do not play a significant role in HDL binding and uptake. Instead, as previously proposed, HDL appears to bind randomly on the cell surface and its internalization is not facilitated by the specific mechanism that internalizes LDL. The small but significant abnormalities in HDL binding and internalization, however, suggest that there may be additional primary or secondary abnormalities of membrane structure and function in HFH cells. Finally, the observed overall rate of uptake of LDL (that internalized plus that degraded) by HFH fibroblasts was considerably greater than that expected from fluid endocytosis alone. This implies that adsorptive endocytosis, associated with binding to low-affinity sites on the cell surface, may play a significant role in LDL degradation by HFH cells, even though it does not regulate endogenous cholesterol synthesis in these cells.     

Oleic,linoleic and linolenic acids enhance receptor-mediated uptake of low density lipoproteins in Hep-G2 cells

In the present study, the binding, internalization and degradation of low-density lipoprotein (LDL) was investigated in Hep-G2 cells treated with 18:0, 18:1, 18:2 and 18:3. In non-treated control cells, the surface binding (heparin-releasable) of 125I-LDL progressed in a saturable manner reaching equilibrium within 2 h, amounting 24.0 +/- 1.1, 29.5 +/- 1.3 and 31.4 +/- 2.8 (ng/mg cell protein) at 1, 2 and 4 h, respectively. The cells rapidly internalized 125I-LDL reaching a plateau at 2 h (72.4 +/- 6.3/1 h, 96.7 +/- 4.3/2 h and 100.8 +/- 4.6 ng/mg protein/4 h, respectively). The degradation of internalized LDL progressed slowly during the first hour of incubation reflecting the time required to an uptake and delivery of LDL to the cellular lysosomes. The levels of degraded LDL discharged into the medium then increased rapidly in a linear manner after the initial lag period, amounting 16.8 +/- 1.2, 51.8 +/- 7.0 and 118.2 +/- 5.7 ng/mg protein at 1, 2 and 4 h, respectively. The treatment of cells with of 1.0 mM of fatty acids for 4 h resulted in a significant increase in the surface binding of 125I-LDL compared to the control (34.9 +/- 3.0), but it was significantly lower in cells exposed to 18:0 (48.2 +/- 2.0) than to 18:1 (56.8 +/- 5.1), 18:2 (56.0 +/- 3.5) and 18:3 (57.8 +/- 6.0 ng/mg protein/4 h) (P < 0.05). The levels of degraded LDL in cells remained nearly the same regardless of fatty acid treatments, but degraded LDL levels in the medium were much higher in cells exposed to 18:1 (167.6 +/- 10.1), 18:2 (159.8 +/- 7.7) and 18:3 (165.1 +/- 14.7) than to 18:0 (142.1 +/- 8.4) and the control (121.2 +/- 3.4 ng/mg protein/4 h) (P < 0.05). The present finding that 18:1 is equally effective in enhancing the receptor-mediated LDL uptake and its degradation as those of 18:2 and 18:3 suggests that the major action of 18:1 in lowering LDL-cholesterol levels also involves an increased clearance of LDL via hepatic LDL-receptors.     

Free fatty acids activate a high-affinity saturable pathway for degradation of low-density lipoproteins in fibroblasts from a subject homozygous for familial hypercholesterolemia.

This paper describes a mechanism for degradation of low-density lipoprotein (LDL) in fibroblasts unable to synthesize the LDL receptor. In this cell line, long-chain free fatty acids (FFA) activated 125I-LDL uptake; unsaturated FFA were the most efficient. The first step of this pathway was the binding of LDL apoB to a single class of sites on the plasma membrane and was reversible in the presence of greater than or equal to 10 mM suramin. Binding equilibrium was achieved after a 60-90-min incubation at 37 degrees C with 1 mM oleate; under these conditions, the apparent Kd for 125I-LDL binding was 12.3 micrograms/mL. Both cholesterol-rich (LDL and beta-VLDL) and triglyceride-rich (VLDL) lipoproteins, but not apoE-free HDL, efficiently competed with 125I-LDL for this FFA-induced binding site. After LDL bound to the cell surface, they were internalized and delivered to lysosomes; chloroquine inhibited subsequent proteolysis of LDL and thereby increased the cellular content of the particles. A physiological oleate to albumin molar ratio, i.e., 1:1 (25 microM oleate and 2 mg/mL albumin), was sufficient to significantly (p less than 0.01) activate all three steps of this alternate pathway: for example, 644 +/- 217 (25 microM oleate) versus 33 +/- 57 (no oleate) ng of LDL/mg of cell protein was degraded after incubation (2 h, 37 degrees C) with 50 micrograms/mL 125I-LDL. We speculate that this pathway could contribute to the clearance of both chylomicron remnants and LDL.     

The fatty acid composition of chylomicron remnants influences their binding and internalization by isolated hepatocytes.

The binding and internalization of (125)I-labelled chylomicron remnants derived from palm, olive, corn, or fish oil (rich in saturated, monounsaturated, n-6, or n-3 polyunsaturated fatty acids, respectively) by hepatocytes from rats fed a low-fat diet or a diet supplemented with the corresponding fat for 21 days was investigated. In hepatocytes from rats fed the low-fat diet, the association of radioactivity with the cells at 4 degrees C (a measure of initial binding only) was similar with all types of remnants tested, but was more rapid at 37 degrees C (a measure of binding plus internalization) when fish oil, as compared to olive, corn or palm oil remnants, was used, and similar differences in the internalization of the particles were observed. In contrast, when hepatocytes from rats fed the fat-supplemented diets were used, the rate of association at 37 degrees C of remnants with cells from rats fed palm, corn or fish oil was similar, and higher than that found with cells from animals fed olive oil, and in this case these differences were mainly due to changes in the binding of the particles to the cells at 4 degrees C. Both excess low-density lipoprotein (LDL), which inhibits remnant uptake by the LDL receptor, and lactoferrin, which blocks the LDL receptor-related protein (LRP), were found to decrease the association of the remnants with cells from rats fed the low-fat and high-fat diets. However, in hepatocytes from animals given the low-fat diet, most of the differences between the various types of particle were retained in the presence of lactoferrin, but abolished in the presence of LDL. In contrast, in cells from rats fed the high-fat diets, the differences were reduced by both lactoferrin and LDL. These findings demonstrate that the hepatic uptake of chylomicron remnants is influenced both by the fatty acid composition of the particles, and by longer-term adaptive changes in liver tissue, and suggest that the former effects are mediated mainly by the LDL receptor, while the latter may involve both the LDL receptor and the LRP.     

Effect of the proteolysis of low-density serum lipoproteins on their interaction with macrophages

125I-labelled human serum low density lipoproteins (LDL) were incubated with cultured mouse peritoneal macrophages at 37 degrees C, with the following study of cellular uptake and 125I-LDL degradation by measuring the content of TCA-soluble products of LDL hydrolysis in the cultural medium. It was shown that limited pepsin proteolysis of LDL (10%) led to a more effective LDL uptake and degradation by macrophages. The data suggest that enzyme-induced modification of LDL may increase their atherogenicity.     

Uptake and degradation of human very-low-density lipoproteins by rat liver endothelial cells in culture

Rat liver endothelial cells in primary cultures take up and degrade 125I-labelled human very-low-density lipoproteins (VLDL) in a saturable fashion at physiological triacylglycerol concentrations. The iodinated VLDL are readily taken up by the freshly isolated endothelial cells and degradation products appear in the medium about 30 min after the addition of VLDL to the cultures. Uptake and degradation at 37 degrees C are effectively inhibited by unlabelled human VLDL, low-density lipoproteins (LDL), high-density lipoproteins and lymph chylomicrons, but only modestly by acetylated LDL. Purified apolipoproteins E and C-III:1 also compete with the uptake of iodinated VLDL, but when degradation was studied for longer periods of time, such a competition could not be demonstrated. This may be due to the fact that the added apolipoproteins become associated with the lipoproteins. In binding experiments at 7 degrees C, iodinated apolipoprotein C III:1 bound to the liver endothelial cells in a manner characteristic of receptor binding with a dissociation constant of 0.5 microM. This binding could not only be inhibited by unlabelled apolipoprotein C-III:1 but also by unlabelled apolipoprotein E. The results indicate that rat liver endothelial cells carry receptors for VLDL and that these recognize the apolipoproteins E, C-III and B on the lipoprotein surface. Considering the large endothelial surface and high blood flow through the liver, significant quantities of lipoproteins can be taken up and degraded, thus influencing the levels of circulating lipoproteins in the in vivo situation.     

Regulation of the uptake and degradation of beta-very low density lipoprotein in human monocyte macrophages

In normal human monocyte macrophages 125I-labeled beta-migrating very low density lipoproteins (125I-beta-VLDL), isolated from the plasma of cholesterol-fed rabbits, and 125I-human low density lipoprotein (LDL) were degraded at similar rates at protein concentrations up to 50 micrograms/ml. The high affinity degradation of 125I-labeled human LDL saturated at approximately 50 micrograms/ml; however, 125I-labeled rabbit beta-VLDL high affinity degradation saturated at 100-120 micrograms/ml. The activity of the beta-VLDL receptor was 3-fold higher than LDL receptor activity on freshly isolated normal monocyte macrophages, but with time-in-culture both receptor activities decreased and were similar after several days. The degradations of both beta-VLDL and LDL were Ca2+ sensitive, were markedly down regulated by sterols, and were up regulated by preincubation of the cells in a lipoprotein-free medium. The beta-VLDL receptor is genetically distinct from the LDL receptor as indicated by its presence on monocyte macrophages from a familial hypercholesterolemic homozygote. Human thoracic duct lymph chylomicrons as well as lipoproteins of Sf 20-5000 from fat-fed normal subjects inhibited the degradation of 125I-labeled rabbit beta-VLDL as effectively as nonradioactive rabbit beta-VLDL. We conclude: 1) the beta-VLDL receptor is genetically distinct from the LDL receptor, and 2) intestinally derived human lipoproteins are recognized by the beta-VLDL receptor on macrophages.     

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.     

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

The binding of human 125I-labeled HDL3 (high-density lipoproteins, rho 1.125-1.210 g/cm3) 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 (Kd), at both 37 and 4 degrees C, was 2.9 micrograms protein/ml (approx. 2.9 X 10(-8) M); the capacity of the binding sites was 490 ng HDL3 (approx. 4.9 pmol) per mg membrane protein at 37 degrees C and 115 at 4 degrees C. Human low-density lipoproteins (LDL) and very-low-density lipoproteins (VLDL) also bound to these sites (Kd = 41 micrograms protein/ml, approx. 6.7 X 10(-8) M for LDL, and Kd = 5.7 micrograms protein/ml, approx. 7.0 X 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 HDL3 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-labeled HDL3 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 (Kd = 130 micrograms protein/ml) than did the other subclasses of HDL.     

Human hepatic low-density lipoprotein receptors: associations of receptor activities in vitro with plasma lipid and apolipoprotein concentrations in vivo

The relationships of plasma lipid and apolipoprotein (apo) concentrations to hepatic low-density lipoprotein (LDL) receptor activity were examined in 21 subjects (16 females, 5 males), who were undergoing laparotomy for non-neoplastic disease (cholecystectomy in 16). None had familial hypercholesterolemia, or renal, endocrine or hepatic disease. Ages were 37-77 years (mean, 58 years), plasma cholesterol concentrations 4.09-6.72 mmol/l (5.38) and plasma triacylglycerol concentrations 0.75-2.35 mmol/l (1.36). Receptor activity was quantified in vitro as the total saturable binding and EDTA-suppressible binding (representing apoB,E receptors) of 125I-labelled human LDL (15 micrograms protein/ml) by liver homogenate at 37 degrees C. There were no significant differences between men and women in 125I-labeled LDL binding. In the pooled data, EDTA-suppressible binding averaged 50 ng 125I-LDL protein/mg cell protein (S.D., 15). Total saturable binding averaged 2-fold greater (mean, 101 ng/mg; S.D., 32). Plasma cholesterol, LDL cholesterol and apoB concentrations were negative functions of both EDTA-suppressible binding and total saturable binding, but the correlations with EDTA-suppressible binding were stronger (cholesterol: r = -0.59, P less than 0.01; LDL cholesterol: r = -0.48, P less than 0.05; apoB: r = -0.61, P less than 0.01). Plasma triacylglycerol, high-density lipoprotein cholesterol and apoA-I concentrations were not related to either measure of receptor activity. These results provide evidence that the activity of apoB,E receptors in the liver is a major determinant of the plasma LDL concentration in middle-aged and elderly humans.     

A saturable, high-affinity binding site for human low density lipoprotein on freshly isolated rat hepatocytes

Freshly isolated rat hepatocytes bind the solely apolipoprotein B-containing human low density lipoprotein (LDL) with a high-affinity component. After 1 h of incubation less than 30% of the cell-associated human LDL is internalized and no evidence for any subsequent high-affinity degradation was obtained. Scatchard analysis of the binding data for human 125I-labeled LDL indicates that the high-affinity receptor for human LDL on rat hepatocytes possesses a Kd of 2.6 x 10(-8)M, while the binding is dependent on the extracellular Ca2+ concentration. Competition experiments indicate that both the apolipoprotein B-containing lipoproteins (human LDL and rat LDL) as well as the apolipoprotein E-containing lipoproteins (human HDL and rat HDL) do compete for the same surface receptor. It is concluded that hepatocytes freshly isolated from untreated rats do contain, in addition to the earlier described rat lipoprotein receptor which does not interact with human apolipoprotein B-containing LDL, a high-affinity receptor which interacts both with solely apolipoprotein B-containing human LDL and apolipoprotein E-containing lipoproteins.     

Scavenger lipoprotein receptors are more effective in ligand internalization than low density lipoprotein receptors in human monocyte-macrophages

Human monocyte-macrophages in culture express specific receptors for low density lipoproteins (LDL receptor) and human acetylated LDL (AcLDL receptors or scavenger receptors). After 24 h in lipoprotein-deficient serum, the cells expressed 2-3 fold more AcLDL receptors than LDL receptors as measured by trypsin releasable radioactivity after exposure to 125I-LDL or 125I-AcLDL at 37 degrees C. The efficiency of intracellular ligand delivery by the two receptors was evaluated as an internalization index (defined as intracellular + degraded/bound ligand). This index was several fold greater for 125I-AcLDL than for 125I-LDL, in the same cells exposed to either ligand under identical conditions. These results suggest that the scavenger receptors recycle more rapidly than do LDL receptors.