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.     

Low density lipoprotein receptors and catabolism in primary cultures of rabbit hepatocytes.

Rabbit 125I-labelled low density lipoproteins (LDL) were incubated with primary monolayer cultures of rabbit hepatocytes in studies designed to assess the role of liver in LDL catabolism at the cellular level. After hepatocytes were preincubated for 20 h in lipoprotein-free medium, they exhibited time- and concentration-dependent interaction with 125I-labelled DLD at concentrations to 1 mg LDL protein/ml and times to 24 h. After a 3 h (37 degrees C) incubation with 50 microgram LDL protein/ml, hepatocytes bound 400 ng (LDL protein)/mg (cell protein), internalized 280 ng/mg, and degraded 660 ng/mg. Internalization and degradation may be greater than indicated by these values since pulse studies suggested the presence of a deiodinase which attacks cell associated 125I-labelled LDL. The amounts of LDL bound to hepatocytes after 3 h (37 degrees C) were similar to amounts for fibroblasts, but DLD internalization and degradation were considerably less. Rabbit hyperlipidemic 125I-labelled DLD showed the same amount of binding but 1.39 times more internalization and degradation than normolipidemic 125I-labelled LDL. Binding of both control and hyperlipidemic LDL was 3-fold greater at 24 and 42 h than at O or 3 h but addition of a 50-fold molar excess of high density lipoproteins (HDL) prevented increased LDL binding with time. Induction of specific high affinity receptors for binding LDL was shown to occur by preincubation of hepatocytes for increasing periods in lipoprotein-free medium and then measuring 125I-labelled LDL binding at 4 degrees C in the presence and absence of excess unlabelled LDL. Finally, hepatocytes took up 40 times more LDL than sucrose or dextran over a 24-h period, an indication that the uptake of LDL occurs via some mechanism other than simple bulk fluid endocytosis.     

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.     

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 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.     

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.     

Abnormal high density lipoproteins from patients with liver disease regulate cholesterol metabolism in cultured human skin fibroblasts

Apolipoprotein B (apoB) of plasma low density lipoproteins (LDL) binds to high affinity receptors on many cell types. A minor subclass of high density lipoproteins (HDL), termed HDL1, which contains apoE but lacks apoB, binds to the same receptor. Bound lipoproteins are engulfed, degraded, and regulate intracellular cholesterol metabolism and receptor activity. The HDL of many patients with liver disease is rich in apoE. We tested the hypothesis that such patient HDL would reduce LDL binding and would themselves regulate cellular cholesterol metabolism. Normal HDL had little effect on binding, uptake, and degradation of 125I-labeled LDL by cultured human skin fibroblasts. Patient HDL (d 1.063-1.21 g/ml) inhibited these processes, and in 15 of the 25 samples studied there was more than 50% inhibition at 125I-labeled LDL and HDL protein concentrations of 10 micrograms/ml and 25 micrograms/ml, respectively. There was a significant negative correlation between the percentage of 125I-labeled LDL bound and the apoE content of the competing HDL (r = -0.54, P less than 0.01). Patient 125I-labeled HDL was also taken up and degraded by the fibroblasts, apparently through the LDL-receptor pathway, stimulated cellular cholesterol esterification, increased cell cholesteryl ester content, and suppressed cholesterol synthesis and receptor activity. We conclude that LDL catabolism by the receptor-mediated pathway may be impaired in liver disease and that patient HDL may deliver cholesterol to cells.     

A hepatocyte receptor for high-density lipoproteins specific for apolipoprotein A-I

Apolipoprotein (apo) E-deficient rat high-density lipoproteins (HDL) bind to isolated rat hepatocytes at 4 degrees C by a process shown to be saturable and competed for by an excess of unlabeled HDL. Uptake (binding and internalization) at 37 degrees C was also saturable and competed for by an excess of unlabeled HDL. At 37 degrees C the HDL apoprotein was degraded as evidenced by the appearance of trichloroacetic acid-soluble radioactivity in the incubation media. The binding of a constant amount of 125I-apo-E-deficient HDL was measured in the presence of increasing concentrations of various lipoproteins. HDL and dimyristoyl phosphatidylcholine (DMPC) X apo-A-I complexes decreased binding by 80 and 65%, respectively. Human low-density lipoproteins, DMPC X apo-E complexes, and DMPC vesicles alone did not compete for apo-E-deficient HDL binding. However, DMPC X apo-E complexes did compete for the binding of the total HDL fraction that contained apo-E but to a lesser extent than did DMPC X apo-A-I. DMPC X 125I-apo-A-I complexes also bound to hepatocytes, and this binding was competed for by excess HDL (70%) and DMPC X apo-A-I complexes (65%), but there was no competition for binding by DMPC vesicles or DMPC X apo-E complexes. It thus appears that hepatocytes have a specific receptor for HDL and that apo-A-I is the ligand for this receptor.     

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.     

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.     

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.     

Receptor-mediated uptake of low density lipoprotein stimulates bile acid synthesis by cultured rat hepatocytes

The cellular mechanisms responsible for the lipoprotein-mediated stimulation of bile acid synthesis in cultured rat hepatocytes were investigated. Adding 280 micrograms/ml of cholesterol in the form of human or rat low density lipoprotein (LDL) to the culture medium increased bile acid synthesis by 1.8- and 1.6-fold, respectively. As a result of the uptake of LDL, the synthesis of [14C]cholesterol from [2-14C]acetate was decreased and cellular cholesteryl ester mass was increased. Further studies demonstrated that rat apoE-free LDL and apoE-rich high density lipoprotein (HDL) both stimulated bile acid synthesis 1.5-fold, as well as inhibited the formation of [14C]cholesterol from [2-14C]acetate. Reductive methylation of LDL blocked the inhibition of cholesterol synthesis, as well as the stimulation of bile acid synthesis, suggesting that these processes require receptor-mediated uptake. To identify the receptors responsible, competitive binding studies using 125I-labeled apoE-free LDL and 125I-labeled apoE-rich HDL were performed. Both apoE-free LDL and apoE-rich HDL displayed an equal ability to compete for binding of the other, suggesting that a receptor or a group of receptors that recognizes both apolipoproteins is involved. Additional studies show that hepatocytes from cholestyramine-treated rats displayed 2.2- and 3.4-fold increases in the binding of apoE-free LDL and apoE-rich HDL, respectively. These data show for the first time that receptor-mediated uptake of LDL by the liver is intimately linked to processes activating bile acid synthesis.     

Proteolytic degradation of HDL1 on the fat cell surface is nutritionally regulated

The importance of plasma HDL apolipoprotein concentration as a predictor of atherosclerotic risk is well recognized, yet the processes of HDL modification and degradation in various cells are not clearly understood. We examined the characteristics of HDL1 apolipoprotein degradation and cellular uptake by rat adipocytes and determined the effects of fasting on these processes. Epididymal and perirenal adipocytes were isolated from male Wistar rats (310 +/- 4 g) fed ad libidum and incubated with 5 micrograms of rat 125I-labeled HDL1 (d: 1.07-1.10 g/mL) mL-1 for 2 h at 37 degrees C. Cellular uptake of HDL1 was calculated as the trichloroacetic acid precipitable radioactivity associated with adipocytes following incubation. Intracellular and medium degradation of HDL1 were determined as trichloroacetic acid soluble 125I counts associated with cells and measured in the postincubation medium, respectively. Fifty to sixty percent of cellular uptake and degradation of HDL1 was inhibited by the addition of 25-fold excess unlabeled HDL. HDL1 degradation measured in the medium was 10- to 12-fold greater than cellular uptake of HDL1 apolipoproteins. Intracellular degradation of HDL1 was negligible. The presence of EDTA in the incubation medium reduced HDL1 degradation measured in the medium, but enhanced HDL1 cellular uptake. Conditioned medium separated from cells after 2 h of incubation at 37 degrees C in the absence of HDL and subsequently incubated with 125I-labeled HDL1 for an additional 2 h at 37 degrees C, degraded less than 5% of HDL compared with degradation in the presence of cells. These results suggest that rat adipocytes degrade, or modify, HDL1 particles, possibly by interactions with cell surface proteases.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interactions of high density lipoprotein subclasses (HDL2 and HDLc) with dog adipocytes: selective effects of cholesterol and saturated fat feeding

Adipose tissue is a cholesterol storage organ and derives its cholesterol primarily from circulating lipoproteins. The present study shows that adipocytes isolated from canine omental fat tissue interact specifically with high density lipoprotein subfractions lacking or enriched in apolipoprotein E, namely canine high density lipoprotein-2 (HDL2) and HDLc, respectively. While 125I-labeled HDL2 binding was inhibited similarly by both excess unlabeled HDLc and HDL2, 125I-labeled HDLc interaction was inhibited by its homologous ligand only. Paired studies showed that the amount of HDLc associated with adipocytes was significantly higher compared to HDL2. The effect of a short-term cholesterol and saturated fat feeding on adipocyte-HDL interaction was examined using fat cells obtained from dogs before and again 3 weeks after a diet supplemented with cholesterol (1% w/w) and saturated fat (30% lard, w/w). Significant increases in body weight and omental fat cell weight occurred after fat feeding. The amount of 125I-labeled HDL2 that could be bound to adipocytes increased after the diet, whether expressed on a per cell basis (P less than 0.005) or per unit cell surface (P less than 0.025). The amount of cell-associated 125I-labeled HDLc, however, was not significantly affected by the cholesterol-rich diet. The characteristics of HDLc and HDL2 dissociation were assessed by examining the release of labeled lipoproteins from adipocytes preincubated with 125I-labeled HDLc and 125I-labeled HDL2. HDL2 dissociation from adipocytes was significantly decreased (P less than 0.05) following the diet and may explain in part the apparent increase in cell-associated 125I-labeled HDL2.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

Metabolism of low-density lipoprotein-proteoglycan complex by macrophages: further evidence for a receptor pathway

Earlier, we (Vijayagopal, P., et al. (1985) Biochim. Biophys. Acta 837-251) have shown that complexes of plasma low-density lipoproteins (LDL) and arterial chondroitin sulfate-dermatan sulfate proteoglycan aggregate promote LDL degradation and cholesteryl ester accumulation in mouse peritoneal macrophages. Further studies were conducted to determine whether LDL-proteoglycan complex is metabolized by a receptor-mediated process. Native proteoglycan aggregate was isolated from bovine aorta by associative CsCl isopycnic centrifugation. Complex of 125I-labeled LDL and proteoglycan aggregate formed in the presence of 30 mM Ca2+ was incubated with macrophages, and the binding at 4 degrees C and degradation at 37 degrees C of 125I-labeled LDL in the complex was monitored. Both binding and degradation of the complex were specific and saturable, suggesting that the processes are receptor mediated. The Kd for binding was 23 micrograms LDL protein per ml in the complex. Degradation of 125I-labeled LDL-proteoglycan complex was not suppressed by preincubation of macrophages with excess unlabeled complex, suggesting that the receptor for the complex is not subject to down regulation. Both binding and degradation of the complex and the resultant stimulation of cholesteryl ester synthesis were inhibited by limited treatment of cells with low doses of trypsin and pronase, indicating that the binding sites are protein or glycoprotein in nature. Binding was not inhibited by an excess of native LDL and beta-VLDL and exhibited only partial competition by excess unlabeled acetyl-LDL; however, polyinosinic acid, fucoidin and dextran sulfate, known inhibitors of acetyl-LDL binding and degradation in macrophages, did not affect LDL-proteoglycan complex binding and degradation. Similarly, excess unlabeled LDL-proteoglycan complex produced only partial inhibition of the binding and degradation of 125I-labeled acetyl-LDL by macrophages, suggesting that the binding sites for acetyl-LDL and LDL-proteoglycan complex are probably not identical. These studies provide evidence for a receptor-mediated pathway for the metabolism of LDL-proteoglycan complex in macrophages.     

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.     

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

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