Uptake of canine beta-very low density lipoproteins by mouse peritoneal macrophages is mediated by a low density lipoprotein receptor

The receptor on mouse peritoneal macrophages that mediates the uptake of canine beta-very low density lipoproteins (beta-VLDL) has been identified in this study as an unusual apolipoprotein (apo-) B,E(LDL) receptor. Ligand blots of Triton X-100 extracts of mouse peritoneal macrophages using 125I-beta-VLDL identified a single protein. This protein cross-reacted with antibodies against bovine apo-B,E(LDL) receptors, but its apparent Mr was approximately 5,000 less than that of the human apo-B,E(LDL) receptor. Binding studies at 4 degrees C demonstrated specific and saturable binding of low density lipoproteins (LDL), beta-VLDL, and cholesterol-induced high density lipoproteins in plasma that contain apo-E as their only protein constituent (apo-E HDLc) to mouse macrophages. Apolipoprotein E-containing lipoproteins (beta-VLDL and apo-E HDLc) bound to mouse macrophages and human fibroblasts with the same high affinity. However, LDL bound to mouse macrophages with an 18-fold lower affinity than to human fibroblasts. Mouse fibroblasts also bound LDL with a similar low affinity. Compared with the apo-B,E(LDL) receptors on human fibroblasts, the apo-B,E(LDL) receptors on mouse macrophages were resistant to down-regulation by incubation of the cells with LDL or beta-VLDL. There are three lines of evidence that an unusual apo-B,E(LDL) receptor on mouse peritoneal macrophages mediates the binding and uptake of beta-VLDL: LDL with residual apo-E removed displaced completely the 125I-beta-VLDL binding to mouse macrophages, preincubation of the mouse macrophages with apo-B,E(LDL) receptor antibody inhibited both the binding of beta-VLDL and LDL to the cells and the formation of beta-VLDL- and LDL-induced cholesteryl esters, and binding of 125I-beta-VLDL to the cells after down-regulation correlated directly with the amount of mouse macrophage apo-B,E(LDL) receptor as determined on immunoblots. This unusual receptor binds LDL poorly, but binds apo-E-containing lipoproteins with normal very high affinity and is resistant to down-regulation by extracellular cholesterol.     

Disparities in the interaction of rat and human lipoproteins with cultured rat fibroblasts and smooth muscle cells. Requirements for homology for receptor binding activity

This study characterizes the interactions of various rat and human lipoproteins with the lipoprotein cell surface receptors of rat and human cells. Iodinated rat very low density lipoproteins (VLDL), rat chylomicron remnants, rat low density lipoproteins (LDL), and rat high density lipoproteins containing predominantly apoprotein E (HDL1) bound to high affinity cell surface receptors of cultured rat fibroblasts and smooth muscle cells. Rat VLDL and chylomicron remnants were most avidly bound; the B-containing LDL and the E-containing HDL1 displayed lesser but similar binding. Rat HDL (d = 1.125 to 1.21) exhibited weak receptor binding; however, after recentrifugation to remove apoprotein E, they were devoid of binding activity. Competitive binding studies at 4 degrees C confirmed these results for normal lipoproteins and indicated that VLDL (B-VLDL), LDL, and HDLc (cholesterol-rich HDL1) isolated from hypercholesterolemic rats had increased affinity for the rat receptors compared with their normal counterparts, the most pronounced change being in the LDL. The cell surface receptor pathway in rat fibroblasts and smooth muscle cells resembled the system described for human fibroblasts as follows: 1) lipoproteins containing either the B or E apoproteins interacted with the receptors; 2) receptor binding activity was abolished by acetoacetylation or reductive methylation of a limited number of lysine residues of the lipoproteins; 3) receptor binding initiated the process of internalization and degradation of the apo-B- and apo-E-containing lipoproteins; 4) the lipoprotein cholesterol was re-esterified as determined by [14C]oleate incorporation into the cellular cholesteryl esters; and 5) receptor-mediated uptake (receptor number) was lipoprotein cholesterol. An important difference between rat and human fibroblasts was the inability of human LDL to interact with the cell surface receptors of rat fibroblasts. Rat lipoproteins did, however, react with human fibroblasts. Furthermore, the rat VLDL were the most avidly bound of the rat lipoproteins to rat fibroblasts. When the direct binding of 125I-VLDL was subjected to Scatchard analysis, the very high affinity of rat VLDL was apparent (Kd = 1 X 10(-11) M). Moreover, compared with data for rat LDL, the data suggested each VLDL particle bound to four to nine lipoprotein receptors. This multiple receptor binding could explain the enhanced binding affinity of the rat VLDL. The Scatchard plot of rat 125I-VLDL revealed a biphasic binding curve in rat and human fibroblast cells and in rat smooth muscle cells, suggesting two populations of rat VLDL. These results indicate that rat cells have a receptor pathway similar to, but not identical with, the LDL pathway of human cells. Since human LDL bind poorly to rat cell receptors on cultured rat fibroblasts and smooth muscle cells, metabolic studies using human lipoproteins in rats must be interpreted cautiously.     

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.     

Metabolism by cells in culture of low-density lipoproteins of abnormal composition from non-human primates with diet-induced hypercholesterolemia

Diet-induced hypercholesterolemia in non-human primates results in the production of a low-density lipoprotein (LDL) of abnormal size and composition. This LDL from hypercholesterolemic monkeys has been shown to be more atherogenic than the same amount of LDL from normocholesterolemic animals. Previous studies have demonstrated that hypercholesterolemic LDL is approximately twice as effective as normal LDL in stimulating cholesterol accumulation and esterification in arterial smooth muscle cells in culture. The purpose of the present study was determine whether this effect was secondary to differences in metabolism of the normal and hypercholesterolemic LDL. for this, the metabolism of 125I-labeled normal and hypercholesterolemic LDL from rhesus and cynomolgus monkeys was compared in several lines of skin fibroblasts and smooth muscle cells. Both normal and hypercholesterolemic LDL bound with high affinity to the same cell surface receptor. However, the affinity for binding of hypercholesterolemic LDL was about twice that of normal LDL (apparent dissociation constant for binding, Kd, was 2.63 micrograms protein/ml and 4.35 micrograms protein/ml, respectively). Conversely, only about 50% as many particles of hypercholesterolemic were able to bind to the receptor, compared with normal LDL. Those cells with the greatest capacity to metabolize LD generally accumulated the most cholesterol with either hypercholesterolemic or normal LDL. In all cell lines, nearly twice as much cholesterol accumulated in cells incubated with hypercholesterolemic LDL compared with normal LDL, and this differential could not be explained by differences in metabolism of the two lipoproteins, suggesting that some cholesterol entered the cells independent of the uptake of the intact LDL molecule. LDL receptors appear necessary for this to occur, since no difference in cholesterol accumulation was observed in cells genetically deficient in LDL receptors.     

Lipid metabolism in cultured cells. XVIII. Comparative uptake of low density and high density lipoproteins by normal, hypercholesterolemic and tumor virus-transformed human fibroblasts.

Serum lipoproteins control cell cholesterol content by regulating its uptake, biosynthesis, and excretion. Monolayers of cultured fibroblasts were used to study interactions with human high density (HDL) and low density (LDL) lipoproteins doubly labeled with [(3)H]cholesterol and (125)I in the apoprotein moiety. In the binding assay for LDL, the absence of specific LDL receptors in type II hypercholesterolemic fibroblasts was confirmed, whereas monolayers of virus-transformed human lung fibroblasts (VA-4) exhibited LDL binding characteristics essentially the same as normal lung fibroblasts. In the studies of HDL binding, specific HDL binding sites were demonstrated in normal and virus-transformed fibroblasts. In addition, type II hypercholesterolemic cells, despite the loss of LDL receptors, retained normal HDL binding sites. No significant competition was displayed between the two lipoprotein classes for their respective binding sites over a 5-fold concentration range. In VA-4 cells, the amount of lipoprotein required to saturate half the receptor sites was 3.5 micro g/ml (9 x 10(-9) M) for LDL and 9.1 micro g/ml (9 x 10(-8) M) for HDL. Pronase treatment reduced LDL binding by more than half but had no effect on HDL binding. Chloroquine, a lysomal enzyme inhibitor, stimulated net LDL uptake 3.5-fold by increasing internalized LDL but had essentially no effect on HDL uptake. Further experiments were conducted using doubly labeled lipoproteins to characterize the interaction of LDL and HDL with cells. While the cholesterol and protein moieties of LDL were incorporated into cells at similar rates, the uptake of the cholesterol moiety of HDL was 5 to 10 times more rapid than that of the protein component. Furthermore, the apoprotein component of LDL is extensively degraded following exposure, whereas the apoprotein moiety of HDL retains its macromolecular chromatographic characteristics. These results indicate that HDL and LDL bind to cultured cells at separate sites and that further processing of the two lipoprotein classes appears to take place by fundamentally different mechanisms.-Wu, J-D., J. Butler, and J. M. Bailey. Lipid metabolism in cultured cells XVIII. Comparative uptake of low density and high density lipoproteins by normal, hypercholesterolemic, and tumor virus-transformed human fibroblasts.     

Regulation of the activity of the low density lipoprotein receptor in human fibroblasts.

A specific receptor on the surface of cultured human fibroblasts binds plasma low density lipoprotein (LDL) with high affinity, and thereby initiates a cellular process by which the LDL is internalized and degraded within lysosomes and its cholesterol component is made available for cellular membrane synthesis. Current studies demonstrate that the activity of this LDL receptor is under feedback regulation. Prior incubation of fibroblast monolayers with cholesterol, 25-hydroxycholesterol, or LDL progressively reduced the ability of the cells to bind 125I-labeled LDL at the high affinity site. A series of kinetic studies indicated that this reduction in binding was due to a decrease in the number of LDL receptors. From measurements of the rate of decline in 125I-LDL binding activity after administration of cycloheximide, the LDL receptor was calculated to have a half-life of about 25 hr. LDL appeared to reduce 125I-LDL-binding activity by suppressing the synthesis of receptor molecules. Thus cultured human fibroblasts regulate their intracellular cholesterol content by regulating the activity of the LDL receptor, which in turn controls the rate of cellular entry of cholesterol derived from plasma LDL contained within the culture medium.     

Lipoprotein and cholesterol metabolism in rabbit arterial endothelial cells in culture.

Like all other peripheral cells types thus far studied in culture, endothelial cells derived from the rabbit aorta bind, internalize and degrade low density lipoprotein (LDL) at a significant rate. At any given LDL concentration, the metabolism by rabbit endothelial cells was slower than that by fibroblasts or smooth muscle cells. Thus, longer incubations were required to achieve a net increment in cell cholesterol content or to suppress endogenous sterol synthesis; after 18-24 h incubation in the presence of LDL at 100 microgram LDL protein/ml inhibition was greater than 80% relative to the rate in cells incubated in the absence of lipoproteins. High density lipoproteins (HDL) were also taken up and degraded but did not inhibit sterol synthesis. Studies of LDL binding to the cell surface suggested the presence of at least two classes of binding sites; the high-affinity binding sites were fully saturated at very low LDL concentrations (about 5 microgram LDL protein/ml). However, the degree of inhibition of endogenous sterol synthesis increased progressively with increasing LDL concentrations from 5 to 100 microgram LDL/ml, suggesting that uptake from the low affinity sites in this cell line contributes to the suppression of endogenous sterol synthesis. The internalization and degradation of LDL also increased with concentrations as high as 700 microgram/ml. Thus, in vivo, where the cells are exposed to LDL concentrations far above that needed to saturate the high affinity sites, most of the LDL degradation would be attributable to LDL taken up from low affinity sites. As noted previously in swine arterial smooth muscle cells and in human skin fibroblasts, unlabeled HDL reduced the binding, internalization and degradation of labeled LDL. Cells incubated for 24 h in the presence of high concentrations of LDL alone showed a net increment in cell cholesterol content; the simultaneous presence of HDL in the medium significantly reduced this LDL-induced increment in cell cholesterol content. The possible relationship between LDL uptake and degradation by these cells in vitro is discussed in relationship to their transport function in vivo.     

Regulation of low-density lipoprotein receptors in the human hepatoma cell line Hep G2

The human hepatoma cell line Hep G2 can be maintained in continuous culture and secretes numerous plasma proteins and lipoproteins into the medium. To better characterize cholesterol homeostasis in these cells we have examined the binding, internalization and degradation of [125I]LDL by cultured Hep G2 cells. Hep G2 cells express high-affinity low-density lipoprotein (LDL) receptors which facilitate the binding, internalization and degradation of [125I]LDL; these receptors can be induced by growth in LDL-depleted medium and repressed by further incubation in medium supplemented with LDL. The degradation of [125I]LDL by derepressed Hep G2 cells was inhibited by greater than 90% by monensin. Incubation of Hep G2 cells in the presence of increasing concentrations of LDL also inhibited cholesterol biosynthesis. Our results indicate that Hep G2 cells possess high affinity LDL receptors which are subject to metabolic regulation and suggest that this cell line affords a valuable model to further examine cholesterol and lipoprotein metabolism in human liver cells.     

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.     

Ligand size as a determinant for catabolism by the low density lipoprotein (LDL) receptor pathway. A lattice model for LDL binding

Low density lipoproteins (LDL) are large (Mr = 2.5 x 10(6)) in comparison to LDL receptors (Mr = 115,000). Since most LDL receptors are clustered in coated pits, we tested the hypothesis that crowding of receptor-bound LDL particles would cause steric effects. The apparent affinity of LDL for receptors on cultured fibroblasts decreased near saturation causing concave-upward Scatchard plots. Both the higher and lower affinity components of binding were up-regulated by the cholesterol synthesis inhibitor, lovastatin, indicating that the entire binding curve was sterol-responsive. In contrast, neither component of LDL binding was present on lovastatin-treated or untreated null fibroblasts which are incapable of expressing LDL receptors. Therefore, the concave-upward Scatchard plots were entirely due to binding to LDL receptors. These results are consistent with a lattice model in which receptor-bound LDL are large enough to decrease binding to adjacent receptors. A lattice model implies that large LDL should produce steric effects at a lower receptor occupancy than should small LDL. This was tested using seven LDL fractions that differed in diameter from 20 to 27 nm. Fewer large than small LDL were bound to the cell surface at 4 degrees C and 37 degrees C, and fewer were internalized and degraded at 37 degrees C. Since large LDL bound via both apolipoprotein (apo) E and apoB100, receptor cross-linking could have caused fewer large LDL to be bound at saturation. However, when the potential for cross-linking was prevented by an apo-E-specific monoclonal antibody (1D7), the difference in binding by large versus small LDL was not eliminated; instead, it was exaggerated. Taken together, these results support a lattice model for LDL binding and indicate that steric hindrance associated with crowding of LDL particles on receptor lattices is a major determinant for catabolism by the LDL receptor pathway in vitro.     

Mathematical modelling of competitive LDL/VLDL binding and uptake by hepatocytes

Elevated levels of low-density-lipoprotein cholesterol (LDL-C) in the plasma are a well-established risk factor for the development of coronary heart disease. Plasma LDL-C levels are in part determined by the rate at which LDL particles are removed from the bloodstream by hepatic uptake. The uptake of LDL by mammalian liver cells occurs mainly via receptor-mediated endocytosis, a process which entails the binding of these particles to specific receptors in specialised areas of the cell surface, the subsequent internalization of the receptor–lipoprotein complex, and ultimately the degradation and release of the ingested lipoproteins’ constituent parts. We formulate a mathematical model to study the binding and internalization (endocytosis) of LDL and VLDL particles by hepatocytes in culture. The system of ordinary differential equations, which includes a cholesterol-dependent pit production term representing feedback regulation of surface receptors in response to intracellular cholesterol levels, is analysed using numerical simulations and steady-state analysis. Our numerical results show good agreement with in vitro experimental data describing LDL uptake by cultured hepatocytes following delivery of a single bolus of lipoprotein. Our model is adapted in order to reflect the in vivo situation, in which lipoproteins are continuously delivered to the hepatocyte. In this case, our model suggests that the competition between the LDL and VLDL particles for binding to the pits on the cell surface affects the intracellular cholesterol concentration. In particular, we predict that when there is continuous delivery of low levels of lipoproteins to the cell surface, more VLDL than LDL occupies the pit, since VLDL are better competitors for receptor binding. VLDL have a cholesterol content comparable to LDL particles; however, due to the larger size of VLDL, one pit-bound VLDL particle blocks binding of several LDLs, and there is a resultant drop in the intracellular cholesterol level. When there is continuous delivery of lipoprotein at high levels to the hepatocytes, VLDL particles still out-compete LDL particles for receptor binding, and consequently more VLDL than LDL particles occupy the pit. Although the maximum intracellular cholesterol level is similar for high and low levels of lipoprotein delivery, the maximum is reached more rapidly when the lipoprotein delivery rates are high. The implications of these results for the design of in vitro experiments is discussed.      

Regulation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity in mouse peritoneal macrophages.

The lipoprotein-mediated regulation of 3-hydroxy-3-methylglutaryl-(HMG-) CoA reductase in cultured mouse peritoneal macrophages has been investigated. In contrast to what has been reported for other cells, HMG-CoA reductase activity is not suppressed by normal serum or by normal low density lipoproteins (LDL) from humans or dogs. Suppression of reductase activity occurred when cells were cultured in the presence of beta-migrating very low density lipoproteins (beta-VLDL) or LDL from hypercholesterolaemic dogs, or LDL modified by acetoacetylation. Human beta-VLDL from an atypical type III hyperlipoproteinaemic patient was also effective, as was apolipoprotein (apo) E-containing high density lipoproteins (HDL) from cholesterol-fed dogs (apo-E HDLc). The results indicate that cholesterol biosynthesis in mouse peritoneal macrophages is regulated by lipoprotein cholesterol entering via receptor-mediated endocytosis. Normal LDL were not effective because of the poor binding and uptake of these lipoproteins by the apo-B, E (LDL) receptor. Only beta-VLDL, apo-E HDLc, and hypercholesterolaemic LDL were avidly taken up by this receptor and were able to suppress HMG-CoA reductase. Acetoacetylated LDL were internalized via the acetyl-LDL (scavenger) receptor. Thus, mouse macrophages differ from human fibroblasts and smooth muscle cells in their physiological regulation of cholesterogenesis.     

Lipid metabolism in cultured cells. XVI. Lipoprotein binding and HMG CoA reductase levels in normal and tumor virus-transformed human fibroblasts.

The loss in feedback control of cholesterol biosynthesis in tumor cells was examined in tissue culture. Human fibroblasts from normal subjects, SV40 tumor virus-transformed cell lines, and homozygous familial hypercholesterolemic cells as reference, were grown in tissue culture. Experiments were conducted to relate the regulatory enzyme for cholesterol biosynthesis, HMG CoA reductase, and the membrane-located binding receptors for low density lipoproteins (LDL) that mediate feedback control in normal cells. Monolayers of virus-transformed tumor cells exhibited specific (125)I-labeled LDL binding of 152 +/- 21 ng/mg cell protein, which was essentially the same as that of normal fibroblasts (135 +/- 20 ng/mg). Binding of LDL by familial hypercholesterolemic cells used as controls was only 8 +/- 3 ng/mg under the same test conditions. Basal levels of HMG CoA reductase in tumor cells of 45.2 +/- 6.5 units/mg cell protein were about twice those of normal cells. However, in contrast to the lack of feedback control of this enzyme observed with tumors in vivo, in both the normal and the transformed cells in vitro, activity of the enzyme decreased about fourfold when serum lipids were added. These findings demonstrate that tumor cells growing in vitro contain a normal complement of the membrane-located binding receptors for low density lipoproteins and, although the basal levels are higher than normal, an effective feedback regulation of the enzyme HMG CoA reductase is retained.     

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.     

Cultured human hepatocytes. Evidence for metabolism of low density lipoproteins by a pathway independent of the classical low density lipoprotein receptor

Studies of low density lipoprotein (LDL) metabolism in nonhuman model systems have indicated that the mammalian liver has dual mechanisms for the uptake and regulation of the concentration of plasma LDL. Heretofore, direct evaluation of lipoprotein uptake mechanisms in human hepatocytes has not been possible. In order to compare hepatocyte LDL uptake with fibroblast LDL metabolism, human hepatocytes were isolated and cultured from small biopsy specimens obtained from normolipidemic and homozygous familial hypercholesterolemic patients. Cells cultured in serum-free culture medium retained the morphological and biochemical characteristics of hepatocytes for at least 7 days. The uptake and degradation of LDL by hepatocytes was compared to that of the cultured human fibroblasts. Like fibroblasts, hepatocytes bound, internalized, and degraded LDL. In both cell types, uptake approached saturation at a concentration of 50 micrograms of LDL protein/ml. Competition for LDL binding by LDL, high density lipoprotein, and modified LD revealed that the hepatocyte binding was specific for LDL. Cellular cholesterol loading by incubation in LDL-enriched culture medium resulted in diminished LDL uptake in both cell types. Chemical modification of LDL by acetoacetylation, acetylation, and reductive methylation abolished LDL uptake and degradation by fibroblasts. However, hepatocytes bound and degraded the modified LDL at 30-50% the level of native LDL. Homozygous familial hypercholesterolemic hepatocytes were devoid of the LDL receptor pathway but metabolized native LDL to the extent observed with modified LDL uptake by normal hepatocytes. In contrast to the classic LDL receptor pathway, the second or alternate pathway does not lead to regulation of 3-hydroxy-3-methylglutaryl-CoA reductase activity. These findings indicate the presence of two separate pathways of LDL uptake in human hepatocytes which have different effects on hepatocytic cholesterol metabolism.     

Thyrotropin modulates low density lipoprotein binding activity in FRTL-5 thyroid cells

FRTL-5 cells possess high affinity low density lipoprotein (LDL) receptors which bind, internalize, and degrade LDL. When FRTL-5 cells are deprived of thyrotropin (TSH) the binding of LDL increases more than 2-fold. Upon addition of TSH, at a concentration of 1 x 10(-10) M or greater, LDL binding decreases rapidly and within 24 h reaches the level which is typical of FRTL-5 cells chronically stimulated by TSH. The data available suggest that TSH-dependent down-regulation of LDL receptor activity is exerted through a reduction of the number of active LDL receptors, with no change in affinity. It is unlikely that the synthesis of LDL receptors is impaired, since LDL receptor messenger RNA is not decreased by TSH. The effect of the hormone on LDL receptor activity can be mimicked by 8-Br-cAMP and is completely abolished by the protein synthesis inhibitor cycloheximide but not by actinomycin D. TSH regulation of LDL receptor activity is lost in v-ras Ki-transformed FRTL-5 cells (Ki Mol) which also have lost TSH dependence for adenylate cyclase activation and growth. However, 8-Br-cAMP decreases LDL binding in Ki Mol FRTL-5 cells. The reduced availability of LDL receptor in TSH-stimulated FRTL-5 cells may be related to the increased membrane fluidity (Beguinot, F., Beguinot, L., Tramontano, D., Duilio, C., Formisano, S., Bifulco, M., Ambesi-Impiombato, F. S., and Aloj, S. M. (1987) J. Biol. Chem. 262, 1575-1582) or may reflect increased degradation of LDL receptors. We propose that a lower cholesterol uptake is needed in an actively proliferating cell population, to increase the production of isoprenoids whether it be for cholesterol biosynthesis or for the synthesis of other compounds requiring isoprenoid precursors.     

Characterization of lipoprotein receptors on rat Fu5AH hepatoma cells

The rat hepatoma cell line Fu5AH has the unusual property of accumulating massive amounts of cholesteryl ester upon incubation with hypercholesterolemic serum, and especially when incubated with beta-very low density lipoproteins (beta-VLDL) from cholesterol-fed dogs. The present study was designed to identify and characterize the lipoprotein receptors that mediate the cholesteryl ester accumulation. The beta-VLDL and cholesterol-induced apolipoprotein (apo) E-containing high density lipoproteins (apoE HDLc) bound to Fu5AH cells with very high affinity (Kd approximately equal to 10(-10) M), whereas low density lipoproteins (LDL) bound with unusually low affinity (Kd approximately equal to 10(-8) M). Receptor binding activity of 125I-labeled beta-VLDL, 125I-labeled apoE HDLc, and 125I-labeled LDL was abolished by incubation in the presence of an excess of unlabeled LDL or of a polyclonal antibody to the bovine adrenal apoB,E(LDL) receptor. The receptors were completely down-regulated by preincubating Fu5AH cells with beta-VLDL, but much higher levels of beta-VLDL were required than for down-regulation of fibroblast apoB,E(LDL) receptors. Receptor binding was abolished by reductive methylation of the lysyl residues of the apolipoprotein of the beta-VLDL and by an apoE monoclonal antibody (1D7) that blocks receptor binding. The Fu5AH receptor was further characterized by using the bovine adrenal apoB,E(LDL) receptor antibody. A single protein (Mr approximately equal to 130,000) was identified in Triton extracts of whole cells, and two proteins (Mr approximately equal to 130,000 and 115,000) were found in Fu5AH cell membranes disrupted by homogenization. The Mr approximately equal to 115,000 protein was released from the membranes and did not react with an antibody to the carboxyl-terminal (cytoplasmic) domain of the apoB,E(LDL) receptors. These studies indicate that Fu5AH cells express apoB,E(LDL) receptors that have unusually low affinity for apoB-continuing lipoproteins, require large amounts of cholesterol to induce down-regulation, and are susceptible to specific proteolysis in cell homogenates. These apoB,E(LDL) receptors are responsible for the receptor-mediated uptake of beta-VLDL and chylomicron remnants by Fu5AH cells.     

Receptors for homologous plasma lipoproteins on a rat hepatoma cell line

Hepatocytes express on their surfaces more than one class of receptors capable of mediating the internalization of lipoproteins. However, relatively little is known about the binding characteristics of hepatic receptors for various lipoproteins, about the regulation of the receptors, and about the consequences for intracellular lipid metabolism of uptake of lipoproteins via different classes of receptors. The aim of the present studies was to characterize the binding and degradation of various lipoproteins and their mutual competition for cellular processing. Since these kinds of studies may be more easily carried out in continuous established hepatoma cell lines than in nondividing primary hepatocyte cultures, we examined the lipoprotein receptor functions of a well differentiated rat hepatoma (H-35). Cells were grown to confluence in Eagle's minimal essential medium in 15% newborn calf serum. Medium then was changed to 15% lipoprotein-deficient serum for 44 hr before experiments. External binding of 125I-labeled rat plasma and intestinal lymph lipoproteins was assessed at 4 degrees C. Cellular uptake and degradation were assessed at 37 degrees C. Lipoproteins were isolated by fixed angle or zonal ultracentrifugation or by heparin affinity column chromatography and characterized as to their lipid and apoprotein compositions. Labeled low density (LDL), high density (HDL2), non-apoE-HDL, very low density lipoproteins (VLDL), and chylomicron remnants (CM-R) each manifested specific and saturable binding and degradation by the hepatoma cells. Competition experiments indicated that separate receptors were present for LDL, HDL2, and CM-R. Most of HDL2 appeared to be bound to the non-apoE-HDL receptor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interactions between human and carp (Cyprimus carpio) low density lipoproteins (LDL) and LDL receptors

1. We have compared the concentration and chemical composition of carp and human plasma lipoproteins and studied their interaction with human fibroblast LDL receptors. 2. The main lipoproteins in carp are of high density (HDL) in contrast to low density lipoproteins (LDL) in human. 3. Carp lipoproteins are devoid of apolipoprotein (apo) E, a major ligand for interaction with LDL receptors in mammals. 4. Carp very low density lipoproteins (VLDL) and LDL but not HDL nor apoA-I cross react with human LDL in their interaction with LDL receptors on human cultured fibroblasts. 5. Carp liver membranes possess high affinity receptors that are saturable and have calcium dependent ligand specificity (apoB and apoE) similar to human LDL receptor. Carp VLDL and LDL but not HDL nor its major apolipoprotein complexed to L-alpha-phosphatidylcholine dimyristoyl (apoA-I-DMPC) competed with the specific binding of human LDL to this receptor.     

Apolipoprotein B: its role in the control of fibroblast cholesterol biosynthesis and in the regulation of its own binding to cellular receptors.

Apolipoprotein B transports cholesterol in plasma as low density lipoprotein (LDL) and targets its delivery to cells by binding to a specific plasma membrane receptor. The cellular consequences of apoB binding to its receptor were investigated to determine whether it suppresses cholesterol biosynthesis and reduces the number of cellular receptors for the apoprotein. Upon preincubation of fibroblasts with lipoprotein-deficient medium alone or supplemented with either LDL or apoB complexed to BSA (apoB-BSA), LDL suppressed cholesterol biosynthesis, but apoB enhanced it. Similarly, fibroblasts preincubated in medium supplemented with LDL bound decreased amounts of either (125)I-labeled LDL or (125)I-labeled apoB-BSA to their receptors, while preincubation with apoB-BSA increased the binding relative to the controls. These latter results occurred in association with a decrease in cellular cholesterol content, indicating that apoB in the medium bound cholesterol and removed it from the cells, thus stimulating both cholesterol synthesis and cellular binding of apoB. Accordingly, fibroblast cholesterol synthesis and the number of functional LDL receptors are not suppressed by the binding of the apoprotein to the receptor, and the known role of apoB remains that of transporting cholesterol in plasma and delivering it to the cell. A possible physiologic role for apoB in depleting cells of cholesterol is presently unknown since apoB is not known to exist free in plasma; however, these findings demonstrate such a functional capability for this apoprotein.-Shireman, R. B., and W. R. Fisher. Apolipoprotein B: its role in the control of fibroblast cholesterol biosynthesis and in the regulation of its own binding to cellular receptors.