VLDL apoprotein secretion and apo-B mRNA level in primary culture of cholesterol-loaded rabbit hepatocytes

Incubation of cultured rabbit hepatocytes with beta very low density lipoproteins (beta-VLDL) induces a dose-dependent increase in cell cholesterol (CH) content and VLDL apoprotein (apo) B and E secretion without change in apo-B mRNA level. These data suggest that beta-VLDL may exert a stimulatory effect on hepatic apo-B production at the co-translational and/or posttranslational level.     

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.     

Endocytosed beta-VLDL and LDL are delivered to different intracellular vesicles in mouse peritoneal macrophages

Hypercholesterolemic rabbit beta-VLDL and human LDL are both internalized by mouse peritoneal macrophages by receptor-mediated endocytosis. However, only beta-VLDL (which binds to the cells with a much higher affinity than LDL) markedly stimulates acyl-CoA/cholesterol acyl transferase (ACAT) and induces foam cell formation in these cells. As an initial step to test whether the two lipoproteins might be targeted to different organelles (which might differ in their ability to deliver cholesterol to microsomal ACAT), we studied the endocytic pathways of beta-VLDL and LDL. Lipoproteins were labeled with the non-transferable fluorescent label, DiI. When the macrophages were incubated with DiI-LDL for 10 min at 37 degrees C, the fluorescence was concentrated near the center of the cell both in heavily labeled vesicles and in a diffuse pattern. The pattern with DiI-beta-VLDL was quite different: an array of bright vesicles throughout the cytoplasm was the predominant feature. Differences in distribution were seen as early as 2 min of incubation and persisted throughout a 10-min chase period. By using a procedure in which photobleaching of DiI fluorescence converts diaminobenzidine into an electron-dense marker, we were able to identify at the ultrastructural level vesicles containing electron-dense material in cells incubated with DiI-beta-VLDL. Human E2/E2 beta-VLDL (from a patient with familial dysbetalipoproteinemia), which has a binding affinity and ACAT-stimulatory potential similar to LDL, gave a pattern of fluorescence virtually identical to LDL. Pulse-chase studies with 125I-labeled and [3H]cholesteryl ester-labeled lipoproteins disclosed that both protein degradation and cholesteryl ester hydrolysis were markedly retarded in beta-VLDL compared with LDL. Thus, in mouse peritoneal macrophages, endocytosed beta-VLDL appears in a distinct set of widely-distributed vesicles not seen with LDL (or with E2-beta-VLDL) and, compared with LDL, has a markedly diminished rate of protein degradation and cholesteryl ester hydrolysis. The differential routing of LDL and beta-VLDL may provide a mechanism for differences in ACAT-stimulatory potential between the two lipoproteins.     

Apolipoprotein C-I modulates the interaction of apolipoprotein E with beta-migrating very low density lipoproteins (beta-VLDL) and inhibits binding of beta-VLDL to low density lipoprotein receptor-related protein

The binding of native rabbit beta-very low density lipoproteins (beta-VLDL) to the low density lipoprotein receptor-related protein (LRP) requires incubation with exogenous apolipoprotein (apo) E. Inclusion of a mixture of the C apolipoproteins in the incubation inhibits this binding. In the present study, the ability of the individual C apolipoproteins (C-I, C-II, and C-III) to block binding of beta-VLDL to the LRP was examined by measuring cholesteryl ester formation in mutant fibroblasts that lack low density lipoprotein receptors or by measuring binding to the LRP using ligand blotting. In each assay, both apoC-I and apoC-II inhibited binding; apoC-I was the more effective inhibitor. Apolipoprotein C-III had no effect on binding activity, regardless of its sialylation level. Binding of human apoE to rabbit beta-VLDL in the absence or presence of human apoC-I, apoC-II, and monosialo-apoC-III was also determined, by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The results of these studies are consistent with a mechanism in which exogenous human apoE displaces the endogenous apoE and the beta-VLDL particle becomes enriched with apoE (by 4.2-fold in this study). At this higher apoE content, the beta-VLDL bound to the LRP. Inclusion of apoC-I, apoC-II, or apoC-III in the incubation mixture resulted in a differential displacement of apoE from the beta-VLDL; however, at the concentrations examined, only apoC-I and apoC-II were capable of displacing sufficient apoE to abolish binding to LRP.     

Complete down-regulation of low-density-lipoprotein-receptor activity in the human hepatoma cell line Hep G2 by beta-migrating very-low-density lipoprotein and non-lipoprotein cholesterol. Different cellular regulatory pools of cholesterol.

Regulation of low-density-lipoprotein-receptor activity by low-density lipoprotein (LDL), cholesteryl-ester-rich beta-migrating very-low-density lipoprotein (beta-VLDL) and non-lipoprotein cholesterol was investigated in the human hepatoma cell line Hep G2. Competition studies indicate that LDL and beta-VLDL are bound to the same recognition site, tentatively the LDL receptor. The regulatory response of the LDL receptor upon prolonged incubation with LDL or beta-VLDL was, however, markedly different. 22 h preincubation of Hep G2 cells with excess LDL caused a partial down regulation to 31% of the initial level of the high-affinity association of LDL and 26% of the high-affinity degradation of LDL, while with beta-VLDL a complete down regulation of the LDL-receptor activity is observed. Preincubation of Hep G2 cells with beta-VLDL for 22 h led to a fourfold increase in intracellular cholesterol esters and a twofold increase in acyl-coA:cholesterol acyltransferase activity. With LDL, the amount of intracellular cholesterol esters is increased 1.6-fold. The more effective down regulation of LDL receptors by beta-VLDL as compared to LDL can be explained by the more effective intracellular cholesterol delivery with beta-VLDL than with LDL. Preincubation of Hep G2 cells for 22 h with acetylated LDL hardly influenced the LDL-receptor activity. Non-lipoprotein cholesterol, however, caused a complete down regulation of LDL-receptor activity at even lower extracellular cholesterol concentrations than with beta-VLDL. The complete down regulation of LDL receptors by non-lipoprotein cholesterol is not accompanied by a significant increase in acyl-coA:cholesterol acyltransferase activity, while the intracellular cholesterol ester concentration is only increased 1.6-fold. It is suggested that the effectiveness of non-lipoprotein cholesterol to regulate LDL receptors is caused by its efficiency to reach the sterol regulatory site. The inability of LDL to down regulate its receptor completely can thus be explained by the inability of LDL to deliver cholesterol adequately at the intracellular regulatory site of the LDL receptor. The observed complete down regulation of the LDL receptor by beta-VLDL may be responsible for the cholesterol-rich-diet induced, complete down regulation of LDL-receptor-mediated clearance of LDL in vivo.     

The effect of khellin and timefurone on secretion and catabolism of lipoproteins by cultured human and rabbit hepatocytes

Using human and rabbit hepatocyte cultures, the effects of khellin and timefurone on lipoprotein metabolism were studied with special reference to the following parameters: i) binding and degradation of 125I-labeled low density lipoproteins (LDL); ii) apoprotein B (apo-B) secretion measured by immunoenzymatic assay, iii) [35S]methionine labeled apo-B and apo-E within the composition of very low density lipoproteins (VLDL); iiii) total cholesterol synthesis and cholesterol secretion within the composition of VLDL. The therapeutic concentrations (0.1-10 micrograms/ml) of the above drugs had no appreciable effect on the binding and degradation of 125I-LDL but inhibited the secretion of apo-B VLDL, leaving the apo-E VLDL unaffected. This was paralleled with inhibition of cholesterol synthesis (by 30-50%) and VLDL secretion. These results suggest that khellin and timefurone mediate the hypolipidemic effect via the reduction of the intracellular synthesis of cholesterol and secretion of apo-B containing VLDL by hepatocytes.     

Loci of catabolism of beta-very low density lipoprotein in vivo delineated with a residualizing label, 125I-dilactitol tyramine

beta-Very low density lipoprotein (beta-VLDL) may be a major atherogenic lipoprotein, and knowledge of the sites of its catabolism should facilitate elucidation of mechanisms important in the regulation of its plasma concentrations. In this study, catabolic sites of beta-VLDL have been delineated in normolipidemic rabbits with a novel, radioiodinated, residualizing label, 125I-dilactitol tyramine (125I-DLT). Comparative studies of beta-VLDL and low density lipoprotein catabolism were performed with 125I-DLT conjugated to each lipoprotein and with lipoproteins iodine-labeled conventionally. Conjugation did not alter size distributions or charge characteristics of lipoprotein particles. The overall processing (binding and degradation) of lipoproteins by cultured rabbit skin fibroblasts was not influenced by 125I-DLT derivatization, suggesting that attachment of the label did not influence cell receptor-lipoprotein interactions. Furthermore, although degradation products of 125I-lipoproteins leaked out of the cells and into the medium, the degradation products of 125I-DLT lipoproteins were retained by the cells. The principal catabolic site of beta-VLDL in normolipidemic rabbits was found to be the liver with 54 +/- 4% of injected 125I retained in this organ 24 h after injection of 125I-DLT-beta-VLDL. When catabolism was normalized to tissue weight, the liver and adrenals were found to be approximately equally active in the metabolism of beta-VLDL. In agreement with results of other studies with residualizing labels, the principal organ of catabolism of 125I-DLT-LDL in vivo was the liver. The adrenals were the most highly catabolizing organ when results were normalized for tissue weight. The quantitative differences observed in the tissue distributions of injected 125I-DLT-beta-VLDL and 125I-DLT-low density lipoprotein suggested that a significant proportion of beta-VLDL is removed by tissues before conversion to low density lipoprotein.     

Cholesterol metabolism in ExHC (exogenous hypercholesterolemic) rats.

Exogenous hypercholesterolemic (ExHC) rats, that develop hypercholesterolemia for exogenous cholesterol, are an established strain Isolated from Sprague-Dawley (SD) rats by Imai and Matsumura ((1973) Atherosclerosis, 18, 59-64). The present study was carried out to clarify the cause of hyperresponsivity in ExHC rats to dietary cholesterol. As early as one day after feeding a high cholesterol diet (1%) serum cholesterol level was doubled in ExHC rats, while the level of hepatic cholesterol was two-thirds of SD rats. The elevation of serum cholesterol was mainly attributed to the d less than 1.006 g/ml fractions. Cholesterol feeding increased fecal bile acid excretion in both strains, but to a more greater extent in SD rats. Absorption of dietary cholesterol and synthesis of cholesterol in vivo were similar between the strains. The uptake of beta-very-low-density-lipoproteins (beta-VLDL) in vivo and the primary cultured hepatocytes was lower in ExHC rats, when a high-cholesterol diet was fed. Even without feeding of a high-cholesterol diet, preincubation with cholesterol-rich lipoproteins caused a lower association and degradation of beta-VLDL by the hepatocytes from ExHC rats. Incubation of hepatocytes with cholesterol-rich lipoproteins did not affect the secretion of [14C]cholesterol into the density less than 1.006 g/ml fraction, but suppressed the secretion into the medium density greater than 1.006 g/ml fractions. These results suggest that ExHC rats, as compared to SD rats, are defective of hepatic uptake and processing cholesterol to bile acids.     

The effect of pure and long-stored commercial cholesterol on the binding of very low density and low density lipoproteins by rabbit hepatocytes (in vitro research)

Effects of pure and long-stored commercial cholesterol feeding on rabbit plasma cholesterol level, on rabbit hepatocyte cholesterol esters levels, and on receptor activity of rabbit hepatocytes were investigated in three experimental groups. In comparison with control, the cholesterol levels in plasma of rabbits, fed with pure and long-stored cholesterol, were 3 and 15 times higher accordingly. Free cholesterol and cholesterol esters concentrations were enhanced in hepatocytes of rabbits fed with pure cholesterol (1.4 and 2.3 times, accordingly, p 0.05) and much more enhanced in hepatocytes of rabbits fed with long-stored cholesterol (4.5 and 24 times, accordingly, p less than 0.05). Specific binding of 125-1-labeled VLDL and LDL to rabbit hepatocytes was decreased in experimental groups by 20% and 32%, accordingly in the first group and by 40% and 77%, accordingly in the second group.     

Regulation of hepatic receptor-dependent degradation of LDL by mevinolin in rabbits with hypercholesterolemia induced by a wheat starch-casein diet

Rabbits fed a wheat starch-casein diet develop a marked hypercholesterolemia and have a slower rate of removal of rabbit 125I-labeled low density lipoproteins (LDL) from plasma. Treating rabbits with mevinolin, a highly potent competitive inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase, at a daily dose of 20 mg per animal prevents the increase in plasma and LDL cholesterol. The mevinolin effect is mediated through an increased rate of removal of rabbit 125I-labeled LDL from plasma. To study the role of mevinolin on the regulation of the hepatic LDL receptor in rabbits, the binding of 125I-labeled LDL and 125I-labeled beta-VLDL (beta-migrating very-low-density lipoproteins) to liver membranes prepared from rabbits fed the wheat starch-casein diet with or without mevinolin was investigated. Liver membranes from wheat starch-casein-fed rabbits have no demonstrable EDTA-sensitive binding activity of 125I-labeled LDL and low (37 ng/mg protein) binding activity of 125I-labeled beta-VLDL. Treatment of the wheat starch-casein fed rabbits with mevinolin results in high levels of specific EDTA-sensitive binding of 125I-labeled LDL (28.7 ng/mg protein) and 125I-labeled beta-VLDL (120 ng/mg protein). To assess the functional role of the hepatic LDL receptor in response to mevinolin, the catabolism of 125I-labeled LDL by perfused rabbit livers was studied. Perfused livers from mevinolin-treated rabbits show a 3.3-fold increase in the rate of receptor-dependent catabolism of 125I-labeled LDL (4.6% X h-1) when compared with that of livers from rabbits not treated with mevinolin (1.4% X h-1). Thus, these studies demonstrate that mevinolin prevents the increase of plasma LDL cholesterol level in rabbits fed a wheat starch-casein diet by regulating the levels of hepatic LDL-binding sites and the rate of receptor-dependent catabolism of LDL by the liver.     

Failure of the intracellular itinerary of beta very low density lipoproteins to augment cholesterol esterification in macrophages from Watanabe heritable hyperlipidemic rabbits

beta very low density lipoproteins (beta-VLDL) interact with mouse peritoneal macrophages via specific receptors leading to pronounced stimulation of cholesterol esterification. The present study has defined an alternative pathway for the processing of beta-VLDL in alveolar macrophages from Watanabe heritable hyperlipidemic (WHHL) rabbits. Macrophages from either New Zealand (NZ) or WHHL rabbits degraded 125I-beta-VLDL to an equivalent extent. Degradation was competed to a similar extent in both cell types by either excess unlabeled beta-VLDL or low density lipoprotein, indicative of a specific receptor involvement. Accumulation of intracellular degradation products of beta-VLDL labeled with the residualizing label, dilactitol-125I-tyramine, was similar in both cell types demonstrating that degradation was not due to secreted proteolytic enzymes. beta-VLDL promoted the incorporation of [3H]oleate into cholesteryl-[3H]oleate and increased the cellular mass of cholesterol in NZ macrophages. In contrast, beta-VLDL did not augment cholesteryl-[3H]oleate deposition in WHHL macrophages. This lack of cholesterol esterification occurred despite equivalent acyl-CoA:cholesterol acyltransferase activity in microsomal fractions of both cell types, and similar augmentations in cholesteryl-[3H]oleate during incubation with phospholipase C-treated LDL. Incubation of WHHL macrophages with beta-VLDL increased cellular cholesterol mass, although the response was attenuated compared to NZ cells. To determine whether these disparities in cholesterol esterification were related to the catabolic fate of beta-VLDL-derived cholesterol esters, [3H]cholesteryl oleate was exchanged into the core of beta-VLDL and incubated with macrophages in medium containing [14C]oleate. NZ macrophages accumulated both [3H]cholesterol and [3H]cholesteryl-[14C]oleate after 5 h, indicating hydrolysis and re-esterification of cholesterol esters. In contrast, WHHL macrophages only accumulated [3H]cholesterol esters, suggesting uptake of cholesterol esters without subsequent hydrolysis. These data demonstrate that WHHL macrophages possess a pathway for the intracellular processing of beta-VLDL that permits internalization of the particle without stimulation of cholesterol esterification.     

Intimal smooth muscle cells up-regulate beta-very low density lipoprotein-mediated cholesterol accumulation by enhancing beta-very low density lipoprotein uptake and decreasing cholesterol efflux

To clarify the mechanism of smooth muscle cell (SMC)-derived foam cell formation, we investigated beta-very low density lipoprotein (beta-VLDL) cholesterol metabolism in vascular medial SMCs (M-SMCs) from normal rabbits compared with intimal SMCs (I-SMCs) from normal rabbits fed a high-cholesterol diet and LDL receptor-deficient rabbits. For both types of I-SMCs, uptake of [3H]cholesteryl oleate labeled beta-VLDL increased 1.6 times and release of [3H]cholesterol decreased 40% compared with M-SMCs. M-SMCs took up part of the beta-VLDL through the LDL receptor but I-SMCs did not. mRNAs for the VLDL receptor and the LDL receptor relative with 11 ligand binding repeats were expressed at similar levels in all SMCs. M-SMCs expressed more LDL receptor-related protein than I-SMCs. Ligand blotting analysis revealed greater 125I-beta-VLDL binding to a 700-kDa protein in I-SMCs compared with M-SMCs. I-SMCs had higher activities of acid cholesterol esterase and acyl-CoA:cholesterol acyltransferase, and lower activity of neutral cholesterol esterase than M-SMCs in both the absence and the presence of beta-VLDL. These results indicate that I-SMCs accumulate more cholesteryl ester than M-SMCs by taking up more beta-VLDL and by effluxing less cholesterol.     

Opposing effects of apolipoproteins E and C on lipoprotein binding to low density lipoprotein receptor-related protein

The low density lipoprotein receptor-related protein (LRP) from rat liver membranes binds apoprotein E (apoE)-enriched rabbit beta-migrating very low density lipoproteins (beta-VLDL) in a ligand blotting assay on nitrocellulose membranes. Binding was markedly activated when the beta-VLDL was preincubated with recombinant human apoE-3, native human apoE-3 or E-4, or native rabbit apoE. Human apoE-2, which binds poorly (1-2% of apo E-3 binding) to low density lipoprotein receptors, was approximately 40% as effective as apoE-3 or apoE-4 in binding to LRP. Stimulation of apoE-dependent binding to LRP was blocked by the inclusion of a mixture of human apoC proteins, but not apoA-I or A-II, in the preincubation reaction. High concentrations of apoE did not overcome the apoC inhibition. The effects of apoE and apoC on the ligand blotting assay were paralleled by similar effects in the ability of beta-VLDL to stimulate cholesteryl ester synthesis in mutant human fibroblasts that lack low density lipoprotein receptors. These properties of LRP are consistent with the known effects of apoE and apoC on uptake of chylomicron and very low density lipoprotein remnants in the liver and raise the possibility that LRP functions as a receptor for apoE-enriched forms of these lipoproteins in intact animals.     

Distribution of apolipoprotein E in the plasma of insulin-dependent and noninsulin-dependent diabetics and its relation to cholesterol net transport

Noninsulin-dependent diabetics, whose plasma contained no detectable beta-VLDL (very low density lipoprotein), had a proportion (0.23 +/- 0.04) of plasma apolipoprotein E in the form of an abnormal lipoprotein not recognized by antibodies to apoB-100 from LDL (low density lipoprotein) or apoA-I from HDL (high density lipoprotein). This lipoprotein, abnormally rich in free cholesterol and apoE, had a calculated particle density within the low density lipoprotein range. It competed with LDL at the apoB,E receptor of normal fibroblasts and stimulated cholesteryl ester accumulation in mouse peritoneal macrophages. However, it did not compete with the binding of labeled rabbit beta-VLDL to macrophages. A much lower proportion of apoE (0.04 +/- 0.03) was in this form in the plasma of patients with insulin-dependent diabetes who had a comparable degree of hyperglycemia. The diabetic lipoprotein was absent in normoglycemic control subjects. The net transport of cholesterol from cell membranes to the plasma of noninsulin-dependent diabetics (and to a lesser extent, insulin-dependent diabetics) was inhibited relative to control values, and the magnitude of this inhibition was well correlated with the concentration of the abnormal lipoprotein of diabetes in plasma (r = 0.66 and 0.75, respectively). These findings suggest that diabetic plasma contains an abnormal and novel low density lipoprotein that mediates the abnormal cholesterol transport characteristic of human diabetes mellitus.     

Receptor-mediated uptake of remnant lipoproteins by cholesterol-loaded human monocyte-macrophages

Normal human monocyte-macrophages were cholesterol-loaded, and the rates of uptake and degradation of several lipoproteins were measured and compared to rates in control cells. Receptor activities for 125I-rabbit beta-very low density lipoproteins (beta-VLDL), 125I-human low density lipoprotein, and 125I-human chylomicrons were down-regulated in cholesterol-loaded cells; however, the rate of uptake and degradation of 125I-human chylomicron remnants was unchanged from control cells. Cholesterol-loaded alveolar macrophages from a Watanabe heritable hyperlipidemic rabbit, which lack low density lipoprotein receptors, showed receptor down-regulation for 125I-beta-VLDL but not for 125I-human chylomicron remnants. In addition to chylomicron remnants, apo-E-phospholipid complexes competed for 125I-chylomicron remnant uptake, but apo-A-I-phospholipid complexes did not. Chylomicrons competed for lipoprotein uptake in control cells but were not recognized under conditions of cholesterol loading. Chylomicron remnants and beta-VLDL were equally effective in competing for 125I-beta-VLDL and 125I-chylomicron remnant uptake in cholesterol-loaded macrophages. When normal human monocyte-macrophages were incubated in serum supplemented with chylomicron remnants, the cholesteryl ester content increased 4-fold over cells incubated in serum with low density lipoprotein added. We conclude: 1) specific lipoprotein receptor activity persists in cholesterol-loaded cells; 2) this receptor activity recognizes lipo-proteins (at least in part) by their apo-E content; and 3) cholesteryl ester accumulation can occur in monocyte-macrophages incubated with chylomicron remnants.     

Study of HDL2-dependent synthesis of bile acids in culture of rabbit hepatocytes: effects of oxidized cholesterol derivatives

The effect of individual oxysterols--products of auto-oxidation of cholesterol on bile acid synthesis by cultivated rabbit hepatocytes was studied. Relative rates of bile acid synthesis were measured as the conversion of 4-14C cholesterol-HDL2 into total 4-14C labeled bile acids. 7 beta-hydroxycholesterol and 3,5-cholestane-7-dione strongly inhibited bile acid synthesis at concentrations 1-10 micrograms/ml. These data support the hypothesis that oxidized cholesterol derivatives accelerate the development of hypercholesterolemia in rabbits fed on cholesterol containing diet.     

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.     

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.     

The beta very low density lipoprotein present in hepatic lipase deficiency competitively inhibits low density lipoprotein binding to fibroblasts and stimulates fibroblast acyl-CoA:cholesterol acyltransferase

Beta very low density lipoprotein (VLDL) was isolated from a patient with hepatic lipase deficiency. The particles were found to contain apolipoprotein B-100 (apoB) and apolipoprotein E (apoE) and were rich in cholesterol and cholesteryl ester relative to VLDL with pre beta electrophoretic mobility. These particles were active in displacing human low density lipoprotein (LDL) from the fibroblast apoB,E receptor and produced a marked stimulation of acyl-CoA:cholesterol acyltransferase. Treatment of intact beta-VLDL with trypsin abolished its ability to displace LDL from fibroblasts. Incubation of trypsin treated beta-VLDL with fibroblasts resulted in a significant stimulation of acyl-CoA:cholesterol acyltransferase activity. beta-VLDL isolated from a patient with Type III hyperlipoproteinemia and an apoE2/E2 phenotype had a higher cholesteryl ester/triglyceride ratio than the beta-VLDL of hepatic lipase deficiency and contained apoB48. It displaced LDL from fibroblasts to a small but significant extent. The Type III beta-VLDL stimulated acyl-CoA:cholesterol acyltransferase to a level similar to that of trypsin-treated beta-VLDL isolated from the hepatic lipase-deficient patient. These results demonstrate that the cholesterol-rich beta-VLDL particles present in patients with hepatic lipase deficiency are capable of interacting with fibroblasts via the apoB,E receptor and that this interaction is completely due to trypsin-sensitive components of the beta-VLDL. These particles were very effective in stimulating fibroblast acyl-CoA:cholesterol acyltransferase. This stimulation was due to both trypsin-sensitive and trypsin-insensitive components.     

Binding of proprotein convertase subtilisin/kexin type 9 to epidermal growth factor-like repeat A of low density lipoprotein receptor decreases receptor recycling and increases degradation

Proprotein convertase subtilisin/kexin type 9 (PCSK9) promotes degradation of hepatic low density lipoprotein receptors (LDLR), the major route of clearance of circulating cholesterol. Gain-of-function mutations in PCSK9 cause hypercholesterolemia and premature atherosclerosis, whereas loss-of-function mutations result in hypocholesterolemia and protection from heart disease. Recombinant human PCSK9 binds the LDLR on the surface of cultured hepatocytes and promotes degradation of the receptor after internalization. Here we localized the site of binding of PCSK9 within the extracellular domain of the LDLR and determined the fate of the receptor after PCSK9 binding. Recombinant human PCSK9 interacted in a sequence-specific manner with the first epidermal growth factor-like repeat (EGF-A) in the EGF homology domain of the human LDLR. Similar binding specificity was observed between PCSK9 and purified EGF-A. Binding to EGF-A was calcium-dependent and increased dramatically with reduction in pH from 7 to 5.2. The addition of PCSK9, but not heat-inactivated PCSK9, to the medium of cultured hepatocytes resulted in redistribution of the receptor from the plasma membrane to lysosomes. These data are consistent with a model in which PCSK9 binding to EGF-A interferes with an acid-dependent conformational change required for receptor recycling. As a consequence, the LDLR is rerouted from the endosome to the lysosome where it is degraded.