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.     

Regulation of acetylated low density lipoprotein uptake in macrophages by pertussis toxin-sensitive G proteins

Class A scavenger receptors (SR-A) mediate the uptake of modified low density lipoprotein (LDL) by macrophages. Although not typically associated with the activation of intracellular signaling cascades, results with peritoneal macrophages indicate that the SR-A ligand acetylated LDL (AcLDL) promotes activation of cytosolic kinases and phospholipases. These signaling responses were blocked by the treatment of cells with pertussis toxin (PTX) indicating that SR-A activates G(i/o)-linked signaling pathways. The functional significance of SR-A-mediated G(i/o) activation is not clear. In this study, we investigated the potential role of G(i/o) activation in regulating SR-A-mediated lipoprotein uptake. Treatment of mouse peritoneal macrophages with PTX decreased association of fluorescently labeled AcLDL with cells. This inhibition was dependent on the catalytic activity of the toxin confirming that the decrease in AcLDL uptake involved inhibiting G(i/o) activation. In contrast to the inhibitory effect on AcLDL uptake, PTX treatment did not alter beta-VLDL-induced cholesterol esterification or deposition of cholesterol. The ability of polyinosine to completely inhibit AcLDL uptake, and the lack of PTX effect on beta-VLDL uptake, demonstrated that the inhibitory effect is specific for SR-A and not the result of non-specific effects on lipoprotein metabolism. Despite having an effect on an SR-A-mediated lipoprotein uptake, there was no change in the relative abundance of SR-A protein after PTX treatment.These results demonstrate that activation of a PTX-sensitive G protein is involved in a feedback process that positively regulates SR-A function.     

Human platelets exclusively bind oxidized low density lipoprotein showing no specificity for acetylated low density lipoprotein.

The widely studied macrophage scavenger receptor system is known to bind both acetylated low density lipoprotein and oxidized low density lipoprotein. Although only the latter ligand has been shown to occur in vivo, acetylated low density lipoprotein is often used to evaluate the contribution of scavenger receptors to different (patho)physiologic processes, assuming that all existing subtypes of scavenger receptors recognise both lipoproteins. In the present work, we identify human platelets as the first natural cell type to bind oxidized low density lipoprotein without showing specificity for acetylated low density lipoprotein. Consequently, platelets possess exclusive receptor(s) for oxidized low density lipoprotein distinct from the 'classical' scavenger receptor AI/AII. From the data presented in this work, we conclude that the class B scavenger receptor CD36 (GPIV) is responsible for this exclusive oxidized low density lipoprotein binding.     

The fatty acid distribution in low density lipoprotein in diabetes.

Atherosclerosis is commonly found in diabetes. There is an association between small dense low density lipoprotein (LDL) phenotype, which is more prevalent in the diabetic state, and atherosclerosis. Small dense LDL is more easily oxidised and it is possible that fatty acid compositional changes, particularly an increase in polyunsaturated fatty acids, could underlie this association. However, there is little information about fatty acids in the different LDL phenotypes in the literature. This study examined LDL subfraction composition in 18 non-insulin-dependent diabetic (NIDDM) patients and 11 control subjects. LDL was isolated and fractionated into LDL 1, 2 and 3 by density gradient ultracentrifugation. NIDDM patients had significantly more fatty acids in all LDL subfractions than control subjects (P<0.01). Palmitic and linoleic acid were significantly greater in all subfractions in the diabetic patients compared to control subjects (P<0.01) and palmitoleic and oleic acids were also greater in LDL1 and LDL2 in diabetic patients (P<0.01). We conclude that in NIDDM fatty acids are increased in all LDL subfractions and this may be the reason for the increased atherosclerosis in diabetes irrespective of phenotype.     

Exposure of rat peritoneal macrophages to acetylated low density lipoprotein results in release of plasma membrane cholesterol. An efficient substrate for esterification by acyl-CoA:cholesterol acyltransferase.

In J774 macrophages and murine macrophages stimulated with acetylated low density lipoprotein (acetyl-LDL), the plasma membrane free cholesterol (FC) became accessible to acyl-CoA:cholesterol acyltransferase (ACAT) as substrate, the result being an accumulation of cholesteryl esters (CE) (Tabas, I., Rosoff, W. J., and Boykow, G. C (1988) J. Biol. Chem. 263, 1266-1272). As the route of delivery of FC to ACAT was not well characterized, we examined this route in the present study. In foam cells derived from rat peritoneal macrophages by preincubation with acetyl-LDL, esterification of the exogenously labeled [3H]FC was low (1.3% of total labeled cholesterol). In contrast, when cells were first labeled with exogenous [3H]FC and then chased with acetyl-LDL, the esterification was more extensive (9.2% of the total labeled cholesterol). During this experiment a significant portion of cellular [3H]FC was released into the medium (up to 33.4% of the total labeled cholesterol). In experiments using a two-compartment chamber in which cells in the lower and upper chambers were separated by filter paper yet the cells in both compartments could communicate without direct contact, [3H]FC released into the medium was biologically active and could serve as an efficient substrate for ACAT. Thus, when acetyl-LDL is not included in culture medium, FC delivery from the macrophage plasma membrane to ACAT is not enhanced, whereas in the presence of acetyl-LDL, plasma membrane FC released and bound to acetyl-LDL may re-enter the cells, possibly through the scavenger receptor. This would provide a significant route for CE synthesis in macrophages.     

Saturated and unsaturated fatty acids independently regulate low density lipoprotein receptor activity and production rate.

These studies examine the regulation of plasma low density lipoprotein (LDL)-cholesterol levels by varying quantities of dietary saturated and polyunsaturated triacylglycerols. At a constant load of 0.12% cholesterol and 20% triacylglycerol, substitution of polyunsaturated for saturated triacylglycerols caused LDL receptor activity to increase from 25% to 80% of control and reduced the LDL-cholesterol production rate from nearly 200% to 155%. These changes caused the plasma LDL-cholesterol concentration to decrease from nearly 190 to 50 mg/dl. When the dietary content of each triacylglycerol alone was incrementally increased, the saturated lipid suppressed receptor activity while the polyunsaturated triacylglycerol increased receptor-dependent LDL transport. The magnitude of these effects was quantitatively similar, although oppositely directed. However, the saturated triacylglycerol also caused a dose-dependent increase in the LDL-cholesterol production rate and markedly increased the plasma LDL-cholesterol level while the polyunsaturated lipid did not affect either of these. These independent effects were also evident in experiments where it was found that substituting polyunsaturated triacylglycerol for saturated lipid increased receptor activity significantly more than did simply reducing the dietary content of saturated triacylglycerol. Thus, these studies show that triacylglycerols containing saturated or polyunsaturated fatty acids have effects on the major processes that regulate the plasma LDL-cholesterol level that are qualitatively and quantitatively distinct.     

Beta-very low density lipoprotein is sequestered in surface-connected tubules in mouse peritoneal macrophages

beta-very low density lipoprotein (VLDL) is a large lipoprotein with multiple apoprotein E (apoE) molecules that bind to the LDL receptors on mouse macrophages. Even though they bind to the same receptor, the endocytic processing of beta-VLDL differs from low density lipoprotein (LDL). LDL is rapidly delivered to perinuclear lysosomes and degraded, but much of the beta-VLDL is retained in peripheral compartments for several minutes. We have investigated the properties of these peripheral compartments. Measurement of the pH was made using FITC- phosphatidylethanolamine incorporated into the beta-VLDL, and we found that the peripheral compartments were near neutral in pH. These peripheral, beta-VLDL containing compartments were poorly accessible to antibodies, but a low molecular weight fluorescence quencher (trypan blue) entered the compartments within a few seconds. Intermediate voltage EM of cells labeled with colloidal-gold-beta-VLDL revealed that the peripheral compartments are tubular, surface-connected invaginations. Kinetic studies with fluorescent beta-VLDL showed that the compartments become fully sealed with a half-time of 6 min, and the beta-VLDL is then delivered rapidly to perinuclear lysosomes. By monitoring fluorescence energy transfer between lipid analogs incorporated into the beta-VLDL, some processing of the lipoprotein in the peripheral tubular compartments is demonstrated. The novel mode of uptake of beta-VLDL may account for the high cholesterol ester accumulation induced by this lipoprotein.     

The fatty acid distribution in low density lipoprotein in diabetes

Atherosclerosis is commonly found in diabetes. There is an association between small dense low density lipoprotein (LDL) phenotype, which is more prevalent in the diabetic state, and atherosclerosis. Small dense LDL is more easily oxidised and it is possible that fatty acid compositional changes, particularly an increase in polyunsaturated fatty acids, could underlie this association. However, there is little information about fatty acids in the different LDL phenotypes in the literature. This study examined LDL subfraction composition in 18 non-insulin-dependent diabetic (NIDDM) patients and 11 control subjects. LDL was isolated and fractionated into LDL 1, 2 and 3 by density gradient ultracentrifugation. NIDDM patients had significantly more fatty acids in all LDL subfractions than control subjects (P<0.01). Palmitic and linoleic acid were significantly greater in all subfractions in the diabetic patients compared to control subjects (P<0.01) and palmitoleic and oleic acids were also greater in LDL1 and LDL2 in diabetic patients (P<0.01). We conclude that in NIDDM fatty acids are increased in all LDL subfractions and this may be the reason for the increased atherosclerosis in diabetes irrespective of phenotype.     

Induction of acetylated low density lipoprotein receptor and suppression of low density lipoprotein receptor on the cells of human monocytic leukemia cell line (THP-1 cell)

THP-1 cells, a human cell line established from acute monocytic leukemia cells, degraded native human low density lipoprotein (LDL) through a LDL-specific pathway, but had no ability to degrade acetylated LDL. When the cells were treated with 12-o-tetradecanoyl-phorbol-13-acetate (TPA) to differentiate into the macrophage-like stage, those acquired the ability to degrade acetylated LDL through its specific pathway and lost the ability to degrade native LDL. Degradation of acetylated LDL by the differentiated cells was not reduced by preincubation with either acetylated LDL or native LDL.     

Essential involvement of 12-lipoxygenase in regiospecific andstereospecific oxidation of low density lipoprotein by macrophages.

To establish a role of the 12-lipoxygenase on the generation of oxidized low density lipoprotein (LDL) in macrophages that leads to foam cell formation in atherosclerosis, we overexpressed 12-lipoxygenases in a macrophage-like cell line, J774A.1, that does not show intrinsic enzyme activity. When the 12-lipoxygenase-expressing cells were incubated with 400 microg.mL-1 LDL in Dulbecco's modified Eagle's medium at 37 degrees C for 12 h, LDL oxidation, as determined by thiobarbituric acid reactive substance, was markedly increased compared with the mock-transfected cells. Oxygenated products in the modified LDL were examined by HPLC before and after alkaline hydrolysis. Most of the oxygenated derivatives were of an esterified form, and the major product was identified as 13S-hydroxyoctadeca-9Z,11E-dienoic acid. These results clearly demonstrate that esterified fatty acids in LDL are oxygenated by the 12-lipoxygenases expressed in the J774A.1 cells. Furthermore, the oxidized LDL generated by intracellular 12-lipoxygenases was recognized by a scavenger receptor as assessed by macrophage degradation assay.     

Cholesteryl ester handling by RAW264 macrophages: response to native and acetylated low density lipoprotein

Summary The effects of LDL and Ac-LDL on the growth properties, morphology, and cholesteryl ester (CE) metabolism of the RAW264 macrophage cell line have been characterized. Cells were grown in media supplemented by a defined media (DM) mixture or fetal bovine serum (FBS). The addition of LDL or Ac-LDL to the culture media did not significantly alter cell growth properties. Cytoplasmic deposition of CE was observed by fluorescence microscopy in macrophages treated with LDL or Ac-LDL but not in untreated controls. Dose-response studies have shown that cholesteryl ester (CE) can accumulate in RAW264 treated with LDL. Cellular cholesterol content saturated at 4 hours with 50 g/ml LDL; this effect may be associated with receptor saturation. Dose-response studies conducted with Ac-LDL in DM have shown dramatic increases in total cell cholesterol content. However, deposition of CE was not observed below Ac-LDL concentrations of 100 g/ml. This indicates that a critical concentration of Ac-LDL must be reached to trigger deposition in DM. In contrast, no critical concentration of Ac-LDL was observed in macrophages grown in medium supplemented with 10% FBS. Cholesterol esterification in response to LDL and Ac-LDL was examined by ¹⁴C-oleic acid incorporation into CE. These results confirmed the mass cellular cholesterol and CE measurements. Kinetic studies conducted with RAW264 cells treated with 50 or 100 g/ml Ac-LDL resulted in a cholesterol efflux from the cells at 6–12 hours of incubation. Therefore, these studies show that (1) the nature of CE deposition is highly dependent upon the incubation media and (2) CE deposition is very sensitive to Ac-LDL concentration under certain conditions.Abbreviations LDL Low Density Lipoprotein - Ac-LDL Acetylated Low Density Lipoprotein - FBS Fetal Bovine Serum - DM Defined Medium - PBS Phosphate Buffered Saline - CE Cholesteryl Ester     

Unesterified fatty acids inhibit the binding of low density lipoproteins to the human fibroblast low density lipoprotein receptor

Micromolar concentrations of oleate were found to inhibit reversibly the binding of low density lipoprotein (LDL) to the human fibroblast LDL receptor. The decrease in LDL binding caused a parallel reduction of both 125I-LDL uptake and degradation at 37 degrees C. At 4 degrees C, oleate was also found to displace 125I-LDL already bound to the LDL receptor. The effect of oleate was rapid, reaching 70-80% of maximum displacement with 5-10 min of incubation, and was closely correlated to oleate-albumin molar ratios. Partition analysis of unesterified fatty acids between cells and LDL showed that the inhibitory effect of oleate resulted mainly from an interaction of unesterified fatty acids with the cell surface rather than with the LDL particles. Using different unesterified fatty acids and fatty acid analogs, we found that the inhibitory effect was modulated by both the length and the conformation of the monomeric carbon chain and was directly dependent on the presence of a negative charge on the carboxylic group. At 4 degrees C, the inhibitory effect of oleate never exceeded half of maximum binding capacity. This limitation was associated with the ability of oleate to interact only with part of the population of LDL receptors which spontaneously recycles in the absence of ligand, as demonstrated by the fact that oleate did not induce any reduction of LDL binding after cell treatment with monensin in the absence of LDL. Our results indicate that unesterified fatty acids could participate in the control of LDL catabolism in vivo by direct modulation of the ability of LDL receptor to bind LDL.     

Recognition of oxidized low density lipoprotein by the scavenger receptor of macrophages results from derivatization of apolipoprotein B by products of fatty acid peroxidation

Uptake of cholesterol-containing lipoproteins by macrophages in the arterial intima is believed to be an important step in the pathogenesis of atherosclerosis. There are a number of possible mechanisms by which macrophages might accumulate cholesterol, and one that has attracted much interest recently involves the uptake of oxidatively modified low density lipoprotein (LDL) via a specific cell surface receptor, termed the scavenger or acetyl-LDL receptor. Previous studies have shown that chemical derivatization of LDL with reagents that result in neutralization of the charge of lysine amino groups also allows recognition by this receptor. As well, it has been shown that oxidation of LDL is accompanied by a decrease in free lysine groups and binding of lipid products to apolipoprotein B. The present studies were done to further characterize the receptor-binding domain on oxidized LDL. It was found that LDL could be modified by incubation with water-soluble products derived from autoxidized unsaturated fatty acids under conditions that inhibited oxidation of the LDL itself. The LDL modified in this way had increased electrophoretic mobility but showed no evidence of the oxidative damage that typifies LDL oxidized by exposure to metal ions. Furthermore, the oxidation product-modified LDL was rapidly degraded by cultured macrophages through the scavenger receptor pathway. Bovine albumin modified by oxidation products also showed greatly accelerated degradation by macrophages. When analyzed by reverse-phase high pressure liquid chromatography, the reactive oxidation products appeared less polar than fatty acids or simple medium-chain aldehydes. When treated with the carbonyl reagent 2,4-dinitrophenylhydrazine, the reactive fractions yielded derivatives, some of which were identified by mass spectrometry as hydrazones of nonenal, heptenal, pentenal, and crotonaldehyde. A series of 2-unsaturated aldehydes (acrolein to 2-nonenal) were all found to modify LDL, but none of these aldehyde-modified LDLs were recognized by the scavenger receptor of macrophages and all were degraded much more slowly by these cells than LDL modified with oxidation products. Furthermore, copper-oxidized LDL had only very slight immunoreactivity toward a panel of antibodies specific for adducts of simple 2-unsaturated aldehydes. Analysis of underivatized autoxidized fatty acids by coupled liquid chromatography/thermospray mass spectrometry revealed compounds with m/z corresponding to M+17, M+31, and 2M+31 in fractions that were capable of modifying LDL. The unoxidized fatty acids showed a dominant peak at M-1. These results indicate that the scavenger receptor of macrophages can recogn     

Reconstitution of the low density lipoprotein receptor

The receptor for low density lipoprotein was purified from bovine adrenal cortex in the presence of the nonionic detergent octylglucoside. Receptors were incorporated into the bilayer of egg phosphatidylcholine vesicles by a detergent-dialysis method. Reconstituted receptors were functional in that they bound low density lipoprotein as well as a monoclonal antibody directed against the receptor in a specific, saturable fashion. Binding activity of reconstituted receptors was measured by a gel chromatography assay. The orientation of the receptor molecule within the phospholipid bilayer was investigated by binding assays following proteolytic digestion. Reconstituted receptors showed an orientation that was functionally indistinguishable from that of low density lipoprotein receptors in the plasma membrane of intact human fibroblasts.     

The very low density lipoprotein receptor is not necessary for maintaining brain polyunsaturated fatty acid concentrations

Polyunsaturated fatty acids (PUFA), especially docosahexaenoic and arachidonic acids, as well as cholesterol are important for neural development and maintaining brain function. However, in contrast to cholesterol, the brain is unable to synthesize the required amounts of these PUFA de novo and requires a constant supply from plasma. Suggested pools of uptake include plasma unesterified PUFA or the uptake of PUFA-containing lipoproteins via lipoprotein receptors into endothelial cells of the blood brain barrier. Our study tested whether the very low density lipoprotein receptor (VLDLr) is necessary for maintaining brain PUFA and cholesterol concentrations. Moreover, since VLDLr knockout (VLDLr^?/?) mice have been reported to have behavioural deficits, this study asked the question whether altered brain PUFA and cholesterol concentrations might be related to these deficits. VLDLr^?/? and wild-type mice had ad libitum access to chow. At 7 weeks of age the mice were sacrificed, and the cortex, cerebellum, hippocampus, and the remainder of the brain were isolated for total fatty acid and cholesterol analyses. There were no differences in total lipid PUFA or cholesterol concentrations in any of the four brain regions between VLDLr^?/? and wild-type mice. These findings demonstrate that the VLDLr is not necessary for maintaining brain PUFA concentrations and suggest that other mechanisms to transport PUFA into the brain must exist.     

Inhibition of fatty acid synthesis decreases very low density lipoprotein secretion in the hamster.

The hamster was developed as a model to study very low density lipoprotein (VLDL) metabolism, since, as is the case in humans, the hamster liver was found to synthesize apoB-100 and not apoB-48. The effect of inhibiting fatty acid synthesis on the hepatic secretion of VLDL triglyceride (TG) and apolipoprotein (apo) B-100 in this model was then investigated. In an in vivo study, hamsters were fed a chow diet containing 0.15% TOFA (5-tetradecyloxy-2-furancarboxylic acid), an inhibitor of acetyl-CoA carboxylase. After 6 days of treatment, plasma triglyceride and cholesterol levels were decreased by 30.2% and 11.6%, respectively. When the secretion of VLDL-TG by the liver was measured in vivo after injection of Triton WR 1339, TOFA treatment was found to decrease VLDL-TG secretion by 40%. In subsequent in vitro studies utilizing cultured primary hamster hepatocytes, incubation with 20 microM TOFA for 4 h resulted in 98% and 76% inhibition in fatty acid and triglyceride synthesis, respectively; VLDL-TG secretion was decreased by 90%. When hepatocytes were pulsed with [3H]leucine, incubation with TOFA resulted in a 50% decrease in the incorporation of radiolabel into secreted VLDL apoB-100. The results of this study indicate that inhibition of intracellular triglyceride synthesis decreases the secretion of VLDL-TG and apoB-100, and does not result in the secretion of a dense, triglyceride-depleted lipoprotein.     

Characterization of binding sites for acetylated low density lipoprotein in the rat liver in vivo and in vitro.

Acetylated low density lipoprotein (acetyl-LDL) binding to hepatic membrane proteins of rats was analysed in vitro by ligand blotting. Specific binding could be demonstrated to two hepatic proteins with an apparent mol. wt. of 250 kd and 220 kd. Polyanionic competitors and maleylated bovine serum albumin inhibited the binding of acetyl-LDL effectively. To determine the sites of the catabolism of acetyl-LDL, [131I]-acetyl-LDL was injected intravenously into control rats and rats pre-treated with the known competitors of the acetyl-LDL binding. Distribution of the radiolabelled acetyl-LDL was followed by a scintillation camera. Six minutes after injection, the radioactivity was concentrated in the liver. The competitors and unlabelled acetyl-LDL but not native LDL reduced the hepatic uptake of [131I]acetyl-LDL dramatically. Thus, the sensitivity of the 220- and 250-kd membrane binding sites to the competitors for the acetyl-LDL binding resembled that of the hepatic compartment in vivo. Finally, an application of scintigraphy with radiolabelled low density lipoproteins for diagnostic evaluation of tumor compartments is presented.