Unlabeled Low Density Lipoprotein Research Articles

24] Quantification of cholesteryl ester transfer protein: Activity and immunochemical assay

The assay for cholesteryl ester transfer protein (CETP) activity common to most publications consists of incubating [ 3 H] high-density lipoprotein (HDL) (1-10 μg cholesterol) and unlabeled low-density lipoprotein (LDL) (1-10μg cholesterol) at various donor-to-acceptor ratios from 1:1 to 1:10, with a source of CETP activity for 2 to 24 hr. Aliquots are removed from the reaction mixture at timed intervals or at the end of the incubation period and the LDL fraction is precipitated by the addition of heparin/MnCl 2 .The amount of CE transferred from HDL to LDL is measured as the difference between the amount of [ 3 H]CE radioactivity, remaining in the supernatant HDL fraction and the amount of [ 3 H]CE HDL in a control reaction lacking CETP. This chapter describes a rapid, high sample capacity CETP activity assay, utilizing the Packard (Downer Grove, IL) topCount 96-well plate liquid scintillation counter and Millipore (Bedford, MA) multiScreen filtration plates that permit direct measurement of CETP-mediated transfer of cholesteryl ester (CE) into LDL, by counting the radioactivity in the precipitated LDL. In this filter plate-based CETP activity assay, higher or lower levels of CETP activity directly correspond to higher or lower amounts of labeled CE incorporated into the precipitated LDL.

Degradation of Chylomicron Remnants by Macrophages Occurs via Phagocytosis

Chylomicron remnants bound to rabbit alveolar macrophages with high-affinity (Kd = 3.3 +/- 0.71 microgram of protein/mL). The binding of chylomicron remnants was competitively inhibited in the presence of unlabeled remnants and to a lesser extent by unlabeled low-density lipoproteins. Pretreatment of cells with either trypsin or pronase inhibited degradation in a dose and time dependent manner, suggesting involvement of a cell surface protein. Chylomicron remnants were degraded by alveolar macrophages from Watanabe heritable hyperlipidemic (WHHL) rabbits, which are devoid of LDL receptor activity. Moreover, colchicine and monensin which are endocytotic and lysozomal inhibitors, respectively, did not have any effect on the degradation of chylomicron remnants by macrophages from normal rabbits. The absence of divalent cations was found to enhance chylomicron remnant degradation by macrophages. Activated alpha 2-macroglobulin and lactoferrin had no effect on chylomicron remnant degradation, indicating that the low-density lipoprotein receptor-related protein was not involved. In addition, the scavenger receptor inhibitors polyinosinic acid and fucoidan increased degradation of chylomicron remnant-ruling out uptake as a consequence of lipoprotein modification. Rather, the phagocytotic inhibitor cytochalasan D was found to significantly decrease chylomicron remnant degradation. Collectively, our data show that chylomicron remnants are metabolized by phagocytotic pathways initiated after binding to a cell surface protein which is distinct from the LDL receptor, LRP, or scavenger receptors.

糖化低比重リポ蛋白(LDL)のヒトマクロファージ(Mφ)における異化機構に関する検討

Using glycolaldehyde (two carbons, C2) and monosaccharides of the aldose D series, i. e., glyceraldehyde (C3), erythrose (C4), arabinose (C5), and glucose (C6), new modified low density lipoproteins (LDLs) were produced to determine if degradation of modified LDLs by the human monocyte-derived macrophage (Mφ) scavenger receptors is based on the molecular weight of the substance used to modify the LDL free lysine.By using a trinitrobenzensulfonic acid (TNBS) assay, it was found that the smaller the molecular weight of the substances used for LDL modification, the more rapid and extensive is the decrease of free lysine, which plateaued after 24h. Based on findings of glycolaldehyde-LDL, glyceraldehyde-LDL and erythrose-LDL, double reciprocal plots were made. Results revealed that all three reactions occured through the same mechanism, although the Km values in the three modified LDLs differed. Further, the electrophoretic mobility of these modified LDLs correlated with a decrease in the percentage of TNBS reactivity.Using Mφ, degradation studies of mildly modified LDLs revealed that, in contrast to native LDL, little degradation occured. However, after extensive modifications, less degradation was seen in glycolaldehyde-LDL, glyceraldehyde-LDL and erythrose-LDL than in the acetyl-LDL, but more degradation occured in these new modified LDLs than in native LDL. Further, degradation of 125-I-glyceraldehyde-LDL was remarkedly inhibited in unlabeled acetyl-LDL, 125-Iglycolaldehyde-LDL and fucoidin, whereas in unlabeled oxidized LDL and advanced glycoslation end products(AGE)-modified bovine serum albumin, degradation of 125-I-glyceraldehyde-LDL was extremely slight. In contrast, unlabeled native LDL produced no inhibitory effect on the degradation of 125-I-glyceraldehyde-LDL. These findings would seem to suggest that after extensive modification, receptor recognition of glyceraldehyde-LDL changes from the LDL receptor to the scavenger one.Degradation of new modified LDLs appeared to depend on a decrease in the percentage of TNBS reactivity. Further, even though these modified LDLs showed the same ΔTNBS decrease, these degradation progressively increased in the following order: erythrose-LDL. glyceraldehyde-LDL, and glycolaldehyde-LDL, These findings suggest that the degree of LDL degradation is not only associated with an increase in the negative charge and decrease in the percentage of TNBS reactivity, but also with the molecular weight of the substance used to modify the LDLs.Arabinose-LDL and glycosylated LDL showed less degradation than native LDL, although LDLs were incubated for 5 days with a high concentration of their respective modifying substance. Degradation decreased even further when NaBH3CN was used for extensive LDL modification. These findings suggest that the particle size and/or the stereoscopic strucure of modified LDLs may play an important role in becoming the ligand of the scavenger receptor.Glucose promotes oxidative LDL modification. Further, glycosylated LDL are easily oxidized by metalic ions, and oxidized LDL are extensively degraded by the scavenger receptors. However, our results have demonstrated that glycosylated LDL is not recognized by the scavenger receptors even after extensive modification. We thus have speculated that under hyperglycemic conditions, LDLs may promote the formation of foam cells as not only AGE but also oxidized and/or glycoxidized LDLs.

An improved method for detection of low density lipoprotein receptor defects in human T lymphocytes.

Familial hypercholesterolemia (FH) results from an inherited functional defect of the low density lipoprotein (LDL) receptor and is complicated by premature atherosclerosis. FH diagnosis is obtained by sophisticated techniques or is suggested by clinical criteria. We have developed a technique based on flow cytometry for the measurement of DiI-labeled LDL uptake in human peripheral blood T lymphocytes left for 2 days in a lipoprotein-deficient culture medium. Flow cytometry allowed us to discriminate the uptake of DiI-LDL by T lymphocytes subpopulation from the uptake by the whole mononuclear population using a T cell-specific anti-CD3 antibody. The method appeared to be highly specific for the receptor-mediated pathway of LDL uptake as DiI-LDL uptake was inhibited in the presence of a 10-fold excess of unlabeled LDL and by EDTA. A good relationship was found between the uptake of DiI-LDL and 125I-labeled LDL degradation. The test was applied in three groups of patients: patients with normal cholesterol levels, patients with heterozygous FH, and patients with high cholesterol levels but without clinical criteria of FH. The mean fluorescence intensities were 23.1 +/- 8.9, 6.3 +/- 1.7, and 17.1 +/- 3.5 (mean +/- standard deviation), respectively. The ability to measure the fluorescence in T lymphocytes improved the discrimination between FH and non-FH subjects when compared with values obtained from the whole mononuclear cell population. These results suggest that our method could be useful for LDL receptor defects screening.

Receptor-Mediated Interaction of the Low-Density Lipoprotein Complex with Dextran Sulfate: Potential as a Multi-Biological Coupler

We have investigated the receptor-mediated binding and uptake of low-density lipoprotein (LDL) and its insoluble complex with dextran sulfate to determine the contribution of positively charged sites in LDL to receptor-mediated interactions. Rabbit plasma LDL derivatized with FITC retained its sedimentation ability with dextran sulfate, as well as intact LDL, and binding of fluorescent LDL and its complex to liver cells was assayed by flow cytometry. Flow cytometry revealed that the binding of complex LDL with dextran sulfate, as well as that of pure LDL, increased on rat liver parenchymal cells treated with estrogen, which enhanced the expression of LDL receptors, and decreased on Hep G2 and Chang Liver cells treated with a monoclonal antibody against LDL receptors. The binding of pure LDL and complex LDL to hepatocytes was depressed by pretreatment with unlabeled LDL in a similar manner, but not with asialoglycoprotein, a ligand of asialoglycoprotein receptors on liver cells. Furthermore, we carried out a stable primary culture of rat hepatocytes, and then pure LDL and complex LDL were applied to the cultured hepatocytes. Hepatic-differentiated functions such as albumin and bile acid secretion decreased on the uptake of pure LDL and complex LDL in a similar manner. Consequently, comparative studies using pure LDL and complex LDL allowed us to determine that the complex formation with dextran sulfate had no influence on the receptor-mediated binding or uptake of LDL, and that LDL possessed binding domains for LDL receptors and sulfonic carbohydrates, containing positively charged amino acids.

Increased uptake of low density lipoprotein by SV40-transformed smooth muscle cells

We compared the metabolism of low density lipoprotein (LDL) in SV40-transformed smooth muscle cells (TSMCs) to that in nontransformed smooth muscle cells (SMCs). When SMCs were incubated in medium with 100 μg/ml LDL for 24 hours, they did not accumulate sudanophilic lipid droplets. On the other hand, when TSMCs were incubated in medium containing more than 100 μg/ml LDL, they accumulated a large amount of lipid droplets in their cytoplasm. When cells were incubated with 200 μg/ml LDL for 24 hours, cholesteryl ester levels significantly increased in TSMCs (18.3 ± 3.53 μg/mg protein), as compared with SMCs (2.40 ± 0.85 μg/mg protein). However, there was no difference in the cellular level of free cholesterol between the TSMCs and SMCs. Although the TSMCs and SMCs had a similar number of binding sites for LDL, the TSMCs demonstrated a markedly higher uptake of LDL labeled with 1,1′-dioctadecyl-3,3,3′,3′-tetramethyl indocarbocyanine perchlorate (Dil-LDL), compared with the SMCs. SMCs that had been pretreated with 100 μg/ml of unlabeled LDL for 24 hours showed a decreased uptake of Dil-LDL. In contrast, TSMCs incorporated Dil-LDL independently of the preincubation with 100 μg/ml LDL. The presence of brefeldin A, which may block the transport of glycoproteins from the ER to Golgi apparatus, had less of an effect on the uptake of LDL in the TSMCs than in the SMCs. These results suggest that SV40-transformed smooth muscle cells show an increased uptake of LDL independent of the cellular cholesterol level, which may induce the accumulation of lipid droplets in their cytoplasm. A LDL receptor-independent pathway may be related to the increased uptake of LDL in SV40-transformed smooth muscle cells.

Inhibitory Effect on the Lipolysis-stimulated Receptor of the 39-kDa Receptor-associated Protein

Adenovirus vector-mediated transfer of the receptor-associated protein (RAP) gene into low density lipoprotein (LDL) receptor-deficient mice was shown to achieve plasma concentrations ranging between 20 and 200 micrograms/ml and to result in the accumulation of remnant lipoproteins (Willnow, T. E., Sheng, Z., Ishibashi, S., and Herz, J. (1994) Science 264, 1471-1474). Both this finding and the observation that in addition to various other members of the LDL receptor gene family, RAP binds to a yet unidentified protein of apparent molecular mass of 105 kDa prompted us to examine the effect of high concentrations of RAP on the lipolysis-stimulated receptor (LSR). LSR is a receptor distinct from the LDL receptor and the LDL receptor-related protein and is capable of binding apoB and apoE when activated by free fatty acids. Data reported here show that in fibroblasts isolated from a subject homozygous for familial hypercholesterolemia, RAP fusion protein inhibited LSR-mediated binding of 125I-LDL and the subsequent internalization and degradation of the particles. Studies on the interaction of RAP with LSR in isolated rat liver membranes revealed that at concentrations > or = 10 micrograms/ml, RAP inhibited in a dose-dependent manner the binding of LDL to LSR; half-maximum inhibition was obtained with 20 micrograms/ml RAP. Ligand blotting studies revealed that RAP bound directly to two rat liver membrane proteins of apparent molecular masses identical to those that bind 125I-LDL after preincubation with oleate. However, unlike LDL, binding of 125I-RAP to LSR did not require preincubation with oleate. Preincubation of nitrocellulose membranes with an excess of unlabeled RAP fusion protein decreased oleate-induced binding of 125I-LDL to LSR candidate proteins, whereas preincubation with excess unlabeled LDL was unable to prevent the subsequent binding of 125I-RAP to the LSR proteins. Both the latter data and analysis of the mechanism of inhibition were consistent with the RAP inhibitory effect on LSR being achieved by interference with a site distinct from the oleate-induced LDL binding site. In conclusion, this study shows that at concentrations reported to delay chylomicron remnant removal in LDL receptor-deficient mice, RAP exerted a significant inhibitory effect on LSR.

Host Plasma Low Density Lipoprotein Particles as an Essential Source of Lipids for the Bloodstream Forms of Trypanosoma brucei

In contrast to mammalian cells, bloodstream forms of Trypanosoma brucei show no activity for fatty acid and sterol synthesis and critically depend on plasma low density lipoprotein (LDL) particles for their rapid growth. We report here that these parasites acquire such lipids by receptor-mediated endocytosis of LDL, subsequent lysosomal degradation of apoprotein B-LDL, and utilization of these lipids. Uptake of LDL-associated [3H]sphingomyelin and of LDL-associated [3H]cholesteryl oleate paralleled each other, and that of 125I-apoprotein B-LDL showed saturation and could be inhibited by unlabeled LDL or by anti-LDL receptor antibodies. Metabolism of lipids carried by LDL was abolished by chloroquine and by the thiol protease inhibitor, leupeptin. Sphingomyelin was cleaved by an acid sphingomyelinase to yield ceramide, which was itself split up into sphingosine and fatty acids. The latter were further incorporated into phosphatidylcholine, triacylglycerols, or cholesteryl esters. Similarly, cholesteryl oleate was hydrolyzed by an acid lipase to yield free cholesterol, which was reesterified with fatty acids, presumably in the cytosol. Like free cholesterol, LDL provided substrate for cholesterol esterification. In the culture-adapted procyclic form of T. brucei, which is capable of sterol synthesis, exogenous LDL-cholesterol rather than endogenously synthesized sterol was utilized for sterol esterification. Interference with exogenous supply of lipids via receptor-mediated endocytosis of LDL should be explored to fight against trypanosomiasis.

Immunohistochemical and Biochemical Detection of Low Density Lipoprotein Receptors in Cultured Rat Mesangial Cells

The expression of low density lipoprotein (LDL) receptors by cultured rat mesangial cells was demonstrated using immunohistochemical and biochemical methods. Using a mouse monoclonal anti-human LDL receptor antibody, the immunofluorescence technique was applied to cultured mesangial cells and showed granular staining. Immunoblotting analysis of the membrane fraction of cultured mesangial cells showed a single band with a protein of approximately 130,000 molecular weight. In addition, [125I]LDL binding and the uptake of [125I]LDL by cultured mesangial cells were studied. Binding and uptake both reached an equilibrium after about 30 min of incubation. A 50% reduction in [125I]LDL (20 micrograms protein/ml) binding and uptake occurred when unlabelled LDL was added to cultures at 20-40 micrograms protein/ml. Addition of high density lipoprotein without apoE to the culture medium did not induce competitive inhibition of [125I]LDL binding and uptake by mesangial cells. These findings suggest that cultured mesangial cells have the capacity to express an LDL receptor that regulates the cellular uptake of LDL.

Photosensitizer targeting in photodynamic therapy II. Conjugates of haematoporphyrin with serum lipoproteins

Conjugates between haematoporphyrin (HP) and human low-density lipoprotein (LDL), human high-density lipoprotein (HDL) and bovine HDL have been prepared, purified and characterized. HP-LDL is aggregated possibly via interparticle apoB protein cross-linking. HP-HDL human and bovine conjugates show different degrees of intraparticle apoA polypeptide cross-linking. Receptor-mediated endocytosis of HP-LDL by NIH 3T3 cells is inferred from the increased uptake observed when LDL receptors are upregulated. HP-LDL uptake into HT29 cells faces competition from unlabelled LDL, albeit at rather high doses. HP-HDL uptake is also inhibited by LDL, suggesting that both lipoprotein conjugates may have cell-surface binding sites in addition to the specific LDL (apoB) receptor. J774.2 macrophages avidly accumulate HP-LDL, retaining most of the fluorescence and some of the protein while degrading the remainder. Oxidized LDL species compete in these processes, with the major effect on protein degradation. Chloroquine has little effect on the fluorescence uptake but inhibits protein degradation (and hence enhances protein accumulation). HP-HDL is also avidly taken up by J774.2 cells, but in the case of the bovine material with a sigmoidal concentration dependence. This is consistent with prior aggregation before the particles can be endocytosed. P388.D1 cells, which appear to be less activated than the J774.2 line, take up less fluorescence and retain and degrade less protein, but still to higher extents than observed for non-phagocytic cells. We conclude that photosensitizer-lipoprotein conjugates can be taken up in large amounts by cells possessing scavenger receptors and/or phagocytic activity, and that this may be a means of targeting photodynamic therapy.

Oxidative modification of low-density lipoprotein by human polymorphonuclear leucocytes to a form recognised by the lipoprotein scavenger pathway

The oxidative modification of low-density lipoprotein (LDL) results in the formation of cytotoxic and chemotactic lipids which are thought to be of importance in the development of atherosclerotic lesions. In the present study we show that polymorphonuclear leucocytes (PMNs) can modify LDL to a form which is rapidly incorporated by macrophages via a scavenger receptor pathway. Incubation of 125I-labelled LDL with PMNs in Ham's F-10 medium resulted in oxidation as shown by the appearance of thiobarbituric acid-reactive substances, increased electrophoretic mobility of the LDL and increased degradatation of the LDL by mouse peritoneal macrophages. The presence of the anti-oxidant butylated hydroxytoluene or the metal ion chelator, EDTA inhibited the PMN-mediated modification. The degradation of 125I-labelled PMN modified LDL by macrophages was competitively inhibited by unlabelled copper-oxidised LDL but not by native LDL, indicating that the degradation was mediated by the scavenger receptor. The oxidative modification of LDL by PMNs could be of pathophysiological importance in inflammation and in the accelerated atherosclerosis seen following cardiac reperfusion injury.

Gold-labelled Low Density Lipoproteins Bind to Washed Human Platelets

There is still disagreement about the presence of receptors for low density lipoproteins (LDL) in human platelets. Therefore, washed human platelets in suspension were incubated with gold-labelled LDL, and the binding sites for LDL were revealed by transmission electron microscopy on ultrathin sections and on surface replicas. The LDL-gold complexes bound to the platelet membrane and appeared in the open canalicular system. Gold particles were also found within the coated vesicles. The predominant labelling pattern, showed by the surface replicas, was that of scattered single gold particles randomly distributed over the platelet surface. Some small clusters of gold particles were also observed, uniformly distributed between the individual particles. Competitive experiments using an excess of unlabelled LDL suggested that the binding was due to specific binding sites for LDL on the platelet membrane, possibly the LDL receptor. However, the binding sites are not expected to be the classical apolipoprotein B/E receptor, since the binding was inhibited by preincubating the platelets with anti-glycoprotein IIb-IIIa.

Measurement of total hepatic low density lipoprotein receptor levels in the hamster.

The ability to measure the total concentration of low density lipoprotein (LDL) receptors in hepatic tissues is of crucial importance to understanding changes in hepatic cholesterol metabolism. Such measurements can be made in conjunction with estimates of LDL receptor transcriptional activity, cell surface LDL receptor number, and rates of hepatic LDL uptake to evaluate the mechanisms controlling cellular LDL receptor expression. Current methods for assessing hepatic LDL receptor levels use microsomes as a source of LDL receptor, and thus rely on consistent contamination of the microsomal preparation with LDL receptor-containing plasma membranes, endocytic vesicles, and/or secretory vesicles. Because this contamination is variable, and may vary with alterations in either the distribution of LDL receptors among the various cellular membrane fractions or in the composition of the intracellular membranes, measurement of LDL receptor concentration in microsomal fractions may not accurately reflect the total compliment of LDL receptors within the cell. We have developed the methodology for isolating the full complement of hepatic LDL receptor containing membranes by discontinuous sucrose density gradient centrifugation, and for quantitating LDL receptor concentration using a Western immunoblotting procedure that uses an anti-C-terminal LDL receptor peptide polyclonal antiserum and assesses the intensity of color formation by reflectance densitometry. Using this methodology, we observed a 126 kDa immunoreactive band for the bovine adrenal cortex LDL receptor that also exhibited LDL binding activity as visualized by biotinylated LDL-ligand blotting, and a doublet of 140 kDa for the hamster liver LDL receptor. These bands were not observed when ligand blotting was conducted in the presence of either 10 mM EDTA or a 5-fold excess of unlabeled LDL, or when immunoblotting was conducted using either preimmune serum or antiserum that had been preabsorbed with LDL receptor peptide. The intensity of color formation was a linear function of the amount of membrane extract separated by electrophoresis. Intra-assay variation averaged 7%, and inter-animal variation averaged 20%. Cholestyramine, tiqueside, CP-88488, 17 alpha-ethinyl estradiol, mevinolin, and the combination of cholestyramine plus mevinolin, pharmacological interventions known to increase LDL receptor activity in experimental animals, produced the predicted increases in hamster total hepatic LDL receptor concentration that were highly correlated with concomitant increases in HMG-CoA reductase activity and reductions in serum cholesterol.(ABSTRACT TRUNCATED AT 400 WORDS)

Fluorescence flow cytometry of human leukocytes in the detection of LDL receptor defects in the differential diagnosis of hypercholesterolemia.

A flow-cytometric method with fluorescence-labeled monoclonal antibodies (MABs) against the low density lipoprotein (LDL) receptor (C7A MAB) or 3,3'-dioctadecylindocarbocyanin-iodide (DiI) LDL has been developed that allows the quantification of LDL receptors on leukocytes and the identification of patients with familial hypercholesterolemia (FH) within 48 hours. Leukocytes were isolated from 10 mL anticoagulated blood by density gradient centrifugation. To induce maximal expression of LDL receptors, mononuclear cells were preincubated with either phytohemagglutinine (PHA) or lipoprotein-deficient serum (LPDS). LPDS-treated monocytes provided a more homogeneous cell population with regard to LDL receptor activity than did the PHA-treated lymphocytes; they also provided a greater discrimination between the fluorescence of the receptor probes and cellular autofluorescence. The C7A MAB was able to compete for DiI LDL binding by about 40%. In competition with unlabeled LDL, DiI LDL revealed linear binding, indicating an affinity similar to native LDL. The binding characteristics of DiI LDL were also similar to 125I-LDL binding. LDL isolated from familial defective apolipoprotein B-100 was not able to compete for DiI LDL binding on monocytes, whereas native LDL reduced it by about 80%. In monocytes from FH heterozygous patients, the cellular mean fluorescence using either C7A MAB or DiI LDL at 4 degrees C was 30% to 70%; in FH homozygotes, cellular mean fluorescence was less than 20% of that in monocytes from normal individuals. In patients with familial defective apolipoprotein B-100 antibody binding was normal, but one patient's own LDL failed to compete with normal DiI LDL for 4 degrees C binding on U937 test monocytes. Patient monocytes having internalization defects showed normal 4 degrees C DiI LDL binding, but at 20 degrees C cell-associated fluorescence was reduced by about 40%. In our study 384 hypercholesterolemic patients (preselected according to serum cholesterol levels, clinical symptoms, and family history) were analyzed for LDL receptor expression using the C7A MAB-based assay. In 71.8% of the patients with cholesterol levels higher than 300 mg/dL, an LDL receptor deficiency was observed. Apolipoprotein E isoforms and lipoprotein[a] were found to be independent from the LDL receptor status. In some patients with high cholesterol levels but normal LDL receptor expression with the C7A MAB assay, LDL receptor defects could be diagnosed when either reduced binding or internalization of DiI LDL or familial defective apolipoprotein B-100 was detected.(ABSTRACT TRUNCATED AT 400 WORDS)

Identification of high density lipoprotein binding proteins in mature adipocyte plasma membranes.

High density lipoprotein (HDL) binding proteins were identified in nonreduced detergent extracts of plasma membranes or crude membrane fractions of rat adipocytes by ligand blotting. Using 125I-labelled human apolipoprotein-E-free HDL ([125I]HDL3), two binding proteins in adipocyte membranes were detected with apparent molecular masses of 122 and 88 kilodaltons (kDa), respectively. The binding of HDL3 to both binding proteins was abolished by pronase treatment and was inhibited by excess unlabelled HDL3. Excessive unlabelled low density lipoprotein reduced the binding of [125I]HDL3 to the 122-kDa binding protein relatively less than that to the 88-kDa binding protein. Polyclonal antisera against purified rat apolipoprotein A-I (apoA-I) effectively inhibited the binding of HDL3 to adipocyte membranes. Affinity-purified antibodies against rat apoA-I also revealed two HDL-binding proteins in rat adipocyte and liver plasma membranes preincubated with rat HDL. The sizes of the HDL-binding proteins in adipocyte plasma membranes detected by anti-apoA-I were similar to those detected by radiolabelled ligand blotting and their counterparts in rat liver plasma membranes. The study demonstrates two HDL-binding proteins, distinguishable by apparent molecular masses and ligand binding affinity, in plasma membrane proteins of mature rat adipocytes using radiolabelled ligand and immunoligand blotting techniques. The results suggest that apoA-I is involved in the interactions between HDL and both variants of HDL-binding proteins.

Tumor necrosis factor up-regulates expression of low-density lipoprotein receptors on HepG2 cells

Tumor necrosis factor mediates most biological activities of endotoxin and also, in part, mediates endotoxin-induced disturbances in lipid metabolism. In this study, the effect of tumor necrosis factor on low-density lipoprotein receptor activity was investigated in cells of HepG2, a well-differentiated human hepatoma cell line. Pretreatment of the cells with tumor necrosis factor leads to enhanced binding, uptake and degradation of 125I-labeled low-density lipoprotein. This effect of tumor necrosis factor was dose and time dependent. Tumor necrosis factor-stimulated enhancement of low-density lipoprotein binding occurred at all stages of cell growth. However, addition of an excess of unlabeled low-density lipoprotein, to down-regulate low-density lipoprotein receptors before exposure to tumor necrosis factor of the cells, completely abolished the effects of tumor necrosis factor. Competition experiments using unlabeled low-density lipoprotein and blockage experiments with a monoclonal low-density lipoprotein receptor antibody showed that tumor necrosis factor-stimulated low-density lipoprotein binding takes place through stimulation of low-density lipoprotein receptors. Comparison of the kinetics of specific low-density lipoprotein binding in the unstimulated cells and in the tumor necrosis factor-stimulated cells indicated that tumor necrosis factor caused a 30% increase in maximum velocity with no significant change in Michaelis constant, suggesting that tumor necrosis factor increases the number of low-density lipoprotein receptors on the cells rather than changing binding affinity. Preincubation of the cells with cycloheximide or actinomycin D totally abolished the up-regulatory effect of tumor necrosis factor on low-density lipoprotein receptors. Tumor necrosis factor did not stimulate proliferation of HepG2 cells, as judged by cell protein determination or by [3H]thymidine incorporation. In conclusion, this study suggests that tumor necrosis factor up-regulates expression of low-density lipoprotein receptors on HepG2 cells by stimulation of de novo synthesis of receptors, independent of cell growth.

Tumor necrosis factor up-regulates expression of low-density lipoprotein receptors on HepG2 cells

Tumor necrosis factor mediates most biological activities of endotoxin and also, in part, mediates endotoxin-induced disturbances in lipid metabolism. In this study, the effect of tumor necrosis factor on low density lipoprotein receptor activity was investigated in cells of HepG2, a well-differentiated human hepatoma cell line. Pretreatment of the cells with tumor necrosis factor leads to enhanced binding, uptake and degradation of 125I-labeled low-density lipoprotein. This effect of tumor necrosis factor was dose and time dependent. Tumor necrosis factor-stimulated enhancement of low-density lipoprotein binding occurred at all stages of cell growth. However, addition of an excess of unlabeled low-density lipoprotein, to down-regulate low-density lipoprotein receptors before exposure to tumor necrosis factor of the cells, completely abolished the effects of tumor necrosis factor. Competition experiments using unlabeled low-density lipoprotein and blockage experiments with a monoclonal low-density lipoprotein recentor antibody showed that tumor necrosis factor-stimulated low-density lipoprotein binding takes place through stimulation of low-density lipoprotein receptors. Comparison of the kinetics of specific lowdensity lipoprotein binding in the unstimulated cells and in the tumor necrosis factor-stimulated cells indicated that tumor necrosis factor caused a 30% increase in maximum velocity with no significant change in Michaelis constant, suggesting that tumor necrosis factor increases the number of low-density lipoprotein receptors on the cells rather than changing binding affinity. Preincubation of the cells with cycloheximide or actinomycin D totally abolished the up-regulatory effect of tumor necrosis factor on low-density lipoprotein receptors. Tumor necrosis factor did not stimulate proliferation of HepG2 cells, as judged by cell protein determination or by [ 3H]thymidine incorporation. In conclusion, this study suggests that tumor necrosis factor up-regulates expression of low-density lipoprotein receptors on HepG2 cells by stimulation of de novo synthesis of receptors, independent of cell growth.

Polymyxin B complexes with and cationizes low density lipoproteins: The cause of polymyxin B-induced enhancement of endocytotic catabolism of low density lipoproteins

We previously reported that polymyxin B (PMB) enhances cellular catabolism of low density lipoproteins (LDLs) through a non-LDL receptor-mediated endocytotic pathway. These data were obtained mainly by using Hep G2 cells, a well differentiated human hepatoma cell line. In the current study, we explore the mechanisms of PMB-mediated endocytotic catabolism of LDL. We found that PMB enhanced LDL catabolism also in homozygous familial hypercholesterolemia fibroblasts, thereby establishing that PMB-mediated cellular catabolism of LDL does not involve LDL receptors. By using [ 14C]sucrose, and ligands for the asialoglycoprotein (ASGP) receptors, possibilities were excluded that PMB enhances cellular endocytosis of LDL, by inducing a general increase of cellular pinocytic activity or by causing endocytosis of LDL via the ASGP receptors in Hep G2 cells. We further show, by using polymyxin B coupled Sepharose 4B (PMB-Sepharose 4B) beads, that PMB binds to LDL to form a complex. This binding was tight, and changes in pH and salt concentrations had no significant effect on the binding, but unlabelled LDL competed with 125I-LDL to bind to PMB-Sepharose 4B. Urea and endotoxins decreased this binding, suggesting that PMB binds to LDL at least partially through hydrophobic interactions. Agarose gel electrophoresis of PMB-LDL indicates that PMB cationizes LDL. In conclusion, PMB binds to LDL to form a PMB-LDL complex presumably through interactions between lipid groups. This endows LDL with positive charges, which enhances LDL binding to negatively charged cell membranes, and such bound LDL is rapidly internalized through absorptive endocytosis.

Cell-derived unesterified cholesterol cycles between different HDLs and LDL for its effective esterification in plasma.

Pulse-chase incubations of human plasma with [3H]cholesterol-laden skin fibroblasts or low density lipoproteins (LDL) and nondenaturing two-dimensional electrophoresis were used to study the transfer and esterification of cell-derived unesterified cholesterol (UC) in human plasma lipoproteins. Specific radioactivities ([3H]UC per microgram of UC) were calculated, and net cholesterol mass transfer was quantified using a fluoro-enzymatic assay to validate productive transfers of UC between high density lipoprotein (HDL) and LDL. Cellular UC was initially taken up by pre-beta 1-HDL and subsequently transferred in the sequence pre-beta 2-HDL-->pre-beta 3-HDL-->alpha-HDL-->LDL. During the first 5 minutes of this process, only 5% of cellular cholesterol was esterified in pre-beta 3-HDL and alpha-HDL; the remainder reached LDL as UC. Cellular UC accumulating in LDL was then redistributed to various HDL particles via two pathways: 1) the partially LDL receptor-mediated uptake and re-secretion of UC by cells and 2) the direct transfer of UC to HDL, mostly to alpha-HDL and a small amount to pre-beta-HDL. UC was not transferred from LDL to HDL after inhibition of lecithin:cholesterol acyltransferase (LCAT). The esterification of cellular [3H]cholesterol in plasma was competitively inhibited by the addition of excess unlabeled LDL but not of excess HDL. However, both excess LDL and excess HDL prevented the esterification of cell-derived cholesterol in apolipoprotein B-free plasma. This demonstrated that LDL is the major source of UC to the LCAT reaction and that the transfer of UC from LDL to HDL is LCAT dependent. In conclusion, the effective esterification of cell-derived cholesterol in plasma involves a rapid transfer of UC via HDL particles to LDL, from which it is distributed to pre-beta-HDL and alpha-HDL. Furthermore, we hypothesize that the transfer per se of cellular UC to LDL forms a cholesterol concentration gradient between cell membranes and HDL and thus a second, reverse cholesterol transport mechanism in addition to the esterification of cholesterol by LCAT.

The effect of serum on low density lipoprotein metabolism of human monocyte-derived macrophages.

Macrophages oxidatively modify low density lipoprotein (LDL). These cells then recognize and take up oxidized LDL (OxLDL) via the scavenger receptor. In the present study, we examined the effect of lipoprotein depleted serum (LPDS) on the oxidative modification of LDL and its uptake and degradation by human monocyte-derived macrophages. LPDS inhibited LDL oxidation in a concentration-dependent manner and the complete inhibition was observed in the concentration from 2.5 to 10.0%. In the presence of 5% of LPDS, cell-associated 125I-LDL was reduced by 70%, compared with that in the absence of LPDS. In contrast, 125I-LDL degradation increased in the presence of LPDS. The ratio of degradation products to cell-associated radioactivity in the presence of LPDS was five-fold greater than that in the absence of LPDS. In the presence of LPDS, unlabeled LDL competed for about 50% of cellular uptake and degradation of 125I-LDL. In the absence of LPDS, however, unlabeled LDL competed for only 20% of uptake and only 30% of degradation of 125I-LDL. Unlabeled OxLDL competed for about 80% of uptake and degradation in the absence of LPDS. These results suggest that LPDS inhibited the production of OxLDL which was taken up by macrophages via the pathway for the OxLDL.