The influence of the triglyceride content of low density lipoprotein on the interaction of apolipoprotein B-100 with cells

To study the effect of triglyceride content of low density lipoprotein (LDL) on its physicochemical and biological properties, we have depleted the triglyceride by incubation with hepatic lipase (HL-LDL) and raised the triglyceride by incubation of HL-LDL with very low density lipoprotein and lipoprotein-deficient serum. HL-LDL was taken up by human monocyte-derived macrophages and by human skin fibroblasts at an increased rate compared to untreated LDL. Incubation of the various LDL preparations revealed that cellular LDL degradation as well as LDL-mediated cholesterol esterification were inversely related to the triglyceride content of the LDL preparation. Modification of the triglyceride content of LDL also was associated with changes in the free fatty acid content, but the interaction of the LDL with cells was unaffected by the level of this component. The triglyceride content of LDL was found to be reciprocally related to the number of free lysine amino groups of LDL apolipoprotein B (apoB) which could be labeled with trinitrobenzenesulfonic acid. 13C-Nuclear magnetic resonance (NMR) spectra of native LDL and HL-LDL samples containing [13CH3]2 lysine residues formed by reductive methylation (11-13% modification) showed that the arrangement of apoB lysines is perturbed by the exposure to hepatic lipase. The ratio of labeled lysines with pK 8.9 to those with pK 10.5 exposed on the surface of LDL particles was decreased by about 40% by lipase treatment. These effects are apparently due to changes in local apoB conformation because circular dichroism spectra revealed that the average secondary structure of the entire apoB molecule is the same in native LDL and HL-LDL. The triglyceride content of LDL reciprocally affected its binding to a monoclonal antibody which recognizes epitopes around the LDL receptor binding domain of apoB. The above evidence indicates that modulation of the core triglyceride and possibly also surface phospholipid content of LDL can alter the conformation of apoB on the surface of the particle, thereby influencing the interaction with cell surface LDL receptors.     

Reduced uptake of cholesterol esterase-modified low density lipoprotein by macrophages.

Changes in low density lipoprotein (LDL) lipid composition were shown to alter its interaction with the LDL receptor, thus affecting its cellular uptake. Upon incubation of LDL with 5 units/ml cholesterol esterase (CEase) for 1 h at 37 degrees C, there was a 33% reduction in lipoprotein cholesteryl ester content, paralleled by an increment in its unesterified cholesterol. CEase-LDL, in comparison to native LDL, was smaller in size, possessed fewer free lysine amino groups (by 14%), and demonstrated reduced binding to heparin (by 83%) and reduced immunoreactivity against monoclonal antibodies directed toward epitopes along the LDL apoB-100. Incubation of CEase-LDL with the J-774 macrophage-like cell line resulted in about a 30% reduction in lipoprotein binding and degradation in comparison to native LDL, and this was associated with a 20% reduction in macrophage cholesterol mass. Similarly, CEase-LDL degradation by mouse peritoneal macrophages, human monocyte-derived macrophages, and human skin fibroblasts was reduced by 20-44% in comparison to native LDL. CEase-LDL uptake by macrophages was mediated via the LDL receptor and not the scavenger receptor. CEase activity toward LDL was demonstrated in plasma and in cells of the arterial wall such as macrophages and endothelial cells. Thus, CEase modification of LDL may take place in vivo, and this phenomenon may have a role in atherosclerosis.     

Low density lipoprotein modification by cholesterol oxidase induces enhanced uptake and cholesterol accumulation in cells.

Oxidation of low density lipoprotein (LDL) by cells of the arterial wall or in the presence of copper ions was shown to result in the peroxidation of its fatty acids as well as its cholesterol moiety. LDL incubation with cholesterol oxidase (CO) resulted in the conversion of up to 85% of the lipoprotein unesterified cholesterol (cholest-5-en-3-ol) to cholestenone (cholest-4-en-3-one) in a dose- and time-dependent pattern. Plasma very low density lipoprotein (VLDL) and high density lipoprotein (HDL) could be similarly modified by CO. In cholesterol oxidase-modified LDL (CO-LDL), unlike copper ion-induced oxidized LDL (Cu-Ox-LDL), there was no fatty acids peroxidation, and lipoprotein size or charge as well as LDL cholesteryl ester, phospholipids, and triglycerides content were not affected. CO-LDL, however, demonstrated enhanced susceptibility to oxidation by copper ions in comparison to native LDL. Upon incubation of CO-LDL with J-774 A.1 macrophage-like cell line, cellular uptake and degradation of the lipoprotein was increased by up to 62% in comparison to native LDL but was 15% lower than that of Cu-Ox-LDL. Similarly, the binding of CO-LDL to macrophages increased by up to 80%, and cellular cholesterol mass was increased 51% more than the mass obtained with native LDL. Several lines of evidence indicate that CO-LDL was taken up via the LDL receptor: 1) Excess amounts of unlabeled LDL, but not acetyl-LDL (Ac-LDL), effectively competed with 125I-CO-LDL for the uptake by cells. 2) The degradation of CO-LDL by various types of macrophages and by fibroblasts could be dissociated from that of Ac-LDL and was always higher than that of native LDL. 3) A monoclonal antibody to the LDL receptor (IgG-C7) and a monoclonal antibody to the LDL receptor binding domains on apoB-100 (B1B6) inhibited macrophage degradation of CO-LDL. The receptor for Cu-Ox-LDL, which is not shared with Ac-LDL, was also partially involved in macrophage uptake of CO-LDL, since Cu-Ox-LDL demonstrated some competition capability with CO-125I-LDL for its cellular degradation. CO-LDL cellular degradation was inhibited by chloroquine, thus implying lysosomal involvement in the cellular processing of the lipoprotein. Incubation of macrophages with LDL in the presence of increasing concentrations of cholestenone resulted in up to 52% enhanced lipoprotein cellular degradation suggesting that the cholestenone in CO-LDL might be involved in the enhanced cellular uptake of the modified lipoprotein.(ABSTRACT TRUNCATED AT 400 WORDS)     

Selective release of extrahepatic lipoprotein lipase by polymetaphosphate

We have studied the lipase released into the circulation by polymetaphosphate injection into rats. Lipase release was in proportion to the dose injected. The post-polymetaphosphate lipase was almost completely inhibited by high salt concentrations or by addition of protamine sulfate to the assay system suggesting that this compound released lipoprotein lipase and not hepatic triglyceride lipase. The lipases released by polymetaphosphate and by heparin were compared using a heparin-sepharose affinity column technique which separates lipoprotein lipase from hepatic triglyceride lipase. While heparin released both lipoprotein lipase and hepatic triglyceride lipase, polymetaphosphate released almost exclusively lipoprotein lipase. Other experiments showed that neither polymetaphosphate nor heparin inhibited the hepatic lipase when added to the assay. These results suggest that lipoprotein lipase may be released by the negative charge on these high-charge polymers while hepatic triglyceride lipase release may require the specific sugar configuration of heparin.     

Preheparin lipoprotein lipolytic activities: relationship to plasma lipoproteins and postheparin lipolytic activities.

To determine the putative metabolic relevance of preheparin versus postheparin lipoprotein lipases, the relationships of both pre- and postheparin lipoprotein lipase (LPL) and hepatic triglyceride lipase (HTGL) to plasma triglycerides, low density lipoprotein (LDL) cholesterol, and high density lipoprotein (HDL) cholesterol were determined in 93 men. Relationships of preheparin lipases to their respective postheparin lipases were also examined. Although relationships between the preheparin lipases and plasma triglycerides and HDL cholesterol were not apparent, both preheparin LPL (rs = 0.306, P = 0.0036) and HTGL (rs = 0.348, P = 0.0008) correlated with LDL cholesterol, a relationship not seen with either postheparin lipase. Both postheparin LPL (rs = 0.515, P = 0.0001) and postheparin HTGL (rs = -0.228, P = 0.0028), however, correlated with HDL cholesterol. In addition, postheparin LPL was inversely correlated with postheparin HTGL (rs = -0.363, P = 0.0003), whereas the relationship between preheparin LPL and preheparin HTGL was positive (rs = 0.228, P = 0.0009). Overall, these data point to differences between pre- and postheparin lipases in their relationships to lipoproteins, and one to another. The relationships of LDL cholesterol to both preheparin LPL and HTGL suggest that displacement of active forms of both lipases from their endothelial binding sites may mark triglyceride-rich lipoproteins or their remnants for metabolic pathways that lead to LDL.     

Experimental hyperthyroidism in man: effects on plasma lipoproteins, lipoprotein lipase and hepatic lipase

We have studied the effects of triiodothyronine administration (20-40 micrograms three times daily over one week) in six healthy young men, on the activities of lipoprotein lipase and hepatic lipase and on plasma lipoprotein concentrations. Hepatic lipase activity in post-heparin plasma rose by 46 +/- 25% (p less than 0.025), whereas the activity of lipoprotein lipase did not change significantly. Plasma cholesterol concentrations decreased by about 20% (p less than 0.025), whereas there was no change in plasma triglyceride levels. The fall in plasma cholesterol could be accounted for by a reduction of HDL cholesterol (-11%, p less than 0.025) as well as LDL cholesterol (-27%, p less than 0.025). The data emphasize the role of hepatic lipase in the lipoprotein alterations associated with thyroid dysfunction.     

Modification of LDL by platelet secretory products induces enhanced uptake and cholesterol accumulation in macrophages

LDL modified by incubation with platelet secretory products caused cholesterol accumulation and stimulation of cholesterol esterification in mouse peritoneal macrophages. Its uptake by the macrophages was a receptor-mediated process, not susceptible to competition by acetyl-LDL or polyanions suggesting independence of the scavenger receptor. Stimulation of the esterification process in macrophages by this modified LDL was inhibited by the lysosomal inhibitor chloroquine, indicating requirement for cellular uptake and lysosomal hydrolysis of the lipoprotein. Within the cell, the modified LDL inhibited cellular biosynthesis of triglycerides in a manner similar to the action of acetyl-LDL but different to the effect of native LDL. In the presence of HDL, acting in the medium as an acceptor for cholesterol, a low rate of cholesterol efflux from cells incubated with this modified LDL as well as with acetyl-LDL was demonstrated. A small reduction in cholesteryl ester synthesis was found in these cells, compared to a 60% reduction in cells incubated with native LDL. Thus it was demonstrated that LDL modified by platelet secretory products could induce macrophage cholesterol accumulation even though it was recognized and taken up via the regulatory LDL receptor.     

A Chinese hamster ovarian cell line imports cholesterol by high density lipoprotein degradation

Plasma high density lipoprotein (HDL) is inversely associated with the development of atherosclerosis. HDL exerts its atheroprotective role through involvement in reverse cholesterol transport in which HDL is loaded with cholesterol at the periphery and transports its lipid load back to the liver for disposal. In this pathway, HDL is not completely dismantled but only transfers its lipids to the cell. Here we present evidence that a Chinese hamster ovarian cell line (CHO7) adapted to grow in lipoprotein-deficient media degrades HDL and concomitantly internalizes HDL-derived cholesterol. Delivery of HDL cholesterol to the cell was demonstrated by a down-regulation of cholesterol biosynthesis, an increase in total cellular cholesterol content and by stimulation of cholesterol esterification after HDL treatment. This HDL degradation pathway is distinct from the low density lipoprotein (LDL) receptor pathway but also degrades LDL. 25-Hydroxycholesterol, a potent inhibitor of the LDL receptor pathway, down-regulated LDL degradation in CHO7 cells only in part and did not down-regulate HDL degradation. Dextran sulfate released HDL bound to the cell surface of CHO7 cells, and heparin treatment released protein(s) contributing to HDL degradation. The involvement of heparan sulfate proteoglycans and lipases in this HDL degradation was further tested by two inhibitors genistein and tetrahydrolipstatin. Both blocked HDL degradation significantly. Thus, we demonstrate that CHO7 cells degrade HDL and LDL to supply themselves with cholesterol via a novel degradation pathway. Interestingly, HDL degradation with similar properties was also observed in a human placental cell line.     

Suppression of endothelial or lipoprotein lipase in THP-1 macrophages attenuates proinflammatory cytokine secretion

LPL and endothelial lipase (EL) are associated with macrophages in human atherosclerotic lesions, and overexpression of LPL in mouse macrophages is associated with a greater extent of atherosclerosis. To investigate potential mechanisms by which macrophage-derived lipase expression may mediate proatherogenic effects, we used lentivirus-mediated RNA interference to suppress the expression of either LPL or EL within THP-1 macrophages. After suppression of either LPL or EL, significant decreases in the concentration of interleukin-1beta, interleukin-6, monocyte chemoattractant protein-1, and tumor necrosis factor-alpha were observed. Incubation of THP-1 macrophages with either mildly or extensively oxidized LDL consistently decreased cytokine expression, which was additive to that contributed by lipase suppression. Decreased lipase expression was also associated with an altered lipid composition, with reduced percentages of cholesterol (unesterified and esterified), triglycerides, and lysophosphatidylcholine. Microarray data indicated a decreased expression of proinflammatory genes, growth factors, and antiapoptotic genes. By contrast, there was an increased expression of lipoprotein receptors (scavenger receptor 1, low density lipoprotein receptor, scavenger receptor class B type I, and CD36). Thus, we conclude that the suppression of either LPL or EL decreases proinflammatory cytokine expression and influences the lipid composition of THP-1 macrophages. These results provide further insight into the specific metabolic and potential pathological roles of LPL and EL in human macrophages.     

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.     

Hepatic lipase stimulates the uptake of high density lipoprotein cholesterol by hepatoma cells

The objective of this study was to determine whether high density lipoproteins (HDL) that have been treated with hepatic lipase have an enhanced ability to deliver cholesterol to cells. Human HDL was incubated with rat hepatic lipase, reisolated, and subjected to compositional analysis. Approximately 28% of the HDL phosphatidylcholine was hydrolyzed by the hepatic lipase but no change was detected in the cholesterol or apoprotein content of the HDL compared to HDL incubated with heat-inactivated hepatic lipase. Cultured rat hepatoma cells exposed to hepatic lipase-modified HDL showed an increased uptake of HDL free cholesterol relative to cells exposed to control HDL. This increased delivery of HDL free cholesterol was demonstrated by both isotopic and mass determinations and it contributed to a 1.6-fold increase in total cellular cholesterol content relative to cells treated with control HDL. The free cholesterol delivered by the HDL is functionally available to the cell as evidenced by the conversion of radiolabeled free cholesterol to cholesteryl ester. The stimulation of free cholesterol delivery was dose-dependent up to a level of 100 micrograms of HDL free cholesterol/ml of extracellular medium, and was directly related to the extent of phosphatidylcholine hydrolysis. The enhanced cellular accumulation of HDL free cholesterol observed with hepatic lipase appears to be due to the phospholipase activity of this enzyme, since similar results were obtained with HDL that had been modified by snake venom phospholipase A2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differences in the metabolism of oxidatively modified low density lipoprotein and acetylated low density lipoprotein by human endothelial cells: inhibition of cholesterol esterification by oxidatively modified low density lipoprotein

The rate of degradation of oxidatively modified low density lipoprotein (Ox-LDL) by human endothelial cells was similar to that of unmodified low density lipoprotein (LDL), and was approximately 2-fold greater than the rate of degradation of acetylated LDL (Ac-LDL). While LDL and Ac-LDL both stimulated cholesterol esterification in endothelial cells, Ox-LDL inhibited cholesterol esterification by 34%, demonstrating a dissociation between the degradation of Ox-LDL and its ability to stimulate cholesterol esterification. Further, while LDL and Ac-LDL resulted in a 5- and 15-fold increase in cholesteryl ester accumulation, respectively, Ox-LDL caused only a 1.3-fold increase in cholesteryl ester mass. These differences could be accounted for, in part, by the reduced cholesteryl ester content of Ox-LDL. However, when endothelial cells were incubated with Ac-LDL in the presence and absence of Ox-LDL, Ox-LDL led to a dose-dependent inhibition of cholesterol esterification without affecting the degradation of Ac-LDL. This inhibitory effect of Ox-LDL on cholesteryl ester synthesis was also manifest in normal human skin fibroblasts incubated with LDL and in LDL-receptor-negative fibroblasts incubated with unesterified cholesterol to stimulate cholesterol esterification. Further, the lipid extract from Ox-LDL inhibited cholesterol esterification in LDL-receptor negative fibroblasts. These findings suggest that the inhibition of cholesterol esterification by oxidized LDL is independent of the LDL and scavenger receptors and may be a result of translocation of a lipid component of oxidatively modified LDL across the cell membrane.     

High-cholesterol diet-induced lipoproteins stimulate lipoprotein lipase secretion in cultured rat alveolar macrophages

We have previously shown that cultured rat alveolar macrophages synthesize and secrete lipoprotein lipase into the medium. The purpose of the present experiments is to examine whether cholesterol-enriched lipoproteins from cholesterol-fed animals have any effects on the lipoprotein lipase secretion and the lipid accumulation in macrophages. Macrophages incubated with the VLDL obtained from rats fed a normal diet secreted 2-fold higher amounts of lipoprotein lipase than those without lipoproteins. Intermediate-, low- and very-low-density lipoproteins from rats fed a high-cholesterol diet also enhanced the lipoprotein lipase secretion. Normal high- and low-density lipoproteins, and high-density lipoproteins from hypercholesterolemic animals did not cause any increase in the lipoprotein lipase secretion. The lipoproteins which stimulated the lipoprotein lipase secretion caused intracellular accumulation of both triacylglycerol and cholesterol. It is speculated that macrophages residing in the environment rich in lipoproteins, especially hypercholesterolemic lipoproteins, take them up and accumulate lipids intracellularly, and that this process links with the lipoprotein lipase secretion. The secreted lipoprotein lipase could facilitate, by degrading lipoproteins, the uptake of lipoprotein lipase-modified lipoproteins. Probably such a series of events is of importance in the foam cell formation of macrophages.     

Sphingomyelinase enhances low density lipoprotein uptake and ability to induce cholesteryl ester accumulation in macrophages.

Cholesteryl ester-loaded macrophages, or foam cells, are a prominent feature of atherosclerotic lesions. Low density lipoprotein (LDL) receptor-mediated endocytosis of native LDL is a relatively poor inducer of macrophage cholesteryl ester accumulation. However, the data herein show that in the presence of a very small amount of sphingomyelinase, LDL receptor-mediated endocytosis of 125I-LDL was enhanced and led to a 2-6-fold increase in 125I-LDL degradation and up to a 10-fold increase in cholesteryl ester accumulation in macrophages. The enhanced lipoprotein uptake and cholesterol esterification was seen after only approximately 12% hydrolysis of LDL phospholipids, was specific for sphingomyelin hydrolysis, and appeared to be related to the formation of fused or aggregated spherical particles up to 100 nm in diameter. Sphingomyelinase-treated LDL was bound by the macrophage LDL receptor. However, when unlabeled acetyl-LDL, a scavenger receptor ligand, was present during or after sphingomyelinase treatment of 125I-LDL, 125I-LDL binding and degradation were enhanced further through the formation of LDL-acetyl-LDL mixed aggregates. Experiments with cytochalasin D suggested that endocytosis, not phagocytosis, was involved in internalization of sphingomyelinase-treated LDL. Nonetheless, the sphingomyelinase effect on LDL uptake was macrophage-specific. These data illustrate that LDL receptor-mediated endocytosis of fused LDL particles can lead to foam cell formation in cultured macrophages. Furthermore, since both LDL and sphingomyelinase are present in atherosclerotic lesions and since some lesion LDL probably is fused or aggregated, there is a possibility that sphingomyelinase-treated LDL is a physiologically important atherogenic lipoprotein.     

Comparative binding and degradation of lipoprotein(a) and low density lipoprotein by human monocyte-derived macrophages.

The binding and degradation of equimolar concentrations of lipoprotein(a) (Lp(a)) and low density lipoprotein (LDL) isolated from the same individual were studied in primary cultures of human monocyte-derived macrophages (HMDM). At 4 degrees C, LDL receptor-mediated binding of both Lp(a) and LDL was of low affinity, being 0.8 and 0.23 microM, respectively. Competitive binding studies indicated that the binding of Lp(a) to HMDM was competed 63% by excess LDL. In contrast to the 4 degrees C binding data, the degradation of Lp(a) at 37 degrees C was mainly nonspecific because the amount of Lp(a) processed by the LDL receptor pathway in 5 h was 17% that of LDL. According to pulse-chase experiments, this phenomenon may be accounted for by the facts that less Lp(a) is bound to HMDM at 37 degrees C and that Lp(a) has a lower intrinsic degradation rate and was not due to increased intracellular accumulation or retroendocytosis of the lipoprotein. Degradation of both lipoproteins was primarily lysosomal and only modestly affected by up- or down-regulation of the LDL receptor. The rate of retroendocytosis in HMDM was approximately equal to the degradation rate and appeared to be independent of the type of lipoprotein used, up- or down-regulation of the LDL receptor, or the presence of the lysosomotropic agent chloroquine. Overall, the results indicate that HMDM degrade Lp(a) mainly via a nonspecific pathway with only 25% of total Lp(a) degradation occurring through the LDL receptor pathway. As both 37 degrees C degradation and 4 degrees C binding of LDL are mainly LDL receptor specific, the different metabolic behavior observed at 37 degrees C suggests that Lp(a) undergoes temperature-induced conformational changes on cooling to 4 degrees C that allows better recognition of Lp(a) by the LDL receptor at a temperature lower than the physiological temperature of 37 degrees C. How apo(a) affects these structural changes remains to be established.     

Role of pre-existing redox profile of human macrophages on lipid synthesis and cholesteryl ester cycle in presence of native, acetylated and oxidised low density lipoprotein

The importance of the interactions of modified lipids and macrophages in foam cell generation is clear; however, little attention has been paid to the role of intra-macrophagic redox potential as a modulator of their lipid synthesis and metabolism. In this study, the effects of previously induced non-toxic manipulations of intracellular redox balance on lipid synthesis in human monocyte-derived macrophages (HMDM) was evaluated. Cells, pre-treated with 2.5 microM of the pro-oxidising agent CuSO(4) or with 5 mM of the antioxidant and thiol supplier N-acetylcysteine (NAC), were exposed to radiolabelled oleic acid alone or in combination with native low density lipoprotein (LDL) or modified LDL to evaluate the incorporation of radioactivity into cholesteryl ester, triacylglycerols and phospholipids. CuSO(4)-treated macrophages synthesised more lipids than NAC-treated cells in absence of exogenous lipid, and, generally, in the presence of native or acetylated, but oxidised LDL. In addition, the activities of the enzymes involved in cholesteryl ester storage were also influenced by the pro-oxidant condition. The ratio values between acyl-coenzyme A:cholesterol acyl transferase and cholesteryl ester hydrolase activity suggest that in CuSO(4)-treated macrophages the hydrolysis of cholesteryl ester is favoured with respect to esterification. The interaction of HMDM with oxidised LDL showed a significant different pattern in term of lipid synthesis with respect to those induced by native or acetylated LDL, disrespectful of the initial redox profile of the cells. On the whole, these results suggest that the pre-existing internal redox condition is a further parameter able to modulate the effects of native or acetylated LDL-cell interaction, influencing both HMDM lipid synthesis profile and cholesterol storage. Moreover, oxidised LDL represent a carrier of additional factor(s) able per se to introduce perturbation in the synthetic pathway of lipids, which is not influenced by the redox potential of the macrophage.     

Uptake of type IV hypertriglyceridemic VLDL by cultured macrophages is enhanced by interferon-gamma.

Hypertriglyceridemic (HTG) very low density lipoproteins (VLDL) from subjects with type IV hyperlipoproteinemia induce both cholesteryl ester (CE) and triglyceride (TG) accumulation in cultured J774 macrophages. We examined whether the cytokine interferon-gamma (IFN-gamma), which is expressed by lymphocytes in atherosclerotic lesions, would modulate macrophage uptake of HTG -VLDL. Incubation of cells with HTG -VLDL alone significantly increased cellular CE and TG mass 17- and 4.3-fold, respectively, while cellular free cholesterol (FC) was unaffected. Pre-incubation of cells with IFN-gamma (50 U/ml) prior to incubation with HTG -VLDL caused a marked enhancement in cellular CE and TG 27- and 6-fold over no additions (controls), respectively, and a 1.5-fold increase in FC. IFN-gamma increased low density lipoprotein (LDL)-induced cellular CE 2-fold compared to LDL alone. IFN-gamma did not enhance the uptake of type III (apoE2/E2) HTG -VLDL or VLDL from apoE knock-out mice. Incubations in the presence of a lipoprotein lipase (LPL) inhibitor or an acylCoA:cholesterol acyltransferase (ACAT) inhibitor demonstrated that the IFN-gamma-enhanced HTG -VLDL uptake was dependent on LPL and ACAT activities. IFN-gamma significantly increased the binding and degradation of 125I-labeled LDL. Binding studies with 125I-labeled alpha2-macroglobulin, a known LDL receptor-related protein (LRP) ligand, and experiments with copper-oxidized LDL indicated that the IFN-gamma-enhanced uptake was not due to increased expression of the LRP or scavenger receptors. Thus, IFN-gamma may promote foam cell formation by accelerating macrophage uptake of native lipoproteins. IFN-gamma-stimulated CE accumulation in the presence of HTG -VLDL occurs via a process that requires receptor binding-competent apoE and active LPL. IFN-gamma-enhanced uptake of both HTG -VLDL and LDL is mediated by the LDL-receptor and requires ACAT-mediated cholesterol esterification.     

Mechanism of the hepatic lipase induced accumulation of high-density lipoprotein cholesterol by cells in culture

Hepatic lipase can enhance the delivery of high-density lipoprotein (HDL) cholesterol to cells by a process which does not involve apoprotein catabolism. The incorporation of HDL-free (unesterified) cholesterol, phospholipid, and cholesteryl ester by cells has been compared to establish the mechanism of this delivery process. Human HDL was reconstituted with 3H-free cholesterol and [14C]sphingomyelin, treated with hepatic lipase in the presence of albumin to remove the products of lipolysis, reisolated, and then incubated with cultured rat hepatoma cells. Relative to control HDL, modification of HDL with hepatic lipase stimulated both the amount of HDL-free cholesterol taken up by the cell and the esterification of HDL-free cholesterol but did not affect the delivery of sphingomyelin. Experiments utilizing HDL reconstituted with 14C-free cholesterol and [3H]cholesteryl oleoyl ether suggest that hepatic lipase enhances the incorporation of HDL-esterified cholesterol. However, the amount of free cholesterol delivered as a result of treatment with hepatic lipase was 4-fold that of esterified cholesterol. On the basis of HDL composition, the cellular incorporation of free cholesterol was about 10 times that which would occur by the uptake and degradation of intact particles. The preferential incorporation of HDL-free cholesterol did not require the presence of lysophosphatidylcholine. To correlate the events observed at the cellular level with alterations in lipoprotein structure, high-resolution, proton-decoupled 13C nuclear magnetic resonance spectroscopy (90.55 MHz) was performed on HDL3 in which the cholesterol molecules were replaced with [4-13C]cholesterol by particle reconstitution.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and characterization of human lipoprotein lipase and hepatic triglyceride lipase. Reactivity with monoclonal antibodies to hepatic triglyceride lipase

Human lipoprotein lipase and hepatic triglyceride lipase were purified to homogeneity from post-heparin plasma. These enzymes were purified 250,000- and 100,000-fold with yields of 27 +/- 15 and 19 +/- 6%, respectively. Molecular weight determination by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and reducing agents yielded Mr of 60,500 +/- 1,800 and 65,200 +/- 400, respectively, for lipoprotein lipase and hepatic triglyceride lipase. These lipase preparations were shown to be free of detectable antithrombin by measuring its activity and by probing of Western blots of lipases with a monospecific antibody against antithrombin. In additions, probing of Western blots with concanavalin A revealed no glycoproteins corresponding to the molecular weight of antithrombin. Four stable hybridoma-producing distinct monoclonal antibodies (mAb) to hepatic triglyceride lipase were isolated. The specificity of one mAb, HL3-5, was established by its ability to immunoprecipitate hepatic triglyceride lipase catalytic activity. Interaction of HL3-5 with this lipase did not inhibit catalytic activity. The three other mAb interacted with hepatic triglyceride lipase only after denaturation of the enzyme with detergents. The relatedness of these two enzymes was examined by comparing under the same conditions the thermal inactivation, the sensitivity to sulfhydryl and reducing agents, amino acid composition, and the mobility of peptide fragments generated by cyanogen bromide cleavage. The results of these studies strongly support the view that the two enzymes are different proteins. Immunological studies confirm this conclusion. Four mAb to hepatic triglyceride lipase did not interact with lipoprotein lipase in Western blots, enzyme-linked immunosorbent assay, and immunoprecipitation experiments. These immunological studies demonstrate that several epitopes of the hepatic triglyceride lipase protein moiety are not present in the lipoprotein lipase molecule.     

Effects of diet and age on lipoprotein lipase and hepatic triglyceride lipase activities in the rat

Plasma clearance of triglyceride-rich lipoproteins appears decreased in aged humans and rats and may be due to lowered activities of the lipases responsible for lipid degradation. This study was designed to examine differential effects of age and diet on lipoprotein lipase (LPL) activity of adipose and heart tissue and hepatic triglyceride lipase (HTGL) activity. LPL and HTGL activities were examined in 3- and 13-month-old Sprague-Dawley rats after they had consumed either a high-carbohydrate or a high-fat diet for 14 days. The data were analyzed for age and diet differences by two-way analysis of variance. Although animals in the two age groups consumed diets of equal caloric content, the older rats gained less weight. Rats on the high-carbohydrate diet consumed less calories and gained less weight than the fat fed rats in both age groups. Neither heart nor adipose tissue LPL activity differed when examined for age or diet. HTGL activity levels, while not affected by age, were higher in the carbohydrate fed rats (P = 0.014). Regardless of age group, fasting plasma cholesterol levels were significantly higher in the carbohydrate-fed rats than fat-fed rats (P = 0.002). Thus, the diet effect was much stronger than the age effect for HTGL and plasma cholesterol levels.