Cholesteryl Ester Cycle Research Articles

Perilipin 2 (PLIN2)-Deficiency Does Not Increase Cholesterol-Induced Toxicity in Macrophages

Interventions on macrophages/foam cells to redirect intracellular cholesterol towards efflux pathways could become a very valuable addition to our therapeutic arsenal against atherosclerosis. However, certain manipulations of the cholesteryl ester cycle, such as the inhibition of ACAT1, an ER-resident enzyme that re-esterifies cholesterol, are not well tolerated. Previously we showed that targeting perilipin-2 (PLIN2), a major lipid droplet (LD)-associated protein in macrophages, prevents foam cell formation and protects against atherosclerosis. Here we have assessed the tolerance of PLIN2-deficient bone marrow derived macrophages (BMM) to several lipid loading conditions similar to the found during atherosclerosis development, including exposure to modified low-density lipoprotein (mLDL) and 7-ketocholesterol (7-KC), a free cholesterol (FC) metabolite, in media with or without cholesterol acceptors. BMM isolated from mice that do or do not express PLIN2 were tested for apoptosis (TUNEL and cleaved caspase-3), ER stress (CHOP induction and XBP-1 splicing), and inflammation (TNF-α and IL-6 mRNA levels). Like in other cell types, PLIN2 deficiency impairs LD buildup in BMM. However, while most stress parameters were elevated in macrophages under ACAT inhibition and 7-KC loading, PLIN2 inactivation was well tolerated. The data support the safety of targeting PLIN2 to prevent foam cell formation and atherosclerosis.

Cholesteryl ester hydrolase activity is abolished in HSL macrophages but unchanged in macrophages lacking KIAA1363

Cholesteryl ester (CE) accumulation in macrophages represents a crucial event during foam cell formation, a hallmark of atherogenesis. Here we investigated the role of two previously described CE hydrolases, hormone-sensitive lipase (HSL) and KIAA1363, in macrophage CE hydrolysis. HSL and KIAA1363 exhibited marked differences in their abilities to hydrolyze CE, triacylglycerol (TG), diacylglycerol (DG), and 2-acetyl monoalkylglycerol ether (AcMAGE), a precursor for biosynthesis of platelet-activating factor (PAF). HSL efficiently cleaved all four substrates, whereas KIAA1363 hydrolyzed only AcMAGE. This contradicts previous studies suggesting that KIAA1363 is a neutral CE hydrolase. Macrophages of KIAA1363−/− and wild-type mice exhibited identical neutral CE hydrolase activity, which was almost abolished in tissues and macrophages of HSL−/− mice. Conversely, AcMAGE hydrolase activity was diminished in macrophages and some tissues of KIAA1363−/− but unchanged in HSL−/− mice. CE turnover was unaffected in macrophages lacking KIAA1363 and HSL, whereas cAMP-dependent cholesterol efflux was influenced by HSL but not by KIAA1363. Despite decreased CE hydrolase activities, HSL−/− macrophages exhibited CE accumulation similar to wild-type (WT) macrophages. We conclude that additional enzymes must exist that cooperate with HSL to regulate CE levels in macrophages. KIAA1363 affects AcMAGE hydrolase activity but is of minor importance as a direct CE hydrolase in macrophages.

Reduced atherosclerosis in hormone-sensitive lipase transgenic mice overexpressing cholesterol acceptors

Macrophage-specific overexpression of cholesteryl ester hydrolysis in hormone-sensitive lipase transgenic (HSL Tg) female mice paradoxically increases cholesterol esterification and cholesteryl ester accumulation in macrophages, and thus susceptibility to diet-induced atherosclerosis compared to nontransgenic C57BL/6 mice. The current studies suggest that whereas increased cholesterol uptake could contribute to transgenic foam cell formation, there are no differences in cholesterol synthesis and the expression of cholesterol efflux mediators (ABCA1, ABCG1, apoE, PPARγ, and LXRα) compared to wild-type macrophages. HSL Tg macrophages exhibit twofold greater efflux of cholesterol to apoA–I in vitro, suggesting the potential rate-limiting role of cholesteryl ester hydrolysis in efflux. However, macrophage cholesteryl ester levels appear to depend on the relative efficacy of alternate pathways for free cholesterol in either efflux or re-esterification. Thus, increased atherosclerosis in HSL Tg mice appears to be due to the coupling of the efficient re-esterification of excess free cholesterol to its limited removal mediated by the cholesterol acceptors in these mice. The overexpression of cholesterol acceptors in HSL-apoA-IV double-transgenic mice increases plasma HDL levels and decreases diet-induced atherosclerosis compared to HSL Tg mice, with aortic lesions reduced to sizes in nontransgenic littermates. The results in vivo are consistent with the effective efflux from HSL Tg macrophages supplemented with HDL and apoA-I in vitro, and highlight the importance of cholesterol acceptors in inhibiting atherosclerosis caused by imbalances in the cholesteryl ester cycle.

Influence of thiol balance on micellar cholesterol handling by polarized Caco-2 intestinal cells

The in vitro thiol redox modulation of cholesterol homeostasis was investigated in polarized Caco-2 intestinal cells. Cells were pre-incubated with the pro-oxidant compound CuSO 4 or with the antioxidant N-acetylcysteine (NAC), to induce a mild shift of the intracellular redox potential toward, respectively, a more oxidizing or a more reducing equilibrium, via a manipulation of intracellular soluble thiols (glutathione). Then, monolayers were exposed to micellar cholesterol and both the cholesterol uptake and export, as well as the cholesteryl ester cycle, were analyzed. We found that pro-oxidizing conditions induced a significant cholesterol retention within the cells, particularly in the unesterified form (FC), as a result of an augmented sterol incorporation coupled with a reduced rate of FC esterification. A reduction in FC export was also evident. Furthermore, the combination of FC retention and the oxidative imbalance leads to significant alterations of the monolayer integrity, evidenced by both the enhanced tight junction permeability and the lactate dehydrogenase release into the basolateral medium. In contrast, a more reducing environment generated by NAC pre-treatment favors the limitation of the resident time of FC into the cells, via a reduced cholesterol uptake and a concomitant increased cholesterol esterification. In addition, a significant higher FC extrusion into the basolateral medium was also appreciable. Our results indicate that the thiol balance of intestinal cells may be critical for the regulation of cholesterol homeostasis at the intestinal level, influencing the lipid transport throughout the intestinal barrier.

17beta-estradiol enhances the flux of cholesterol through the cholesteryl ester cycle in human macrophages.

Estrogens have been shown to have many positive effects on the function of arterial wall, and recent evidence suggest that 17beta-estradiol has a direct action in reducing the accumulation of cholesteryl ester in macrophages. The mechanisms underlying the effects of 17beta-estradiol on foam cell formation, however are poorly understood. The aim of this study is to investigate the role of 17beta-estradiol in the regulation of the cholesteryl ester cycle and cholesterol efflux in human macrophages. In addition, the influence of 17beta-estradiol on apolipoprotein E (apoE) and lipoprotein lipase (LDL) secretion by the cells was also tested. Human Monocyte Derived Macrophages (HMDM), matured in the presence or the absence of 17beta-estradiol, were loaded with [3H]-cholesteryl ester-labeled-acetyl LDL (low density lipoprotein) and the efflux of radioactivity into the medium was measured. The effect of 17beta-estradiol on cellular activities of acyl coenzyme A: cholesterol acyl transferase (ACAT), and both neutral and acid cholesteryl ester hydrolase (CEH) and the secretion of apoE and LDL into the medium, were also studied. The results indicate that 17beta-estradiol induces an increase in the amount of labeled cholesterol released from the cells and, the data obtained from the measurements of ACAT and CEH activities showed that, in estrogen-treated HMDM, the cholesteryl ester cycle favors the hydrolysis of lipoprotein cholesterol by CEH in comparison with its acylation by ACAT. In particular, for the first time a strong enhancement of neutral and acid CEH in human macrophages by 17beta-estradiol, was demonstrated. ApoE and LDL secretion increased during the maturation of monocytes to macrophages, and was not modified by 17beta-estradiol. In contrast, loading the cells with cholesterol by incubation in the presence of acetylated or oxidized LDL produced an increase in the levels of apoE secreted by both estrogen-treated and control macrophages. The activity of LPL found in the cell medium, on the other hand, in lipid loaded cells tended to be increased only in estrogen treated macrophages, suggesting that the effects of estrogen on unloaded macrophages are different from those produced on lipid-loaded macrophages. On the whole, the present findings suggest that one of the mechanisms by which 17beta-estradiol acts to reduce cholesterol accumulation in macrophages is by increasing reverse cholesterol transport through the enhancement of the cholesteryl ester cycle, so that the generation of intracellular unesterified cholesterol for excretion from the cells is favored.

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 μM 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.

ACAT inhibitor F-1394 prevents intimal hyperplasia induced by balloon injury in rabbits

Acyl-CoA:cholesterol acyltransferase (ACAT) is thought to contribute significantly to lipid deposition in macrophages, which subsequently leads to the initiation and progression of atherosclerosis. The aim of the present study was to examine the influence of hypercholesterolemia on arterial hyperplasia induced by endothelial denudation and the direct effect of ACAT inhibition on lesion formation. Rabbits were fed either a cholesterol diet or a regular diet for 4 weeks, and then the left common carotid arteries were denuded of endothelium. After the operation, all rabbits were kept on the regular diet for 2;-6 weeks. Two weeks after the denudation, the degree of intimal thickening and the number of proliferating cells (which were immunohistologically identified to be smooth muscle cells) were similar in hypercholesterolemic and normolipidemic rabbits. After that, both parameters progressively increased in hypercholesterolemic rabbits but declined in normolipidemic rabbits. Macrophages were apparent in the lesions only in hypercholesterolemic rabbits. Next, the effect of the ACAT inhibitor, (1S,2S)-2-[3-(2,2-dimethylpropyl)-3-nonylureido] cyclohexane-1-yl 3-[(4R)-N-(2,2,5,5-tetramethyl-1,3-dioxane-4-carbonyl)amino]propionate (F-1394), on neointimal formation in hypercholesterolemic rabbits was examined. Oral administration of F-1394 significantly reduced neointimal thickening and the extent of macrophages in lesions without affecting serum cholesterol levels. These results suggest that hypercholesterolemia causes macrophage-derived foam cell accumulation in lesions, and that the progression of lesions is accelerated by the presence of macrophages. Moreover, the study shows that F-1394 prevents neointimal formation even in the presence of hypercholesterolemia, indicating that F-1394 may be useful for treating restenosis after percutaneous translumenal coronary angioplasty in hyperlipidemic patients.

Reduction in apolipoprotein-mediated removal of cellular lipids by immortalization of human fibroblasts and its reversion by cAMP: lack of effect with Tangier disease cells

High density lipoprotein (HDL) phospholipids and apolipoproteins remove cellular lipids by two distinct mechanisms, but their relative contribution to reverse cholesterol transport is unknown. Whereas phospholipid-mediated cholesterol efflux from cultured cells reflects the activity of the HDL receptor SR-BI, apolipoprotein-mediated lipid removal is regulated in response to changes in cellular cholesterol content (positive) and cell proliferation rates (negative). Here we show that immortalization of human skin fibroblast lines with the papillomavirus E6/E7 oncogenes increased their proliferation rates and selectively reduced the activity of the apolipoprotein-mediated lipid removal pathway. This reduction was accompanied by a decrease in cellular cAMP levels and was reversed by treatment with a cAMP analog. The stimulatory effect of cAMP was independent of changes in cellular phenotype or activities of cholesteryl ester cycle enzymes. The severely impaired apolipoprotein-mediated lipid removal pathway in Tangier disease fibroblasts, which persisted after immortalization, was not improved by treatment with a cAMP analog, implying that the cellular defect in Tangier disease is upstream from this cAMP-dependent signaling pathway.These results indicate that papillomavirus-induced immortalization of fibroblasts selectively reduces the activity of the apolipoprotein-mediated lipid removal pathway by a cAMP-dependent process, perhaps to prevent loss of cellular lipids needed for continual membrane synthesis.

Limited proteolysis of high density lipoprotein abolishes its interaction with cell-surface binding sites that promote cholesterol efflux

High-density lipoprotein (HDL) components remove cholesterol from cells by two independent mechanisms. Whereas HDL phospholipids pick up cholesterol that desorbs from the plasma membranes, HDL apolipoproteins appear to interact with cell-surface binding sites that target for removal pools of cellular cholesterol that feed into the cholesteryl ester cycle. Here we show that mild trypsin treatment of HDL almost completely abolishes this apolipoprotein-mediated cholesterol removal process. When HDL was treated with trypsin for various periods of time and then incubated with cholesterol-loaded fibroblasts, treatment for only 5 min reduced the ability of HDL to remove excess cholesterol from cellular pools that were accessible to esterification by the enzyme acyl CoA:cholesterol acyltransferase. This mild treatment digested less than 20% of HDL apolipoproteins and did not alter the lipid composition, size distribution, or electrophoretic mobility of the particles. Trypsin treatment of HDL for up to 1 h caused no further reduction in its ability to remove cellular cholesterol despite a greater than 2-fold increase in apolipoprotein digestion. Trypsin treatment of HDL also reduced its ability to deplete the cholesteryl ester content of sterol-laden macrophages. Promotion of cholesterol efflux from the plasma membrane by HDL phospholipids was unaffected by even extensive proteolysis. In parallel to the loss of cholesterol transport-stimulating activity, trypsin treatment of HDL for only 5 min nearly abolished its interaction with high-affinity binding sites on cholesterol-loaded fibroblasts. Reconstitution of trypsin-modified HDL with isolated apo A-I or apo A-II restored the cholesterol transport-stimulating activity of the particles. Thus a minor trypsin-labile fraction of HDL apolipoproteins is almost exclusively responsible for the apolipoprotein-dependent component of cholesterol efflux mediated by HDL particles.

Apolipoprotein-mediated removal of cellular cholesterol and phospholipids

It is widely believed that high density lipoprotein (HDL) protects against cardiovascular disease by removing excess cholesterol from cells of the artery wall. Recent cell culture studies have provided evidence that a major pathway for removing cholesterol and phospholipids from cells is mediated by the direct interactions of HDL apolipoproteins (apo) with plasma membrane domains. These interactions efficiently clear cells of excess sterol by targeting for removal pools of cholesterol that feed into the cholesteryl ester cycle. The precursors for this pathway in vivo are likely to be lipid-free or lipid-poor apolipoproteins generated either by dissociation from the surface of HDL particles or by de novo synthesis. Fibroblasts from subjects with a severe HDL deficiency syndrome called Tangier disease have a cellular defect that prevents apolipoproteins from removing both cholesterol and phospholipids from cells. This defect is associated with a near absence of plasma HDL, markedly below normal low density lipoprotein (LDL) levels, and the appearance of macrophage foam cells in tissues. Thus, an inability of nascent apoA-I to acquire cellular lipids results in a rapid clearance of apoA-I from the plasma, decreased production and increased clearance of LDL, and sterol deposition in tissue macrophages. Although the molecular properties of this pathway are still poorly understood, these studies imply that the apolipoprotein-mediated pathway for removal of cellular lipids is a major source of plasma cholesterol and phospholipids and plays an important role in clearing excess cholesterol from macrophages in vivo.

Cholesterol efflux from macrophages and other cells.

Foam cell formation by lipid accumulation in macrophages is a prominent finding in atherosclerotic plaques. Since macrophages cannot limit the uptake of lipids, cholesterol efflux is probably essential to inhibit progression and cause regression of atherosclerosis. Cholesterol efflux is generally attributed to HDL in the extracellular space. Slow bidirectional fluxes of cholesterol occur between plasma membrane and lipid-rich HDL subclasses. Esterification of cholesterol in HDL by lecithin: cholesterol acyltransferase causes net cholesterol efflux. In contrast, some lipid-free apolipoproteins (especially apolipoprotein A-I) and lipid-poor HDL subclasses such as prebeta 1-apolipoprotein A-I containing lipoprotein mediate rapid and unidirectional cholesterol efflux from specific cholesterol domains in the plasma membrane. Extracellular presence of HDL or apolipoprotein A-I moreover facilitates the translocation of cholesterol from intracellular pools to the plasma membrane, probably via signal transduction. The activated transfer machinery appears to involve the Golgi apparatus and diverts cholesterol from the shuttle between acylcoenzyme A: cholesterol acyltransferase and neutral cholesteryl ester hydrolase (cholesteryl ester cycle). Endogenously synthesized apolipoprotein E facilitates HDL-mediated cholesterol efflux from macrophages. Moreover, at least in human monocyte-derived macrophages, apolipoprotein E appears to be involved in the export of cholesterol independently from extracellular acceptors. Cholesterol efflux can be inhibited by some oxysterols that are found in macrophages of atherosclerotic plaques and macrophages that are loaded in vitro with oxidized LDL.

Dehydroepiandrosterone markedly inhibits the accumulation of cholesteryl ester in mouse macrophage J774-1 cells

To clarify the antiatherogenic mechanism of action of dehydroepiandrosterone (DHEA), we investigated the effects of DHEA on the accumulation of cholesteryl ester (CE) in cultured mouse macrophage J774-1 cells. The accumulation of CE in J774-1 cells in the presence of acetyl low density lipoprotein (AcLDL) and 10 −5 mol/1 DHEA was significantly reduced to 30% of the control values for 24 h. The marked effect of DHEA was observed as early as 6 h and continued at least for 48 h. This reduction by DHEA was dose-dependent and occurred starting at a DHEA dose of 5 × 10 −7 mol/1 for 24 h. DHEA treatment did not induced any changes in the cell surface binding, cell-association, or degradation of AcLDL. In comparison, the DHEA analogues, 8354 and 8356, which are known to be much stronger inhibitors of glucose 6-phosphate dehydrogenase than DHEA, did not show as marked an effect as DHEA on the accumulation of CE during the first 6 h. However, after 24–48 h of incubation, both 8354 and 8356 caused a marked reduction in the accumulation of CE similar to that observed with DHEA. A quantitative analysis of the cellular cholesterol content revealed that DHEA caused a marked reduction in CE with a concomitant continuous increase in free cholesterol (FC), while the DHEA analogues caused a marked reduction in CE with no change in FC. DHEA demonstrated little inhibitory effect on 25-hydroxycholesterol-driven esterification. Moreover, 10 −5 mol/1 DHEA induced a CE reduction in the foam cells induced by AcLDL. The CE-reducing capacity was also observed in the DHEA analogues. This CE-reducing capacity disappeared, however, when acyl CoA:cholesterol acyltransferase inhibitor, 58-035, was also present. Based on these findings, it can be concluded that the inhibitory effect of DHEA on the CE storage in response to AcLDL can be explained, at least in part, by two mechanisms. First, a recently published mechanism, namely, the inhibitory action of DHEA on lysosomal cholesterol transport, correlates well with the inhibition against foam cell transformation by DHEA in the early phase (at 6 h) observed in our study. With regard to the second mechanism, the CE-reducing capacity of DHEA from CE-laden foam cells, which appears to be related to a decreased cholesteryl ester cycle, may contribute to the inhibitory effect on the CE storage in the late phase (at 24 h and 48 h). These phase-specific inhibitory mechanisms of DHEA on the CE-storage may therefore partly explain the antiatherogenic action of DHEA.

Dehydroepiandrosterone markedly inhibits the accumulation of cholesteryl ester in mouse macrophage J774-1 cells

<h2>Abstract</h2> To clarify the antiatherogenic mechanism of action of dehydroepiandrosterone (DHEA), we investigated the effects of DHEA on the accumulation of cholesteryl ester (CE) in cultured mouse macrophage J774-1 cells. The accumulation of CE in J774-1 cells in the presence of acetyl low density lipoprotein (AcLDL) and 10⁻⁵ mol/1 DHEA was significantly reduced to 30% of the control values for 24 h. The marked effect of DHEA was observed as early as 6 h and continued at least for 48 h. This reduction by DHEA was dose-dependent and occurred starting at a DHEA dose of 5 × 10⁻⁷ mol/1 for 24 h. DHEA treatment did not induced any changes in the cell surface binding, cell-association, or degradation of AcLDL. In comparison, the DHEA analogues, 8354 and 8356, which are known to be much stronger inhibitors of glucose 6-phosphate dehydrogenase than DHEA, did not show as marked an effect as DHEA on the accumulation of CE during the first 6 h. However, after 24–48 h of incubation, both 8354 and 8356 caused a marked reduction in the accumulation of CE similar to that observed with DHEA. A quantitative analysis of the cellular cholesterol content revealed that DHEA caused a marked reduction in CE with a concomitant continuous increase in free cholesterol (FC), while the DHEA analogues caused a marked reduction in CE with no change in FC. DHEA demonstrated little inhibitory effect on 25-hydroxycholesterol-driven esterification. Moreover, 10⁻⁵ mol/1 DHEA induced a CE reduction in the foam cells induced by AcLDL. The CE-reducing capacity was also observed in the DHEA analogues. This CE-reducing capacity disappeared, however, when acyl CoA:cholesterol acyltransferase inhibitor, 58-035, was also present. Based on these findings, it can be concluded that the inhibitory effect of DHEA on the CE storage in response to AcLDL can be explained, at least in part, by two mechanisms. First, a recently published mechanism, namely, the inhibitory action of DHEA on lysosomal cholesterol transport, correlates well with the inhibition against foam cell transformation by DHEA in the early phase (at 6 h) observed in our study. With regard to the second mechanism, the CE-reducing capacity of DHEA from CE-laden foam cells, which appears to be related to a decreased cholesteryl ester cycle, may contribute to the inhibitory effect on the CE storage in the late phase (at 24 h and 48 h). These phase-specific inhibitory mechanisms of DHEA on the CE-storage may therefore partly explain the antiatherogenic action of DHEA.

Compartmental Isolation of Cholesterol Participating in the Cytoplasmic Cholesteryl Ester Cycle in Chinese Hamster Ovary 25-RA Cells

Using the Chinese hamster ovary cell line, 25-RA, we have demonstrated that lipoprotein-derived cholesterol and endogenously synthesized cholesterol are selectively differentiated with respect to their cellular locations. These cells lack sterol-mediated regulation, spontaneously storing large amounts of esterified cholesterol, which turns over with a half-time of 7.5 h. When [3H]cholesterol was provided to the cells in serum to trace cellular cholesterol, the specific activities of cellular free and esterified cholesterol (6238 +/- 273 and 5128 +/- 277 cpm/ microg, respectively) failed to equilibrate, indicating that bulk cellular free cholesterol is isolated from that participating in the cholesteryl ester cycle. Using [3H]acetate to trace the fate of endogenously synthesized cholesterol, a failure of equilibration was also observed (specific activities of free and esterified cholesterol = 280 +/- 37 and 458 +/- 8 cpm/ microg, respectively). The lower specific activity of the precursor indicates that endogenously synthesized cholesterol is preferentially esterified. When cells radiolabeled with [3H]acetate were post-incubated in the absence of radiolabel, the specific activity of the esterified cholesterol pool remained significantly higher than that of the free cholesterol, suggesting that cholesterol derived from hydrolysis of esterified cholesterol is preferentially re-esterified.

Oxidative tyrosylation of HDL enhances the depletion of cellular cholesteryl esters by a mechanism independent of passive sterol desorption.

It is believed that HDL protects against atherosclerosis by removing excess cholesteryl esters from cells of the artery wall. Previous studies have suggested that HDL depletes cells of cholesteryl esters both by stimulating cholesterol efflux from the plasma membrane and by activating transport processes that divert cholesterol from the cholesteryl ester cycle, but it is unknown if these are independent processes. We previously found that HDL oxidized by tyrosyl radical has a markedly enhanced ability to promote the removal of cholesterol from cultured cells [Francis, G. A., et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 6631-6635]. Here we show that incubation of cholesterol-loaded human fibroblasts with low concentrations of tyrosylated HDL depleted cells of cholesteryl esters and increased cellular free cholesterol without increasing efflux of cholesterol into the medium as compared to incubation with untreated HDL. Cells preincubated with tyrosylated HDL and then exposed to a variety of cholesterol acceptors exhibited significantly higher rates of free cholesterol efflux than did cells preincubated with HDL. This effect was observed in the presence or absence of an inhibitor of acyl CoA:cholesterol acyltransferase (ACAT) and was independent of cholesteryl ester hydrolysis, suggesting that alterations in cholesteryl ester cycle enzymes were not responsible for the loss of cholesteryl esters. In contrast to the reduction of cholesteryl esters, the rates of cholesterol and phospholipid efflux from the plasma membranes of cells exposed to tyrosylated HDL and HDL were identical. These results suggest for the first time that a mechanism exists to deplete cellular cholesteryl esters and the cholesterol substrate pool for esterification by ACAT prior to the removal of cholesterol from the plasma membrane. Identification of products in tyrosylated HDL responsible for this redistribution of cellular cholesterol may provide important insights into mechanisms of intracellular cholesterol trafficking and the ability of modified forms of HDL to protect the artery against wall pathological cholesterol accumulation.

Progesterone blocks intracellular translocation of free cholesterol derived from cholesteryl ester in macrophages.

Macrophage foam cells must accommodate continuing fluxes of free cholesterol in spite of a greatly expanded store of cholesteryl ester. Though endogenous free cholesterol synthesis is suppressed, free cholesterol continues to enter the cell via endocytosis of oxidized/modified lipoproteins. It has been shown previously that this free cholesterol is released into the lysosomal compartment and rapidly transported to the plasma membrane prior to its esterification. A substantial amount of free cholesterol is also presented via the continuous hydrolysis of cholesteryl ester during the cholesteryl ester cycle. We addressed the question of whether the intracellular free cholesterol derived from the hydrolysis of cholesteryl ester formed a protected pool for rapid re-esterification. Incubation of macrophage foam cells with cyclic AMP to enhance cholesteryl ester hydrolysis, and with S58035 to inhibit acyl-CoA:cholesterol acyltransferase (ACAT) activity, led to conversion of cellular cholesteryl ester to free cholesterol and transport of this free cholesterol to the plasma membrane. Addition of progesterone, previously demonstrated to be an inhibitor of free cholesterol transport in other cell types, also led to conversion of cholesteryl ester to free cholesterol even though progesterone was only a weak inhibitor of ACAT activity. Free cholesterol in the plasma membrane was an important source of ACAT substrate to balance the constitutive hydrolysis of cholesteryl ester in cholesterol-loaded macrophages. Treatment of cells with progesterone, however, prevented free cholesterol derived from cholesteryl ester hydrolysis from moving to the plasma membrane. The sequestration of free cholesterol by progesterone could be reversed by incubation with human HDL3.(ABSTRACT TRUNCATED AT 250 WORDS)

Species difference in cholesteryl ester cycle and HDL-induced cholesterol efflux from macrophage foam cells.

The species difference in the turnover rates of the cholesteryl ester (CE) cycle in macrophage foam cells (MFC) was examined in mice and rats. MFC were induced by acetyl-LDL and pulsed with [3H]oleate, followed by a chase with [14C]oleate. The replacement of the initial amount of cholesteryl [3H]oleate by cholesteryl [14C]oleate within 24 hours was 63% in mouse MFC, whereas it was 33% in rat MFC. The corresponding replacement in rabbit MFC was < 10%. In addition, HDL removed 41% of the CE mass from mouse MFC but only 22% from rat MFC. HDL-induced CE reduction from mouse MFC was enhanced by 40% by the inhibitor for acyl-coenzyme A:cholesterol acyltransferase (58-035), whereas the enhancing effect was not observed with rat MFC. These results indicate that the rate of CE turnover may serve as a critical factor to determine the capacity of MFC to respond to HDL-induced CE reduction, suggesting the possibility that the species difference in the turnover rates of the CE cycle in MFC might explain, in part, the species difference in susceptibility to experimental atherosclerosis.

Sphingosine inhibits sphingomyelinase-induced cholesteryl ester formation in cultured fibroblasts

We have in this study examined the effects of sphingosine, a possible secondary degradation product following sphingomyelin hydrolysis, on cholesterol homeostasis in cultured human fibroblasts treated with sphingomyelinase. The activation of cholesterol esterification, which resulted from the degradation of plasma membrane sphingomyelin (by sphingomyelinase), was observed to be effectively blocked by sphingosine (half-maximal dose 6–7 μM). The inhibitory action of sphingosine could not be reproduced with other amines (e.g., dodecyl amine or imipramine). The onset of inhibition of cholesteryl ester formation by sphingosine was rapid (maximal effect within 15 min). Sphingosine itself had no spontaneous effects on the distribution of cellular cholesterol. At concentrations below 10 μM, sphingosine was not cytotoxic, as determined by cellular trypan blue permeability. The inhibitory action of sphingosine on cholesteryl ester formation apparently did not result from a direct inhibition of acyl-CoA cholesterol acyltransferase (ACAT), since the activity of this enzyme was unaffected by sphingosine (10 μM) in a cell-free homogenate, using [ 14C]oleoyl-CoA as a co-substrate. Sphingosine was also unable to prevent the formation of activated fatty acids (oleoyl-CoA), since acyl-CoA synthetase activity in a cell-free homogenate was not inhibited by sphingosine (at 5 μM). The cellular cholesteryl ester cycle (i.e., the neutral cholesteryl ester hydrolase) was unaffected by sphingosine (at 5 μM). Down-regulation of PKC activity (24 h exposure of cells to 100 nM (62 ng/ml) phorbol ester) did not affect the sphingomyelinase-induced stimulation of [ 3H]cholesteryl ester formation. In addition, the sphingosine-induced inhibition of [ 3H]cholesteryl ester formation was not reversed in the presence of phorbol ester (short-term exposures), suggesting that the effect of sphingosine was not mediated via PKC. In conclusion, we have shown that sphingosine is an inhibitor of cholesteryl ester formation in fibroblasts. The inhibition is only seen with intact cells, which may suggest that a secondary metabolite of sphingosine was responsible for the observed inhibition of cholesteryl ester formation.

The effect of cyclic AMP analogues and glucagon on cholesteryl ester synthesis and hydrolysis in cultured hamster hepatocytes

Two cyclic AMP analogues, 8-chloro cyclic AMP and 8-(4 ehlorophenylthio) cyclic AMP, were found to increase the incorporation of [ 3H]oleate into cholereryl ester in cultured hamster hapatocytes (30–40%), while incorporation into triacylglycerol was unaffected. An increase of a similar magnitude was observed in the presence of glucagon and the phosphodiesterase inhibitor, theophylline. The cyclic AMP analogues also stimulated the activity of neutral cholesteryl ester hydrolase in the cells, and this effect was mimicked by glucagon and theophylline. These results show that cyclic AMP can affect the cholesteryl ester cycle in hamster hepatocytes, and support the idea that the enzymes involved may be co-ordinately regulated.

Regulation of cholesteryl ester metabolism in the hamster liver

Neutral cholesteryl ester hydrolase activity has been described in the cytosolic and microsomal fraction of rat liver, but the relationship of these activities to other parameters of hepatic cholesterol metabolism is not known. We have studied this in the hamster by manipulating the flux of cholesterol across the liver by dietary modifications. A bile acid séquestrant was used to stimulate LDL receptor activity and hence flux of cholesterol into the liver. A cholesterol-rich diet caused a hypercholesterolaemia and substantial uptake of cholesterol and deposition in the liver. Hypercholesterolaemia was also induced by a saturated fat-rich diet, but in contrast this reduced the flux of cholesterol into the liver. Animals were fed these diets for 1 week and then the livers removed and enzyme activities determined. These were 3-hydroxy-3-methylglutaryl-CoA reductase, cholesterol 7α-hydroxylase, acyl-CoA:cholesterol acyltransferase, microsomal cholesteryl ester hydrolase and cytosolic cholesteryl ester hydrolase. Feeding the bile acid séquestrant increased the hydrolysis of cholesteryl ester in the liver over its synthesis. In contrast, both fat feeding and cholesterol feeding caused a reduction in the relative rate of hydrolysis of cholesteryl ester compared with synthesis. This was particularly marked with the cholesterol-rich diet. These results show that the hydrolysis of cholesteryl ester in hamster liver responds to dietary manipulation in a way that reflects the needs of the cell for cholesterol or the presence of an excess. It is suggested that a metabolically significant cholesteryl ester cycle may operate in the liver to a greater extent that had previously been thought.