Incorporation of lipoxygenase products into cholesteryl esters by acyl-CoA:cholesterol acyltransferase in cholesterol-rich macrophages.

Macrophages which were incubated with acetylated low-density lipoproteins, resulting in cholesteryl ester accumulation, incorporated the monohydroxyeicosatetraenoic acids (5-, 15-, and 12-HETEs) into cholesteryl esters. The esterification of these hydroxy fatty acids to cholesterol by total membrane preparations of cholesterol-rich macrophages was dependent on the synthesis of the fatty acyl-CoA derivative, and was catalysed by acyl-CoA:cholesterol acyltransferase (ACAT). Stimulation of membrane ACAT activity by 25-hydroxycholesterol increased the synthesis of cholesteryl 12-HETE by 40%. In contrast, inhibiting ACAT activity by progesterone and compound 58-035 decreased cholesteryl 12-HETE production by 60% and 90% respectively. Although 5-, 15- and 12-HETE were esterified to cholesterol by ACAT, these monohydroxy fatty acids were less optimal as substrates compared with oleic acid or arachidonic acid. The hydrolysis and release of 12-HETE and the other monohydroxyeicosatetraenoic acids from intracellular cholesteryl esters and phospholipids occurred at a faster rate than for the more conventional fatty acids, oleate and arachidonate. Cholesteryl esters which contain hydroxy fatty acids therefore provide only a transient storage for lipoxygenase products, as these fatty acids are released into the medium as readily as hydroxy fatty acids found in phospholipids and triacylglycerols. The data provide evidence, for the first time, of an ACAT-dependent esterification of the lipoxygenase products 5-, 15- and 12-HETEs to cholesterol in the macrophage-derived foam cell. The channelling of these monohydroxy fatty acids to cholesteryl esters provides a mechanism which can alter the amount of lipoxygenase products incorporated into cellular phospholipids, thus averting deleterious changes to cell membranes. ACAT, by catalysing the esterification of monohydroxyeicosatetraenoic acids to cholesterol, could play a key role in regulating the amount of lipoxygenase products in the pericellular space of the cholesterol-enriched macrophage.     

Macrophage‐mediated cholesterol handling in atherosclerosis

Formation of foam cells is a hallmark at the initial stages of atherosclerosis. Monocytes attracted by pro‐inflammatory stimuli attach to the inflamed vascular endothelium and penetrate to the arterial intima where they differentiate to macrophages. Intimal macrophages phagocytize oxidized low‐density lipoproteins (oxLDL). Several scavenger receptors (SR), including CD36, SR‐A1 and lectin‐like oxLDL receptor‐1 (LOX‐1), mediate oxLDL uptake. In late endosomes/lysosomes of macrophages, oxLDL are catabolysed. Lysosomal acid lipase (LAL) hydrolyses cholesterol esters that are enriched in LDL to free cholesterol and free fatty acids. In the endoplasmic reticulum (ER), acyl coenzyme A: cholesterol acyltransferase‐1 (ACAT1) in turn catalyses esterification of cholesterol to store cholesterol esters as lipid droplets in the ER of macrophages. Neutral cholesteryl ester hydrolases nCEH and NCEH1 are involved in a secondary hydrolysis of cholesterol esters to liberate free cholesterol that could be then out‐flowed from macrophages by cholesterol ATP‐binding cassette (ABC) transporters ABCA1 and ABCG1 and SR‐BI. In atherosclerosis, disruption of lipid homoeostasis in macrophages leads to cholesterol accumulation and formation of foam cells.     

Preferential incorporation of eicosanoid precursor fatty acids into human umbilical vein endothelial cell phospholipids

We have examined the preferential incorporation of specific fatty acids into phospholipid classes of cultured human umbilical vein endothelial cells. Pulse-labeling of human umbilical vein endothelial cell phospholipids with radiolabeled fatty acids and inhibition of radiolabeled fatty acid incorporation by competition with excess, unlabeled fatty acids in pair-wise combinations revealed two distinct classes of esterification systems into human umbilical vein endothelial cell phospholipids. The eicosanoid precursor fatty acids, including arachidonate, 8,11,14-eicosatrienoate (ETA) and 5,8,11,14,17-eicosapentaenoate (EPA), exhibited high affinity incorporation into total phospholipids, whereas other fatty acids, including docosahexaenoate and monohydroxy eicosatetraenoates, showed low affinity incorporation. The relative degree of incorporation of eicosanoid precursor fatty acids into phospholipid classes was phosphatidylcholine (PC) greater than phosphatidylethanolamine (PE) greater than phosphatidylinositol (PI) greater than phosphatidylserine (PS). The specific activity of [14C]arachidonic acid-labeled PI was two times higher than that of any other radiolabeled phospholipids. When competitive incorporation of eicosanoid precursor fatty acids into phospholipid classes was studied, they were found to be acylated into different phospholipid classes at different rates. Although eicosanoid precursor fatty acids were not preferentially incorporated into PC, arachidonic acid was preferentially incorporated into the other phospholipids and exhibited particular selectivity in comparison with the other eicosanoid precursor fatty acids for incorporation into PI. These results demonstrate that human umbilical vein endothelial cells possess selective incorporation mechanisms for specific fatty acids into various phospholipids via the deacylation-reacylation pathway.     

n-3 and n-6 polyunsaturated fatty acids have different effects on acyl-CoA:cholesterol acyltransferase in J774 macrophages.

The effects of incubating J774 mouse macrophages with different fatty acids on cholesterol esterification were investigated. In cells incubated with n-3 polyunsaturated fatty acids, the rate of cholesterol esterification was significantly reduced compared with cells incubated with n-6 polyunsaturated fatty acids or with oleic acid. This change in cholesterol esterification appears to be the result of reductions in the activity of acyl-CoA:cholesterol acyltransferase (ACAT) in the endoplasmic reticulum of the macrophages incubated with the n-3 polyunsaturated fatty acids. No differences in microsomal cholesterol were observed among cells incubated with different fatty acids. However, cellular cholesterol levels were lower in cells incubated with n-3 polyunsaturated fatty acids. In microsomes from cells incubated with n-3 polyunsaturated fatty acids, both the Km and the Vmax of ACAT were lower than in microsomes from cells incubated with n-6 fatty acids or oleic acid. These findings may explain some of the reduction in atherosclerotic lesions that are observed with dietary fish oils that contain high levels of n-3 polyunsaturated fatty acids.     

Cholesterol and phospholipid metabolism in macrophages

Cholesterol-loaded macrophages are present at all stages of atherogenesis, and recent in vivo data indicate that these cells play important roles in both early lesion development and late lesion complications. To understand how these cells promote atherogenesis, it is critical that we understand how lesional macrophages interact with subendothelial lipoproteins, the consequences of this interaction, and the impact of subsequent intracellular metabolic events. In the arterial wall, macrophages likely interact with both soluble and matrix-retained lipoproteins, and a new challenge is to understand how certain consequences of these two processes might differ. Initially, the major intracellular metabolic route of the lipoprotein-derived cholesterol is esterification to fatty acids, but macrophages in advanced atherosclerotic lesions progressively accumulate large amounts of unesterified, or free, cholesterol (FC). In cultured macrophages, excess FC accumulation stimulates phospholipid biosynthesis, which is an adaptive response to protect the macrophage from FC-induced cytotoxicity. This phospholipid response eventually decreases with continued FC loading, leading to a series of cellular death reactions involving both death receptor-induced signaling and mitochondrial dysfunction. Because macrophage death in advanced lesions is thought to promote plaque instability, these intracellular processes involving cholesterol, phospholipid, and death pathways may play a critical role in the acute clinical manifestations of advanced atherosclerotic lesions.     

Cholesterol and oxysterol metabolism and subcellular distribution in macrophage foam cells. Accumulation of oxidized esters in lysosomes

Cholesterol- and cholesteryl ester-rich macrophage foam cells, characteristic of atherosclerotic lesions, are often generated in vitro using oxidized low density lipoprotein (OxLDL). However, relatively little is known of the nature and extent of sterol deposition in these cells or of its relationship to the foam cells formed in atherosclerotic lesions. The purpose of this study was to examine the content and cellular processing of sterols in OxLDL-loaded macrophages, and to compare this with macrophages loaded with acetylated LDL (AcLDL; cholesteryl ester-loaded cells containing no oxidized lipids) or 7-ketocholesterol-enriched acetylated LDL (7KCAcLDL; cholesteryl ester-loaded cells selectively supplemented with 7-ketocholesterol (7KC), the major oxysterol present in OxLDL). Both cholesterol and 7KC and their esters were measured in macrophages after uptake of these modified lipoproteins. Oxysterols comprised up to 50% of total sterol content of OxLDL-loaded cells. Unesterified 7KC and cholesterol partitioned into cell membranes, with no evidence of retention of either free sterol within lysosomes. The cells also contained cytosolic, ACAT-derived, cholesteryl and 7-ketocholesteryl esters. The proportion of free cholesterol and 7KC esterified by ACAT was 10-fold less in OxLDL-loaded cells than in AcLDL or 7KCAcLDL-loaded cells. This poor esterification rate in OxLDL-loaded cells was partly caused by fatty acid limitation. OxLDL-loaded macrophages also contained large (approximately 40-50% total cell sterol content) pools of oxidized esters, containing cholesterol or 7KC esterified to oxidized fatty acids. These were insensitive to ACAT inhibition, very stable and located in lysosomes, indicating resistance to lysosomal esterases. Macrophages loaded with OxLDL do not accumulate free sterols in their lysosomal compartment, but do accumulate lysosomal deposits of OxLDL-derived cholesterol and 7-ketocholesterol esterified to oxidized fatty acids. The presence of similar deposits in lesion foam cells would represent a pool of sterols that is particularly resistant to removal.     

Effects of cholesterol depletion and increased lipid unsaturation on the properties of endocytic membranes

Lipid analogs with dialkylindocarbocyanine (DiI) head groups and short or unsaturated hydrocarbon chains (e.g. DiIC(12) and FAST DiI) enter the endocytic recycling compartment efficiently, whereas lipid analogs with long, saturated tails (e.g. DiIC(16) and DiIC(18)) are sorted out of this pathway and targeted to the late endosomes/lysosomes (Mukherjee, S., Soe, T. T., and Maxfield, F. R. (1999) J. Cell Biol. 144, 1271-1284). This differential trafficking of lipid analogs with the same polar head group was interpreted to result from differential partitioning to different types of domains with varying membrane order and/or curvature. Here we investigate the system further by monitoring the trafficking behavior of these lipid analogs under conditions that alter domain properties. There was a marked effect of cholesterol depletion on the cell-surface distribution and degree of internalization of the lipid probes. Furthermore, instead of going to the late endosomes/lysosomes as in control cells, long chain DiI analogs, such as DiIC(16), were sorted to the recycling pathway in cholesterol-depleted cells. We confirmed that this difference was due to a change in overall membrane properties, and not cholesterol levels per se, by utilizing a Chinese hamster ovary cell line that overexpressed transfected stearoyl-CoA desaturase 1, a rate-limiting enzyme in the production of monounsaturated fatty acids. These cells have a decrease in membrane order because they contain a much larger fraction of unsaturated fatty acids. These cells showed alteration of DiI trafficking very similar to cholesterol-depleted cells. By using cold Triton X-100 extractability of different lipids as a criterion to determine the membrane properties of intracellular organelles, we found that the endocytic recycling compartment has abundant detergent-resistant membranes, in contrast to the late endosomes and lysosomes.     

Effect of fatty acid supplementation on cholesterol and retinol esterification in J774 macrophages

J774 macrophages exposed to medium containing cholesterol-rich phospholipid dispersions accumulate cholesteryl ester. Supplementing this medium with 100 micrograms oleate/ml increased cellular cholesteryl ester contents 3-fold. Cell retinyl ester contents increased 8-fold when medium containing retinol dispersed in dimethyl sulfoxide was supplemented with oleate. These increases were not the result of increases in total lipid uptake by the cells but rather of redistribution of cholesterol and retinol into their respective ester pools. Effective oleate concentration of 15-30 micrograms/ml increased cellular retinyl and cholesteryl ester contents. The effective oleate concentration was reduced to 5 micrograms/ml when the fatty acid/albumin molar ratio was increased. The oleate-stimulated increase in cholesterol esterification was blocked by incubating cells with Sandoz 58-035, a specific inhibitor of acyl-CoA:cholesterol acyltransferase (ACAT), indicating that the effect of fatty acid exposure is mediated through changes in ACAT activity. When cholesterol or retinol was added to cells which had been exposed to oleate for 24 h to provide a triacylglycerol store, the cellular contents of cholesteryl or retinyl ester were also significantly increased compared to cells not previously exposed to oleate. The oleate-stimulated increase in the esterification of cholesterol and/or retinol was also observed in P388D1 macrophages, human (HepG2) and rat (Fu5AH) hepatomas, human fibroblasts, rabbit aortic smooth muscle cells and MCF-7 breast carcinoma cells. In addition to oleate, a number of other fatty acids increased retinol esterification in J774 macrophages; however, cellular cholesterol esterification in these cells was increased only by unsaturated fatty acids and was inhibited in the presence of saturated fatty acids. Although the cellular uptake of radiolabeled oleate and palmitate was similar, a significant difference in the distribution of these fatty acids among the lipid classes was observed. These data demonstrate that exogenous fatty acids are one factor that regulate cellular cholesteryl and retinyl ester contents in cultured cells.     

Homoeostatic control of membrane cholesterol and fatty acid metabolism in the rat liver.

Experiments were designed to assess the effect of cholesterol feeding, with or without high levels of either saturated (coconut oil) or unsaturated (sunflower-seed oil) fat on the fatty acid composition of hepatic microsomal membrane lipids, as well as on the activities of several membrane-bound enzymes of cholesterol synthesis and metabolism. Administration of 2% (w/w) cholesterol in the rat diet inhibited hydroxymethylglutaryl-CoA reductase activity, and this inhibition was much more pronounced when cholesterol was fed in combination with unsaturated rather than with saturated fat. Cholesterol 7 alpha-hydroxylase activity was increased by all the high-cholesterol diets and inhibited by both the high-fat diets. Cholesterol esterification, as assessed by acyl-CoA:cholesterol acyltransferase (ACAT) activity, was enhanced after unsaturated-fat feeding. Cholesterol supplement, without any added fat, failed to elicit any significant increase in ACAT activity, whereas consumption of cholesterol in combination with unsaturated fat led to the greatest increase in ACAT activity. After cholesterol feeding, C18:1 and C18:2 fatty acids in the microsomal phospholipids were increased, with concomitant decreases in C18:0, C20:4 and C22:6 fatty acids, leading to an overall decrease in membrane unsaturation, irrespective of the particular fat supplement. It can be concluded that the inhibition of cholesterol biosynthesis and the enhancement of cholesterol utilization, either by increased bile formation or by increased cholesterol esterification, after cholesterol feeding, may not be enough to prevent cholesterol accumulation in the microsomal membranes. Then, to compensate for the altered fluidity resulting from cholesterol enrichment, the unsaturation of membrane phospholipids is decreased, which would in turn have an effect on membrane lipid fluidity opposite to that of increased cholesterol.     

Modification of CaCo-2 cell membrane fatty acid composition by eicosapentaenoic acid and palmitic acid: effect on cholesterol metabolism

Membrane fatty acid composition of CaCo-2 cells was modified by incubating the cells for 8 days in medium containing 100 microM eicosapentaenoic acid or palmitic acid. The effect of membrane fatty acid changes on cholesterol metabolism was then studied. Cells incubated with eicosapentaenoic acid had significant changes in membrane fatty acid composition with an accumulation of 20:5 and 22:5 and a reduction in monoenoic fatty acids compared to cells grown in palmitic acid. Intracellular cholesteryl esters could not be detected in CaCo-2 cells grown in the presence of the n-3 polyunsaturated fatty acid. In contrast, cells incubated with the saturated fatty acid contained 2 micrograms/mg protein of cholesteryl esters. Cells grown in eicosapentaenoic acid, however, accumulated significantly more triglycerides compared to cells modified with palmitic acid. The rate of oleic acid incorporation into triglycerides was significantly increased in cells incubated with eicosapentaenoic acid. CaCo-2 cells modified by eicosapentaenoic acid had lower rates of HMG-CoA reductase and ACAT activities compared to cells modified with palmitic acid. The incorporation of the two fatty acids into cellular lipids also differed. Palmitic acid was predominantly incorporated into cellular triglycerides, whereas eicosapentaenoic acid was preferentially incorporated into phospholipids with 60% of it in the phosphatidylethanolamine fraction. The data indicate that membrane fatty acid composition is significantly altered by growing CaCo-2 cells in eicosapentaenoic acid. These modifications in membrane fatty acid saturation are accompanied by a decrease in the rates of cholesterol synthesis and cholesterol esterification.     

Fatty acid incorporation is decreased in astrocytes cultured from alpha-synuclein gene-ablated mice

Because alpha-synuclein may function as a fatty acid binding protein, we measured fatty acid incorporation into astrocytes isolated from wild-type and alpha-synuclein gene-ablated mice. alpha-Synuclein deficiency decreased palmitic acid (16:0) incorporation 31% and arachidonic acid [20:4 (n-6)] incorporation 39%, whereas 22:6 (n-3) incorporation was unaffected. In neutral lipids, fatty acid targeting of 20:4 (n-6) and 22:6 (n-3) (docosahexaenoic acid) to the neutral lipid fraction was increased 1.7-fold and 1.6-fold, respectively, with an increase in each of the major neutral lipids. This was consistent with a 3.4- to 3.8-fold increase in cholesteryl ester and triacylglycerol mass. In the phospholipid fraction, alpha-synuclein deficiency decreased 16:0 esterification 39% and 20:4 (n-6) esterification 43% and decreased the distribution of these fatty acids, including 22:6 (n-3), into this lipid pool. alpha-Synuclein gene-ablation significantly decreased the trafficking of these fatty acids to phosphatidylinositol. This observation is consistent with changes in phospholipid fatty acid composition in the alpha-synuclein-deficient astrocytes, including decreased 22:6 (n-3) content in the four major phospholipid classes. In summary, these studies demonstrate that alpha-synuclein deficiency significantly disrupted astrocyte fatty acid uptake and trafficking, with a marked increase in fatty acid trafficking to cholesteryl esters and triacylglycerols and decreased trafficking to phospholipids, including phosphatidylinositol.     

Preferential ATP-binding cassette transporter A1-mediated cholesterol efflux from late endosomes/lysosomes

Recently, ATP-binding cassette transporter A1 (ABCA1), the defective molecule in Tangier disease, has been shown to stimulate phospholipid and cholesterol efflux to apolipoprotein A-I (apoA-I); however, little is known concerning the cellular cholesterol pools that act as the source of cholesterol for ABCA1-mediated efflux. We observed a higher level of isotopic and mass cholesterol efflux from mouse peritoneal macrophages labeled with [(3)H]cholesterol/acetyl low density lipoprotein (where cholesterol accumulates in late endosomes and lysosomes) compared with cells labeled with [(3)H]cholesterol with 10% fetal bovine serum, suggesting that late endosomes/lysosomes act as a preferential source of cholesterol for ABCA1-mediated efflux. Consistent with this idea, macrophages from Niemann-Pick C1 mice that have an inability to exit cholesterol from late endosomes/lysosomes showed a profound defect in cholesterol efflux to apoA-I. In contrast, phospholipid efflux to apoA-I was normal in Niemann-Pick C1 macrophages, as was cholesterol efflux following plasma membrane cholesterol labeling. These results suggest that cholesterol deposited in late endosomes/lysosomes preferentially acts as a source of cholesterol for ABCA1-mediated cholesterol efflux.     

Esterification of exogenously derived free cholesterol by cultured fibroblasts

The incorporation and esterification by cultured human fibroblasts of vesicle- or low density lipoprotein-derived free [3H]cholesterol was examined. The rate of the cellular uptake of free [3H]cholesterol from lipid vesicles was similar in both LDL-receptor positive lung fibroblasts and in LDL-receptor negative fibroblasts. When human LDL was used as the carrier of free [3H]cholesterol, however, the LDL-receptor positive lung fibroblasts incorporated significantly more [3H]cholesterol than did the LDL-receptor negative cells. The exchangeable free [3H]cholesterol was available for intracellular esterification. The formation of [3H]cholesteryl esters was markedly inhibited by lysosomotropic drugs, either indicating a partly lysosomal esterification reaction, or implying that free [3H]cholesterol moves through the lysosomal compartment on its way to intracellular esterification sites. Either way, the lysosomes appear to have a metabolic role in the metabolism of exchangeable free [3H]cholesterol.     

A PEST deletion mutant of ABCA1 shows impaired internalization and defective cholesterol efflux from late endosomes

ATP-binding cassette transporter A1 (ABCA1) promotes the efflux of cellular cholesterol and phospholipids to apoA-I. We described previously a cytoplasmic PEST sequence in ABCA1 and showed that deletion of the PEST sequence results in a prominent increase in the cell surface concentration of ABCA1. In the current study we evaluated the hypothesis that the PEST sequence-deleted ABCA1 might display defective internalization and trafficking to the late endosomes/lysosomes. As assessed by monensin treatment and cell surface biotinylation, the internalization rate of PEST sequence-deleted ABCA1 (ABCA1-dPEST) was markedly decreased compared with wild-type ABCA1 (ABCA1-wt). Immunofluorescence confocal microscopy of ABCA1-wt showed both plasma membrane localization and substantial co-localization with LAMP2 in late endosomes. In contrast, ABCA1-dPEST showed more prominent plasma membrane localization but little co-localization with LAMP2. To assess cholesterol efflux from late endosomes, HEK293 cells were transiently co-transfected with scavenger receptor A (SR-A) and incubated with [3H]cholesterol/acetyl low density lipoprotein (acLDL). Although ABCA1-dPEST showed higher cholesterol efflux than did ABCA1-wt following cell surface labeling ([3H]cholesterol/acLDL in the absence of SR-A co-transfection), it showed impaired cholesterol efflux after late endosomal labeling ([3H]cholesterol/acLDL in the presence of SR-A). Thus, deletion of the PEST sequence leads to a decrease in the internalization of ABCA1 and decreased cholesterol efflux from late endosomal cholesterol pools, providing evidence that the internalization and trafficking of ABCA1 is functionally important in mediating cholesterol efflux from intracellular cholesterol pools.     

Monoclonal antibody detection of plasma membrane cholesterol microdomains responsive to cholesterol trafficking

The hypothesis of lipid domains in cellular plasma membranes is well established. However, direct visualization of the domains has been difficult. Here we report direct visualization of plasma membrane cholesterol microdomains modulated by agents that affect cholesterol trafficking to and from the plasma membrane. The cholesterol microdomains were visualized with a monoclonal antibody that specifically detects ordered cholesterol arrays. These unique cholesterol microdomains were induced on macrophages and fibroblasts when they were enriched with cholesterol in the presence of an ACAT inhibitor, to block esterification of excess cellular cholesterol. Induction of the plasma membrane cholesterol microdomains could be blocked by agents that inhibit trafficking of cholesterol to the plasma membrane and by cholesterol acceptors that remove cholesterol from the plasma membrane. In addition, plasma membrane cholesterol microdomains did not develop in mutant Niemann-Pick type C fibroblasts, consistent with the defect in cholesterol trafficking reported for these cells.The induction of plasma membrane cholesterol microdomains on inhibition of ACAT helps explain how ACAT inhibition promotes cholesterol efflux from cells in the presence of cholesterol acceptors such as HDL. The anti-cholesterol monoclonal antibody also detected extracellular cholesterol-containing particles that accumulated most prominently during cholesterol enrichment of less differentiated human monocyte-macrophages. For the first time, cholesterol microdomains have been visualized that function in cholesterol trafficking to and from the plasma membrane.     

Acid sphingomyelinase activity is regulated by membrane lipids and facilitates cholesterol transfer by NPC2

During endocytosis, membrane components move to intraluminal vesicles of the endolysosomal compartment for digestion. At the late endosomes, cholesterol is sorted out mainly by two sterol-binding proteins, Niemann-Pick protein type C (NPC)1 and NPC2. To study the NPC2-mediated intervesicular cholesterol transfer, we developed a liposomal assay system. (Abdul-Hammed, M., B. Breiden, M. A. Adebayo, J. O. Babalola, G. Schwarzmann, and K. Sandhoff. 2010. Role of endosomal membrane lipids and NPC2 in cholesterol transfer and membrane fusion. J. Lipid Res. 51: 1747–1760.) Anionic lipids stimulate cholesterol transfer between liposomes while SM inhibits it, even in the presence of anionic bis(monoacylglycero)phosphate (BMP). Preincubation of vesicles containing SM with acid sphingomyelinase (ASM) (SM phosphodiesterase, EC 3.1.4.12) results in hydrolysis of SM to ceramide (Cer), which enhances cholesterol transfer. Besides SM, ASM also cleaves liposomal phosphatidylcholine. Anionic phospholipids derived from the plasma membrane (phosphatidylglycerol and phosphatidic acid) stimulate SM and phosphatidylcholine hydrolysis by ASM more effectively than BMP, which is generated during endocytosis. ASM-mediated hydrolysis of liposomal SM was also stimulated by incorporation of diacylglycerol (DAG), Cer, and free fatty acids into the liposomal membranes. Conversely, phosphatidylcholine hydrolysis was inhibited by incorporation of cholesterol, Cer, DAG, monoacylglycerol, and fatty acids. Our data suggest that SM degradation by ASM is required for physiological secretion of cholesterol from the late endosomal compartment, and is a key regulator of endolysosomal lipid digestion.     

Effects of different types of polyunsaturated fatty acids on cholesterol esterification in human fibroblasts.

We have enriched human fibroblasts with oleic acid, with linoleic acid and with eicosapentaenoic acid. The accumulation of cholesteryl esters in the cells and the rate of esterification of cholesterol by microsomal acyl-CoA:cholesterol acyltransferase (ACAT) were measured in these cells. Cholesteryl ester levels were lower in cells enriched with eicosapentaenoic acid compared with cells enriched with oleate or linoleate. We also observed significantly lower ACAT activities in the microsomes from fibroblasts enriched with the n-3 polyunsaturated fatty acids relative to cells enriched with oleic acid or linoleic acid. We suggest that the presence of n-3 polyunsaturated fatty acids might suppress cholesteryl ester accumulation and inhibit atherogenesis.     

Acid sphingomyelinase-deficient macrophages have defective cholesterol trafficking and efflux

Cholesterol efflux from macrophage foam cells, a key step in reverse cholesterol transport, requires trafficking of cholesterol from intracellular sites to the plasma membrane. Sphingomyelin is a cholesterol-binding molecule that transiently exists with cholesterol in endosomes and lysosomes but is rapidly hydrolyzed by lysosomal sphingomyelinase (L-SMase), a product of the acid sphingomyelinase (ASM) gene. We therefore hypothesized that sphingomyelin hydrolysis by L-SMase enables cholesterol efflux by preventing cholesterol sequestration by sphingomyelin. Macrophages from wild-type and ASM knockout mice were incubated with [(3)H]cholesteryl ester-labeled acetyl-LDL and then exposed to apolipoprotein A-I or high density lipoprotein. In both cases, [(3)H]cholesterol efflux was decreased substantially in the ASM knockout macrophages. Similar results were shown for ASM knockout macrophages labeled long-term with [(3)H]cholesterol added directly to medium, but not for those labeled for a short period, suggesting defective efflux from intracellular stores but not from the plasma membrane. Cholesterol trafficking to acyl-coenzyme A:cholesterol acyltransferase (ACAT) was also defective in ASM knockout macrophages. Using filipin to probe cholesterol in macrophages incubated with acetyl-LDL, we found there was modest staining in the plasma membrane of wild-type macrophages but bright, perinuclear fluorescence in ASM knockout macrophages. Last, when wild-type macrophages were incubated with excess sphingomyelin to "saturate" L-SMase, [(3)H]cholesterol efflux was decreased. Thus, sphingomyelin accumulation due to L-SMase deficiency leads to defective cholesterol trafficking and efflux, which we propose is due to sequestration of cholesterol by sphingomyelin and possibly other mechanisms. This model may explain the low plasma high density lipoprotein found in ASM-deficient humans and may implicate L-SMase deficiency and/or sphingomyelin enrichment of lipoproteins as novel atherosclerosis risk factors.     

Biosynthesis of prostaglandin E 2 in human skin: subcellular localization and inhibition by unsaturated fatty acids and anti-inflammatory drugs

The biosynthesis of prostaglandin E(2) (PGE(2)) from [1-(14)C]arachidonic acid has been demonstrated in homogenates and subcellular fractions of human epidermis. This biosynthetic capacity is localized in the microsomal fraction, indicating the presence of an active prostaglandin synthetase system associated with membranes of the skin. The incorporation of (14)C from [1-(14)C]arachidonic acid into PGE(2) by the microsomal fraction was enhanced by EDTA. This apparent increase in (14)C incorporation into PGE(2) in the presence of EDTA could be due at least in part to its chelating properties of removing the divalent cations in the homogenate that enhance the selective formation of PGF(2alpha) and the suppression of the activity of epidermal phospholipase A, which causes the release of nonradioactive fatty acid precursors from endogenous phospholipids. This study has also demonstrated that the formation of PGE(2) from arachidonic acid by the microsomal fraction from human skin could be inhibited by polyunsaturated fatty acids, suggesting a possible regulatory role of fatty acids released from endogenous phospholipids on prostaglandin synthesis in this tissue. The inhibitory effects of some anti-inflammatory drugs on skin microsomal prostaglandin synthetase were also demonstrated in these studies. Results from these studies indicate that the skin is therefore a useful tissue for the study of mechanisms of prostaglandin biosynthesis and the mode of action of various anti-inflammatory drugs.