Influence of cellular arachidonic acid levels on phospholipid remodeling and CoA-independent transacylase activity in human monocytes and U937 cells

The availability of free arachidonic acid (AA) constitutes a limiting step in the synthesis of biologically active eicosanoids. Free AA levels in cells are regulated by a deacylation/reacylation cycle of membrane phospholipids, the so-called Lands cycle, as well as by further remodeling reactions catalyzed by CoA-independent transacylase. In this work, we have comparatively investigated the process of AA incorporation into and remodeling between the various phospholipid classes of human monocytes and monocyte-like U937 cells. AA incorporation into phospholipids was similar in both cell types, but a marked difference in the rate of remodeling was appreciated. U937 cells remodeled AA at a much faster rate than human monocytes. This difference was found not to be related to the differentiation state of the U937 cells, but rather to the low levels of esterified arachidonate found in U937 cells compared to human monocytes. Incubating the U937 cells in AA-rich media increased the cellular content of this fatty acid and led to a substantial decrease of the rate of phospholipid AA remodeling, which was due to reduced CoA-independent transacylase activity. Collectively, these findings provide the first evidence that cellular AA levels determine the amount of CoA-independent transacylase activity expressed by cells and provide support to the notion that CoA-IT is a major regulator of AA metabolism in human monocytes.     

Phospholipid remodeling/generation in Giardia: the role of the Lands cycle.

Recent results suggest that Giardia is able to carry out deacylation/reacylation reactions (the Lands cycle) to generate new phospholipids, effectively bypassing the de novo synthesis of the entire phospholipid molecule. The successful operation of this deacylation/reacylation cycle is important for Giardia because this protozoan parasite possesses limited lipid synthesis ability. This article discusses how Giardia might use the Lands cycle to alter phospholipids acquired from the host during its colonization in the human small intestine.     

The yeast acylglycerol acyltransferase LCA1 is a key component of Lands cycle for phosphatidylcholine turnover

Cellular phospholipids undergo deacylation and reacylation through a process known as Lands cycle. In this report, we provide evidence demonstrating that yeast YOR175c, herein designated as LCA1, encodes a key component of the Lands cycle, the acyl-CoA: lysophosphatidylcholine acyltransferase (LPCAT). Deletion of LCA1 resulted in a drastic reduction in LPCAT activity, while over expression led to a several fold increase in enzyme activity. We further show that disruption of LCA1 caused an enhanced production of glycerophosphorylcholine, a product of phosphatidylcholine (PC) deacylation and that the lysophosphatidic acid acyltransferase SLC1 was not involved in this process. Identification of LCA1 provides an essential molecular tool for further study of Lands cycle in PC turnover.     

IL-1 alpha increases arachidonyl-CoA: lysophospholipid acyltransferase activity and stimulates [3H]arachidonate incorporation into phospholipids in rat mesangial cells.

The proinflammatory cytokine interleukin-1 alpha is a potent stimulus of prostaglandin synthesis. We have previously shown that IL-1 amplifies mesangial cell prostaglandin synthesis by inducing synthesis of a non-pancreatic phospholipase A2. Phospholipase A2 activation results in the formation of lysophospholipids and free fatty acids. We now investigate the effects of IL-1 alpha on reacylation of lysophospholipids. Incubations with IL-1 alpha for 24 hours significantly stimulated mesangial cell [3H]arachidonic acid incorporation but not [3H]oleic acid incorporation into phosphatidylinositol and phosphatidylethanolamine. Lysophospholipid acyltransferase activity was measured in vitro. Cytokine treatment increased enzyme activity when lysophosphatidylcholine, lysophosphatidylethanolamine and lysophosphatidylinositol were used as exogenous substrates. We conclude that IL-1 promotes cellular phospholipid remodeling by stimulating the deacylation and reacylation of phospholipids.     

LPGAT1 controls the stearate/palmitate ratio of phosphatidylethanolamine and phosphatidylcholine in sn-1 specific remodeling

Most mammalian phospholipids contain a saturated fatty acid at the sn-1 carbon atom and an unsaturated fatty acid at the sn-2 carbon atom of the glycerol backbone group. While the sn-2 linked chains undergo extensive remodeling by deacylation and reacylation (Lands cycle), it is not known how the composition of saturated fatty acids is controlled at the sn-1 position. Here, we demonstrate that lysophosphatidylglycerol acyltransferase 1 (LPGAT1) is an sn-1 specific acyltransferase that controls the stearate/palmitate ratio of phosphatidylethanolamine (PE) and phosphatidylcholine. Bacterially expressed murine LPGAT1 transferred saturated acyl-CoAs specifically into the sn-1 position of lysophosphatidylethanolamine (LPE) rather than lysophosphatidylglycerol and preferred stearoyl-CoA over palmitoyl-CoA as the substrate. In addition, genetic ablation of LPGAT1 in mice abolished 1-LPE:stearoyl-CoA acyltransferase activity and caused a shift from stearate to palmitate species in PE, dimethyl-PE, and phosphatidylcholine. Lysophosphatidylglycerol acyltransferase 1 KO mice were leaner and had a shorter life span than their littermate controls. Finally, we show that total lipid synthesis was reduced in isolated hepatocytes of LPGAT1 knockout mice. Thus, we conclude that LPGAT1 is an sn-1 specific LPE acyltransferase that controls the stearate/palmitate homeostasis of PE and the metabolites of the PE methylation pathway and that LPGAT1 plays a central role in the regulation of lipid biosynthesis with implications for body fat content and longevity.     

Identification and characterization of a major liver lysophosphatidylcholine acyltransferase

Phosphatidylcholine (PC) is synthesized through the Kennedy pathway, but more than 50% of PC is remodeled through the Lands cycle, i.e. the deacylation and reacylation of PC to attain the final and proper fatty acids within PC. The reacylation step is catalyzed by lysophosphatidylcholine acyltransferase (LPCAT), and we report here the identification of a novel LPCAT, which we named LPCAT3. LPCAT3 belongs to the membrane-bound O-acyltransferase (MBOAT) family and encodes a protein of 487 amino acids with a calculated molecular mass of 56 kDa. Membranes from HEK293 cells overexpressing LPCAT3 showed significantly increased LPCAT activity as assessed by thin layer chromatography analysis with substrate preference toward unsaturated fatty acids. LPCAT3 is localized within the endoplasmic reticulum and is primarily expressed in metabolic tissues including liver, adipose, and pancreas. In a human hepatoma Huh7 cells, RNA interference-mediated knockdown of LPCAT3 resulted in virtually complete loss of membrane LPCAT activity, suggesting that LPCAT3 is primarily responsible for hepatic LPCAT activity. Furthermore, peroxisome proliferator-activated receptor alpha agonists dose-dependently regulated LPCAT3 in liver in a peroxisome proliferator-activated receptor alpha-dependent fashion, implicating a role of LPCAT3 in lipid homeostasis. Our studies identify a long-sought enzyme that plays a critical role in PC remodeling in metabolic tissues and provide an invaluable tool for future investigations on how PC remodeling may potentially impact glucose and lipid homeostasis.     

Docosahexaenoic acid enrichment can reduce L929 cell necrosis induced by tumor necrosis factor

We previously reported that docosahexaenoic acid (DHA) attenuated tumor necrosis factor (TNF)-induced apoptosis in human monocytic U937 cells (J. Nutr. 130: 1095-1101, 2000). In the present study, we examined the effects of DHA and other polyunsaturated fatty acids (PUFA) on TNF-induced necrosis, another mode of cell death, using L929 murine fibrosarcoma cells. After preincubation with PUFA conjugated with BSA for 24 h, cells were treated with TNF or TNF+actinomycin D (Act D). Preincubation of cells with DHA enriched this polyunsaturated acid in the phospholipids and attenuated cell death induced by either TNF or TNF+Act D. When cells were treated with TNF alone, DNA laddering was not detected, and cells were coincidently stained with both annexin V-FITC and propidium iodide, indicating that the death mode was necrotic. TNF+Act D predominantly induced necrosis, although concurrent apoptotic cell death was also observed in this case. Preincubation with oleic acid, linoleic acid or 20:3(n-3) did not affect TNF-induced necrosis. Conversely, supplementation with n-3 docosapentaenoic acid (DPAn-3) or eicosapentaenoic acid (EPA) reduced necrotic cell death, but to a lesser extent in comparison with DHA. Unlike the case of U937 cell apoptosis, arachidonic acid (AA) significantly attenuated L929 cell necrosis, and 20:3(n-6) or 22:4(n-6) showed similar or less activity, respectively. Statistical evaluation indicated that the order of effective PUFA activity was DHA>DPAn-3> or =EPA>AA approximately 20:3(n-6)> or =22:4(n-6). One step desaturation, C2 elongation or C2 cleavage within the n-6 or n-3 fatty acid group was probably very active in L929 cells, because AA, synthesized from 20:3(n-6) or 22:4(n-6), and C22 fatty acids, synthesized from AA or EPA, were preferentially retained in cellular phospholipids. These observations suggested that attenuation of TNF-induced necrosis by the supplementation of various C20 or C22 polyunsaturated fatty acids is mainly attributable to the enrichment of three kinds of polyunsaturated fatty acids, i.e., DHA, DPAn-3 or AA, in phospholipids. Among these fatty acids, DHA was the most effective in the reduction of L929 necrosis as observed in the case of U937 apoptosis. This suggests that DHA-enriched membranes can protect cell against TNF irrespective of death modes and that membranous DHA may abrogate the death signaling common to necrosis and apoptosis.     

Control of arachidonic acid accumulation in bone marrow-derived macrophages by acyltransferases

The turnover of phospholipid fatty acid moieties of bone marrow-derived macrophages was analyzed by separate determination of degrading and acylating activities. Acylating activities were followed in intact cells by incubation with excess arachidonic acid and degradation of phospholipids was followed in cells prelabeled with fatty acids. Significant phospholipase A2 activity was detectable only if the reutilization of liberated fatty acid was inhibited , e.g. by p-chloromercuribenzoate. It was of interest that the divalent cation ionophore A 23187 and various antiphlogistic drugs like indomethacin, diclofenac, and acetylsalicylic acid were found to inhibit the acylation reaction. These compounds led to increased levels of free arachidonic acid in stimulated, as well as in unstimulated cells. Increased activities of phospholipase A2 were achieved by treatment with the bivalent cation ionophore A 23187 and with zymosan. The effect of zymosan obtained from various sources was found to be exclusively due to contamination of tee zymosan particles with phospholipase A2 activity. Even when the cellular phospholipase activity was increased by the addition of exogenous phospholipase activity contained in the zymosan particles, degradation of cellular phospholipids was not measurable unless the reacylation was inhibited. These results suggest that in the cells studied, the level of free arachidonic acid is mainly controlled by the activity of the lysophosphatide acyltransferase.     

Fatty acid modulation of MCF-7 human breast cancer cell proliferation,apoptosis and differentiation

Epidemiological studies suggest that dietary polyunsaturated fatty acids (PUFA) may influence breast cancer progression and prognosis. In order to study potential mechanisms of action of fatty acid modulation of tumor growth, we studied, in vitro, the influence of n-3 and n-6 fatty acids on proliferation, cell cycle, differentiation and apoptosis of MCF-7 human breast cancer cells. Both eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) inhibited the MCF-7 cell growth by 30% and 54%, respectively, while linoleic acid (LA) had no effect and arachidonic acid (AA) inhibited the cell growth by 30% (p < 0.05). The addition of vitamin E (10uM) to cancer cells slightly restored cell growth. The incubation of MCF-7 cells with PUFAs did not alter the cell cycle parameters or induce cell apoptosis. However, the growth inhibitory effects of EPA, DHA and AA were associated with cell differentiation as indicated by positive Oil-Red-O staining of the cells. Lipid droplet accumulation was increased by 65%, 30% and 15% in the presence of DHA, EPA and AA, respectively; (p < 0.05). These observations suggest that fatty acids may influence cellular processes at a molecular level, capable of modulating breast cancer cell growth.     

Inhibitors of arachidonic acid metabolism act synergistically to signal apoptosis in neoplastic cells

Arachidonic acid (AA) and its metabolites are intimately linked to carcinogenesis. Inhibitors of AA metabolic enzymes have demonstrated anti-carcinogenic effects in vivo and induce apoptosis of many cancer cell lines in vitro. The mechanism by which AA influences carcinogenesis, however, remains unresolved. The current study explores the growth inhibitory potential of Triacsin C, PLT-98625, and NS-398 which inhibit three distinct metabolic enzymes that control intracellular AA levels: fatty acid coenzyme-A ligase 4 (FACL-4), coenzyme-A independent transacylase (CoA-IT), and cyclooxygenase (COX), respectively. Results reveal the anti-proliferative effects of these inhibitors in a number of human cancer cell lines. Further studies in the SK-MES-1 cell line demonstrate that all three inhibitors induce accumulation of unesterified AA which correlates with induction of apoptosis. Addition of exogenous AA also induces apoptosis. Furthermore, in combination, these inhibitors act cooperatively to induce AA accumulation which correlates to a synergistic reduction in cell viability. Taken together, these results suggest that accumulation of unesterified AA is a common mechanism in the induction of cancer cell apoptosis by various inhibitors of AA metabolism, confirm that previously described AA remodeling pathways are valid in cancer cells, and indicate that combination treatment strategies utilizing these inhibitors may represent a novel approach to blocking cancer cell growth. Further study is required to determine the downstream pathway(s) whereby high cellular burdens of unesterified AA promote apoptosis.     

Phospholipid remodeling in human neutrophils. Parallel activation of a deacylation/reacylation cycle and platelet-activating factor synthesis

A23187 stimulated two enzymatic activities of human neutrophils (polymorphonuclear leukocytes), phospholipase A2 and fatty acyl-CoA acyltransferase, which resulted in a stimulated deacylation/reacylation cycle. The incorporation of fatty acids, other than arachidonic or eicosapentaenoic acid, into diacyl and alkylacyl species of choline phosphoglycerides was stimulated by 10-fold by A23187. These fatty acids were exclusively incorporated into the sn-2 position, and [3H]glycerol labeling showed there was no stimulation of de novo synthesis. A23187 also stimulated fatty acid incorporation into other phospholipids, but de novo synthesis accounted for a portion of this uptake. Inhibitors of protein kinase C prevented the stimulated recycling of phosphatidylcholine, and the simultaneous induction of platelet-activating factor synthesis, by inhibiting phospholipase A2 activation. They inhibited [3H]arachidonate release from prelabeled polymorphonuclear leukocytes, but had no effect on in vitro fatty acyl-CoA acyltransferase or acetyl-CoA acetyltransferase activity. Extracts from A23187-treated cells contained a fatty acyl-CoA acyltransferase, which did not utilize arachidonoyl-CoA, that was 2.3-fold more active than that of control extracts. Phosphatase treatment decreased this stimulated activity by 66%. Thus, A23187 stimulated a phospholipase A2 activity that generated both 1-alkyl and 1-acyl lysophosphatidylcholines. A stimulated acetyltransferase used a portion of the alkyl species for platelet-activating factor synthesis, while the acyl species and residual alkyl species were rapidly reacylated to phosphatidylcholine by a stimulated acyl-transferase. Arachidonate, an eicosanoid precursor, was spared by this process.     

Evidence for the Involvement of Carnitine-Dependent Long-Chain Acyltransferases in Neuronal Triglyceride and Phospholipid Fatty Acid Turnover

Abstract: This study focuses on the potential involvement of carnitine palmitoyltransferase (CRT) on the phospholipid and triglyceride fatty acid turnover in neurons. This category of enzymes, which has been identified in several rat brain tissues, is well known for its role in modulating cellular fatty acid oxidation. Neuronal cell cultures from rat brain cortex incorporated radioactive palmitate or oleate into phospholipids and triglycerides. The largest fraction of radioactive fatty acids was recovered in phosphatidyl- choline followed by triglycerides and, to a lesser extent, phosphatidylethanolamine. CPT activity measured in neuronal lysates obtained from neurons treated with 40 μ M 2-tetradecylglycidic acid (TDGA) was almost completely abolished. Furthermore, between 2 and 10 μ M TDGA CPT activity dropped more rapidly than between 10 and 40 μ M. When the cells were pretreated with TDGA, the incorporation process of either radioactive fatty acid into triglycerides was dose-dependently suppressed. Radioactive fatty acid incorporation into phosphatidylcholine was significantly decreased in cells treated with TDGA. In contrast, phosphatidylethanolamine reacylation was essentially not affected by the CpT inhibitor. Similar results on the fatty acid incorporation into triglycerides and phospholipids were observed with neurons treated with palmitoyl- dl - aminocarnitine (PAC), a reversible CPT inhibitor, which does not consume free CoA. These effects do not seem to be the result of an inhibitory activity toward one of the steps involved in the acylation-deacylation process of triglycerides or phospholipids, as cellular lysates from TDGA-treated cells or lysates containing PAC incorporated radioactive fatty acids at rates comparable to controls. Our results suggest that CRT may be an important partner in the pathway of phospholipid and triglyceride fatty acid turnover in neurons.     

CWH43 is required for the introduction of ceramides into GPI anchors in Saccharomyces cerevisiae

After glycosylphosphatidylinositols (GPIs) are added to GPI proteins of Saccharomyces cerevisiae, the fatty acid in sn-2 of the diacylglycerol moiety can be replaced by a C26:0 fatty acid by a deacylation-reacylation cycle catalysed by Per1p and Gup1p. Furthermore the diacylglycerol moiety of the yeast GPI anchor can also be replaced by ceramides. CWH43 of yeast is homologous to PGAP2, a gene that recently was implicated in a similar deacylation reacylation cycle of GPI proteins in mammalian cells, where PGAP2 is required for the reacylation of monoradylglycerol-type GPI anchors. Here we show that mutants lacking CWH43 are unable to synthesize ceramide-containing GPI anchors, while the replacement of C18 by C26 fatty acids on the primary diacylglycerol anchor by Per1p and Gup1p is still intact. CWH43 contains the COG3568 metal hydrolase motif, which is found in many eukaryotic and prokaryotic enzymes. The conserved His 802 residue of this motif was identified as being essential for ceramide remodelling. Ceramide remodelling is not required for the normal integration of GPI proteins into the cell wall. All remodelling reactions are dependent on prior removal of the inositol-linked fatty acid by Bst1p.     

Role of cytochrome P450 in phospholipase A2- and arachidonic acid-mediated cytotoxicity

Phospholipases A2 (PLA2) comprise a set of extracellular and intracellular enzymes that catalyze the hydrolysis of the sn-2 fatty acyl bond of phospholipids to yield fatty acids and lysophospholipids. The PLA2 reaction is the primary pathway through which arachidonic acid (AA) is released from phospholipids. PLA2s have an important role in cellular death that occurs via necrosis or apoptosis. Several reports support the hypothesis that unesterified arachidonic acid in cells is a signal for the induction of apoptosis. However, most of the biological effects of arachidonic acid are attributable to its metabolism by mainly three different groups of enzymes: cytochromes P450, cyclooxygenases, and lipoxygenases. In this review we will focus on the role of cytochrome P450 in AA metabolism and toxicity. The major pathways of arachidonic acid metabolism catalyzed by cytochrome P450 generate metabolites that are subdivided into two groups: the epoxyeicosatrienoic acids, formed by CYP epoxygenases, and the arachidonic acid derivatives that are hydroxylated at or near the omega-terminus by CYP omega-oxidases. In addition, autoxidation of AA by cytochrome P450-derived reactive oxygen species produces lipid hydroperoxides as primary oxidation products. In some cellular models of toxicity, cytochrome P450 activity exacerbates PLA2- and AA-dependent injury, mainly through the production of oxygen radicals that promote lipid peroxidation or production of metabolites that alter Ca2+ homeostasis. In contrast, in other situations, cytochrome P450 metabolism of AA is protective, mainly by lowering levels of unesterified AA and by production of metabolites that activate antiapoptotic pathways. Several lines of evidence point to the combined action of phospholipase A2 and cytochrome P450 as central in the mechanism of cellular injury in several human diseases, such as alcoholic liver disease and myocardial reperfusion injury. Inhibition of specific PLA2 and cytochrome P450 isoforms may represent novel therapeutic strategies against these diseases.     

Differential turnover of phospholipid acyl groups in mouse peritoneal macrophages

Phospholipid acyl turnover was assessed in mouse peritoneal exudate cells which consisted primarily of macrophages. The cells were incubated for up to 5 h in media containing 40% H218O, and uptake of 18O into ester carbonyls of phospholipids was determined by gas chromatography-mass spectrometry of hydrogenated methyl esters. The uptake was highest in choline phospholipids and phosphatidylinositol, less in ethanolamine phospholipids, and much less in phosphatidylserine. Acyl groups at the sn-1 and sn-2 positions of diacyl glycerophospholipids, including arachidonic and other long-chain polyunsaturated fatty acids, acquired 18O at about the same rate. Acyl groups of alkylacyl glycerophosphocholine exhibited lower rates of 18O uptake, and acyl groups of ethanolamine plasmalogens (alkenylacyl glycerophosphoethanolamines) acquired only minimal amounts of 18O within 5 h, indicating a low average acyl turnover via free fatty acids. Pulse experiments with exogenous 3H-labeled arachidonic acid supported the concept that acylation of alkenyl glycerophosphoethanolamine occurs by acyl transfer from other phospholipids rather than via free fatty acids and acyl-CoA. The 18O content of intracellular free fatty acids increased gradually over a 5-h period, whereas in extracellular free fatty acids it reached maximal 18O levels within the first hour. Arachidonate and other long-chain polyunsaturated fatty acids were found to participate readily in deacylation-reacylation reactions but were present only in trace amounts in the free fatty acid pools inside and outside the cells. We conclude that acyl turnover of macrophage phospholipids through hydrolysis and reacylation is rapid but tightly controlled so that appreciable concentrations of free arachidonic acid do not occur.     

Role of carnitine and carnitine palmitoyltransferase as integral components of the pathway for membrane phospholipid fatty acid turnover in intact human erythrocytes.

The deacylation and reacylation process of phospholipids is the major pathway of turnover and repair in erythrocyte membranes. In this paper, we have investigated the role of carnitine palmitoyltransferase in erythrocyte membrane phospholipid fatty acid turnover. The role of acyl-L-carnitine as a reservoir of activated acyl groups, the buffer function of carnitine, and the importance of the acyl-CoA/free CoA ratio in the reacylation process of erythrocyte membrane phospholipids have also been addressed. In intact erythrocytes, the incorporation of [1-14C]palmitic acid into acyl-L-carnitine, phosphatidylcholine, and phosphatidylethanolamine was linear with time for at least 3 h. The greatest proportion of the radioactivity was found in acyl-L-carnitine. Competition experiments using [1-14C]palmitic and [9,10-3H]oleic acid demonstrated that [9,10-3H]oleic acid was incorporated preferentially into the phospholipids and less into acyl-L-carnitine. When an erythrocyte suspension was incubated with [1-14C]palmitoyl-L-carnitine, radiolabeled palmitate was recovered in the phospholipid fraction, and the carnitine palmitoyltransferase inhibitor, 2-tetradecylglycidic acid, completely abolished the incorporation. ATP depletion decreased incorporation of [1-14C]palmitic and/or [9,10-3H]oleic acid into acyl-L-carnitine, but the incorporation into phosphatidylcholine and phosphatidylethanolamine was unaffected. In contrast, ATP depletion enhanced the incorporation into phosphatidylcholine and phosphatidylethanolamine of the radiolabeled fatty acid from [1-14C]palmitoyl-L-carnitine. These data are suggestive of the existence of an acyl-L-carnitine pool, in equilibrium with the acyl-CoA pool, which serves as a reservoir of activated acyl groups. The carnitine palmitoyltransferase inhibition by 2-tetradecylglycidic acid or palmitoyl-D-carnitine caused a significant reduction of radiolabeled fatty acid incorporation into membrane phospholipids, only when intact erythrocytes were incubated with [9,10-3H]oleic acid. These latter data may be explained by the differences in rates and substrates specificities between acyl-CoA synthetase and the reacylating enzymes for palmitate and oleate, which support the importance of carnitine palmitoyltransferase in modulating the optimal acyl-CoA/free CoA ratio for the physiological expression of the membrane phospholipids fatty acid turnover.     

Fatty acid oxidation and signaling in apoptosis

It is well established that fatty acid metabolites of cyclooxygenase, lipoxygenase (LOX), and cytochrome P450 are implicated in essential aspects of cellular signaling including the induction of programmed cell death. Here we review the roles of enzymatic and non-enzymatic products of polyunsaturated fatty acids in controlling cell growth and apoptosis. Also, the spontaneous oxidation of polyunsaturated fatty acids yields reactive aldehydes and other products of lipid peroxidation that are potentially toxic to cells and may also signal apoptosis. Significant conflicting data in terms of the role of LOX enzymes are highlighted, prompting a re-evaluation of the relationship between LOX and prostate cancer cell survival. We include new data showing that LNCaP, PC3, and Du145 cells express much lower levels of 5-LOX mRNA and protein compared with normal prostate epithelial cells (NHP2) and primary prostate carcinoma cells (TP1). Although the 5-LOX activating protein inhibitor MK886 killed these cells, another 5-LOX inhibitor AA861 hardly showed any effect. These observations suggest that 5-LOX is unlikely to be a prostate cancer cell survival factor, implying that the mechanisms by which LOX inhibitors induce apoptosis are more complex than expected. This review also suggests several mechanisms involving peroxisome proliferator activated receptor activation, BCL proteins, thiol regulation, and mitochondrial and kinase signaling by which cell death may be produced in response to changes in non-esterified and non-protein bound fatty acid levels. Overall, this review provides a context within which the effects of fatty acids and fatty acid oxidation products on signal transduction pathways, particularly those involved in apoptosis, can be considered in terms of their overall importance relative to the much better studied protein or peptide signaling factors.     

Fatty acid remodelling of phosphatidylinositol under conditions of de novo synthesis in rat liver microsomes

Phosphatidylinositol (PI) is initially synthesized in mammalian cells with a fatty acid composition similar to that of its precursor, primarily monounsaturated forms of cytidine diphosphodiglyceride (CDP-DAG). However, at the steady state, over 80% of PI exists in the 1-stearoyl, 2-arachidonoyl form. The fatty acid remodelling of PI is due to a number of deacylation/reacylation mechanisms. In the preceding paper we demonstrated that de novo synthesized PI is rapidly deacylated and subsequently reacylated. In this report we present further evidence that cycles of deacylation and reacylation are involved in the remodelling of PI. Incubation of microsomes with CDP-DAG of different fatty acid composition results in quantitative and qualitative differences in lysoPI formation. Additionally, analyses of the resulting lysoPI and PI species reveal that multiple species of fatty acids are incorporated into the 1-position of both PI and lysoPI. Addition of acylation cofactors (fatty acyl CoAs or ATP plus CoA) potentiate reacylation in this system. The addition of stearoyl or myristoyl CoA during de novo synthesis of PI results in the incorporation of these added fatty acids into the I-positive of PI. In addition, some evidence is presented that multiple mechanisms for remodelling of the 1-position of PI may be active in the microsomes, including ATP- and CoA-dependent acylation, ATP-independent, CoA-dependent acylation and CoA-independent mechanisms. Finally, the disappearance of only a subset of lysoPI species upon the addition of acylation cofactors suggests that the reacylation step exhibits some substrate specificity.     

Control of free arachidonic acid levels by phospholipases A2 and lysophospholipid acyltransferases

Arachidonic acid (AA) and its oxygenated derivatives, collectively known as the eicosanoids, are key mediators of a wide variety of physiological and pathophysiological states. AA, obtained from the diet or synthesized from linoleic acid, is rapidly incorporated into cellular phospholipids by the concerted action of arachidonoyl-CoA synthetase and lysophospholipid acyltransferases. Under the appropriate conditions, AA is liberated from its phospholipid storage sites by the action of one or various phospholipase A₂ enzymes. Thus, cellular availability of AA, and hence the amount of eicosanoids produced, depends on an exquisite balance between phospholipid reacylation and hydrolysis reactions. This review focuses on the enzyme families that are involved in these reactions in resting and stimulated cells.     

Degradation of Phospholipids and Liquefaction of Pressed Baker’s Yeast by Acetic Acid Vapour

When pressed baker’s yeast (Saccharomyces cerevisiae) was exposed to the vapour of acetic acid, autolysis of yeast cells was induced in 3 or 4 hr. In order to elucidate the mechanism of the autolysis caused by the AcOH-treatment, we investigated variations in the lipid content of yeast cells during the treatment. The degradation of phospholipids and the accumulation of free fatty acids occurred within 3 hr. Formic acid exerted a similar effect on the pressed yeast. The effect of propionic acid was not seen in 3hr but was after 18 hr. When the homogenate of fresh yeast cells was incubated in the acidic region below pH 4.5 for 1 hr, phospholipids were hydrolyzed and free fatty acids were accumulated. Such deacylation of phospholipids was observed even at pH 6 on incubation for 12hr, but not observed at pH 7 or above pH 9. At pH 8, although phospholipids were somewhat degraded, free fatty acids almost never accumulated but diacylglycerol did accumulate.

Therefore, yeast cells have inherently phospholipid-acylhydrolases and, on AcOH-treatment, such enzymes may degrade membrane phospholipids to induce the autolysis of pressed yeast.