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.     

Human lysophosphatidylcholine acyltransferases 1 and 2 are located in lipid droplets where they catalyze the formation of phosphatidylcholine

Phosphatidylcholine (PC) is synthesized by two different pathways, the Lands cycle and the Kennedy pathway. The recently identified key enzymes of the Lands cycle, lysophosphatidylcholine acyltransferase 1 and 2 (LPCAT1 and -2), were reported to localize to the endoplasmic reticulum and to function in lung surfactant production and in inflammation response. Here, we show in various mammalian cell lines that both enzymes additionally localize to lipid droplets (LDs), which consist of a core of neutral lipids surrounded by a monolayer of phospholipid, mainly PC. This dual localization is enabled by the monotopic topology of these enzymes demonstrated in this study. Furthermore, we show that LDs have the ability to locally synthesize PC and that this activity correlates with the LPCAT1 and -2 expression level. This suggests that LPCAT1 and -2 have, in addition to their known function in specialized cells, a ubiquitous role in LD-associated lipid metabolism.     

Lysophosphatidylcholine acyltransferase 3 deficiency impairs 3T3L1 cell adipogenesis through activating Wnt/β-catenin pathway

Levels of polyunsaturated phosphatidylcholine (PC) influence plasma membrane structure and function. Phosphatidylcholine (PC) is synthesized de novo in the Kennedy pathway and then undergoes extensive deacylation/reacylation remodeling via Lands' cycle (non-Kennedy pathway). The reacylation is catalyzed by lysophosphatidylcholine acyltransferase (LPCAT), which adds a polyunsaturated fatty acid at the sn-2 position. Four LPCAT isoforms have been described to date, among which we found LPCAT3 to be the major isoform in adipose tissue, but its exact role in adipogenesis is unclear. In this study, we aimed to investigate whether LPCAT3 activity affects 3T3L1 cell adipogenic differentiation potential and its underline mechanism. Lentivirus-mediated LPCAT3 shRNA expression stably knocked down LPCAT3 in 3T3L1 preadipocytes and LPCAT3 deficiency dramatically reduced the levels of cellular polyunsaturated PCs. Importantly, we found that this deficiency activated the β-catenin dependent Wnt signaling pathway, which suppressed the expression of adipogenesis-related genes, thereby inhibiting 3T3L1 preadipocyte differentiation and lipid accumulation. Moreover, three different Wnt/β-catenin pathway inhibitors reversed the effect of LPCAP3 deficiency, suggesting that Wnt/β-catenin pathway activation is one of the causes for the observed phenotypes. To the best of our knowledge, we show here for the first time that PC remodeling is an important regulator of adipocyte differentiation.     

Selective inhibitors of a PAF biosynthetic enzyme lysophosphatidylcholine acyltransferase 2

Platelet-activating factor (PAF) is a potent pro-inflammatory phospholipid mediator. In response to extracellular stimuli, PAF is rapidly biosynthesized by lyso-PAF acetyltransferase (lyso-PAFAT). Previously, we identified two types of lyso-PAFATs: lysophosphatidylcholine acyltransferase (LPCAT)1, mostly expressed in the lungs where it produces PAF and dipalmitoyl-phosphatidylcholine essential for respiration, and LPCAT2, which biosynthesizes PAF and phosphatidylcholine (PC) in the inflammatory cells. Under inflammatory conditions, LPCAT2, but not LPCAT1, is activated and upregulated to produce PAF. Thus, it is important to develop inhibitors specific for LPCAT2 in order to ameliorate PAF-related inflammatory diseases. Here, we report the first identification of LPCAT2-specific inhibitors, N-phenylmaleimide derivatives, selected from a 174,000-compound library using fluorescence-based high-throughput screening followed by the evaluation of the effects on LPCAT1 and LPCAT2 activities, cell viability, and cellular PAF production. Selected compounds competed with acetyl-CoA for the inhibition of LPCAT2 lyso-PAFAT activity and suppressed PAF biosynthesis in mouse peritoneal macrophages stimulated with a calcium ionophore. These compounds had low inhibitory effects on LPCAT1 activity, indicating that adverse effects on respiratory functions may be avoided. The identified compounds and their derivatives will contribute to the development of novel drugs for PAF-related diseases and facilitate the analysis of LPCAT2 functions in phospholipid metabolism in vivo.     

LYCAT, a homologue of C. elegans acl-8, acl-9, and acl-10, determines the fatty acid composition of phosphatidylinositol in mice

Mammalian phosphatidylinositol (PI) has a unique fatty acid composition in that 1-stearoyl-2-arachidonoyl species is predominant. This fatty acid composition is formed through fatty acid remodeling by sequential deacylation and reacylation. We recently identified three Caenorhabditis elegans acyltransferases (ACL-8, ACL-9, and ACL-10) that incorporate stearic acid into the sn-1 position of PI. Mammalian LYCAT, which is the closest homolog of ACL-8, ACL-9, and ACL-10, was originally identified as a lysocardiolipin acyltransferase by an in vitro assay and was subsequently reported to possess acyltransferase activity toward various anionic lysophospholipids. However, the in vivo role of mammalian LYCAT in phospholipid fatty acid metabolism has not been well elucidated. In this study, we generated LYCAT-deficient mice and demonstrated that LYCAT determined the fatty acid composition of PI in vivo. LYCAT-deficient mice were outwardly healthy and fertile. In the mice, stearoyl-CoA acyltransferase activity toward the sn-1 position of PI was reduced, and the fatty acid composition of PI, but not those of other major phospholipids, was altered. Furthermore, expression of mouse LYCAT rescued the phenotype of C. elegans acl-8 acl-9 acl-10 triple mutants. Our data indicate that LYCAT is a determinant of PI molecular species and its function is conserved in C. elegans and mammals.     

In Vivo and in Vitro Evidence for Biochemical Coupling of Reactions Catalyzed by Lysophosphatidylcholine Acyltransferase and Diacylglycerol Acyltransferase

Seed oils of flax (Linum usitatissimum L.) and many other plant species contain substantial amounts of polyunsaturated fatty acids (PUFAs). Phosphatidylcholine (PC) is the major site for PUFA synthesis. The exact mechanisms of how these PUFAs are channeled from PC into triacylglycerol (TAG) needs to be further explored. By using in vivo and in vitro approaches, we demonstrated that the PC deacylation reaction catalyzed by the reverse action of acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT) can transfer PUFAs on PC directly into the acyl-CoA pool, making these PUFAs available for the diacylglycerol acyltransferase (DGAT)-catalyzed reaction for TAG production. Two types of yeast mutants were generated for in vivo and in vitro experiments, respectively. Both mutants provide a null background with no endogenous TAG forming capacity and an extremely low LPCAT activity. In vivo experiments showed that co-expressing flax DGAT1-1 and LPCAT1 in the yeast quintuple mutant significantly increased 18-carbon PUFAs in TAG with a concomitant decrease of 18-carbon PUFAs in phospholipid. We further showed that after incubation of sn-2-[¹⁴C]acyl-PC, formation of [¹⁴C]TAG was only possible with yeast microsomes containing both LPCAT1 and DGAT1-1. Moreover, the specific activity of overall LPCAT1 and DGAT1-1 coupling process exhibited a preference for transferring ¹⁴C-labeled linoleoyl or linolenoyl than oleoyl moieties from the sn-2 position of PC to TAG. Together, our data support the hypothesis of biochemical coupling of the LPCAT1-catalyzed reverse reaction with the DGAT1-1-catalyzed reaction for incorporating PUFAs into TAG. This process represents a potential route for enriching TAG in PUFA content during seed development in flax.     

Identification of Brassica napus lysophosphatidylcholine acyltransferase genes through yeast functional screening

Acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT), which acylates lysophosphatidylcholine (LPC) to produce phosphatidylcholine (PC), is a key enzyme in the Lands cycle. There is evidence that acyl exchange involving LPCAT is a prevailing metabolic process during triacylglycerol (TAG) synthesis in seeds. In this study, by complementing the yeast lca1Δ mutant deficient in LPCAT activity with an Arabidopsis seedling cDNA library, it was found that the previously reported lysophospholipid acyltransferases (LPLATs), At1g12640 and At1g63050, were the only two acyltransferase genes that restored hyposensitivity of the lca1Δ mutant to lyso-platelet-activating factor (lyso-PAF). A developing seed cDNA library from Brassica napus L. cv Hero was constructed to further explore the heterologous yeast complementation approach. Three B. napus LPCAT homologs were identified, of which BnLPCAT1-1 and BnLPCAT1-2 are orthologous to Arabidopsis AtLPLAT1 (At1g12640) while BnLPCAT2 is an ortholog of AtLPLAT2 (At1g63050). The proteins encoded by BnLPCAT1-1 and BnLPCAT2 were chosen for further study. Enzymatic assays demonstrated that both proteins exhibited a substrate preference for LPCs and unsaturated fatty acyl-CoAs. In addition to the enzymatic properties of plant lysophosphatidylcholine acyltransferases uncovered in this study, this report describes a useful technique that facilitates subsequent analyses into the role of LPCATs in PC turnover and seed oil biosynthesis.     

A family of eukaryotic lysophospholipid acyltransferases with broad specificity

The budding yeast ALE1 gene encodes a lysophospholipid acyltransferase (LPLAT) with broad specificity. We show that yeast LPLAT (ScLPLAT) belongs to a distinct protein family that includes human MBOAT1, MBOAT2, MBOAT4, and several closely related proteins from other eukaryotes. We further show that two plant proteins within this family, the Arabidopsis proteins AtLPLAT1 and AtLPLAT2, possess lysophospholipid acyltransferase activities similar to ScLPLAT. We propose that other members of this protein family, which we refer to as the LPLAT family, also are likely to possess LPLAT activity. Finally, we show that ScLPLAT differs from the specific lysophosphatidic acid acyltransferase that is encoded by SLC1 in that it cannot efficiently use lysophosphatidic acid produced by acylation of glycerol-3-phosphate in vitro.     

Biosynthesis of phosphatidylcholine by human lysophosphatidylcholine acyltransferase 1

Pulmonary surfactant is a complex of phospholipids and proteins lining the alveolar walls of the lung. It reduces surface tension in the alveoli, and is critical for normal respiration. Pulmonary surfactant phospholipids consist mainly of phosphatidylcholine (PC) and phosphatidylglycerol (PG). Although the phospholipid composition of pulmonary surfactant is well known, the enzyme(s) involved in its biosynthesis have remained obscure. We previously reported the cloning of murine lysophosphatidylcholine acyltransferase 1 (mLPCAT1) as a potential biosynthetic enzyme of pulmonary surfactant phospholipids. mLPCAT1 exhibits lysophosphatidylcholine acyltransferase (LPCAT) and lysophosphatidylglycerol acyltransferase (LPGAT) activities, generating PC and PG, respectively. However, the enzymatic activity of human LPCAT1 (hLPCAT1) remains controversial. We report here that hLPCAT1 possesses LPCAT and LPGAT activities. The activity of hLPCAT1 was inhibited by N-ethylmaleimide, indicating the importance of some cysteine residue(s) for the catalysis. We found a conserved cysteine (Cys²¹¹) in hLPCAT1 that is crucial for its activity. Evolutionary analyses of the close homologs of LPCAT1 suggest that it appeared before the evolution of teleosts and indicate that LPCAT1 may have evolved along with the lung to facilitate respiration. hLPCAT1 mRNA is highly expressed in the human lung. We propose that hLPCAT1 is the biosynthetic enzyme of pulmonary surfactant 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 of a novel GPCAT activity and a new pathway for phosphatidylcholine biosynthesis in S. cerevisiae

Turnover of phospholipids in the yeast Saccharomyces cerevisiae generates intracellular glycerophosphocholine (GPC). Here we show that GPC can be reacylated in an acyl-CoA-dependent reaction by yeast microsomal membranes. The lysophosphatidylcholine that is formed in this reaction is efficiently further acylated to phosphatidylcholine (PC) by yeast microsomes, thus providing a new pathway for PC biosynthesis that can either recycle endogenously generated GPC or utilize externally provided GPC. Genetic and biochemical evidence suggests that this new enzymatic activity, which we call GPC acyltransferase (GPCAT), is not mediated by any of the previously known acyltransferases in yeast. The GPCAT activity has an apparent V(max) of 8.7 nmol/min/mg protein and an apparent K(m) of 2.5 mM. It has a neutral pH optimum, similar to yeast glycerol-3-phosphate acyltransferase, but differs from the latter in being more heat stable. The GPCAT activity is sensitive to N-ethylmaleimide, phenanthroline, and Zn(2+) ions. In vivo experiments showed that PC is efficiently labeled when yeast cells are fed with [(3)H]choline-GPC, and that this reaction occurs also in pct1 knockout strains, where de novo synthesis of PC by the CDP-choline pathway is blocked. This suggests that GPCAT can provide an alternative pathway for PC biosynthesis in vivo.     

Deciphering the roles of Arabidopsis LPCAT and PAH in phosphatidylcholine homeostasis and pathway coordination for chloroplast lipid synthesis

Phosphatidylcholine (PC) is a key intermediate in the metabolic network of glycerolipid biosynthesis. Lysophosphatidylcholine acyltransferase (LPCAT) and phosphatidic acid phosphatase (PAH) are two key enzymes of PC homeostasis. We report that LPCAT activity is markedly induced in the Arabidopsis pah mutant. The quadruple pah lpcat mutant, with dual defects in PAH and LPCAT, had a level of lysophosphatidylcholine (LPC) that was much higher than that in the lpcat mutants and a PC content that was higher than that in the pah mutant. Comparative molecular profile analysis of monogalactosyldiacylglycerol and digalactosyldiacylglycerol revealed that both the pah and pah lpcat mutants had increased proportions of 34:6 from the prokaryotic pathway despite differing levels of LPCAT activity. We show that a decreased representation of the C_16:0C_18:2 diacylglycerol moiety in PC was a shared feature of pah and pah lpcat, and that this change in PC metabolic profile correlated with the increased prokaryotic contribution to chloroplast lipid synthesis. We detected increased PC deacylation in the pah lpcat mutant that was attributable at least in part to the induced phospholipases. Increased LPC generation was also evident in the pah mutant, but the phospholipases were not induced, raising the possibility that PC deacylation is mediated by the reverse reaction of LPCAT. We discuss possible roles of LPCAT and PAH in PC turnover that impacts lipid pathway coordination for chloroplast lipid synthesis.     

In vitro study of docosahexaenoic acid incorporation into phosphatidylcholine by enzymes of rat heart

Several studies have shown that in animals fed fish oils, docosahexaenoic acid (DHA) is incorporated into cardiac phosphatidylcholines (PC) mainly at the expense of arachidonic acid. In this study we were interested in examining if the enzymatic system involved in the remodeling of membrane PC presented any selectivity for DHA in rat heart. The enzymes that were studied from sequential incubations carried out in parallel, were acyl-CoA synthetase (EC 6.2.1.3) and acyl-CoA:lysophosphatidylcholine acyltransferase (EC 2.3.1.23) (ACLAT). The heart preparations examined were homogenates of whole heart and of purified cultured rat ventricular myocytes.Results showed that ACLAT tended to preferentially incorporate into PC the polyunsaturated fatty acids of the n-6 series (+30%) rather than those of the n-3 series. DHA, however, inhibited the incorporation of arachidonic acid (AA) into PC by 50% at a molar ratio (DHA/AA) of 1.5. This phenomenon seems to be related to the competitive inhibition exerted by DHA on the thio-esterification of AA, a reaction catalyzed by acyl-CoA synthetase. This inhibitory effect appears to be dependent on the kinetic properties of the acyl-CoA synthetase toward DHA which, among the fatty acids examined, exhibited the lowest apparent Km and Vmax.It is suggested that the intracellular pool of DHA-CoA is the determinant species in altering the DHA composition of cardiac PC in animals given fish oils.     

A novel lysophosphatidic acid acyltransferase enzyme (LPAAT4) with a possible role for incorporating docosahexaenoic acid into brain glycerophospholipids

Glycerophospholipids are important components of cellular membranes, required for constructing structural barriers, and for providing precursors of bioactive lipid mediators. Lysophosphatidic acid acyltransferases (LPAATs) are enzymes known to function in the de novo glycerophospholipid biosynthetic pathway (Kennedy pathway), using lysophosphatidic acid (LPA) and acyl-CoA to form phosphatidic acid (PA). Until now, three LPAATs (LPAAT1, 2, and 3) have been reported from the 1-acyl-glycerol-3-phosphate O-acyltransferase (AGPAT) family. In this study, we identified a fourth LPAAT enzyme, LPAAT4, previously known as an uncharacterized enzyme AGPAT4 (LPAATδ), from the AGPAT family. Although LPAAT4 was known to contain AGPAT motifs essential for acyltransferase activities, detailed biochemical properties were unknown. Here, we found that mouse LPAAT4 (mLPAAT4) possesses LPAAT activity with high acyl-CoA specificity for polyunsaturated fatty acyl-CoA, especially docosahexaenoyl-CoA (22:6-CoA, DHA-CoA). mLPAAT4 was distributed in many tissues, with relatively high expression in the brain, rich in docosahexaenoic acid (DHA, 22:6). mLPAAT4 siRNA in a neuronal cell line, Neuro 2A, caused a decrease in LPAAT activity with 22:6-CoA, suggesting that mLPAAT4 functions endogenously. siRNA in Neuro 2A cells caused a decrease in 18:0–22:6 PC, whereas mLPAAT4 overexpression in Chinese hamster ovary (CHO)-K1 cells caused an increase in this species. Although DHA is considered to have many important functions for the brain, the mechanism of its incorporation into glycerophospholipids is unknown. LPAAT4 might have a significant role for maintaining DHA in neural membranes. Identification of LPAAT4 will possibly contribute to understanding the regulation and the biological roles of DHA-containing glycerophospholipids in the brain.     

Lysophosphatidylcholine acyltransferase 3 knockdown-mediated liver lysophosphatidylcholine accumulation promotes very low density lipoprotein production by enhancing microsomal triglyceride transfer protein expression

After de novo biosynthesis phospholipids undergo extensive remodeling by the Lands' cycle. Enzymes involved in phospholipid biosynthesis have been studied extensively but not those involved in reacylation of lysophosphopholipids. One key enzyme in the Lands' cycle is fatty acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT), which utilizes lysophosphatidylcholine (LysoPC) and fatty acyl-CoA to produce various phosphatidylcholine (PC) species. Four isoforms of LPCAT have been identified. In this study we found that LPCAT3 is the major hepatic isoform, and its knockdown significantly reduces hepatic LPCAT activity. Moreover, we report that hepatic LPCAT3 knockdown increases certain species of LysoPCs and decreases certain species of PC. A surprising observation was that LPCAT3 knockdown significantly reduces hepatic triglycerides. Despite this, these mice had higher plasma triglyceride and apoB levels. Lipoprotein production studies indicated that reductions in LPCAT3 enhanced assembly and secretion of triglyceride-rich apoB-containing lipoproteins. Furthermore, these mice had higher microsomal triglyceride transfer protein (MTP) mRNA and protein levels. Mechanistic studies in hepatoma cells revealed that LysoPC enhances secretion of apoB but not apoA-I in a concentration-dependent manner. Moreover, LysoPC increased MTP mRNA, protein, and activity. In short, these results indicate that hepatic LPCAT3 modulates VLDL production by regulating LysoPC levels and MTP expression.     

Cloning and characterization of mouse lung-type acyl-CoA:lysophosphatidylcholine acyltransferase 1 (LPCAT1). Expression in alveolar type II cells and possible involvement in surfactant production

Phosphatidylcholine (1,2-diacyl-sn-glycero-3-phosphocholine, PC), is an important constituent of biological membranes. It is also the major component of serum lipoproteins and pulmonary surfactant. In the remodeling pathway of PC biosynthesis, 1-acyl-sn-glycero-3-phosphocholine (LPC) is converted to PC by acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT, EC 2.3.1.23). Whereas LPCAT activity has been detected in several tissues, the structure and detailed biochemical information on the enzyme have not yet been reported. Here, we present the cloning and characterization of a cDNA for mouse lung-type LPCAT (LPCAT1). The cDNA encodes an enzyme of 60 kDa, with three putative transmembrane domains. When expressed in Chinese hamster ovary cells, mouse LPCAT1 exhibited Ca(2+)-independent activity with a pH optimum between 7.4 and 10. LPCAT1 demonstrated a clear preference for saturated fatty acyl-CoAs, and 1-myristoyl- or 1-palmitoyl-LPC as acyl donors and acceptors, respectively. Furthermore, the enzyme was predominantly expressed in the lung, in particular in alveolar type II cells. Thus, the enzyme might synthesize phosphatidylcholine in pulmonary surfactant and play a pivotal role in respiratory physiology.     

Identification of Yju3p as functional orthologue of mammalian monoglyceride lipase in the yeast Saccharomycescerevisiae

Monoacylglycerols (MAGs) are short-lived intermediates of glycerolipid metabolism. Specific molecular species, such as 2-arachidonoylglycerol, which is a potent activator of cannabinoid receptors, may also function as lipid signaling molecules. In mammals, enzymes hydrolyzing MAG to glycerol and fatty acids, resembling the final step in lipolysis, or esterifying MAG to diacylglycerol, are well known; however, despite the high level of conservation of lipolysis, the corresponding activities in yeast have not been characterized yet. Here we provide evidence that the protein Yju3p functions as a potent MAG hydrolase in yeast. Cellular MAG hydrolase activity was decreased by more than 90% in extracts of Yju3p-deficient cells, indicating that Yju3p accounts for the vast majority of this activity in yeast. Loss of this activity was restored by heterologous expression of murine monoglyceride lipase (MGL). Since yju3Δ mutants accumulated MAG in vivo only at very low concentrations, we considered the possibility that MAGs are re-esterified into DAG by acyltransferases. Indeed, cellular MAG levels were further increased in mutant cells lacking Yju3p and Dga1p or Lro1p acyltransferase activities. In conclusion, our studies suggest that catabolic and anabolic reactions affect cellular MAG levels. Yju3p is the functional orthologue of mammalian MGL and is required for efficient degradation of MAG in yeast.     

Phospholipid dependence of rat liver microsomal acyl: CoA synthetase and acyl-CoA : 1-Acyl-sn-glycero-3-phosphocholine O-acyltransferase

Summary Investigations were performed on the influence of the phospholipid composition and physicochemical properties of the rat liver microsomal membranes on acyl-CoA synthetase and acyl-CoA : 1-acyl-sn-glycero-3-phosphocholine O-acyltransferase activities. The phospholipid composition of the membranes was modified by incubation with different phospholipids in the presence of lipid transfer proteins or by partial delipidation with exogenous phospholipase C and subsequent enrichment with phospholipids. The results indicated that the incorporation of phosphatidylglycerol, phosphatidylserine and phosphatidylethanolamine induced a marked activation of acyl-CoA synthetase for both substrates used—palmitic and oleic acids. Sphingomyelin occurred as specific inhibitor for this activity especially for palmitic acid. Palmitoyl-CoA: and oleoyl-CoA : lacyl-sn-glycero-3-phosphocholine acyltransferase activities were found to depend on the physical state of the membrane lipids. The alterations in the membrane physical state were estimated using two different fluorescent probes—1,6-diphenyl-1,3,5-hexatriene and pyrene. In all cases of membrane fluidization this activity was elevated. On the contrary, in more rigid membranes obtained by incorporation of sphingomyelin and dipalmitoylphosphatidylcholine, acyltransferase activity was reduced for both palmitoyl-CoA and oleoyl-CoA. We suggest a certain similarity in the way of regulation of membrane-bound acyltransferase and phospholipase A₂ which both participate in the deacylation-reacylation cycle.     

The activities of monocyte lysophosphatidylcholine acyltransferase and coenzyme A-independent transacylase are changed by the inflammatory cytokines tumor necrosis factor alpha and interferon gamma

Alteration of membrane phospholipid fatty acid compositions has been shown to be important for leukocyte inflammatory responses. Such modification of the molecular species of these lipid classes requires deacylation and reacylation reactions and for phosphatidylcholines, lysophosphatidylcholine acyltransferase (LPCAT) and a coenzyme A-independent transacylase (CoAIT) can each be involved. Since previous studies have shown a significant IFNgamma- and TNFalpha-induced modification of phosphatidylcholine species, we have examined whether these inflammatory cytokines alter the activity of reacylation enzymes in the human monocyte cell line MonoMac 6 (MM6). IFN-gamma caused a significant increase in the activity of the LPCAT and CoAIT enzymes in the microsomal fraction at concentrations and over a time-course consistent with an important role for these enzymes in the sensitization (priming) of monocytes. In contrast, TNFalpha was found to significantly increase the activity of the CoAIT by 50% over controls in MM6 cells after 30 min incubation with the cytokine, but decreased LPCAT activity by 65% after 24 h incubation. Such data imply that CoAIT is important for the remodelling of phospholipid composition, which is seen during the acute response of cells to TNFalpha. The results provide further information to emphasise the role of acyltransferases as part of the molecular mechanism underlying inflammation.     

Metabolic Interactions between the Lands Cycle and the Kennedy Pathway of Glycerolipid Synthesis in Arabidopsis Developing Seeds

It has been widely accepted that the primary function of the Lands cycle is to provide a route for acyl remodeling to modify fatty acid (FA) composition of phospholipids derived from the Kennedy pathway. Lysophosphatidylcholine acyltransferase (LPCAT) is an evolutionarily conserved key enzyme in the Lands cycle. In this study, we provide direct evidence that the Arabidopsis thaliana LPCATs, LPCAT1 and LPCAT2, participate in the Lands cycle in developing seeds. In spite of a substantially reduced initial rate of nascent FA incorporation into phosphatidylcholine (PC), the PC level in the double mutant lpcat1 lpcat2-2 remained unchanged. LPCAT deficiency triggered a compensatory response of de novo PC synthesis and a concomitant acceleration of PC turnover that were attributable at least in part to PC deacylation. Acyl-CoA profile analysis revealed complicated metabolic alterations rather than merely reduced acyl group shuffling from PC in the mutant. Shifts in FA stereo-specific distribution in triacylglycerol of the mutant seed suggested a preferential retention of saturated acyl chains at the stereospecific numbering (sn)-1 position from PC and likely a channeling of lysophosphatidic acid, derived from PC, into the Kennedy pathway. Our study thus illustrates an intricate relationship between the Lands cycle and the Kennedy pathway.