Metabolism of host lysophosphatidylcholine in Plasmodium falciparum-infected erythrocytes.

The human malaria parasite Plasmodium falciparum requires exogenous fatty acids to support its growth during the pathogenic, asexual erythrocytic stage. Host serum lysophosphatidylcholine (LPC) is a significant fatty acid source, yet the metabolic processes responsible for the liberation of free fatty acids from exogenous LPC are unknown. Using an assay for LPC hydrolysis in P. falciparum-infected erythrocytes, we have identified small-molecule inhibitors of key in situ lysophospholipase activities. Competitive activity-based profiling and generation of a panel of single-to-quadruple knockout parasite lines revealed that two enzymes of the serine hydrolase superfamily, termed exported lipase (XL) 2 and exported lipase homolog (XLH) 4, constitute the dominant lysophospholipase activities in parasite-infected erythrocytes. The parasite ensures efficient exogenous LPC hydrolysis by directing these two enzymes to distinct locations: XL2 is exported to the erythrocyte, while XLH4 is retained within the parasite. While XL2 and XLH4 were individually dispensable with little effect on LPC hydrolysis in situ, loss of both enzymes resulted in a strong reduction in fatty acid scavenging from LPC, hyperproduction of phosphatidylcholine, and an enhanced sensitivity to LPC toxicity. Notably, growth of XL/XLH-deficient parasites was severely impaired when cultured in media containing LPC as the sole exogenous fatty acid source. Furthermore, when XL2 and XLH4 activities were ablated by genetic or pharmacologic means, parasites were unable to proliferate in human serum, a physiologically relevant fatty acid source, revealing the essentiality of LPC hydrolysis in the host environment and its potential as a target for anti-malarial therapy.

Lysophospholipid remodeling mediated by the LplT and Aas protein complex in the bacterial envelope.

Lysophospholipid transporter LplT and acyltransferase Aas consist of a lysophospholipid-remodeling system ubiquitously found in gram-negative microorganisms. LplT flips lysophospholipid across the inner membrane which is subsequently acylated by Aas on the cytoplasmic membrane surface. Our previous study showed that the proper functioning of this system is important to protecting Escherichia coli from phospholipase-mediated host attack by maintaining the integrity of the bacterial cell envelope. However, the working mechanism of this system is still unclear. Herein, we report that LplT and Aas form a membrane protein complex in E. coli which allows these two enzymes to cooperate efficiently to move lysophospholipids across the bacterial membrane and catalyze their acylation. The direct interaction of LplT and Aas was demonstrated both in vivo and in vitro with a binding affinity of 2.3 µM. We found that a cytoplasmic loop of LplT adjacent to the exit of the substrate translocation pathway plays an important role in maintaining its interaction with Aas. Aas contains an acyl-acyl carrier protein synthase domain and an acyl-transferase domain. Its interaction with LplT is mediated exclusively by its transferase domain. Mutations within the three loops near the putative catalytic site of the transferase domain, respectively, disrupt its interaction with LplT and lysophospholipid acylation activity. These results support a hypothesis of the functional coupling mechanism, in which LplT directly interacts with the transferase domain of Aas for specific substrate membrane migration, providing synchronization of substrate translocation and biosynthetic events.

Overexpression of lysophospholipid acyltransferase, LPLAT10/LPCAT4/LPEAT2, in the mouse liver increases glucose-stimulated insulin secretion.

Postprandial hyperglycemia is an early indicator of impaired glucose tolerance that leads to type 2 diabetes mellitus (T2DM). Alterations in the fatty acid composition of phospholipids have been implicated in diseases such as T2DM and nonalcoholic fatty liver disease. Lysophospholipid acyltransferase 10 (LPLAT10, also called LPCAT4 and LPEAT2) plays a role in remodeling fatty acyl chains of phospholipids; however, its relationship with metabolic diseases has not been fully elucidated. LPLAT10 expression is low in the liver, the main organ that regulates metabolism, under normal conditions. Here, we investigated whether overexpression of LPLAT10 in the liver leads to improved glucose metabolism. For overexpression, we generated an LPLAT10-expressing adenovirus (Ad) vector (Ad-LPLAT10) using an improved Ad vector. Postprandial hyperglycemia was suppressed by the induction of glucose-stimulated insulin secretion in Ad-LPLAT10-treated mice compared with that in control Ad vector-treated mice. Hepatic and serum levels of phosphatidylcholine 40:7, containing C18:1 and C22:6, were increased in Ad-LPLAT10-treated mice. Serum from Ad-LPLAT10-treated mice showed increased glucose-stimulated insulin secretion in mouse insulinoma MIN6 cells. These results indicate that changes in hepatic phosphatidylcholine species due to liver-specific LPLAT10 overexpression affect the pancreas and increase glucose-stimulated insulin secretion. Our findings highlight LPLAT10 as a potential novel therapeutic target for T2DM.

LPCAT3/LPLAT12 deficiency in the liver ameliorates acetaminophen-induced acute liver injury.

Acetaminophen (APAP) is a double-edged sword, mainly depending on the dosage. A moderate dose of APAP is effective for fever and pain relief; however, an overdose induces acute liver injury. The mechanism underlying APAP-induced acute liver failure is unclear, and its treatment is limited. A recent report has shown that several oxidized phospholipids are associated with APAP-induced acute liver failure. Lysophosphatidylcholine acyltransferase 3 (Lpcat3, Lplat12), which is highly expressed in the liver, preferentially catalyzes the incorporation of arachidonate into lysophospholipids (PLs). In the present study, we investigated the roles of Lpcat3 on APAP-induced acute liver injury using liver-specific Lpcat3-knockout mice. Hepatic Lpcat3 deficiency reduced the degree of APAP-induced necrosis of hepatocytes around Zone 3 and ameliorated the elevation of hepatic injury serum marker levels, and prolonged survival. Lipidomic analysis showed that the accumulation of oxidized and hydroperoxidized phospholipids was suppressed in Lpcat3-knockout mice. The amelioration of APAP-induced acute liver injury was due not only to the reduction in the lipid synthesis of arachidonic acid PLs because of Lpcat3 deficiency, but also to the promotion of the APAP detoxification pathway by facilitating the conjugation of glutathione and N-acetyl-p-benzoquinone imine. Our findings suggest that Lpcat3 is a potential therapeutic target for treating APAP-induced acute liver injury.

Lysophospholipid Acyltransferase 9 Promotes Emphysema Formation via Platelet-activating Factor.

Cigarette smoking is known to be the leading cause of chronic obstructive pulmonary disease (COPD). However, the detailed mechanisms have not been elucidated. PAF (platelet-activating factor), a potent inflammatory mediator, is involved in the pathogenesis of various respiratory diseases such as bronchial asthma and COPD. We focused on LPLAT9 (lysophospholipid acyltransferase 9), a biosynthetic enzyme of PAF, in the pathogenesis of COPD. LPLAT9 gene expression was observed in excised COPD lungs and single-cell RNA sequencing data of alveolar macrophages (AMs). LPLAT9 was predominant and upregulated in AMs, particularly monocyte-derived AMs, in patients with COPD. To identify the function of LPLAT9/PAF in AMs in the pathogenesis of COPD, we exposed systemic LPLAT9-knockout (LPALT9-/-) mice to cigarette smoke (CS). CS increased the number of AMs, especially the monocyte-derived fraction, which secreted MMP12 (matrix metalloprotease 12). Also, CS augmented LPLAT9 phosphorylation/activation on macrophages and, subsequently, PAF synthesis in the lung. The LPLAT9-/- mouse lung showed reduced PAF production after CS exposure. Intratracheal PAF administration accumulated AMs by increasing MCP1 (monocyte chemoattractant protein-1). After CS exposure, AM accumulation and subsequent pulmonary emphysema, a primary pathologic change of COPD, were reduced in LPALT9-/- mice compared with LPLAT9+/+ mice. Notably, these phenotypes were again worsened by LPLAT9+/+ bone marrow transplantation in LPALT9-/- mice. Thus, CS-induced LPLAT9 activation in monocyte-derived AMs aggravated pulmonary emphysema via PAF-induced further accumulation of AMs. These results suggest that PAF synthesized by LPLAT9 has an important role in the pathogenesis of COPD.

Plasma C24:0- and C26:0-lysophosphatidylcholines are reliable biomarkers for the diagnosis of peroxisomal ß-oxidation disorders.

The gold-standard diagnostic test for peroxisomal disorders (PDs) is plasma concentration analysis of very long-chain fatty acids (VLCFAs). However, this method's time-consuming nature and limitations in cases which present normal VLCFA levels necessitates alternative approaches. The analysis of C26:0-lysophosphatydylcholine (C26:0-LPC) in dried blood spot samples by tandem-mass spectrometry (MS/MS) has successfully been implemented in certain newborn screening programs to diagnose X-linked adrenoleukodystrophy (ALD). However, the diagnostic potential of very long-chain LPCs concentrations in plasma remains poorly understood. This study sought to evaluate the diagnostic performance of C26:0-LPC and other very long-chain LPCs, comparing them to VLCFA analysis in plasma. The study, which included 330 individuals affected by a peroxisomal ß-oxidation deficiency and 407 control individuals, revealed that C26:0- and C24:0-LPC concentrations demonstrated the highest diagnostic accuracy (98.8% and 98.4%, respectively), outperforming VLCFA when C26:0/C22:0 and C24:0/C22:0 ratios were combined (98.1%). Combining C24:0- and C26:0-LPC gave the highest sensitivity (99.7%), with ALD females exhibiting notably higher sensitivity compared with the VLCFA ratio combination (98.7% vs. 93.5%, respectively). In contrast, C22:0-LPC exhibited suboptimal performance, primarily due to its low sensitivity (75%), but we identified a potential use to help distinguish between ALD and Zellweger spectrum disorders. In summary, MS/MS analysis of plasma C24:0- and C26:0-LPC concentrations represents a rapid and straightforward approach to diagnose PDs, demonstrating superior diagnostic accuracy, particularly in ALD females, compared with conventional VLCFA biomarkers. We strongly recommend integrating very-long chain LPC plasma analysis in the diagnostic evaluation of individuals suspected of having a PD.

Mycobacterium tuberculosis encodes a YhhN family membrane protein with lysoplasmalogenase activity that protects against toxic host lysolipids.

The pathogen Mycobacterium tuberculosis (M.tb) resides in human macrophages, wherein it exploits host lipids for survival. However, little is known about the interaction between M.tb and macrophage plasmalogens, a subclass of glycerophospholipids with a vinyl ether bond at the sn-1 position of the glycerol backbone. Lysoplasmalogens, produced from plasmalogens by hydrolysis at the sn-2 carbon by phospholipase A2, are potentially toxic but can be broken down by host lysoplasmalogenase, an integral membrane protein of the YhhN family that hydrolyzes the vinyl ether bond to release a fatty aldehyde and glycerophospho-ethanolamine or glycerophospho-choline. Curiously, M.tb encodes its own YhhN protein (MtbYhhN), despite having no endogenous plasmalogens. To understand the purpose of this protein, the gene for MtbYhhN (Rv1401) was cloned and expressed in Mycobacterium smegmatis (M.smeg). We found the partially purified protein exhibited abundant lysoplasmalogenase activity specific for lysoplasmenylethanolamine or lysoplasmenylcholine (pLPC) (Vmax∼15.5 µmol/min/mg; Km∼83 µM). Based on cell density, we determined that lysoplasmenylethanolamine, pLPC, lysophosphatidylcholine, and lysophosphatidylethanolamine were not toxic to M.smeg cells, but pLPC and LPC were highly toxic to M.smeg spheroplasts, which are cell wall-deficient mycobacterial forms. Importantly, spheroplasts prepared from M.smeg cells overexpressing MtbYhhN were protected from membrane disruption/lysis by pLPC, which was rapidly depleted from the media. Finally, we found that overexpression of full-length MtbYhhN in M.smeg increased its survival within human macrophages by 2.6-fold compared to vector controls. These data support the hypothesis that MtbYhhN protein confers a growth advantage for mycobacteria in macrophages by cleaving toxic host pLPC into potentially energy-producing products.

Dietary omega-3 fatty acid does not improve male infertility caused by lysophospholipid acyltransferase 3 (LPLAT3/AGPAT3) deficiency.

Docosahexaenoic acid (DHA), an omega-3 fatty acid, usually presents as a constituent of phospholipids in the cellular membrane. Lysophospholipid acyltransferase 3 (LPLAT3; AGPAT3) is the primary enzyme that incorporates DHA into phospholipids. LPLAT3-KO mice show male infertility and visual dysfunction accompanied by decreased phospholipids (PLs) containing DHA (PL-DHA) in the testis and retina, respectively. In this study, we evaluated the effect of diets consisting mainly of triacylglycerol-bound DHA (fish oil) and PL-bound DHA (salmon roe oil) on the amount of PL-DHA in a broad range of tissues and on reproductive functions. Both diets elevated phosphatidylcholines (PCs)-containing DHA in most tissues of wild type (WT) mice. Although LPLAT3-KO mice acquired a minimal amount of PC-DHA in the testes and sperm by eating either of the diets, reproductive function did not improve. The present study suggests that DHA-rich diets do not restore sufficient PL-DHA to improve male infertility in LPLAT3-KO mice. Alternatively, PL-DHA can be biosynthesized by LPLAT3 but not by external supplementation, which may be necessary for normal reproductive function.

Lysophospholipids transport across blood-brain barrier in an in vitro reconstruction model.

This study builds on our previous study, which highlighted the need for further research on the potential use of lysophospholipid (LPL) supplementation to prevent chronic and age-related diseases. We aimed to evaluate the transmembrane transport of LPL across rat and monkey blood-brain barrier (BBB) models. An in vitro monkey BBB model is required to elucidate the differences between rat and primate BBB-related data and to measure the permeability of LPLs being researched in relation to the human BBB. Based on our previous experiment, porcine liver decomposition product-derived phospholipids (PEL) strongly inhibit α-synuclein (α-Syn) aggregation. We have identified several candidates potentially relevant for the inhibition of α-Syn aggregation, such as LPC18:1, LPE18:1, and LPI18:0; however, the BBB permeability of these LPLs remains unclear. In the present study, we assessed the ability of these LPLs to pass through the in vitro rat and monkey BBB models. LPC18:1 showed high BBB permeability, LPI18:0 showed medium permeability, and the BBB permeation of LPE18:1 was negligible. Our results suggest that LPC18:1 and LPI18:0 are functional food factors that can cross the BBB.

Ponesimod inhibits astrocyte-mediated neuroinflammation and protects against cingulum demyelination via S1P1 -selective modulation.

Ponesimod is a sphingosine 1-phosphate (S1P) receptor (S1PR) modulator that was recently approved for treating relapsing forms of multiple sclerosis (MS). Three other FDA-approved S1PR modulators for MS-fingolimod, siponimod, and ozanimod-share peripheral immunological effects via common S1P1 interactions, yet ponesimod may access distinct central nervous system (CNS) mechanisms through its selectivity for the S1P1 receptor. Here, ponesimod was examined for S1PR internalization and binding, human astrocyte signaling and single-cell RNA-seq (scRNA-seq) gene expression, and in vivo using murine cuprizone-mediated demyelination. Studies confirmed ponesimod's selectivity for S1P1 without comparable engagement to the other S1PR subtypes (S1P2,3,4,5 ). Ponesimod showed pharmacological properties of acute agonism followed by chronic functional antagonism of S1P1 . A major locus of S1P1 expression in the CNS is on astrocytes, and scRNA-seq of primary human astrocytes exposed to ponesimod identified a gene ontology relationship of reduced neuroinflammation and reduction in known astrocyte disease-related genes including those of immediate early astrocytes that have been strongly associated with disease progression in MS animal models. Remarkably, ponesimod prevented cuprizone-induced demyelination selectively in the cingulum, but not in the corpus callosum. These data support the CNS activities of ponesimod through S1P1 , including protective, and likely selective, effects against demyelination in a major connection pathway of the brain, the limbic fibers of the cingulum, lesions of which have been associated with several neurologic impairments including MS fatigue.

Effect of lysophospholipids on the growth performance, nutrient digestibility, lipid metabolism and meat quality of fattening rabbits.

The present study aimed to investigate the effect of emulsifier lysophospholipids (LP), enzymatically modified from soy phospholipids, on the growth performance, nutrient digestibility, lipid metabolism and meat quality of fattening rabbits. The LP was added in control (CON), LP1, LP2 and LP3 at 0, 200, 400 and 600 mg/kg, respectively. A total of 240 rabbits at approximately 52 d of age were divided into 4 groups with 6 replicates of 10 rabbits each. The feeding trial lasted for 42 d. Results showed that compared to CON, LP1, LP2 and LP3 increased (p < 0.05) body weight gain, feed efficiency, the apparent faecal digestibility of gross energy, crude protein and ether extract, the percentages of dissectible fat and ether extract in the longissimus and legs, the serum contents of apolipoprotein B, free fatty acid and total phospholipids in the longissimus, but decreased (p < 0.05) serum total triglyceride and total cholesterol. Meanwhile, LP1, LP2 and LP3 had higher (p < 0.05) carcass weight, longissimus weight and percentages of foreleg and hindleg than the CON; and the three LP diets also increased (p < 0.05) the tenderness, lightness and redness of longissimus. It is concluded that soy LP as an emulsifier can improve the growth, digestibility and meat quality of fattening rabbits.

Dietary threonine and lysophospholipid supplement in broiler chickens: effect on productive performance, carcass variables, cecal microbiota activity, and jejunal morphology.

The objective of this experiment was to investigate the efficacy of lysophospholipid (LPL) in combination with different levels of threonine (Thr) on productive performance, jejunal morphometric variables, cecal microbial population, and carcass characteristics of male broiler chickens. Four hundred 1-day-old male broiler chicks were distributed into eight experimental groups with five replicates, each of ten birds. Factors were two levels of 0 or 0.1% Lipidol as LPL supplement and four inclusion amount of Thr (100, 105, 110, or 115% of requirements) in the diets. During 1 to 35 days, inclusion of LPL supplementation in diets improved the body weight gain (BWG) and feed conversion ratio (FCR) of broilers (P < 0.05). Besides, FCR was significantly greater in the birds fed with 100% Thr compared to the other inclusion rates of Thr (P < 0.05). The jejuna villus length (VL) and crypt depth (CD) were greater in the birds fed with LPL-supplemented diets (P < 0.05) while the greatest VH/CD and villus surface was observed in the birds fed with 105% of dietary Thr (P < 0.05). In cecal microbiota composition, Lactobacillus population was lower in broilers which received diet containing 100% Thr compared to the other group receiving more than 100% Thr (P < 0.05). In conclusion, inclusion of LPL supplements and amounts greater than Thr requirement in diet improved productive performance and jejunal morphology of male broiler chickens.

Identification of two lipid phosphatases that regulate sphingosine-1-phosphate cellular uptake and recycling.

Sphingosine-1-phosphate (S1P) is a sphingolipid metabolite that serves as a potent extracellular signaling molecule. Metabolic regulation of extracellular S1P levels impacts key cellular activities through altered S1P receptor signaling. Although the pathway through which S1P is degraded within the cell and thereby eliminated from reuse has been previously described, the mechanism used for S1P cellular uptake and the subsequent recycling of its sphingoid base into the sphingolipid synthesis pathway is not completely understood. To identify the genes within this S1P uptake and recycling pathway, we performed a genome-wide CRISPR/Cas9 KO screen using a positive-selection scheme with Shiga toxin, which binds a cell-surface glycosphingolipid receptor, globotriaosylceramide (Gb3), and causes lethality upon internalization. The screen was performed in HeLa cells with their sphingolipid de novo pathway disabled so that Gb3 cell-surface expression was dependent on salvage of the sphingoid base of S1P taken up from the medium. The screen identified a suite of genes necessary for S1P uptake and the recycling of its sphingoid base to synthesize Gb3, including two lipid phosphatases, PLPP3 (phospholipid phosphatase 3) and SGPP1 (S1P phosphatase 1). The results delineate a pathway in which plasma membrane-bound PLPP3 dephosphorylates extracellular S1P to sphingosine, which then enters cells and is rephosphorylated to S1P by the sphingosine kinases. This rephosphorylation step is important to regenerate intracellular S1P as a branch-point substrate that can be routed either for dephosphorylation to salvage sphingosine for recycling into complex sphingolipid synthesis or for degradation to remove it from the sphingolipid synthesis pathway.

Integrative analysis of transcriptomics and metabolomics to reveal the melanogenesis pathway of muscle and related meat characters in Wuliangshan black-boned chickens.

BACKGROUND: Melanin is an important antioxidant in food and has been used in medicine and cosmetology. Chicken meat with high melanin content from black-boned chickens have been considered a high nutritious food with potential medicinal properties. The molecular mechanism of melanogenesis of skeletal muscle in black-boned chickens remain poorly understood. This study investigated the biological gene-metabolite associations regulating the muscle melanogenesis pathways in Wuliangshan black-boned chickens with two normal boned chicken breeds as control. RESULTS: We identified 25 differentially expressed genes and 11 transcription factors in the melanogenesis pathways. High levels of the meat flavor compounds inosine monophosphate, hypoxanthine, lysophospholipid, hydroxyoctadecadienoic acid, and nicotinamide mononucleotide were found in Wuliangshan black-boned chickens. CONCLUSION: Integrative analysis of transcriptomics and metabolomics revealed the dual physiological functions of the PDZK1 gene, involved in pigmentation and/or melanogenesis and regulating the phospholipid signaling processes in muscle of black boned chickens.

Low lysophosphatidylcholine induces skeletal muscle myopathy that is aggravated by high-fat diet feeding.

Obesity alters skeletal muscle lipidome and promotes myopathy, but it is unknown whether aberrant muscle lipidome contributes to the reduction in skeletal muscle contractile force-generating capacity. Comprehensive lipidomic analyses of mouse skeletal muscle revealed a very strong positive correlation between the abundance of lysophosphatidylcholine (lyso-PC), a class of lipids that is known to be downregulated with obesity, with maximal tetanic force production. The level of lyso-PC is regulated primarily by lyso-PC acyltransferase 3 (LPCAT3), which acylates lyso-PC to form phosphatidylcholine. Tamoxifen-inducible skeletal muscle-specific overexpression of LPCAT3 (LPCAT3-MKI) was sufficient to reduce muscle lyso-PC content in both standard chow diet- and high-fat diet (HFD)-fed conditions. Strikingly, the assessment of skeletal muscle force-generating capacity ex vivo revealed that muscles from LPCAT3-MKI mice were weaker regardless of diet. Defects in force production were more apparent in HFD-fed condition, where tetanic force production was 40% lower in muscles from LPCAT3-MKI compared to that of control mice. These observations were partly explained by reductions in the cross-sectional area in type IIa and IIx fibers, and signs of muscle edema in the absence of fibrosis. Future studies will pursue the mechanism by which LPCAT3 may alter protein turnover to promote myopathy.

The Many Roles of Lysophospholipid Mediators and Japanese Contributions to This Field.

Lysophospholipids are phospholipids with only one fatty acid. During the past two decades, it has become apparent that lysophospholipids are not merely degradation products but have various physiological and pathological functions in vivo via G protein-coupled receptor (GPCR)-type receptors. These include lysophosphatidic acid (LPA), sphingosine 1-phosphate (S1P), lysophosphatidylinositol/lysophosphatidylglucose (LPI/LPtdGlc), and lysophosphatidylserine (LysoPS). This review focuses on identifying the functions of the receptors, enzymes, transporters, and carrier proteins required for these four lysophospholipids to function as lipid mediators. We also note that many of advances in this field have been made by Japanese pharmaceutical scientists.

Lysophosphatidylinositol Induced Morphological Changes and Stress Fiber Formation through the GPR55-RhoA-ROCK Pathway.

We previously reported that lysophosphatidylinositol (LPI) functions as an endogenous agonist of GPR55, a novel cannabinoid receptor. However, the physiological roles of LPI-GPR55 have not yet been elucidated in detail. In the present study, we found that LPI induced morphological changes in GPR55-expressing HEK293 cells. LPI induced the cell rounding of GPR55-expressing HEK293 cells but not of empty-vector-transfected cells. LPI also induced the activation of small GTP-binding protein RhoA and increased stress fiber formation in GPR55-expressing HEK293 cells. The inhibition of RhoA and Rho kinase ROCK by the C3 exoenzyme and the ROCK inhibitor reduced LPI-induced cell rounding and stress fiber formation. These results clearly indicated that the LPI-induced morphological changes and the assembly of the cytoskeletons were mediated through the GPR55-RhoA-ROCK pathway.

Current Knowledge on Mammalian Phospholipase A1, Brief History, Structures, Biochemical and Pathophysiological Roles.

Phospholipase A1 (PLA1) is an enzyme that cleaves an ester bond at the sn-1 position of glycerophospholipids, producing a free fatty acid and a lysophospholipid. PLA1 activities have been detected both extracellularly and intracellularly, which are well conserved in higher eukaryotes, including fish and mammals. All extracellular PLA1s belong to the lipase family. In addition to PLA1 activity, most mammalian extracellular PLA1s exhibit lipase activity to hydrolyze triacylglycerol, cleaving the fatty acid and contributing to its absorption into the intestinal tract and tissues. Some extracellular PLA1s exhibit PLA1 activities specific to phosphatidic acid (PA) or phosphatidylserine (PS) and serve to produce lysophospholipid mediators such as lysophosphatidic acid (LPA) and lysophosphatidylserine (LysoPS). A high level of PLA1 activity has been detected in the cytosol fractions, where PA-PLA1/DDHD1/iPLA1 was responsible for the activity. Many homologs of PA-PLA1 and PLA2 have been shown to exhibit PLA1 activity. Although much has been learned about the pathophysiological roles of PLA1 molecules through studies of knockout mice and human genetic diseases, many questions regarding their biochemical properties, including their genuine in vivo substrate, remain elusive.

Lysophospholipids Contribute to Oxaliplatin-Induced Acute Peripheral Pain.

Oxaliplatin, a platinum-based chemotherapeutic drug, which is used as first-line treatment for some types of colorectal carcinoma, causes peripheral neuropathic pain in patients. In addition, an acute peripheral pain syndrome develop in almost 90% of patients immediately after oxaliplatin treatment, which is poorly understood mechanistically but correlates with incidence and severity of the later-occurring neuropathy. Here we investigated the effects of acute oxaliplatin treatment in a murine model, showing that male and female mice develop mechanical hypersensitivity 24 h after oxaliplatin treatment. Interestingly, we found that the levels of several lipids were significantly altered in nervous tissue during oxaliplatin-induced acute pain. Specifically, the linoleic acid metabolite 9,10-EpOME (epoxide of linoleic acid) as well as the lysophospholipids lysophosphatidylcholine (LPC) 18:1 and LPC 16:0 were significantly increased 24 h after oxaliplatin treatment in sciatic nerve, DRGs, or spinal cord tissue as revealed by untargeted and targeted lipidomics. In contrast, inflammatory markers including cytokines and chemokines, ROS markers, and growth factors are unchanged in the respective nervous system tissues. Importantly, LPC 18:1 and LPC 16:0 can induce Ca2+ transients in primary sensory neurons, and we identify LPC 18:1 as a previously unknown endogenous activator of the ligand-gated calcium channels transient receptor potential V1 and M8 (transient receptor potential vanilloid 1 and transient receptor potential melastatin 8) in primary sensory neurons using both pharmacological inhibition and genetic knockout. Additionally, a peripheral LPC 18:1 injection was sufficient to induce mechanical hypersensitivity in naive mice. Hence, targeting signaling lipid pathways may ameliorate oxaliplatin-induced acute peripheral pain and the subsequent long-lasting neuropathy.SIGNIFICANCE STATEMENT The first-line cytostatic drug oxaliplatin can cause acute peripheral pain and chronic neuropathic pain. The former is causally connected with the chronic neuropathic pain, but its mechanisms are poorly understood. Here, we performed a broad unbiased analysis of cytokines, chemokines, growth factors, and ∼200 lipids in nervous system tissues 24 h after oxaliplatin treatment, which revealed a crucial role of lysophospholipids lysophosphatidylcholine (LPC) 18:1, LPC 16:0, and 9,10-EpOME in oxaliplatin-induced acute pain. We demonstrate for the first time that LPC 18:1 contributes to the activation of the ion channels transient receptor potential vanilloid 1 and transient receptor potential melastatin 8 in sensory neurons and causes mechanical hypersensitivity after peripheral injection in vivo These findings suggest that the LPC-mediated lipid signaling is involved in oxaliplatin-induced acute peripheral pain.

Development of a selective fluorescence-based enzyme assay for glycerophosphodiesterase family members GDE4 and GDE7.

Lysophosphatidic acid (LPA) is a lipid mediator that regulates various processes, including cell migration and cancer progression. Autotaxin (ATX) is a lysophospholipase D-type exoenzyme that produces extracellular LPA. In contrast, glycerophosphodiesterase (GDE) family members GDE4 and GDE7 are intracellular lysophospholipases D that form LPA, depending on Mg2+ and Ca2+, respectively. Since no fluorescent substrate for these GDEs has been reported, in the present study, we examined whether a fluorescent ATX substrate, FS-3, could be applied to study GDE activity. We found that the membrane fractions of human GDE4- and GDE7-overexpressing human embryonic kidney 293T cells hydrolyzed FS-3 in a manner almost exclusively dependent on Mg2+ and Ca2+, respectively. Using these assay systems, we found that several ATX inhibitors, including α-bromomethylene phosphonate analog of LPA and 3-carbacyclic phosphatidic acid, also potently inhibited GDE4 and GDE7 activities. In contrast, the ATX inhibitor S32826 hardly inhibited these activities. Furthermore, FS-3 was hydrolyzed in a Mg2+-dependent manner by the membrane fraction of human prostate cancer LNCaP cells that express GDE4 endogenously but not by those of GDE4-deficient LNCaP cells. Similar Ca2+-dependent GDE7 activity was observed in human breast cancer MCF-7 cells but not in GDE7-deficient MCF-7 cells. Finally, our assay system could selectively measure GDE4 and GDE7 activities in a mixture of the membrane fractions of GDE4- and GDE7-overexpressing human embryonic kidney 293T cells in the presence of S32826. These findings allow high-throughput assays of GDE4 and GDE7 activities, which could lead to the development of selective inhibitors and stimulators as well as a better understanding of the biological roles of these enzymes.