Molecular Species Of Phosphatidic Acid Research Articles

Simple Does Not Mean Trivial: Behavior of Phosphatidic Acid in Lipid Mono- and Bilayers.

Phosphatidic acid (PA) is one of the simplest membrane phospholipids, yet it plays a crucial role in various biologically relevant processes that take place in cells. Since PA generation may be triggered by a variety of factors, very often of antagonistic character, the specific nature of physiological responses driven by PA is not clear. In order to shed more light on these issues, we carried out a systematic characterization of membranes containing one of the three biologically significant PA molecular species. The effect of these molecules on the properties of membranes composed of phosphatidylcholine and/or cholesterol was assessed in a multidisciplinary approach, including molecular dynamic simulations, flicker noise spectroscopy, and Langmuir monolayer isotherms. The first enables the determination of various macroscopic and microscopic parameters such as lateral diffusion, membrane thickness, and defect analysis. The obtained data revealed a strong interaction between unsaturated PA species and phosphatidylcholine. On the other hand, the behavior of saturated PA was greatly influenced by cholesterol. Additionally, a strong effect on mechanical properties was observed in the case of three-component systems, which could not be explained by the simple extrapolation of parameters of the corresponding two-component systems. Our data show that various PA species are not equivalent in terms of their influence on lipid mono- and bilayers and that membrane composition/properties, particularly those related to the presence of cholesterol, may strongly modulate PA behavior.

Recovery from chilling modulates the acyl-editing of phosphatidic acid molecular species in barley roots (Hordeum vulgare L.)

In plants, lipid metabolism and remodelling are key mechanisms for survival under temperature stress. The present study attempted to compare the lipid profile in barley roots both under chilling stress treatment and in the subsequent recovery to stress. Lipids were obtained through a single-extraction method with a polar solvent mixture, followed by mass spectrometry analysis. The results indicate that lipid metabolism was significantly affected by chilling. Most of the glycerolipids analysed returned to control values during short- and long-term recovery, whereas several representative phosphatidic acid (PA) molecular species were edited during long-term recovery. Most of the PA molecular species that increased in the long-term had the same acyl chains as the phosphatidylcholine (PC) species that decreased. C34:2 and C36:4 underwent the most remarkable changes. Given that the mechanisms underlying the acyl-editing of PC in barley roots remain elusive, we also evaluated the contribution of lysophosphatidylcholine acyltransferases (HvLPCAT) and phospholipase A (HvPLA). In line with the aforementioned results, the expression of the HvLPCAT and HvPLA genes was up-regulated during recovery from chilling. The differential acyl-editing of PA during recovery, which involves the remodelling of PC, might therefore be a regulatory mechanism of cold tolerance in barley.

Sphingomyelin synthase‐related protein generates diacylglycerol via hydrolysis of phosphatidic acid, phosphatidylcholine, phosphatidylinositol and phosphatidylethanolamine without ceramide

Introduction Sphingomyelin synthase-related protein (SMSr) produces diacylglycerol (DG) and ceramide phosphoethanolamine (CPE) by the transfer of phosphoethanolamine from phosphatidylethanolamine (PE) to ceramide. We previously reported that SMSr functionally interacts with DG kinase (DGK) δ via steraile α motif domains (SAMD), protein-protein interaction module [Murakami et al. J. Biol. Chem. 295, 2932–47, 2020]. Moreover, SMSr provided DG to DGKδ. Puzzlingly, mammalian SMSr produces only trace amounts of CPE and DG. However, we previously reported that DG levels, particularly saturated fatty acid (SFA)- and/or monounsaturated fatty acid (MUFA)-containing DG molecular species, were significantly increased in COS-7 cells overexpressing human SMSr. To clarify this discrepancy, we purified human SMSr and determined whether purified SMSr showed other DG-generating activity via hydrolyis of phospholipids without ceramide in the present study. Results and Conclusion Human SMSr was expressed using the baculovirus-insect cell expression system and highly purified. Interestingly, purified SMSr showed DG-generating activity by hydrolysis of PE, phosphatidylcholine (PC), phosphatidylinositol (PI) and phosphatidic acid (PA) in the absence of ceramide. PA phosphatase (PAP) activity of SMSr was about 300-fold stronger than CPE synthase activity. Moreover, SMSr showed substrate selectivity for SFA- and/or MUFA-containing PA molecular species, but not arachidonic acid-containing PA, which is exclusively generated in the PI(4,5)P2 cycle. We confirmed that SMSr expressed in COS-7 cells showed PAP and PI-phospholipase C (PLC) activities. Collectively, our study indicated that SMSr possesses previously unrecognized enzyme activities (multi-glycerophospholipid PLC hydrolase) and constitutes a novel DG signaling pathway together with DGKd independent of the PI(4,5)P2 cycle.

Sphingomyelin synthase-related protein generates diacylglycerol via the hydrolysis of glycerophospholipids in the absence of ceramide

Diacylglycerol (DG) is a well-established lipid second messenger. Sphingomyelin synthase (SMS)-related protein (SMSr) produces DG and ceramide phosphoethanolamine (CPE) by the transfer of phosphoethanolamine from phosphatidylethanolamine (PE) to ceramide. We previously reported that human SMSr overexpressed in COS-7 cells significantly increased DG levels, particularly saturated and/or monounsaturated fatty acid–containing DG molecular species, and provided DG to DG kinase (DGK) δ, which regulates various pathophysiological events, including epidermal growth factor–dependent cell proliferation, type 2 diabetes, and obsessive–compulsive disorder. However, mammalian SMSr puzzlingly produces only trace amounts of CPE/DG. To clarify this discrepancy, we highly purified SMSr and examined its activities other than CPE synthase. Intriguingly, purified SMSr showed a DG-generating activity via hydrolysis of PE, phosphatidic acid (PA), phosphatidylinositol (PI), and phosphatidylcholine (PC) in the absence of ceramide. DG generation through the PA phosphatase (PAP) activity of SMSr was approximately 300-fold higher than that with PE and ceramide. SMSr hydrolyzed PI ten times stronger than PI(4,5)bisphosphate (PI(4,5)P2). The PAP and PC-phospholipase C (PLC) activities of SMSr were inhibited by propranolol, a PAP inhibitor, and by D609, an SMS/PC-PLC inhibitor. Moreover, SMSr showed substrate selectivity for saturated and/or monounsaturated fatty acid–containing PA molecular species, but not arachidonic-acid-containing PA, which is exclusively generated in the PI(4,5)P2 cycle. We confirmed that SMSr expressed in COS-7 cells showed PAP and PI-PLC activities. Taken together, our study indicated that SMSr possesses previously unrecognized enzyme activities, PAP and PI/PE/PC-PLC, and constitutes a novel DG/PA signaling pathway together with DGKδ, which is independent of the PI(4,5)P2 cycle.

Polyunsaturated fatty acid-containing phosphatidic acids selectively interact with L-lactate dehydrogenase A and induce its secondary structural change and inactivation

Phosphatidic acid (PA) consists of various molecular species that have different fatty acyl chains at the sn-1 and sn-2 positions; and consequently, mammalian cells contain at least 50 structurally distinct PA molecular species. However, the different roles of each PA species are poorly understood. In the present study, we attempted to identify dipalmitoyl (16:0/16:0)-PA-binding proteins from mouse skeletal muscle using liposome precipitation and tandem mass spectrometry analysis. We identified L-lactate dehydrogenase (LDH) A, which catalyzes conversion of pyruvate to lactate and is a key checkpoint of anaerobic glycolysis critical for tumor growth, as a 16:0/16:0-PA-binding protein. LDHA did not substantially associate with other phospholipids, such as phosphatidylcholine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, phosphoinositides and cardiolipin at physiological pH (7.4), indicating that LDHA specifically bound to PA. Interestingly, 18:0/18:0-, 18:0/20:4- and 18:0/22:6-PA also interacted with LDHA, and their binding activities were stronger than 16:0/16:0-PA at pH 7.4. Moreover, circular dichroism spectrometry showed that 18:0/20:4- and 18:0/22:6-PA, but not 16:0/16:0- or 18:0/18:0-PA, significantly reduced the α-helical structure of LDHA. Furthermore, 18:0/20:4- and 18:0/22:6-PA attenuated LDH activity. Taken together, we demonstrated for the first time that LDHA is a PA-binding protein and is a unique PA-binding protein that is structurally and functionally controlled by associating with 18:0/20:4- and 18:0/22:6-PA.

Functional relationship between sphingomyelin synthase‐related protein and diacylglycerol kinase δ

IntroductionThe δ isozyme of diacyglycerol kinase (DGKδ) plays critical roles in lipid signaling by converting diacylglycerol (DG) to phosphatidic acid (PA). Intriguingly, we recently demonstrated that DGKδ preferably metabolized palmitic acid (16:0)‐ and/or palmitoleic acid (16:1)‐containing DG molecular species in response to high glucose stimulation in myoblasts. However, it is still unclear where 16:0‐ and/or 16:1‐containing DG molecular species come from, although another species arachidonic acid (20:4)‐containing DGs are derived from phosphatidylinositol (PI) turnover. Sphingomyelin synthase (SMS) 1 and SMS‐related protein (SMSr) synthesize the sphingomyelin and ceramide‐phosohoethanolamine by the transfer of phosphocholine/phosphoethanolamine to ceramide, respectively. These are also assumed to function as DG‐generating enzymes. SMS1, SMSr and DGKδ contain a sterile α motif domain (SAM), which is a putative protein interaction module. Interestingly, we found that DGKδ interacted with SMSr via their SAMs directly. In the present study, we investigated the functional relationship between DGKδ and SMSr.Results and ConclusionWe first investigated the changes in the amounts of PA molecular species in COS‐7 cells overexpressing DGKδ and/or SMSr using LC‐MS/MS. We found that the overexpression of SMSr and DGKδ significantly enhanced the production of 16:0‐ and/or 16:1‐containing PA species such as 16:0/16:1‐, 16:0/16:0‐, 16:0/18:1‐ and 16:0/18:2‐PA in COS‐7 cells. It is possible that DGKδ and SMSr are functionally linked and that SMSr acts in an upstream pathway of DGKδ by providing 16:0‐ and/or 16:1‐DG. To support the possibility, we determined DG levels in SMSr‐overexpressing cells. Compared with control cells, total DG levels in SMSr‐overexpressing cells were significantly increased. In particular, 16:0‐ and/or 16:1‐containig DG species, such as 14:0/16:0 (30:0)‐, 16:0/16:1 (32:1)‐, 16:1/18:2 (34:3)‐, and 16:1/18:1 (34:2)‐DG were significantly increased. These results further suggest the functional relationship (SMSr supplies DG to DGKδ) between DGKδ and SMSr. To examine whether forming SMSr‐DGKδ heteromeric complex affects DGKδ activity, we measured purified DGKδ activity in vitro using DG and ATP as substrates. Interestingly, when presence of purified SMSr with purified DGKδ, DGK activity was significantly increased. However, the deletion of SAM of SMSr did not significantly augment the DGKδ activity. Taken together, these results suggest that SMSr is one of the candidates of upstream DG‐providing enzymes of DGKδ and that SMSr enhances DGKδ activity by forming heteromeric complex via their SAMs.Support or Funding InformationThis work was supported in part by grants from MEXT/JSPS KAKENHI (Grant Numbers: JP18J20003 to C.M.); The Chiba Bank (to C.M.); the Chiba University Venture Business Laboratory (to C.M.); Chiba University Open Recruitment for International Exchange Program at the Institute for Global Prominent Research (to C.M.); and the American Society for Biochemistry and Molecular Biology (ASBMB 2019 Graduate/Postdoctoral Travel Award to C.M)

Regulation of DGKε Activity and Substrate Acyl Chain Specificity by Negatively Charged Phospholipids

Diacylglycerol kinase ε (DGKε) is a membrane-bound enzyme that catalyzes the ATP-dependent phosphorylation of diacylglycerol to form phosphatidic acid (PA) in the phosphatidylinositol cycle. DGKε lacks a putative regulatory domain and has recently been reported to be regulated by highly curved membranes. To further study the effect of other membrane properties as a regulatory mechanism of DGKε, our work reports the effect of negatively charged phospholipids on DGKε activity and substrate acyl chain specificity. These studies were conducted using purified DGKε and detergent-free phospholipid aggregates, which present a more suitable model system to access the impact of membrane physical properties on membrane-active enzymes. The structural properties of the different model membranes were studied by means of differential scanning calorimetry and 31P-NMR. It is shown that the enzyme is inhibited by a variety of negatively charged phospholipids. However, PA, which is a negatively charged phospholipid and the product of DGKε catalyzed reaction, showed a varied regulatory effect on the enzyme from being an activator to an inhibitor. The type of feedback regulation of DGKε by PA depends on the particular PA molecular species as well as the physical properties of the membrane that the enzyme binds to. In the presence of highly packed PA-rich domains, the enzyme is activated. However, its acyl chain specificity is only observed in liposomes containing 1,2-dioleoyl PA in the presence of Ca2+. It is proposed that to endow the enzyme with its substrate acyl chain specificity, a highly dehydrated (hydrophobic) membrane interface is needed. The presence of an overlap of mechanisms to regulate DGKε ensures proper phosphatidylinositol cycle function regardless of the trigged stimulus and represents a sophisticated and specialized manner of membrane-enzyme regulation.

Thioesterase superfamily member 2 promotes hepatic insulin resistance in the setting of glycerol-3-phosphate acyltransferase 1–induced steatosis

Hepatic insulin resistance in the setting of steatosis is attributable at least in part to the accumulation of bioactive lipids that suppress insulin signaling. The mitochondria-associated glycerol-3-phosphate acyltransferase 1 (GPAT1) catalyzes the first committed step in glycerolipid synthesis, and its activity diverts fatty acids from mitochondrial β-oxidation. GPAT1 overexpression in mouse liver leads to hepatic steatosis even in the absence of overnutrition. The mice develop insulin resistance owing to the generation of saturated diacylglycerol and phosphatidic acid molecular species that reduce insulin signaling by activating PKCϵ and by suppressing mTORC2, respectively. Them2, a mitochondria-associated acyl-CoA thioesterase, also participates in the trafficking of fatty acids into oxidative versus glycerolipid biosynthetic pathways. Them2-/- mice are protected against diet-induced hepatic steatosis and insulin resistance. To determine whether Them2 contributes to hepatic insulin resistance due to hepatic overexpression of GPAT1, recombinant adenovirus was used to overexpress GPAT1 in livers of chow-fed Them2+/+ and Them2-/- mice. Hepatic GPAT1 overexpression led to steatosis in both genotypes. In the setting of GPAT1 overexpression, glucose tolerance was reduced in Them2+/+ but not Them2-/- mice, without influencing whole-body insulin sensitivity or basal hepatic glucose production. Improved glucose tolerance in Them2-/- mice was associated with reduced PKCϵ translocation. Preserved insulin receptor activity was supported by Thr-308 phosphorylation of Akt following GPAT1 overexpression in Them2-/- hepatocytes. These findings suggest a pathogenic role of Them2 in the biosynthesis of glycerolipid metabolites that promote hepatic insulin resistance.

Diacylglycerol kinase α-selective inhibitors induce apoptosis and reduce viability of melanoma and several other cancer cell lines.

Diacylglycerol (DG) kinase (DGK), which phosphorylates DG to generate phosphatidic acid (PA), consists of ten isozymes (α-к). Recently, we identified a novel small molecule inhibitor, CU-3, that selectively inhibits the activity of the α isozyme. In addition, we newly obtained Compound A, which selectively and strongly inhibits type I DGKs (α, β, and γ). In the present study, we demonstrated that both CU-3 and Compound A induced apoptosis (caspase 3/7 activity and DNA fragmentation) and viability reduction of AKI melanoma cells. Liquid chromatography-mass spectrometry revealed that the production of 32:0- and 34:0-PA species was commonly attenuated by CU-3 and Compound A, suggesting that lower levels of these PA molecular species are involved in the apoptosis induction and viability reduction of AKI cells. We determined the effects of the DGKα inhibitors on several other cancer cell lines derived from refractory cancers. In addition to melanoma, the DGKα inhibitors enhanced caspase 3/7 activity and reduced the viability of hepatocellular carcinoma, glioblastoma, and pancreatic cancer cells, but not breast adenocarcinoma cells. Interestingly, Western blot analysis indicated that the DGKα expression levels were positively correlated with the sensitivity to the DGK inhibitors. Because both CU-3 and Compound A induced interleukin-2 production by T cells, it is believed that these two compounds can enhance cancer immunity. Taken together, our results suggest that DGKα inhibitors are promising anticancer drugs.

Differential phosphatidic acid metabolism in barley leaves and roots induced by chilling temperature

Phosphatidic acid (PA) is an important bioactive lipid that mediates chilling responses in barley. Modifications in the lipid composition of cellular membranes during chilling are essential to maintain their integrity and fluidity. First, we investigated the molecular species of PA present in leaves and roots by ESI-MS/MS, to evaluate the modifications that occur in response to chilling. We demonstrated that PA pools in leaves differ from PA fatty acid composition in roots. Compared with plants grown at 25 °C, the short-term and long-term chilling for 3 h and 36 h at 4 °C not produced significant changes in PA molecular species. The endogenous DAG and PA phosphorylation by in vitro DAG and PA kinase activities showed higher activity in leaves compared with that in root, and they showed contrasting responses to chilling. Similarly, PA removal by phosphatidate phosphohydrolase was tested, showing that this activity was specifically increased in response to chilling in roots. The findings presented here may be helpful to understand how the PA signal is modulated between tissues, and may serve to highlight the importance of knowing the basal PA pools in different plant organs.

Lipidomic Adaptations in White and Brown Adipose Tissue in Response to Exercise Demonstrate Molecular Species-Specific Remodeling

Exercise improves whole-body metabolic health through adaptations to various tissues, including adipose tissue, but the effects of exercise training on the lipidome of white adipose tissue (WAT) and brown adipose tissue (BAT) are unknown. Here, we utilize MS/MSALL shotgun lipidomics to determine the molecular signatures of exercise-induced adaptations to subcutaneous WAT (scWAT) and BAT. Three weeks of exercise training decrease specific molecular species of phosphatidic acid (PA), phosphatidylcholines (PC), phosphatidylethanolamines (PE), and phosphatidylserines (PS) in scWAT and increase specific molecular species of PC and PE in BAT. Exercise also decreases most triacylglycerols (TAGs) in scWAT and BAT. In summary, exercise-induced changes to the scWAT and BAT lipidome are highly specific to certain molecular lipid species, indicating that changes in tissue lipid content reflect selective remodeling in scWAT and BAT of both phospholipids and glycerol lipids in response to exercise training, thus providing a comprehensive resource for future studies of lipid metabolism pathways.

Diacylglycerol kinase ζ generates dipalmitoyl-phosphatidic acid species during neuroblastoma cell differentiation

Phosphatidic acid (PA) is one of the phospholipids composing the plasma membrane and acts as a second messenger to regulate a wide variety of important cellular events, including mitogenesis, migration and differentiation. PA consists of various molecular species with different acyl chains at the sn-1 and sn-2 positions. However, it has been poorly understood what PA molecular species are produced during such cellular events. Here we identified the PA molecular species generated during retinoic acid (RA)-induced neuroblastoma cell differentiation using a newly established liquid chromatography/mass spectrometry (LC/MS) method. Intriguingly, the amount of 32:0-PA species was dramatically and transiently increased in Neuro-2a neuroblastoma cells 24–48h after RA-treatment. In addition, 30:0- and 34:0-PA species were also moderately increased. Moreover, similar results were obtained when Neuro-2a cells were differentiated for 24h by serum starvation. MS/MS analysis revealed that 32:0-PA species contains two palmitic acids (16:0s). RT-PCR analysis showed that diacylglycerol kinase (DGK) δ and DGKζ were highly expressed in Neuro-2a cells. The silencing of DGKζ expression significantly decreased the production of 32:0-PA species, whereas DGKδ-siRNA did not. Moreover, neurite outgrowth was also markedly attenuated by the deficiency of DGKζ. Taken together, these results indicate that DGKζ exclusively generates very restricted PA species, 16:0/16:0-PA, and up-regulates neurite outgrowth during the initial/early stage of neuroblastoma cell differentiation.

Lipidomic profiling analysis reveals the dynamics of phospholipid molecules in Arabidopsis thaliana seedling growth.

High-throughput lipidomic profiling provides a sensitive approach for discovering minor lipid species. By using an advance in electrospray ionization tandem mass spectrometry, a large set of phospholipid molecular species (126 species) with high resolution were identified from Arabidopsis seedling; of them 31 species are newly identified (16 are unique in plants), including 13 species of phosphatidic acid (PA), nine phosphatidylcholine, six phosphatidylinositol and three phosphatidylserine. Further analysis of the lipidomic profile reveals dynamics of phospholipids and distinct species alterations during seedling development. PA molecules are found at the lowest levels in imbibition and follow an increasing trend during seedling growth, while phosphatidylethanolamine (PE) molecules show the opposite pattern with highest levels at imbibition and a general decreasing trend at later stages. Of PA molecular species, 34:2-, 34:3-, 36:4-, 36:5-, 38:3- and 38:4-PA increase during radicle emergence, and 34:2- and 34:3-PA reach highest levels during hypocotyl and cotyledon emergence from the seed coat. Conversely, molecular species of PE show higher levels in imbibition and decrease in later stages. These results suggest the crucial roles of specific molecular species and homeostasis of phospholipid molecules in seedling growth and provide insights into the mechanisms of how phospholipid molecules are involved in regulating plant development.

Diacylglycerol Kinase ζ Produces 16:0‐containing Phosphatidic Acid Molecular Species during Neuroblastoma Cell Differentiation

Phosphatidic acid (PA) is one of the phospholipids composing the plasma membrane and acts as a second messenger to regulate a wide variety of cellular functions, including cell growth, migration and differentiation. PA consists of various molecular species with different acyl chains at the sn‐1 and sn‐2 positions. However, it was unclear which PA molecular species were produced during cell stimulation. In this study, we identified the PA molecular species produced during neuroblastoma cell differentiation and the PA species production pathway using our previously developed liquid chromatography (LC)/mass spectrometry (MS) method (1).First, mouse neuroblastoma Neuro‐2a cells were differentiated by treatment with 20 μM retinoic acid (RA) for 24 hours. LC/MS analysis showed that the amount of total PA was significantly increased in RA treated Neuro‐2a cells (1.69‐fold). Notably, the amount of 30:0‐ and 32:0‐PA species were dramatically increased in RA treated Neuro‐2a cells (2.13‐, 4.67‐ and 1.85‐fold, respectively). Similar results were obtained when Neuro‐2a cells were treated with serum starvation for 24 hours. MS/MS analysis revealed that these PA species commonly contain palmitic acid (16:0). Next, we examined the production pathway of 30:0‐ and 32:0‐PA. FIPI, phospholipase D‐specific inhibitor, did not inhibit the production of these PA species. RT‐PCR analysis showed that diacylglycerol kinase (DGK) δ and DGKζ were highly expressed in Neuro‐2a cells. The silencing of DGKδ expression did not decrease the production of the PA species. However, DGKζ‐specific siRNA significantly decreased the production of 30:0‐ and 32:0‐PA. In addition, DGKζ‐specific siRNA substantially attenuated neurite outgrowth/differentiation. These results suggest that DGKζ produces 16:0‐containing 30:0‐ and 32:0‐PA species and regulates neurite outgrowth during neuroblastoma cell differentiation.Support or Funding Information

Triacylglycerol and Glycerophospholipid Identification and Accumulation During Ripening of Pistacia lentiscus L. (Lentisc) Fruit

AbstractTriacylglycerol (TAG) and phospholipid (PL) compositions of vegetable oils are considered a marker of quality and are often used in industry to control the purity of the oils and to detect adulteration. In this study, the TAG and PL composition of developing fruit of Pistacia lentiscus were investigated for the first time. The total TAG content was found to increase rapidly during fruit ripening from 105 to 966 mg/100 g of oil respectively between the 35th and the 175th day after fructification (DAF). During this period, 16 different molecular species of TAG were identified and quantified. POO was the major TAG from the second stage of maturation. Only four classes of PL were identified in the P. lentiscus oil: the phosphatidic acid (PA), the phosphatidylethanolamine (PE), the phosphatidylglycerol (PG) and the phosphatidylinositol (PI). The mass spectra obtained showed the presence of nine molecular species of PA, five species of PE and seven molecular species for each PG and PI classes. The total phospholipid content decreased rapidly during fruit ripening, from 45.5 % at the 15th DAF to 6.88 % at the 175th DAF.

Effect of oral administration of soy-derived phosphatidic acid on concentrations of phosphatidic acid and lyso-phosphatidic acid molecular species in human plasma

Background The glycerophospholipid Phosphatidic acid (PA) has been identified as a potential nutritional treatment for gastrointestinal disorders. Dietary food sources rich in PA include cabbage and radish leaves as well as Mallotus japonicas, a Japanese edible herb historically used for the treatment of stomach ulcers. The mammalian target of rapamycin (mTOR) has been shown to regulate rates of muscle protein synthesis and a mechanical stimulus (resistance exercise) has been shown to activate mTOR with PA playing a key role. Supplementation with soy-derived PA significantly increases responses in skeletal muscle hypertrophy, lean body mass, and maximal strength to resistance exercise. PA accounts for less than 0.1% of the total glycerophospholipid concentration of 201 mg/dl in the human plasma. 15 of the more than 600 distinct molecular lipid species quantified in human plasma are PA, 6 are lysophosphatidic acid (LPA). Orally administered PA can be metabolized to LPA and glycerophosphate by pancreatic phospholipases A1 and A2, which hydrolyze the fatty acid at the sn-1 position and the sn-2 position, respectively. Lysophospholipids are absorbed by the mucosal cells of the gastrointestinal tract and are rapidly re-acylated with fatty acids of the body pool resulting in a newly-formed phospholipid-molecule whose fatty acid composition is determined by the physiological and nutritional status and not by its source. This study sought to assess the effect of soy-derived PA supplementation on concentrations LPA and PA molecular species in human plasma. Methods After a 12 hour overnight fast one subject (20 years of age, bodyweight of 82 kg, and height of 178 cm) was assigned to receive 1.5 grams of soy-derived PA (Mediator, Chemi Nutra, White Bear Lake, MN). Blood draws were taken immediately prior to, and at 30 min, 1, 2, 3, and 7 hours following supplementation. The samples were analyzed by an ultra-performance liquid chromatograph with triple quadrupole mass spectrometry (LC/MS/MS) using 17:1-LPA and 37:4-PA as internal standards to determine the concentration of LPA and PA molecular species in human plasma. Results At baseline, 19 PA (highest concentrations: C34:2 (15%), C40:4 (11%), and C36:4 (10%)) and 5 LPA (16:0 (45%), 18:2 (19%), 20:4 (17%), 14:0 (11%) and 18:1 (8%)) molecular species could be quantified with total concentrations of PA of 2.66 nmol/ml, and LPA of 0.11 nmol/ml. Plasma concentrations of PA peaked at 3 hours (+32%) after ingestion and stayed elevated even after 7h ours (+18%). LPA showed a bimodal absorption kinetic with peaks after 1 hour (+500%) and 3 hours (+264%), after almost dropping back to baseline levels after 2 hours. On an individual fatty acid level, most prominent was a 23-fold increase in 20:4-LPA after 1 hour compared to baseline. The increase in 20:4-LPA does not result from the administration of PA, since soy-derived PA does not contain any arachidonic acid (fatty acids distribution of soy-PA: 18:2 (66.1%), 18:1 (12.6%), 16:0 (11.7%), 18:3 (6.1%) and 18:0 (3.4%)). Absorption of soy-derived PA must yield glycerophosphate which is re-acylated with arachidonic acid.

Diacylglycerol kinase-dependent formation of phosphatidic acid molecular species during interleukin-2 activation in CTLL-2 T-lymphocytes

Although effective liquid chromatography (LC)/mass spectrometry (MS) methods enabling the separation of phospholipid molecular species have been developed, there are still problems with an intracellular signaling molecule, phosphatidic acid (PA). In this study, we optimized LC/MS conditions to improve the quantitative detection of PA molecular species from a cellular lipid mixture. Using the newly developed LC/MS method, we showed that stimulation of CTLL-2 murine T-lymphocytes by interleukin-2 (IL-2) induced a significant increase of 36:1-, 36:2-, 40:5- and 40:6-diacyl-PA. A diacylglycerol kinase (DGK) inhibitor, R59949, attenuated the increase of 36:1-, 40:5-, 40:6-diacyl-PA, suggesting that DGK IL-2-dependently and selectively generated these diacyl-PA species.

Phosphatidic Acid Binds and Stimulates Arabidopsis Sphingosine Kinases

Phosphatidic acid (PA) and phytosphingosine-1-phosphate (phyto-S1P) have both been identified as lipid messengers mediating plant response to abscisic acid (ABA). To determine the relationship of these messengers, we investigated the direct interaction of PA with Arabidopsis sphingosine kinases (SPHKs) that phosphorylate phytosphingosine to generate phyto-S1P. Two unique SPHK cDNAs were cloned from the annotated At4g21540 locus of Arabidopsis, and the two transcripts are differentially expressed in Arabidopsis tissues. Both SPHKs are catalytically active, phosphorylating various long-chain sphingoid bases (LCBs) and are associated with the tonoplast. They both interact with PA as demonstrated by lipid-filter binding, liposome binding, and surface plasmon resonance (SPR). SPHK1 and SPHK2 exhibited strong binding to 18:1/18:1, 16:0/18:1, and 16:0/18:2 PA, but poor binding to 16:0/16:0, 8:0/8:0, 18:0/18:0, and 18:2/18:2 PA. Surface dilution kinetics analysis indicates that PA stimulates SPHK activity by increasing the specificity constant through decreasing K(m)(B). The results show that the annotated At4g21540 locus is actually comprised of two separate SPHK genes. PA binds to both SPHKs, and the interaction promotes lipid substrate binding to the catalytic site of the enzyme. The PA-SPHK interaction depends on the PA molecular species. The data suggest that these two Arabidopsis SPHKs are molecular targets of PA, and the PA stimulation of SPHK is part of the signaling networks in Arabidopsis.

Multiple Functions as Lipase, Steryl Ester Hydrolase, Phospholipase, and Acyltransferase of Tgl4p from the Yeast Saccharomyces cerevisiae

Triacylglycerol (TAG) hydrolysis, membrane lipid biosynthesis, and lipid turnover are largely interlinked processes. In yeast, TAG is mobilized by three TAG lipases named Tgl3p, Tgl4p, and Tgl5p, which are localized to lipid particles/droplets. These TAG lipases posses a conserved GXSXG motif that is characteristic of hydrolytic enzymes. Here, we demonstrated that the yeast TAG lipase Tgl4p, the functional ortholog of the adipose TAG lipase, ATGL, catalyzes multiple functions in lipid metabolism. An extended domain and motif search analysis revealed that Tgl4p bears not only a lipase consensus domain but also a conserved motif for calcium-independent phospholipase A(2). We show that Tgl4p exhibits TAG lipase, steryl ester hydrolase, and phospholipase A(2) activities, but surprisingly it also catalyzed the acyl-CoA-dependent acylation of lysophosphatidic acid to phosphatidic acid (PA). Heterologous overexpression of Tgl4p in Pichia pastoris increased total phospholipid and specifically PA synthesis. Moreover, deletion of TGL4 in Saccharomyces cerevisiae showed an altered pattern of phosphatidylcholine and PA molecular species. Altogether, our data suggest that yeast Tgl4p functions as a hydrolytic enzyme in lipid degradation but also contributes to fatty acid channeling and phospholipid remodeling.

Activation of the Chloroplast Monogalactosyldiacylglycerol Synthase MGD1 by Phosphatidic Acid and Phosphatidylglycerol

One of the major characteristics of chloroplast membranes is their enrichment in galactoglycerolipids, monogalactosyldiacylglycerol (MGDG), and digalactosyldiacylglycerol (DGDG), whereas phospholipids are poorly represented, mainly as phosphatidylglycerol (PG). All these lipids are synthesized in the chloroplast envelope, but galactolipid synthesis is also partially dependent on phospholipid synthesis localized in non-plastidial membranes. MGDG synthesis was previously shown essential for chloroplast development. In this report, we analyze the regulation of MGDG synthesis by phosphatidic acid (PA), which is a general precursor in the synthesis of all glycerolipids and is also a signaling molecule in plants. We demonstrate that under physiological conditions, MGDG synthesis is not active when the MGDG synthase enzyme is supplied with its substrates only, i.e. diacylglycerol and UDP-gal. In contrast, PA activates the enzyme when supplied. This is shown in leaf homogenates, in the chloroplast envelope, as well as on the recombinant MGDG synthase, MGD1. PG can also activate the enzyme, but comparison of PA and PG effects on MGD1 activity indicates that PA and PG proceed through different mechanisms, which are further differentiated by enzymatic analysis of point-mutated recombinant MGD1s. Activation of MGD1 by PA and PG is proposed as an important mechanism coupling phospholipid and galactolipid syntheses in plants.