Diacylglycerol Molecular Species Research Articles

Membrane morphology determines diacylglycerol kinase α substrate acyl chain specificity.

Diacylglycerol kinases catalyze the ATP-dependent phosphorylation of diacylglycerol (DAG) to produce phosphatidic acid (PA). In humans, the alpha isoform (DGKα) has emerged as a potential target in the treatment of cancer due to its anti-tumor and pro-immune responses. However, its mechanism of action at a molecular level is not fully understood. In this work, a systematic investigation of the role played by the membrane in the regulation of the enzymatic properties of human DGKα is presented. By using a cell-free system with purified DGKα and model membranes of variable physical and chemical properties, it is shown that membrane physical properties determine human DGKα substrate acyl chain specificity. In model membranes with a flat morphology; DGKα presents high enzymatic activity, but it is not able to differentiate DAG molecular species. Furthermore, DGKα enzymatic properties are insensitive to membrane intrinsic curvature. However, in the presence of model membranes with altered morphology, specifically the presence of physically curved membrane structures, DGKα bears substrate acyl chain specificity for palmitic acid-containing DAG. The present results identify changes in membrane morphology as one possible mechanism for the depletion of specific pools of DAG as well as the production of specific pools of PA by DGKα, adding an extra layer of regulation on the interconversion of these two potent lipid-signaling molecules. It is proposed that the interplay between membrane physical (shape) and chemical (lipid composition) properties guarantee a fine-tuned signal transduction system dependent on the levels and molecular species of DAG and PA.

Diacylglycerol kinase ζ interacts with sphingomyelin synthase 1 and sphingomyelin synthase‐related protein

Introduction Mammalian sphingomyelin (SM) synthase (SMS) consists of two isoform (SMS1, SMS2). SMS produces diacylglycerol (DG) by the transfer of phosphocholine from phosphatidylcholine (PC) to ceramide. SMS-related protein (SMSr) also generates DG via synthesis of ceramide phosphoethanolamine, a SM analog, using phosphatidylethanolamine. Mammalian DG kinase (DGK) consists of ten isozymes (α, β, γ, δ, η, ϵ, κ, ι, ζ, θ) and governs a wide range of physiological and pathological events such as type 2 diabetes. Among them, SMS1, SMSr, DGKδ and DGKη contain a steraile a motif domain (SAMD), a protein-protein interaction module. We previously reported that DGKδ interacts with SMSr, but not SMS1, via SAMDs [Murakami et al. J. Biol. Chem. 295, 2932–47, 2020]. Moreover, SMSr provided DG to DGKδ. However, it is still unclear whether other DGK isozymes interact with SMSs. In the present study, we analyzed interaction between DGKs and SMSs by co-immunoprecipitation analysis. Results and Conclusion First, we determined whether SAMD-containing DGKs (DGKδ and DGKη) interact with SAMD-containing SMSs (SMS1 and SMSr). Although DGKη-SAMD co-sedimented with DGKη-SAMD itself, SMS1- and SMSr-SAMD failed to interact with DGKη-SAMD. Furthermore, only the combination of full-length DGKδ and full-length SMSr could form a heteromeric complex. These results suggest that the interaction via heteromerization of SAMDs is specific to DGKδ and SMSr. Next, we analyzed whether other DGKs, which contain no SAMD, interact with SMSs. We found that DGKζ was strongly co-immunoprecipitated with SMSr and SMS1, but not SMS2. These results suggest that DGKζ can interact with SMS1 and SMSr without heteromerization between SAMDs. Taken together, these results suggest that, in addition to DGKδ, DGKζ is a new candidate that acts downstream of SMSr and SMS1. SMSr produces palmitic acid (16:0)-containing DG molecular species and SMS1 generates PC-derived DG species including 16:0-containing DGs. Moreover, DGKζ preferably phosphorylates 16:0-containing DG species during neuronal cell differentiation. Therefore, it is possible that these enzymes compose a new pathway independent of the phosphatidylinositol turnover, which primary utilizes arachidonic acid (20:4)-containing DG species.

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.

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)

Diacylglycerol kinase δ and sphingomyelin synthase–related protein functionally interact via their sterile α motif domains

The δ isozyme of diacylglycerol kinase (DGKδ) plays critical roles in lipid signaling by converting diacylglycerol (DG) to phosphatidic acid (PA). We previously demonstrated that DGKδ preferably phosphorylates palmitic acid (16:0)- and/or palmitoleic acid (16:1)-containing DG molecular species, but not arachidonic acid (20:4)-containing DG species, which are recognized as DGK substrates derived from phosphatidylinositol turnover, in high glucose-stimulated myoblasts. However, little is known about the origin of these DG molecular species. DGKδ and two DG-generating enzymes, sphingomyelin synthase (SMS) 1 and SMS-related protein (SMSr), contain a sterile α motif domain (SAMD). In this study, we found that SMSr-SAMD, but not SMS1-SAMD, co-immunoprecipitates with DGKδ-SAMD. Full-length DGKδ co-precipitated with full-length SMSr more strongly than with SMS1. However, SAMD-deleted variants of SMSr and DGKδ interacted only weakly with full-length DGKδ and SMSr, respectively. These results strongly suggested that DGKδ interacts with SMSr through their respective SAMDs. To determine the functional outcomes of the relationship between DGKδ and SMSr, we used LC-MS/MS to investigate whether overexpression of DGKδ and/or SMSr in COS-7 cells alters the levels of PA species. We found that SMSr overexpression significantly enhances the production of 16:0- or 16:1-containing PA species such as 14:0/16:0-, 16:0/16:0-, 16:0/18:1-, and/or 16:1/18:1-PA in DGKδ-overexpressing COS-7 cells. Moreover, SMSr enhanced DGKδ activity via their SAMDs in vitro Taken together, these results strongly suggest that SMSr is a candidate DG-providing enzyme upstream of DGKδ and that the two enzymes represent a new pathway independent of phosphatidylinositol turnover.

Endurance exercise training and high-fat diet differentially affect composition of diacylglycerol molecular species in rat skeletal muscle.

Insulin resistance of peripheral muscle is implicated in the etiology of metabolic syndrome in obesity. Although accumulation of glycerolipids, such as triacylglycerol and diacylglycerol (DAG), in muscle contributes to insulin resistance in obese individuals, endurance-trained athletes also have higher glycerolipid levels but normal insulin sensitivity. We hypothesized that the difference in insulin sensitivity of skeletal muscle between athletes and obese individuals stems from changes in fatty acid composition of accumulated lipids. Here, we evaluated the effects of intense endurance exercise and high-fat diet (HFD) on the accumulation and composition of lipid molecular species in rat skeletal muscle using a lipidomic approach. Sprague-Dawley female rats were randomly assigned to three groups and received either normal diet (ND) in sedentary conditions, ND plus endurance exercise training, or HFD in sedentary conditions. Rats were fed ND or HFD between 4 and 12 wk of age. Rats in the exercise group ran on a treadmill for 120 min/day, 5 days/wk, for 8 wk. Soleus muscle lipidomic profiles were obtained using liquid chromatography/tandem mass spectrometry. Total DAG levels, particularly those of palmitoleate-containing species, were increased in muscle by exercise training. However, whereas the total DAG level in the muscle was also increased by HFD, the levels of DAG molecular species containing palmitoleate were decreased by HFD. The concentration of phosphatidylethanolamine molecular species containing palmitoleate was increased by exercise but decreased by HFD. Our results indicate that although DAG accumulation was similar levels in trained and sedentary obese rats, specific changes in molecular species containing palmitoleate were opposite.

Where do substrates of diacylglycerol kinases come from? Diacylglycerol kinases utilize diacylglycerol species supplied from phosphatidylinositol turnover-independent pathways.

Diacylglycerol kinase (DGK) phosphorylates diacylglycerol (DG) to produce phosphatidic acid (PA). Mammalian DGK comprises ten isozymes (α-κ) and regulates a wide variety of physiological and pathological events, such as cancer, type II diabetes, neuronal disorders and immune responses. DG and PA consist of various molecular species that have different acyl chains at the sn-1 and sn-2 positions, and consequently, mammalian cells contain at least 50 structurally distinct DG/PA species. Because DGK is one of the components of phosphatidylinositol (PI) turnover, the generally accepted dogma is that all DGK isozymes utilize 18:0/20:4-DG derived from PI turnover. We recently established a specific liquid chromatography-mass spectrometry method to analyze which PA species were generated by DGK isozymes in a cell stimulation-dependent manner. Interestingly, we determined that DGKδ, which is closely related to the pathogenesis of type II diabetes, preferentially utilized 14:0/16:0-, 14:0/16:1-, 16:0/16:0-, 16:0/16:1-, 16:0/18:0- and 16:0/18:1-DG species (X:Y=the total number of carbon atoms: the total number of double bonds) supplied from the phosphatidylcholine-specific phospholipase C pathway, but not 18:0/20:4-DG, in high glucose-stimulated C2C12 myoblasts. Moreover, DGKα mainly consumed 14:0/16:0-, 16:0/18:1-, 18:0/18:1- and 18:1/18:1-DG species during cell proliferation in AKI melanoma cells. Furthermore, we found that 16:0/16:0-PA was specifically produced by DGKζ in Neuro-2a cells during retinoic acid- and serum starvation-induced neuronal differentiation. These results indicate that DGK isozymes utilize a variety of DG molecular species derived from PI turnover-independent pathways as substrates in different stimuli and cells. DGK isozymes phosphorylate various DG species to generate various PA species. It was revealed that the modes of activation of conventional and novel protein kinase isoforms by DG molecular species varied considerably. However, PA species-selective binding proteins have not been found to date. Therefore, we next attempted to identify PA species-selective binding proteins from the mouse brain and identified α-synuclein, which has causal links to Parkinson's disease. Intriguingly, we determined that among phospholipids, including several PA species (16:0/16:0-PA, 16:0/18:1-PA, 18:1/18:1-PA, 18:0/18:0-PA and 18:0/20:4-PA); 18:1/18:1-PA was the most strongly bound PA to α-synuclein. Moreover, 18:1/18:1-PA strongly enhanced secondary structural changes from the random coil form to the α-helix form and generated a multimeric and proteinase K-resistant α-synuclein protein. In contrast with the dogma described above, our recent studies strongly suggest that PI turnover-derived DG species and also various DG species derived from PI turnover-independent pathways are utilized by DGK isozymes. DG species supplied from distinct pathways may be utilized by DGK isozymes based on different stimuli present in different types of cells, and individual PA molecular species would have specific targets and exert their own physiological functions.

Identification of the molecular species of acylglycerols containing hydroxy fatty acids in wild edible mushroom Ganoderma lucidum

Abstract Edible Philippine mushrooms including Ganoderma lucidum have many health benefits. Seventy-two molecular species of triacylglycerols and five molecular species of diacylglycerols containing hydroxy fatty acids (FA) in the lipid extract of this mushroom were identified by HPLC and MS. The mono-, di- and tri-hydroxy FA constituents of the molecular species of acylglycerols were identified by various degrees of dehydration of [FA + Li] + . Many odd carbon numbered hydroxy FA constituents of acylglycerols were also identified. The contents of the 77 molecular species of acylglyerols and their constituent hydroxy FA in the lipid extract were estimated by the relative ion signal intensities of the molecular species of acylglycerols and the HPLC peak area % (ELSD). The contents (%) of the molecular species of acylglycerols containing hydroxy FA in the lipid extract were as: P-19:0-2OH18:2, 0.69%; 2OH18:2-2OH18:2-2OH18:2, 0.60%; P-OH19:1-OH19:1, 0.48%; S-OH19:1-OH19:1, 0.43%; 19:0-19:0-2OH18:2, 0.27% and the total of about 5.8%. “2OH18:2” stands for dihydroxy FA with 18 carbon atoms and two double bonds. The mushroom lipid extract was 1.67% of freeze-dried mycelium. The contents (%) of individual hydroxy FA in the mushroom lipid extract in decreasing order were: 2OH18:2, 1.4%; OH19:1, 1.1%; OH18:1, 0.38%; 2OH19:0, 0.19%; 2OH18:0, 0.13% and the total of about 3.5%. The odd carbon numbered hydroxy FA was about 1.44% of the mushroom lipid extract. The high contents of acylglycerols containing hydroxy FA, odd carbon numbered FA and odd carbon numbered hydroxy FA in edible mushroom might be related to one or more of its health benefits. As far as we are aware of, this mushroom contains the highest amounts of hydroxy FA and odd carbon numbered FA among the human foods.

Activation of conventional and novel protein kinase C isozymes by different diacylglycerol molecular species

A variety of diacylglycerol (DG) molecular species are produced in stimulated cells. Conventional (α, βII and γ) and novel (δ, ε, η and θ) protein kinase C (PKC) isoforms are known to be activated by DG. However, a comprehensive analysis has not been performed. In this study, we analyzed activation of the PKC isozymes in the presence of 2–2000mmol% 16:0/16:0-, 16:0/18:1-, 18:1/18:1-, 18:0/20:4- or 18:0/22:6-DG species. PKCα activity was strongly increased by DG and exhibited less of a preference for 18:0/22:6-DG at 2mmol%. PKCβII activity was moderately increased by DG and did not have significant preference for DG species. PKCγ activity was moderately increased by DG and exhibited a moderate preference for 18:0/22:6-DG at 2mmol%. PKCδ activity was moderately increased by DG and exhibited a preference for 18:0/22:6-DG at 20 and 200mmol%. PKCε activity moderately increased by DG and showed a moderate preference for 18:0/22:6-DG at 2000mmol%. PKCη was not markedly activated by DG. PKCθ activity was the most strongly increased by DG and exhibited a preference for 18:0/22:6-DG at 2 and 20mmol% DG. These results indicate that conventional and novel PKCs have different sensitivities and dependences on DG and a distinct preference for shorter and saturated fatty acid-containing and longer and polyunsaturated fatty acid-containing DG species, respectively. This differential regulation would be important for their physiological functions.

Multidimensional mass spectrometry-based shotgun lipidomics analysis of vinyl ether diglycerides.

Diglycerides play a central role in lipid metabolism and signaling in mammalian cells. Although diacylglycerol molecular species comprise the majority of cellular diglycerides that are commonly measured using a variety of approaches, identification of extremely low abundance vinyl ether diglycerides has remained challenging. In this work, representative molecular species from the three diglyceride subclasses (diacyl, vinyl ether, and alkyl ether diglycerides; hereafter referred to as diradylglycerols) were interrogated by mass spectrometric analysis. Product ion mass spectra of the synthesized diradylglycerols with varied chain lengths and degrees of unsaturation demonstrated diagnostic fragmentation patterns indicative of each subclass. Multidimensional mass spectrometry-based shotgun lipidomics (MDMS-SL) analysis of mouse brain and heart lipid extracts were performed using the identified informative signature product ions. Through an array of tandem mass spectrometric analyses utilizing the orthogonal characteristics of neutral loss scanning and precursor ion scanning, the differential fragmentation of each subclass was exploited for high-yield structural analyses. Although molecular ion mass spectra readily identified diacylglycerol molecular species directly from the hexane fractions of tissue extracts enriched in nonpolar lipids, molecular ion peaks corresponding to ether-linked diglycerides were not observable. The power of MDMS-SL utilizing the tandem mass spectrometric array analysis was demonstrated by identification and profiling of individual molecular species of vinyl ether diglycerides in mouse brain and heart from their undetectable molecular ion peaks during MS(1) analysis. Collectively, this technology enabled the identification and profiling of previously inaccessible vinyl ether diglyceride molecular species in mammalian tissues directly from extracts of biologic tissues.

Lipidomic analysis of Arabidopsis seed genetically engineered to contain DHA.

Metabolic engineering of omega-3 long-chain (≥C20) polyunsaturated fatty acids (ω3 LC-PUFA) in oilseeds has been one of the key targets in recent years. By expressing a transgenic pathway for enhancing the synthesis of the ω3 LC-PUFA docosahexaenoic acid (DHA) from endogenous α-linolenic acid (ALA), we obtained the production of fish oil-like proportions of DHA in Arabidopsis seed oil. Liquid chromatography-mass spectrometry (LC-MS) was used to characterize the triacylglycerol (TAG), diacylglycerol (DAG) and phospholipid (PL) lipid classes in the transgenic and wild type Arabidopsis seeds at both developing and mature stages. The analysis identified the appearance of several abundant DHA-containing phosphatidylcholine (PC), DAG and TAG molecular species in mature seeds. The relative abundances of PL, DAG, and TAG species showed a preferred combination of LC-PUFA with ALA in the transgenic seeds, where LC-PUFA were esterified in positions usually occupied by 20:1ω9. Trace amounts of di-DHA PC and tri-DHA TAG were identified and confirmed by high resolution MS/MS. Studying the lipidome in transgenic seeds provided insights into where DHA accumulated and combined with other fatty acids of neutral and phospholipids from the developing and mature seeds.

Comparative Analysis of Biological Sphingolipids with Glycerophospholipids and Diacylglycerol by LC-MS/MS.

Liquid chromatography-electrospray ionization mass spectrometry (LC-MS) is an effective and popular technique used in lipid metabolomic studies. Although many LC-MS methods enabling the determination of sphingolipid molecular species have been reported, they do not cover a broad range of sphingolipid metabolites with expanding glycerophospholipids (GPLs) and diacylglycerol (DAG). In this study, we developed an approach for the comprehensive analysis of sphingolipids, GPLs and DAG molecular species in a biological sample, without alkaline hydrolysis. After validating the reliability of this approach, we analyzed tissue lipids of sphingomyelin synthase 2-knockout mice and found that changes in sphingolipid metabolism in the liver affect the level of docosahexaenoic acid-containing GPLs. Our method analyzes GPLs and DAG, as well as sphingolipids within biological samples and, thus, will facilitate more comprehensive studies of sphingolipid metabolism in pathology and diagnostics.

Identification of TAG and DAG and their FA Constituents in Lesquerella (Physaria fendleri) Oil by HPLC and MS

AbstractCastor oil has many industrial uses because of its high content (90 %) of the hydroxy fatty acid, ricinoleic acid (OH1218:19). Lesquerella oil containing lesquerolic acid (Ls, OH1420:111) is potentially useful in industry. Ten molecular species of diacylglycerols and 74 molecular species of triacylglycerols in lesquerella (Physaria fendleri) oil were identified by electrospray ionization mass spectrometry as lithium adducts of acylglycerols in the HPLC fractions of lesquerella oil. Among them were: LsLsO, LsLsLn, LsLsL, LsLn–OH20:2, LsO–OH20:2 and LsL–OH20:2. The structures of the four new hydroxy fatty acid constituents of acylglycerols were proposed by the MS of the lithium adducts of fatty acids as (comparing to those in castor oil): OH1218:29,14 (OH1218:29,13 in castor oil), OH1218:39,14,16 (OH18:3 not detected in castor oil), diOH12,1318:29,14 (diOH11,1218:29,13 in castor oil) and diOH13,1420:111 (diOH20:1 not detected in castor oil, diOH11,1218:19 in castor oil). Trihydroxy fatty acids were not detected in lesquerella oil. The differences in the structures of these C18 hydroxy fatty acids between lesquerella and castor oils indicated that the polyhydroxy fatty acids were biosynthesized and were not the result of autoxidation products.

Measurements of Diacylglycerols in Skeletal Muscle by Atmospheric Pressure Chemical Ionization Mass Spectrometry

Diacylglycerol (DAG) is an intermediate lipid involved in the synthesis of phospholipids and triglycerides. As signaling regulators, DAG activate novel protein kinase C leading to decreased response to insulin in skeletal muscle. Alteration of DAG contents correlates with development of metabolic dysregulation in obese and diabetic conditions. Recent advances in lipidomics using mass spectrometry allow expanded measurements of various lipid species. This study describes a rapid measurement of DAG species using the triple quadrupole mass spectrometry using atmospheric pressure chemical ionization in a positive ion mode. DAG in the cells and muscle tissues were separated depending on differences in chain lengths and degree of unsaturation. The limit of detection and quantification for DAG was 0.2 to 17pmol for this method. When C2C12 cells were treated with palmitate or oleate, we found a 12-fold and 2-fold DAG increase respectively compared to the no-treatment control. In the muscles of obese db/db mice, DAG levels were elevated by 6-fold compared to those of wild-type skeletal muscles. The present analytical method provides a rapid and sensitive quantification of DAG molecular species from various biological samples and can be used to correlate the degree of metabolic dysregulation with lipotoxic metabolites.

Long-term Expression of Apolipoprotein B mRNA-specific Hammerhead Ribozyme via scAAV8.2 Vector Inhibits Atherosclerosis in Mice

Target substrate-specific hammerhead ribozyme cleaves the specific mRNA efficiently and results in the inhibition of gene expression. In humans, overproduction of apolipoprotein B (apoB) is positively associated with premature coronary artery diseases. The goal of this study is to demonstrate that long-term reduction of apoB gene expression using hammerhead ribozyme would result in inhibition of atherosclerosis development. We designed two hammerhead ribozymes targeted at the nucleotides of apoB mRNA GUC2326 (designated RB1) and GUA6679 (designated RB15), and we used self-complementary adeno-associated virus 8.2 (scAAV8.2) vector to deliver these active ribozymes of RB1, RB15, combination of RB1/RB15, and an inactive hammerhead ribozyme RB15 mutant to atherosclerosis-prone LDb mice (Ldlr−/−Apobec1−/−). LDb mice lack both low density lipoproteins (LDL) receptor (Ldlr−/−) and apoB mRNA editing enzyme (Apobec1−/−) genes and develop atherosclerosis spontaneously. After the RB1, RB15, or combination of RB1/RB15 ribozymes treatment, the LDb mice had significantly decreased plasma triglyceride and apoB levels, resulting in markedly decreased of atherosclerotic lesions, Furthermore, the active ribozymes treatment decreased the levels of diacylglycerol acyltransferase 1 (Dgat1) mRNA and the levels of multiple diacylglycerol (DAG) molecular species. These results provide the first evidence that decreased apoB levels results to reduction of Dgat1 expression and triglyceride levels (TAG), which had a significant impact on the development of atherosclerosis.

Methods for microbial screening and production of polyol oils from soybean oil through bioprocess

Abstract Soypolyol oils (oxygenated acylglycerols) are important starting materials for the manufacture of polymers such as polyurethane. Currently, they are produced by a two-step chemical process involving epoxidation and the subsequent opening of the oxirane ring. The objective of this study is to develop a bioprocess to produce polyol oils directly from soybean oil. For product separation, we found that TLC with a two step development solvent systems could separate the polyol products from substrate soybean oil. The products and substrates were separated in the following order: substrate triacylglycerols (Rf 0.8), free fatty acids (FA, Rf 0.7), product dihydroxy TAG (Rf 0.4), trihydroxy TAG (Rf 0.3), monohydroxy FA (Rf 0.1) and dihydroxy FA (Rf 0.05). We also found that HPLC with a C18 reverse phase column and a linear gradient of 100% methanol to 100% 2-propanol over 60 min at 1 mL/min flow rate was able to separate product polyol oils and substrate soybean oil. Free FA and polyol oils (diacylglycerols (DAG) containing hydroxyl FA) were eluted between 5 min and 15 min. DAG containing two normal FA was eluted between 15 min and 28 min and the substrate soybean oil was eluted between 36 min and 45 min. A total of 400 microbial cultures were screened and we identified 25 hits. Polyol oils' products were purified through a silica gel column chromatography, fractionated by HPLC and then analyzed by MS. A total of 57 molecular species of DAG containing tri-, di-, monohydroxy FA and normal FA were identified by MS. HPLC chromatogram of evaporative light scattering detector was used for semi-quantification of these DAG in the purified polyol oils. The total content of the DAG containing two normal FA was about 25% and the total content of DAG containing hydroxy FA might be about 75%. The yield of bioconversion by culture A01-35 from soybean oil to polyol oil products (DAG containing hydroxyl FA) plus DAG containing normal FA was 31% by weight. Bioconversion of soybean oil to polyol oils is a new research area without available methodology to follow. Here we report a new bioprocess for the production of soypolyol oils directly from soybean oil.

Quantification of Signaling Lipids by Nano-Electrospray Ionization Tandem Mass Spectrometry (Nano-ESI MS/MS).

Lipids, such as phosphoinositides (PIPs) and diacylglycerol (DAG), are important signaling intermediates involved in cellular processes such as T cell receptor (TCR)-mediated signal transduction. Here we report identification and quantification of PIP, PIP2 and DAG from crude lipid extracts. Capitalizing on the different extraction properties of PIPs and DAGs allowed us to efficiently recover both lipid classes from one sample. Rapid analysis of endogenous signaling molecules was performed by nano-electrospray ionization tandem mass spectrometry (nano-ESI MS/MS), employing lipid class-specific neutral loss and multiple precursor ion scanning for their identification and quantification. Profiling of DAG, PIP and PIP2 molecular species in primary human T cells before and after TCR stimulation resulted in a two-fold increase in DAG levels with a shift towards 1-stearoyl-2-arachidonoyl-DAG in stimulated cells. PIP2 levels were slightly reduced, while PIP levels remained unchanged.

Localisation and composition of skeletal muscle diacylglycerol predicts insulin resistance in humans.

We sought to evaluate if the cellular localisation and molecular species of diacylglycerol (DAG) were related to insulin sensitivity in human skeletal muscle. Healthy sedentary obese controls (Ob; n = 6; mean±SEM age 39.5 ± 2.3 years; mean ± SEM BMI 33.3 ± 1.4 kg/m(2)), individuals with type 2 diabetes (T2D; n = 6; age 44 ± 1.8 years; BMI 30.1 ± 2.3 kg/m(2)), and lean endurance-trained athletes (Ath; n = 10; age 35.4 ± 3.1 years; BMI 23.3 ± 0.8 kg/m(2)) were studied. Insulin sensitivity was determined using an IVGTT. Muscle biopsy specimens were taken after an overnight fast, fractionated using ultracentrifugation, and DAG species measured using liquid chromatography/MS/MS. Total muscle DAG concentration was higher in the Ob (mean ± SEM 13.3 ± 1.0 pmol/μg protein) and T2D (15.2 ± 1.0 pmol/μg protein) groups than the Ath group (10.0 ± 0.78 pmol/μg protein, p = 0.002). The majority (76-86%) DAG was localised in the membrane fraction for all groups, but was lowest in the Ath group (Ob, 86.2 ± 0.98%; T2D, 84.2 ± 1.2%; Ath, 75.9 ± 2.7%; p = 0.008). There were no differences in cytoplasmic DAG species (p > 0.12). Membrane DAG species C18:0/C20:4, Di-C16:0 and Di-C18:0 were significantly more abundant in the T2D group. Cytosolic DAG species were negatively related to activation of protein kinase C (PKC)ε but not PKCθ, whereas membrane DAG species were positively related to activation of PKCε, but not PKCθ. Only total membrane DAG (r = -0.624, p = 0.003) and Di-C18:0 (r = -0.595, p = 0.004) correlated with insulin sensitivity. Disaturated DAG species were significantly lower in the Ath group (p = 0.001), and significantly related to insulin sensitivity (r = -0.642, p = 0.002). These data indicate that both cellular localisation and composition of DAG influence the relationship to insulin sensitivity. Our results suggest that only saturated DAG in skeletal muscle membranes are related to insulin resistance in humans.

Analysis of cholesteryl esters and diacylglycerols using lithiated adducts and electrospray ionization-tandem mass spectrometry

Cholesteryl ester (CE) and diacylglycerol (DAG) molecular species are important lipid storage and signaling molecules. Mass spectrometric analyses of these lipids are complicated by the presence of isobaric molecular ions shared by these lipid classes and by relatively poor electrospray ionization, which is a consequence of an inherently weak dipole moment in these lipid classes. The current study demonstrates that lithiated adducts of CE and DAG molecular ions have enhanced ionization and lipid class-specific fragmentation in tandem mass spectrometry (MS/MS) scan modes, thereby allowing the implementation of strategies capable of lipid class-specific detection. Using neutral loss (NL) mode for the loss of cholestane from cholesterol esters (NL 368.5) and specific selected reaction monitoring for DAG molecular species, the response of specific molecular species to that of internal standards was determined. CE and DAG molecular species were quantified in human coronary artery endothelial cells (HCAECs) incubated with both palmitic acid and oleic acid. Furthermore, NL 368.5 spectra revealed the oxidation of the aliphatic fatty acid residues of CE molecular species. Taken together, these studies demonstrate a new analytical approach to assessing CE and DAG molecular species that exploits the utility of lithiated adducts in conjunction with MS/MS approaches.