Formation Of Phosphatidylcholine Research Articles

Relationship between flavor characteristics and lipid oxidation in air-dried beef at different roasting stages

Lipid oxidation plays a key role in the formation of flavor during hot processing. The purpose of this study was to investigate the lipid oxidation, lipid molecular changes and their effects on flavor of air-dried beef at different roasting stages. The results revealed that the degree of lipid oxidation increased and the unsaturation of fatty acids decreased from 47.30% to 30.77%. A total of 48 volatile compounds were identified by headspace-solid phase microextraction gas chromatography-mass spectrometry (HS-SPME-GC-MS). Notably, the types and contents of volatile compounds in S3 group exceeded those in S1 and S2, showing significant differences in the degree of separation among all groups. The aroma differences among the groups were associated with aromatic compounds formed by the oxidation of lipids. Moreover, a total of 59 lipids with significant differences were identified through liquid chromatography-tandem mass spectrometry (LC-MS/MS). These lipids may have major roles in the formation of aromatic compounds, particularly triglyceride, phosphatidylcholine, and lysophosphatidylcholine. This investigation elucidated the mechanisms for the flavor developments of air-dried beef during roasting stages.

Lysophospholipid acyltransferase-mediated formation of saturated glycerophospholipids maintained cell membrane integrity for hypoxic adaptation.

Adaptation to hypoxia has attracted much public interest because of its clinical significance. However, hypoxic adaptation in the body is complicated and difficult to fully explore. To explore previously unknown conserved mechanisms and key proteins involved in hypoxic adaptation in different species, we first used a yeast model for mechanistic screening. Further multi-omics analyses in multiple species including yeast, zebrafish and mice revealed that glycerophospholipid metabolism was significantly involved in hypoxic adaptation with up-regulation of lysophospholipid acyltransferase (ALE1) in yeast, a key protein for the formation of dipalmitoyl phosphatidylcholine [DPPC (16:0/16:0)], which is a saturated phosphatidylcholine. Importantly, a mammalian homolog of ALE1, lysophosphatidylcholine acyltransferase 1 (LPCAT1), enhanced DPPC levels at the cell membrane and exhibited the same protective effect in mammalian cells under hypoxic conditions. DPPC supplementation effectively attenuated growth restriction, maintained cell membrane integrity and increased the expression of epidermal growth factor receptor under hypoxic conditions, but unsaturated phosphatidylcholine did not. In agreement with these findings, DPPC treatment could also repair hypoxic injury of intestinal mucosa in mice. Taken together, ALE1/LPCAT1-mediated DPPC formation, a key pathway of glycerophospholipid metabolism, is crucial for cell viability under hypoxic conditions. Moreover, we found that ALE1 was also involved in glycolysis to maintain sufficient survival conditions for yeast. The present study offers a novel approach to understanding lipid metabolism under hypoxia and provides new insights into treating hypoxia-related diseases.

Characterization of multiple lysophosphatidic acid acyltransferases in the plant pathogen Xanthomonas campestris.

Phosphatidic acid (PA) is the precursor of most phospholipids like phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin. In bacteria, its biosynthesis begins with the acylation of glycerol-3-phosphate to lysophosphatidic acid (LPA), which is further acylated to PA by the PlsC enzyme. Some bacteria, like the plant pathogen Xanthomonas campestris, use a similar pathway to acylate lysophosphatidylcholine to phosphatidylcholine (PC). Previous studies assigned two acyltransferases to PC formation. Here, we set out to study their activity and found a second much more prominent function of these enzymes in LPA to PA conversion. This PlsC-like activity was supported by the functional complementation of a temperature-sensitive plsC-deficient Escherichia coli strain. Biocomputational analysis revealed two further PlsC homologs in X. campestris. The cellular levels of the four PlsC-like proteins varied with respect to growth phase and growth temperature. To address the question whether these enzymes have redundant or specific functions, we purified two recombinant, detergent-solubilized enzymes in their active form, which enabled the first direct biochemical comparison of PlsC isoenzymes from the same organism. Overlapping but not identical acyl acceptor and acyl donor preferences suggest redundant and specialized functions of the X. campestris PlsC enzymes. The altered fatty acid composition in plsC mutant strains further supports the functional differentiation of these enzymes.

The endoplasmic reticulum membrane protein complex localizes to the mitochondrial - endoplasmic reticulum interface and its subunits modulate phospholipid biosynthesis in Trypanosoma brucei.

The endoplasmic reticulum membrane complex (EMC) is a versatile complex that plays a key role in membrane protein biogenesis in the ER. Deletion of the complex has wide-ranging consequences including ER stress, disturbance in lipid transport and organelle tethering, among others. Here we report the function and organization of the evolutionarily conserved EMC (TbEMC) in the highly diverged eukaryote, Trypanosoma brucei. Using (co-) immunoprecipitation experiments in combination with mass spectrometry and whole cell proteomic analyses of parasites after depletion of select TbEMC subunits, we demonstrate that the TbEMC is composed of 9 subunits that are present in a high molecular mass complex localizing to the mitochondrial-endoplasmic reticulum interface. Knocking out or knocking down of single TbEMC subunits led to growth defects of T. brucei procyclic forms in culture. Interestingly, we found that depletion of individual TbEMC subunits lead to disruption of de novo synthesis of phosphatidylcholine (PC) or phosphatidylethanolamine (PE), the two most abundant phospholipid classes in T. brucei. Downregulation of TbEMC1 or TbEMC3 inhibited formation of PC while depletion of TbEMC8 inhibited PE synthesis, pointing to a role of the TbEMC in phospholipid synthesis. In addition, we found that in TbEMC7 knock-out parasites, TbEMC3 is released from the complex, implying that TbEMC7 is essential for the formation or the maintenance of the TbEMC.

Choline and methionine supplementation in layer hens fed flaxseed: effects on hen production performance, egg fatty acid composition, tocopherol content, and oxidative stability

Choline is an essential nutrient in laying hen diets and is needed for the formation of phosphatidylcholine (PC), that serves as a rich source of long chain (≥20 C) n-3 fatty acids (FA) in eggs. Methionine (Met) is the first limiting amino acid in layer hen diets and serves as a lipotropic agent with antioxidant properties. The objectives of the current study is based on the hypothesis that choline and Met supplementation will enhance egg PC and n-3 FA status, lipid stability, and production indices in layer hens fed flaxseed. Ninety-six, 40-wk-old laying hens (W-36 White Leghorns) were randomly allocated to 4 treatment groups, with 6 replicates containing four hens per cage. Hens were fed corn-soybean meal-based diet containing 0% flaxseed (Control), 15/100 g flaxseed (Flax), Flax+50% more methionine requirement for W-36 White Leghorns (Flax+Met), or Flax+0.15g/100g choline chloride (Cho) (Flax+Cho). All experimental diets were isocaloric and isonitrogenous and fed for a period of 120 d. Egg production and egg mass (g/hen/d) was higher for Flax+Met and Flax+Cho when compared to Flax and Control (P < 0.05). Egg weight was greater (P < 0.05) among hens fed the Control and Flax+Cho diets compared to Flax diet. Feeding flaxseed to hens led to over 6-fold increase in total n-3 FA. Choline supplementation increased egg α-tocopherol content (P < 0.05) while reducing lipid oxidation products measured as thiobarituric acid reactive substances in egg yolk (P < 0.05). Neither Met nor Cho had any impact on docosahexaenoic (22:6 n-3) acid concentration in eggs from hens fed flaxseed. However, addition of Met and Cho to layer diets increased docosapentaenoic acid (22:5 n-3) levels in eggs from hens fed flaxseed (P < 0.05). The PC content was lower in Control and Flax+Met (P < 0.05) when compared to Flax+Cho group. No difference was found in total lipid or phosphatidylethanolamine content of eggs (P > 0.05). The results from the current study suggest that n-3 FA content of egg yolk can be greatly increased by feeding flaxseed but reduced egg production. However, dietary Met and Cho can improve production performance in hens fed flaxseed-containing diets. Addition of Cho to flaxseed increased in egg weight, yolk α-tocopherol levels, PC content and oxidative stability of eggs when compared to hens fed flaxseed. Met and choline could be used in flaxseed (>15%) to increase egg production and egg mas.

Potential Neuroregenerative and Neuroprotective Effects of Uridine/Choline-Enriched Multinutrient Dietary Intervention for Mild Cognitive Impairment: A Narrative Review.

In mild cognitive impairment (MCI) due to Alzheimer disease (AD), also known as prodromal AD, there is evidence for a pathologic shortage of uridine, choline, and docosahexaenoic acid [DHA]), which are key nutrients needed by the brain. Preclinical and clinical evidence shows the importance of nutrient bioavailability to support the development and maintenance of brain structure and function in MCI and AD. Availability of key nutrients is limited in MCI, creating a distinct nutritional need for uridine, choline, and DHA. Evidence suggests that metabolic derangements associated with ageing and disease-related pathology can affect the body’s ability to generate and utilize nutrients. This is reflected in lower levels of nutrients measured in the plasma and brains of individuals with MCI and AD dementia, and progressive loss of cognitive performance. The uridine shortage cannot be corrected by normal diet, making uridine a conditionally essential nutrient in affected individuals. It is also challenging to correct the choline shortfall through diet alone, because brain uptake from the plasma significantly decreases with ageing. There is no strong evidence to support the use of single-agent supplements in the management of MCI due to AD. As uridine and choline work synergistically with DHA to increase phosphatidylcholine formation, there is a compelling rationale to combine these nutrients. A multinutrient enriched with uridine, choline, and DHA developed to support brain function has been evaluated in randomized controlled trials covering a spectrum of dementia from MCI to moderate AD. A randomized controlled trial in subjects with prodromal AD showed that multinutrient intervention slowed brain atrophy and improved some measures of cognition. Based on the available clinical evidence, nutritional intervention should be considered as a part of the approach to the management of individuals with MCI due to AD, including adherence to a healthy, balanced diet, and consideration of evidence-based multinutrient supplements.

The Plastid Lipase PLIP1 Is Critical for Seed Viability in diacylglycerol acyltransferase1 Mutant Seed.

In developing Arabidopsis (Arabidopsis thaliana) seeds, the synthesis of triacylglycerol (TAG) is mediated primarily by the acyl-CoA-dependent enzyme diacylglycerol acyltransferase1 (DGAT1). In the absence of DGAT1 activity, phospholipid:diacylglycerol acyltransferase (PDAT1) plays an important role in TAG synthesis, consistent with the higher-than-expected oil content and altered fatty acid composition of dgat1 seed. Transcript profiling of developing wild type (Columbia-0) and dgat1-1 mutant seed identified 602 differentially expressed genes. Expression of genes important for the formation of phosphatidylcholine, including LYSOPHOSPHATIDYLCHOLINE ACYLTRANSFERASE2, and REDUCED OLEATE DESATURATION1 were strongly upregulated, consistent with increased substrate supply for PDAT1. In addition, several genes lacking a defined role in TAG biosynthesis were also upregulated, including the α/β-hydrolase family gene PLIP1, which encodes a plastid-localized lipase. In most tissues, PLIP1 was expressed at equivalent levels in wild-type and dgat1 plants, except for developing seed, where transcript levels were higher in the dgat1 mutant. Seeds from plip1 mutant plants possessed a 20% reduction in oil content and were smaller than seed from wild-type plants. Crosses between dgat1 and plip1 failed to generate double-homozygous mutant plants. Reciprocal crossing with wild-type plants demonstrated that both male and female gametophytes could transmit the dgat1 plip1 double-mutant genotype. Double-homozygous dgat1 plip1 seed formed but was green and failed to germinate. The synthetic lethal phenotype of dgat1 with plip1 indicates an important role for PLIP1 in the absence of DGAT1 activity, likely by supplying polyunsaturated fatty acid substrates for PDAT1.

Localization and expression of CTP: Phosphocholine cytidylyltransferase in rat brain following cocaine exposure

Phosphatidylcholine (PC) is a primary phospholipid and major source of secondary lipid messengers and also serves as a biosynthetic precursor for other membrane phospholipids. Phosphocholine cytidylyltransferase (CCT) is the rate-limiting enzyme responsible for catalyzing the formation of PC. Changes in CCT activity have been associated with lipid dysregulation across various neurological disorders. Additionally, intermediates in PC synthesis, such as CDP-choline, have been suggested to attenuate drug craving during cocaine addiction. Recent work from our group demonstrated that cocaine exposure and conditioning alter the level of PC in the brain, specifically in the cerebellum and hippocampus. The present study examines the role of CCT expression in the brain and determines the effect of cocaine exposure on CCT expression. Immunohistochemical analysis (IHC) was performed to assess region-specific expression of CCT, including both of its isoforms; alpha (CCTα) and beta (CCTβ). IHC did not detect any staining of CCTα throughout the rat brain. In contrast, CCTβ expression was detected in the Purkinje cells of the cerebellum with decreases in expression following cocaine exposure. Collectively, these data demonstrate the region- and cell-specific localization of CCTα and CCTβ in the rat brain, as well as the altered expression of CCTβ in the cerebellum following cocaine exposure.

Lipid Conversion by Cell-Free Synthesized Phospholipid Methyltransferase Opi3 in Defined Nanodisc Membranes Supports an in Trans Mechanism.

Biomembranes composed of lipids and proteins play central roles in physiological processes, and the precise balance between different lipid species is crucial for maintaining membrane function. One pathway for the biosynthesis of the abundant lipid phosphatidylcholine in eukaryotes involves a membrane-integrated phospholipid methyltransferase named Opi3 in yeast. A still unanswered question is whether Opi3 can catalyze phosphatidylcholine synthesis in trans, at membrane contact sites. While evidence for this activity was obtained from studies with complex in vitro-reconstituted systems based on endoplasmic reticulum membranes, isolated and purified Opi3 could not be analyzed. We present new insights into Opi3 activity by characterizing the in vitro-synthesized enzyme in defined hydrophobic environments. Saccharomyces cerevisiae Opi3 was cell-free synthesized and either solubilized in detergent micelles or co-translationally inserted into preformed nanodisc membranes of different lipid compositions. While detergent-solubilized Opi3 was inactive, the enzyme inserted into nanodisc membranes showed activity and stayed monomeric as revealed by native mass spectrometry. The methylation of its lipid substrate dioleoylphosphatidylmonomethylethanolamine to phosphatidylcholine was monitored by one-dimensional 31P nuclear magnetic resonance. Phosphatidylcholine formation was observed not only in nanodiscs containing inserted Opi3 but also in nanodiscs devoid of the enzyme containing the lipid substrate. This result gives a clear indication for in trans catalysis by Opi3; i.e., it acts on the substrate in juxtaposed membranes, while in cis lipid conversion may also contribute. Our established system for the characterization of pure Opi3 in defined lipid environments may be applicable to other lipid biosynthetic enzymes and help in understanding the subcellular organization of lipid synthesis.

Roles of phospholipid methyltransferases in pycnidia development, stress tolerance and secondary metabolism in the taxol-producing fungus Pestalotiopsis microspore

Phosphatidylcholine (PC) is an important membrane component of the eukaryotic cell. In yeast fungi, two phospholipid methyltransferases catalyze consecutive steps of methylation in the formation of phosphatidylcholine from phosphatidylethanolamine. However, roles of phospholipid methyltransferases in filamentous fungi remains less investigated. We report here the characterization of two genes, choA and choC, that putatively encoded phospholipid methyltransferases in the taxol-producing fungus Pestalotiopsis microspora. Deletion of choC resulted in defects in PC production, vegetative growth and development of asexual structure. The mutant strains exhibited multiple morphological abnormalities, e.g. swollen hyphal tips and enhanced hyphal branching, and even mycelial autolysis. Some novel roles for the genes were also revealed, for instance, the deletion of either choC or choA impaired the development of pycnidia and conidia, the cell wall integrity. The mutant strains displayed a hypersensitivity to stress conditions, e.g. osmotic stress, cold and metal ions. The osmotic hypersensitivity indicates a crosstalk of PC pathways to other signaling pathways, such as the HOG pathway. Still more, choA, but not choC, was required for the production of secondary metabolites, e.g. pestalotiollide B, suggesting distinct roles of the two genes. This work would contribute to better understanding the function of phospholipid methyltransferases in fungi.

Dissection of membrane-binding and -remodeling regions in two classes of bacterial phospholipid N-methyltransferases

Bacterial phospholipid N-methyltransferases (Pmts) catalyze the formation of phosphatidylcholine (PC) via successive N-methylation of phosphatidylethanolamine (PE). They are classified into Sinorhizobium-type and Rhodobacter-type enzymes. The Sinorhizobium-type PmtA protein from the plant pathogen Agrobacterium tumefaciens is recruited to anionic lipids in the cytoplasmic membrane via two amphipathic helices called αA and αF. Besides its enzymatic activity, PmtA is able to remodel membranes mediated by the αA domain. According to the Heliquest program, αA- and αF-like amphipathic helices are also present in other Sinorhizobium- and Rhodobacter-type Pmt enzymes suggesting a conserved architecture of α-helical membrane-binding regions in these methyltransferases. As representatives of the two Pmt families, we investigated the membrane binding and remodeling capacity of Bradyrhizobium japonicum PmtA (Sinorhizobium-type) and PmtX1 (Rhodobacter-type), which act cooperatively to produce PC in consecutive methylation steps. We found that the αA regions in both enzymes bind anionic lipids similar to αA of A. tumefaciens PmtA. Membrane binding of PmtX1 αA is enhanced by its substrate monomethyl-PE indicating a substrate-controlled membrane association. The αA regions of all investigated enzymes remodel spherical liposomes into tubular filaments suggesting a conserved membrane-remodeling capacity of bacterial Pmts. Based on these results we propose that the molecular details of membrane-binding and remodeling are conserved among bacterial Pmts.

Metabolic Effects of Clenbuterol and Salbutamol on Pork Meat Studied Using Internal Extractive Electrospray Ionization Mass Spectrometry

Direct mass spectrometry analysis of metabolic effects of clenbuterol and salbutamol on pork quality at the molecular level is incredibly beneficial for food regulations, public health and the development of new anti-obesity drugs. With internal extractive electrospray ionization mass spectrometry (iEESI-MS), nutrients including creatine, amino acids, L-carnitine, vitamin B6, carnosine and phosphatidylcholines in pork tissue were identified, without sample pretreatment, using collision-induced dissociation (CID) experiments and by comparison with authentic compounds. Furthermore, normal pork samples were clearly differentiated from pork samples with clenbuterol and salbutamol via principal component analysis (PCA). Correlation analysis performed on the spectral data revealed that the above-mentioned nutrients strongly correlated with pork quality, and the absolute intensity of phosphatidylcholines in normal pork was much higher than pork contaminated by clenbuterol and salbutamol. Our findings suggested that clenbuterol and salbutamol may render effects on the activity of carnitine acyltransferase I, hence the process that L-carnitine transports long-chain fatty acids into mitochondria and the formation of phosphatidylcholines might be affected. However, the underlying metabolic mechanisms of clenbuterol and salbutamol on carnitine acyltransferase I requires more comprehensive studies in future work.

1,2-Diacylglycerol choline phosphotransferase catalyzes the final step in the unique Treponema denticola phosphatidylcholine biosynthesis pathway.

Treponema denticola synthesizes phosphatidylcholine through a licCA-dependent CDP-choline pathway identified only in the genus Treponema. However, the mechanism of conversion of CDP-choline to phosphatidylcholine remained unclear. We report here characterization of TDE0021 (herein designated cpt) encoding a 1,2-diacylglycerol choline phosphotransferase homologous to choline phosphotransferases that catalyze the final step of the highly conserved Kennedy pathway for phosphatidylcholine synthesis in eukaryotes. T. denticola Cpt catalyzed in vitro phosphatidylcholine formation from CDP-choline and diacylglycerol, and full activity required divalent manganese. Allelic replacement mutagenesis of cpt in T. denticola resulted in abrogation of phosphatidylcholine synthesis. T. denticola Cpt complemented a Saccharomyces cerevisiae CPT1 mutant, and expression of the entire T. denticola LicCA-Cpt pathway in E. coli resulted in phosphatidylcholine biosynthesis. Our findings show that T. denticola possesses a unique phosphatidylcholine synthesis pathway combining conserved prokaryotic choline kinase and CTP:phosphocholine cytidylyltransferase activities with a 1,2-diacylglycerol choline phosphotransferase that is common in eukaryotes. Other than in a subset of mammalian host-associated Treponema that includes T. pallidum, this pathway is found in neither bacteria nor Archaea. Molecular dating analysis of the Cpt gene family suggests that a horizontal gene transfer event introduced this gene into an ancestral Treponema well after its divergence from other spirochetes.

Impaired biosynthesis of the non-bilayer lipids phosphatidylethanolamine or cardiolipin does not affect peroxisome biogenesis and proliferation in Saccharomyces cerevisiae

The non-bilayer forming lipids cardiolipin (CL) and phosphatidylethanolamine (PE) modulate membrane curvature, facilitate membrane fusion and affect the stability and function of membrane proteins. Yeast peroxisomal membranes contain significant amounts of CL and PE.We analysed the effect of CL deficiency and PE depletion on peroxisome biogenesis and proliferation in Saccharomyces cerevisiae. Our data indicate that deletion of CRD1, which encodes cardiolipin synthase, does not affect peroxisome biogenesis or abundance, both at peroxisome repressing (glucose) or inducing (oleate) growth conditions. Analysis of strains deficient in one of the three PE biosynthesis pathways (psd1, psd2 or the triple deletion strain eki1 cki1 dpl1) revealed that in all three strains peroxisome numbers were reduced upon growth of cells on oleic acid, whereas the psd1 strain also showed a reduction in peroxisome abundance upon growth on glucose. Because PE is an intermediate of the phosphatidylcholine (PC) biosynthesis pathway, PE depletion affects PC formation. PC however can be synthesized by an alternative pathway when choline is supplemented to the growth medium. Because the addition of choline resulted in suppression of the peroxisome phenotypes in phosphatidylserine decarboxylase mutant strains, we conclude that peroxisome biogenesis and proliferation are not crucially dependent on CL or PE.

Ligand binding to the ACBD6 protein regulates the acyl-CoA transferase reactions in membranes

The binding determinants of the human acyl-CoA binding domain-containing protein (ACBD) 6 and its function in lipid renewal of membranes were investigated. ACBD6 binds acyl-CoAs of a chain length of 6 to 20 carbons. The stoichiometry of the association could not be fitted to a 1-to-1 model. Saturation of ACBD6 by C16:0-CoA required higher concentration than less abundant acyl-CoAs. In contrast to ACBD1 and ACBD3, ligand binding did not result in the dimerization of ACBD6. The presence of fatty acids affected the binding of C18:1-CoA to ACBD6, dependent on the length, the degree of unsaturation, and the stereoisomeric conformation of their aliphatic chain. ACBD1 and ACBD6 negatively affected the formation of phosphatidylcholine (PC) and phosphatidylethanolamine in the red blood cell membrane. The acylation rate of lysophosphatidylcholine into PC catalyzed by the red cell lysophosphatidylcholine-acyltransferase 1 protein was limited by the transfer of the acyl-CoA substrate from ACBD6 to the acyltransferase enzyme. These findings provide evidence that the binding properties of ACBD6 are adapted to prevent its constant saturation by the very abundant C16:0-CoA and protect membrane systems from the detergent nature of free acyl-CoAs by controlling their release to acyl-CoA-utilizing enzymes.

A Yeast Mutant Deleted of GPH1 Bears Defects in Lipid Metabolism.

In a previous study we demonstrated up-regulation of the yeast GPH1 gene under conditions of phosphatidylethanolamine (PE) depletion caused by deletion of the mitochondrial (M) phosphatidylserine decarboxylase 1 (PSD1) (Gsell et al., 2013, PLoS One. 8(10):e77380. doi: 10.1371/journal.pone.0077380). Gph1p has originally been identified as a glycogen phosphorylase catalyzing degradation of glycogen to glucose in the stationary growth phase of the yeast. Here we show that deletion of this gene also causes decreased levels of phosphatidylcholine (PC), triacylglycerols and steryl esters. Depletion of the two non-polar lipids in a Δgph1 strain leads to lack of lipid droplets, and decrease of the PC level results in instability of the plasma membrane. In vivo labeling experiments revealed that formation of PC via both pathways of biosynthesis, the cytidine diphosphate (CDP)-choline and the methylation route, is negatively affected by a Δgph1 mutation, although expression of genes involved is not down regulated. Altogether, Gph1p besides its function as a glycogen mobilizing enzyme appears to play a regulatory role in yeast lipid metabolism.

Enzymatic properties and substrate specificity of a bacterial phosphatidylcholine synthase.

Pcs and Pcs bind by molecular sieving (1, 2, 3).

Folic Acid Supplementation Alters Adipose Tissue Lipid Metabolism

Mandatory folate fortification polices in Canada and the United States has resulted in high levels of circulating folate in a large proportion of the population. At the same time, a trend toward increased energy intake and reduced energy expenditure has lead to an increased prevalence of obesity, characterized by an accumulation of TAG in adipose tissue. Folate plays an essential role in maintaining S‐adenosylmethionine (AdoMet), an important biological methyl‐donor. AdoMet functions in the formation of phosphatidylcholine from phosphatidylethanolamine catalyzed by the enzyme phosphatidylethanolamine N‐methyltransferase (PEMT). PEMT activity contributes to storage of triacylglycerol (TAG) in adipocytes.The objective of this study was to investigate the effect of dietary folic acid supplementation on adipose tissue lipid metabolism. Sprague Dawley rats were fed a high fat diet containing 0.75 mg or 7.5 mg folic acid for 12 weeks.Adipose tissue weight was significantly greater in rats fed excess folic acid. This was accompanied by an increased expression of genes involved in re‐esterification of fatty acids to glycerol, suggesting elevated TAG formation. Up‐regulation of genes involved in phospholipid biosynthesis supports increased storage of TAG in adipocytes upon folate supplementation.In summary, folate supplementation enhances TAG storage in adipose tissue of high fat diet‐fed rats.Grant Funding Source : CIHR, Food and Health Innovation Initiative

Phosphatidylcholine formation by LPCAT1 is regulated by Ca2+ and the redox status of the cell

BackgroundUnsaturated fatty acids are susceptible to oxidation and damaged chains are removed from glycerophospholipids by phospholipase A2. De-acylated lipids are then re-acylated by lysophospholipid acyltransferase enzymes such as LPCAT1 which catalyses the formation of phosphatidylcholine (PC) from lysoPC and long-chain acyl-CoA.ResultsActivity of LPCAT1 is inhibited by Ca2+, and a Ca2+-binding motif of the EF-hand type, EFh-1, was identified in the carboxyl-terminal domain of the protein. The residues Asp-392 and Glu-403 define the loop of the hairpin structure formed by EFh-1. Substitution of D392 and E403 to alanine rendered an enzyme insensitive to Ca2+, which established that Ca2+ binding to that region negatively regulates the activity of the acyltransferase amino-terminal domain. Residue Cys-211 of the conserved motif III is not essential for catalysis and not sufficient for sensitivity to treatment by sulfhydryl-modifier agents. Among the several active cysteine-substitution mutants of LPCAT1 generated, we identified one to be resistant to treatment by sulfhydryl-alkylating and sulfhydryl-oxidizer agents.ConclusionMutant forms of LPCAT1 that are not inhibited by Ca2+ and sulfhydryl-alkylating and –oxidizing agents will provide a better understanding of the physiological function of a mechanism that places the formation of PC, and the disposal of the bioactive species lysoPC, under the control of the redox status and Ca2+ concentration of the cell.

Human Lysophosphatidylcholine Acyltransferases 1 and 2 Are Located in Lipid Droplets Where They Catalyze the Formation of Phosphatidylcholine

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