Mechanisms of hepatic phosphatidylcholine synthesis in the developing guinea pig: contributions of acyl remodelling and of N-methylation of phosphatidylethanolamine.
ABSTRACT: Hepatic phosphatidylcholine (PC) from the immature fetal guinea pig at day 55 of gestation comprised mainly unsaturated molecular species containing C18:2(n-6) and C22:6(n-3) at the sn-2 position, reflecting placental permeability to essential fatty acids. At both day 55 and term (day 68), [Me-14C]choline was incorporated in utero over 3 h largely into sn-1-C16:0 PC species, with incorporation into sn-1-C18:0 PC species increasing by 18 h of incubation. Comparison of specific radioactivities after 3 h and 18 h suggests PC acyl remodelling by phospholipase A1. No incorporation into C20:4(n-6)-containing PC species could be detected of either [Me-14C]choline in vivo or CDP-[Me-14C]choline in isolated microsomes. The major phosphatidylethanolamine (PE) species were 16:0/22:6 and 18:0/22:6. Although [14C]ethanolamine was initially incorporated mainly into sn-1-C16:0 species, specific-radioactivity analysis suggested differential turnover rather than acyl remodelling. [1,2-14C]Ethanolamine and [Me-14C]methionine incorporation into PC molecular species indicated that both newly synthesized and total PE pools were available for N-methylation. Since the PC pool synthesized from PE included C20:4- and C22:6-containing species, N-methylation may provide a mechanism for supplying essential long-chain fatty acids to developing tissues that can be regulated independently from bulk PC synthesis.
Project description:Late pregnancy in the rat (gestational ages 16-21 days) was accompanied by a specific increase in hepatic phosphatidylcholine (PC) and phosphatidylethanolamine (PE) molecular species containing C16:0 at the sn-1 position and polyunsaturated essential fatty acids (PUFA), in particular C22:6(n-3), at the sn-2 position. Incorporation of either CDP:[Me-14C]choline or CDP:[1,2-14C]-ethanolamine into hepatic microsomal sn-1 C16:0 PC or PE molecular species in vitro was greater at term than in non-pregnant animals, suggesting modifications to the composition of specific diacylglycerol (DAG) pools destined for synthesis of either PC or PE. Also, incorporation of [Me-14C]choline or [Me-14C]methionine into hepatic PC in vivo over 6 h in term pregnant rats was consistent with decreased phospholipase A1-dependent acyl remodelling of sn-1 C16:0 to sn-1 C18:0 molecular species. There was, however, no evidence to support any change to the specificity of acyl remodelling. The rate of PC synthesis by the de novo pathway in vivo was increased in term liver compared with non-pregnant animals, accompanied by increased choline-phosphotransferase activity in vitro in d21 liver microsomes. The rate of PC synthesis by PE N-methylation did not appear to change during pregnancy. Changes in composition of plasma PC species at term reflected those of newly synthesized hepatic PC. Our data suggest supply of PUFA to the developing fetal rat is the result of specific adaptations to maternal hepatic phospholipid biosynthesis rather than passive transfer from the maternal diet.
Project description:To expand our knowledge of lipid and fatty acid (FA) biosynthesis in marine cnidarians, polar lipidomes of hydrocorals were studied for the first time and then compared with those of soft corals from tropical and boreal regions. The structure and content of FAs and molecular species of ethanolamine, choline, serine, and inositol glycerophospholipids (PE, PC, PS, and PI, respectively), and ceramide aminoethylphosphonate (CAEP) in tropical hydrocorals (Millepora platyphylla, M. dichotoma) and the cold-water hydrocoral Allopora steinegeri were determined by chromatography and mass spectrometry. All soft corals and cold-water hydrocorals are characterized by a considerable amount of C20 polyunsaturated FAs (PUFAs) elongated into C22 PUFAs. In the Millepora species, the high level of 22:5n-6 and 22:6n-3 against the background of the extremely low level of C20 PUFAs may be explained by a high activity of rare ?4 desaturase. In contrast to hydrocorals, soft corals are able to elongate and further desaturate C22 PUFAs into C24 PUFAs. Allopora and soft corals use C20 PUFAs mainly for the synthesis of PE and PC. The molecular species of PS of soft corals concentrate C24 PUFAs, while in Allopora and Millepora the PS molecules are mainly based on 22:4n-6 and 22:5n-6 acyl groups, respectively. Short acyl groups (C14) dominate the CAEP molecules of Allopora. In all the animals compared, most molecular species of PE and PC are ether lipids, but diacyl molecular species dominate PI. Hydrocorals and tropical soft corals contain diacyl and ether PS molecules, respectively, whereas cold-water soft corals contain a mixture of these PS forms. The high similarity of the alkyl/acyl compositions indicates a possible biosynthetic relationship between PS and PI in hydrocorals. The data obtained in our study will provide a resource to further investigate the lipid metabolism in marine invertebrates.
Project description:The molecular-species compositions of the diacyl classes of the major phospholipids from the brain and retina of rainbow trout (Salmo gairdneri) were determined. A total of 46 possible species was identified. Didocosahexaenoyl species were major components of phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylserine (PS) from retina, comprising 14.1, 41.3 and 28.3% of the respective totals. This species was also abundant in PE and PS from brain, accounting for 14.9 and 19.9% of the totals respectively. Small amounts of di-polyunsaturated fatty acid species [C22:6(n-3) with C20:5(n-3), and C22:6(n-3) with C22:5(n-3)] were also found in these phospholipids. Phosphatidylinositol (PI) from both tissues contained no di-polyunsaturated fatty acid species. Retinal PI contained 40.1% C18:0-C20:4(n-6) with 14.9% of C18:0-C20:5(n-3); brain PI contained 42.3% of C18:0-C20:5 and 10.4% of C18:0-C20:4 species. Brain PC contained a substantial amount of nervonic acid-containing species with the pair C18:1-C24:1/C24:1-C18:1 comprising 8.9% of the total.
Project description:Previous studies with electropermeabilized cells have suggested the occurrence of metabolic compartmentation and Ca2+-dependent channeling of intermediates of phosphatidylcholine (PC) biosynthesis in C6 rat glioma cells. With a more accessible permeabilization technique, we investigated whether this is a more general phenomenon also occurring in other cell types and whether channeling is involved in phosphatidylethanolamine (PE) synthesis as well. C6 rat glioma cells, C3H10T12 fibroblasts and rat hepatocytes were permeabilized with Staphylococcus aureus alpha-toxin, and the incorporation of the radiolabelled precursors choline, phosphocholine (P-choline), ethanolamine and phosphoethanolamine (P-EA) into PC and PE were measured both at high and low Ca2+ concentrations. In glioma cells, permeabilization at high Ca2+ concentration did not affect [14C]choline or [14C]P-choline incorporation into PC. However, reduction of free Ca2+ in the medium from 1.8 mM to <1 nM resulted in a dramatic increase in [14C]P-choline incorporation into permeabilized cells, whereas [14C]choline incorporation remained unaffected. Also, in fibroblasts, reduction of extracellular Ca2+ increased [14C]P-choline and [14C]P-EA incorporation into PC and PE respectively. In hepatocytes, a combination of alpha-toxin and low Ca2+ concentration severely impaired [14C]choline incorporation into PC. Therefore, alpha-toxin-permeabilized hepatocytes are not a good model in which to study channeling of intermediates in PC biosynthesis. In conclusion, our results indicate that channeling is involved in PC synthesis in glioma cells and fibroblasts. PE synthesis in fibroblasts is also at least partly dependent on channeling.
Project description:Mass changes in the incorporation of linoleic (C18:2), eicosapentaenoic (C20:5) and docosahexaenoic (C22:6) acids in human blood platelet phospholipids were induced by incubating the cells and these fatty acids complexed to albumin. The remodelling of [14C]C18:2, [14C]C20:5 and [14C]C22:6 in classes, subclasses and molecular species of platelet phospholipids was studied in resting and thrombin-stimulated cells. More than 85% of the incorporation was located in phospholipids, representing 5-fold and 2.5-fold increases in the phospholipid C20:5 and C22:6 endogenous content respectively. Thrombin stimulation induced a 30% degradation of 1-acyl-2-C20:5-glycerophosphocholine (GPC) and 1-acyl-2-C22:6-GPC, but did not induce significant release of C18:2 from 1-acyl-2-C18:2-GPC. There was no change in the [14C]fatty acid composition of 1-alkyl-2-acyl-GPC. Thrombin-dependent increases in 1-alkenyl-2-C20:5-glycerophosphoethanolamine (GPE) and 1-alkenyl-2-C22:6-GPE of 2.1-fold and 2.5-fold respectively accounted for the rise in GPE radioactivity and partly compensated for the loss of these fatty acids from 1,2-diacyl-GPC: transfer to 1-alkenyl-2-acyl-GPE was 0.4 and 1.5 nmol/10(9) platelets for C20:5 and C22:6 respectively. [14C]C20:5 and [14C]C22:6 were incorporated into six different species of 1,2-diacyl-GPC, with acylation in the major endogenous forms (C18:1 +C16:0 and C18:0 species) representing 76% and 66% respectively of the total radioactivity present in 1,2-diacyl-GPC. Stimulation by thrombin induced significant release of these fatty acids from the main molecular species of 1,2-diacyl-GPC, but significantly stimulated the synthesis of alkenyl forms of GPE containing C18:1/C22:6 +C16:0/C22:6, C18:0/C22:6 and C18:0/C20:5. C18:0/C18:2, the major endogenous C18:2 molecular species, represented only 10.5% of the incorporation; none of the [14C]C18:2 molecular species was a substrate for transfer towards 1-alkenyl-2-acyl-GPE. It is concluded that when C20:5 and C22:6, but not C18:2, are acylated in 1,2-diacyl-GPC, they participate in thrombin-dependent phospholipid remodelling, and might compete with the turnover and release of arachidonic acid from platelet phospholipids and the subsequent activation of the cells.
Project description:Abnormal lipid metabolisms are closely associated with cancers. In this study, mass spectrometry was employed to in situ investigate the associations of membrane lipid phenotypes of six human lung cancer cell lines (i.e., A549, H1650, H1975 from adenocarcinoma, H157 and H1703 from squamous cell carcinomas, and H460 from a large cell carcinoma) with cancer cell types and finally total 230 lipids were detected. Based these 230 lipids, partial least-square discriminant analysis indicated that fifteen lipids (i.e., PE 18:0_18:1, PI 18:0_20:4, SM 42:2, PE 16:0_20:4, PE 36:2, PC 36:2, SM 34:1, PA 38:3,C18:0, C22:4, PA 34:2, C20:5, C20:2, C18:2, and CerP 36:2) with variable importance in the projection (VIP) value of > 1.0 could be used to differentiate six cancer cell lines with the Predicted Residual Sum of Square (PRESS) score of 0.1974. Positive correlation between polyunsaturated fatty acids (i.e., C20:4, C22:4, C22:5, and C22:6) and polyunsaturated phospholipids (PE 16:0_20:4, PE 38:4, and PI 18:0_20:4) was observed in lung adenocarcinoma cells, especially for H1975 cells. Three adenocarcinoma cell lines (i.e., A549, H1650, and H1975) could be differentiated from other lung cancer cell lines based on the expression of C18:1, C20:1, C20:2, C20:5, and C22:6.
Project description:The present study was undertaken to study the role of AMP-activated kinase (AMPK) in the biosynthesis of two major membrane phospholipids, phosphatidylcholine (PC) and phosphatidylethanolamine (PE). Incubation of rat hepatocytes with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), an activator of AMPK, produced dose-dependent inhibition of the incorporation of [(3)H]choline and [(3)H]ethanolamine into PC and PE, respectively. Determination of the cellular uptake of choline and ethanolamine showed that the reduced synthesis of PC and PE did not result from impaired uptake of these two precursors. The decreased synthesis of PC was not mirrored by a reduction in the activities of the enzymes of the CDP-choline pathway. The diminution of PE biosynthesis, however, was paralleled by a depressed activity of CTP:phosphoethanolamine cytidylyltransferase (ET), the pace-setting enzyme of the CDP-ethanolamine pathway. AICAR treatment of hepatocytes stimulated the conversion of choline into betaine, indicating that reduced PC synthesis most probably resulted from a decrease in the availability of choline. In addition, AICAR induced a 50% reduction in the cellular level of diacylglycerols, which may further impair the synthesis of PC and PE. The results thus indicate that AICAR inhibits the biosynthesis of PC and PE and that the effect is exerted at different sites in the two pathways. Increased oxidation of choline to betaine is the main target of AICAR in the PC pathway, whereas inhibition of ET activity is the locus of AICAR action in the PE pathway.
Project description:Phosphatidylethanolamine (PE) and phosphatidylcholine (PC) are among the most abundant phospholipids in biological membranes. In many eukaryotes, the CDP-ethanolamine and CDP-choline branches of the Kennedy pathway represent major and often essential routes for the production of PE and PC, with ethanolamine and choline/ethanolamine phosphotransferases (EPT and CEPT, respectively) catalysing the last reactions in the respective pathways. Although the site of PE and PC synthesis is commonly known to be the endoplasmic reticulum (ER), detailed information on the localization of the different phosphotransferases is lacking. In the unicellular parasite, Trypanosoma brucei, both branches of the Kennedy pathway are essential for cell growth in culture. We have previously reported that T. brucei EPT (TbEPT) catalyses the production of ether-type PE molecular species while T. brucei CEPT (TbCEPT) synthesizes diacyl-type PE and PC molecular species. We now show that the two enzymes localize to different sub-compartments of the ER. By expressing a series of tagged forms of the two enzymes in T. brucei parasites, in combination with sub-cellular fractionation and enzyme activity measurements, TbEPT was found exclusively in the perinuclear ER, a distinct area located close to but distinct from the nuclear membrane. In contrast, TbCEPT was detected in the bulk ER.
Project description:The conversion of phosphatidylethanolamine (PE) into phosphatidylcholine (PC) by a sequence of three transmethylation reactions is shown to be stimulated by the apolipoprotein E-free subclass of high-density lipoprotein (HDL3) in isolated bovine brain capillary (BBC) membranes, HDL3-induced stimulation of BBC membranes pulsed with [methyl-14C]methionine causes a transient increase in each methylated phospholipid, i.e. phosphatidyl-N-monomethylethanolamine (PMME), phosphatidyl-NN-dimethylethanolamine (PDME) and PC. PC substrate arising from the activation of PE N-methyltransferase (PEMT) is hydrolysed by a phospholipase A2 (PLA2), as demonstrated by the accumulation of lysophosphatidylcholine (lyso-PC). When PE containing [14C]arachidonic acid in the sn-2 position ([14C]PAPE) is incorporated into BBC membranes, HDL3 stimulation induces the formation of PMME, PDME, PC and lyso-PC and the release of [14C]arachidonic acid, which correlates with the previous production of lyso-PC, suggesting that HDL3 stimulates a PLA2 that can release polyunsaturated fatty acids (PUFA). Both PEMT and PLA2 activities depend on a HDL3 concentration in the range 0-50 micrograms/ml and are strictly dependent on HDL3 binding, because HDL3 modified by tetranitromethane is no longer able to bind to specific receptors and to trigger PEMT and PLA2 activation. Moreover, HDL3 prelabelled with [14C]PAPE can stimulate PDME and lyso-PC synthesis in BBC membranes in the presence of S-adenosylmethionine, suggesting that HDL3 can supply BBC membranes in polyunsaturated PE and can activate enzymes involved in PE N-methylation and PUFA release. The results support the hypothesis of a close relationship between HDL3 binding, PE methylation and PUFA release, and suggest that the PC pool arising from PE could be used as a pathway for the supply of PUFA to the brain.
Project description:Platelet stimulation by thrombin or Ca2+ ionophore induces mobilization of arachidonate from lipid stores. We have previously shown that, in [14C]arachidonic acid-prelabelled resting platelets, [14C]arachidonate was transferred from diacyl-sn-glycerophosphocholine to ethanolamine and choline-containing ether phospholipids. This transfer reached an equilibrium after 5 h incubation [Colard, Breton & Bereziat (1984a) Biochem. J. 222, 657-662]. [14C]Arachidonate-prelabelled platelets having reached this transfer equilibrium were used to study the mobilization of arachidonate in etheracyl and diacyl phospholipids. Upon thrombin stimulation, arachidonate decreased in diacyl-sn-glycero-3-phosphoinositol, in alkylacyl- and diacyl-sn-glycero-3-phosphocholine and increased in alkenylacyl- and diacyl-sn-glycero-3-phosphoethanolamine. Upon challenge with Ca2+ ionophore A23187, arachidonate decreased in diacyl-sn-glycero-3-phosphoethanolamine, in diacyl- and alkylacyl-sn-glycero-3-phosphocholine and increased in alkenylacyl-sn-glycero-3-phosphoethanolamine. We also compared arachidonate mobilization in platelets stimulated immediately after [14C]arachidonic acid chase with platelets stimulated after 5 h reincubation. We observed that the arachidonate newly incorporated into diacyl-sn-glycero-3-phosphocholine and triacylglycerols was rapidly released upon stimulation. This suggests the presence in these two lipids of a rapidly-turning-over arachidonate pool.