Mechanisms of accumulation of arachidonate in phosphatidylinositol in yellowtail. A comparative study of acylation systems of phospholipids in rat and the fish species Seriola quinqueradiata.

It is known that phosphatidylinositol (PtdIns) contains abundant arachidonate and is composed mainly of 1-stearoyl-2-arachidonoyl species in mammals. We investigated if this characteristic of PtdIns applies to the PtdIns from yellowtail (Seriola quinqueradiata), a marine fish. In common with phosphatidylcholine (PtdCho), phosphatidylethanolamine (PtdEtn) and phosphatidylserine (PtdSer) from brain, heart, liver, spleen, kidney and ovary, the predominant polyunsaturated fatty acid was docosahexaenoic acid, and levels of arachidonic acid were less than 4.5% (PtdCho), 7.5% (PtdEtn) and 3.0% (PtdSer) in these tissues. In striking contrast, arachidonic acid made up 17.6%, 31.8%, 27.8%, 26.1%, 25.4% and 33.5% of the fatty acid composition of PtdIns from brain, heart, liver, spleen, kidney and ovary, respectively. The most abundant molecular species of PtdIns in all these tissues was 1-stearoyl-2-arachidonoyl. Assay of acyltransferase in liver microsomes of yellowtail showed that arachidonic acid was incorporated into PtdIns more effectively than docosahexaenoic acid and that the latter inhibited incorporation of arachidonic acid into PtdCho without inhibiting the utilization of arachidonic acid for PtdIns. This effect of docosahexaenoic acid was not observed in similar experiments using rat liver microsomes and is thought to contribute to the exclusive utilization of arachidonic acid for acylation to PtdIns in yellowtail. Inositolphospholipids and their hydrolysates are known to act as signaling molecules in cells. The conserved hydrophobic structure of PtdIns (the 1-stearoyl-2-arachidonoyl moiety) may have physiological significance not only in mammals but also in fish.     

Metabolic characterization of sciadonic acid (5c,11c,14c-eicosatrienoic acid) as an effective substitute for arachidonate of phosphatidylinositol.

Sciadonic acid (20:3 Delta-5,11,14) is an n-6 series trienoic acid that lacks the Delta8 double bond of arachidonic acid. This fatty acid is not converted to arachidonic acid in higher animals. In this study, we characterized the metabolic behavior of sciadonic acid in the process of acylation to phospholipid of HepG2 cells. One of the characteristics of fatty acid compositions of phospholipids in sciadonic acid-supplemented cells is a higher proportion of sciadonic acid in phosphatidylinositol (PtdIns) (27.4%) than in phosphatidylethanolamine (PtdEtn) (23.2%), phosphatidylcholine (PtdCho) (17.3%) and phosphatidylserine (PtdSer) (20.1%). Similarly, the proportion of arachidonic acid was higher in PtdIns (35.8%) than in PtdEtn (29.1%), PtdSer (18.2%) and PtdCho (20.2%) in arachidonic-acid-supplemented cells. The extensive accumulation of sciadonic acid in PtdIns resulted in the enrichment of newly formed 1-stearoyl-2-sciadonoyl molecular species (38%) in PtdIns and caused the reduction in the level of pre-existing arachidonic-acid-containing molecular species. The kinetics of incorporation of sciadonic acid to PtdEtn, PtdSer and PtdIns of cells were similar to those of arachidonic acid. In contrast to sciadonic acid, neither eicosapentaenoic acid (20:5 Delta-5,8,11,14,17) nor juniperonic acid (20:4 Delta-5,11,14,17) accumulated in the PtdIns fraction. Rather, these n-3 series polyunsaturated fatty acids, once incorporated into PtdIns, tended to be excluded from PtdIns. In addition, the level of arachidonic-acid-containing PtdIns molecular species remained unchanged by eicosapentaenoic-acid-supplementation. These results suggest that sciadonic acid or sciadonic-acid-containing glycerides are metabolized in a similar manner to arachidonic acid or arachidonic-acid-containing glyceride in the biosynthesis of PtdIns and that sciadonic acid can effectively modify the molecular species composition of PtdIns in HepG2 cells. In this regard, sciadonic acid will be an interesting experimental tool to clarify the significance of arachidonic acid-residue of PtdIns-origin bioactive lipids.     

Mass‐spectrometric characterization of phospholipids and their primary peroxidation products in rat cortical neurons during staurosporine‐induced apoptosis

The molecular diversity of phospholipids is essential for their structural and signaling functions in cell membranes. In the current work, we present, the results of mass spectrometric characterization of individual molecular species in major classes of phospholipids – phosphatidylcholine (PtdCho), phosphatidylethanolamine (PtdEtn), phosphatidylserine (PtdSer), phosphatidylinositol (PtdIns), sphingomyelin (CerPCho), and cardiolipin (Ptd₂Gro) – and their oxidation products during apoptosis induced in neurons by staurosporine (STS). The diversity of molecular species of phospholipids in rat cortical neurons followed the order Ptd₂Gro > PtdEtn >> PtdCho >> PtdSer > PtdIns > CerPCho. The number of polyunsaturated oxidizable species decreased in the order Ptd₂Gro >> PtdEtn > PtdCho > PtdSer > PtdIns > CerPCho. Thus a relatively minor class of phospholipids, Ptd₂Gro, was represented in cortical neurons by the greatest variety of both total and peroxidizable molecular species. Quantitative fluorescence HPLC analysis employed to assess the oxidation of different classes of phospholipids in neuronal cells during intrinsic apoptosis induced by STS revealed that three anionic phospholipids – Ptd₂Gro >> PtdSer > PtdIns – underwent robust oxidation. No significant oxidation in the most dominant phospholipid classes – PtdCho and PtdEtn – was detected. MS‐studies revealed the presence of hydroxy‐, hydroperoxy‐ as well as hydroxy‐/hydroperoxy‐species of Ptd₂Gro, PtdSer, and PtdIns. Experiments in model systems where total cortex Ptd₂Gro and PtdSer fractions were incubated in the presence of cytochrome c (cyt c) and H₂O₂, confirmed that molecular identities of the products formed were similar to the ones generated during STS‐induced neuronal apoptosis. The temporal sequence of biomarkers of STS‐induced apoptosis and phospholipid peroxidation combined with recently demonstrated redox catalytic properties of cyt c realized through its interactions with Ptd₂Gro and PtdSer suggest that cyt c acts as a catalyst of selective peroxidation of anionic phospholipids yielding Ptd₂Gro and PtdSer peroxidation products. These oxidation products participate in mitochondrial membrane permeability transition and in PtdSer externalization leading to recognition and uptake of apoptotic cells by professional phagocytes.     

Lipid alterations in transient forebrain ischemia: possible new mechanisms of CDP-choline neuroprotection

We have previously demonstrated that cytidine 5'-diphosphocholine (CDP-choline or citicoline) attenuated arachidonic acid (ArAc) release and provided significant protection for the vulnerable hippocampal CA(1) neurons of the cornu ammonis after transient forebrain ischemia of gerbil. ArAc is released by the activation of phospholipases and the alteration of phosphatidylcholine (PtdCho) synthesis. Released ArAc is metabolized by cyclooxygenases/lipoxygenases to form eicosanoids and reactive oxygen species (ROS). ROS contribute to neurotoxicity through generation of lipid peroxides and the cytotoxic byproducts 4-hydroxynonenal and acrolein. ArAc can also stimulate sphingomyelinase to produce ceramide, a potent pro-apoptotic agent. In the present study, we examined the changes and effect of CDP-choline on ceramide and phospholipids including PtdCho, phosphatidylethanolamine (PtdEtn), phosphatidylinositol (PtdIns), phosphatidylserine (PtdSer), sphingomyelin, and cardiolipin (an exclusive inner mitochondrial membrane lipid essential for electron transport) following ischemia/1-day reperfusion. Our studies indicated significant decreases in total PtdCho, PtdIns, PtdSer, sphingomyelin, and cardiolipin and loss of ArAc from PtdEtn in gerbil hippocampus after 10-min forebrain ischemia/1-day reperfusion. CDP-choline (500 mg/kg i.p. immediately after ischemia and at 3-h reperfusion) significantly restored the PtdCho, sphingomyelin, and cardiolipin levels as well as the ArAc content of PtdCho and PtdEtn but did not affect PtdIns and PtdSer. These data suggest multiple beneficial effects of CDP-choline: (1) stabilizing the cell membrane by restoring PtdCho and sphingomyelin (prominent components of outer cell membrane), (2) attenuating the release of ArAc and limiting its oxidative metabolism, and (3) restoring cardiolipin levels.     

Ca2(+)-dependence of arachidonic acid redistribution among phospholipids of cultured mouse keratinocytes

Mouse keratinocytes cultured in a medium containing less than 0.1 mM Ca2+ (low Ca2+) incorporated [1-14C]arachidonic acid (AA) into phospholipids by kinetics including; (i) a rapid labelling of phosphatidylinositol (PtdIns), phosphatidylserine (PtdSer) and both acid-stable and alkenylacyl forms of phosphatidylcholine (PtdCho); and (ii) a slow but long-lasting radiolabel incorporation into both acid-stable and alkenylacyl forms of phosphatidylethanolamine (PtdEtn), partly associated with a net radioactivity loss from acid stable-PtdCho. Under low Ca2+ conditions no radioactivity transfer apparently occurred between PtdIns and other phospholipid classes. When cells were prelabelled for 24 h with [1-14C]AA and reincubated in label-free medium containing 1.2 mM Ca2+ (normal Ca2+), an early and extensive loss of radioactivity from PtdIns was observed, reasonably in connection with Ca2+ stimulation of phosphoinositide turnover. Cell shift to normal Ca2+ did not result in an increased synthesis of labelled eicosanoids, but was consistent with an increase of radioactivity incorporation into diacylglycerol (DAG) and with a complex pattern of [1-14C]AA redistribution, eventually leading to a marked radioactivity incorporation into acid stable-PtdEtn (but not into alkenylacyl-PtdEtn) and to a labelling decrease of acid stable-PtdCho. The possible mechanisms driving AA recycling after cell shift to normal Ca2+ are discussed.     

Contribution of different pathways to the supply of phosphatidylethanolamine and phosphatidylcholine to mitochondrial membranes of the yeast Saccharomyces cerevisiae

In the yeast, three biosynthetic pathways lead to the formation of phosphatidylethanolamine (PtdEtn): (i) decarboxylation of phosphatidylserine (PtdSer) by phosphatidylserine decarboxylase 1 (Psd1p) in mitochondria; (ii) decarboxylation of PtdSer by Psd2p in a Golgi/vacuolar compartment; and (iii) the CDP-ethanolamine (CDP-Etn) branch of the Kennedy pathway. The major phospholipid of the yeast, phosphatidylcholine (PtdCho), is formed either by methylation of PtdEtn or via the CDP-choline branch of the Kennedy pathway. To study the contribution of these pathways to the supply of PtdEtn and PtdCho to mitochondrial membranes, labeling experiments in vivo with [(3)H]serine and [(14)C]ethanolamine, or with [(3)H]serine and [(14)C]choline, respectively, and subsequent cell fractionation were performed with psd1Delta and psd2Delta mutants. As shown by comparison of the labeling patterns of the different strains, the major source of cellular and mitochondrial PtdEtn is Psd1p. PtdEtn formed by Psd2p or the CDP-Etn pathway, however, can be imported into mitochondria, although with moderate efficiency. In contrast to mitochondria, microsomal PtdEtn is mainly derived from the CDP-Etn pathway. PtdEtn formed by Psd2p is the preferred substrate for PtdCho synthesis. PtdCho derived from the different pathways appears to be supplied to subcellular membranes from a single PtdCho pool. Thus, the different pathways of PtdEtn biosynthesis play different roles in the assembly of PtdEtn into cellular membranes.     

Arachidonic Acid Incorporation and Redistribution in Human Neuroblastoma (SK-N-BE) Cell Phospholipids

The incorporation and redistribution of [1-14C]arachidonic acid in SK-N-BE human neuroblastoma cell phospholipids were investigated. By continuous labelling in serum-enriched medium, a rapid radioactivity incorporation into phosphatidylcholine (PtdCho), phosphatidylinositol, and phosphatidylserine was observed; initially, phosphatidylethanolamine (PtdEtn) was poorly labelled, but at later stages it displayed the highest level of arachidonic acid incorporation, in comparison with other phospholipid classes. Labelling of triacylglycerols was also observed. When cells were pulse-labelled with [1-14C]arachidonic acid and then reincubated in label-free medium, a decrease of the radioactivity in triacylglycerols was observed initially, paralleled by an increase of phospholipid labelling; thereafter, arachidonic acid redistribution was consistent with a net decrease of the radioactivity associated with PtdCho acid-stable forms (i.e., diacyl plus alkylacyl forms), concomitantly with a net labelling increase of both acid-stable PtdEtn and alkenylacyl-PtdEtn. Data indicate the following: (a) neuroblastoma cells incorporate arachidonic acid into phospholipids through complex kinetics involving transfer of the fatty acid from acid-stable PtdCho to both alkenylacyl-PtdEtn and acid-stable PtdEtn; and (b) triacylglycerols act as storage molecules for arachidonic acid which is subsequently incorporated into phospholipids. The possibility that arachidonic acid transfer to PtdEtn subclasses is driven by distinct mechanisms is discussed.     

Myelination process in the rat sciatic nerve during regeneration and development: Molecular species composition and acyl group biosynthesis of choline-, ethanolamine-, and serine-glycerophospholipids of myelin fractions

The content of alkenyl-acyl, alkyl-acyl and diacyl types of the three major myelin glycerophospholipids such as PtdCho, PtdEtn and PtdSer was determined in myelin fractions prepared from sciatic nerve segments of rats at 12, 25 and 45 days after birth, and of adult rats (6-month-old) 90 days after crush injury. The biosynthesis and metabolic heterogeneity of lipid classes and types were also studied by incubation with [1-¹⁴C] acetate of nerve segments of young rats at different ages as well as crushed and sham-operated control nerve segments of adult rats. The analysis of composition and positional distribution in major individual molecular species extracted from light myelin and myelin-related fraction suggest that the metabolism of alkenyl-acyl-glycerophosphorylethanolamines and unsaturated species of PtdCho and PtdSer may not be regulated in the same manner during peripheral nerve myelination of developing rat and remyelination of regenerating nerve in the adult animal. The¹⁴C-radioactivity incorporation into lipid classes and alkyl and acyl moieties of the three major phospholipids of sciatic nerve segments during the developmental period investigated revealed that Schwann cells were capable of synthesizing acyl-linked fatty acids in both myelin fractions at a decreasing rate and with different patterns during development. In regenerating sciatic nerve of adult animals the labeling of myelin lipid classes and types of remyelinating nerve segment distal to the crush site was markedly higher than that of sham-operated normal one; however, the magnitude and the pattern of the specific radioactivity never approached those observed during active myelination of the nerve in young animals. These observations show that the remyelinating process of injured nerve during regeneration seems not to recapitulate nerve myelin ensheathment occurring during development.Abbreviations used PtdEtn Phosphatidylethanolamine - PtdCho Phosphatidylcholine - PtdSer Phosphatidylserine - GPE Glycero(3)phosphoethanolamine - GPC Glycero(3)phosphocholine - GPS Glycero(3)phosphoserine - DG-acetates 1,2-diradyl-3-acetyl-sn-glycerols - HPLC High performance liquid chromatography - TLC Thin-layer chromatography - BHT 2,6-di-tert-butyl-4-methylphenol     

Identification of Two Molecular Species of Rat Brain Phosphatidylcholine that Rapidly Incorporate and Turn Over Arachidonic Acid In Vivo

Abstract: In vivo rates of arachidonic acid incorporation and turnover were determined for molecular species of rat brain phosphatidylcholine (PtdCho) and phosphatidylinositol (PtdIns). [³H]Arachidonic acid was infused intravenously in pentobarbital-anesthetized rats at a programmed rate to maintain constant plasma specific activity for 2–10 min. At the end of infusion, animals were killed by microwave irradiation, and brain phospholipids were isolated, converted to diacylglycerobenzoates, and resolved as molecular species by reversed-phase HPLC. Most [³H]arachidonate (>87%) was incorporated into PtdCho and PtdIns, with arachidonic acid at the sn -2 position and with oleic acid (18:1), palmitic acid (16:0), or stearic acid (18:0) at the sn -1 position. However, 10–15% of labeled brain PtdCho eluted in a small peak containing two molecular species with arachidonic acid at the sn -2 position and palmitoleic acid (16:1) or linoleic acid (18:2) at the sn -1 position. Analysis demonstrated that tracer was present in both the 16:1–20:4 and 18:2–20:4 PtdCho species at specific activities 10–40 times that of the other phospholipids. Based on the measured mass of arachidonate in each phospholipid molecular species, half-lives were calculated for arachidonate of <10 min in 16:1–20:4 and 18:2–20:4 PtdCho and 1–3 h in 16:0–20:4, 18:0–20:4, and 18:1–20:4 PtdCho and PtdIns. The very short half-lives for arachidonate in the 16:1–20:4 and 18:2–20:4 PtdCho molecular species suggest important roles for these molecules in brain phospholipid metabolism and signal transduction.     

Alterations by perfluorooctanoic acid of glycerolipid metabolism in rat liver

The effects of perfluorooctanoic acid (PFOA) feeding on hepatic levels of glycerolipids and the underlying mechanism were investigated. Feeding of rats with 0.01% of PFOA in the diet for 1 week caused an increase in the contents of phosphatidylcholine (PtdCho), phosphatidylethanolamine (PtdEtn), phosphatidylinositol (PtdIns), phosphatidylserine (PtdSer) and triglyceride (TG), which were 2.2, 2.4, 2.4, 1.6 and 5.2 times over control, respectively, on the basis of whole liver. The activities of glycerol-3-phosphate acyltransferase, diacylglycerol kinase and PtdSer decarboxylase were significantly increased upon PFOA feeding, whereas the activities of CTP:phosphoethanolamine cytidylyltransferase and PtdEtn N-methyltransferase were decreased. On the other hand, the activity of CTP:phosphocholine cytidylyltransferase was not increased by PFOA. Upon PFOA feeding, hepatic level of 16:0-18:1 PtdCho was markedly increased and, by contrast, the levels of molecular species of PtdCho which contain 18:2 were decreased, resulting in the reduced concentration of molecular species of serum PtdCho containing 18:2. The increase in the level of hepatic 16:0-18:1 PtdCho seemed to be due to 3-fold increase in the activities of both delta9 desaturase and 1-acylglycerophosphocholine (1-acyl-GPC) acyltransferase. The mechanism by which PFOA causes the accumulation of glycerolipids in liver was discussed.     

Stimulation of arachidonic acid mobilization by adherence of resident peritoneal macrophages to plastic substrate

To interpret results of studies on arachidonic acid (AA) mobilization and metabolism in vitro, it is essential that the influence of culture and conditions should be well defined. Thus, we investigated the effects of murine resident peritoneal macrophage adherence and the presence of foetal calf serum in culture medium on arachidonic acid mobilization. The present data demonstrate that [³H] AA mobilization was triggered simply by contact between cell and substrate. The presence of serum can modulate cell-substrate interactions but not AA mobilization. Protein kinase C, and calmodulin inhibitors failed to inhibit [³H] AA release induced by cell adherence. Finally, low molecular weight PLA₂ inhibitors were not able to inhibit [³H] AA mobilization stimulated by cell adherence.     

Effects of carbachol and pancreozymin (cholecystokinin-octapeptide) on polyphosphoinositide metabolism in the rat pancreas in vitro.

We studied the possibility that hydrolysis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] may be the initiating event for the increase in [32P]Pi incorporation into phosphatidic acid (PtdA) and phosphatidylinositol (PtdIns) during carbachol and pancreozymin (cholecystokinin-octapeptide) action in the rat pancreas. After prelabelling acini for 2h, [32P]Pi incorporation into PtdA, PtdIns(4,5)P2 and phosphatidylinositol 4-phosphate (PtdIns4P) had reached equilibrium. Subsequent addition of carbachol or pancreozymin caused 32P in PtdIns(4,5)P2 to decrease by 30-50% within 10-15 s, and this was followed by sequential increases in [32P]Pi incorporation into PtdA and PtdIns. Similar changes in 32P-labelling of PtdIns4P were not consistently observed. Confirmation that the decrease in 32P in chromatographically-purified PtdIns(4,5)P2 reflected an actual decrease in this substance was provided by the fact that similar results were obtained (a) when PtdIns(4,5)P2 was prelabelled with [2-3H]inositol, and (b) when PtdIns(4,5)P2 was measured as its specific product (glycerophosphoinositol bisphosphate) after methanolic alkaline hydrolysis and ion-exchange chromatography. The secretogogue-induced breakdown of PtdIns(4,5)P2 was not inhibited by Ca2+ deficiency (severe enough to inhibit amylase secretion and Ca2+-dependent hydrolysis of PtdIns), and ionophore A23187 treatment did not provoke PtdIns(4,5)P2 hydrolysis. The increase in the hydrolysis of PtdIns(4,5)P2 and the increase in [32P]Pi incorporation into PtdA commenced at the same concentration of carbachol in dose-response studies. Our findings suggest that the hydrolysis of PtdIns(4,5)P2 is an early event in the action of pancreatic secretogogues that mobilize Ca2+, and it is possible that this hydrolysis may initiate the Ca2+-independent labelling of PtdA and PtdIns. Ca2+ mobilization may follow these responses, and subsequently cause Ca2+-dependent hydrolysis of PtdIns and exocytosis.     

Effects of calmodulin antagonists on serine phospholipid base-exchange reaction in rabbit platelets

Effects of the calmodulin antagonists chlorpromazine, trifluoperazine, and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide on phospholipid metabolism were examined in rabbit platelets using [3H]serine, [3H]ethanolamine, [3H]choline, and [3H]glycerol. All these drugs markedly stimulated the incorporation of [3H]serine into phosphatidylserine. On the other hand, these drugs had only a slight effect on the rate of incorporation of [3H]ethanolamine and [3H]choline into the corresponding phospholipid. When [3H]glycerol was used as a precursor of the phospholipids, 3H-labeled phospholipids were mainly composed of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol. Although the phosphorus content of phosphatidylserine was about 40% of that of phosphatidylcholine in rabbit platelets, the amount of phosphatidylserine labeled with [3H]glycerol was less than 2% of that of the labeled phosphatidylcholine, and calmodulin antagonists slightly stimulated the incorporation of [3H]glycerol into phosphatidylserine. Treatment with calmodulin antagonists caused a marked decrease in the content of endogenous free serine with concomitant increase in the contents of endogenous free ethanolamine and choline. On the other hand, the contents of other free amino acids, including essential and non-essential amino acids, were unchanged. These results suggest that the calmodulin antagonists we used did not affect de novo synthesis of phosphatidylserine, but did stimulate the serine phospholipid base-exchange reaction in rabbit platelets.     

Mobilization of arachidonic acid from phosphatidylethanolamine fraction to phosphatidylcholine fraction in platelets

Rabbit platelets rapidly incorporated methyl groups of [³H] methionine to phosphatidylcholine (PC). Rabbit platelets also incorporated [³H]choline to PC, but the rate of incorporation was far lower than that of [³H]methionine. Further fractionation of labeled PC revealed that a considerable amount of arachidonyl PC was synthesized via the N-methylation pathway. Thrombin stimulation resulted in a release of arachidonic acid from PC, and not from phosphatidylethanolamine (PE). These observations suggest that the N-methylation pathway plays an important role in the intracellular mobilization of arachidonic acid from the PE fraction to the PC fraction, this fraction being more sensitive to the hydrolysis with phospholipase A₂ during platelet activation.     

Characterization of phosphatidylserine synthesis and translocation in permeabilized animal cells

The synthesis of phosphatidylserine and its translocation to the mitochondria were examined in permeabilized Chinese hamster ovary (CHO)-K1 cells by following the metabolism of a [3H]serine precursor to [3H] phosphatidylserine (PtdSer) and [3H]phosphatidylethanolamine (PtdEtn). In physiological salt solutions approximating the intracellular ionic composition, both the synthesis of PtdSer and its translocation required ATP. The ATP requirement for PtdSer synthesis could be completely bypassed, and that for translocation could be partially bypassed at Ca2+ concentrations 10(3)-10(4) times the intracellular physiological level (i.e. 1 mM). The ATP-dependent synthesis of PtdSer could be inhibited by chelation of Ca2+ with EGTA, inhibition of Ca2+ sequestration with 2,5-di(tert-butyl)hydroquinone, mobilization of sequestered Ca2+ with ionomycin, and competition for [3H]serine with ethanolamine. The inhibition of the ATP-dependent synthesis of PtdSer by the aforementioned inhibitors provided an efficient method to rapidly arrest the incorporation of [3H]serine into [3H]PtdSer. By pulse-labeling the [3H]PtdSer pool and arresting further synthesis with inhibitors, the translocation of nascent PtdSer could be uncoupled from synthesis. The results of these pulse-labeling-arrest experiments provide unambiguous evidence that PtdSer translocation to the mitochondria is not driven by PtdSer synthesis. The addition of apyrase to ATP-supplemented, permeabilized cells abruptly terminates [3H]serine incorporation into [3H]PtdSer and the decarboxylation of [3H]PtdSer to [3H]PtdEtn, thereby demonstrating that a specific ATP requirement exists for the translocation of nascent PtdSer to the mitochondria in permeabilized cells. The translocation of nascent PtdSer to the mitochondria was unaffected by 45-fold dilution of the standard reaction thus indicating that the translocation intermediate was unlikely to be a freely diffusible complex. The requirements for translocation of nascent phosphatidylserine are different from those for the vesicular movement of proteins insofar as the lipid movement does not require cytosol and is unaffected by the addition of Ca2+, GTP, or GTP gamma S. From these studies, we conclude that: 1) the synthesis and translocation of PtdSer can be readily studied in permeabilized cells, 2) the ATP-dependent synthesis of PtdSer is functionally coupled to the ATP-dependent sequestration of Ca2+ by the endoplasmic reticulum or closely related membranes, 3) PtdSer translocation is independent of its synthesis, and 4) there is a specific requirement for ATP in the translocation of PtdSer to the mitochondria.     

Phosphatidylthreonine and Lipid-Mediated Control of Parasite Virulence

The major membrane phospholipid classes, described thus far, include phosphatidylcholine (PtdCho), phosphatidylethanolamine (PtdEtn), phosphatidylserine (PtdSer), and phosphatidylinositol (PtdIns). Here, we demonstrate the natural occurrence and genetic origin of an exclusive and rather abundant lipid, phosphatidylthreonine (PtdThr), in a common eukaryotic model parasite, Toxoplasma gondii. The parasite expresses a novel enzyme PtdThr synthase (TgPTS) to produce this lipid in its endoplasmic reticulum. Genetic disruption of TgPTS abrogates de novo synthesis of PtdThr and impairs the lytic cycle and virulence of T. gondii. The observed phenotype is caused by a reduced gliding motility, which blights the parasite egress and ensuing host cell invasion. Notably, the PTS mutant can prevent acute as well as yet-incurable chronic toxoplasmosis in a mouse model, which endorses its potential clinical utility as a metabolically attenuated vaccine. Together, the work also illustrates the functional speciation of two evolutionarily related membrane phospholipids, i.e., PtdThr and PtdSer.     

Preferential externalization of newly synthesized phosphatidylserine in apoptotic U937 cells is dependent on caspase-mediated pathways

Externalization of phosphatidylserine (PtdSer) is a common feature of programmed cell death and plays an important role in the recognition and removal of apoptotic cells. In this study with U937 cells, PtdSer synthesis from [(3)H]serine was stimulated and newly synthesized PtdSer was transferred preferentially to cell-free medium vesicles (CFMV) from cells when apoptosis was induced with a topoisomerase I inhibitor, camptothecin (CAM). When CAM-induced apoptosis was blocked by a caspase inhibitor, z-VAD-fmk, stimulation of PtdSer synthesis and movement to CFMV were abolished. In contrast, changes in synthesis and transport of sphingomyelin (SM) or phosphatidylethanolamine (PtdEtn) were minor; total phosphatidylcholine (PtdCho) synthesis was below control levels. All phospholipids appeared in CFMV but PtdSer displayed a 6-fold increase relative to controls compared to 3-fold for SM, 2-fold for PtdCho and 1.8-fold for PtdEtn. Even greater effects on specificity of PtdSer synthesis, movement to CFMV and inhibition by z-VAD-fmk were observed in apoptotic cells induced by UV irradiation or tumor necrosis factor-alpha/cycloheximide treatment. Thus, PtdSer biosynthesis stimulated during apoptosis in U937 cells was specific for this phospholipid and was correlated with caspase-mediated exposure of PtdSer at the cell surface and preferential movement to vesicles during apoptosis.     

The influence of fermentation conditions and recycling on the phospholipid and fatty acid composition of the brewer’s yeast plasma membranes

Phospholipid (PL) and fatty acid (FA) compositions of the plasma membrane (PM), as well as the FA composition of the PM phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEtn) in the pure culture (zero generation) and the first three recycled generations of the bottom-fermenting brewer’s yeast, have been determined. The PL composition differed markedly among the generations; in the zero generation, phosphatidylinositol (PtdIns) was the main PL, accounting for 27% of total PLs, followed by phosphatidic acid and PtdCho. In all recycled generations, the main PL was PtdCho with a marked increase in the first generation compared with the zero (32% and 20%, respectively), followed by PtdIns in the first and second generations. In the FA composition of the PM, 22 FAs were identified, ranging from C₁₀ to C₂₆. The compositions of the PM FAs, as well as those of PtdCho and PtdEtn, were characterised by a high preponderance of C₁₆ acids. Saturated FAs prevailed in the zero generation, whilst unsaturated prevailed in the first and second generation. Although the profiles of FAs in PtdCho and PtdEtn were similar, some marked differences were observed, pointing out to their specific functions in the regulation of membrane properties.     

Metformin Decouples Phospholipid Metabolism in Breast Cancer Cells

Introduction

The antidiabetic drug metformin, currently undergoing trials for cancer treatment, modulates lipid and glucose metabolism both crucial in phospholipid synthesis. Here the effect of treatment of breast tumour cells with metformin on phosphatidylcholine (PtdCho) metabolism which plays a key role in membrane synthesis and intracellular signalling has been examined.

Methods

MDA-MB-468, BT474 and SKBr₃ breast cancer cell lines were treated with metformin and [³H-methyl]choline and [¹⁴C(U)]glucose incorporation and lipid accumulation determined in the presence and absence of lipase inhibitors. Activities of choline kinase (CK), CTP:phosphocholine cytidylyl transferase (CCT) and PtdCho-phospholipase C (PLC) were also measured. [³H] Radiolabelled metabolites were determined using thin layer chromatography.

Results

Metformin-treated cells exhibited decreased formation of [³H]phosphocholine but increased accumulation of [³H]choline by PtdCho. CK and PLC activities were decreased and CCT activity increased by metformin-treatment. [¹⁴C] incorporation into fatty acids was decreased and into glycerol was increased in breast cancer cells treated with metformin incubated with [¹⁴C(U)]glucose.

Conclusion

This is the first study to show that treatment of breast cancer cells with metformin induces profound changes in phospholipid metabolism.     

Serine and ethanolamine incorporation into different plasmalogen pools: Subcellular analyses of phosphoglyceride synthesis in cultured glioma cells

In cultured glioma cells, plasma membrane (PM) is enriched in phosphatidylserine (PtdSer) and plasmalogens (1-O-alk-1-enyl-2-acyl-sn-glycero-3-phosphoethanolamine). Serine can be a precursor of headgroups of both ptdSer and ethanolamine phosphoglycerides (PE) including plasmalogens and non-plasmalogen PE (NP-PE). Synthesis of phospholipids was investigated at the subcellular level using established fractionation procedures and incorporation of [³H(G)]L-serine and [1,2-¹⁴C]ethanolamine. Specific radioactivity of PtdSer from [³H]serine was 2-fold greater in PM than in microsomes, reaching maximum by 2–4 h. Labeled plasmalogen from [³H]serine appeared in PM by 4 h and increased to 48 h, whereas almost no plasmalogen accumulated in microsomes within 12 h. In contrast, labeled plasmalogen from [1,2-¹⁴C]ethanolamine appeared in both PM and microsomes at early incubation times and became enriched in PM beyond 12 h. Thus, in glioma cells: (1) greater and faster accumulation of labeled PtdSer in PM may reflect direct synthesis from serine within PM; (2) PM is a major source of PtdSer for decarboxylation and PE synthesis; (3) NP-PE in both PM and microsome provides headgroup for synthesis of plasmalogen; and, (4) plasmalogen synthesis may involve different intracellular pools depending on headgroup origin.Abbreviations NP-PE nonplasmenylethanolamine phosphoglycerides including both diacyl and alkylacyl species - PE total ethanolamine phosphoglycerides: plasmalogen-plasmenylethanolamine or alkenylacyl ethanolamine phosphoglyceride (1-O-alk-1-enyl-2-acyl-sn-glycero-3-phosphoethanolamine) - PL phospholipid - PM plasma membrane - PtdCho phosphatidylcholine - PtdSer phosphatidylserine