Accumulation of arachidonic acid-rich triacylglycerols in the microalga Parietochloris incisa (Trebuxiophyceae,Chlorophyta)

The freshwater green microalga Parietochloris incisa is the richest known plant source of the polyunsaturated fatty acid (PUFA), arachidonic acid (20:4omega6, AA). While many microalgae accumulate triacylglycerols (TAG) in the stationary phase or under certain stress conditions, these TAG are generally made of saturated and monounsaturated fatty acids. In contrast, most cellular AA of P. incisa resides in TAG. Using various inhibitors, we have attempted to find out if the induction of the biosynthesis of AA and the accumulation of TAG are codependent. Salicylhydroxamic acid (SHAM) affected a growth reduction that was accompanied with an increase in the content of TAG from 3.0 to 6.2% of dry weight. The proportion of 18:1 increased sharply in all lipids while that of 18:2 and its down stream products, 18:3omega6, 20:3omega6 and AA, decreased, indicating an inhibition of the Delta12 desaturation of 18:1. Treatment with the herbicide SAN 9785 significantly reduced the proportion of TAG. However, the proportion of AA in TAG, as well as in the polar lipids, increased. These findings indicate that while there is a preference for AA as a building block of TAG, the latter can be produced using other fatty acids, when the production of AA is inhibited. On the other hand, inhibiting TAG construction did not affect the production of AA. In order to elucidate the possible role of AA in TAG we have labeled exponential cultures of P. incisa kept at 25 degrees C with [1-14C]arachidonic acid and cultivated the cultures for another 12 h at 25, 12 or 4 degrees C. At the lower temperatures, labeled AA was transferred from TAG to polar lipids, indicating that TAG of P. incisa may have a role as a depot of AA that can be incorporated into the membranes, enabling the organism to quickly respond to low temperature-induced stress.     

Lipid and fatty acid composition of the green oleaginous alga Parietochloris incisa,the richest plant source of arachidonic acid

We have hypothesized that among algae of alpine environment there could be strains particularly rich in long chain polyunsaturated fatty acids (LC-PUFA). Indeed, the chlorophyte (Trebuxiophyceae) Parietochloris incisa isolated from Mt. Tateyama, Japan, was found to be the richest plant source of the pharmaceutically valuable LC-PUFA, arachidonic acid (AA, 20:4omega6). The alga is also extremely rich in triacylglycerols (TAG), which reaches 43% (of total fatty acids) in the logarithmic phase and up to 77% in the stationary phase. In contrast to most algae whose TAG are made of mainly saturated and monounsaturated fatty acids, TAG of P. incisa are the major lipid class where AA is deposited, reaching up to 47% in the stationary phase. Except for the presence of AA, the PUFA composition of the chloroplastic lipids resembled that of green algae, consisting predominantly of C(16) and C(18) PUFAs. The composition of the extrachloroplastic lipids is rare, including phosphatidylcholine (PC), phosphatidylethanolamine (PE) as well as diacylglyceryltrimethylhomoserine (DGTS). PC and PE are particularly rich in AA and are also the major depots of the presumed precursors of AA, l8:3omega6 and 20:3omega6, respectively.     

The role of triacylglycerol as a reservoir of polyunsaturated fatty acids for the rapid production of chloroplastic lipids in certain microalgae

Many microalgae are known to accumulate triacylglycerols (TAGs), especially under nitrate starvation. Generally, these TAGs are predominantly constructed of saturated and monounsaturated fatty acids. In contrast, the TAGs of the red microalga Porphyridium cruentum are rich in arachidonic acid (AA, 20:4 omega6) and eicosapentaenoic acid (EPA, 20:5 omega3). A mutant of this alga, impaired of growth at suboptimal temperatures, was shown to have reduced levels of EPA and of the eukaryotic molecular species of monogalactosyldiacylglycerol (MGDG) and an elevated level of TAG. Labelling experiments have shown that labelling of wild-type TAGs decreased, whereas that of the mutant remained high. Contemporarily, eukaryotic MGDG of the mutant was less labelled. Similarly, TAGs of the green alga Tl2, which can grow at a low temperature, are extremely rich in AA. We have labelled exponentially growing cultures of T12 kept at 25 degrees C with radioactive AA and cultivated the cultures for a further 12 h at 25 degrees C, 12 degrees C or 4 degrees C. At low temperatures, labelled AA was transferred from TAGs to polar lipids. These findings may indicate that polyunsaturated fatty acids that can be incorporated into the membranes, enabling the organism to quickly respond to low-temperature-induced stress.     

A block in endoplasmic reticulum-to-Golgi trafficking inhibits phospholipid synthesis and induces neutral lipid accumulation

Seeking to better understand how membrane trafficking is coordinated with phospholipid synthesis in yeast, we investigated lipid synthesis in several Sec(-) temperature-sensitive mutants, including sec13-1. Upon shift of sec13-1 cells to the restrictive temperature of 37 degrees C, phospholipid synthesis decreased dramatically relative to the wild type control, whereas synthesis of neutral lipids, especially triacylglycerol (TAG), increased. When examined by fluorescence microscopy, the number of lipid droplets appeared to increase and formed aggregates in sec13-1 cells shifted to 37 degrees C. Electron microscopy confirmed the increase in lipid droplet number and revealed that many were associated with the vacuole. Analysis of lipid metabolism in strains lacking TAG synthase genes demonstrated that the activities of the products of these genes contribute to accumulation of TAG in sec13-1 cells after the shift to 37 degrees C. Furthermore, the permissive temperature for growth of the sec13-1 strain lacking TAG synthase genes was 3 degrees C lower than sec13-1 on several different growth media, indicating that the synthesis of TAG has physiological significance under conditions of secretory stress. Together these results suggest that following a block in membrane trafficking, yeast cells channel lipid metabolism from phospholipid synthesis into synthesis of TAG and other neutral lipids to form lipid droplets. We conclude that this metabolic switch provides a degree of protection to cells during secretory stress.     

Distribution and metabolism of arachidonic and docosahexaenoic acids in rat pineal cells. Effect of norepinephrine

The time-course incorporation of 10 μM [¹⁴C]arachidonic (AA) and docosahexaenoic (DHA) acids into glycerolipids was studied in rat pineal cells. The incorporation of both labeled fatty acids into total lipids was approximately equal, but their distribution profiles among the various cell lipids showed marked differences. The esterification of [¹⁴C]DHA in the neutral lipids, triacylglycerols (TAG) and cholesterol esters (CE), was 2-fold higher than that of [¹⁴C]AA whereas the opposite could be observed in total phospholipids (PL). The order of incorporation into PL was phosphatidylcholine (PC) > phosphatidylinositol (PI) = phosphatidylethanolamine (PE) for [¹⁴C]AA and PC = PE for [¹⁴C]DHA, the incorporation of both fatty acids being not detected in phosphatidylserine (PS) and that of DHA not in PI. When using 0.5 μM [³H] fatty acids, the respective distribution patterns resembled that of fatty acids at 10 μM, except for a lower proportion in TAG. The stimulation of ³H-labeled cells by 100 μM norepinephrine induced a 170% increase of basal release of [³H]AA into the medium, while [³H]DHA was virtually not released. However, the analysis of cell labeling revealed that both [³H] fatty acid levels were decreased in PL and increased in TAG. These findings suggest different involvement for AA and DHA in the pineal function. The preferential incorporation of DHA in TAG suggests that TAG might play an important role in the pineal enrichment with DHA. The absence of DHA release after NE stimulation, which however cannot be ascertained, may raise the question of the role of DHA in NE transduction.     

Culture age and carbamoylcholine increase the incorporation of endogenously synthesized linoleic acid in lipids of Trypanosoma cruzi epimastigotes

Physiological and cellular adaptations to environmental changes are known to be related to modifications in membrane lipids. This work provides metabolic and compositional evidence that Trypanosoma cruzi epimastigotes are able to synthesize and desaturate fatty acids, to incorporate them into their lipids, and to modify this incorporation when carbamoylcholine is present in the medium. The fatty acids formed from [2-(14)C]acetate in the period from 2 to 9 days were mostly (70%) incorporated in phospholipids, the remainder 30% being recovered in neutral lipids, such as triacylglycerols (TAG) and diacylglycerols (DAG). The main fatty acids formed from [2-(14)C]acetate were saturates (16:0, 18:0), monoenes (16:1, 18:1) and dienes (mostly 18:2). The ratios between labelled unsaturated and saturated fatty acids increased continuously with growth, consistent with a precursor-product relationship between the main fatty acids, and with the occurrence in T. cruzi of Delta(9)- and Delta(12)-desaturases. From days 2 to 5, [(14)C]18:2 was the main fatty acid produced. Accordingly, the fatty acid profiles showed a significant increase in the percentage of 18:2 in all lipids in the period under study, especially in the first 2 to 5 days. In the presence of carbamoylcholine, the labelling of DAG and TAG with [(14)C]18:2 augmented. The results indicate that T cruzi is able to synthesize the main types of fatty acids required to form its membrane lipids, and to exchange them actively in response to environmental stimuli.     

NITROGEN STARVATION INDUCES THE ACCUMULATION OF ARACHIDONIC ACID IN THE FRESHWATER GREEN ALGA PARIETOCHLORIS INCISA (TREBUXIOPHYCEAE)1

The Chlorophyte Parietochloris incisa comb. nov (Trebuxiophyceae) was found to be the richest plant source of the pharmaceutically valuable long‐chain polyunsaturated fatty acid (PUFA), arachidonic acid (20:4ω6, AA). Over 90% of total AA are deposited in triacylglycerols (TAG). Under nitrogen starvation, the fatty acid content constituted over 35% of dry weight and the proportion of AA exceeded 60% of total fatty acids. Consequently, we obtained an AA content of over 20%. This is, to the best of our knowledge, the highest reported content of any PUFA in algae. Increasing the biomass concentration resulted in an enhancement of both the proportion of AA and the fatty acid content. We hypothesize that one of the roles of TAG in P. incisa is to serve as a reservoir of AA that can be used for the construction of membranal lipids.     

Regulation of triacylglycerol biosynthesis in embryos and microsomal preparations from the developing seeds of Cuphea lanceolata.

Embryos of Cuphea lanceolata have more than 80 mol% of decanoic acid ('capric acid') in their triacylglycerols, while this fatty acid is virtually absent in phosphatidylcholine (PtdCho). Seed development was complete 25-27 days after pollination, with rapid triacylglycerol deposition occurring between 9 and 24 days. PtdCho amounts increased until day 15 after pollination. Analysis of embryo lipids showed that the diacylglycerol (DAG) pool consisted of mainly long-chain molecular species, with a very small amount of mixed medium-chain/long-chain glycerols. Almost 100% of the fatty acid at position sn-2 in triacylglycerols (TAG) was decanoic acid. When equimolar mixtures of [14C]decanoic and [14C]oleic acid were fed to whole detached embryos, over half of the radioactivity in the DAG resided in [14C]oleate, whereas [14C]decanoic acid accounted for 93% of the label in the TAG. Microsomal preparations from developing embryos at the mid-stage of TAG accumulation catalysed the acylation of [14C]glycerol 3-phosphate with either decanoyl-CoA or oleoyl-CoA, resulting in the formation of phosphatidic acid (PtdOH), DAG and TAG. Very little [14C]glycerol entered PtdCho. In combined incubations, with an equimolar supply of [14C]oleoyl-CoA and [14C]decanoyl-CoA in the presence of glycerol 3-phosphate, the synthesized PtdCho species consisted to 95% of didecanoic and dioleic species. The didecanoyl-glycerols were very selectively utilized over the dioleoylglycerols in the production of TAG. Substantial amounts of [14C]oleate, but not [14C]decanoate, entered PtdCho. The microsomal preparations of developing embryos were used to assess the acyl specificities of the acyl-CoA:sn-glycerol-3-phosphate acyltransferase (GPAT, EC 2.3.1.15) and the acyl-CoA:sn-1-acyl-glycerol-3-phosphate acyltransferase (LPAAT, EC 2.3.1.51) in Cuphea lanceolata embryos. The efficiency of acyl-CoA utilization by the GPAT was in the order decanoyl = dodecanoyl greater than linoleoyl greater than myristoyl = oleoyl greater than palmitoyl. Decanoyl-CoA was the only acyl donor to be utilized to any extent by the LPAAT when sn-decanoylglycerol 3-phosphate was the acyl acceptor. sn-1-Acylglycerol 3-phosphates with acyl groups shorter than 16 carbon atoms did not serve as acyl acceptors for long-chain (greater than or equal to 16 carbon atoms) acyl-CoA species. On the basis of the results obtained, we propose a schematic model for triacylglycerol assembly and PtdCho synthesis in a tissue specialized in the synthesis of high amounts of medium-chain fatty acids.     

Enhancement of extraplastidic oil synthesis in Chlamydomonas reinhardtii using a type‐2 diacylglycerol acyltransferase with a phosphorus starvation–inducible promoter

When cultivated under stress conditions, many plants and algae accumulate oil. The unicellular green microalga Chlamydomonas reinhardtii accumulates neutral lipids (triacylglycerols; TAGs) during nutrient stress conditions. Temporal changes in TAG levels in nitrogen (N)‐ and phosphorus (P)‐starved cells were examined to compare the effects of nutrient depletion on TAG accumulation in C. reinhardtii. TAG accumulation and fatty acid composition were substantially changed depending on the cultivation stage before nutrient starvation. Profiles of TAG accumulation also differed between N and P starvation. Logarithmic‐growth‐phase cells diluted into fresh medium showed substantial TAG accumulation with both N and P deprivation. N deprivation induced formation of oil droplets concomitant with the breakdown of thylakoid membranes. In contrast, P deprivation substantially induced accumulation of oil droplets in the cytosol and maintaining thylakoid membranes. As a consequence, P limitation accumulated more TAG both per cell and per culture medium under these conditions. To enhance oil accumulation under P deprivation, we constructed a P deprivation‐dependent overexpressor of a Chlamydomonas type‐2 diacylglycerol acyl‐CoA acyltransferase (DGTT4) using a sulphoquinovosyldiacylglycerol 2 (SQD2) promoter, which was up‐regulated during P starvation. The transformant strongly enhanced TAG accumulation with a slight increase in 18 : 1 content, which is a preferred substrate of DGTT4. These results demonstrated enhanced TAG accumulation using a P starvation–inducible promoter.     

Incorporation of [14C] arachidonic acid into ovine conceptus and endometrial lipids.

The purpose of this study was to quantitate conceptus and endometrial incorporation of [14C]arachidonic acid (AA) into individual neutral and polar lipids. Endometrium and conceptuses from pregnant ewes and endometrium from nonbred ewes were collected 14 and 16 d after onset of estrus (d 0). Tissues were incubated for 8 h at 37 degrees C in medium containing 1 microCi of [14C]AA. Thin-layer chromatographic procedures were used to separate 12 lipids. Radioactivity was measured in each lipid, and the amount (ng) of [14C]AA incorporated into each lipid was calculated. Conceptuses and endometrium incorporated more [14C]AA into triacylglycerols than into any other lipid. Day and tissue type affected differentially (i.e., day X tissue interaction) the incorporation of [14C]AA into several lipids; d-14 conceptuses incorporated [14C]AA more actively than did any other day-tissue combination. Results indicate that triacylglycerols may be an important reservoir for conceptus and endometrial AA. The remarkable ability of d-14 conceptuses to incorporate [14C]AA into various lipids may be important for their accelerated elongation and active prostaglandin synthetic system.     

Glucosylation of Teichoic Acid: Solubilization and Partial Characterization of the Uridine Diphosphoglucose:Polyglycerolteichoic Acid Glucosyl Transferase from Membranes of Bacillus subtilis

Polyglycerolteichoic acid:glucosyl transferase (TAG transferase), one of the three enzymes involved in the pathway leading to the glucosylation of teichoic acid in Bacillus subtilis 168, was investigated. During the early stages of the growth of B. subtilis, TAG transferase is predominantly a soluble enzyme found in the cytoplasm. As growth proceeds, the amount of soluble enzyme decreases and the proportion of insoluble, membrane-bound TAG transferase increases, reaching a maximal value at the close of the logarithmic phase. Data are presented which suggest that these are two forms of the same enzyme, or have some common component. The effects of chaotropic agents, such as sodium trichloroacetate and sodium perchlorate, on the cytoplasmic membrane were also studied. These data show that such compounds can effectively remove the TAG transferase from the membrane in a water-soluble form. A study of some of the physical properties of this solubilized enzyme suggests that there is little difference between the two forms of the enzyme. Experiments are described which indicate that the glucosyl transfer by both the membrane-bound and soluble enzymes is not mediated by lipids.     

Separation and preparative purification of arachidonic acid from microbial lipids by urea inclusion reaction and HPLC

Arachidonic acid (AA) was separated and purified from microbial lipids by the combined method of urea inclusion reaction and reversed-phase high performance liquid chromatography. At first, AA was concentrated from free fatty acids made from microbial lipids by a urea inclusion reaction. The optimum conditions were as follows: methanol was the suitable solvent, the ratio of free fatty acids to urea to methanol was 1:2:8 (wt/wt), and the temperature of the urea inclusion reaction was -10 degrees C. The AA content was increased from 38% to 79%, and then AA was purified on a C(18) preparative column (300 mm x 30 mm I.D., d(p)=15 microm), using methanol-water (95:5, v/v) as the mobile phase, at a flow rate of 5 mL/min. The purity of AA after two steps purification reached 99%. This result indicates that the combined method of the urea inclusion reaction and reversed-phase high performance liquid chromatography is a promising technique for purification of AA.     

Triacylglycerol accumulation of Phaeodactylum tricornutum with different supply of inorganic carbon

The diatom Phaeodactylum tricornutum produces large quantities of lipids, especially triacylglycerols (TAGs) under nitrogen or phosphorus limitation. In this study, production of lipids and TAGs during this process was compared under conditions with different inputs of inorganic carbon. With an abundant supply of inorganic carbon, considerable accumulation of biomass, lipids, and TAGs was identified after a nitrogen/phosphorus-limiting “induction incubation.” TAGs were still synthesized and accumulated even under inorganic carbon limitation with a cessation in the production of biomass and cellular lipids. This part of accumulated TAGs could be synthesized through recycling and transformation of other lipids such as glycolipids and phospholipids. Additionally, some alterations in the fatty acid profile following TAG accumulation were found. The content of the C16:0 fatty acid increased with decreases in C16:3 and C20:5, which could have been caused by enzymatic selectivity for these fatty acids during the process of TAG synthesis. It was concluded that nitrogen and phosphorus metabolism regulates the synthesis of TAG, while carbon metabolism promotes it by providing sufficient substrates.     

三角褐指藻甘油酯对氮胁迫的响应

【背景】三角褐指藻作为生物燃料潜在的生产者,在胁迫条件下能通过改变其甘油酯组成来适应外部环境的变化,同时伴随着生物燃料原料甘油三酯(TAG)的积累,研究三角褐指藻甘油酯对氮胁迫的响应机制有利于深入认识TAG的积累过程。【目的】通过分析三角褐指藻在正常和氮胁迫条件下各类脂质含量及其脂肪酸成分的变化,揭示氮胁迫诱导积累的TAG酰基主要来源,以及在胁迫前生成的各极性甘油酯脂肪酸的去向,从而为进一步认识三角褐指藻对氮胁迫的响应机制提供新信息。【方法】利用高效薄层色谱结合气相色谱法分析三角褐指藻在正常和氮胁迫条件下的脂肪酸及甘油酯组分的变化。【结果】三角褐指藻在氮胁迫条件下TAG含量增加至57.8 mg/g时,总甘油酯含量几乎不变,但各甘油酯含量变化差异很大,表现为各极性脂含量显著降低。在此期间,各类甘油酯脂肪酸组成含量的变化表明,三角褐指藻TAG主要积累饱和及单不饱和脂肪酸,即16:0和16:1n7,分别以从头合成及原有极性脂转化为主,极性脂的部分二十碳五烯酸(EPA)作为酰基供体也向TAG发生了转化;此外组成极性脂的多不饱和脂肪酸16:2n4、16:3n4及EPA分解导致其含量显著下降。【结论】当氮胁迫诱导的三角褐指藻TAG含量为57.8 mg/g时,积累的TAG酰基中有48%来自从头合成,52%来自极性脂转化;而氮胁迫诱导所减少的极性脂酰基中有54%转化成TAG,46%发生了分解。     

Differential changes in lipid metabolism of myeloid and lymphoid cell lines induced by treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA)

Treatment of the myeloid cell lines, U-937 or HL-60, with 10 nM of the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), for 24 h increased the rate of incorporation of [3H]glycerol into total chloroform extracts. A proportionally greater labeling of the non-polar lipid (NL) fraction compared to the polar, phospholipid (PL), fraction was observed. Chromatographic analysis showed a 6-fold increase in the labeling of triacylglycerols (TAG), a 2-fold increase in diacylglycerols, and no changes in monoacylglycerols. PL labeling showed a 3-fold increase in phosphatidylcholine (PC). The effect of TPA on TAG labeling was selectively observed in myeloid cell lines. No such a change was found in the lymphoid cell line. MOLT-3, which did respond to TPA with increased PC labeling. Incorporation of [3H]arachidonic acid (AA) into TAG by U-937 cells was selectively increased (2.5-fold) after treatment with TPA for 24 h. Treatment of U-937 cells with TPA in serum-free medium resulted in no increased labeling of TAG. These studies suggest that changes in TAG metabolism may be characteristic of myeloid differentiation and depend on the presence of serum factor(s).     

Influence of cold temperatures on leaf lipids of Hibiscus rosa-sinensis

Triaclyglycerols (TAG) accounted for 15% of the leaf acyl lipids in a Hibiscus rosa-sinensis plant that survived the January 1986 freeze in Florida, U.S.A. This high TAG level suggested that the plant had cold hardened. The TAG concentration in leaves on new shoots from this plant was greater than that in new leaves on new shoots from five plants frozen to the roots during the freeze. However, five months later, the amounts of TAG in the six Hibiscus plants were nearly the same. The minor differences in TAG levels, however, related to the survival rate of these six plants during subsequent freezes. In addition to linoleic acid, two cyclopropene fatty acids, malvalic and sterculic, were major constituents of the leaf TAG. Hibiscus plants placed in environmental chambers under control and cold-hardening regimes optimized for Citrus showed TAG concentrations of 7% in control and 20% in hardened plants. A survey of neutral lipids in Hibiscus and other plants showed that plastoquinone A (B) and α-tocopherol decreased and plastoquinone C increased under cold-hardening conditions. Polyprenols, a major component of Hibiscus leaves under normal conditions, declined greatly under cold-hardening regimes.     

Selective degradation with phospholipases D and C of radioactive isomeric spin-labelled lipids bound to guinea pig liver microsomal membranes.

Membrane-bound lipids of isolated guinea pig liver microsomal membranes were selectively enzymatically labelled with isomeric (5-, 12-, and 16-)doxyl stearic acid. After reisolation, the membranes were degraded with phospholipases D and C under conditions not requiring detergents or organic solvent activators. The degradation of membrane-bound lipids occurred according to the recognized specificity of phospholipases D and C. Temperature-induced changes of degraded membranes containing radioactive spin-labelled isomeric lipids were followed by the electron spin resonance and spectral changes correlated with the lipid composition of membranes. Discontinuities in plots of experimental spectral parameters versus temperature detected in the case of microsomal membranes before and after degradation with phospholipases D and C were attributed to lipid-protein and lipid-lipid interaction(s). On the basis of these and control experiments, discontinuity at around 10-12 degrees C was attributed to the microsomal membrane phosphatidylcholine intrinsic microsomal membrane protein interaction(s), while discontinuities detected at 19-21 degrees C approximately and at 20-30 degrees C approximately were attributed to the phase separation of Ca or Zn salts of membranous phosphatidic acid and to the similar phenomenon involving membrane-bound diglycerides respectively.     

Apparent Role of Phosphatidylcholine in the Metabolism of Petroselinic Acid in Developing Umbelliferae Endosperm

Studies were conducted to characterize the metabolism of the unusual fatty acid petroselinic acid (18:1cis[delta]6) in developing endosperm of the Umbelliferae species coriander (Coriandrum sativum L.) and carrot (Daucus carota L.). Analyses of fatty acid compositions of glycerolipids of these tissues revealed a dissimilar distribution of petroselinic acid in triacylglycerols (TAG) and the major polar lipids phosphatidylcholine (PC) and phosphatidylethanolamine (PE). Petroselinic acid comprised 70 to 75 mol% of the fatty acids of TAG but only 9 to 20 mol% of the fatty acids of PC and PE. Although such data appeared to suggest that petroselinic acid is at least partially excluded from polar lipids, results of [1-14C]acetate radiolabeling experiments gave a much different picture of the metabolism of this fatty acid. In time-course labeling of carrot endosperm, [1-14C]acetate was rapidly incorporated into PC in high levels. Through 30 min, radiolabel was most concentrated in PC, and of this, 80 to 85% was in the form of petroselinic acid. One explanation for the large disparity in amounts of petroselinic acid in PC as determined by fatty acid mass analyses and 14C radiolabeling is that turnover of these lipids or the fatty acids of these lipids results in relatively low accumulation of petroselinic acid mass. Consistent with this, the kinetics of [1-14C]acetate time-course labeling of carrot endosperm and "pulse-chase" labeling of coriander endosperm suggested a possible flux of fatty acids from PC into TAG. In time-course experiments, radiolabel initially entered PC at the highest rates but accumulated in TAG at later time points. Similarly, in pulse-chase studies, losses in absolute amounts of radioactivity from PC were accompanied by significant increases of radiolabel in TAG. In addition, stereospecific analyses of unlabeled and [1-14C]acetate-labeled PC of coriander endosperm indicated that petroselinic acid can be readily incorporated into both the sn-1 and sn-2 positions of this lipid. Because petroselinic acid is neither synthesized nor further modified on polar lipids, the apparent metabolism of this fatty acid through PC (and possibly through other polar lipids) may define a function of PC in TAG assembly apart from its involvement in fatty acid modification reactions.     

Dopaminergic Neurons Respond to Iron-Induced Oxidative Stress by Modulating Lipid Acylation and Deacylation Cycles

Metal-imbalance has been reported as a contributor factor for the degeneration of dopaminergic neurons in Parkinson Disease (PD). Specifically, iron (Fe)-overload and copper (Cu) mis-compartmentalization have been reported to be involved in the injury of dopaminergic neurons in this pathology. The aim of this work was to characterize the mechanisms of membrane repair by studying lipid acylation and deacylation reactions and their role in oxidative injury in N27 dopaminergic neurons exposed to Fe-overload and Cu-supplementation. N27 dopaminergic neurons incubated with Fe (1mM) for 24 hs displayed increased levels of reactive oxygen species (ROS), lipid peroxidation and elevated plasma membrane permeability. Cu-supplemented neurons (10, 50 μM) showed no evidence of oxidative stress markers. A different lipid acylation profile was observed in N27 neurons pre-labeled with [³H] arachidonic acid (AA) or [³H] oleic acid (OA). In Fe-exposed neurons, AA uptake was increased in triacylglycerols (TAG) whereas its incorporation into the phospholipid (PL) fraction was diminished. TAG content was 40% higher in Fe-exposed neurons than in controls. This increase was accompanied by the appearance of Nile red positive lipid bodies. Contrariwise, OA incorporation increased in the PL fractions and showed no changes in TAG. Lipid acylation profile in Cu-supplemented neurons showed AA accumulation into phosphatidylserine and no changes in TAG. The inhibition of deacylation/acylation reactions prompted an increase in oxidative stress markers and mitochondrial dysfunction in Fe-overloaded neurons. These findings provide evidence about the participation of lipid acylation mechanisms against Fe-induced oxidative injury and postulate that dopaminergic neurons cleverly preserve AA in TAG in response to oxidative stress.