Analysis of Acyl Fluxes through Multiple Pathways of Triacylglycerol Synthesis in Developing Soybean Embryos

The reactions leading to triacylglycerol (TAG) synthesis in oilseeds have been well characterized. However, quantitative analyses of acyl group and glycerol backbone fluxes that comprise extraplastidic phospholipid and TAG synthesis, including acyl editing and phosphatidylcholine-diacylglycerol interconversion, are lacking. To investigate these fluxes, we rapidly labeled developing soybean (Glycine max) embryos with [¹⁴C]acetate and [¹⁴C]glycerol. Cultured intact embryos that mimic in planta growth were used. The initial kinetics of newly synthesized acyl chain and glycerol backbone incorporation into phosphatidylcholine (PC), 1,2-sn-diacylglycerol (DAG), and TAG were analyzed along with their initial labeled molecular species and positional distributions. Almost 60% of the newly synthesized fatty acids first enter glycerolipids through PC acyl editing, largely at the sn-2 position. This flux, mostly of oleate, was over three times the flux of nascent [¹⁴C]fatty acids incorporated into the sn-1 and sn-2 positions of DAG through glycerol-3-phosphate acylation. Furthermore, the total flux for PC acyl editing, which includes both nascent and preexisting fatty acids, was estimated to be 1.5 to 5 times the flux of fatty acid synthesis. Thus, recycled acyl groups (16:0, 18:1, 18:2, and 18:3) in the acyl-coenzyme A pool provide most of the acyl chains for de novo glycerol-3-phosphate acylation. Our results also show kinetically distinct DAG pools. DAG used for TAG synthesis is mostly derived from PC, whereas de novo synthesized DAG is mostly used for PC synthesis. In addition, two kinetically distinct sn-3 acylations of DAG were observed, providing TAG molecular species enriched in saturated or polyunsaturated fatty acids.     

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.     

Lipids of Ectocarpus fasciculatus (Phaeophyceae). Incorporation of [l-14C]Oleate and the Role of TAG and MGDG in Lipid Metabolism

Lipids and fatty acids of Ectocarpus fasciculatus (Ectocarpales,Phaeophyceae) were analyzed. Major polar lipids are monogalactosyldiacylglycerol(MGDG), digalactosyldiacylglycerol (DGDG), sulfoquinovosyldiacylglycerol(SQDG), diacylglycerylhydroxymethyl-N,N,N-trimethyl-rß-alanine(DGTA), phosphatidylcholine (PC), phospha-tidylethanolamine(PE), phosphatidylglycerol (PG) and phosphatidylinositol (PI).Diphosphatidylglycerol (DPG), phosphatidic acid (PA) and phosphatidyl-O-[N-(2-hydroxy-ethyl)glycine](PHEG) were also present in small amounts. Nonpolar lipids mainlyconsist of triacylglycerol (TAG) and diacylglycerol (DAG). Majorfatty acids are 16:0,18:1, 18:3, 18:4, 20:4 and 20:5. The positionaldistribution of fatty acids showed that molecular species ofeukaryotic structure account for 99% in MGDG, 98% in DGDG, 62%in PG and 23% in SQDG. On incubation with [1-¹⁴C]18:1 for 30min, 33% of the total label was detected in TAG, 16% in PG,14% in PE, 10% in PC and 8% in MGDG. During 7 days of chase,the label in TAG, PG, PE and PC decreased and simultaneouslyincreased in MGDG up to 41% of the total. In SQDG, labelledfatty acids were found in prokaryotic as well as in eukaryoticmolecular species. During the experiment, the label shiftedfrom 18:1 to 18:2, 18:3, 18:4 and, to a minor extent, to 20:4and 20:5 acids indicating 18:1 to be processed by elongationand/or desaturation. These results suggest TAG to act as a majorprimary acceptor of exogenous oleate and to be involved in thetransfer of fatty acids to MGDG and other polar lipids. (Received March 24, 1997; Accepted June 11, 1997)     

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.     

Saturated, but not n-6 polyunsaturated, fatty acids induce insulin resistance: role of intramuscular accumulation of lipid metabolites.

Consumption of a Western diet rich in saturated fats is associated with obesity and insulin resistance. In some insulin-resistant phenotypes this is associated with accumulation of skeletal muscle fatty acids. We examined the effects of diets high in saturated fatty acids (Sat) or n-6 polyunsaturated fatty acids (PUFA) on skeletal muscle fatty acid metabolite accumulation and whole-body insulin sensitivity. Male Sprague-Dawley rats were fed a chow diet (16% calories from fat, Con) or a diet high (53%) in Sat or PUFA for 8 wk. Insulin sensitivity was assessed by fasting plasma glucose and insulin and glucose tolerance via an oral glucose tolerance test. Muscle ceramide and diacylglycerol (DAG) levels and triacylglycerol (TAG) fatty acids were also measured. Both high-fat diets increased plasma free fatty acid levels by 30%. Compared with Con, Sat-fed rats were insulin resistant, whereas PUFA-treated rats showed improved insulin sensitivity. Sat caused a 125% increase in muscle DAG and a small increase in TAG. Although PUFA also resulted in a small increase in DAG, the excess fatty acids were primarily directed toward TAG storage (105% above Con). Ceramide content was unaffected by either high-fat diet. To examine the effects of fatty acids on cellular lipid storage and glucose uptake in vitro, rat L6 myotubes were incubated for 5 h with saturated and polyunsaturated fatty acids. After treatment of L6 myotubes with palmitate (C16:0), the ceramide and DAG content were increased by two- and fivefold, respectively, concomitant with reduced insulin-stimulated glucose uptake. In contrast, treatment of these cells with linoleate (C18:2) did not alter DAG, ceramide levels, and glucose uptake compared with controls (no added fatty acids). Both 16:0 and 18:2 treatments increased myotube TAG levels (C18:2 vs. C16:0, P < 0.05). These results indicate that increasing dietary Sat induces insulin resistance with concomitant increases in muscle DAG. Diets rich in n-6 PUFA appear to prevent insulin resistance by directing fat into TAG, rather than other lipid metabolites.     

Mitochondrial lipids in Bufo arenarum full-grown oocytes

Both the content and composition of polar and neutral lipids from the mitochondrial fraction of ovarian full-grown Bufo arenarum oocytes were analysed in the present study. Triacylglycerols (TAG) represent 33% of the total lipids, followed by phosphatidylcholine (PC), free fatty acids (FFA) and phosphatidylethanolamine (PE). Diphosphatidylglycerol (DPG) or cardiolipin, a specific component of the inner mitochondrial membrane, represents about 4% of the total lipid content. Palmitic (16:0) and arachidonic (20:4n6) acids are the most abundant fatty acids in PC and PE, respectively. DPG is enriched in fatty acids with carbon chain lengths of 18, the principal component being linoleic acid. In phosphatidylinositol (PI), 20:4n6 and stearic acid (18:0) represent about 72 mol% of the total acyl group level. The main fatty acids in TAG are linoleic (18:2), oleic (18:1), and palmitic acids. The fatty acid composition of FFA and diacylglycerols (DAG) is similar, 16:0 being the most abundant acyl group. PE is the most unsaturated lipid and sphingomyelin (SM) has the lowest unsaturation index.     

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.     

Metabolic pathways for dietary lipids in the midgut of hematophagous Panstrongylus megistus (Hemiptera: Reduviidae)

The metabolism of dietary lipids in the anterior midgut of Panstrongylus megistus during blood digestion was studied. Fifth instar nymphs were fed a blood meal containing 7.1 +/- 0.4 mg of lipids, consisting mainly of triacylglycerol (TAG), and completed the overall process of digestion in about 20 days. Lipolysis of TAG and pathways for diacylglycerol (DAG) biosynthesis in the midgut were investigated by feeding the insects with [9,10-3H]-oleic acid-labeled triolein. Lumenal [3H]-triacylglycerol was hydrolyzed, generating mainly fatty acids (FA) and glycerol and to lesser extent, DAG. Almost no radioactivity associated with monoacylglycerol was found at any time. In midgut tissue, labeled fatty acids were incorporated into phosphatidic acid, DAG and TAG, whereas no significantly labeled monoacylglycerol was observed. In addition, the activities of enzymes related to DAG metabolism were assayed in non-blood fed midgut homogenates and at different times after feeding on a blood meal. Significant changes in the activities of phosphatidate phosphohydrolase (EC 3.1.3.4) and triacylglycerol lipase (EC 3.1.1.3) were observed during blood digestion, suggesting that these enzymes are important in regulating intracellular DAG synthesis and mobilization in midgut cells. Finally, the histological changes of lipid stores observed in anterior midgut confirmed the active process of uptake and trafficking of lipids performed by the enterocytes during blood digestion.     

Triacylglycerol and phospholipid fatty acids of the silverleaf whitefly: composition and biosynthesis

The identification and composition of the fatty acids of the major lipid classes (triacylglycerols and phospholipids) within Bemisia argentifolii Bellows and Perring (Homoptera: Aleyrodidae) nymphs were determined. Comparisons were made to fatty acids from the internal lipids of B. argentifolii adults. The fatty acids, as ester derivatives, were analyzed by capillary gas chromatography (CGC) and CGC-mass spectrometry (MS). All lipid classes contained variable distributions of eight fatty acids: the saturated fatty acids, myristic acid (14:0), palmitic acid (16:0), stearic acid (18:0), arachidic acid (20:0); the monounsaturated fatty acids, palmitoleic acid (16:1), oleic acid (18:1); the polyunsaturated fatty acids, linoleic acid (18:2), linolenic acid (18:3). Fourth instar nymphs had 5-10 times the quantities of fatty acids as compared to third instar nymphs and 1-3 times the quantities from adults. The fatty acid quantity differences between fourth and third instar nymphs were related to their size and weight differences. The percentage compositions for fatty acids from each lipid class were the same for the pooled groups of third and fourth instar nymphs. For nymphs and adults, triacylglycerols were the major source of fatty acids, with 18:1 and 16:0 acids as major components and the majority of the polyunsaturated fatty acids, 18:2 and 18:3 were present in the two phospholipid fractions, phosphatidylethanolamine and phosphatidylcholine. Evidence was obtained that whiteflies indeed synthesize linoleic acid and linolenic acid de novo: radiolabel from [2-(14)C] acetate was incorporated into 18:2 and 18:3 fatty acids of B. argentifolii adults and CGC-MS of pyrrolidide derivatives established double bonds in the Delta(9,12) and Delta(9,12,15) positions, respectively.     

Polyunsaturated fatty acid biosynthesis from [1-14C]20:3 n-6 acid in rat cultured Sertoli cells. Linoleic acid effect

Primary culture is a suitable system to study lipid metabolism and polyunsaturated fatty acid biosynthesis. Sertoli cell-enriched preparations were used to determine the fatty acid composition after 5 and 7 days in culture (serum free) as well as the uptake and metabolism of [1-14C]eicosa-8,11,14-trienoic acid. The addition of unlabeled linoleic acid (0.2 and 2.0 microg/ml) was also evaluated. Fatty acid methyl esters derived from cellular lipids were analyzed by gas liquid chromatography and radiochromatography. After 5 days in culture, cells had significantly less 18:2, 20:4, 22:5 and 24:5 and more 18:3, 20:3, 22:4 and 24:4 n-6 fatty acids than non-cultured cells. On day 7, an additional increment in 22:4 n-6 and a decrease in linoleic, gamma-linoleic and 24:4 n-6 fatty acids were observed. The presence of linoleic acid (low dose) produced a significant decrease in saturated and monounsaturated acids and an increase in 18:2, 20:4 and 22:5 n-6 fatty acids. At a high concentration almost all fatty acids belonging to 18:2 n-6 increased significantly. The drop in 20:4 n-6/20:3 n-6 ratio was considered as an indirect evidence of a Delta 5 desaturase activity depression. This assumption was corroborated by studying the transformation of [1-14C]eicosa-8,11,14-trienoic acid into 20:4, 22:4, 22:5, 24:4 and 24:5 n-6 fatty acids. We conclude that Sertoli cells after 7 days in culture evidenced changes in the fatty acid profile similar to those described under fat deprivation. The addition of linoleic acid reverted this pattern and indicated that the Delta 5 desaturase activity is a limiting step in the polyunsaturated fatty acid biosynthesis.     

Influence of testosterone on polyunsaturated fatty acid biosynthesis in Sertoli cells in culture

Sertoli cells play a central role in spermatogenesis, its development and regulation. They are target cells for androgen action in the seminiferous tubules. The Sertoli cell is considered to be the most important cell type in the testis with regard to essential fatty acid metabolism. We studied the response to testosterone of cultured Sertoli cells from immature rats by determining the fatty acid composition of total cellular lipids as well as by the biosynthesis of polyunsaturated fatty acids. Fatty acid methyl esters were analysed by gas liquid chromatography and radiochromatography. Two doses of testosterone were tested (150 and 300 ng ml(-1)). Significant differences were found in fatty acids derived from total cellular lipids after 8 days in culture in the presence of testosterone (300 ng ml(-1), for 48 h). Compared to controls, the hormone produced a significant increase of 16:1 and 18:1 n-9, and of 18:2 n-6, and a decrease of 20:4 and 22:5 n-6 in total cellular lipids. The decrease in the n-6 fatty acid ratios 20:4/20:3, 20:4/18:2 and 24:5/24:4, and the increase in 18:1n-9/18:0 and 16:1n-9/16:0 ratios were taken as an indirect signal of testosterone effects on Delta5, Delta6 and Delta9 desaturase activities. The drop in Delta5 and Delta6 desaturase activities was corroborated by analysing the transformation of [1-14C]20:3 n-6 into its higher homologues. We concluded that testosterone modifies the fatty acid pattern of cultured Sertoli cells, and this hormone is involved in polyunsaturated fatty acid biosynthesis, modulating Delta5 and Delta6 desaturases activity.     

Effets physiologiques de ľ atrazine à doses sublétales sur Lemna minor, VII. Incorporation ďacétate-l,2-[14C] dans les groupes de lipides et leurs acides gras

Physiological effects of sublethal doses of atrazine on Lemna minor. VII. 1,2-[¹⁴C] acetate incorporation into the groups of lipids and their fatty acids. The lipids and the fatty acids of ten-day old duckweed (Lemna minor L.), cultivated aseptically in mineral solution containing sublethal concentrations of 0,10 and 0,50 ppm (0.46 and 2.3 μM, respectively) of atrazine, were analyzed by thin-layer chromatography and gas-liquid radiochromatography after 1,2-[¹⁴C] acetate feeding. Sublethal concentrations of atrazine increased the incorporation of radioactivity in total lipids, diacylgalactosylglycerol (DGG), diacyldigalactosylglycerol (DDG), sul-folipids (SL), phosphatidylglycerol (PG), diacylglycerol (DAG) and triacylglycerol + steroll esters (TAG+SE). The incorporation of acetate-1,2-[¹⁴C] decreased in phos-phatidylcholine (PC) and in phosphatidylethanolamine (PE) in the presence of atrazine. The radioactivity increased in total Transic-hexadecenoic, linoleic and α -linolenic acids while it decreased in the other fatty acids. This indicates that the sublethal concentrations of atrazine stimulate the desaturation of fatty acids of L. minor. The radioactivity was strongly incorporated in the α -linolenic acid of DGG in the presence of atrazine. The specific radioactivity of α-linolenic acid was greater in DAG than in PG > TAG + SE > PC > PE > DGG > SL > DDG and it increased in all groupd of lipids analyzed under the influence of sublethal doses of atrazine. The labelling of Translchexadecenoic acid of PG and its specific radioactivity increased in the presence of atrazine. These changes suggest that the sublethal concentrations of atrazine stimulate especially the lipid metabolism of the chloroplasts of L. minor and they could explain the increase in the number of grana per chloroplast in treated L. minor. The results are discussed in relation to the biosynthesis of galactolipids.     

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.     

Radiolabelling Studies on the Lipid Metabolism in the Marine Brown Alga Dictyopteris membranacea

The lipid metabolism of the marine brown alga D. membranaceawas investigated using [2–¹⁴C]acetate, [1–¹⁴C]myristate,[l–^I4C]oleate and [l–¹⁴C]arachidonate as precursors.On incubation with [2–¹⁴C]acetate, 18:1 and 16:0 werethe main products formed by de novo synthesis and incorporatedinto polar lipids. With all the exogenous substrates used, DGTAwas strongly labelled and the subsequent rapid turnover of radioactivitysuggested a key role for this lipid in the redistribution ofacyl chains and most likely also in the biosynthesis of theeukaryotic galacto-lipids produced in the absence of PC. Inthe glycolipids a continuous accumulation of radioactivity wasobserved with all the substrates used. The labelling kineticsof molecular species of MGDG suggested the desaturation of 18:1to 18:4 and of 20:4 (n-6) to 20:5 (n–3) acids on thislipid. Both PG and PE were primary acceptors of de novo synthesizedfatty acids and exogenous [l–¹⁴C]oleate, but no evidenceexists for a further processing of acyl chains on these lipids.TAG, although strongly labelled with all exogenous [l–¹⁴CJacids,was not labelled when [2–¹⁴C]acetate was used as a precursorindicating the flux of endogenous fatty acids to be differentof that of exogenously supplied fatty acids. (Received November 4, 1997; Accepted February 23, 1998)     

The pathway of triacylglycerol synthesis through phosphatidylcholine in Arabidopsis produces a bottleneck for the accumulation of unusual fatty acids in transgenic seeds

Engineering of oilseed plants to accumulate unusual fatty acids (FAs) in seed triacylglycerol (TAG) requires not only the biosynthetic enzymes for unusual FAs but also efficient utilization of the unusual FAs by the host-plant TAG biosynthetic pathways. Competing pathways of diacylglycerol (DAG) and subsequent TAG synthesis ultimately affect TAG FA composition. The membrane lipid phosphatidylcholine (PC) is the substrate for many FA-modifying enzymes (desaturases, hydroxylases, etc.) and DAG can be derived from PC for TAG synthesis. The relative proportion of PC-derived DAG versus de novo synthesized DAG utilized for TAG synthesis, and the ability of each pathway to utilize unusual FA substrates, are unknown for most oilseed plants, including Arabidopsis thaliana. Through metabolic labeling experiments we demonstrate that the relative flux of de novo DAG into the PC-derived DAG pathway versus direct conversion to TAG is ～14/1 in wild-type Arabidopsis. Expression of the Ricinus communis FA hydroxylase reduced the flux of de novo DAG into PC by ～70%. Synthesis of TAG directly from de novo DAG did not increase, resulting in lower total synthesis of labeled lipids. Hydroxy-FA containing de novo DAG was rapidly synthesized, but it was not efficiently accumulated or converted to PC and TAG, and appeared to be in a futile cycle of synthesis and degradation. However, FA hydroxylation on PC and conversion to DAG allowed some hydroxy-FA to accumulate in sn-2 TAG. Therefore, the flux of DAG through PC represents a major bottleneck for the accumulation of unusual FAs in TAG of transgenic Arabidopsis seeds.     

Elongation of polyunsaturated fatty acids in trypanosomatids

Leishmania major synthesizes polyunsaturated fatty acids by using Delta6, Delta5 and Delta4 front-end desaturases, which have recently been characterized [Tripodi KE, Buttigliero LV, Altabe SG & Uttaro AD (2006) FEBS J273, 271-280], and two predicted elongases specific for C18 Delta6 and C20 Delta5 polyunsaturated fatty acids, respectively. Trypanosoma brucei and Trypanosoma cruzi lack Delta6 and Delta5 desaturases but contain Delta4 desaturases, implying that trypanosomes use exogenous polyunsaturated fatty acids to produce C22 Delta4 fatty acids. In order to identify putative precursors of these C22 fatty acids and to completely describe the pathways for polyunsaturated fatty acid biosynthesis in trypanosomatids, we have performed a search in the three genomes and identified four different elongase genes in T. brucei, five in T. cruzi and 14 in L. major. After a phylogenetic analysis of the encoded proteins together with elongases from a variety of other organisms, we selected four candidate polyunsaturated fatty acid elongases. Leishmania major CAJ02037, T. brucei AAX69821 and T. cruzi XP_808770 share 57-52% identity, and group together with C20 Delta5 polyunsaturated fatty acid elongases from algae. The predicted activity was corroborated by functional characterization after expression in yeast. T. brucei elongase was also able to elongate Delta8 and Delta11 C20 polyunsaturated fatty acids. L. major CAJ08636, which shares 33% identity with Mortierella alpinaDelta6 elongase, showed a high specificity for C18 Delta6 polyunsaturated fatty acids. In all cases, a preference for n6 polyunsaturated fatty acids was observed. This indicates that L. major has, as predicted, Delta6 and Delta5 elongases and a complete pathway for polyunsaturated fatty acid biosynthesis. Trypanosomes contain only Delta5 elongases, which, together with Delta4 desaturases, allow them to use eicosapentaenoic acid and arachidonic acid, a precursor that is relatively abundant in the host, for C22 polyunsaturated fatty acid biosynthesis.     

Fatty acid utilisation and metabolism in caecal enterocytes of rainbow trout (Oncorhynchus mykiss) fed dietary fish or copepod oil

A combined fatty acid metabolism assay was employed to determine fatty acid uptake and relative utilisation in enterocytes isolated from the pyloric caeca of rainbow trout. In addition, the effect of a diet high in long-chain monoenoic fatty alcohols present as wax esters in oil derived from Calanus finmarchicus, compared to a standard fish oil diet, on caecal enterocyte fatty acid metabolism was investigated. The diets were fed for 8 weeks before caecal enterocytes from each dietary group were isolated and incubated with [1-14C]fatty acids: 16:0, 18:1n-9, 18:2n-6, 18:3n-3, 20:1n-9, 20:4n-6, 20:5n-3, and 22:6n-3. Uptake was measured over 2 h with relative utilisation of different [1-14C]fatty acids calculated as a percentage of uptake. Differences in uptake were observed, with 18:1n-9 and 18:2n-6 showing the highest rates. Esterification into cellular lipids was highest with 16:0 and C18 fatty acids, accounting for over one-third of total uptake, through predominant incorporation in triacylglycerol (TAG). The overall utilisation of fatty acids in phospholipid synthesis was low, but highest with 16:0, the most prevalent fatty acid recovered in intracellular phosphatidylcholine (PC) and phosphatidylinositol (PI), although exported PC exhibited higher proportions of C20/C22 polyunsaturated fatty acids (PUFA). Other than 16:0, incorporation into PC and PI was highest with C20/C22 PUFA and 20:4n-6 respectively. Recovery of labelled 18:1n-9 in exported TAG was 3-fold greater than any other fatty acid which could be due to multiple esterification on the glycerol 'backbone' and/or increased export. Approximately 20-40% of fatty acids taken up were beta-oxidised, and was highest with 20:4n-6. Oxidation of 20:5n-3 and 22:6n-3 was also surprisingly high, although 22:6n-3 oxidation was mainly attributed to retroconversion to 20:5n-3. Metabolic modification of fatty acids by elongation-desaturation was generally low at <10% of [1-14C]fatty acid uptake. Dietary copepod oil had generally little effect on fatty acid metabolism in enterocytes, although it stimulated the elongation and desaturation of 16:0 and elongation of 18:1n-9, with radioactivity recovered in longer n-9 monoenes. The monoenoic fatty acid, 20:1n-9, abundant in copepod oil as the homologous alcohol, was poorly utilised with 80% of uptake remaining unesterified in the enterocyte. However, the fatty acid composition of pyloric caeca was not influenced by dietary copepod oil.     

Lipids shed into the culture medium by trypomastigotes of Trypanosoma cruzi

Trypomastigote forms of Trypanosoma cruzi were metabolically labeled with [14C]-ethanolamine and [3H]-palmitic acid. Lipids shed to the culture medium were analyzed and compared with the parasite components. Phosphatidylcholine and lysophosphatidylcholine accounted for 53% of the total incorporated precursor. Interestingly, phosphatidylethanolamine and its lyso derivative lysophosphatidylethanolamine, although present in significant amounts in the parasites, could not be detected in the shed material. Shed lipids were highly enriched in the desaturated fatty acids C16:1 and C18:1 when compared to the total fatty acid pool isolated from the parasites.     

Conversion of alpha-linolenic acid to palmitic,palmitoleic, stearic and oleic acids in men and women

The purpose of this study was to determine whether adult humans can recycle carbon from alpha-linolenic acid (18:3n-3) into saturated (SFA) and monounsaturated (MUFA) fatty acids. Six men and six women consumed 700 mg [U-13C]-18:3n-3. Blood was collected over 21 days and breath over 24h. [13C]-labelled SFA and MUFA were detected in plasma phosphatidylcholine (PC) and triacylglycerol (TAG). Total labelled fatty acid incorporation into SFA and MUFA was five- and 25-fold greater in PC than TAG in men and women, respectively. [13C]-16:0 was the major labelled fatty acid in both fractions. Total [13C] incorporation into SFA and MUFA was 20% greater in men than women, and related positively (r(2) = 0.35, P<0.05) to the fractional recovery of labelled 18:3n-3 as 13CO2 on breath. These results suggest that the extent of partitioning towards beta-oxidation and carbon recycling may regulate the availability of 18:3n-3 for conversion to longer-chain fatty acids.     

Brugia malayi: microfilarial polyunsaturated fatty acid composition and synthesis

The polyunsaturated fatty acid composition of Brugia malayi microfilariae was analyzed by gas chromatography and compared to that of sera from B. malayi-infected jirds. The essential fatty acid, linoleic acid (18:2 omega 6), was the most abundant fatty acid present in both microfilarial total lipids and phospholipids as well as in jird sera. In contrast, arachidonic acid (20:4 omega 6), as well as the 18:3 omega 6, 20:2 omega 6, and 20:3 omega 6 intermediates that are formed in the enzymatic conversion of linoleic acid to arachidonic acid, were proportionally more abundant in microfilariae than in jird sera. To assess the capacity of microfilariae to transform linoleic acid into arachidonic acid, B. malayi microfilariae were incubated with [14C]linoleic acid. Microfilarial lipids were extracted and resolved by high-pressure liquid chromatography and thin-layer chromatography. A portion of [14C]linoleic acid incorporated by microfilariae was converted to [14C]arachidonic acid. Thus, microfilariae can not only incorporate exogenous arachidonic acid, as previously demonstrated, but can also synthesize arachidonic acid from exogenous linoleic acid. The capacity of microfilariae to utilize specific host polyunsaturated fatty acids raises the possibility that intravascular filarial parasites may synthesize eicosanoid metabolites of arachidonic acid that could mediate filarial-host cell interactions.