Accumulation Of Polyunsaturated Fatty Acids Research Articles

Reconstitution of EPA and DHA biosynthesis in Arabidopsis: Iterative metabolic engineering for the synthesis of n−3 LC-PUFAs in transgenic plants

An iterative approach to optimising the accumulation of non-native long chain polyunsaturated fatty acids in transgenic plants was undertaken in Arabidopsis thaliana. The contribution of a number of different transgene enzyme activities was systematically determined, as was the contribution of endogenous fatty acid metabolism. Successive iterations were informed by lipidomic analysis of neutral, polar and acyl-CoA pools. This approach allowed for a four-fold improvement on levels previously reported for the accumulation of eicosapentaenoic acid in Arabidopsis seeds and also facilitated the successful engineering of the high value polyunsaturated fatty acid docosahexaenoic acid to 10-fold higher levels. Our studies identify the minimal gene set required to direct the efficient synthesis of these fatty acids in transgenic seed oil.

Metabolomics Reveals Metabolic Targets and Biphasic Responses in Breast Cancer Cells Treated by Curcumin Alone and in Association with Docetaxel

BackgroundCurcumin (CUR) has deserved extensive research due to its anti-inflammatory properties, of interest in human diseases including cancer. However, pleiotropic even paradoxical responses of tumor cells have been reported, and the mechanisms of action of CUR remain uncompletely elucidated.Methodology/Principal Findings 1H-NMR spectroscopy-based metabolomics was applied to get novel insight into responses of MCF7 and MDA-MB-231 breast cancer cells to CUR alone, and MCF7 cells to CUR in cotreatment with docetaxel (DTX). In both cell types, a major target of CUR was glutathione metabolism. Total glutathione (GSx) increased at low dose CUR (≤ 10 mg.l−1–28 µM-) (up to +121% in MCF7 cells, P<0.01, and +138% in MDA-MB-231 cells, P<0.01), but decreased at high dose (≥ 25 mg.l−1 −70 µM-) (−49%, in MCF7 cells, P<0.02, and −56% in MDA-MB-231 cells, P<0.025). At high dose, in both cell types, GSx-related metabolites decreased, including homocystein, creatine and taurine (−60 to −80%, all, P<0.05). Together with glutathione-S-transferase actvity, data established that GSx biosynthesis was upregulated at low dose, and GSx consumption activated at high dose. Another major target, in both cell types, was lipid metabolism involving, at high doses, accumulation of polyunsaturated and total free fatty acids (between ×4.5 and ×11, P<0.025), and decrease of glycerophospho-ethanolamine and -choline (about −60%, P<0.025). Multivariate statistical analyses showed a metabolic transition, even a biphasic behavior of some metabolites including GSx, between low and high doses. In addition, CUR at 10 mg.l−1 in cotreatment with DTX induced modifications in glutathione metabolism, lipid metabolism, and glucose utilization. Some of these changes were biphasic depending on the duration of exposure to CUR.Conclusions/SignificanceMetabolomics reveals major metabolic targets of CUR in breast cancer cells, and biphasic responses that challenge the widely accepted beneficial effects of the phytochemical.

Effect of polyunsaturated fatty acids on tight junctions in a model of the human intestinal epithelium under normal and inflammatory conditions

Owing to their immune-modulatory action on the intestinal mucosa immune cells, the n-3 and n-6 polyunsaturated fatty acids (PUFA) have been suggested to modulate the risk and development of inflammatory bowel diseases. Failure in the intestinal barrier is an important hallmark of inflammatory bowel diseases. This study aimed at evaluating the impact of dietary PUFA on tight junction protein localisation and on the modulation of epithelial permeability under physiological conditions or under an inflammatory stress. For this purpose, we first confirmed the accumulation of PUFA in phospholipid fractions of Caco-2 cells upon 7 days of incubation with specific PUFA. Thereafter, Caco-2 cells were cultured in inserts, which provide a model of the human intestinal barrier. Accumulation of dietary n-3 PUFA in phospholipids did not affect the presence of occludin in tight junction complexes, while that of dietary n-6 PUFA decreased it. Whatever the PUFA, at 30 μM, no distortion of the Caco-2 barrier function was observed. Otherwise, 150 μM of docosahexaenoic acid (DHA) affected ZO-1 intensity under normal conditions, but not occludin or the barrier function parameters. Finally, to simulate an inflammatory state, cells were exposed for 24 h to interleukin-1β, tumor necrosis factor-α, interferon-γ at their basolateral side and to lypopolysaccharides at both sides. DHA limited the effect of inflammatory stimulus on occludin, ZO-1 and barrier function. In conclusion, this study has evidenced the specific effect of individual PUFA to modulate occludin and ZO-1 localization, according to the inflammatory status of this in vitro model of the intestinal barrier.

Seasonal changes in the accumulation of polyunsaturated fatty acids in zooplankton

In aquatic food webs, consumers, such as daphnids and copepods, differ regarding their accumulation of polyunsaturated fatty acids (PUFAs). We tested if the accumulation of PUFAs in a seston size fraction containing different consumers and in Daphnia as a separate consumer is subject to seasonal changes in a large deep lake due to changes in the dietary PUFA supply and specific demands of different consumers. We found that the accumulation of arachidonic acid (ARA) in Daphnia increased from early summer to late summer and autumn. However, ARA requirements of Daphnia appeared to be constant throughout the year, because the accumulation of ARA increased when the dietary ARA supply decreased. In the size fraction .140 mm, we found an increased accumulation of docosahexaenoic acid (DHA) during late summer and autumn. These seasonal changes in DHA accumulation were linked to changes in the proportion of copepods in this size fraction, which may have increasingly accumulated DHA for active overwintering. We show that consumer-specific PUFA demands can result in seasonal changes in PUFA accumulation, which may influence the trophic transfer of PUFAs within the food web.

Dietary alpha-linolenic acid does not enhance accumulation of omega-3 long-chain polyunsaturated fatty acids in barramundi (Lates calcarifer)

This study examined the effects of substituting fish oil and fish meal with a blend of alpha-linolenic acid (ALA, 18:3 n-3) rich vegetable oils (14%, w/w) and defatted poultry meal (34%, w/w) in a formulated diet, on growth and tissue fatty acid profiles in barramundi fingerlings. Results indicated that on average, while the ALA levels of the barramundi liver and fillet increased with increasing dietary ALA, there was no corresponding increase in the levels of the omega-3 (n-3) long chain polyunsaturated fatty acid (LCPUFA). Compared to fish consuming a commercial feed, which contained fish meal and fish oil, fish on the ALA diets grew slower, had a lower feed intake and lower n-3 LCPUFA levels in the tissues. Hepatic mRNA expression of Δ6 desaturase (FADS2) and elongase (ELOVL5/2) was ~10 fold and ~3 fold higher, respectively, in all the ALA dietary groups, relative to those fed the commercial feed. However, the level of expression of the two genes was not different between fish fed differing ALA levels. These data demonstrate that increasing the ALA level of the diet is not an appropriate strategy for replacing marine sources of n-3 LCPUFA in barramundi. It was also noted, however, that within the different ALA dietary groups there was a large amount of variation between individual fish in their tissue DHA levels, suggesting a significant heterogeneity in their capacity for conversion of ALA and/or retention of n-3 LCPUFA. When dietary ALA intakes were greater than 0.8% en, tissue DHA levels were inversely related to ALA intake, suggesting that high intake of dietary ALA may inhibit DHA synthesis.

Pressure Overload Selectively Increases n-3 PUFA in Myocardial Phospholipids During Early Postnatal Period

Increasing hemodynamic load during early postnatal development leads to rapid growth of the left ventricular (LV) myocardium, which is associated with membrane phospholipid (PL) remodeling characterized by n-3 polyunsaturated fatty acids (PUFA) accumulation. The aim of this study was to examine the influence of additional workload imposed early after birth when ventricular myocytes are still able to proliferate. Male Wistar rats were subjected to abdominal aortic constriction (AC) at postnatal day 2. Concentrations of PL and their fatty acid (FA) profiles in the LV were analyzed in AC, sham-operated (SO) and intact animals on postnatal days 2 (intact only), 5 and 10. AC resulted in LV enlargement by 22 % and 67 % at days 5 and 10, respectively, compared with age-matched SO littermates. Concentrations of phosphatidylcholine, cardiolipin, phosphatidylinositol, phosphatidylethanolamine, phosphatidylserine and sphingomyelin decreased in AC myocardium, albeit with different time course and extent. The main effect of AC on FA remodeling consisted in the accumulation of n-3 PUFA in PL. The most striking effect of AC on FA composition was observed in phosphatidylinositol and cardiolipin. We conclude that excess workload imposed by AC inhibited the normal postnatal increase of PL concentration while further potentiating the accumulation of n-3 PUFA as an adaptive response of the developing myocardium to accelerated growth.

Cold-Induced Accumulation of ω-3 Polyunsaturated Fatty Acid in a Liverwort,Marchantia polymorphaL.

The liverwort Marchantia polymorpha L. synthesizes various long-chain polyunsaturated fatty acids including arachidonic acid and eicosapentaenoic acid, neither of which is produced by higher plants. Here we report the effects of temperature on long-chain polyunsaturated fatty acid accumulation in the liverwort. The accumulation of ω-3 polyunsaturated fatty acids increased significantly as the growth temperature decreased. Specifically, the relative content of eicosapentaenoic acid to total fatty acids at 5 °C was approximately 3-fold higher than at 25 °C. On the other hand, the accumulation of ω-6 polyunsaturated fatty acids decreased at low temperatures. An analysis of gene expression indicated that the mRNA of the MpFAD3 gene for ER ω-3 desaturase increased significantly at 5 °C. These results indicate that in the liverwort the n-3 pathway was enhanced at low temperature, mainly via expression of the cold-induced ω-3 desaturase gene, leading to increased accumulation of eicosapentaenoic acid.

Enhancing the accumulation of omega-3 long chain polyunsaturated fatty acids in transgenic Arabidopsis thaliana via iterative metabolic engineering and genetic crossing

The synthesis and accumulation of long chain polyunsaturated fatty acids such as eicosapentaenoic acid has previously been demonstrated in the seeds of transgenic plants. However, the obtained levels are relatively low, indicating the need for further studies and the better definition of the interplay between endogenous lipid synthesis and the non-native transgene-encoded activities. In this study we have systematically compared three different transgenic configurations of the biosynthetic pathway for eicosapentaenoic acid, using lipidomic profiling to identify metabolic bottlenecks. We have also used genetic crossing to stack up to ten transgenes in Arabidopsis. These studies indicate several potential approaches to optimize the accumulation of target fatty acids in transgenic plants. Our data show the unexpected channeling of heterologous C20 polyunsaturated fatty acids into minor phospholipid species, and also the apparent negative metabolic regulation of phospholipid-dependent Δ6-desaturases. Collectively, this study confirms the benefits of iterative approaches to metabolic engineering of plant lipid synthesis.

Accumulation of polyunsaturated fatty acids by cladocerans: effects of taxonomy, temperature and food

Summary1. Zooplankton are important in transferring dietary nutrients, including polyunsaturated fatty acids (PUFA), up through aquatic food webs.2. We tested the hypothesis that the taxonomic composition of zooplankton affects the retention and subsequent transfer of PUFA from upwards through the food web. Using laboratory experiments, we investigated dietary PUFA accumulation and bioconversion capacities of six cladoceran species (Ceriodaphnia sp., Daphnia longispina, Daphnia magna, Daphnia pulex, Scapholeberis mucronata and Simocephalus vetulus) fed on two diets (Scenedesmus obliquus and Cryptomonas sp.) that differed in their PUFA profiles. We performed experiments at two different temperatures (14 and 20 °C) to assess the role of temperature in the trophic transfer of PUFA.3. There was little variation in the concentrations of PUFA in these cladocerans which were controlled by dietary PUFA supply. Moreover, as expected, the concentrations of PUFA in all cladoceran species were higher at low temperature.4. However, even if the composition of PUFA in the cladoceran species generally corresponded to that in their diet, preferential accumulation of some PUFA was recorded in all these taxa. When fed on a highly unsaturated fatty acid‐deficient diet, all the cladocerans showed some ability to convert C18‐PUFA into arachidonic acid and eicosapentaenoic acid. Interspecific variation in the ability to accumulate and bioconvert PUFA in cladocerans was more pronounced at low temperature (14 °C) for both diets.5. Our results strongly suggest that in heterogeneous habitats with food partitioning between co‐existing cladocerans, foraging behaviour may affect the transfer of PUFA more strongly than interspecific variation in accumulating and/or bioconverting dietary PUFA.

Polyunsaturated fatty acids are incorporated into maturating male mouse germ cells by lysophosphatidic acid acyltransferase 3

Long-chain polyunsaturated fatty acids (PUFAs) accumulate in mammalian testis during puberty and are essential for fertility. To investigate whether lysophospholipid acyltransferases determine the PUFA composition of testicular phospholipids during pubertal development, we compared their mRNA expression, in vitro activity, and specificity with the lipidomic profile of major phospholipids. The accumulation of PUFAs in phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine correlated with an induced lysophosphatidic acid acyltransferase (LPAAT)3 mRNA expression, increased microsomal LPAAT3 activity, and shift of LPAAT specificity to PUFA-coenzyme A. LPAAT3 was induced during germ cell maturation, as shown by immunofluorescence microscopy. Accordingly, differentiation of mouse GC-2spd(ts) spermatocytes into spermatides up-regulated LPAAT3 mRNA, increased the amount of polyunsaturated phospholipids, and shifted the specificity for the incorporation of deuterium-labeled docosahexaenoic acid toward phosphatidylcholine and phosphatidylethanolamine. Stable knockdown of LPAAT3 in GC-2spd(ts) cells significantly decreased microsomal LPAAT3 activity, reduced levels of polyunsaturated phosphatidylethanolamine species, and impaired cell proliferation/survival during geneticin selection. We conclude that the induction of LPAAT3 during germ cell development critically contributes to the accumulation of PUFAs in testicular phospholipids, thereby possibly affecting sperm cell production.

Up-regulated Desaturase and Elongase Gene Expression Promoted Accumulation of Polyunsaturated Fatty Acid (PUFA) but Not Long-Chain PUFA in Lates calcarifer, a Tropical Euryhaline Fish, Fed a Stearidonic Acid- and γ-Linoleic Acid-Enriched Diet

The limited activity of Δ6 fatty acid desaturase (FAD6) on α-linolenic (ALA, 18:3n-3) and linoleic (LA, 18:2n-6) acids in marine fish alters the long-chain (≥C(20)) polyunsaturated fatty acid (LC-PUFA) concentration in fish muscle and liver when vegetable oils replace fish oil (FO) in aquafeeds. Echium oil (EO), rich in stearidonic acid (SDA, 18:4n-3) and γ-linoleic acid (GLA, 18:3n-6), may enhance the biosynthesis of n-3 and n-6 LC-PUFA by bypassing the rate-limiting FAD6 step. Nutritional and environmental modulation of the mechanisms in LC-PUFA biosynthesis was examined in barramundi, Lates calcarifer , a tropical euryhaline fish. Juveniles were maintained in either freshwater or seawater and fed different dietary LC-PUFA precursors present in EO or rapeseed oil (RO) and compared with FO. After 8 weeks, growth of fish fed EO was slower compared to the FO and RO treatments. Irrespective of salinity, expression of the FAD6 and elongase was up-regulated in fish fed EO and RO diets, but did not lead to significant accumulation of LC-PUFA in the neutral lipid of fish tissues as occurred in the FO treatment. However, significant concentrations of eicosapentaenoic acid (EPA, 20:5n-3) and arachidonic acid (ARA, 20:4n-6), but not docosahexaenoic acid (DHA, 22:6n-3), appeared in liver and, to a lesser extent, in muscle of fish fed EO with marked increases in the phospholipid fraction. Fish in the EO treatment had higher EPA and ARA in their liver phospholipids than fish fed FO. Endogenous conversion of dietary precursors into neutral lipid LC-PUFA appears to be limited by factors other than the initial rate-limiting step. In contrast, phospholipid LC-PUFA had higher biosynthesis, or selective retention, in barramundi fed EO rather than RO.

Food sources and lipid retention of zooplankton in subarctic ponds

SUMMARY 1. Subarctic ponds are seasonal aquatic habitats subject to short summers but often have surprisingly numerous planktonic consumers relative to phytoplankton productivity. Because subarctic ponds have low pelagic productivity but a high biomass of benthic algae, we hypothesised that benthic mats provide a complementary and important food source for the zooplankton. To test this, we used a combination of fatty acid and stable isotope analyses to evaluate the nutritional content of benthic and pelagic food and their contributions to the diets of crustacean zooplankton in 10 Finnish subarctic ponds. 2. Benthic mats and seston differed significantly in total lipids, with seston (62.5 l gm g )1 ) having approximately eight times higher total lipid concentrations than benthic mats (7.0 l gm g )1 ). Moreover, the two potential food sources differed in their lipid quality, with benthic organic matter completely lacking some nutritionally important polyunsaturated fatty acids (PUFA), most notably docosahexaenoic acid and arachidonic acid. 3. Zooplankton had higher PUFA concentrations (27–67 l gm g )1 ) than either of the food sources (mean benthic mats: 1.2 l gm g )1 ; mean seston: 9.9 l gm g )1 ), indicating that zooplankton metabolically regulate their accumulation of PUFA. In addition, when each pond was evaluated independently, the zooplankton was consistently more 13 C-depleted (d 13 C )20 to )33&) than seston ()23 to )29&) or benthic ()15 to )27&) food sources. In three ponds, a subset of the zooplankton (Eudiaptomus graciloides, Bosmina sp., Daphnia sp. and Branchinecta paludosa) showed evidence of feeding on both benthic and planktonic resources, whereas in most (seven out of 10) ponds the zooplankton appeared to feed primarily on plankton. 4. Our results indicate that pelagic primary production was consistently the principal food resource of most metazoans. While benthic mats were highly productive, they did not appear to be a major food source for zooplankton. The pond zooplankton, faced by strong seasonal food limitation, acquires particular dietary elements selectively.

Composition of fatty acids in plasma and erythrocytes and eicosanoids level in patients with metabolic syndrome.

BackgroundDisturbances of the fatty acids composition in plasma and red blood cells and eicosanoid synthesis play an important role in the metabolic syndrome (MS) formation.MethodsThe observation group included 61 people with metabolic syndrome (30 patients with MS and normal levels of insulin, 31 people with MS and insulin resistance - IR). The parameters of carbohydrate and lipid metabolism in blood serum were examined. The composition of nonesterified fatty acids (NEFA), fatty acid (FA) of red blood cells lipids was analyzed by gas-liquid chromatography. Eicosanoids level in MS patients blood serum was studied by enzyme immunoassay.ResultsIn MS patients in the absence of glucose-insulin homeostasis disturbances and in patients with IR the accumulation of polyunsaturated fatty acids (18:2 n6, 18:3 n3, 22:4 n6) and lower pool of saturated FA (12:0, 14:0, 16: 0, 17:0) in plasma were discovered. A deficit of polyunsaturated FA (18:3 n3, 20:4 n6) with a predominance of on-saturated FA (14:0, 18:0) in erythrocyte membranes was revealed. In MS patients regardless of the carbohydrate metabolism status high levels of leukotriene B4 and 6-keto-prostaglandin-F1α in serum were found. The development of IR in MS patients leads to increased synthesis of thromboxane A2.ConclusionThe results revealed a disturbance in nonesterified fatty acids of plasma lipids and red blood cells, eicosanoid synthesis in MS patients. The breach of the plasma and cell membranes fatty acids compositions, synthesis of vasoactive and proinflammatory eicosanoids is an important pathogenetic part of the MS development.

Nonsymbiotic Hemoglobin-2 Leads to an Elevated Energy State and to a Combined Increase in Polyunsaturated Fatty Acids and Total Oil Content When Overexpressed in Developing Seeds of Transgenic Arabidopsis Plants

Nonsymbiotic hemoglobins are ubiquitously expressed in plants and divided into two different classes based on gene expression pattern and oxygen-binding properties. Most of the published research has been on the function of class 1 hemoglobins. To investigate the role of class 2 hemoglobins, transgenic Arabidopsis (Arabidopsis thaliana) plants were generated overexpressing Arabidopsis hemoglobin-2 (AHb2) under the control of a seed-specific promoter. Overexpression of AHb2 led to a 40% increase in the total fatty acid content of developing and mature seeds in three subsequent generations. This was mainly due to an increase in the polyunsaturated C18:2 (ω-6) linoleic and C18:3 (ω-3) α-linolenic acids. Moreover, AHb2 overexpression led to an increase in the C18:2/C18:1 and C18:3/C18:2 ratios as well as in the C18:3 content in mol % of total fatty acids and in the unsaturation/saturation index of total seed lipids. The increase in fatty acid content was mainly due to a stimulation of the rate of triacylglycerol synthesis, which was attributable to a 3-fold higher energy state and a 2-fold higher sucrose content of the seeds. Under low external oxygen, AHb2 overexpression maintained an up to 5-fold higher energy state and prevented fermentation. This is consistent with AHb2 overexpression results in improved oxygen availability within developing seeds. In contrast to this, overexpression of class 1 hemoglobin did not lead to any significant increase in the metabolic performance of the seeds. These results provide evidence for a specific function of class 2 hemoglobin in seed oil production and in promoting the accumulation of polyunsaturated fatty acids by facilitating oxygen supply in developing seeds.

CO2 biofixation and fatty acid composition of Scenedesmus obliquus and Chlorella pyrenoidosa in response to different CO2 levels

In this study, Scenedesmus obliquus SJTU-3 and Chlorella pyrenoidosa SJTU-2 were cultivated with 0.03%, 5%, 10%, 20%, 30%, 50% CO(2). The two microalgae could grow at 50% CO(2) (>0.69 g L(-1)) and grew well (>1.22 g L(-1)) under CO(2) concentrations ranging from 5% to 20%. Both of the two examined microalgae showed best growth potential at 10% CO(2). The maximum biomass concentration and CO(2) biofixation rate were 1.84 g L(-1) and 0.288 g L(-1) d(-1) for S. obliquus SJTU-3 and 1.55 g L(-1) and 0.260 g L(-1) d(-1) for C. pyrenoidosa SJTU-2, respectively. The main fatty acid compositions of the two examined microalgae were fatty acids with C(16)-C(18) (>94%) under different CO(2) levels. High CO(2) levels (30-50%) were favorable for the accumulation of total lipids and polyunsaturated fatty acids. The present results suggested that the two microalgae be appropriate for mitigating CO(2) in the flue gases and biodiesel production.

The ABC Transporter PXA1 and Peroxisomal β-Oxidation Are Vital for Metabolism in Mature Leaves ofArabidopsisduring Extended Darkness

Fatty acid beta-oxidation is essential for seedling establishment of oilseed plants, but little is known about its role in leaf metabolism of adult plants. Arabidopsis thaliana plants with loss-of-function mutations in the peroxisomal ABC-transporter1 (PXA1) or the core beta-oxidation enzyme keto-acyl-thiolase 2 (KAT2) have impaired peroxisomal beta-oxidation. pxa1 and kat2 plants developed severe leaf necrosis, bleached rapidly when returned to light, and died after extended dark treatment, whereas the wild type was unaffected. Dark-treated pxa1 plants showed a decrease in photosystem II efficiency early on and accumulation of free fatty acids, mostly alpha-linolenic acid [18:3(n-3)] and pheophorbide a, a phototoxic chlorophyll catabolite causing the rapid bleaching. Isolated wild-type and pxa1 chloroplasts challenged with comparable alpha-linolenic acid concentrations both showed an 80% reduction in photosynthetic electron transport, whereas intact pxa1 plants were more susceptible to the toxic effects of alpha-linolenic acid than the wild type. Furthermore, starch-free mutants with impaired PXA1 function showed the phenotype more quickly, indicating a link between energy metabolism and beta-oxidation. We conclude that the accumulation of free polyunsaturated fatty acids causes membrane damage in pxa1 and kat2 plants and propose a model in which fatty acid respiration via peroxisomal beta-oxidation plays a major role in dark-treated plants after depletion of starch reserves.

Proteomic, metabolomic and lipidomic approaches to unravel the roles of polyunsaturated fatty acid nutrition in the developing liver

Polyunsaturated fatty acids (PUFA) regulate metabolism in adult liver; PUFA accumulation in fetal and infant liver depends on diet. We used combined proteomics, metabolomics and lipidomics to address whether PUFA nutrition impacts metabolic development in neonatal rat liver. Rats were fed as % energy, 3.9% 18:2n‐6 and 1.5% 18:3n‐3 (high PUFA), or 1 % 18:2n‐6 and &lt;0.1% 18:3n‐3 (low PUFA) in gestation and lactation. On day 3 postnatal, lipidomics using HPLC and GLC showed lower n‐3/n‐6 PUFA, but no difference in lipid classes in low compared to high PUFA offspring. Liver proteins were resolved on 2D gels, resolving over 800 proteins. PDQuest analysis showed 24 proteins up‐regulated and 1 down‐regulated over 3 fold in the high compared to low PUFA group, and these were identified by MALDI‐TOF MS. Up‐regulated proteins included F‐1,6‐biphosphatase 1, G‐3‐P dehydrogenase, galactokinase 1, catalase and 60 kDa heat shock protein, with argininosuccinate synthase down‐regulated. GC‐MS profiling of small molecules in liver extracts, with principal component analysis to address changes in flux through metabolic pathways showed higher gluconeogenic amino acids in the high PUFA group. Finally, integration of results from the proteomic and metabolomic analyses into metabolic pathways shows that maternal lipid nutrition impacts pathways of gluconeogenesis and oxidative stress in developing liver. Funded by CIHRGrant Funding SourceMichael Smith Fdn Health Research Studentship

Member of the membrane‐bound O‐acyltransferase (MBOAT) family encodes a lysophospholipid acyltransferase with broad substrate specificity

Glycerophospholipids in biological membranes are metabolically active and participate in a series of deacylation-reacylation reactions, which may lead to accumulation of polyunsaturated fatty acids (PUFAs) at the sn-2 position of the glycerol backbone. The reacylation reaction is believed to be catalyzed by acyl-coenzyme A (acyl-CoA):lysophospholipid acyltransferase. Very recently, we have shown that Caenorhabditis elegans mboa-7, which belongs to the membrane-bound O-acyltransferase (MBOAT) family, encodes lysophosphatidylinositol (LPI)-specific acyltransferase (LPIAT). In this study, we found that knockdown of another member of the MBOAT family in C. elegans, named mboa-6, reduced incorporation of exogenous PUFAs into phosphatidylcholine (PC), phosphatidylserine (PS) and phosphatidylethanolamine (PE) in C. elegans. Knockdown of a human mboa-6 homologue, referred to as MBOAT5, also impaired the incorporation of PUFAs into PC, PS and PE in HeLa cells. In in vitro assays, lysoPC (LPC), lysoPS (LPS) and lysoPE (LPE) acyltransferase activities using [(14)C]arachidonoyl-CoA were significantly reduced in the microsomes of MBOAT5 knockdown cells. Conversely, over-expression of MBOAT5 in human embryonic kidney (HEK) 293 cells resulted in great increases in LPC, LPS and LPE acyltransferase activities but not in LPIAT or lysophosphatidic acid (LPA) acyltransferase (LPAAT) activities. These results indicate that human MBOAT5 is a lysophospholipid acyltransferase acting preferentially on LPC, LPS and LPE.

Investigation of the physiological properties and synthesis of PUFAs from Thraustochytrids and its electrophoretic karyotypes

Physiological properties of organism, such as the number of chromosomes, genome size, fatty acid profile and the activities of desaturases and elongases were investigated for four differentThraustochytrium species. The investigation revealed thatThraustochytrium aureum could synthesize a significant level of polyunsaturated fatty acids (PUFAs), particularly docosa-hexaenoic acid (DHA), when compared to the other threeThraustochytrium species tested. A higher level of saturated fatty acids was observed byT. striatum followed byThraustochytrium sp. ATCC 26185. The PUFA accumulation rate was higher in the n-3 than in the n-6 pathway. A comparison of the activities for these desaturases and elongases of the four different species were also studied. Further, the electrophoretic karyotypes of Thraustochytrids were separated by pulsed-field gel electrophoresis (PFGE). The separation condition of PFGE was developed to obtain the different chromosomes from the variousThraustochytrium species. The number of chromosomes inT. aureum, T. striatum, Thraustochytrium sp. ATCC 20891 andThraustochytrium sp. ATCC 26185 were 13, 17, 10. 8, and the whole genome size of those species were estimated to be 12.9, 11.7, 11.3 and 9.93 Mbp, respectively.

Δ6 Desaturase mRNA Abundance in HepG2 Cells Is Suppressed by Unsaturated Fatty Acids

The effect of unsaturated fatty acids on the abundance of Delta6 desaturase (D6D) mRNA and the fatty acid composition of HepG2 cell membranes was examined. Supplementation of HepG2 cells with oleic acid (18:1n-9, OA), linoleic acid (18:2n-6, LA), alpha-linolenic acid (18:3n-3, ALA), arachidonic acid (20:4n-6, AA) or eicosapentaenoic acid (20:5n-3, EPA) reduced D6D mRNA abundance by 39 +/- 6.6, 40 +/- 2.2, 31 +/- 5.2, 55 +/- 4.8, and 52 +/- 5.0%, respectively, compared with control cells. Despite the reduction in D6D mRNA abundance, the level of D6D conversion products (20:3n-9, EPA and AA) in OA, ALA and LA supplemented cells, respectively, was elevated above that in control cells. Our results suggest that although unsaturated fatty acids decrease the abundance of D6D mRNA by as much as 50%, the conversion of polyunsaturated fatty acids and accumulation of long chain polyunsaturated fatty acids (LCPUFA) in HepG2 cell phospholipids continues to occur.