Δ6 Desaturation Research Articles

Biosynthetic mechanisms of omega-3 polyunsaturated fatty acids in microalgae

Marine microalgae are the primary producers of ω3 polyunsaturated fatty acids (PUFAs), such as octadecapentaenoic acid (OPA, 18:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) for food chains. However, the biosynthetic mechanisms of these PUFAs in the algae remain elusive. To study how these fatty acids are synthesized in microalgae, a series of radiolabeled precursors were used to trace the biosynthetic process of PUFAs in Emiliania huxleyi. Feeding the alga with 14C-labeled acetic acid in a time course showed that OPA was solely found in glycoglycerolipids such as monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) synthesized plastidically by sequential desaturations while DHA was exclusively found in phospholipids synthesized extraplastidically. Feeding the alga with 14C-labeled α-linolenic acid (ALA), linoleic acid (LA) and oleic acid (OA) showed that DHA was synthesized extraplastidically from fed ALA and LA, but not from OA, implying that the aerobic pathway of DHA biosynthesis is incomplete with missing a Δ12 desaturation step. The in vitro enzymatic assays with 14C-labeled malonyl-CoA showed that DHA was synthesized from acetic acid by a PUFA synthase. These results provide the first and conclusive biochemistry evidence that OPA is synthesized by a plastidic aerobic pathway through sequential desaturations with the last step of Δ3 desaturation, while DHA is synthesized by an extraplastidic anaerobic pathway catalyzed by a PUFA synthase in the microalga.

Characterization and Functional Analysis of Fads Reveals Δ5 Desaturation Activity during Long-Chain Polyunsaturated Fatty Acid Biosynthesis in Dwarf Surf Clam Mulinia lateralis.

Fatty acid desaturases (Fads), as key enzymes in the biosynthesis of long-chain polyunsaturated fatty acids (LC-PUFAs), catalyze the desaturation between defined carbons of fatty acyl chains and control the degree of unsaturation of fatty acids. In the present study, two Fads genes, designated MulFadsA and MulFadsB, were identified from the genome of the dwarf surf clam Mulinia lateralis (Mollusca, Mactridae), and their spatiotemporal expression was examined. MulFadsA and MulFadsB contained the corresponding conserved functional domains and clustered closely with their respective orthologs from other mollusks. Both genes were expressed in the developmental stages and all tested adult tissues of M. lateralis, with MulFadsA exhibiting significantly higher expression levels in adult tissues than MulFadsB. Subsequently, the effects of dietary microalgae on Fads expressions in the dwarf surf clam were investigated by feeding clams with two types of unialgal diets varying in fatty acid content, i.e., Chlorella pyrenoidosa (Cp) and Platymonas helgolandica (Ph). The results show that the expressions of MulFads were significantly upregulated among adult tissues in the Cp group compared with those in the Ph group. In addition, we observed the desaturation activity of MulFadsA via heterologous expression in yeasts, revealing Δ5 desaturation activity toward PUFA substrates. Taken together, these results provide a novel perspective on M. lateralis LC-PUFA biosynthesis, expanding our understanding of fatty acid synthesis in marine mollusks.

Effects of Dietary Oxidized Phytosterol on Lipid Metabolism in Rats.

Many in vitro studies have revealed the toxic effects of oxidized phytosterols (OPSs); however, their effects on lipid metabolism are not well understood in vivo. Therefore, we examined the bioavailability of OPS and compared the effects of dietary phytosterols (PSs) or OPS on lipid metabolism in rats. OPS was detected in the plasma and liver of rats administered 50 mg of OPS for 3 days. Rats were fed the AIN76 diet (C group), basal diet plus 0.25% PS (P group), or 0.25% OPS (O group) for 4 weeks. Dietary OPS but not PS reduced hepatic fatty acid synthase activity. Liver triacylglycerol (TG) levels tended to be lower in the P group than in the C group and were significantly lower in the O group. The mRNA expression level of HMG-CoA reductase in the liver was the lowest in the O group, whereas that of CYP27A1 was the highest in the O group. The mRNA expression levels of NPC1L1 in the intestinal mucosa were significantly lower in the P and O groups than in the C group. Consistent with these modulations, plasma total cholesterol (TC) and HDL-C levels were similar between the C and P groups but tended to be higher or significantly higher in the O group. Liver TC levels were significantly lower in the P and O groups than in the C group. Moreover, fecal neutral and acidic steroid levels were the highest in the O group. The mRNA expression level of Δ6 desaturase in the liver was significantly higher in both the P and the O groups than in the C group. The Δ6 desaturation indices of fatty acids in the total liver lipids were the highest in the O group. Thus, dietary OPS may modulate lipid metabolism in the liver.

Functional characterization reveals a diverse array of metazoan fatty acid biosynthesis genes.

Long-chain (≥C20 ) polyunsaturated fatty acids (LC-PUFAs) are physiologically important fatty acids for most animals, including humans. Although most LC-PUFA production occurs in aquatic primary producers such as microalgae, recent research indicates the ability of certain groups of (mainly marine) invertebrates for endogenous LC-PUFA biosynthesis and/or bioconversion from dietary precursors. The genetic pathways for and mechanisms behind LC-PUFA biosynthesis remain unknown in many invertebrates to date, especially in non-model species. However, the numerous genomic and transcriptomic resources currently available can contribute to our knowledge of the LC-PUFA biosynthetic capabilities of metazoans. Within our previously generated transcriptome of the benthic harpacticoid copepod Platychelipus littoralis, we detected expression of one methyl-end desaturase, one front-end desaturase, and seven elongases, key enzymes responsible for LC-PUFA biosynthesis. To demonstrate their functionality, we characterized eight of them using heterologous expression in yeast. The P. littoralis methyl-end desaturase has Δ15/17/19 desaturation activity, enabling biosynthesis of α-linolenic acid, eicosapentaenoic acid and docosahexaenoic acid (DHA) from 18:2 n-6, 20:4 n-6 and 22:5 n-6, respectively. Its front-end desaturase has Δ4 desaturation activity from 22:5 n-3 to DHA, implying that P. littoralis has multiple pathways to produce this physiologically important fatty acid. All studied P. littoralis elongases possess varying degrees of elongation activity for saturated and unsaturated fatty acids, producing aliphatic hydrocarbon chains with lengths of up to 30 carbons. Our investigation revealed a functionally diverse range of fatty acid biosynthesis genes in copepods, which highlights the need to scrutinize the role that primary consumers could perform in providing essential nutrients to upper trophic levels.

LC-PUFA endogenous biosynthesis is improved in Nile tilapia fed plant-oil sources at cold suboptimal temperature

Nile tilapia, though a tropical species, is cultured throughout most subtropical regions of the globe, in areas with large seasonal thermal variations. The maintenance of sustained productivity during adverse environmental conditions, such as winter, is a growing area of research effort, including nutritional interventions and seasonal specific diets. The present study aimed at evaluating how the in vivo fatty acid metabolism is modulated by the rearing temperature in juvenile Nile tilapia fed different lipid sources. Four experimental diets were tested in two trials at optimal (28 °C) and suboptimal (22 °C) temperature, and the dietary treatments were: a diet containing fish oil (FO), and therefore rich in n-3 LC-PUFA (long-chain PUFA of 20 or 22 carbons), and three plant-oil based diets rich in C18 PUFA (PUFA of 18 carbons), containing formulated blends including sunflower oil (rich in 18:2n-6) and linseed oil (rich in 18:3n-3), to achieve a gradient of C18 PUFA n-3/n-6 ratio. The results showed that the in vivo fatty acid metabolism was substantially modified by the diets and the environmental conditions. Among dietary treatments, differences were noted in the enzyme activities for SFA (saturated fatty acids) and MUFA (monounsaturated fatty acids) neogenesis and bioconversion. The activities of the enzymes involved in C18 PUFA bioconversion towards LC-PUFA, namely elongases, Δ5Δ6-desaturase and Δ4-desaturase, were low in Nile tilapia fed FO diet, whereas in fish fed the plant-oil based diets the bioconversion towards n-6 and n-3 LC-PUFA was proportional to substrate availability. Temperature was responsible for important modifications, including a clear metabolic attempt towards increasing LC-PUFA content, and a simultaneous reduction of other classes, in fish exposed to cold suboptimal conditions. In fact, Nile tilapia fed plant-oil diets at 22 °C increased the activities of PUFA elongases and desaturases, whilst decreased activities of SFA neogenesis, Δ9 desaturation, and elongation of SFA or MUFA. Interestingly, at 22 °C, Δ4 desaturation increased with dietary 18:3n-3 increase, while at 28 °C, fish fed the diet poor in 18:3n-3 had no apparent activity of this enzyme. This study also showed a preference of enzymes towards n-3 substrates over n-6 substrates, but the possible essentiality of n-6 LC-PUFA, as well as n-3 LC-PUFA, at the lower temperature for the Nile tilapia, could not be excluded. In summary, dietary fatty acids directly influence body fatty acid accumulation, but environmental temperatures play major roles in modulating the overall in vivo fatty acid metabolism in Nile tilapia juveniles.

A 13CO2 Enrichment Experiment to Study the Synthesis Pathways of Polyunsaturated Fatty Acids of the Haptophyte Tisochrysis lutea.

The production of polyunsaturated fatty acids (PUFA) in Tisochrysis lutea was studied using the gradual incorporation of a 13C-enriched isotopic marker, 13CO2, for 24 h during the exponential growth of the algae. The 13C enrichment of eleven fatty acids was followed to understand the synthetic pathways the most likely to form the essential polyunsaturated fatty acids 20:5n-3 (EPA) and 22:6n-3 (DHA) in T. lutea. The fatty acids 16:0, 18:1n-9 + 18:3n-3, 18:2n-6, and 22:5n-6 were the most enriched in 13C. On the contrary, 18:4n-3 and 18:5n-3 were the least enriched in 13C after long chain polyunsaturated fatty acids such as 20:5n-3 or 22:5n-3. The algae appeared to use different routes in parallel to form its polyunsaturated fatty acids. The use of the PKS pathway was hypothesized for polyunsaturated fatty acids with n-6 configuration (such as 22:5n-6) but might also exist for n-3 PUFA (especially 20:5n-3). With regard to the conventional n-3 PUFA pathway, Δ6 desaturation of 18:3n-3 appeared to be the most limiting step for T. lutea, “stopping” at the synthesis of 18:4n-3 and 18:5n-3. These two fatty acids were hypothesized to not undergo any further reaction of elongation and desaturation after being formed and were therefore considered “end-products”. To circumvent this limiting synthetic route, Tisochrysis lutea seemed to have developed an alternative route via Δ8 desaturation to produce longer chain fatty acids such as 20:5n-3 and 22:5n-3. 22:6n-3 presented a lower enrichment and appeared to be produced by a combination of different pathways: the conventional n-3 PUFA pathway by desaturation of 22:5n-3, the alternative route of ω-3 desaturase using 22:5n-6 as precursor, and possibly the PKS pathway. In this study, PKS synthesis looked particularly effective for producing long chain polyunsaturated fatty acids. The rate of enrichment of these compounds hypothetically synthesized by PKS is remarkably fast, making undetectable the 13C incorporation into their precursors. Finally, we identified a protein cluster gathering PKS sequences of proteins that are hypothesized allowing n-3 PUFA synthesis.

New understandings of the pathway of long-chain polyunsaturated fatty acid biosynthesis.

Molecular studies have clarified the roles of the fatty acid desaturase (FADSx) and elongation of very long chain fatty acid (ELOVLx) genes, as well as acyl-coenzyme A synthase long-chain isoforms (ACSLx) required for entry to long-chain polyunsaturated fatty acid (LCPUFA) biosynthetic pathways. FADS1 and FADS2 but not FADS3 are active toward PUFA. FADS1 is a Δ5-desaturase operating on five C20 PUFA, and is strongly regulated by human genetic polymorphisms, modulating circulating arachidonic acid (20:4n-6) levels. In contrast, FADS2 operates on at least 16 substrates, including five saturates, and catalyzes Δ6, Δ4, and Δ8 desaturation. FADS2 silencing in cancer cells leads to FADS1 synthesis of unusual fatty acids. ACSL6 and ACSL4 are required to maintain tissue 22:6n-3 and 20:4n-6, respectively. FADS2AT2, is the first transcript to differentially inhibit desaturation, attenuating 18:3n-3 but not 18:2n-6 desaturation. The PUFA elongases ELOVL5, 2, and 4 are implicated in cancer, age-related methylation, and retinal degeneration, respectively. The mixture of fatty acids available to FADS2 in any tissue defines the product mixture available for further synthesis of membrane lipids and signaling molecules and may be relevant in many clinical conditions including cancer. Functional genetic variants define the levels of circulating arachidonic acid via FADS1 regulation; genotypes that drive high arachidonic acid may predispose to disease.

Regioselectivity mechanism of the Thunbergia alata Δ6-16:0-acyl carrier protein desaturase

Abstract Plant plastidial acyl–acyl carrier protein (ACP) desaturases are a soluble class of diiron-containing enzymes that are distinct from the diiron-containing integral membrane desaturases found in plants and other organisms. The archetype of this class is the stearoyl-ACP desaturase which converts stearoyl-ACP into oleoyl (18:1Δ9cis)-ACP. Several variants expressing distinct regioselectivity have been described including a Δ6-16:0-ACP desaturase from black-eyed Susan vine (Thunbergia alata). We solved a crystal structure of the T. alata desaturase at 2.05 Å resolution. Using molecular dynamics (MD) simulations, we identified a low-energy complex between 16:0-ACP and the desaturase that would position C6 and C7 of the acyl chain adjacent to the diiron active site. The model complex was used to identify mutant variants that could convert the T. alata Δ6 desaturase to Δ9 regioselectivity. Additional modeling between ACP and the mutant variants confirmed the predicted regioselectivity. To validate the in-silico predictions, we synthesized two variants of the T. alata desaturase and analyzed their reaction products using gas chromatography-coupled mass spectrometry. Assay results confirmed that mutants designed to convert T. alata Δ6 to Δ9 selectivity exhibited the predicted changes. In complementary experiments, variants of the castor desaturase designed to convert Δ9 to Δ6 selectivity lost some of their Δ9 desaturation ability and gained the ability to desaturate at the Δ6 position. The computational workflow for revealing the mechanistic understanding of regioselectivity presented herein lays a foundation for designing acyl-ACP desaturases with novel selectivities to increase the diversity of monoenes available for bioproduct applications.

The Malaysian Mahseer, Tor tambroides possess all required biosynthesis enzymes for the conversion of C18 polyunsaturated fatty acids to long-chain polyunsaturated fatty acids

The Malaysian mahseer (Tor tambroides) is an iconic and highly-priced freshwater large cyprinid with a natural dietary preference towards riverine fruits. Previous works indicate that dietary C18 polyunsaturated fatty acids can fulfill mahseer's essential fatty acid requirements. Given the need to reduce the use of fish meal and fish oil in aquafeed, there is considerable interest to sustain healthy levels of the beneficial n-3 long-chain polyunsaturated fatty acids (LC-PUFA) in farmed fish. A prerequisite for a functional LC-PUFA biosynthesis pathway is an entire repertoire of desaturase (Fads) and elongase (Elovl) enzymes required for specific steps along the path. We showed here the presence of a Fads2 and two elongases, Elovl5 and Elovl2, in mahseer. These enzymes function collectively to convert C18 PUFA to the C20 LC-PUFA through Δ6 and Δ5 desaturation processes working in tandem with the Elovl5 elongation activities. Subsequently, further conversion to docosahexaenoic acid (DHA; 22:6n-3) involves Δ6 desaturation and Elovl2 and Elovl5. These enzymes displayed a typical expression pattern of freshwater teleost at the mRNA level, with significant transcripts in liver, intestine, and brain tissues. Under limited dietary LC-PUFA intakes, the expression of these genes in the liver was elevated. Tissue fatty acid composition also suggests the conversion of dietary α-linolenic acid (ALA; 18:3n-3) and linoleic acid (LA; 18:2n-6) to their respective LC-PUFA for deposition in brain and muscle tissues. Although the highest concentration of n-3 LC-PUFA is obtained using fish oil-based diet, our results showed considerable accumulation of EPA and DHA with dietary ALA provision. Taken together, we show the existence of complete machinery for the biosynthesis of LC-PUFA from C18 PUFA in mahseer and validates the mahseer's capacity to utilize dietary vegetable oils (VO) both growth and n-3 LC-PUFA deposition.

Apocryphal FADS2 activity promotes fatty acid diversification in cancer

Canonical fatty acid metabolism describes specific enzyme-substrate interactions that result in products with well-defined chain lengths, degree(s), and positions of unsaturation. Deep profiling of lipids across a range of prostate cancer cell lines reveals a variety of fatty acids with unusual site(s) of unsaturation that are not described by canonical pathways. The structure and abundance of these unusual lipids correlate with changes in desaturase expression and are strong indicators of cellular phenotype. Gene silencing and stable isotope tracing demonstrate that direct Δ6 and Δ8 desaturation of 14:0 (myristic), 16:0 (palmitic), and 18:0 (stearic) acids by FADS2 generate new families of unsaturated fatty acids (including n-8, n-10, and n-12) that have rarely-if ever-been reported in human-derived cells. Isomer-resolved lipidomics reveals theselective incorporation of these unusual fatty acids into complex structural lipids and identifies their presence in cancer tissues, indicating functional roles in membrane structure and signaling.

Identification and Functional Characterization of a Novel Δ12 Fatty Acid Desaturase Gene from Haematococcus pluvialis

The freshwater microalga Haematococcus pluvialis accumulates large amounts of fatty acids in response to adverse conditions. However, the key fatty acid desaturase genes in H. pluvialis remain unknown. In this study, we cloned and functionally characterized a A12 fatty acid desaturase gene, and designated it as HpFAD2. The open reading frame of HpFAD2 consisted of 1137 base pairs and encoded 378 amino acids. The deduced polypeptide showed 70% identity to other endoplasmic reticulum Δ12 fatty acid desaturases, whereas it had only 44% identity to plastid Δ12 fatty acid desaturases. The PSORT algorithm and phylogenetic analysis further confirmed its affiliation to the endoplasmic reticulum Δ12 fatty acid desaturases. Heterologous expression was performed in Saccharomyces cerevisiae cells transformed with the recombinant plasmid pYES2-HpFAD2. Two additional fatty acids (C16:2 and C18:2) were detected in the yeast transformants. The results indicated Δ12 desaturation activity and substrate preference for C18:1 over C16:1. The transcriptional levels of H. pluvialis HpFAD2 at different growth stages were measured by quantitative polymerase chain reaction (PCR), indicating that the HpFAD2 transcriptional levels were significantly higher in red cells than those in green cells. Our study brings more insight into the fatty acid biosynthetic pathway of H. pluvialis.

A conserved evolutionary mechanism permits Δ9 desaturation of very-long-chain fatty acyl lipids

Δ9 fatty acyl desaturases introduce a cis-double bond between C9 and C10 of saturated fatty acyl chains. From the crystal structure of the mouse stearoyl-CoA desaturase (mSCD1) it was proposed that Tyr-104, a surface residue located at the distal end of the fatty acyl binding pocket plays a key role in specifying 18C selectivity. We created mSCD1-Y104G to test the hypothesis that eliminating this bulky side chain would create an opening and permit the substrate's methyl end to protrude through the enzyme into the lipid bilayer, facilitating the desaturation of very-long-chain (VLC) substrates. Consistent with this hypothesis, Y104G acquired the ability to desaturate 24C and 26C acyl-CoAs while maintaining its Δ9-regioselectivity. We also investigated two distantly related very-long-chain fatty acyl (VLCFA) desaturases from Arabidopsis, ADS1.2 and ADS1.4, which have Ala and Gly, respectively, in place of the gatekeeping Tyr found in mSCD1. Substitution of Tyr for Ala and Gly in ADS1.2 and ADS1.4, respectively, blocked their ability to desaturate VLCFAs. Further, we identified a pair of fungal desaturase homologs which contained either an Ile or a Gly at this location and showed that only the Gly-containing desaturase was capable of very-long-chain desaturation. The conserved desaturase architecture wherein a surface residue with a single bulky side chain forms the end of the substrate binding cavity predisposes them to single amino acid substitutions that enable a switch between long- and very-long-chain selectivity. The data presented here show that such changes have independently occurred multiple times during evolution.

Letrozole – The Antiestrogen Drug Increases FADS2 Function

Abstract Objectives Our main aim is to test the effect of estrogen and antiestrogen (letrozole) on the modulation of fatty acid desaturation and ScA levels in vitro using human cancer cells. Methods We used two sets of cells, MCF7 cells stably expressing FADS1 and FADS2 genes and a series of wild type (MCF7, HepG2, SK-N-SH, Caco2 and Y79) cancer cells. Cells were treated with estrogen (0 to 200 ppm) or letrozole (0 to 100 ppm) at the time of seeding and at confluence 50 μM albumin bound 20:2n-6 was added to FBS-free media. FAME was quantified by GC-flame ionization detector (GC-FID) and structures were identified by gas chromatography (GC) – covalent adduct chemical ionization mass spectrometry (CACI-MS/MS). Results Estrogen caused a dose dependent decrease in ScA via apparent inhibition of FADS1 activity in all wild type and had no effect on FADS2 (Δ8 desaturation) mediated synthesis of DGLA. In MCF7 cells, letrozole caused a dose dependent increase in FADS2 catalyzed DGLA and a decrease in ScA. Conclusions We provide the first biochemical evidence demonstrating MCF7 cells treated with letrozole increase DGLA, the immediate precursor to the anti-inflammatory eicosanoid PGE1. Letrozole is the first hormone-active agent to have opposing effects on FADS1 and FADS2. Funding Sources NIH grant R01 AT007003.

The links between supplementary tannin levels and conjugated linoleic acid (CLA) formation in ruminants: A systematic review and meta-analysis.

A systematic review and meta-analysis were conducted to predict and identify ways to increase conjugated linoleic acid (CLA) formation in ruminant-derived products to treat human health issues with dietary tannins. The objective was to compare and confirm the effects of dietary tannins on CLA formation by analyzing in vitro and/or in vivo studies. We reported the results of the meta-analysis based on numerical data from 38 selected publications consisting of 3712 treatments. Generally, via multiple pathways, the CLA formation increased when dietary tannins increased. Concurrently, dietary tannins increased Δ9 desaturation and the CLA indices in milk and meat (P < 0.05 and P < 0.001, with average R2 values of 0.23 and 0.44, respectively), but they did not change the rumen fermentation characteristics, including total volatile fatty acids (mmol/L) and their acid components. In vitro observations may accurately predict in vivo results. Unfortunately, there was no relationship between in vitro observations and in vivo results (R2 < 0.10), indicating that it is difficult to predict CLA formation in vivo considering in vitro observations. According to the statistical meta-analysis results regarding animal aspects, the ranges of tannin levels required for CLA formation in vitro and in vivo were approximately 0.1–20 g/kg dry matter (DM) (P < 0.001) and 2.1–80 g/kg DM (P < 0.001), respectively. In conclusion, the in vivo method was more suitable for the direct observation of fatty acid transformation than the in vitro method.

Endocrine Hormone Beta-estradiol and Anti-estrogen Letrozole Modulate 20:3 Isomer Production from 20:2n-6 in Human Cancer Cells (P08-119-19)

ObjectivesThe product of linoleic acid elongation, 20:2n-6, is the only known substrate for both FADS1 and FADS2. FADS1 catalyzes 20:2n-6 à 5Z,11Z,14Z-20:3 (sciadonic acid, SA), whereas FADS2 action yields 8Z,11Z,14Z-20:3 (DGLA). In certain cancer cell types, non-functional FADS2 activity unmasks 18:2n-6 elongation to 20:2n-6 and Δ5 desaturation by FADS1 to yield SA. Estrogen is known to inhibit Δ5-desaturation activity. Our objective was to examine the effect of 17-β-estradiol (estrogen) and the antiestrogen drug letrozole on the modulation of fatty acid desaturation and SA levels in vitro. MethodsEstrogen doses (0 to 200 ppm) and letrozole doses (0 to 100 ppm) studies were performed using stably transformed MCF7 cells with either FADS1 or FADS2 or empty vector and five different (MCF7, HepG2, SK-N-SH, Y79 and Caco2) wild type cancer cells. Fatty acid methyl esters (FAME) were prepared from harvested cells and analyzed quantitatively by gas chromatograph-flame ionization detector (GC-FID). The peak structures were positively identified by GC-covalent adduct chemical ionization tandem mass spectrometry (GC-CACI-MS/MS). ResultsIncreasing estrogen caused a decrease in the 5-desaturation product 5,11,14–20:3 in a dose-response manner in all cell types, but had no effect on DGLA. Letrozole exerted its effects most prominently in MCF7 cells. In response to increasing letrozole doses, DGLA increased, and SA decreased. ConclusionsWe provide the first biochemical evidence demonstrating letrozole enhancing FADS2-mediated DGLA levels in MCF7 cells. In all cell types estrogen reduced FADS1 mediated synthesis of SA. Both product fatty acids have anti-inflammatory activity, DGLA as a precursor to PGE1 and SA in displacing the largely proinflammatory arachidonic acid. Funding SourcesNIH grant R01 AT007003.

Molecular cloning and functional characterization of elongase (elovl5) and fatty acyl desaturase (fads2) in sciaenid, Nibea diacanthus (Lacepède, 1802)

In the present paper, we investigated the molecular cloning and functional characterization of elongase of very long chain fatty acid (elovl) and fatty acyl desaturase (fads) genes in a marine teleost, Nibea diacanthus. The elongase cDNA sequence encoded a polypeptide of 294 amino acids exhibiting Elovl5 activity, which effectively elongated both C18 (18:2n−6, 18:3n−3 and 18:3n−6) and C20 (20:4n−6 and 20:5n−3) polyunsaturated fatty acids. The desaturase cDNA sequence specified a polypeptide of 445 amino acids indicating Δ6 desaturation activity, which coul converted C18:2n−6 and C18:3n−3 to C18:3n−6 and C18:4n−3, respectively. Tissue distribution analysis by quantitative real-time PCR revealed that the elovl5 was primarily expressed in intestine and liver, while the fads2 was mainly expressed in liver and brain. These results increase our knowledge of the ability of endogenous highly unsaturated fatty acids (HUFA) biosynthesis in marine carnivorous fish.

Modulation of sphingolipid long-chain base composition and gene expression during early olive-fruit development, and putative role of brassinosteroid

Sphingolipids are abundant membrane components and signalling molecules in various aspects of plant development. However, the role of sphingolipids in early fleshy-fruit growth has rarely been investigated. In this study, we first investigated the temporal changes in sphingolipid long-chain base (LCB) content, composition, and gene expression that occurred during flower opening and early fruit development in olive (Olea europaea L. cv Picual). Moreover, the interaction between sphingolipid and the plant hormone, brassinosteroid (BR), during the early fruit development was also explored. For this, BR levels were manipulated through the application of exogenous BRs (24-epibrassinolide, EBR) or a BR biosynthesis inhibitor (brassinazole, Brz) and their effects on early fruit development, sphingolipid LCB content, and gene expression were examined in olive fruit at 14 days post-anthesis (DPA). We here show that sphingolipid with C-4 hydroxylation and Δ8 desaturation with a preference for (E)-isomer formation are quantitatively the most important sphingolipids in olive reproductive organs. In this work, the total LCB amount significantly decreased at the anthesis stage, but olive sphingosine-1-phosphate lyase (OeSPL) gene was expressed exclusively in flower and upregulated during the anthesis, revealing an association with the d18:1(8E) accumulation. However, the LCB content increased in parallel with the upregulation of the expression of genes for key sphingolipid biosynthetic and LCB modification enzymes during early fruit development in olive. Likewise, we found that EBR exogenously applied to olive trees significantly stimulated the fruit growth rate whereas Brz inhibited fruit growth rate after 7 and 14 days of treatment. In addition, this inhibitory effect could be counteracted by the application of EBR. The promotion of early fruit growth was accompanied by the down-regulation of sphingolipid LCB content and gene expression in olive fruit, whereas Brz application raised levels of sphingolipid LCB content and gene expression in olive fruit after 7 and 14 days of treatment. Thus, our data indicate that endogenous sphingolipid LCB and gene-expression levels are intricately controlled during early fruit development and also suggest a possible link between BR, the sphingolipid content/gene expression, and early fruit development in olive.

Capacity for eicosapentaenoic acid and arachidonic acid biosynthesis in silver barb (Barbonymus gonionotus): Functional characterisation of a Δ6/Δ8/Δ5 Fads2 desaturase and Elovl5 elongase

The silver barb (Barbonymus gonionotus), a widely distributed cyprinid in Southeast Asia, has a reasonable deposition of the physiologically important omega-3 (n-3) long-chain polyunsaturated fatty acids (LC-PUFA), implying the existence of a functional LC-PUFA biosynthesis pathway. In relation to this, the cloning and functional characterisation of a fatty acyl desaturase (Fads) and fatty acyl elongase (Elovl) were examined to elucidate the capacity of silver barb in converting α-linolenic acid (ALA; 18:3n-3) into eicosapentaenoic acid (EPA; 20:5n-3) and subsequently docosahexaenoic acid (DHA; 22:6n-3), and linoleic acid (LA; 18:2n-6) into arachidonic acid (ARA; 20:4n-6). Sequence analysis showed that the cloned cDNAs correspond to fads2 and elovl5. In terms of function, the Fads2 showed Δ6, Δ5 and Δ8 desaturation activities, while the Elovl5 showed activities towards polyunsaturated fatty acid (PUFA) substrates at preference of C18 > C20 > C22. Both enzymes showed higher activities towards n-3 PUFAs as compared to n-6 PUFAs. Taken together, these results showed the presence of all the required enzymes for biosynthesis of EPA and ARA from ALA and LA, respectively in silver barb fish. Both genes are highly expressed in brain and liver. Juvenile fish fed with ALA-rich diet showed higher expression of hepatic fads2 and elovl5 than to those fed with LC-PUFA-rich diet. Dietary ALA also contributed to the biosynthesis and deposition of EPA and DHA in fish tissues, albeit lower than levels achieved with the fish oil (FO)-based diet. Overall, this study shows the presence of functional Fads2 and Elovl5 in a farmed cyprinid species, which contributes to LC-PUFA biosynthesis during limited dietary LC-PUFA intake.

Deletion of Stearoyl-CoA Desaturase-1 From the Intestinal Epithelium Promotes Inflammation and Tumorigenesis, Reversed by Dietary Oleate

The enzyme stearoyl-coenzyme A desaturase 1 (SCD or SCD1) produces monounsaturated fatty acids by introducing double bonds into saturated bonds between carbons 9 and 10, with oleic acid as the main product. SCD1 is present in the intestinal epithelium, and fatty acids regulate cell proliferation, so we investigated the effects of SCD1-induced production of oleic acid in enterocytes in mice. We generated mice with disruption of Scd1 selectively in the intestinal epithelium (iScd1-/- mice) on a C57BL/6 background; iScd1+/+ mice were used as controls. We also generated iScd1-/-ApcMin/+ mice and studied cancer susceptibility. Mice were fed a chow, oleic acid-deficient, or oleic acid-rich diet. Intestinal tissues were collected and analyzed by histology, reverse transcription quantitative polymerase chain reaction, immunohistochemistry, and mass spectrometry, and tumors were quantified and measured. Compared with control mice, the ileal mucosa of iScd1-/- mice had a lower proportion of palmitoleic (C16:1 n-7) and oleic acids (C18:1 n-9), with accumulation of stearicacid (C18:0); this resulted a reduction of the Δ9 desaturation ratio between monounsaturated (C16:1 n-7 and C18:1 n-9) and saturated (C16:0 and C18:0) fatty acids. Ileal tissues from iScd1-/- mice had increased expression of markers of inflammation activation and crypt proliferative genes compared with control mice. The iScd1-/-ApcMin/+ mice developed more and larger tumors than iScd1+/+ApcMin/+ mice. iScd1-/-ApcMin/+ mice fed the oleic acid-rich diet had reduced intestinal inflammation and significantly lower tumor burden compared with mice fed a chow diet. In studies of mice,we found intestinal SCD1 to be required for synthesis of oleate in the enterocytes and maintenance of fatty acid homeostasis. Dietary supplementation with oleic acid reduces intestinal inflammation and tumor development in mice.

Trace elements in wild edible Aplysia species: Relationship with the desaturation–elongation indexes of fatty acids

The profile of essential and non-essential elements was traced in the edible sea hares Aplysia depilans Gmelin, Aplysia fasciata Poiret and Aplysia punctata Cuvier. Manganese (Mn), iron (Fe), zinc (Zn), copper (Cu) and selenium (Se) were identified as the major essential elements. Risk assessment evidenced that the levels of cadmium (Cd) and lead (Pb) did not exceed the maximum limit value established by the European Regulation, the contents of chromium (Cr), nickel (Ni) and arsenic (As) being also below the levels established by the FDA guide. A correlation between trace elements levels and desaturation–elongation indexes of fatty acids was found. While Cd, Se and molybdenum (Mo) seem to promote the desaturation–elongation process involved on the production of C20:4n-6c, Ni, Cr and Fe may potentiate the conversion of C18:3n-3c to C20:5n-3c. Furthermore, cobalt (Co), Ni and Cu appear to decrease Δ9 desaturation index. Besides the suggested biosynthetic switch modulated by trace elements, the nutritional value of the species is further strengthened.