Bovine Brain Region‐Specific Stearoyl‐CoA Desaturase Expression and Fatty Acid Composition

In this study, we sought to determine the relationship between stearoyl‐CoA desaturase (SCD) gene isoform expression in the bovine brain and the accumulation of 18:1n‐9. Two SCD gene isoforms are found in cows—SCD1 and SCD5. Samples of six brain regions (cerebellum, frontal cortex, hippocampus, hypothalamus, midbrain, and pons) were collected from animals at four different ages (neonates, weanlings, yearlings, and adults) for mRNA isolation and fatty acid analysis. Expression of SCD1 and SCD5 mRNA was compared across age groups to determine its developmental regulation. Fatty acid composition and SCD isoform mRNA expression were compared to examine the correlation of SCD1 and SCD5 with 18:1n‐9 content in different brain regions. We found statistically significant correlations between SCD1 and SCD5 mRNA expression and the ratio of 18:1n‐9 to 18:0 across age groups, with stronger correlations observed for SCD5. Similarly, there was a significant correlation between the ratio of 18:1n‐9 to 18:0 and SCD5 mRNA expression across brain regions. SCD1 mRNA and the 18:1n‐9 to 18:0 ratio were negatively correlated in the hippocampus. There was no trend of increasing 18:1n‐9 content or SCD expression with age. Correlations indicated a stronger relationship between SCD5 mRNA expression and the 18:1n‐9 to 18:0 ratio, potentially indicating a strong contribution of the SCD5 isoform to brain 18:1n‐9 content. This is the first study examining a potential role for SCD5 in providing 18:1n‐9 for brain lipids.     

Fibroblast Growth Factor-21 and the Beneficial Effects of Long-Chain n-3 Polyunsaturated Fatty Acids

Long‐chain n‐3 polyunsaturated fatty acids (LC n‐3 PUFA) in the diet protect against insulin resistance and obesity. Fibroblast growth factor‐21 (Fgf21) is a hormonal factor released mainly by the liver that has powerful anti‐diabetic effects. Here, we tested whether the beneficial metabolic effects of LC n‐3 PUFA involve the induction of Fgf21. C57BL/6 J mice were exposed to an obesogenic, corn‐oil‐based, high‐fat diet (cHF), or a diet in which corn oil was replaced with a fish‐derived LC n‐3 PUFA concentrate (cHF + F) using two experimental settings: short‐term (3 weeks) and long‐term treatment (8 weeks). CHF + F reduced body weight gain, insulinemia, and triglyceridemia compared to cHF. cHF increased plasma Fgf21 levels and hepatic Fgf21 gene expression compared with controls, but these effects were less pronounced or absent in cHF + F‐fed mice. In contrast, hepatic expression of peroxisome proliferator‐activated receptor (PPAR)‐α target genes were more strongly induced by cHF + F than cHF, especially in the short‐term treatment setting. The expression of genes encoding Fgf21, its receptors, and Fgf21 targets was unaltered by short‐term LC n‐3 PUFA treatment, with the exception of Ucp1 (uncoupling protein 1) and adiponectin genes, which were specifically up‐regulated in white fat. In the long‐term treatment setting, the expression of Fgf21 target genes and receptors was not differentially affected by LC n‐3 PUFA. Collectively, our findings indicate that increased Fgf21 levels do not appear to be a major mechanism through which LC n‐3 PUFA ameliorates high‐fat‐diet‐associated metabolic disorders.     

Efficacy of Polyunsaturated Fatty Acids (PUFAs) on Impulsive Behaviours and Aggressiveness in Psychiatric Disorders

It is the focus of increasing interest to investigate the effects of long-chain n-3 and long-chain n-6 polyunsaturated fatty acids (LC n-3 PUFAs; LC n-6 PUFAs) on psychiatric symptoms in a transdiagnostic perspective. There is some evidence that low levels of LC n-3 PUFAs and a higher ratio of LC n-6 to LC n-3 PUFAs in plasma and blood cells are associated with aggressive and impulsive behaviours. Therefore, implementation of LC n-3 PUFAs may produce positive effects on hostility, aggression, and impulsivity in both psychiatric and non-psychiatric samples across different stages of life. A possible mechanism of action of LC n-3 PUFAs in conditions characterized by a high level of impulsivity and aggression is due to the effect of these compounds on the serotonin system and membrane stability. Studies that evaluated the effects of LC n-3 PUFAs on impulsivity and aggressiveness indicated that addition of rather low doses of these agents to antipsychotic treatment might reduce agitation and violent behaviours in psychosis, attention deficit hyperactivity disorder, personality disorders, and impulsive control and conduct disorders. The present review is aimed at examining and discussing available data from recent trials on this topic.     

The Influence of Polyunsaturated Fatty Acids on the Phospholipase D Isoforms Trafficking and Activity in Mast Cells

The impact of polyunsaturated fatty acid (PUFA) supplementation on phospholipase D (PLD) trafficking and activity in mast cells was investigated. The enrichment of mast cells with different PUFA including α-linolenic acid (LNA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), linoleic acid (LA) or arachidonic acid (AA) revealed a PUFA-mediated modulation of the mastoparan-stimulated PLD trafficking and activity. All PUFA examined, except AA, prevented the migration of the PLD1 to the plasma membrane. For PLD2 no PUFA effects on trafficking could be observed. Moreover, PUFA supplementation resulted in an increase of mastoparan-stimulated total PLD activity, which correlated with the number of double bonds of the supplemented fatty acids. To investigate, which PLD isoform was affected by PUFA, stimulated mast cells were supplemented with DHA or AA in the presence of specific PLD-isoform inhibitors. It was found that both DHA and AA diminished the inhibition of PLD activity in the presence of a PLD1 inhibitor. By contrast, only AA diminished the inhibition of PLD activity in the presence of a PLD2 inhibitor. Thus, PUFA modulate the trafficking and activity of PLD isoforms in mast cells differently. This may, in part, account for the immunomodulatory effect of unsaturated fatty acids and contributes to our understanding of the modulation of mast cell activity by PUFA.     

Essential Polyunsaturated Fatty Acids in Blood from Patients with and without Catheter-Proven Coronary Artery Disease

Coronary artery disease (CAD) is the leading cause of death worldwide. Statins reduce morbidity and mortality of CAD. Intake of n-3 polyunsaturated fatty acid (n-3 PUFAs), particularly eicosapentaenoic acid (EPA), is associated with reduced morbidity and mortality in patients with CAD. Previous data indicate that a higher conversion of precursor fatty acids (FAs) to arachidonic acid (AA) is associated with increased CAD prevalence. Our study explored the FA composition in blood to assess n-3 PUFA levels from patients with and without CAD. We analyzed blood samples from 273 patients undergoing cardiac catheterization. Patients were stratified according to clinically relevant CAD (n = 192) and those without (n = 81). FA analysis in full blood was performed by gas chromatography. Indicating increased formation of AA from precursors, the ratio of dihomo-gamma-linolenic acid (DGLA) to AA, the delta-5 desaturase index (D5D index) was higher in CAD patients. CAD patients had significantly lower levels of omega-6 polyunsaturated FAs (n-6 PUFA) and n-3 PUFA, particularly EPA, in the blood. Thus, our study supports a role of increased EPA levels for cardioprotection.     

Determination of Substrate Preferences for Desaturases and Elongases for Production of Docosahexaenoic Acid from Oleic Acid in Engineered Canola

Production of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in plant seed oils has been pursued to improve availability of these omega‐3 fatty acids that provide important human health benefits. Canola (Brassica napus), through the introduction of 10 enzymes, can convert oleic acid (OLA) into EPA and ultimately DHA through a pathway consisting of two elongation and five desaturation steps. Herein we present an assessment of the substrate specificity of the seven desaturases and three elongases that were introduced into canola by expressing individual proteins in yeast. In vivo feeding experiments were conducted with 14 potential fatty acid intermediates in an OLA to DHA pathway to determine the fatty acid substrate profiles for each enzyme. Membrane fractions were prepared from yeast expression strains and shown to contain active enzymes. The elongases, as expected, extended acyl‐CoA substrates in the presence of malonyl‐CoA. To distinguish between enzymes that desaturate CoA‐ and phosphatidylcholine‐linked fatty acid substrates, we developed a novel in vitro method. We show that a delta‐12 desaturase from Phytophthora sojae, an omega‐3 desaturase from Phytophthora infestans and a delta‐4 desaturase from Thraustochytrium sp., all prefer phosphatidylcholine‐linked acyl substrates with comparatively low use of acyl‐CoA substrates. To further validate our method, a delta‐9 desaturase from Saccharomyces cerevisiae was confirmed to use acyl‐CoA as substrate, but could not use phosphatidylcholine‐linked substrates. The results and the assay methods presented herein will be useful in efforts to improve modeling of fatty acid metabolism and production of EPA and DHA in plants.     

Dietary Fish Oil Alters DNA Methylation of Genes Involved in Polyunsaturated Fatty Acid Biosynthesis in Muscle and Liver of Atlantic Salmon (Salmo salar)

Adequate dietary supply of eicosapentaenoic acid (20:5n-3) and docosahexaenoic acid (22:6n-3) is required to maintain health and growth of Atlantic salmon (Salmo salar). However, salmon can also convert α-linolenic acid (18:3n-3) into eicosapentaenoic acid (20:5n-3) and docosahexaenoic acid (22:6n-3) by sequential desaturation and elongation reactions, which can be modified by 20:5n-3 and 22:6n-3 intake. In mammals, dietary 20:5n-3 + 22:6n-3 intake can modify Fads2 expression (Δ6 desaturase) via altered DNA methylation of its promoter. Decreasing dietary fish oil (FO) has been shown to increase Δ5fad expression in salmon liver. However, it is not known whether this is associated with changes in the DNA methylation of genes involved in polyunsaturated fatty acid synthesis. To address this, we investigated whether changing the proportions of dietary FO and vegetable oil altered the DNA methylation of Δ6fad_b, Δ5fad, Elovl2, and Elovl5_b promoters in liver and muscle from Atlantic salmon and whether any changes were associated with mRNA expression. Higher dietary FO content increased the proportions of 20:5n-3 and 22:6n-3 and decreased Δ6fad_b mRNA expression in liver, but there was no effect on Δ5fad, Elovl2, and Elovl5_b expression. There were significant differences between liver and skeletal muscle in the methylation of individual CpG loci in all four genes studied. Methylation of individual Δ6fad_b CpG loci was negatively related to its expression and to proportions of 20:5n-3 and 22:6n-3 in the liver. These findings suggest variations in dietary FO can induce gene-, CpG locus-, and tissue-related changes in DNA methylation in salmon.     

Divergent Response of Murine and Porcine Adipocytes to Stimulation of Browning Genes by 18‐Carbon Polyunsaturated Fatty Acids and Beta‐Receptor Agonists

Long‐chain fatty acids (LCFA) are known to activate brown and beige adipocytes. However, very little is known about the effects of the number and the position of double bonds in LCFA with the same length on brown fat‐specific gene expression. To determine the specificity of LCFA in the regulation of these genes in different adipocyte models, fully differentiated 10T1/2, 3T3‐L1, murine, or porcine primary adipocytes (obtained from the subcutaneous fat pad of C57BL/6 mice or Landrace × Yorkshire × Duroc crossbred piglets) were treated with 50 μM of the following 18‐carbon fatty acids: stearic acid (STA; 18:0), oleic acid (OLA; 18:1, Δ9), linoleic acid (LNA; 18:2, Δ9,12), α‐linolenic acid (ALA; 18:3, Δ9,12,15), γ‐linolenic acid (GLA; 18:3, Δ6,9,12), or pinolenic acid (PLA; 18:3, Δ5,9,12) for 24 h with or without 4‐h norepinephrine (NE) treatment. Expression levels of thermoregulatory markers were measured by quantitative real‐time PCR. LNA, ALA, GLA, and PLA upregulated Ucp1 expression and tended to upregulate Pgc1a expression in murine primary adipocytes, but not in 10T1/2, 3T3‐L1, and porcine primary adipocytes. In murine primary adipocytes, NE induced a higher expression of Ucp1 and Pgc1a than non‐NE‐treated cells, and PLA augmented the NE effect. In 10T1/2 cells, NE upregulated Ucp1 and Pgc1a expression, but there was no fatty acid effect. However, 3T3‐L1 cells were insensitive to both fatty acid and beta‐adrenergic agonist stimulation. These results indicate that different adipocyte cell types have different levels of sensitivity to both LCFA and beta agonists in regard to induction of brown fat‐specific gene expression.     

Biosynthesis of Essential Polyunsaturated Fatty Acids in Wheat Triggered by Expression of Artificial Gene

The artificial gene D6D encoding the enzyme ∆⁶desaturase was designed and synthesized using the sequence of the same gene from the fungus Thamnidium elegans. The original start codon was replaced by the signal sequence derived from the wheat gene for high-molecular-weight glutenin subunit and the codon usage was completely changed for optimal expression in wheat. Synthesized artificial D6D gene was delivered into plants of the spring wheat line CY-45 and the gene itself, as well as transcribed D6D mRNA were confirmed in plants of T₀ and T₁ generations. The desired product of the wheat genetic modification by artificial D6D gene was the γ-linolenic acid. Its presence was confirmed in mature grains of transgenic wheat plants in the amount 0.04%–0.32% (v/v) of the total amount of fatty acids. Both newly synthesized γ-linolenic acid and stearidonic acid have been detected also in leaves, stems, roots, awns, paleas, rachillas, and immature grains of the T₁ generation as well as in immature and mature grains of the T₂ generation. Contents of γ-linolenic acid and stearidonic acid varied in range 0%–1.40% (v/v) and 0%–1.53% (v/v) from the total amount of fatty acids, respectively. This approach has opened the pathway of desaturation of fatty acids and production of essential polyunsaturated fatty acids in wheat.     

Inhibition of Polyunsaturated Fatty Acids Synthesis Decreases Growth Rate and Membrane Fluidity of <Emphasis Type="Italic">Rhodosporidium kratochvilovae</Emphasis> at Low Temperature

The intention of this study was to investigate the role of polyunsaturated fatty acids (PUFA) in the cold adaptation of Rhodosporidium kratochvilovae YM25235 by knockout of the Δ¹²/Δ¹⁵-fatty acid desaturase gene (RKD12) to inactivate Δ¹²/Δ¹⁵-fatty acid desaturase. Polymerase chain reaction (PCR) amplification was used to detect the genomic structure of RKD12 gene in YM25235. The RKD12 gene was knocked out by DNA homologous recombination to inhibit the biosynthesis of PUFA. Then, the contents of linoleic acid (LNA) and α-linolenic acid (ALA) after gene knockout were investigated using a gas chromatography-mass spectrometer, followed by determination of the growth rate and membrane fluidity of YM25235 at low temperature. After PCR amplification, a 1611 bp genomic fragment was amplified from YM25235. When the RKD12 gene was knocked out, the contents of LNA and ALA in YM25235 significantly decreased. The growth rate and membrane fluidity of YM25235 decreased significantly at low temperature. Inhibition of PUFA biosynthesis by RKD12 gene knockout influenced cold adaptation of YM25235 by decreasing the PUFA content in cell membranes and reducing the growth rate and membrane fluidity of YM25235 at low temperature.     

Elongation of very Long-Chain (>C<Subscript>24</Subscript>) Fatty Acids in <Emphasis Type="Italic">Clarias gariepinus</Emphasis>: Cloning,Functional Characterization and Tissue Expression of <Emphasis Type="Italic">elovl4</Emphasis> Elongases

Elongation of very long‐chain fatty acid 4 (Elovl4) proteins participate in the biosynthesis of very long‐chain (>C₂₄) saturated and polyunsaturated fatty acids (FA). Previous studies have shown that fish possess two different forms of Elovl4, termed Elovl4a and Elovl4b. The present study aimed to characterize both molecularly and functionally two elovl4 cDNA from the African catfish Clarias gariepinus. The results confirmed that C. gariepinus possessed two elovl4‐like elongases with high homology to two previously characterized Elovl4 from Danio rerio, and thus they were termed accordingly as Elovl4a and Elovl4b. The C. gariepinus Elovl4a and Elovl4b have open reading frames (ORF) of 945 and 915 base pairs, respectively, encoding putative proteins of 314 and 304 amino acids, respectively. Functional characterization in yeast showed both Elovl4 enzymes have activity towards all the PUFA substrates assayed (18:4n‐3, 18:3n‐6, 20:5n‐3, 20:4n‐6, 22:5n‐3, 22:4n‐6 and 22:6n‐3), producing elongated products of up to C₃₆. Moreover, the C. gariepinus Elovl4a and Elovl4b were able to elongate very long‐chain saturated FA (VLC‐SFA) as denoted by increased levels of 28:0 and longer FA in yeast transformed with elovl4 ORF compared to control yeast. These results confirmed that C. gariepinus Elovl4 play important roles in the biosynthesis of very long‐chain FA. Tissue distribution analysis of elovl4 mRNAs showed both genes were widely expressed in all tissues analyzed, with high expression of elovl4a in pituitary and brain, whereas female gonad and pituitary had the highest expression levels for elovl4b.     

Identification of n‐6 Monounsaturated Fatty Acids in Acer Seed Oils

Seed oils from Acer species are a potential source of the nutraceutical fatty acids, nervonic acid (cis‐15‐tetracosenoic acid, NA), and γ‐linolenic acid (cis‐6,9,12‐octadecatrienoic acid, GLA). To further characterize the genus, seed fatty acid content and composition were determined for 20 species of Acer. Fatty acid content ranged from 8.2% for Acer macrophyllum to over 36% for A. mono and A. negundo. The presence of very‐long‐chain fatty acids (VLCFA), with chain length of 20‐carbons or greater, and GLA were characteristic features of the seed oils. In all species, erucic acid (cis‐13‐docosenoic acid, EA) was the predominant VLCFA with the highest level of NA being only 8.6% in A. olivianum. Regioselective lipase digestion demonstrated that VLCFA are largely absent from the sn‐2 position of seed triacylglycerol, whereas GLA is primarily located at this position. Five Acer species contained low levels (<2%) of cis‐12‐octadecenoic acid and cis‐14‐eicosenoic acid, uncommon n‐6 fatty acids not previously reported from Acer.     

Mono‐Estolide Synthesis from trans‐8‐Hydroxy‐Fatty Acids by Lipases in Solvent‐Free Media and Their Physical Properties

Pseudomonas aeruginosa 42A2 is known to produce two hydroxy‐fatty acids, 10(S)‐hydroxy‐8(E)‐octadecenoic and 7,10(S,S)‐dihydroxy‐8(E)‐octadecenoic acids, when cultivated in a mineral medium using oleic acid as a single carbon source. These compounds were purified, 91 and 96 % respectively, to produce two new families of estolides: trans‐8‐estolides and saturated estolides from the monohydroxylated monomer. trans‐8‐estolides were produced by three different lipases (Novozym 435, Lipozyme RM IM and Lipozyme TL IM) with reaction yields between 68.4 ± 2.1 and 94.7 ± 2.4 % in a solvent‐free medium at 80 °C in 168 h under vacuum. Novozym 435 was found to be the most efficient biocatalyst for both hydroxy‐fatty acids with reaction yields of 71.7 ± 2.3 and 94.7 ± 2.4 %, respectively. Moreover, saturated estolides were also produced from a saturated 10(S)‐hydroxy‐8(E)‐octadecenoic. These estolides were chemically and enzymatically synthesized with Novozym 435, under the previous described reaction conditions with yields of 60.7 ± 2.1 and 71.2 ± 2.3 % respectively. Finally, viscosity, glass transition temperature, decomposition temperatures and enthalpies were determined to characterize both types of estolides. Thermal applications for both types of polyesters were improved since glass transition temperatures were lowered and decomposition temperatures were increased, with respect to their corresponding substrates.     

Dietary Unsaturated Fatty Acids Modulate Maternal Dyslipidemia‐Induced DNA Methylation and Histone Acetylation in Placenta and Fetal Liver in Rats

The present study assessed the role of dietary unsaturated fatty acids in maternal dyslipidemia‐induced DNA methylation and histone acetylation in placenta and fetal liver and accumulation of lipids in the fetal liver. Weanling female Wistar rats were fed control and experimental diets for 2 months, mated, and continued on their diets during pregnancy. At gestation days of 18–20, rats were euthanized to isolate placenta and fetal liver. DNA methylation, DNA methyl transferase‐1 (DNMT1) activity, acetylation of histones (H2A and H2B), and histone acyl transferase (HAT) activity were evaluated in placenta and fetal liver. Fetal liver lipid accumulation and activation of peroxisome proliferator‐activated receptor‐α (PPAR‐α) were assessed. Maternal dyslipidemia caused significant epigenetic changes in placenta and fetal liver. In the placenta, (1) global DNA methylation increased by 37% and DNMT1 activity by 86%, (2) acetylated H2A and H2B levels decreased by 46% and 24% respectively, and (3) HAT activity decreased by 39%. In fetal liver, (1) global DNA methylation increased by 52% and DNMT1 activity by 78%, (2) acetylated H2A and H2B levels decreased by 28% and 26% respectively, and (3) HAT activity decreased by 37%. Maternal dyslipidemia caused a 4.75‐fold increase in fetal liver triacylglycerol accumulation with a 78% decrease in DNA‐binding ability of PPAR‐α. Incorporation of dietary unsaturated fatty acids in the maternal high‐fat diet significantly (p < 0.05) modulated dyslipidemia‐induced effects in placenta and fetal liver. Eicosapentaenoic acid (EPA, 20:5n‐3) + docosahexaenoic acid (DHA, 22:6n‐3) exhibited a profound effect followed by alpha‐linolenic acid (ALA, 18:3n‐3) than linoleic acid (LNA, 18:2n‐6) in modulating the epigenetic parameters in placenta and fetal liver.     

Matrix Extension Validation of AOCS Ce 2c-11 for Omega-3 Polyunsaturated Fatty Acids in Conventional Foods and Dietary Supplements Containing Added Marine Oil

Marine oils are commonly added to conventional foods and dietary supplements to enhance their contents of omega-3 polyunsaturated fatty acids (PUFA), including eicosapentaenoic acid (EPA; 20:5n-3) and docosahexaenoic acid (DHA; 22:6n-3), which have been associated with numerous potential health benefits. This study compared American Oil Chemists’ Society (AOCS) Official Methods Ce 2b-11 and Ce 2c-11 for determining EPA and DHA in foods and dietary supplements and found that AOCS Ce 2c-11 produces significantly higher analyzed values, which could be attributed to a more comprehensive breakdown of the sample matrix and derivatization of fatty acids. Our subsequent food matrix extension validation of AOCS Ce 2c-11 demonstrated that the method produces true, accurate, sensitive, and precise determinations of EPA, DHA, and total omega-3 PUFA in foods and dietary supplements containing added marine oil, including those formulated with emulsified and microencapsulated oils. The method detection limits for EPA and DHA were 0.012 ± 0.002 and 0.011 ± 0.003 mg g⁻¹, respectively (means ± SD). The analyzed contents of EPA (1.26–386 mg serving⁻¹), DHA (1.37–563 mg serving⁻¹), and total omega-3 PUFA (2.69–1270 mg serving⁻¹) were reported for 27 conventional food and dietary supplement products. Eighteen products declared contents of DHA, EPA + DHA, or total omega-3 PUFA on product labels, and the analyzed contents of those fatty acids varied from 95 to 162% of label declarations for all but two of the products.