Induction of the Staphylococcal Proteolytic Cascade by Antimicrobial Fatty Acids in Community Acquired Methicillin Resistant Staphylococcus aureus

Community acquired methicillin resistant Staphylococcus aureus (CA-MRSA), and the USA300 strain of CA-MRSA in particular, are known for their rapid community transmission, and propensity to cause aggressive skin and soft tissue infections. To assess factors that contribute to these hallmark traits of CA-MRSA, we evaluated how growth of USA300 and production of secreted virulence factors was influenced on exposure to physiologic levels of unsaturated free fatty acids that would be encountered on the skin or anterior nares, which represent the first sites of contact with healthy human hosts. There was a sharp threshold between sub-inhibitory and inhibitory concentrations, such that 100 µM sapienic acid (C16∶1) and linoleic acid (C18∶1) were sufficient to prevent growth after 24 h incubation, while 25 µM allowed unrestricted growth, and 50 µM caused an approximate 10–12 h lag, followed by unimpeded exponential growth. Conversely, saturated palmitic or stearic acids did not affect growth at 100 µM. Although growth was not affected by 25 µM sapienic or linoleic acid, these and other unsaturated C16 and C18 fatty acids, but not their saturated counterparts, promoted robust production of secreted proteases comprising the Staphylococcal proteolytic cascade. This trait was also manifested to varying degrees in other CA-MRSA, and in genetically diverse methicillin susceptible S. aureus strains. Therefore, induction of the Staphylococcal proteolytic cascade by unsaturated fatty acids is another feature that should now be evaluated as a potential contributing factor in the aggressive nature of skin and soft tissue infections caused by USA300, and as a general virulence mechanism of S. aureus.     

Fatty Acids of Extractable and Bound Lipids of Rhodomicrobium vannielii

Cells of Rhodomicrobium vannielii grown at 29 C in a lactate-containing medium were extracted at room temperature with organic solvents. The extractable fraction contained the bulk of the simple lipid (1.87% of cell dry weight) and complex lipids (phospholipids, 4.2%; sulfolipid, 0.01%), coenzyme Q (0.09%), and pigments (carotenoids 1.2%; bacteriochlorophyll, 1.9%). The cell residue contained the bound lipids (nonpolar fatty acid fraction, 1.86%; polar hydroxy fatty acids, 0.49%). The residue also contained poly-β-hydroxybutyric acid (0.2%), which was extracted in boiling chloroform. In both the simple and complex lipids, vaccenic acid (11-octadecenoic acid) was the largest single component (approximately 90% in each fraction). The fatty acids of the bound lipid contained 35% vaccenic acid, even- and odd-numbered saturated and unsaturated straight-chain fatty acids, cyclopropane-, branched-, and α- and β-hydroxy fatty acids. The extractable lipids contained only straight-chain saturated and unsaturated even-numbered fatty acids. Nearly 60% of hydroxy fatty acid fraction was α-hydroxydodecanoic acid (24%) and β-hydroxydodecanoic acid (34.5%). Coenzyme Q was crystallized and identified as Q₉ on the basis of melting point and chromatographic properties. Q₁₀ had been previously reported.     

Subcutaneous Adipose Fatty Acid Profiles and Related Rumen Bacterial Populations of Steers Fed Red Clover or Grass Hay Diets Containing Flax or Sunflower-Seed

Steers were fed 70∶30 forage∶concentrate diets for 205 days, with either grass hay (GH) or red clover silage (RC), and either sunflower-seed (SS) or flaxseed (FS), providing 5.4% oil in the diets. Compared to diets containing SS, FS diets had elevated (P<0.05) subcutaneous trans (t)-18:1 isomers, conjugated linoleic acids and n-6 polyunsaturated fatty acid (PUFA). Forage and oilseed type influenced total n-3 PUFA, especially α-linolenic acid (ALA) and total non-conjugated diene biohydrogenation (BH) in subcutaneous fat with proportions being greater (P<0.05) for FS or GH as compared to SS or RC. Of the 25 bacterial genera impacted by diet, 19 correlated with fatty acids (FA) profile. Clostridium were most abundant when levels of conjugated linolenic acids, and n-3 PUFA''s were found to be the lowest in subcutaneous fat, suggestive of their role in BH. Anerophaga, Fibrobacter, Guggenheimella, Paludibacter and Pseudozobellia were more abundant in the rumen when the levels of VA in subcutaneous fat were low. This study clearly shows the impact of oilseeds and forage source on the deposition of subcutaneous FA in beef cattle. Significant correlations between rumen bacterial genera and the levels of specific FA in subcutaneous fat maybe indicative of their role in determining the FA profile of adipose tissue. However, despite numerous correlations, the dynamics of rumen bacteria in the BH of unsaturated fatty acid and synthesis of PUFA and FA tissue profiles require further experimentation to determine if these correlations are consistent over a range of diets of differing composition. Present results demonstrate that in order to achieve targeted FA profiles in beef, a multifactorial approach will be required that takes into consideration not only the PUFA profile of the diet, but also the non-oil fraction of the diet, type and level of feed processing, and the role of rumen microbes in the BH of unsaturated fatty acid.     

Identification of Unusual Phospholipid Fatty Acyl Compositions of Acanthamoeba castellanii

Acanthamoeba are opportunistic protozoan pathogens that may lead to sight-threatening keratitis and fatal granulomatous encephalitis. The successful prognosis requires early diagnosis and differentiation of pathogenic Acanthamoeba followed by aggressive treatment regimen. The plasma membrane of Acanthamoeba consists of 25% phospholipids (PL). The presence of C20 and, recently reported, 28- and 30-carbon fatty acyl residues is characteristic of amoeba PL. A detailed knowledge about this unusual PL composition could help to differentiate Acanthamoeba from other parasites, e.g. bacteria and develop more efficient treatment strategies. Therefore, the detailed PL composition of Acanthamoeba castellanii was investigated by ³¹P nuclear magnetic resonance spectroscopy, thin-layer chromatography, gas chromatography, high performance liquid chromatography and liquid chromatography-mass spectrometry. Normal and reversed phase liquid chromatography coupled with mass spectrometric detection was used for detailed characterization of the fatty acyl composition of each detected PL. The most abundant fatty acyl residues in each PL class were octadecanoyl (18∶0), octadecenoyl (18∶1 Δ9) and hexadecanoyl (16∶0). However, some selected PLs contained also very long fatty acyl chains: the presence of 28- and 30-carbon fatty acyl residues was confirmed in phosphatidylethanolamine (PE), phosphatidylserine, phosphatidic acid and cardiolipin. The majority of these fatty acyl residues were also identified in PE that resulted in the following composition: 28∶1/20∶2, 30∶2/18∶1, 28∶0/20∶2, 30∶2/20∶4 and 30∶3/20∶3. The PL of amoebae are significantly different in comparison to other cells: we describe here for the first time unusual, very long chain fatty acids with Δ⁵-unsaturation (30∶3^5,21,24) and 30∶2^21,24 localized exclusively in specific phospholipid classes of A. castellanii protozoa that could serve as specific biomarkers for the presence of these microorganisms.     

Heterologous Production of Dihomo-γ-Linolenic Acid in Saccharomyces cerevisiae

To make dihomo-γ-linolenic acid (DGLA) (20:3n-6) in Saccharomyces cerevisiae, we introduced Kluyveromyces lactis Δ12 fatty acid desaturase, rat Δ6 fatty acid desaturase, and rat elongase genes. Because Fad2p is able to convert the endogenous oleic acid to linoleic acid, this allowed DGLA biosynthesis without the need to supply exogenous fatty acids on the media. Medium composition, cultivation temperature, and incubation time were examined to improve the yield of DGLA. Fatty acid content was increased by changing the medium from a standard synthetic dropout medium to a nitrogen-limited minimal medium (NSD). Production of DGLA was higher in the cells grown at 15°C than in those grown at 20°C, and no DGLA production was observed in the cells grown at 30°C. In NSD at 15°C, fatty acid content increased up until day 7 and decreased after day 10. When the cells were grown in NSD for 7 days at 15°C, the yield of DGLA reached 2.19 μg/mg of cells (dry weight) and the composition of DGLA to total fatty acids was 2.74%. To our knowledge, this is the first report describing the production of polyunsaturated fatty acids in S. cerevisiae without supplying the exogenous fatty acids.     

Inhibition of Candida parapsilosis Fatty Acid Synthase (Fas2) Induces Mitochondrial Cell Death in Serum

We have recently observed that a fatty acid auxotrophic mutant (fatty acid synthase, Fas2Δ/Δ) of the emerging human pathogenic yeast Candida parapsilosis dies after incubation in various media including serum. In the present study we describe the mechanism for cell death induced by serum and glucose containing media. We show that Fas2Δ/Δ yeast cells are profoundly susceptible to glucose leading us to propose that yeast cells lacking fatty acids exhibit uncontrolled metabolism in response to glucose. We demonstrate that incubation of Fas2Δ/Δ yeast cells with serum leads to cell death, and this process can be prevented with inhibition of protein or DNA synthesis, indicating that newly synthesized cellular components are detrimental to the mutant cells. Furthermore, we have found that cell death is mediated by mitochondria. Suppression of electron transport enzymes using inhibitors such as cyanide or azide prevents ROS overproduction and Fas2Δ/Δ yeast cell death. Additionally, deletion of mitochondrial DNA, which encodes several subunits for enzymes of the electron transport chain, significantly reduces serum-induced Fas2Δ/Δ yeast cell death. Therefore, our results show that serum and glucose media induce Fas2Δ/Δ yeast cell death by triggering unbalanced metabolism, which is regulated by mitochondria. To our knowledge, this is the first study to critically define a link between cytosolic fatty acid synthesis and mitochondrial function in response to serum stress in C. parapsilosis.     

Interactions of lipoidal materials and a pyridazinone inhibitor of chloroplast development

Formation of chloroplast pigments was inhibited, and free fatty acids accumulated in mustard (Brassica juncea [L.] Coss.) cotyledons and in barley (Hordeum vulgare L.) first leaves developed after treatment with 4-chloro-5- (dimethylamino)-2- (α, α, α-trifluoro-m-tolyl) -3 (2H) -pyridazinone. The inhibitor reduced the amount of fatty acids found in polar lipids (galactolipids) of barley chloroplasts and increased the amount in nonpolar lipids while having little effect on total content of bound fatty acids. The inhibition of chlorophyll formation was circumvented by D-α-tocopherol acetate, phytol, farnesol, and squalene, and by unsaturated fatty acids and their methyl esters. The protective action can be explained partially by an interaction external to the plant whereby 4-chloro-5- (dimethylamino) -2- (α, α, α-trifluoro-m-tolyl) -3 (2H) -pyridazinone partitioned out of the aqueous phase and into the lipid phase, thus limiting availability of the inhibitor to plants. However, the amount of inhibitor reaching the cotyledons of tocopherol-protected mustard seedlngs was still in excess of the amount necessary to cause white foliage, but it failed to produce the effect. Tocopherol treatment did not prevent the 4-chloro-5- (dimethylamino) -2- (α, α, α-trifluoro-m-tolyl) -3 (2H) -pyridazinone-induced buildup of fatty acids in mustard cotyledons but did partially circumvent the effect in barley leaves. The amount of linolenic acid relative to linoleic acid was reduced in barley leaves and chloroplasts by 4-chloro-5- (dimethylamino) -2- (α, α, α-trifluoro-m-tolyl) -3 (2H) -pyridazinone action and this effect was circumvented by tocopherol.     

Expression and Characterization of CYP52 Genes Involved in the Biosynthesis of Sophorolipid and Alkane Metabolism from Starmerella bombicola

Three cytochrome P450 monooxygenase CYP52 gene family members were isolated from the sophorolipid-producing yeast Starmerella bombicola (former Candida bombicola), namely, CYP52E3, CYP52M1, and CYP52N1, and their open reading frames were cloned into the pYES2 vector for expression in Saccharomyces cerevisiae. The functions of the recombinant proteins were analyzed with a variety of alkane and fatty acid substrates using microsome proteins or a whole-cell system. CYP52M1 was found to oxidize C₁₆ to C₂₀ fatty acids preferentially. It converted oleic acid (C_18:1) more efficiently than stearic acid (C_18:0) and linoleic acid (C_18:2) and much more effectively than α-linolenic acid (C_18:3). No products were detected when C₁₀ to C₁₂ fatty acids were used as the substrates. Moreover, CYP52M1 hydroxylated fatty acids at their ω- and ω-1 positions. CYP52N1 oxidized C₁₄ to C₂₀ saturated and unsaturated fatty acids and preferentially oxidized palmitic acid, oleic acid, and linoleic acid. It only catalyzed ω-hydroxylation of fatty acids. Minor ω-hydroxylation activity against myristic acid, palmitic acid, palmitoleic acid, and oleic acid was shown for CYP52E3. Furthermore, the three P450s were coassayed with glucosyltransferase UGTA1. UGTA1 glycosylated all hydroxyl fatty acids generated by CYP52E3, CYP52M1, and CYP52N1. The transformation efficiency of fatty acids into glucolipids by CYP52M1/UGTA1 was much higher than those by CYP52N1/UGTA1 and CYP52E3/UGTA1. Taken together, CYP52M1 is demonstrated to be involved in the biosynthesis of sophorolipid, whereas CYP52E3 and CYP52N1 might be involved in alkane metabolism in S. bombicola but downstream of the initial oxidation steps.     

Loss of FADS2 Function Severely Impairs the Use of HeLa Cells as an In Vitro Model for Host Response Studies Involving Fatty Acid Effects

Scope

Established epithelial cell lines equipped with pattern recognition receptors such as the Toll-like receptor (TLR)-2 are common tools for immune response studies on invading pathogens, e.g. the obligate intracellular species of Chlamydia. Moreover, such models are widely used to elucidate fatty acid-mediated immune effects. In several transformed cell lines, however, unusual loss of metabolic functions was described. The cell lines A549 and HeLa are poorly characterized in this respect. Therefore, we comparatively assessed the metabolic capacity of A549 and HeLa prior to proposed application as in vitro model for fatty acid effects on chlamydial infection.

Methodology/Principal Findings

We incubated both cell lines either with substrates (C18∶2n−6 or C18∶3n−3) or products (C18∶3n−6, C18∶4n−3) of fatty acid desaturase-2 (FADS2), and analysed the fatty acid profiles after 24 h and 72 h by gas chromatography. Based on these data, we suspected that the complete discontinuation of normal biosynthesis of long-chain polyunsaturated fatty acids (LC-PUFA) in HeLa was due to loss of FADS2 function. Consequently, prostaglandin E2 (PGE2) formation was less inducible by TLR2 stimulation in HeLa, likely as a result of not only insufficient supply of precursors but also weak cyclooxygenase-2 (COX-2) response. In accordance, Chlamydia infection rates were consistently lower in HeLa than in A549. Sequence analysis revealed no alteration within the FADS2 gene in HeLa. The FADS2 expression level, however, was significantly lower and, in contrast to A549, not regulated by C18∶2n−6. A549 exhibited regular fatty acid metabolism and enzyme functionality.

Conclusions/Significance

Our data show that HeLa cells considerably differ from A549 at several stages of fatty acid metabolism. The poor metabolic potential of HeLa, mainly concerning FADS2 upstream of COX-2 function, calls into question whether these cells represent a good model to unveil fatty acid or downstream eicosanoid effects in the course of intracellular bacterial infection.     

Selective Enrichment of Omega-3 Fatty Acids in Oils by Phospholipase A1

Omega fatty acids are recognized as key nutrients for healthier ageing. Lipases are used to release ω-3 fatty acids from oils for preparing enriched ω-3 fatty acid supplements. However, use of lipases in enrichment of ω-3 fatty acids is limited due to their insufficient specificity for ω-3 fatty acids. In this study use of phospholipase A1 (PLA1), which possesses both sn-1 specific activity on phospholipids and lipase activity, was explored for hydrolysis of ω-3 fatty acids from anchovy oil. Substrate specificity of PLA1 from Thermomyces lenuginosus was initially tested with synthetic p-nitrophenyl esters along with a lipase from Bacillus subtilis (BSL), as a lipase control. Gas chromatographic characterization of the hydrolysate obtained upon treatment of anchovy oil with these enzymes indicated a selective retention of ω-3 fatty acids in the triglyceride fraction by PLA1 and not by BSL. ¹³C NMR spectroscopy based position analysis of fatty acids in enzyme treated and untreated samples indicated that PLA1 preferably retained ω-3 fatty acids in oil, while saturated fatty acids were hydrolysed irrespective of their position. Hydrolysis of structured triglyceride,1,3-dioleoyl-2-palmitoylglycerol, suggested that both the enzymes hydrolyse the fatty acids at both the positions. The observed discrimination against ω-3 fatty acids by PLA1 appears to be due to its fatty acid selectivity rather than positional specificity. These studies suggest that PLA1 could be used as a potential enzyme for selective concentrationof ω-3 fatty acids.     

Metabolomic Analysis of Cold Acclimation of Arctic Mesorhizobium sp. Strain N33

Arctic Mesorhizobium sp. N₃₃ isolated from nodules of Oxytropis arctobia in Canada’s eastern Arctic has a growth temperature range from 0°C to 30°C and is a well-known cold-adapted rhizobia. The key molecular mechanisms underlying cold adaptation in Arctic rhizobia remains totally unknown. Since the concentration and contents of metabolites are closely related to stress adaptation, we applied GC-MS and NMR to identify and quantify fatty acids and water soluble compounds possibly related to low temperature acclimation in strain N₃₃. Bacterial cells were grown at three different growing temperatures (4°C, 10°C and 21°C). Cells from 21°C were also cold-exposed to 4°C for different times (2, 4, 8, 60 and 240 minutes). We identified that poly-unsaturated linoleic acids 18∶2 (9, 12) & 18∶2 (6, 9) were more abundant in cells growing at 4 or 10°C, than in cells cultivated at 21°C. The mono-unsaturated phospho/neutral fatty acids myristoleic acid 14∶1(11) were the most significantly overexpressed (45-fold) after 1hour of exposure to 4°C. As reported in the literature, these fatty acids play important roles in cold adaptability by supplying cell membrane fluidity, and by providing energy to cells. Analysis of water-soluble compounds revealed that isobutyrate, sarcosine, threonine and valine were more accumulated during exposure to 4°C. These metabolites might play a role in conferring cold acclimation to strain N₃₃ at 4°C, probably by acting as cryoprotectants. Isobutyrate was highly upregulated (19.4-fold) during growth at 4°C, thus suggesting that this compound is a precursor for the cold-regulated fatty acids modification to low temperature adaptation.     

Genome-Wide Association Analyses for Fatty Acid Composition in Porcine Muscle and Abdominal Fat Tissues

Fatty acid composition is an important phenotypic trait in pigs as it affects nutritional, technical and sensory quality of pork. Here, we reported a genome-wide association study (GWAS) for fatty acid composition in the longissimus muscle and abdominal fat tissues of 591 White Duroc×Erhualian F₂ animals and in muscle samples of 282 Chinese Sutai pigs. A total of 46 loci surpassing the suggestive significance level were identified on 15 pig chromosomes (SSC) for 12 fatty acids, revealing the complex genetic architecture of fatty acid composition in pigs. Of the 46 loci, 15 on SSC5, 7, 14 and 16 reached the genome-wide significance level. The two most significant SNPs were ss131535508 (P = 2.48×10⁻²⁵) at 41.39 Mb on SSC16 for C20∶0 in abdominal fat and ss478935891 (P = 3.29×10⁻¹³) at 121.31 Mb on SSC14 for muscle C18∶0. A meta-analysis of GWAS identified 4 novel loci and enhanced the association strength at 6 loci compared to those evidenced in a single population, suggesting the presence of common underlying variants. The longissimus muscle and abdominal fat showed consistent association profiles at most of the identified loci and distinct association signals at several loci. All loci have specific effects on fatty acid composition, except for two loci on SSC4 and SSC7 affecting multiple fatness traits. Several promising candidate genes were found in the neighboring regions of the lead SNPs at the genome-wide significant loci, such as SCD for C18∶0 and C16∶1 on SSC14 and ELOVL7 for C20∶0 on SSC16. The findings provide insights into the molecular basis of fatty acid composition in pigs, and would benefit the final identification of the underlying mutations.     

Fatty Acid and Sterol Composition of Mucor genevensis in Relation to Dimorphism and Anaerobic Growth

Fatty acid and sterol content and composition were determined for the dimorphic mold, Mucor genevensis, grown under a variety of experimental conditions. Fatty acids account for 6 to 9% of the dry weight of aerobically grown mycelium, and 70 to 80% of these are unsaturated. The organism contains γ-linolenic acid which is characteristic for Phycomycetes, and in sporangiospores this compound represents 40% of the total fatty acids. Of the sterols found in mycelium, 80% is ergosterol, and stigmasterol was positively identified as one of the minor components. In anaerobically grown yeastlike cells, sterol content is less than 10% of the level found in aerobically grown cells, and fatty acids amount to less than 2% of the dry weight. These fatty acids are predominantly short chain and less than 10% are unsaturated. Yeastlike cells obtained under aerobic conditions by growth in the presence of phenethyl alcohol have fatty acid and sterol compositions characteristic of aerobically grown mycelium. It is concluded that the dimorphology of the organism is not directly related to lipid composition.     

Fatty Acid Production from Amino Acids and α-Keto Acids by Brevibacterium linens BL2

Low concentrations of branched-chain fatty acids, such as isobutyric and isovaleric acids, develop during the ripening of hard cheeses and contribute to the beneficial flavor profile. Catabolism of amino acids, such as branched-chain amino acids, by bacteria via aminotransferase reactions and α-keto acids is one mechanism to generate these flavorful compounds; however, metabolism of α-keto acids to flavor-associated compounds is controversial. The objective of this study was to determine the ability of Brevibacterium linens BL2 to produce fatty acids from amino acids and α-keto acids and determine the occurrence of the likely genes in the draft genome sequence. BL2 catabolized amino acids to fatty acids only under carbohydrate starvation conditions. The primary fatty acid end products from leucine were isovaleric acid, acetic acid, and propionic acid. In contrast, logarithmic-phase cells of BL2 produced fatty acids from α-keto acids only. BL2 also converted α-keto acids to branched-chain fatty acids after carbohydrate starvation was achieved. At least 100 genes are potentially involved in five different metabolic pathways. The genome of B. linens ATCC 9174 contained these genes for production and degradation of fatty acids. These data indicate that brevibacteria have the ability to produce fatty acids from amino and α-keto acids and that carbon metabolism is important in regulating this event.     

A system for manipulating the membrane Fatty Acid composition of soybean cell cultures by adding tween-Fatty Acid esters to their growth medium : basic parameters and effects on cell growth

The development of a system for modifying the membrane fatty acid composition of cultured soybean cells (Glycine max [L.] Merr.) is described. Tween-fatty acid esters carrying specific fatty acids were synthesized and added to the medium of suspension cultures. Cells transferred large quantities of exogenous fatty acids from Tweens to all acylated membrane lipids; up to 50% of membrane fatty acids were exogenously derived. C15 to C20 saturated fatty acids and C16, C18, and C20 unsaturated fatty acids with either cis or trans double bonds were incorporated into lipids. Cells elongated saturated fatty acids of C16 or less, and unsaturated fatty acids with cis double bonds were further desaturated. No other types of modifications were observed. Growth ceased in cells treated with excessive concentrations of Tween-fatty acid esters, but frequently not for several days. Cessation of cell growth was correlated with changes in membrane fatty acid composition resulting from incorporation of large amounts of exogenous fatty acids into membrane lipids, although cells tolerated large variations in fatty acid composition. Maximum tolerable Tween concentrations varied widely according to the fatty acid supplied. Potential uses of this system and implications of the observed modifications on the pathway of incorporation are discussed.     

DNA Methylation in an Enhancer Region of the FADS Cluster Is Associated with FADS Activity in Human Liver

Levels of omega-6 (n-6) and omega-3 (n-3), long chain polyunsaturated fatty acids (LcPUFAs) such as arachidonic acid (AA; 20∶4, n-6), eicosapentaenoic acid (EPA; 20∶5, n-3) and docosahexaenoic acid (DHA; 22∶6, n-3) impact a wide range of biological activities, including immune signaling, inflammation, and brain development and function. Two desaturase steps (Δ6, encoded by FADS2 and Δ5, encoded by FADS1) are rate limiting in the conversion of dietary essential 18 carbon PUFAs (18C-PUFAs) such as LA (18∶2, n-6) to AA and α-linolenic acid (ALA, 18∶3, n-3) to EPA and DHA. GWAS and candidate gene studies have consistently identified genetic variants within FADS1 and FADS2 as determinants of desaturase efficiencies and levels of LcPUFAs in circulating, cellular and breast milk lipids. Importantly, these same variants are documented determinants of important cardiovascular disease risk factors (total, LDL, and HDL cholesterol, triglycerides, CRP and proinflammatory eicosanoids). FADS1 and FADS2 lie head-to-head (5′ to 5′) in a cluster configuration on chromosome 11 (11q12.2). There is considerable linkage disequilibrium (LD) in this region, where multiple SNPs display association with LcPUFA levels. For instance, rs174537, located ∼15 kb downstream of FADS1, is associated with both FADS1 desaturase activity and with circulating AA levels (p-value for AA levels = 5.95×10⁻⁴⁶) in humans. To determine if DNA methylation variation impacts FADS activities, we performed genome-wide allele-specific methylation (ASM) with rs174537 in 144 human liver samples. This approach identified highly significant ASM with CpG sites between FADS1 and FADS2 in a putative enhancer signature region, leading to the hypothesis that the phenotypic associations of rs174537 are likely due to methylation differences. In support of this hypothesis, methylation levels of the most significant probe were strongly associated with FADS1 and, to a lesser degree, FADS2 activities.     

Turnover of Fatty Acids during Natural Senescence of Arabidopsis, Brachypodium, and Switchgrass and in Arabidopsis β-Oxidation Mutants

During leaf senescence, macromolecule breakdown occurs and nutrients are translocated to support growth of new vegetative tissues, seeds, or other storage organs. In this study, we determined the fatty acid levels and profiles in Arabidopsis (Arabidopsis thaliana), Brachypodium distachyon, and switchgrass (Panicum virgatum) leaves during natural senescence. In young leaves, fatty acids represent 4% to 5% of dry weight and approximately 10% of the chemical energy content of the leaf tissues. In all three species, fatty acid levels in leaves began to decline at the onset of leaf senescence and progressively decreased as senescence advanced, resulting in a greater than 80% decline in fatty acids on a dry weight basis. During senescence, Arabidopsis leaves lost 1.6% of fatty acids per day at a rate of 2.1 μg per leaf (0.6 μg mg⁻¹ dry weight). Triacylglycerol levels remained less than 1% of total lipids at all stages. In contrast to glycerolipids, aliphatic surface waxes of Arabidopsis leaves were much more stable, showing only minor reduction during senescence. We also examined three Arabidopsis mutants, acx1acx2, lacs6lacs7, and kat2, which are blocked in enzyme activities of β-oxidation and are defective in lipid mobilization during seed germination. In each case, no major differences in the fatty acid contents of leaves were observed between these mutants and the wild type, indicating that several mutations in β-oxidation that cause reduced breakdown of reserve oil in seeds do not substantially reduce the degradation of fatty acids during leaf senescence.     

Circulating Docosahexaenoic Acid Levels Are Associated with Fetal Insulin Sensitivity

Background

Arachidonic acid (AA; C20∶4 n-6) and docosahexaenoic acid (DHA; C22∶6 n-3) are important long-chain polyunsaturated fatty acids (LC-PUFA) in maintaining pancreatic beta-cell structure and function. Newborns of gestational diabetic mothers are more susceptible to the development of type 2 diabetes in adulthood. It is not known whether low circulating AA or DHA is involved in perinatally “programming” this susceptibility. This study aimed to assess whether circulating concentrations of AA, DHA and other fatty acids are associated with fetal insulin sensitivity or beta-cell function, and whether low circulating concentrations of AA or DHA are involved in compromised fetal insulin sensitivity in gestational diabetic pregnancies.

Methods and Principal Findings

In a prospective singleton pregnancy cohort, maternal (32-35 weeks gestation) and cord plasma fatty acids were assessed in relation to surrogate indicators of fetal insulin sensitivity (cord plasma glucose-to-insulin ratio, proinsulin concentration) and beta-cell function (proinsulin-to-insulin ratio) in 108 mother-newborn pairs. Cord plasma DHA levels (in percentage of total fatty acids) were lower comparing newborns of gestational diabetic (n = 24) vs. non-diabetic pregnancies (2.9% vs. 3.5%, P = 0.01). Adjusting for gestational age at blood sampling, lower cord plasma DHA levels were associated with lower fetal insulin sensitivity (lower glucose-to-insulin ratio, r = 0.20, P = 0.036; higher proinsulin concentration, r = −0.37, P <0.0001). The associations remained after adjustment for maternal and newborn characteristics. Cord plasma saturated fatty acids C18∶0 and C20∶0 were negatively correlated with fetal insulin sensitivity, but their levels were not different between gestational diabetic and non-diabetic pregnancies. Cord plasma AA levels were not correlated with fetal insulin sensitivity.

Conclusion

Low circulating DHA levels are associated with compromised fetal insulin sensitivity, and may be involved in perinatally “programming” the susceptibility to type 2 diabetes in the offspring of gestational diabetic mothers.     

Mitochondrial 2,4-dienoyl-CoA Reductase Deficiency in Mice Results in Severe Hypoglycemia with Stress Intolerance and Unimpaired Ketogenesis

The mitochondrial β-oxidation system is one of the central metabolic pathways of energy metabolism in mammals. Enzyme defects in this pathway cause fatty acid oxidation disorders. To elucidate the role of 2,4-dienoyl-CoA reductase (DECR) as an auxiliary enzyme in the mitochondrial β-oxidation of unsaturated fatty acids, we created a DECR–deficient mouse line. In Decr^−/− mice, the mitochondrial β-oxidation of unsaturated fatty acids with double bonds is expected to halt at the level of trans-2, cis/trans-4-dienoyl-CoA intermediates. In line with this expectation, fasted Decr^−/− mice displayed increased serum acylcarnitines, especially decadienoylcarnitine, a product of the incomplete oxidation of linoleic acid (C_18:2), urinary excretion of unsaturated dicarboxylic acids, and hepatic steatosis, wherein unsaturated fatty acids accumulate in liver triacylglycerols. Metabolically challenged Decr^−/− mice turned on ketogenesis, but unexpectedly developed hypoglycemia. Induced expression of peroxisomal β-oxidation and microsomal ω-oxidation enzymes reflect the increased lipid load, whereas reduced mRNA levels of PGC-1α and CREB, as well as enzymes in the gluconeogenetic pathway, can contribute to stress-induced hypoglycemia. Furthermore, the thermogenic response was perturbed, as demonstrated by intolerance to acute cold exposure. This study highlights the necessity of DECR and the breakdown of unsaturated fatty acids in the transition of intermediary metabolism from the fed to the fasted state.