Are Fatty Acid Patterns Characteristic of Essential Fatty Acid Deficiency Indicative of Oxidative Stress?

Several unrelated diseases show plasma and tissue fatty acid patterns characteristic of those seen in Essential Fatty Acid Deficiency Disease (EFADD). A common feature occurring in all these diseases is oxidative stress. We hypothesize that reactive oxygen species or products of oxidative damage, particularly those derived from lipids, act as signal molecules to alter desaturase enzymes and induce the fatty acid patterns characteristic of EFADD.     

Effects of ω3 Polyunsaturated Fatty Acids on Growth and Tissue Fatty Acid Compositions of Maternal Rats and Their Progeny

The effects of ω3 polyunsaturated fatty acids [PUFA; mainly eicosapentaenoic acid (20: 5, ω3) and docosahexaenoic acid (22:6, ω3)] on the growth, tissue weights and fatty acid compositions of tissue total lipids in female rats and their progeny were investigated. Female rats of the Wistar strain, weighing 77~94g, were fed a 25% casein diet containing 5% of either corn oil (control), sardine oil or PUFA ethyl ester for 8 ~ 9 weeks prior to mating, and during gestation and lactation, and then for a further 2 weeks. The progeny were weaned to the maternal diet and then the latter was administered for a further 2 weeks. Dietary changes in the body weights of the dams were not generally seen, but the body weights at birth and growth of the offspring from the females supplied with the PUFA diet were inferior compared to those of the other groups. The fertility did not differ among the dietary groups. The weights of several tissues in the dams and the progeny increased in proportion to their body weights but not that of the progeny brain, which remained ' almost unchanged by the dietary fats. As to the fatty acid compositions of total lipids in the tissues, on the whole, decreased levels of ωβ fatty acids and increased percentages of ω3 fatty acids were found in the sardine and PUFA groups, the changes being greater in the PUFA group than in the sardine one. Such findings due to the feeding of PUFA were more remarkable in the progeny compared with in the dams. Eicosatrienoic acid (20: 3, ω9) was almost completely undetectable in the tissue total lipids of all the dietary groups.     

Structural and Functional Analysis of E. coli Cyclopropane Fatty Acid Synthase

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Isolation of a New Intermediary Substance Consisting of Colistin Fatty Acid and l-α,δ-Diaminobutyric Acid from Colistin-producing Cells of Bacillus colistinus Koyama

Several saccharides were found to be significantly effective in providing protection against hydrostatic pressure and high temperature damage in the yeast Saccharomyces cerevisiae. The extent of barotolerance and thermotolerance with seven different sugars showed a linear relationship to their mean number of equatorial OH groups. The same linear relatioship is seen when sugars protect protein molecules against elevated temperatures in vitro. Some sugars were more effective in providing protection against hydrostatic pressure nearly a hundred times than high temperature. Pre-heat shock treatment on yeast cells induce various stress tolerances. In this report, pre-heat shocked cells showed potent protection against elevated temperature, but these cells showed faint protection against elevated pressure.

These results suggest that sugars may protect cells against hydrostatic pressure and high temperature in a similar manner, probably by stabilizing the macromolecule(s), and such type of protection may be suited for pressure stress.     

Diversity of Δ12 Fatty Acid Desaturases in Santalaceae and Their Role in Production of Seed Oil Acetylenic Fatty Acids

Plants in the Santalaceae family, including the native cherry Exocarpos cupressiformis and sweet quandong Santalum acuminatum, accumulate ximenynic acid (trans-11-octadecen-9-ynoic acid) in their seed oil and conjugated polyacetylenic fatty acids in root tissue. Twelve full-length genes coding for microsomal Δ12 fatty acid desaturases (FADs) from the two Santalaceae species were identified by degenerate PCR. Phylogenetic analysis of the predicted amino acid sequences placed five Santalaceae FADs with Δ12 FADs, which include Arabidopsis thaliana FAD2. When expressed in yeast, the major activity of these genes was Δ12 desaturation of oleic acid, but unusual activities were also observed: i.e. Δ15 desaturation of linoleic acid as well as trans-Δ12 and trans-Δ11 desaturations of stearolic acid (9-octadecynoic acid). The trans-12-octadecen-9-ynoic acid product was also detected in quandong seed oil. The two other FAD groups (FADX and FADY) were present in both species; in a phylogenetic tree of microsomal FAD enzymes, FADX and FADY formed a unique clade, suggesting that are highly divergent. The FADX group enzymes had no detectable Δ12 FAD activity but instead catalyzed cis-Δ13 desaturation of stearolic acid when expressed in yeast. No products were detected for the FADY group when expressed recombinantly. Quantitative PCR analysis showed that the FADY genes were expressed in leaf rather than developing seed of the native cherry. FADs with promiscuous and unique activities have been identified in Santalaceae and explain the origin of some of the unusual lipids found in this plant family.     

Substrate Specificity and Regioselectivity of Δ12 and ω3 Fatty Acid Desaturases from Saccharomyces kluyveri

Δ12 and ω3 fatty acid desaturases are key enzymes in the synthesis of polyunsaturated fatty acids (PUFAs), which are important constituents of membrane glycerolipids and also precursors to signaling molecules in many organisms. In this study, we determined the substrate specificity and regioselectivity of the Δ12 and ω3 fatty acid desaturases from Saccharomyces kluyveri (Sk-FAD2 and Sk-FAD3). Based on heterologous expression in Saccharomyces cerevisiae, it was found that Sk-FAD2 converted C16–20 monounsaturated fatty acids to diunsaturated fatty acids by the introduction of a second double bond at the ν+3 position, while Sk-FAD3 recognized the ω3 position of C18 and C20. Furthermore, fatty acid analysis of major phospholipids suggested that Sk-FAD2 and Sk-FAD3 have no strong substrate specificity toward the lipid polar head group or the sn-positions of fatty acyl groups in phospholipids.     

Fatty Acid Composition of Frontal,Temporal and Parietal Neocortex in the Normal Human Brain and in Alzheimer’s Disease

Dietary ω3-polyunsaturated fatty acids are thought to influence the risk of Alzheimer’s disease (AD), and supplemental docosahexaenoic acid (DHA; 22:6n-3) has been reported to reduce neurodegeneration in mouse models of AD. We have analysed the fatty acid composition of frontal, temporal and parietal neocortex in 58 normal and 114 AD brains. Significant reductions were found for stearic acid (18:0) in frontal and temporal cortex and arachidonic acid (20:4n-6) in temporal cortex in AD, and increases in oleic acid in frontal and temporal cortex (18:1n-9) and palmitic acid (16:0) in parietal cortex. DHA level varied more in AD than controls but the mean values were not significantly different. Fatty acid composition was not related to APOE genotype, age, gender or post-mortem delay. Further research is needed to distinguish between alterations that are secondary to AD and those that contribute to the disease process.     

Inflammation and Resolution Are Associated with Upregulation of Fatty Acid β-Oxidation in Zymosan-Induced Peritonitis

Inflammation is a fundamental defensive response to harmful stimuli. However, it can cause damage if it does not subside. To avoid such damage, organisms have developed a mechanism called resolution of inflammation. Here we applied an untargeted metabolomics approach to a sterile and self-resolving animal model of acute inflammation, namely zymosan-induced peritonitis in mice, to examine the effect of inflammation and resolution on the metabolomic profiles. Significant and time-dependent changes in metabolite profiles after zymosan administration were observed in both peritoneal wash fluid (PWF) and plasma. These metabolomic changes correlated well with inflammatory chemokine or cytokine production. In PWF, most of metabolites that could detected increased in zymosan-treated mice, which is suggestive of inflammation, oxidative stress and increased energy demands. In plasma, most metabolites in the central metabolic pathway (glycolysis and TCA cycle) were significantly downregulated after zymosan administration. The concentration of the ketone body 3-hydroxybutyric acid (3-HB) in plasma and PWF increased in zymosan-injected animals indicating upregulation of fatty acid β-oxidation. Increased 3-HB level was observed in the cells that infiltrated into the peritoneal cavity and these infiltrated cells might contribute, at least in part, to the production of 3-HB in the peritoneal cavity.     

Bifidobacterium breve with α-Linolenic Acid and Linoleic Acid Alters Fatty Acid Metabolism in the Maternal Separation Model of Irritable Bowel Syndrome

The aim of this study was to compare the impact of dietary supplementation with a Bifidobacterium breve strain together with linoleic acid & α-linolenic acid, for 7 weeks, on colonic sensitivity and fatty acid metabolism in rats. Maternally separated and non-maternally separated Sprague Dawley rats (n = 15) were orally gavaged with either B. breve DPC6330 (10⁹ microorganisms/day) alone or in combination with 0.5% (w/w) linoleic acid & 0.5% (w/w) α-linolenic acid, daily for 7 weeks and compared with trehalose and bovine serum albumin. Tissue fatty acid composition was assessed by gas-liquid chromatography and visceral hypersensitivity was assessed by colorectal distension. Significant differences in the fatty acid profiles of the non-separated controls and maternally separated controls were observed for α-linolenic acid and arachidonic acid in the liver, oleic acid and eicosenoic acid (c11) in adipose tissue, and for palmitoleic acid and docosahexaenoic acid in serum (p<0.05). Administration of B. breve DPC6330 to MS rats significantly increased palmitoleic acid, arachidonic acid and docosahexaenoic acid in the liver, eicosenoic acid (c11) in adipose tissue and palmitoleic acid in the prefrontal cortex (p<0.05), whereas feeding B. breve DPC6330 to non separated rats significantly increased eicosapentaenoic acid and docosapentaenoic acid in serum (p<0.05) compared with the NS un-supplemented controls. Administration of B. breve DPC6330 in combination with linoleic acid and α-linolenic acid to maternally separated rats significantly increased docosapentaenoic acid in the serum (p<0.01) and α-linolenic acid in adipose tissue (p<0.001), whereas feeding B. breve DPC6330 with fatty acid supplementation to non-separated rats significantly increased liver and serum docosapentaenoic acid (p<0.05), and α-linolenic acid in adipose tissue (p<0.001). B. breve DPC6330 influenced host fatty acid metabolism. Administration of B. breve DPC6330 to maternally separated rats significantly modified the palmitoleic acid, arachidonic acid and docosahexaenoic acid contents in tissues. The effect was not observed in non-separated animals.     

Glycogen Synthase Kinase-3α Promotes Fatty Acid Uptake and Lipotoxic Cardiomyopathy

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β-Arrestin Recruitment and Biased Agonism at Free Fatty Acid Receptor 1

FFAR1/GPR40 is a seven-transmembrane domain receptor (7TMR) expressed in pancreatic β cells and activated by FFAs. Pharmacological activation of GPR40 is a strategy under consideration to increase insulin secretion in type 2 diabetes. GPR40 is known to signal predominantly via the heterotrimeric G proteins G_q/11. However, 7TMRs can also activate functionally distinct G protein-independent signaling via β-arrestins. Further, G protein- and β-arrestin-based signaling can be differentially modulated by different ligands, thus eliciting ligand-specific responses (“biased agonism”). Whether GPR40 engages β-arrestin-dependent mechanisms and is subject to biased agonism is unknown. Using bioluminescence resonance energy transfer-based biosensors for real-time monitoring of cell signaling in living cells, we detected a ligand-induced GPR40-β-arrestin interaction, with the synthetic GPR40 agonist TAK-875 being more effective than palmitate or oleate in recruiting β-arrestins 1 and 2. Conversely, TAK-875 acted as a partial agonist of G_q/11-dependent GPR40 signaling relative to both FFAs. Pharmacological blockade of G_q activity decreased FFA-induced insulin secretion. In contrast, knockdown or genetic ablation of β-arrestin 2 in an insulin-secreting cell line and mouse pancreatic islets, respectively, uniquely attenuated the insulinotropic activity of TAK-875, thus providing functional validation of the biosensor data. Collectively, these data reveal that in addition to coupling to G_q/11, GPR40 is functionally linked to a β-arrestin 2-mediated insulinotropic signaling axis. These observations expose previously unrecognized complexity for GPR40 signal transduction and may guide the development of biased agonists showing improved clinical profile in type 2 diabetes.     

Effects of 20 Standard Amino Acids on the Growth,Total Fatty Acids Production,and γ-Linolenic Acid Yield in Mucor circinelloides

Twenty standard amino acids were examined as single nitrogen source on the growth, total fatty acids production, and yield of γ-linolenic acid (GLA) in Mucor circinelloides. Of the amino acids, tyrosine gave the highest biomass and lipid accumulation and thus resulted in a high GLA yield with respective values of 17.8 g/L, 23 % (w/w, dry cell weight, DCW), and 0.81 g/L, which were 36, 25, and 72 % higher than when the fungus was grown with ammonium tartrate. To find out the potential mechanism underlying the increased lipid accumulation of M. circinelloides when grown on tyrosine, the activity of lipogenic enzymes of the fungus during lipid accumulation phase was measured. The enzyme activities of glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and ATP-citrate lyase were up-regulated, while NADP-isocitrate dehydrogenase was down-regulated by tyrosine during the lipid accumulation phase of the fungus which suggested that these enzymes may be involved in the increased lipid biosynthesis by tyrosine in this fungus.     

Towards an Understanding of Mesocestoides vogae Fatty Acid Binding Proteins’ Roles

Two fatty acid binding proteins, MvFABPa and MvFABPb were identified in the parasite Mesocestoides vogae (Platyhelmithes, Cestoda). Fatty acid binding proteins are small intracellular proteins whose members exhibit great diversity. Proteins of this family have been identified in many organisms, of which Platyhelminthes are among the most primitive. These proteins have particular relevance in flatworms since de novo synthesis of fatty acids is absent. Fatty acids should be captured from the media needing an efficient transport system to uptake and distribute these molecules. While HLBPs could be involved in the shuttle of fatty acids to the surrounding host tissues and convey them into the parasite, FABPs could be responsible for the intracellular trafficking. In an effort to understand the role of MvFABPs in fatty acid transport of M. vogae larvae, we analysed the intracellular localization of both MvFABPs and the co-localization with in vivo uptake of fatty acid analogue BODIPY FL C₁₆. Immunohistochemical studies on larvae sections using specific antibodies, showed a diffuse cytoplasmic distribution of each protein with some expression in nuclei and mitochondria. MvFABPs distribution was confirmed by mass spectrometry identification from 2D-electrophoresis of larvae subcellular fractions. This work is the first report showing intracellular distribution of MvFABPs as well as the co-localization of these proteins with the BODIPY FL C₁₆ incorporated from the media. Our results suggest that fatty acid binding proteins could target fatty acids to cellular compartments including nuclei. In this sense, M. vogae FABPs could participate in several cellular processes fulfilling most of the functions attributed to vertebrate’s counterparts.     

The Synthesis of Dimethyl Esters of dl-O,O′-Dimethylfukiic Acid and dl-O,O′-Dimethylepifukiic Acid

The synthesis of dimethyl esters of dl-O,O′-dimethylfukiic acid (11) and dl-O,O′-dimethylepifukiic acid (12) are described.     

The Emergence and Evolution of Life in a “Fatty Acid World” Based on Quantum Mechanics

Quantum mechanical based electron correlation interactions among molecules are the source of the weak hydrogen and Van der Waals bonds that are critical to the self-assembly of artificial fatty acid micelles. Life on Earth or elsewhere could have emerged in the form of self-reproducing photoactive fatty acid micelles, which gradually evolved into nucleotide-containing micelles due to the enhanced ability of nucleotide-coupled sensitizer molecules to absorb visible light. Comparison of the calculated absorption spectra of micelles with and without nucleotides confirmed this idea and supports the idea of the emergence and evolution of nucleotides in minimal cells of a so-called Fatty Acid World. Furthermore, the nucleotide-caused wavelength shift and broadening of the absorption pattern potentially gives these molecules an additional valuable role, other than a purely genetic one in the early stages of the development of life. From the information theory point of view, the nucleotide sequences in such micelles carry positional information providing better electron transport along the nucleotide-sensitizer chain and, in addition, providing complimentary copies of that information for the next generation. Nucleotide sequences, which in the first period of evolution of fatty acid molecules were useful just for better absorbance of the light in the longer wavelength region, later in the PNA or RNA World, took on the role of genetic information storage.     

Effect of Globotriaosyl Ceramide Fatty Acid α-Hydroxylation on the Binding by Verotoxin 1 and Verotoxin 2

Variation in the lipid moiety of the verotoxin (VT) receptor glycosphingolipid, globotriaosyl ceramide (Gb₃) can modulate toxin binding. The binding of VT1 and VT2 to C18 and C22 ahydroxy and nonhydroxy fatty acid isoforms of Gb₃ were compared using a receptor ELISA and a ¹²⁵l-labeled toxin/glycolipid microtitre plate direct binding assay. Increased binding to the hydroxylated species, particularly C22OH, was observed for both toxins. Increased RELISA binding at low glycolipid concentrations only, suggested the binding affinity is increased following Gb₃ fatty acid hydroxylation. Nonlinear regression analysis of direct binding assay to these Gb₃ isoforms confirmed the increased affinity of both toxins for the C22 hydroxylated Gb₃. The capacity was also significantly increased. The increased binding of VTs for hydroxylated fatty acid Gb₃ isoforms may be a factor in the selective renal pathology which can follow systemic verotoxemia, particularly in the mouse model. The more pronounced effect at lower glycolipid concentrations prompted investigation of VT1 binding affinity at different Gb₃ concentrations. Unexpectedly, the VT1 Kd for Gb₃ was found to decrease as an inverse function of the Gb₃ concentration. This shows that glycolipids have nonclassical receptor properties.     

Cloning of the Fatty Acid Synthetase β Subunit from Fission Yeast, Coexpression with the α Subunit, and Purification of the Intact Multifunctional Enzyme Complex

We have cloned and sequenced the fission yeast (Schizosaccharomyces pombe)fas1⁺gene, which encodes the fatty acid synthetase (FAS) β subunit, by applying a PCR technique to conserved regions in the β subunit of the α₆β₆types of FAS among different organisms. The deduced amino acid sequence of the Fas1 polypeptide, consisting of 2073 amino acids (M_r= 230,616), exhibits the 48.1% identity with the β subunit from the budding yeast (Saccharomyces cerevisiae). This subunit, with five different catalytic activities, bears four distinct domains, while the α subunit, the sequence of which was previously reported by Saitohet al.(S. Saitohet al.,1996,J. Cell Biol.134, 949–961), carries three domains. We have developed a co-expression system of the FAS α and β subunits by cotransformation of two expression vectors, containing thelsd1⁺/fas2⁺gene and thefas1⁺gene, into fission yeast cells. The isolated FAS complex showed quite high specific activity, of more than 4000 mU/mg, suggesting complete purification. Its molecular weight was determined by dynamic light scattering and ultracentrifugation analysis to be 2.1–2.4 × 10⁶, and one molecule of the FAS complex was found to contain approximately six FMN molecules. These results indicate that the FAS complex fromS. pombeforms a heterododecameric α₆β₆structure. Electron micrographs of the negatively stained molecule suggest that the complex adopts a unique barrel-shaped cage architecture.