Anti-inflammatory effects of α-linolenic acid in M1-like macrophages are associated with enhanced production of oxylipins from α-linolenic and linoleic acid

Chronic inflammation, mediated in large part by proinflammatory macrophage populations, contributes directly to the induction and perpetuation of metabolic diseases, including obesity, insulin resistance and type 2 diabetes. Polyunsaturated fatty acids (PUFAs) can have profound effects on inflammation through the formation of bioactive oxygenated metabolites called oxylipins. The objective of this study was to determine if exposure to the dietary omega-3 PUFA α-linolenic acid (ALA) can dampen the inflammatory properties of classically activated (M1-like) macrophages derived from the human THP-1 cell line and to examine the accompanying alterations in oxylipin secretion. We find that ALA treatment leads to a reduction in lipopolysaccharide (LPS)-induced interleukin (IL)-1β, IL-6 and tumor necrosis factor-α production. Although ALA is known to be converted to longer-chain PUFAs eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), DHA oxylipins were reduced overall by ALA treatment, as was LPS-induced secretion of EPA oxylipins. In contrast, we observed profound increases in oxylipins directly derived from ALA. Lipoxygenase products of linoleic acid were also dramatically increased, and LPS-induced production of AA oxylipins, particularly prostaglandin D2, was reduced. These results suggest that ALA may act to dampen the inflammatory phenotype of M1-like macrophages by a unique set of mechanisms distinct from those used by the long-chain omega-3 fatty acids EPA and DHA. Thus, there is strong rationale for investigating the functions of ALA oxylipins and lesser-known LA oxylipins since they hold promise as anti-inflammatory agents.     

Effects of capric acid on rumen methanogenesis and biohydrogenation of linoleic and α-linolenic acid

Capric acid (C10:0), a medium chain fatty acid, was evaluated for its anti-methanogenic activity and its potential to modify the rumen biohydrogenation of linoleic (C18:2n-6) and α-linolenic acids (C18:3n-3). A standard dairy concentrate (0.5 g), supplemented with sunflower oil (10 mg) and linseed oil (10 mg) and increasing doses of capric acid (0, 10, 20 and 30 mg), was incubated with mixed rumen contents and buffer (1 : 4 v/v) for 24 h. The methane inhibitory effect of capric acid was more pronounced at the highest (30 mg) dose compared to the medium (20 mg) (-85% v. -34%), whereas the lower dose (10 mg) did not reduce rumen methanogenesis. A 23% decrease in total short-chain fatty acid (SCFA) production was observed, accompanied by shifts towards increased butyrate at 20 mg and increased propionate at 30 mg of capric acid (P < 0.001). Capric acid linearly decreased the extent of biohydrogenation of C18:2n-6 and C18:3n-3, by up to 60% and 86%, respectively. This reduction was partially due to a lower extent of lipolysis when capric acid was supplemented. Capric acid at 20 and 30 mg completely inhibited the production of C18:0 (P < 0.001), resulting in an accumulation of biohydrogenation intermediates, mainly C18:1t10 + t11 and C18:2t11c15. In contrast to effects on rumen fermentation (methane production and proportions of SCFA), 30 mg of capric acid did not induce major changes in rumen biohydrogenation as compared to the medium (20 mg) dose. This study revealed the dual action of capric acid, being inhibitory to both methane production and biohydrogenation of C18:2n-6 and C18:3n-3.     

One-pot synthesis of bioactive cyclopentenones from α-linolenic acid and docosahexaenoic acid

Oxidation products of the poly-unsaturated fatty acids (PUFAs) arachidonic acid, α-linolenic acid and docosahexaenoic acid are bioactive in plants and animals as shown for the cyclopentenones prostaglandin 15d-PGJ₂ and PGA₂, cis-(+)-12-oxophytodienoic acid (12-OPDA), and 14-A-4 neuroprostane. In this study an inexpensive and simple enzymatic multi-step one-pot synthesis is presented for 12-OPDA, which is derived from α-linolenic acid, and the analogous docosahexaenoic acid (DHA)-derived cyclopentenone [(4Z,7Z,10Z)-12-[[-(1S,5S)-4-oxo-5-(2Z)-pent-2-en-1yl]-cyclopent-2-en-1yl] dodeca-4,7,10-trienoic acid, OCPD]. The three enzymes utilized in this multi-step cascade were crude soybean lipoxygenase or a recombinant lipoxygenase, allene oxide synthase and allene oxide cyclase from Arabidopsis thaliana. The DHA-derived 12-OPDA analog OCPD is predicted to have medicinal potential and signaling properties in planta. With OCPD in hand, it is shown that this compound interacts with chloroplast cyclophilin 20-3 and can be metabolized by 12-oxophytodienoic acid reductase (OPR3) which is an enzyme relevant for substrate bioactivity modulation in planta.     

Conjugation with α-linolenic acid improves cancer cell uptake and cytotoxicity of doxorubicin

The synthetic DOX–LNA conjugate was characterized by proton nuclear magnetic resonance and mass spectrometry. In addition, the purity of the conjugate was analyzed by reverse-phase high-performance liquid chromatography. The cellular uptake, intracellular distribution, and cytotoxicity of DOX–LNA were assessed by flow cytometry, fluorescence microscopy, liquid chromatography/electrospray ionization tandem mass spectrometry, and the tetrazolium dye assay using the in vitro cell models. The DOX–LNA conjugate showed substantially higher tumor-specific cytotoxicity compared with DOX.     

Double hydroperoxidation of α-linolenic acid by potato tuber lipoxygenase

The potato tuber lipoxygenase preparations convert α-linolenic acid not only to 9(S)-HPOTE, but also to some more polar metabolites. Two of these polar products, I and II, with ultraviolet absorbance maxima at 267 nm were purified by HPLC. It was found that metabolites I and II have, respectively, one and two hydroperoxy groups. Products of NaBH₄ reduction of both I and II were identified by their chemical ionization and electron impact mass spectra and by ¹H-NMR spectra as 9,16-dihydroxy-10(E), 12(Z), 14(E)-octadecatrienoic acid. The obtained results suggest that compound II is 9,16-dihydroperoxy-10(E), 12(Z), 14(E)-octadecatrienoic acid and product I is a mixture of two positional isomers, 9-hydroxy-16-hydroperoxy-10(E),12(Z),14(E)-octadecatrienoic and 9-hydroperoxy-16-hydroxy-10(E),12(Z), 14(E)-octadecatrienoic acids. Lipoxygenase converts efficiently [¹⁴C]9-HOTE into product I. Also, both metabolites I and II are the products of double dioxygenation. The second oxygenation at C-16 position as well as the first one at C-9 is controlled by lipoxygenase.     

Effect of α-linolenic acid on oocyte maturation and embryo development of prepubertal sheep oocytes

The purpose of this study was to evaluate the effect of omega-3 α-linolenic acid (ALA) added to the IVM medium on embryo development of prepubertal sheep oocytes. Experiment 1 investigated the effect of ALA at different concentrations (0 [control], 50, 100, and 200 μM) and DMSO (100 μM) in IVM media on cumulus cell expansion and oocyte nuclear maturation and on synthesis of prostaglandins (PGE2 and PGF2α). Experiment 2 investigated the effects of ALA at different concentrations in the IVM medium on oocyte fertilization, cleavage, and developmental potential to blastocyst stage and changes in estradiol and progesterone concentrations in the spent IVM media. IVM oocytes were fertilized with frozen-thawed spermatozoa capacitated in a serum-free sperm medium. Presumptive zygotes were cultured 8 days in synthetic oviductal fluid (SOF) medium without serum. Blastocyst quality was assessed by counting total cell number and the number of apoptotic cells using Hoechst and Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. Nuclear maturation of oocytes and the number of fully expanded cumulus cells were reduced after treatment with 200 μM of ALA compared with other groups (P ≤ 0.05). Supplementation with ALA increased both PGE2 and PGF2α concentrations in the spent media (P ≤ 0.05). No differences were observed in blastocyst development among control (12.2%) and 50, 100, and 200 μM ALA groups (6.9%, 11.5% and 14.0%, respectively). However, the total cell number (46.50 ± 5.85, 67.94 ± 6.71, 45.20 ± 6.37, and 59.80 ± 5.51, respectively; P ≤ 0.05) and apoptotic cell number (6.45 ± 0.89, 2.48 ± 0.81, 4.02 ± 1.15, and 3.67 ± 1.15, respectively; P ≤ 0.05) were significantly improved. After IVM, estradiol concentration was lower and progesterone concentration was higher in ALA groups compared with the control group (P ≤ 0.05). In conclusion, these results revealed that ALA affects prepubertal sheep embryo quality associated with alteration of releasing reproductive hormones.     

Synthesis and biological evaluation of caffeic acid derivatives as potent inhibitors of α-MSH-stimulated melanogenesis

We have disclosed our effort to develop caffeic acid derivatives as potent and non-toxic inhibitors of α-MSH-stimulated melanogenesis to treat pigmentation disorders and skin medication including a cosmetic skin-whitening agent. The SAR studies revealed that cyclohexyl ester and secondary amide derivatives of caffeic acid showed significant inhibitory activities.     

Production of α-linolenic acid in Yarrowia lipolytica using low-temperature fermentation

α-Linolenic acid (ALA) is an essential ω-3 fatty with reported health benefits. However, this molecule is naturally found in plants such as flaxseed and canola which currently limits production. Here, we demonstrate the potential to sustainably produce ALA using the oleaginous yeast Yarrowia lipolytica. Through the use of a recently identified Δ12–15 desaturase (Rk Δ12–15), we were able to enable production in Y. lipolytica. When combined with a previously engineered lipid-overproducing strain with high precursor availability, further improvements of ALA production were achieved. Finally, the cultivation of this strain at lower temperatures significantly increased ALA content, with cells fermented at 20 °C accumulating nearly 30% ALA of the total lipids in this cell. This low-temperature fermentation represents improved ALA titer up to 3.2-fold compared to standard growth conditions. Scale-up into a fed-batch bioreactor produced ALA at 1.4 g/L, representing the highest published titer of this ω-3 fatty acid in a yeast host.

     

In vitro evaluation of glyceric acid and its glucosyl derivative, α-glucosylglyceric acid,as cell proliferation inducers and protective solutes

We demonstrate that 0.78?mm glyceric acid activated the proliferation of human dermal fibroblasts by about 45%, whereas 34?mm α-glucosylglyceric acid (GGA) increased collagen synthesis by the fibroblasts by 1.4-fold compared to that in the absence of GGA. The two substances also exerted protective effects on both DNA scission by the hydroxyl radical and protein aggregation by heat in vitro.     

Structural modifications of 2,3-indolobetulinic acid: Design and synthesis of highly potent α-glucosidase inhibitors

A series of nineteen nitrogen-containing lupane triterpenoids was obtained by modification of C2, C3, C20 and C28 positions of betulonic acid and their α-glucosidase inhibiting activity was investigated. Being a leader compound from our previous study, 2,3-indolo-betulinic acid was used as the main template for different modifications at C-(28)-carboxyl group to obtain cyano-, methylcyanoethoxy-, propargyloxy- and carboxamide derivatives. 20-Oxo- and 29-hydroxy-20-oxo-30-nor-analogues of 2,3-indolo-betulinic acid were synthesized by ozonolysis of betulonic acid followed by Fischer indolization reaction. To compare the influence of the fused indole or the seven-membered A-ring on the inhibitory activity, lupane A-azepanones with different substituents at C28 were synthesized. The structure-activity relationships revealed that the enzyme inhibition activity dramatically increased (up to 4730 times) when the carboxylic group of 2,3-indolo-betulinic acid was converted to the corresponding amide. Thus, the IC₅₀ values for glycine amide and L-phenylalanine amides were 0.04 and 0.05 μM, respectively. This study also revealed that 2,3-indolo-platanic acid is 4.5 times more active than the parent triterpenoid with IC₅₀ of 0.4 μM. Molecular modeling suggested that improved potency is due to additional polar interactions formed between C28 side chain and a sub-pocket of the α-glucosidase allosteric site.     

Physiological effects of γ-linolenic acid and sesamin on hepatic fatty acid synthesis and oxidation

Interrelated effects of γ-linolenic acid (GLA) and sesamin, a sesame lignan, on hepatic fatty acid synthesis and oxidation were examined. Rats were fed experimental diets supplemented with 0 or 2 g/kg sesamin (1:1 mixture of sesamin and episesamin) and containing 100 g/kg of palm oil (saturated fat), safflower oil rich in linoleic acid, or oil of evening primrose origin containing 43% GLA (GLA oil) for 18 days. In rats fed sesamin-free diets, GLA oil, compared with other oils, increased the activity and mRNA levels of various enzymes involved in fatty acid oxidation, except for some instances. Sesamin greatly increased these parameters, and the enhancing effects of sesamin on peroxisomal fatty acid oxidation rate and acyl-CoA oxidase, enoyl-CoA hydratase and acyl-CoA thioesterase activities were more exaggerated in rats fed GLA oil than in the animals fed other oils. The combination of sesamin and GLA oil also synergistically increased the mRNA levels of some peroxisomal fatty acid oxidation enzymes and of several enzymes involved in fatty acid metabolism located in other cell organelles. In the groups fed sesamin-free diets, GLA oil, compared with other oils, markedly reduced the activity and mRNA levels of various lipogenic enzymes. Sesamin reduced all these parameters, except for malic enzyme, in rats fed palm and safflower oils, but the effects were attenuated in the animals fed GLA oil. These changes by sesamin and fat type accompanied profound alterations in serum lipid levels. This may be ascribable to the changes in apolipoprotein-B-containing lipoproteins.     

Identification and evaluation of ω-3 fatty acid desaturase genes for hyperfortifying α-linolenic acid in transgenic rice seed

α-Linolenic acid (ALA) deficiency and a skewed of ω6:ω3 fatty acid ratio in the diet are a major explanation for the prevalence of cardiovascular diseases and inflammatory/autoimmune diseases. There is a need to enhance the ALA content and to reduce the ratio of linoleic acid (LA) to ALA. Six ω-3 (Δ-15) fatty acid desaturase (FAD) genes were cloned from rice and soybean. The subcellular localizations of the proteins were identified. The FAD genes were introduced into rice under the control of an endosperm-specific promoter, GluC, or a Ubi-1 promoter to evaluate their potential in increasing the ALA content in seeds. The ALA contents in the seeds of endoplasmic reticulum (ER)-localized GmFAD3-1 and OsFAD3 overexpression lines increased from 0.36 mg g?1 to 8.57 mg g?1 and 10.06 mg g?1, respectively, which was 23.8- and 27.9-fold higher than that of non-transformants. The trait of high ALA content was stably inheritable over three generations. Homologous OsFAD3 is more active than GmFAD3-1 in catalysing LA conversion to ALA in rice seeds. Overexpression of ER-localized GmFAD3-2/3 and chloroplast-localized OsFAD7/8 had less effect on increasing the ALA content in rice seeds. The GluC promoter is advantageous compared with Ubi-1 in this experimental system. The enhanced ALA was preferentially located at the sn-2 position in triacylglycerols. A meal-size portion of high ALA rice would meet >80% of the daily adult ALA requirement. The ALA-rich rice could be expected to ameliorate much of the global dietary ALA deficiency.     

Biosynthesis and in vitro enzymatic synthesis of the isoleucine conjugate of 12-oxo-phytodienoic acid from the isoleucine conjugate of α-linolenic acid

The isoleucine conjugate of 12-oxo-phytodienoic acid (OPDA-Ile), a new member of the jasmonate family, was recently identified in Arabidopsis thaliana and might be a signaling molecule in plants. However, the biosynthesis and function of OPDA-Ile remains elusive. This study reports an in vitro enzymatic method for synthesizing OPDA-Ile, which is catalyzed by reactions of lipoxygenase (LOX), allene oxide synthase (AOS), and allene oxide cyclase (AOC) using isoleucine conjugates of α -linolenic acid (LA-Ile) as the substrate. A. thaliana fed LA-Ile exhibited a marked increase in the OPDA-Ile concentration. LA-Ile was also detected in A. thaliana. Furthermore, stable isotope labelled LA-Ile was incorporated into OPDA-Ile. Thus, OPDA-Ile is biosynthesized via the cyclization of LA-Ile in A. thaliana.     

Relationship between transport and metabolism of α-naphthaleneacetic acid, β-naphthaleneacetic acid and α-decalylacetic acid in segments of Coleus

Summary Transportand metabolism of -naphthaleneacetic acid -naphthaleneacetic acid, and -decalylacetic acid, all labelled with ¹⁴C in the carboxyl, group, were studied. Only -naphthaleneacetic acid is transported in a polar way. Most of the radioactivity in the tissue is in a low molecular form, either free or as immobilization products. The immobilization of -naphthaleneacetic acid is similar to that of -naphthaleneacetic acid. Immobilization of -decalylacetic acid is typically different. Bioassays showed -naphthaleneacetic acid as the sole biologically active component. It is concluded that stereo requirements necessary for biological activity are also required for polar auxin transport. It is further concluded that the observed specificity of the transport system is not related to the formation of immobilization products.     

The aqueous solubility and thermal stability of α-lipoic acid are enhanced by cyclodextrin

We investigated the effects of various cyclodextrins (CDs) on the aqueous solubility and thermal stability of α-lipoic acid, a compound with low water solubility. α-CD, β-CD, and γ-CD had little effect on the aqueous solubility of α-lipoic acid. In contrast, 6-O-α-maltosyl-CDs increased it in a concentration-dependent manner, 6-O-α-maltosyl-β-CD enhancing solubility the most. The thermal stability of α-lipoic acid in the solid state was improved by the addition of G2-β-CD(?), a commercial product of 6-O-α-maltosyl-β-CD. The thermal stability of α-lipoic acid was also improved by the addition of β-CD. Analysis by differential scanning calorimetry showed that G2-β-CD(?), a mixture of maltosyl-β-CDs, and β-CD efficiently formed complexes with α-lipoic acid. These results suggest that the sizes and shapes of these β-CD compounds are compatible with complexation with α-lipoic acid. Moreover, both the formation of an aqueous complex with G2-β-CD(?) and an insoluble complex with β-CD increased the thermal stability of α-lipoic acid.     

Production and partial purification of γ-linolenic acid and some pigments fromSpirulina platensis

The polyunsaturated fatty acid -linolenic acid (GLA, 18:36) is of potential pharmaceutical value. The cyanobacteriumSpirulina platensis could become an excellent source for this fatty acid, provided that GLA content could be increased and a GLA concentrate could be obtained at a low cost. Increasing the cell concentration inSpirulina platensis enhanced the fatty acid content and thus the GLA content. This effect was used to further enhance the GLA content of GLA-overproducing strains. Separation of the galactolipids and their purification via urea complexes formation, resulted in a GLA concentrate of over 90% purity.     

Substitution of dietary oleic acid for myristic acid increases the tissue storage of α-linolenic acid and the concentration of docosahexaenoic acid in the brain, red blood cells and plasma in the rat

Various strategies have been developed to increase the cellular level of (n-3) polyunsaturated fatty acids in animals and humans. In the present study, we investigated the effect of dietary myristic acid, which represents 9% to 12% of fatty acids in milk fat, on the storage of α-linolenic acid and its conversion to highly unsaturated (n-3) fatty acid derivatives. Five isocaloric diets were designed, containing equal amounts of α-linolenic acid (1.3% of dietary fatty acids, i.e. 0.3% of dietary energy) and linoleic acid (7.0% of fatty acids, i.e. 1.5% of energy). Myristic acid was supplied from traces to high levels (0%, 5%, 10%, 20% and 30% of fatty acids, i.e. 0% to 6.6% of energy). To keep the intake of total fat and other saturated fatty acids constant, substitution was made with decreasing levels of oleic acid (76.1% to 35.5% of fatty acids, i.e. 16.7% to 7.8% of energy) that is considered to be neutral in lipid metabolism. After 8 weeks, results on physiological parameters showed that total cholesterol and low-density lipoprotein-cholesterol did not differ in the diets containing 0%, 5% and 10% myristic acid, but were significantly higher in the diet containing 30% myristic acid. In all the tissues, a significant increasing effect of the substitution of oleic acid for myristic acid was shown on the level of both α-linolenic and linoleic acids. Compared with the rats fed the diet containing no myristic acid, docosahexaenoic acid significantly increased in the brain and red blood cells of the rats fed the diet with 30% myristic acid and in the plasma of the rats fed the diet with 20% myristic acid. Arachidonic acid also increased in the brain of the rats fed the diet with 30% myristic acid. By measuring Δ6-desaturase activity, we found a significant increase in the liver of the rats fed the diet containing 10% of myristic acid but no effect at higher levels of myristic acid. These results suggest that an increase in dietary myristic acid may contribute in increasing significantly the tissue storage of α-linolenic acid and the overall bioavailability of (n-3) polyunsaturated fatty acids in the brain, red blood cells and plasma, and that mechanisms other than the single Δ6-desaturase activity are involved in this effect.     

Metabolism of linoleic and α-linolenic acids in cultured cardiomyocytes: Effect of different N-6 and N-3 fatty acid supplementation

The metabolites of linoleic (LA) and -linolenic (ALA) acids are involved in coronary heart disease. Both n-6 and n-3 essential fatty acids (EFAs) are likely to be important in prevention of atherosclerosis since the common risk factors are associated with their reduced 6-desaturation. We previously demonstrated the ability of heart tissue to desaturate LA. In this study we examined the ability of cultured cardiomyocytes to metabolize both LA and ALA in vivo, in the absence and in the presence of gamma linolenic acid (GLA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) alone or combined together. In control conditions, about 25% of LA and about 90% of ALA were converted in PUFAs. GLA supplementation had no influence on LA conversion to more unsaturated fatty acids, while the addition of n-3 fatty acids, alone or combined together, significantly decreased the formation of interconversion products from LA. Using the combination of n-6 and n-3 PUFAs, GLA seemed to counterbalance partially the inhibitory effect of EPA and DHA on LA desaturation/elongation. The conversion of ALA to more unsaturated metabolites was greatly affected by GLA supplementation. Each supplemented fatty acid was incorporated to a significant extent into cardiomyocyte lipids, as revealed by gas chromatographic analysis. The n-6/n-3 fatty acid ratio was greatly influenced by the different supplementations; the ratio in GLA+EPA+DHA supplemented cardiomyocytes was the most similar to that recorded in control cardiomyocytes. Since important risk factors for coronary disease may be associated with reduced 6-desaturation of the parent EFAs, administration of n-6 or n-3 EFA metabolites alone could cause undesirable effects. Since they appear to have different and synergistic roles, only combined treatment with both n-6 and n-3 metabolites is likely to achieve optimum results.     

Linoleic and α-linolenic acid as precursor and inhibitor for the synthesis of long-chain polyunsaturated fatty acids in liver and brain of growing pigs

Studies suggested that in human adults, linoleic acid (LA) inhibits the biosynthesis of n-3 long-chain polyunsaturated fatty acids (LC-PUFA), but their effects in growing subjects are largely unknown. We used growing pigs as a model to investigate whether high LA intake affects the conversion of n-3 LC-PUFA by determining fatty acid composition and mRNA levels of Δ5- and Δ6 desaturase and elongase 2 and -5 in liver and brain. In a 2 × 2 factorial arrangement, 32 gilts from eight litters were assigned to one of the four dietary treatments, varying in LA and α-linolenic acid (ALA) intakes. Low ALA and LA intakes were 0.15 and 1.31, and high ALA and LA intakes were 1.48 and 2.65 g/kg BW0.75 per day, respectively. LA intake increased arachidonic acid (ARA) in liver. ALA intake increased eicosapentaenoic acid (EPA) concentrations, but decreased docosahexaenoic acid (DHA) (all P < 0.01) in liver. Competition between the n-3 and n-6 LC-PUFA biosynthetic pathways was evidenced by reductions of ARA (>40%) at high ALA intakes. Concentration of EPA (>35%) and DHA (>20%) was decreased by high LA intake (all P < 0.001). Liver mRNA levels of Δ5- and Δ6 desaturase were increased by LA, and that of elongase 2 by both ALA and LA intakes. In contrast, brain DHA was virtually unaffected by dietary LA and ALA. Generally, dietary LA inhibited the biosynthesis of n-3 LC-PUFA in liver. ALA strongly affects the conversion of both hepatic n-3 and n-6 LC-PUFA. DHA levels in brain were irresponsive to these diets. Apart from Δ6 desaturase, elongase 2 may be a rate-limiting enzyme in the formation of DHA.