Synergistic effects of punicic acid and alpha lipoic acid ameliorate inflammatory and metabolic genes expression in C2C12 myoblast cells under oxidative stress condition.

Inflammation is a reaction of the immune system to infection and injury; in fact, it positioned at the center of metabolic disorders, particularly obesity, type 2 diabetes, and cardiovascular diseases. Thus play a major role not only in their development, but also exerts as a crucial linking factor among those diseases. In this regard, one of the strategies for tackling this problem is application of antioxidants to treat such diseases. The present study was performed to evaluate the synergistic effects of punicic acid (PUA) and alpha-lipoic acid (ALA) as antioxidants and radical scavenging reagents on the expression of some inflammatory and metabolism-related genes under oxidative stress in the muscle cells. The experimental treatments consisted of a range of 20, 40, 80, 160, and 320 µM of PUA, and 5, 25, 50, 100, and 200 µM of ALA with a 200 µM concentration of H2 O2 as an oxidative stress inducer. Accordingly, fatty acid treatments were applied for 24 h, and H2 O2 was treated for 1 h. Our results indicated that the simultaneous treatment of PUA and ALA at optimal concentrations (80 and 50 µM, respectively) decreased the expression of inflammation genes and increased the expression of regulatory genes (Pparγ, Pgc-1α) related to metabolism (p < .05). Unexpectedly, H2 O2 treatment increased the Fndc5 expression (p < .05). Maximal upregulation of Pparγ, Pgc-1α were obtained when fatty acids combination (PUA and ALA) were used in the culture of H2 O2 treated cells (p < .05). Therefore, our findings suggest that the simultaneous use of PUA and ALA fatty acids could reduce oxidative stress, and the expression of inflammatory genes, thereby improving the cell metabolism.

The Beneficial Effects of Pine Nuts and Its Major Fatty Acid, Pinolenic Acid, on Inflammation and Metabolic Perturbations in Inflammatory Disorders.

Inflammatory disorders such as atherosclerosis, diabetes and rheumatoid arthritis are regulated by cytokines and other inflammatory mediators. Current treatments for these conditions are associated with significant side effects and do not completely suppress inflammation. The benefits of diet, especially the role of specific components, are poorly understood. Polyunsaturated fatty acids (PUFAs) have several beneficial health effects. The majority of studies on PUFAs have been on omega-3 fatty acids. This review will focus on a less studied fatty acid, pinolenic acid (PNLA) from pine nuts, which typically constitutes up to 20% of its total fatty acids. PNLA is emerging as a dietary PUFA and a promising supplement in the prevention of inflammatory disorders or as an alternative therapy. Some studies have shown the health implications of pine nuts oil (PNO) and PNLA in weight reduction, lipid-lowering and anti-diabetic actions as well as in suppression of cell invasiveness and motility in cancer. However, few reviews have specifically focused on the biological and anti-inflammatory effects of PNLA. Furthermore, in recent bioinformatic studies on human samples, the expression of many mRNAs and microRNAs was regulated by PNLA indicating potential transcriptional and post-transcriptional regulation of inflammatory and metabolic processes. The aim of this review is to summarize, highlight, and evaluate research findings on PNO and PNLA in relation to potential anti-inflammatory benefits and beneficial metabolic changes. In this context, the focus of the review is on the potential actions of PNLA on inflammation along with modulation of lipid metabolism and oxidative stress based on data from both in vitro and in vivo experiments, and human findings, including gene expression analysis.

Pomegranate Seed Oil as a Source of Conjugated Linolenic Acid (CLnA) Has No Effect on Atherosclerosis Development but Improves Lipid Profile and Affects the Expression of Lipid Metabolism Genes in apoE/LDLR-/- Mice.

The aim of this study was to evaluate the anti-atherosclerotic effect of pomegranate seed oil as a source of conjugated linolenic acid (CLnA) (cis-9,trans-11,cis-13; punicic acid) compared to linolenic acid (LnA) and conjugated linoleic acid (CLA) (cis-9,trans-11) in apoE/LDLR-/- mice. In the LONG experiment, 10-week old mice were fed for the 18 weeks. In the SHORT experiment, 18-week old mice were fed for the 10 weeks. Diets were supplied with seed oils equivalent to an amount of 0.5% of studied fatty acids. In the SHORT experiment, plasma TCh and LDL+VLDL cholesterol levels were significantly decreased in animals fed CLnA and CLA compared to the Control. The expression of PPARα in liver was four-fold increased in CLnA group in the SHORT experiment, and as a consequence the expression of its target gene ACO was three-fold increased, whereas the liver's expression of SREBP-1 and FAS were decreased in CLnA mice only in the LONG experiment. Punicic acid and CLA isomers were determined in the adipose tissue and liver in animals receiving pomegranate seed oil. In both experiments, there were no effects on the area of atherosclerotic plaque in aortic roots. However, in the SHORT experiment, the area of atherosclerosis in the entire aorta in the CLA group compared to CLnA and LnA was significantly decreased. In conclusion, CLnA improved the lipid profile and affected the lipid metabolism gene expression, but did not have the impact on the development of atherosclerotic plaque in apoE/LDLR-/- mice.

Anti-inflammatory and immunoregulatory effects of pinolenic acid in rheumatoid arthritis.

OBJECTIVES: In pre-clinical studies, pinolenic acid (PNLA), an omega-6-polyunsaturated fatty acid from pine nuts, has shown anti-inflammatory effects. We aimed to investigate the effect of PNLA in human cell lines and peripheral blood mononuclear cells (PBMCs) from RA patients and healthy controls (HCs). METHODS: A modified Boyden chamber was used to assess chemokine-induced migration of THP-1 monocytes. Macropinocytosis was assessed using Lucifer yellow and oxidized low-density lipoprotein (oxLDL) uptake using DiI-labelled oxLDL in THP-1 macrophages and human monocyte-derived macrophages (HMDMs). IL-6, TNF-α and prostaglandin E2 (PGE2) release by lipopolysaccharide (LPS)-stimulated PBMCs from RA patients and HCs was measured by ELISA. The transcriptomic profile of PNLA-treated, LPS-activated PBMCs was investigated by RNA-sequencing. RESULTS: PNLA reduced THP-1 cell migration by 55% (P < 0.001). Macropinocytosis and DiI-oxLDL uptake were reduced by 50% (P < 0.001) and 40% (P < 0.01), respectively, in THP-1 macrophages and 40% (P < 0.01) and 25% (P < 0.05), respectively, in HMDMs. PNLA reduced IL-6 and TNF-α release from LPS-stimulated PBMCs from RA patients by 60% (P < 0.001) and from HCs by 50% and 35%, respectively (P < 0.01). PNLA also reduced PGE2 levels in such PBMCs from RA patients and HCs (P < 0.0001). Differentially expressed genes whose expression was upregulated included pyruvate dehydrogenase kinase-4, plasminogen activator inhibitor-1, fructose bisphosphatase1 and N-Myc downstream-regulated gene-2, which have potential roles in regulating immune and metabolic pathways. Pathway analysis predicted upstream activation of the nuclear receptors peroxisome proliferator-activated receptors involved in anti-inflammatory processes, and inhibition of nuclear factor-κB and signal transducer and activator of transcription 1. CONCLUSIONS: PNLA has immune-metabolic effects on monocytes and PBMCs that are pathogenic in RA and atherosclerosis. Dietary PNLA supplementation may be beneficial in RA.

Direct effects of linoleic and linolenic acids on bovine uterine function using in vivo and in vitro studies.

The aim of the present study was to evaluate the effects of continuous administration of linoleic acid or linolenic acid into the intra-uterine horn, ipsilateral to the corpus luteum, on the duration of the estrous cycle and plasma progesterone (P4) concentration. The effects of linoleic and linolenic acids on bovine uterine and luteal functions were also studied using a tissue culture system. Intra-uterine administration of linoleic or linolenic acid (5 mg/10 ml of each per day) in cows, between days 12 and 21, resulted in a prolonged estrous cycle compared to the average duration of the last one to three estrous cycles before administration in each group (P < 0.05). Moreover, plasma P4 concentration in cows treated with linoleic or linolenic acid was high between days 19 and 21 (linoleic acid), or on day 20 (linolenic acid), compared to that of the control cows (saline administration; P < 0.05 or lower). Both linoleic (500 µg/ml) and linolenic (5 and 500 µg/ml) acids stimulated prostaglandin (PG) E2 but inhibited PGF2α production by cultured endometrial tissue (P < 0.01), while P4 production by cultured luteal tissue was not affected. These findings suggest that both linoleic and linolenic acids support luteal P4 production by regulating endometrial PG production and, subsequently, prolonging the duration of the estrous cycle in cows.

Lipophilic Compounds and Antibacterial Activity of Opuntia ficus-indica Root Extracts from Algeria.

The chemical composition, investigated by gas chromatography-mass spectrometry, and antibacterial activity of lipophilic extractives of three varieties of Opuntia ficus-indica roots from Algeria are reported in this paper for the first time. The results obtained revealed a total of 55 compounds, including fatty acids, sterols, monoglycerides and long chain aliphatic alcohols that were identified and quantified. ß-Sitosterol was found as the major compound of the roots of the three varieties. Furthermore, considerable amounts of essential fatty acids (ω3, ω6, and ω9) such as oleic, linoleic, and linolenic acids were also identified. The green variety was the richest among the three studied varieties. The antibacterial activity, evaluated with disc diffusion method, revealed that lipophilic extracts were effective mainly against Gram-positive Staphylococcus aureus and methicillin-resistant Staphylococcus aureus (MRSA) (19~23 mm). Gram-negative strains mainly Pseudomonas aeruginosa gave an inhibition zone of 18 mm, which is considered high antibacterial activity. The minimal inhibitory concentrations of the tested bacteria revealed interesting values against the majority of bacteria tested: 75-100 µg mL-1 for Bacillus sp., 250-350 µg/mL for the two Staphylococcus strains, 550-600 µg mL-1 for E. coli, and 750-950 µg mL-1 obtained with Pseudomonas sp. This study allows us to conclude that the lipophilic fractions of cactus roots possess interesting phytochemicals such as steroids, some fatty acids and long chain alcohols that acted as antibiotic-like compounds countering pathogenic strains.

Pinocembrin and its linolenoyl ester derivative induce wound healing activity in HaCaT cell line potentially involving a GPR120/FFA4 mediated pathway.

Wound healing represents an urgent need from the clinical point of view. Several diseases result in wound conditions which are difficult to treat, such as in the case of diabetic foot ulcer. Starting from there, the medicinal research has focused on various targets over the years, including GPCRs as new wound healing drug targets. In line with this, GPR120, known to be an attractive target in type 2 diabetes drug discovery, was studied to finalize the development of new wound healing agents. Pinocembrin (HW0) was evaluated as a suitable compound for interacting with GPR120, and was hybridized with fatty acids, which are known endogenous GPR120 ligands, to enhance the wound healing potential and GPR120 interactions. HW0 and its 7-linolenoyl derivative (HW3) were found to be innovative wound healing agents. Immunofluorescence and functional assays suggested that their activity was mediated by GPR120, and docking simulations showed that the compounds could share the same pocket occupied by the known GPR120 agonist, TUG-891.

Beneficial Impacts of Alpha-Eleostearic Acid from Wild Bitter Melon and Curcumin on Promotion of CDGSH Iron-Sulfur Domain 2: Therapeutic Roles in CNS Injuries and Diseases.

Neuroinflammation and abnormal mitochondrial function are related to the cause of aging, neurodegeneration, and neurotrauma. The activation of nuclear factor κB (NF-κB), exaggerating these two pathologies, underlies the pathogenesis for the aforementioned injuries and diseases in the central nervous system (CNS). CDGSH iron-sulfur domain 2 (CISD2) belongs to the human NEET protein family with the [2Fe-2S] cluster. CISD2 has been verified as an NFκB antagonist through the association with peroxisome proliferator-activated receptor-ß (PPAR-ß). This protective protein can be attenuated under circumstances of CNS injuries and diseases, thereby causing NFκB activation and exaggerating NFκB-provoked neuroinflammation and abnormal mitochondrial function. Consequently, CISD2-elevating plans of action provide pathways in the management of various disease categories. Various bioactive molecules derived from plants exert protective anti-oxidative and anti-inflammatory effects and serve as natural antioxidants, such as conjugated fatty acids and phenolic compounds. Herein, we have summarized pharmacological characters of the two phytochemicals, namely, alpha-eleostearic acid (α-ESA), an isomer of conjugated linolenic acids derived from wild bitter melon (Momordica charantia L. var. abbreviata Ser.), and curcumin, a polyphenol derived from rhizomes of Curcuma longa L. In this review, the unique function of the CISD2-elevating effect of α-ESA and curcumin are particularly emphasized, and these natural compounds are expected to serve as a potential therapeutic target for CNS injuries and diseases.

Linolenic fatty acid hydroperoxide acts as biocide on plant pathogenic bacteria: Biophysical investigation of the mode of action.

Fatty acid hydroperoxides (HPO) are free phyto-oxylipins known for their crucial role as signalling molecules during plant defense mechanisms. They were also demonstrated to have direct biocidal activities against plant pathogens including gram negative bacteria. In the present work, the biocidal effect of one linolenic fatty acid hydroperoxide, namely 13-HPOT has been investigated on three plant pathogen gram negative bacteria: Pectobacterium carotovorum, Pseudomonas syringae and Xanthomonas translucens. We showed that 13-HPOT has a strong dose response effect on those phytopathogens. In a second part, the molecular mechanism behind the antibacterial effect of 13-HPOT was investigated at a molecular level using an integrative biophysical approach combining in vitro and in silico methods. Since other antimicrobial amphiphilic molecules have been shown to target the lipid membrane of the organisms they act on, we focused our study on the interaction of 13-HPOT with biomimetic membranes. In a first step, we hypothesized that the inner membrane of the bacteria was the main site of action of 13-HPOT and hence we used lipids representative of this membrane to form our models. Our results indicated that 13-HPOT can interact with the lipid representative of the inner bacterial plasma membrane. A strong membrane insertion is suggested but no major permeabilization of the membrane is observed. Phosphatidylethanolamine (PE) and cardiolipin (CL), present in the bacterial plasma membrane, appear to play important roles in this interaction. We suggest that the mode of action of 13-HPOT should involve either subtle changes in membrane properties, such as its lateral organization and distribution, and/or interactions with membrane proteins.

Therapeutic Effects of Coccomyxagloeobotrydiformis on the Metabolic Syndrome in Rats.

BACKGROUND/AIMS: The metabolic syndrome (MS) is a cluster of metabolic changes that carry a high risk of cardiovascular disease (CVD). A newly discovered microalga, coccomyxagloeobotrydiformis (CGD), has been reported to improve ischemic stroke and metabolism-related indicators. We observed the therapeutic effects of CGD on MS and postulated the underlying mechanism. METHODS: A diet-induced MS model in rats was used to observe the therapeutic effects of CGD on MS. Blood-glucose and lipid indices were measured using enzymatic colorimetric kits. A biologic data acquisition and analysis system (BL-420F) was used to evaluate cardiac function. Expression of mitochondrial respiratory chain (MRC) enzymes was measured by immunofluorescence staining. The proteins associated with oxidative stress, apoptosis and inflammation were detected by western blotting. RESULTS: Body weight, abdominal circumference, fasting blood glucose , blood pressure as well as serum levels of total cholesterol, triglycerides and low-density lipoprotein-cholesterol were decreased whereas serum levels of high-density lipoprotein-cholesterol was increased in CGD-treated MS rats. CGD increased left-ventricular systolic pressure, left-ventricular end-diastolic pressure, left-ventricular systolic pressure maximum rate of increase and left-ventricular diastolic pressure maximum rate of decrease in MS rats with cardiovascular complications. CGD up-regulated expression of adenosine monophosphate-activated protein kinase and peroxisome proliferator activated receptor gamma coactivator 1-alpha in the heart, adipose tissue and skeletal muscle. Expression of the MRC subunits of ATPase 6, cytochrome b and succinate dehydrogenase complex, subunit-A was increased whereas that of uncoupling protein-2 decreased in different tissues. CGD showed anti-oxidation effects by increasing expression of superoxide dismutase and decreasing that of malondialdehyde. High expression of Bcl-2 and low expression of Bax and caspase-3 supported the anti-apoptotic effect of CGD on the cardiovascular complications of MS. CONCLUSION: CGD has a therapeutic effect on MS and associated cardiovascular complications by eliciting mitochondrial protection and having anti-oxidation and anti-apoptosis effects. CGD could be used for MS treatment.

The anti-adiposity effect of bitter melon seed oil is solely attributed to its fatty acid components.

BACKGROUND: Obesity is the leading chronic disease affecting people of all ages. The objective of this study was to optimize composition of a bitter melon seed oil (BMSO) product to maximize its anti-adiposity effect. METHODS: Bleaching oil, saponifiables and non-saponifiables were prepared from BMSO, with α-eleostearic acid (α-ESA) content in BMSO maintained in bleaching oil and saponifiables. C57BL/6 J mice were allocated into five groups (n = 10/group) to receive diet C [30% soybean oil (SBO)], BM [25% SBO + 5% BMSO], BMS, BMNS or BMD. For the three latter diets, saponifiables (hydrolyzed fatty acids from BMSO), non-saponifiables (excluding fatty acids from BMSO) or bleaching oil (excluding pigments from BMSO), respectively, were added in amount equivalent to their content in 5% BMSO and SBO was added to bring total fat to 30%. After 14 wk., indices associated with adiposity and safety, as well as lipid metabolic signaling in white adipose tissue (WAT), were measured. RESULTS: The body fat percentage of mice in group BM, BMS, BMNS, and BMD were 90 ± 26, 76 ± 21, 115 ± 30 and 95 ± 17% of that in group C. Based on body fat percentage and plasma leptin concentrations, an anti-adiposity effect was evident in groups BM, BMS and BMD (greatest effect in BMS). Histologically, inguinal fat had smaller adipocytes in groups BM, BMS and BMD (P < 0.05), but not in group BMNS, relative to group C. There were no differences among groups in blood pressure or heart rate. Moreover, Sirt1 mRNA levels in inguinal fat were significantly greater in groups BM, BMS and BMD than group C. CONCLUSION: We concluded that the anti-adiposity function of BMSO was solely attributed to the fatty acid fraction, with the free fatty acid form having the greatest effect.

Incorporation and effects of punicic acid on muscle and adipose tissues of rats.

BACKGROUND: This study evaluated the effect of pomegranate seed oil (PSO) supplementation, rich in punicic acid (55 %/C18:3-9c,11 t,13c/CLNA), on the lipid profile and on the biochemical and oxidative parameters in the gastrocnemius muscle and adipose tissues of healthy rats. Linseed oil (LO), rich in linolenic acid (52 %/C18:3-9c12c15c/LNA) was used for comparison. METHODS: Male Wistar rats (n = 56) were distributed in seven groups: control (water); LNA 1 %, 2 % and 4 % (treated with LO); CLNA 1 %, 2 % and 4 % (treated with PSO), po for 40 days. The percentages were compared to the daily feed intake. Fatty acid profile were performed by gas chromatography, antioxidant enzymes activity by spectrophotometer and the adipocytes were isolated by collagenase tissue digestion. Analysis of variance (ANOVA) was applied to check for differences between the groups (control, LNAs and CLNAs) and principal component analysis (PCA) was used to project the groups in the factor-place (PC1 vs PC2) based on the biochemical responses assessed in the study. RESULTS: The fatty acids profile of tissues showed that the LNA percentages were higher in the animals that were fed LO. However, PA was only detected in the adipose tissues. Conjugated linoleic acid (CLA) was present in all the tissues of the animals supplemented with PSO, in a dose dependent manner, and 9c11t-CLA was the predominant isomer. Nevertheless there were no changes in the total weight gain of the animals, the weights of the tissues, and the oxidative stress parameters in the muscle. In addition, there was an increase in the size of the epididymal fat cells in the groups treated with PSO. Principal component analysis (PCA) showed that the CLNAs groups were arranged separately with a cumulative variance of 68.47 %. CONCLUSIONS: The results show that PSO can be used as a source of CLAs but that it does not cause changes in body modulation and does not interfere in the antioxidant activity of healthy rats.

Activity of dietary fatty acids on FFA1 and FFA4 and characterisation of pinolenic acid as a dual FFA1/FFA4 agonist with potential effect against metabolic diseases.

Various foods are associated with effects against metabolic diseases such as insulin resistance and type 2 diabetes; however, their mechanisms of action are mostly unclear. Fatty acids may contribute by acting as precursors of signalling molecules or by direct activity on receptors. The medium- and long-chain NEFA receptor FFA1 (free fatty acid receptor 1, previously known as GPR40) has been linked to enhancement of glucose-stimulated insulin secretion, whereas FFA4 (free fatty acid receptor 4, previously known as GPR120) has been associated with insulin-sensitising and anti-inflammatory effects, and both receptors are reported to protect pancreatic islets and promote secretion of appetite and glucose-regulating hormones. Hypothesising that FFA1 and FFA4 mediate therapeutic effects of dietary components, we screened a broad selection of NEFA on FFA1 and FFA4 and characterised active compounds in concentration-response curves. Of the screened compounds, pinolenic acid, a constituent of pine nut oil, was identified as a relatively potent and efficacious dual FFA1/FFA4 agonist, and its suitability for further studies was confirmed by additional in vitro characterisation. Pine nut oil and free and esterified pure pinolenic acid were tested in an acute glucose tolerance test in mice. Pine nut oil showed a moderately but significantly improved glucose tolerance compared with maize oil. Pure pinolenic acid or ethyl ester gave robust and highly significant improvements of glucose tolerance. In conclusion, the present results indicate that pinolenic acid is a comparatively potent and efficacious dual FFA1/FFA4 agonist that exerts antidiabetic effects in an acute mouse model. The compound thus deserves attention as a potential active dietary ingredient to prevent or counteract metabolic diseases.

[Influence of Oxygenated Derivatives of Linoleic and Linolenic Acids on the Formation of Conidia and Protoperithecia in Wild-Type and Photoreceptor Complex Mutants of Neurospora crassa].

The regulatory effect of two oxyderivatives of unsaturated fatty acids (oxylipins), 18-hydroxy-(9Z,12Z)-octadecadienoic acid (18-HODE) and 18-(9Z,12Z,15Z)-octadecatrienoic acid (18-HOTrE), on the sexual and asexual sporulation of wild-type Neurospora crassa strains and wc-1 and wc-1 mutants was studied. In the wild-type strain, 18-HODE, unlike 18-HOTrE, stimulated protoperithecia formation in the dark and in the light. In the same strain, the studied oxylipins influenced conidiagenesis only under illumination. 18-HODE stimulated and 18-HOTrE inhibited the conidia formation. Oxylipins had no effect on protoperithecia formation in photoreceptor complex mutants, which apparently indicated its involvement in signal transmission in N. crassa. The stimulating action of the studied oxylipins on conidiagenesis in wc-1 and the lack of action in wc-2 may indicate alternative signaling pathways of oxylipins in this process.

Luteolin, ellagic acid and punicic acid are natural products that inhibit prostate cancer metastasis.

Prostate cancer (PCa) is the second cause of cancer deaths in men in the USA. When the cancer recurs, early stages can be controlled with hormone ablation therapy to delay the rate of cancer progression but, over time, the cancer overcomes its hormone dependence, becomes highly aggressive and metastasizes. Clinical trials have shown that pomegranate juice (PJ) inhibits PCa progression. We have previously shown that the PJ components luteolin (L), ellagic acid (E) and punicic acid (P) together inhibit growth of hormone-dependent and -independent PCa cells and inhibit their migration and chemotaxis towards CXCL12, a chemokine that is important in PCa metastasis. On the basis of these findings, we hypothesized that L+E+P inhibit PCa metastasis in vivo. To test this possibility, we used a severe combined immunodeficiency mouse model in which luciferase-expressing human PCa cells were injected subcutaneously near the prostate. Tumor progression was monitored with bioluminescence imaging weekly. We found that L+E+P inhibits PC-3M-luc primary tumor growth, inhibits the CXCL12/CXCR4 axis for metastasis and none of the tumors metastasized. In addition, L+E+P significantly inhibits growth and metastasis of highly invasive Pten (-/-) ;K-ras (G12D) prostate tumors. Furthermore, L+E+P inhibits angiogenesis in vivo, prevents human endothelial cell (EC) tube formation in culture and disrupts preformed EC tubes, indicating inhibition of EC adhesion to each other. L+E+P also inhibits the angiogenic factors interleukin-8 and vascular endothelial growth factor as well as their induced signaling pathways in ECs. In conclusion, these results show that L+E+P inhibits PCa progression and metastasis.

Defense activated by 9-lipoxygenase-derived oxylipins requires specific mitochondrial proteins.

9-Lipoxygenases (9-LOXs) initiate fatty acid oxygenation, resulting in the formation of oxylipins activating plant defense against hemibiotrophic pathogenic bacteria. Previous studies using nonresponding to oxylipins (noxy), a series of Arabidopsis (Arabidopsis thaliana) mutants insensitive to the 9-LOX product 9-hydroxy-10,12,15-octadecatrienoic acid (9-HOT), have demonstrated the importance of cell wall modifications as a component of 9-LOX-induced defense. Here, we show that a majority (71%) of 41 studied noxy mutants have an added insensitivity to isoxaben, an herbicide inhibiting cellulose synthesis and altering the cell wall. The specific mutants noxy2, noxy15, and noxy38, insensitive to both 9-HOT and isoxaben, displayed enhanced susceptibility to Pseudomonas syringae DC3000 as well as reduced activation of salicylic acid-responding genes. Map-based cloning identified the mutation in noxy2 as At5g11630 encoding an uncharacterized mitochondrial protein, designated NOXY2. Moreover, noxy15 and noxy38 were mapped at the DYNAMIN RELATED PROTEIN3A and FRIENDLY MITOCHONDRIA loci, respectively. Fluorescence microscopy and molecular analyses revealed that the three noxy mutants characterized exhibit mitochondrial dysfunction and that 9-HOT added to wild-type Arabidopsis causes mitochondrial aggregation and loss of mitochondrial membrane potential. The results suggest that the defensive responses and cell wall modifications caused by 9-HOT are under mitochondrial retrograde control and that mitochondria play a fundamental role in innate immunity signaling.

Potential anti-inflammatory effects of the hydrophilic fraction of pomegranate (Punica granatum L.) seed oil on breast cancer cell lines.

In this work, we characterized conjugated linolenic acids (e.g., punicic acid) as the major components of the hydrophilic fraction (80% aqueous methanol extract) from pomegranate (Punica granatum L.) seed oil (PSO) and evaluated their anti-inflammatory potential on some human colon (HT29 and HCT116), liver (HepG2 and Huh7), breast (MCF-7 and MDA-MB-231) and prostate (DU145) cancer lines. Our results demonstrated that punicic acid and its congeners induce a significant decrease of cell viability for two breast cell lines with a related increase of the cell cycle G0/G1 phase respect to untreated cells. Moreover, the evaluation of a great panel of cytokines expressed by MCF-7 and MDA-MB-231 cells showed that the levels of VEGF and nine pro-inflammatory cytokines (IL-2, IL-6, IL-12, IL-17, IP-10, MIP-1α, MIP-1ß, MCP-1 and TNF-α) decreased in a dose dependent way with increasing amounts of the hydrophilic extracts of PSO, supporting the evidence of an anti-inflammatory effect. Taken together, the data herein suggest a potential synergistic cytotoxic, anti-inflammatory and anti-oxidant role of the polar compounds from PSO.

Fatty Acids Increase GDF15 and Reduce Food Intake Through a GFRAL Signaling Axis.

In contrast to the well-defined biological feedback loops controlling glucose, the mechanisms by which the body responds to changes in fatty acid availability are less clearly defined. Growth differentiating factor 15 (GDF15) suppresses the consumption of diets high in fat but is paradoxically increased in obese mice fed a high-fat diet. Given this interrelationship, we investigated whether diets high in fat could directly increase GDF15 independently of obesity. We found that fatty acids increase GDF15 levels dose dependently, with the greatest response observed with linolenic acid. GDF15 mRNA expression was modestly increased in the gastrointestinal tract; however, kidney GDF15 mRNA was ∼1,000-fold higher and was increased by more than threefold, with subsequent RNAscope analysis showing elevated expression within the cortex and outer medulla. Treatment of wild-type mice with linolenic acid reduced food intake and body mass; however, this effect disappeared in mice lacking the GDF15 receptor GFRAL. An equal caloric load of glucose did not suppress food intake or reduce body mass in either wild-type or GFRAL-knockout mice. These data indicate that fatty acids such as linolenic acid increase GDF15 and suppress food intake through a mechanism requiring GFRAL. These data suggest that a primary physiological function of GDF15 may be as a fatty acid sensor designed to protect cells from fatty acid overload. ARTICLE HIGHLIGHTS: The mechanisms by which the body responds to changes in fatty acid availability are less clearly defined. We investigated whether diets high in fat could directly increase growth differentiating factor 15 (GDF15) independently of obesity. Fatty acids increase GDF15 and reduce food intake through a GFRAL signaling axis. GDF15 is a sensor of fatty acids that may have important implications for explaining increased satiety after consumption of diets high in fat.

A new integrated strategy for high purity pinolenic acid production from Pinus koraiensis Sieb. et Zucc seed oil and evaluation of its hypolipidemic activity in vivo.

Pinolenic acid is a polyunsaturated fatty acid present only in Pinus koraiensis Sieb. et Zucc seed oil. In order to solve the structural instability problem of polyunsaturated fatty acids, pinolenic acid of P. koraiensis seed oil was effectively isolated and purified by the integrated strategy of ethyl esterification followed by urea inclusion for the first time. Under the optimal conditions after the Box-Benhnken Design experimental, ethyl pinolenate with high purity 94.95% could be obtained, and the average content of PNAEE can still reach 86.18%. Then ethyl pinolenate was characterized by Gas chromatography-mass spectrometry, Fourier transform infrared, and Nuclear magnetic resonance spectra, results showed that ethyl pinolenate was successfully prepared. In addition, the hypolipidemic activity of ethyl pinolenate had been tested in vivo and showed that ethyl pinolenate had obvious hypolipidemic activity. The new strategy for high purity ethyl pinolenate production from P. koraiensis seed oil possesses great potential in food healthy field in the future.

Punicic acid ameliorates obesity-related hyperlipidemia and fatty liver in mice via regulation of intestinal flora and lipopolysaccharide-related signaling pathways.

Punicic acid (PA), mainly found in pomegranate seed oil (PSO), has attracted increasing attention due to its potential to mitigate obesity. The regulation of intestinal microflora was identified as a crucial factor and an effective strategy to reverse obesity-related hyperlipidemia and non-alcoholic fatty liver disease (NAFLD). To assess the impact of PSO on hyperlipidemia related to obesity, we investigated the hepatic lipid status and gut microbiota regulation in mice over 13 weeks of feeding a high-fructose high-fat diet (HFHFD). Serum lipid markers, including TG, TC and LDL-C, were markedly reduced in hyperlipidemic mice. PSO supplementation reduced hepatic lipid accumulation and steatosis, inhibited the expression of pro-inflammatory mediators (including IL-6 and IL-1ß), and restored the normal levels of the anti-inflammatory cytokine IL-10. In addition, PSO also alleviated oxidative stress and increased T-AOC and SOD activities, as well as GSH levels, while reducing the MDA content in the liver of HFHFD-fed mice. The activation of TLR4/MyD88/NF-κB and TLR4/IL-22/STAT3 signaling pathways in the liver due to the HFHFD was also evidently inhibited by PSO. Furthermore, supplementation of PSO ameliorated the HFHFD-induced dysbiosis of intestinal microflora, resulting in a markedly increased proportion of Muribaculaceae, a decreased ratio of Blautia, and elevated levels of microbiota-derived short-chain fatty acids (SCFAs). Moreover, the expression of tight junction proteins correlated with intestinal barrier function was notably restored in the colon. The collected results indicate that PSO may be an effective nutraceutical ingredient for attenuating lipid metabolic disorders.