Linoleic Acid Treatment Research Articles

Abstract 121: A genetic signature of cancer dormancy: implications for human disease derived from a novel murine model

Abstract Pancreatic cancer (PC) is a highly lethal malignancy characterized by local tumor aggressiveness and early metastatic dissemination. While surgical resection is used to treat localized cancers, the majority of patients do recur suggesting the presence of disseminated tumor cells (DTC) at the time of surgery. These DTC's represent minimal residual disease. While most patients recur early, a subset recur late due to the reactivation of dormant tumor cells. Currently, the mechanisms of cancer dormancy are poorly understood in part due to a lack of animal models that reflect human disease. Here we describe a murine model of PC dormancy that mimics outcomes in resected human patients. Using single-cell transcriptomics and an assay for chromatin accessibility, we found dormancy is a distinct cellular state from pre or post-dormant cancer cells. Mechanisms of dormancy include increased expression of transcriptional repressor Dec2, which functionally drives quiescence, and monoallelic suppression of mutant KRAS by DNA methylation. Pathway analysis of dormant tumor cell transcriptomics indicated an up-regulation of enzymes involved in linoleic acid metabolism among others. Linoleic acid treatment of tumor cells resulted in inhibited cell proliferation, decreased tumor growth, and an increase in Dec2 expression. We have identified a dormancy gene signature that is distinct from non-dormant PC cells and found that this correlates with human resected patients with long term survivals, and inversely in patients with short term survivals. We demonstrate that dormancy is characterized by large scale transcriptomic changes and global chromatin remodeling. We developed methods for isolating DTCs from the livers of early stage PC patients undergoing resection. Using these methods we have found that the murine dormancy signature correlated with the gene expression of the DTCs and not primary tumor cells indicating this dormancy signature has human relevance with potential novel therapeutic avenues. Citation Format: Anthony S. Casabianca, Crissy Dudgeon, Chris Harris, Subhajyoto De, Mihir Shah, Arthur Roberts, Eric Collisson, Vinod Balachandran, Darren Carpizo. A genetic signature of cancer dormancy: implications for human disease derived from a novel murine model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 121.

Activation of Hair Cell Growth Factors by Linoleic Acid in Malva verticillata Seed.

Hair loss by excessive stress from work and lifestyle changes has become a growing concern, particularly among young individuals. However, most drugs for alopecia impose a plethora of side effects. We have found the powerful impact of Malva verticillata seed extracts on alleviating hair loss. This study further isolated effective chemicals in M. verticillata seed extracts by liquid silica gel column chromatography. Under the screening for the growth rate (%) of human follicles dermal papilla cells (HFDPCs), we identified linoleic acid (LA) and oleic acid in n-hexane of M. verticillate (MH)2 fraction. LA treatment activated Wnt/β-catenin signaling and induced HFDPCs growth by increasing the expression of cell cycle proteins such as cyclin D1 and cyclin-dependent kinase 2. LA treatment also increased several growth factors, such as vascular endothelial growth factor, insulin-like growth factor-1, hepatocyte growth factor, and keratinocyte growth factor, in a dose-dependent manner. Besides, LA significantly inhibited Dickkopf-related protein expression (DKK-1), a primary alopecia signaling by dihydrotestosterone. Our findings suggest that LA treatment may alleviate a testosterone-induced signaling molecule and induces HFDPCs growth by activating Wnt/β-catenin signaling.

Peroxidized Linoleic Acid, 13-HPODE, Alters Gene Expression Profile in Intestinal Epithelial Cells.

Lipid peroxides (LOOHs) abound in processed food and have been implicated in the pathology of diverse diseases including gut, cardiovascular, and cancer diseases. Recently, RNA Sequencing (RNA-seq) has been widely used to profile gene expression. To characterize gene expression and pathway dysregulation upon exposure to peroxidized linoleic acid, we incubated intestinal epithelial cells (Caco-2) with 100 µM of 13-hydroperoxyoctadecadienoic acid (13-HPODE) or linoleic acid (LA) for 24 h. Total RNA was extracted for library preparation and Illumina HiSeq sequencing. We identified 3094 differentially expressed genes (DEGs) in 13-HPODE-treated cells and 2862 DEGs in LA-treated cells relative to untreated cells. We show that 13-HPODE enhanced lipid metabolic pathways, including steroid hormone biosynthesis, PPAR signaling, and bile secretion, which alter lipid uptake and transport. 13-HPODE and LA treatments promoted detoxification mechanisms including cytochrome-P450. Conversely, both treatments suppressed oxidative phosphorylation. We also show that both treatments may promote absorptive cell differentiation and reduce proliferation by suppressing pathways involved in the cell cycle, DNA synthesis/repair and ribosomes, and enhancing focal adhesion. A qRT-PCR analysis of representative DEGs validated the RNA-seq analysis. This study provides insights into mechanisms by which 13-HPODE alters cellular processes and its possible involvement in mitochondrial dysfunction-related disorders and proposes potential therapeutic strategies to treat LOOH-related pathologies.

Study of the Effect of Linoleic Acid on the Expression Level of MicroRNA-106b and MicroRNA-20a and their Related Target MHC Class I Chain-related Protein A in Docetaxel-treated Gastric Cancer Cells

Background: MicroRNAs are involved in response to therapeutic agents and have the ability to regulate the expression level of the targets associated with cancer growth and progression. As a dangerous signal in tumor cells, increased expression level of MHC class I chain-related protein A (MICA) could activate the immune system and induce responses to tumor cells. We conducted the present research to study the effect of linoleic acid (LA) and docetaxel alone or in combination with miR-106b, miR-20a, and MICA expression level in metastatic gastric cancer (GC) cell line MKN-45. Method: The study was an in vitro study using the gastric cancer cell line MKN- 45, which was cultured and treated with docetaxel and LA. Subsequently, the expression level of miR-106b, miR-20a, and MICA were assessed with quantitative real-time polymerase chain reaction. Results: MiR-106b decreased in LA and LA/docetaxel (P < 0.0001 and P = 0.002),and increased in docetaxel alone (P = 0.01). Meanwhile, miR-20a significantly decreased in docetaxel and LA/docetaxel (P < 0.0001), increased in LA treatment (P = 0.02). Regarding MICA, it significantly decreased in all the treated cells (P < 0.0001, p <0.0001, and P=0.0002 for docetaxel, LA and docetaxel/LA, respectively) but with different reduction intensities. Conclusion: Using LA or docetaxel alone had a different effect on miR-106b, miR-20a, and MICA expression level, yet in a simultaneous treatment, their positive effects were intensified. LA enhanced the effect of docetaxel concerning the expression level of miR-106b, miR-20a, and MICA and vice versa, which suggested that LA could be employed as an effective complementary agent in GC along with docetaxel.

Induction of Beneficial Changes in Glucose Homeostasis, Lipid Profile, Inflammation, Retinopathy, Bile Acids and Gut Microbiota in Streptozotocin-Induced Type 1 Diabetes Mellitus by Essential Fatty Acids

Background: Essential fatty acids (EFAs) n3 alpha-linolenic acid (ALA) and n6 linoleic acid (LA) have been shown to improve lipid metabolism, promote nervous system development, suppress inflammation, and are of benefit in diabetes mellitus and cancer. But the exact mechanism of their action(s) is not clear. In the present study, we evaluated the effects of ALA and LA on glucose homeostasis, circulating lipid profile, inflammatory markers, gut microbiome, and bile acids in streptozotocin (STZ)-induced type 1 diabetes with retinopathy in rats. We also measured vitreal LXA4 levels in patients with diabetic retinopathy since arachidonic acid (AA), the precursor of LXA4, is derived from LA. Methods: The study was performed in male Sprague Dawley rats. Type 1 DM was induced by intraperitoneal injection of STZ. The type 1 DM animals received LA or ALA 100 ug/day via intraperitoneal route on alternate days till the end of the study (21 days after the DM onset). In these animals, glucose homeostasis and lipid profile were evaluated. Plasma and retinal fatty acid profile by gas chromatography (GC), retinal histology by H&E staining, activities of desaturases in the retina and liver tissues and inflammatory markers by qRT-PCR, and composition of the gut microbiota by 16S rDNA sequencing technology of the fecal samples were performed. In addition, short-chain fatty acids (SCFAs) and bile acids (BAs) in the feces and liver of rats were assayed by GC and liquid chromatography coupled with tandem mass spectrometry (UHPLC–MS/MS), respectively. Human vitreal samples obtained from patients with proliferative diabetic retinopathy (PDR) were analyzed for their fatty acids profile and LXA4 levels and compared with those with macular hole (control). Findings: LA and ALA-treated STZ-induced type 1 DM animals showed significant reduction in their plasma glucose levels, pro-inflammatory cytokines (IL-6 and TNF-α), changes in the composition of gut microbiome in the form of an increase in the abundance of Ruminococcaceae (ALA-treated group), Alloprevotella, Prevotellaceae_Ga6A1_group, Ruminococcaceae_UCG_010 and Ruminococcus_1 (in LA-treated group) , an increase in acetic acid and butyric acid, and an increase in the fecal concentrations of CA, CDCA and TUDCA and a decrease in GCA. Concentrations of hepatic CDCA, TCDCA, and TUDCA were significantly increased in ALA treated animals while CDCA were elevated in those with LA treatment. STZ-treated animals showed significant decrease in plasma LA, GLA, and AA and ALA levels with no significant changes in retinal fatty acid composition. All these changes reverted to normal in LA and ALA treated group. The changes in the plasma fatty acid composition were accompanied by significant alterations in the expression of desaturases, COX-2 and 5-LOX and 12-LOX enzymes and pro- and anti-inflammatory cytokines. In addition, retinal LXA4 levels were found to be decreased in patients with PDR. Interpretation: The results of the present study suggest that ALA and LA administration can improve plasma glucose levels, suppress inflammation, and produce significant beneficial alterations in the gut microbiota, plasma lipid profile and EFAs metabolism. Decrease in retinal LXA4 levels in those with PDR agree with the suggestion that EFAs metabolism is altered in T1DM and DR. These results imply a significant role for EFAs and their metabolites in the pathobiology of type 1 DM and diabetic retinopathy. Funding Information: The project was funded by the National Natural Science Foundation of China (No. 82000886), and Natural Science Foundation of Zhejiang Province (No.LQ20H120011). Declaration of Interests: Authors have no conflicts of interest to declare. Ethics Approval Statement: This study was approved by the animal ethics committee of the Second Affiliated Hospital of Zhejiang University school of medicine. All experimental procedures using animals were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. This study was approved by the Clinical Ethical Research Committee of the Second Affiliated Hospital of Zhejiang University school of medicine. The study was performed as per the tenets of Helsinki declaration. Informed consent obtained from all the participating patients.

Symptom Duration Shortened by Early Initiation of Nebulized Isomerized Linoleic Acid (LA) for Outpatient Treatment of COVID-19.

With a total of 6 patients, albeit a small number, we find a statistically significant correlation between 'when beginning nebulizing treatment with LA' and the 'length of reported COVID-related symptoms' (p<0.01; r=0.965). We report the clinical course of four COVID-19 cases, 2 probable cases, and 2 close contacts treated with nebulized Isomerized Linoleic Acid (LA). Comorbidity ranged from none to high risk. No side effects were reported by this group of patients. Isomerized LA was completely effective in the small sample of close contacts. Early treatment onset shortened the duration of symptoms. We define symptom duration as the total length of symptoms that were reported during the course of illness. All patients reported a total resolution of symptoms after treatment, except for one who started nebulized LA 4 days after onset of symptoms of infection, who reported some lingering fatigue. The case study findings could pose a potential timeline for future research into when nebulized LA treatment would be most effective. Complications of COVID-19 appeared to be affected by early onset of treatment. Since global case counts and deaths attributed to COVID-19 are continuing to rise, there needs to be an effective, low cost outpatient therapeutic available. Nebulized LA provides the opportunity for just that, a safe, low cost, and effective option for outpatient COVID-19 treatment. We hope this case study will prompt further and much needed research into COVID-19 outpatient treatment.

Alterations in niban gene expression as a response to stress conditions in 3T3-L1 adipocytes.

Adipocyte death is important in obesity development. Understanding and prevention of adipocyte deaths may be a molecular approach in the treatment. In the study, we aimed to understand role of Niban gene, which acts as an anti-apoptotic molecule as a response to stress conditions, in adipocytes. 3T3-L1 adipocytes were treated with different doses of linoleic acid, hydrogen peroxide and ethanol; and proliferation of the cells examined with real time monitoring iCELLingence system. Gene expression levels were measured by q-PCR. As a response to 24h 480 µM linoleic acid treatment, Niban gene expression was found to be higher than control group (p = 0.008), whereas 24 h 90 mM ethanol treatment was determined to be lower than control group (p = 0.008). The highest value of Niban gene expression among H2O2 treatment groups was detected in 4h 600µM H2O2 in comparison to control group (p = 0.008). To understand role of Niban in adipogenesis, Niban gene expressions were compared between pre-adipocytes and advanced fat accumulated adipocytes and determined to be significantly different (p = 0.042). Our results suggest that Niban might be involved in stress response process in adipocytes. However, the exact molecular role of Niban needs to be investigated in further studies.

Linoleic acid and α-linolenic acid inhibit conjugative transfer of an IncX4 plasmid carrying mcr-1.

The aim of this study was to determine the effects of unsaturated fatty acids on clinical plasmids. Two unsaturated fatty acids, linoleic acid (LA) and α-linolenic acid (ALA) at final concentration 0, 0·03, 0·3 and 3mmoll-1 , respectively, were used to assess the effects on conjugative transfer of a mcr-1-harbouring plasmid pCSZ4 (IncX4) in conjugation experiment. The inhibitory mechanisms were analysed by molecular docking and the gene expression of virB11 was quantitated by qRT-PCR. Target plasmid diversity was carried out by TrwD/VirB11 homology protein sequence prediction analysis. Our results showed that LA and ALA inhibit plasmid pCSZ4 transfer by binding to the amino acid residues (Phe124 and Thr125) of VirB11 with dose-dependent effects. The expression levels of virB11 gene were also significantly inhibited by LA and ALA treatment. Protein homology analysis revealed a wide distribution of TrwD/VirB11-like genes among over 37 classes of plasmids originated from both Gram-negative and Gram-positive bacteria. This study demonstrates representing a diversity of plasmids that may be potentially inhibited by unsaturated fatty acids. Our work reported here provides additional support for application of curbing the spread of multiple plasmids by unsaturated fatty acids.

Topical application of autophagy-activating peptide improved skin barrier function and reduced acne symptoms in acne-prone skin.

Recent studies about the important roles of autophagy signaling in sebaceous lipogenesis and epidermal differentiation suggest potential benefits of autophagy activation in acne. To investigate the effects of an autophagy activator on acne-prone skin. Autophagy signaling in human immortalized SZ95 sebocytes, normal human epidermal keratinocytes, and 3D reconstituted skin was examined. Effects of an autophagy-activating peptide on sebaceous lipogenesis were measured by fluorescence microscopic analysis. The clinical efficacy in acne-prone skin was evaluated through an eight-week, double-blind, randomized, vehicle-controlled study. Changes in skin surface lipid compositions were further analyzed. In cultured sebocytes and keratinocytes, the investigated autophagy-activating peptide increased LC3-II expression, indicating a stimulation of autophagy signaling. Testosterone and linoleic acid treatment induced lipogenesis in cultured sebocytes and is further inhibited by the autophagy activator peptide treatment. Increased expression of differentiation marker proteins in cultured keratinocytes was also observed by autophagy-activating peptide. In clinical study, reduction of closed comedones and the amount of skin surface lipids as well as of trans-epidermal water loss (TEWL) were observed in acne-prone skin after autophagy-activating peptide application. In addition, reduction of squalene and increase in cholesterol were observed after an 8-week application. Topical application of an autophagy activator downregulated sebaceous lipogenesis and improved the skin barrier function. Considering the important roles of sebum and skin barrier function in acne pathogenesis, autophagy activation might represent a new therapeutic option in early forms of acne.

125-OR: Polyunsaturated Omega-6 Fatty Acids Reduce Axonal Mitochondrial Trafficking in Dorsal Root Ganglion Sensory Neurons

Peripheral neuropathy is a prevalent complication of diabetes and prediabetes characterized by distal-to-proximal peripheral nerve damage. Dyslipidemia is a contributory factor that underlies neuropathy development in type 2 diabetes and prediabetes. However, the molecular pathways that underlie sensory neuron dysfunction in peripheral neuropathy associated with diabetes and prediabetes are unknown. Our recent lipidomic and transcriptomic analyses identified altered metabolism of polyunsaturated omega-6 fatty acid linoleic acid in the peripheral nerves of prediabetic mice with neuropathy. However, the molecular effect of linoleic acid on sensory neurons remains unknown. Since sensory dorsal root ganglion (DRG) neurons are dependent on trafficking mechanisms to transport healthy mitochondria throughout the length of the axon, we evaluated the effect of linoleic acid on axonal mitochondrial trafficking and function in DRG neurons. Primary DRG neurons from adult C57BL/6J mice were treated with physiological concentrations of linoleic acid ranging from 31.25-250 µM for 24 hours. Following treatment, we evaluated the effect of linoleic acid treatments on mitochondrial trafficking, mitochondrial membrane potential, ATP level, and apoptosis in DRG neurons. Interestingly, all concentrations of linoleic acid ranging from 31.25-250 µM preserved mitochondrial membrane potential and ATP level in DRG neurons. In line with these results, linoleic acid treatments did not stimulate neuronal apoptosis. However, physiological diabetic concentrations of 250 µM linoleic acid significantly impaired mitochondrial trafficking in the axon of DRG neurons. Overall, these results suggest that elevated levels of linoleic acid impair axonal mitochondrial trafficking without effecting mitochondrial function, and that defects in mitochondrial trafficking of sensory neurons may contribute to neuropathy pathogenesis in diabetes and prediabetes. Disclosure A. Rumora: None. G. LoGrasso: None. J.D. McGrath: None. S.I. Lentz: None. E.L. Feldman: Consultant; Self; Novartis Pharmaceuticals Corporation. Funding American Diabetes Association (7-12-BS-045 to E.L.F.); National Institutes of Health (1R24082841, 1DP3DK094292); National Institute of Diabetes and Digestive and Kidney Diseases (T32DK101357, F32DK112642, K99DK119366, P30DK020572); NeuroNetwork for Emerging Therapies; A. Alfred Taubman Medical Research Institute

Modulatory Effect of Linoleic Acid During Brucella abortus 544 Infection in Murine Macrophage RAW264.7 Cells and Murine Model BALB/c Mice.

In this study, we investigated the effects of linoleic acid (LA) treatment on Brucella abortus infection in professional phagocyte RAW264.7 cells, particularly during the pathogens invasion and intracellular growth in these cells, as well as in murine model BALB/c mice focusing on bacterial splenic proliferation and immunoregulatory activities. LA inhibited the growth of Brucella in a doseand time-dependent manner. The ability of the pathogen to enter the phagocytes was inhibited as was its survival within these cells. This was accompanied by increased nitrite accumulation in these cells at 24 h post-infection. The concentration of LA used in the present study did not affect the total body weight or liver function of the mice. During Brucella infection, the total splenic weight of these animals was not changed; rather, resistance to bacterial proliferation was enhanced in the spleen. Furthermore, mice treated with LA displayed elevated levels of IL-12 and IFN-γ but reduced levels of IL-10 during infection. The findings in this study showed the regulatory role of LA against B. abortus infection suggesting its potential use in designing intervention strategy for brucellosis.

Abstract P3-02-07: Fatty acid metabolism is associated with chromatin remodeling in mammary epithelial cells

Abstract Introduction: Altered metabolism is a hallmark of cancer. We have reported that a set of lipid metabolism (LiMe) genes is over-expressed in the contralateral unaffected breasts (CUBs) of women with unilateral estrogen receptor negative (ER-) breast cancer, who are at high risk for future ER- tumors. We hypothesized that a potential mechanism connecting lipid metabolism to risk of ER- disease may involve epigenetic regulation of gene expression in breast cells by lipids, as shown by McDonnel et. al., Cell reports 2016 in hepatocytes. Methods: Mass spectrometry was performed to profile the composition of lipid species in the sera of patients with ER+ versus ER- disease. MCF-10A cells and mammary organoids from reduction mammoplasty patients were treated with Octanoic (OA) and Linoleic acid (LA) followed by western blot analysis of acetylated histones and sequencing of RNA with qPCR confirmation of significant findings. Partial wave spectroscopic (PWS) microscopy was employed to study change in chromatin. Epithelial RNA from microdissected benign breast samples from CUBs of 84 patients (28 ER+, 28 ER- and 28 healthy controls) were subjected to qPCR. Results: Sera from ER- patients showed higher levels of two triacylglycerides: TAG46:4-FA18:2 (1.25-fold, p=0.05) and TAG 47:2-FA18:2 (1.35-fold, p=0.07), both of which contain LA. MCF10A cells and human mammary organoids showed induction of H3 acetylation at lysine 9 &amp; 14 after treatment with LA and OA treatment at physiological levels. This was accompanied by evidence of chromatin remodeling and 2-fold increase in DNA accessibility on PWS. RNA-Seq of OA treated MCF-10A cells revealed significant upregulation of LiMe genes together with pathways associated with breast malignancy: Notch, Wnt, EMT and adenylate cyclase. The adenylate cyclase pathway (known to be specifically associated with ER- breast cancer risk), was enriched 3.7-fold (p=0.007). In RNA from CUBs of 84 patients, we found overexpression of Notch pathway intermediates that responded to lipid exposure in MCF10A in the ER- group, specifically Notch1 (1.8-fold, p=0.02) and Hey1 (2.9-fold, p=0.05). Conclusion: Specific classes of dietary lipids are the source of acetyl-CoA which acetylates histones and remodels chromatin to modulate gene transcription in ER- MCF10A cells. Thus revved-up fatty acid oxidation may play a role in ER- breast oncogenesis via Notch signaling. Our novel findings point to potential interception strategies that target connections between lipid metabolism and histone modifications. Citation Format: Shivangi Yadav, MiRan Choi, David Van Derway, Greta Braur, Vadim Backman, Seema Ahsan Khan, Susan Clare. Fatty acid metabolism is associated with chromatin remodeling in mammary epithelial cells [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-02-07.

Enhancement of linoleic acid content stimulates astaxanthin esterification in Coelastrum sp.

Most natural astaxanthin is fatty acid-esterified in microalgae to prevent oxidation. However, the factors influencing astaxanthin esterification (AE) are poorly understood. In this study, obstacles to AE in Coelastrum sp. HA-1 were investigated. Only half of the astaxanthin molecules in HA-1 were esterified, but AE was stimulated with exogenous linoleic acid (LA) and ethanol treatment. Astaxanthin esters and total astaxanthin (TA) with exogenous LA were elevated to 3.82-fold and 2.18-fold of control levels, respectively. Treatment with 3% (v/v) ethanol enhanced transcription of the Δ12 fatty acid desaturase gene, which caused more oleic acid (OA) to be converted to LA. Furthermore, the contents of astaxanthin esters and TA were 2.42-fold and 1.61-fold control levels, respectively. These findings confirmed that AE was upregulated by increasing LA content. Thus, a large concentration of OA alone does not increase astaxanthin accumulation in HA-1, and a certain amount of LA was necessary for AE.

Effects of Essential Fatty Acids during In Vitro Maturation of Porcine Oocytes: Hormone Synthesis and Embryonic Developmental Potential

Omega-3 α-linolenic acid and omega-6 linoleic acid are essential fatty acids for health maintenance of human and animals because they are not synthesized in vivo. The purpose of this study was to evaluate the effect of α-linolenic acid and linoleic acid supplementation on in vitro maturation and developmental potential of porcine oocytes. Various concentrations of α-linolenic acid and linoleic acid were added into in vitro maturation medium, and we evaluated the degree of cumulus expansion, oocyte nuclear-maturation rate, blastocyst rate, blastocyst quality, and levels of prostaglandin E2, 17b-estradiol, and progesterone in the spent medium. High doses (100 μM) of α-linolenic acid and linoleic acid supplementation significantly inhibited cumulus expansion and oocyte nuclear maturation, and prostaglandin E2 synthesis also significantly decreased compared with other groups (p < 0.05). Supplementation of 50 μM α-linolenic acid and 10 μM linoleic acid showed higher quality blastocysts in terms of high cell numbers and low apoptosis when compared with other groups (p < 0.05), and synthesis ratio of 17b-estradiol / progesterone also significantly increased compared with control group (3.59 ± 0.22 vs. 2.97 ± 0.22, 3.4 ± 0.28 vs. 2.81 ± 0.19, respectively; p < 0.05). Our results indicated that supplementation with appropriate levels of α-linolenic acid and linoleic acid beneficially affects the change of hormone synthesis (in particular, an appropriate increase in the 17b-estradiol / progesterone synthesis ratio) for controlling oocyte maturation, leading to improved embryo quality. However, high doses of α-linolenic acid and linoleic acid treatment results in detrimental effects.

Regulation of IL-10-producing T cells by polyunsaturated fatty acids

Abstract The modern day western diet has led to an increase of n-6 polyunsaturated fatty acid (n-6 PUFA) intake and is considered an environmental factor resulting in negative health outcomes in different disease settings. In relation to the immune system, it is not well understood how changes in dietary factors like n-6 PUFAs can influence immune responses, for example during autoimmune disease. PUFAs have a variety of roles within cells such as being used as an energy source, incorporation in cell membranes, and conversion into lipid mediators that influence cell signaling and gene expression. We initially sought to understand how n-6 PUFAs influence the phenotype and function of T cell subsets from the Type 1 Diabetes mouse model (NOD) and non-diabetic control mouse model (B6.NOD). By treating splenocytes from these mouse models in vitro with n-6 PUFA linoleic acid we discovered by flow cytometry a dose response inhibition of interleukin 10 (IL-10), an anti-inflammatory cytokine, in both CD4+ and CD8+ T cell subsets. To the contrary, interferon γ production was not diminished after linoleic acid treatment. While IL-10 inhibition was not strain-specific, further studies by gene expression analysis displayed a decrease of IL-10 mRNA expression and of transcription factors involved in regulation of IL-10 (c-maf, IRF4, and Bhlhe40) after linoleic acid treatment. These results demonstrate that dietary n-6 PUFAs regulate T cell cytokine production and can influence the balance of pro- and anti-inflammatory cytokines. Further studies will provide more insight into the regulation of T cell subsets by fatty acids present in the organismal environment, with potential implications for the development of new therapeutics to modulate autoimmune responses.

Effect of linoleic acid and methyl jasmonate on astaxanthin content of Scenedesmus acutus and Chlorella sorokiniana under heterotrophic cultivation and salt shock conditions

Aquatic organisms, especially microalgae, are sources of highly bioactive metabolites which could be used in pharmaceutical, biofuels, food, agricultural, and cosmetics industries. In this research, the effect of linoleic acid (LA), as allelochemical and the precursor of two phytohormones, on astaxanthin production in Scenedesmus acutus and Chlorella sorokiniana was investigated under heterotrophic and salt stress conditions. In the absence of salt addition, addition of 32 μM LA after 96 h post inoculation during C. sorokiniana cultivation enhanced cell astaxanthin concentration 1.6 times compared to the control (no addition of LA), while the addition of 160 μM LA during the logarithmic phase of S. acutus enhanced astaxanthin production 10 times compared to the control. In C. sorokiniana under salt stress conditions, the incremental effect of LA on astaxanthin production is expanded, while salt addition did not have significant impact on astaxanthin production during S. acutus culture. In the presence of 20% salt, methyl jasmonate (MJ) treatment produced approximately threefold astaxanthin compared to LA in C. sorokiniana, while the astaxanthin formation for LA and MJ treatment in the presence of 20% salt were roughly equal during S. acutus culture. It can be therefore concluded that LA, as allelochemical and precursor of jasmonic and traumatic acid in oxylipin pathway, has an important role in the astaxanthin production in microalgae. Moreover, for the first time, the potential of LA as an enhancer of algal astaxanthin production was shown.

Modulatory effect of linoleic and oleic acid on cell proliferation and lipid metabolism gene expressions in primary bovine satellite cells

ABSTRACTThis study was performed to elucidate the effects of linoleic acid (LA), oleic acid (OA) and their combination (LA + OA) on cell proliferation, apoptosis, necrosis, and the lipid metabolism related gene expression in bovine satellite cells (BSCs), isolated from bovine muscles. Cell viability was significantly increased with the OA and LA treatment. Furthermore, LA + OA enhanced cell proliferation in a dose-dependent manner (10 to 100 µM), whereas it lowered at 250 µM. In addition, a cell-cycle analysis showed that 100 µM of LA and OA markedly decreased the G0/G1 phase proportion (62.58% and 61.33%, respectively), compared to controls (68.02%), whereas the S-phase cells’ proportion was increased. The ratio of G2/M phase cells was not significantly different among the groups. Moreover, analyses with AO/EtBr staining showed that no apoptosis occurred. Necrosis were determined by flow cytometry using Annexin V-FITC/PI staining which revealed no early apoptosis in the cells pretreated with LA or OA, but occurred in the LA + OA group. We also analyzed the mRNA expression of lipid metabolizing genes such as peroxisome proliferator receptor alfa (PPARα), peroxisome proliferator receptor gamma (PPARγ), acyl-CoA oxidase (ACOX), lipoprotein lipase (LPL), carnitine palmitoyl transferase (CPT-1), and fatty-acid binding protein4 (FABP4), which were upregulated in LA or OA treated cells compared to the control group. In essence, LA and OA alone promote the cell proliferation without any apoptosis and necrosis, which might upregulate the lipid metabolism related gene expressions, and increase fatty-acid oxidation in the BSCs’ lipid metabolism.

Accumulation of conjugated linoleic acid in Lactobacillus plantarum WU-P19 is enhanced by induction with linoleic acid and chitosan treatment

Production of conjugated linoleic acid (CLA) by the potential probiotic bacterium Lactobacillus plantarum WU-P19 was investigated with the aim of enhancing production. CLA produced using this bacterium may be used to supplement dietary intake. Cultures were fed linoleic acid for conversion to CLA and the CLA produced was measured. In some cases, chitosan was added to cultures to improve cellular uptake of linoleic acid. Under static conditions at 37 °C, the bacterium grew and produced CLA in the pH range of 5.5–6.5. At pH 6.0, a 36-h incubation period maximized the concentration of the dry biomass (0.82 g/L), the CLA content in the biomass (4.1 mg/g), and linoleic acid in the biomass (1.2 mg/g). In comparison with cultures grown without linoleic acid in the medium, supplementing the medium with linoleic acid at 600 μg/mL slowed the production of CLA, but the CLA content in the dry biomass increased to 12–14 mg/g and the linoleic acid content increased to 8–11 mg/g. Supplementing the culture medium with chitosan and linoleic acid enhanced production of CLA in the dry biomass to 21 mg/g within 36 h. Nearly 50% of the CLA was cis-9, trans-11-CLA, and the remainder was trans-10, cis-12-CLA. Linoleic acid content of the dry biomass was increased to 37 mg/g. Accumulation of CLA in the cells was enhanced by feeding linoleic acid. Supplementing the culture with linoleic acid and chitosan further increased accumulation of CLA.

Linoleic acid reduces vascular reactivity and improves the vascular dysfunction of the small mesentery in hypertension

We aimed to investigate the effect of linoleic acid (LA) treatment on the blood pressure and function of mesenteric resistance arteries (MRA) in spontaneous hypertensive rats (SHR). Male SHR were treated daily with LA (15 mg/kg) or vehicle (control) for 15 days. Compared with controls, LA treatment decreased blood pressure and showed the following in MRA: (1) increased lumen and external diameter, (2) decreased wall:lumen ratio and wall thickness, (3) decreased stiffness and (4) less collagen deposition. LA treatment reduced the contractile response to phenylephrine, although there were no changes observed in MRA in regard to the acetylcholine or sodium nitroprusside responses. Incubation with L-NAME left-shifted the reactivity to phenylephrine only in the MRA treated group, suggesting that LA treatment can improve NO bioavailability. This result was accompanied by an increase “in situ” NO production. Incubation with tiron decreased vascular reactivity to phenylephrine in MRA in LA rats, which was accompanied by decreased superoxide anion production. Moreover, incubation with indomethacin (non-selective COX inhibitor, 10 μM), NS 398 (COX-2 specific inhibitor, 1 μM), furegrelate (TXA2 synthase inhibitor, 1 μM), SQ 29.548 (TP receptor antagonist, 1 μM) and SC 19220 (EP1 receptor antagonist, 10 μM) reduced the vasoconstrictor responses to phenylephrine in MRA in the treated group. These results were accompanied by a reduction in COX-2 protein expression. In conclusion, these findings show that LA treatment decreases blood pressure. In addition, the improvement of endothelial dysfunction and structural changes in this hypertension model may be responsible for the reduction in blood pressure.

Phosphorylation of protein kinase B, the key enzyme in insulin-signaling cascade, is enhanced in linoleic and arachidonic acid-treated HT29 and HepG2 cells.

Defects in the insulin-signaling pathway have been implicated in the pathogenesis of impaired glucose uptake, insulin resistance, and type 2 diabetes. However, the specific defects that precipitate these abnormalities are yet to be fully elucidated. After binding to insulin, the plasma membrane-embedded insulin receptor transmembrane protein initiates a cascade of phosphorylation that leads to the activation of protein kinase B (AKT) and subsequently to the initiation of some metabolic actions of insulin. The activities of this receptor, insulin binding, and tyrosine kinase activation is dependent on its plasma lipid environment. Published data on the influence of omega-3 and -6 polyunsaturated fatty acids on insulin response are scarce. Moreover, the findings of the published investigations, most of which used omega-3 and -6, polyunsaturated fatty-acid blends, have been inconclusive. Hence, further, well thought out research is needed. The aim of the current study was to elucidate the effect of treatments with linoleic acid (LNA), arachidonic acid (ARA), alpha-linolenic acid (ALA), docoshexaenoic acid (DHA), and eicosapentaenoic acid (EPA) on cell membrane composition and consequently on the insulin-signaling pathway and specifically AKT phosphorylation. Human colon adenocarcinoma (HT29) and liver hepatocellular (HepG2) cells were treated with or without 40 µM of LNA, ARA, ALA, EPA, or DHA for 48 h, the fatty-acid composition of phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEtn) from the treated cells by capillary gas liquid chromatograph. Cells were incubated for 30 min with or without human insulin (50 ng/mL), and the phosphorylation of AKT was assessed with the use of Western blotting. The fatty acids were incorporated in the PtdCho and PtdEtn of both cell lines, but the level of incorporation was higher in HT29. Phosphorylation of AKT increased when HT29 was treated with LNA (P < 0.05) and ARA (P < 0.01) but not with ALA, EPA, or DHA. A similar but non-significant increase in AKT phosphorylation was observed in LNA- and ARA- treated HepG2 cells. The finding of this investigation demonstrates that plasma membrane lipid bilayer enrichment with LNA or ARA treatment enhances insulin action by AKT activation.