Eicosapentaenoic Acid Treatment Research Articles

Fish oil omega-3 polyunsaturated fatty acids attenuate oxidative stress-induced DNA damage in vascular endothelial cells.

ObjectiveOmega-3 fatty acids, particularly eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), likely prevent cardiovascular disease, however their mechanisms remain unclear. Recently, the role of DNA damage in atherogenesis has been receiving considerable attention. Here, we investigated the effects of EPA and DHA on DNA damage in vascular endothelial cells to clarify their antiatherogenic mechanisms.Methods and resultsWe determined the effect of EPA and DHA on H2O2-induced DNA damage response in human aortic endothelial cells. Immunofluorescence staining showed that γ-H2AX foci formation, a prominent marker of DNA damage, was significantly reduced in the cells treated with EPA and DHA (by 47% and 48%, respectively). H2O2-induced activation of ATM, a major kinase orchestrating DNA damage response, was significantly reduced with EPA and DHA treatment (by 31% and 33%, respectively). These results indicated EPA and DHA attenuated DNA damage independently of the DNA damage response. Thus the effects of EPA and DHA on a source of DNA damage were examined. EPA and DHA significantly reduced intracellular reactive oxygen species under both basal condition and H2O2 stimulation. In addition, the mRNA levels of antioxidant molecules, such as heme oxygenase-1, thioredoxin reductase 1, ferritin light chain, ferritin heavy chain and manganese superoxide dismutase, were significantly increased with EPA and DHA. Silencing nuclear factor erythroid 2-related factor 2 (NRF2) remarkably abrogated the increases in mRNA levels of antioxidant molecules and the decrease in intracellular reactive oxygen species. Furthermore, EPA and DHA significantly reduced H2O2-induced senescence-associated β-galactosidase activity in the cells (by 31% and 22%, respectively), which was revoked by NRF2 silencing.ConclusionsOur results suggested that EPA and DHA attenuate oxidative stress-induced DNA damage in vascular endothelial cells through upregulation of NRF2-mediated antioxidant response. Therefore omega-3 fatty acids likely help prevent cardiovascular disease, at least in part, by their genome protective properties.

Effects of lipoic acid and n-3 long-chain polyunsaturated fatty acid on the liver ovariectomized rat model of menopause.

Bilateral ovariectomy is an experimental model used to analyse the effects of menopause and develop strategies to mitigate the deleterious effects of this condition. Supplementation of the diet with antioxidants has been used to reduce potential oxidative stress caused by menopause. The purpose of the study was to analyse the effects of α-lipoic acid (LA), docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), dietary supplementation on oxidative stress in the livers of ovariectomized rats. In this study, we evaluated the effect of dietary supplementation with LA, DHA and EPA for a period of 16 weeks on oestrogen levels and oxidative stress biomarkers in the livers of ovariectomized 25 three-month-old rats. Serum oestrogen levels were lower after ovariectomy but were not altered by dietary treatments. LA was capable of acting in the liver, recovering the activity of the antioxidant enzymes superoxide dismutase and glutathione peroxidase, and reducing protein oxidative damage. Moreover, LA supplementation reduced nitrite and nitrate levels. DHA and EPA recovered the antioxidant activity of cytosolic and mitochondrial superoxide dismutase, decreasing protein oxidation. Protection against lipid oxidation differed between treatments. The DHA-treated group showed increased levels of the lipid peroxidation biomarker malondialdehyde compared to the ovariectomized group. However, malondialdehyde levels were not altered by EPA treatment. The results suggest that the antioxidant response varies among evaluated supplementations and all supplements were able to alter enzymatic and non-enzymatic antioxidants in the livers of ovariectomized rats. DHA presented the most evident antioxidant effect, decreasing protein and lipid damage.

12-OH-17,18-Epoxyeicosatetraenoic acid alleviates eosinophilic airway inflammation in murine lungs.

Asthma is characterized by airway inflammation and obstruction with eosinophil infiltration into the airway. Arachidonic acid, an omega-6 fatty acid, is metabolized into cysteinyl leukotriene with pro-inflammatory properties for allergic inflammation, whereas the omega-3 fatty acid eicosapentaenoic acid (EPA) and its downstream metabolites are known to have anti-inflammatory effects. In this study, we investigated the mechanism underlying the counter-regulatory roles of EPA in inflamed lungs. Male C57BL6 mice were sensitized and challenged by ovalbumin (OVA). After EPA treatment, we evaluated the cell count of Bronchoalveolar lavage fluid (BALF), mRNA expressions in the lungs by q-PCR, and the amounts of lipid mediators by liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based lipidomics. We investigated the effect of the metabolite of EPA by in vivo and in vitro studies. Eicosapentaenoic acid treatment reduced the accumulation of eosinophils in the airway and decreased mRNA expression of selected inflammatory mediators in the lung. Lipidomics clarified the metabolomic profile in the lungs. Among EPA-derived metabolites, 12-hydroxy-17,18-epoxyeicosatetraenoic acid (12-OH-17,18-EpETE) was identified as one of the major biosynthesized molecules; the production of this molecule was amplified by EPA administration and allergic inflammation. Intravenous administration of 12-OH-17,18-EpETE attenuated airway eosinophilic inflammation through downregulation of C-C chemokine motif 11 (CCL11) mRNA expression in the lungs. In vitro, this molecule also inhibited the release of CCL11 from human airway epithelial cells stimulated with interleukin-4. These results demonstrated that EPA alleviated airway eosinophilic inflammation through its conversion into bioactive metabolites. Additionally, our results suggest that 12-OH-17,18-EpETE is a potential therapeutic target for the management of asthma.

Eicosapentaenoic Acid (EPA) Induced Macrophages Activation through GPR120-Mediated Raf-ERK1/2-IKKβ-NF-κB p65 Signaling Pathways.

Objectives: To investigate the immunomodulatory effect and molecular mechanisms of Eicosapentaenoic acid (EPA, a typical kind of n-3PUFAs) on RAW264.7 cells. Methods: A variety of research methods, including the RAW264.7 cells culture, cell proliferation assays, morphologic observations, measurements of NO production, cytokine assays, nuclear protein extractions, western blot analyses and NF-κB p65 immunofluorescence assays were used in this study. Results: The results showed that EPA could increase the proliferation index and enhance the release of nitric oxide (NO) and cytokines in RAW264.7 cells. Western blotting results revealed that the protein level of GPR120 increased significantly in RAW264.7 cells after EPA treatment. Meanwhile, EPA elevated the phosphorylation status of Raf, which may act as an upstream regulator of EPA-induced phosphorylated ERK1/2. In addition, the phosphorylated ERK1/2 may then promote IKKβ in endochylema and translocate the NF-κB p65 subunit into the nucleus, thus regulating the production of inducible nitric oxide synthase (iNOS) and cytokines. Conclusions: EPA (0.6–3.0 μmol) activates RAW264.7 cells through GPR120-mediated Raf-ERK1/2-IKKβ-NF-κB p65 signaling pathways.

Effect of eicosapentaenoic acid and pitavastatin on electrophysiology and anticoagulant gene expression in mice with rapid atrial pacing.

Atrial remodeling is considered to be any persistent change in atrial structure or function, and is responsible for the development and perpetuation of atrial fibrillation (AF). Oxidative stress and intracellular pH regulation may also be linked to AF; however it remains unclear whether eicosapentaenoic acid (EPA) or statins have beneficial therapeutic effects. The aim of the present study was to investigate the effects of EPA and pitavastatin on the electrophysiology of and gene expressions in mice with rapidly-paced atria. Mice were treated with EPA (10 mg/g/day) or pitavastatin (30 ng/g/day) for 6 weeks, following which AF was simulated by 8-h atrial pacing at 1,800 bpm. The atrial electrophysiological properties and the expression of cardiac genes, potassium voltage-gated channel subfamily A member 5 (Kcna5), Kcn subfamily D member 2 (Kcnd2), Kv channel-interacting protein 2 (KChIP2), solute carrier family 9 member A1, thrombomodulin (TM) and tissue factor pathway inhibitor (TFPI) were examined using reverse transcription-quantitative polymerase chain reaction. In control mice, significant atrial electrical remodeling was observed (P<0.05); however, treatment with either EPA or pitavastatin ameliorated these electrophysiological changes (P>0.05). mRNA levels of Kcnd2, KChIP2 and Kcna5 were significantly upregulated in control mice (P<0.05), whereas treatment with EPA or pitavastatin attenuated this upregulation (P>0.05). Administration of pitavastatin significantly reduced the downregulation of both TFPI and TM (P<0.05). EPA treatment attenuated the TFPI downregulation compared with control mice (P>0.05), however no significant effect on TM expression was observed. In addition, both EPA (P>0.05) and pitavastatin (P<0.05) suppressed the overexpression of endothelial nitric oxide synthase. This was also exhibited in Ras-related C3 botulinum toxin substrate 1 genes (P<0.01 for both treatments). In conclusion, the results of the present study suggested that EPA and pitavastatin are able to prevent atrial electrical remodeling, thrombotic states and oxidative stress in rapidly-paced murine atria.

Abstract 1235: EPHA2-targeted therapy enhances the cytotoxicity of eicosapentaenoic acid against triple-negative inflammatory breast cancer

Abstract Background: Inflammatory breast cancer (IBC) is the most aggressive form of breast cancer. We have previously reported that mediators of inflammation, such as COX-2, promote the growth of Triple-Negative receptor (TN) IBC xenografts; therefore, inflammation in TN-IBC has a unique opportunity as a therapeutic strategy. Eicosapentaenoic acid (EPA), a non-toxic omega-3 fatty acid with anti-inflammatory properties, has partially reduced tumor growth in pre-clinical models of TN-IBC. Therefore, our goal is to develop a novel non-toxic approach that enhances EPA efficacy against TN-IBC in combination with targeted therapy. Methods and Results: Using a high-throughput, siRNA screen (939 genes) in the TN-IBC cell line SUM149PT, we identified Ephrin type-A receptor 2 (EPHA2), an oncogenic cell-surface receptor tyrosine kinase, as a target that modulates the sensitivity of TN-IBC cells to EPA treatment. To determine the clinical relevance of EPHA2, we interrogated a meta-analysis of breast cancer mRNA expression data sets, and found that high EPHA2 tumor expression was significantly correlated with poor overall survival in TN-IBC patients, compared to low EPHA2 expressing tumors (P = 0.01). We observed no significant correlations to other breast cancer subtypes. Similar findings were observed in vitro were EPHA2 expression predominantly occurred in the TN-IBC subtypes (19 of 30) among 49 breast cancer cell lines. Gain/loss-of-expression studies were performed to functionally validate EPHA2 as a synergistic combinational target with EPA in two EPHA2-expressing TN-IBC models, SUM149PT and BCX010, using proliferation and apoptosis assays in vitro and established tumor xenografts in vivo. EPHA2 gene silencing significantly reduced cell growth and induced apoptosis in combination with EPA when compared with untreated control and monotherapy in vitro (P &amp;lt; 0.05) and in vivo (P &amp;lt; 0.001), while vector-induced EPHA2 expression reversed cell growth reduction and apoptosis induction following combination treatment with EPA in vitro (P &amp;lt; 0.05). To translate our findings to the clinic, we validated that dasatinib, a small molecule inhibitor of EPHA2, in combination with EPA significantly enhanced cell death of SUM149PT and BCX010 cells in vitro when compared to non-treated and monotherapy (P &amp;lt; 0.05). Finally, using membrane fluidity assessment and reverse-phase protein array (300 antibodies), we determined that combination treatment efficacy depended on EPA/EPHA2 inhibition-mediated increase in cell membrane rigidity (P &amp;lt; 0.001, compared to monotherapy), which subsequently inhibited receptor tyrosine kinase signaling activity, potentially resulting in induction of apoptosis. Conclusions: Our preclinical findings provide a rationale for the development of a phase 1 clinical trial investigating combination EPA and EPHA2-inhibitors in patients with EPHA2-positive TN-IBC. Citation Format: Angie M. Torres-Adorno, Heidi Vitrac, Yuan Qi, Yiwen Yang, Peiying Yang, Bedrich L. Eckhardt, Naoto T. Ueno. EPHA2-targeted therapy enhances the cytotoxicity of eicosapentaenoic acid against triple-negative inflammatory breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1235. doi:10.1158/1538-7445.AM2017-1235

Lipid remodeling and an altered membrane-associated proteome may drive the differential effects of EPA and DHA treatment on skeletal muscle glucose uptake and protein accretion.

In striated muscle, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have differential effects on the metabolism of glucose and differential effects on the metabolism of protein. We have shown that, despite similar incorporation, treatment of C2C12 myotubes (CM) with EPA but not DHA improves glucose uptake and protein accretion. We hypothesized that these differential effects of EPA and DHA may be due to divergent shifts in lipidomic profiles leading to altered proteomic profiles. We therefore carried out an assessment of the impact of treating CM with EPA and DHA on lipidomic and proteomic profiles. Fatty acid methyl esters (FAME) analysis revealed that both EPA and DHA led to similar but substantials changes in fatty acid profiles with the exception of arachidonic acid, which was decreased only by DHA, and docosapentanoic acid (DPA), which was increased only by EPA treatment. Global lipidomic analysis showed that EPA and DHA induced large alterations in the cellular lipid profiles and in particular, the phospholipid classes. Subsequent targeted analysis confirmed that the most differentially regulated species were phosphatidylcholines and phosphatidylethanolamines containing long-chain fatty acids with five (EPA treatment) or six (DHA treatment) double bonds. As these are typically membrane-associated lipid species we hypothesized that these treatments differentially altered the membrane-associated proteome. Stable isotope labeling by amino acids in cell culture (SILAC)-based proteomics of the membrane fraction revealed significant divergence in the effects of EPA and DHA on the membrane-associated proteome. We conclude that the EPA-specific increase in polyunsaturated long-chain fatty acids in the phospholipid fraction is associated with an altered membrane-associated proteome and these may be critical events in the metabolic remodeling induced by EPA treatment.

Eicosapentaenoic acid modulates the synergistic action of CREB1 and ID/E2A family members in the rat pup brain and mouse embryonic stem cells

The aim of this study was to investigate the molecular mechanism by which eicosapentaenoic acid (EPA) may exert neuroprotective effects through an “EPA-cyclic AMP response element-binding protein (CREB)” signaling pathway. The current study reveals that EPA modulates the exquisite interplay of interaction of CREB1 with the inhibitor of DNA binding (ID) and E2A family members, thereby delivering mechanistic insights into specific neural differentiation program. In this scenario, our work provides evidence for the capability of CREB1 to sequester ID:E2A family members in brain tissues and neural differentiating mouse embryonic stem cells (mESCs) through formation of a [CREB1]2:ID2:E47 tetrameric complex.In essence, the molecular function of CREB1 is to dynamically regulate the location-specific assembly or disassembly of basic-helix-loop-helix (bHLH):HLH protein complexes to mediate the activation of neural/glial target genes. Together, these findings support the one-to-many binding mechanism of CREB1 and indicate that EPA treatment potentiates the integration of CREB dependent signaling with HLH/bHLH transcriptional network, adding specificity to the CREB1-mediated gene regulation during neural/glial differentiation. Our current research on the EPA-CREB axis could reveal new molecular targets for treating neurogenerative disease.

Eicosapentaenoic acid (EPA) promotes differentiation of human brown precursor cells into metabolically active classical brown adipocytes via Gq/11 signaling

Eicosapentaenoic acid (EPA) supplementation exerts a plethora of health benefits. Previously, we demonstrated that EPA promotes brown adipogenesis by mediating G‐coupled protein receptor 120 (GPR120) activation in rodent brown precursor cells. The aim of this study is to establish the role of EPA in regulating brown adipogenesis in human classical brown precursor cells. The immortalized human brown precursor cells were employed to induce differentiation in the presence of oleic acid, palmitic acid, and EPA. EPA treatment significantly increased brown‐specific gene expressions including Ucp1, Prdm16, Cidea, DiO2, UCP1 protein expressions and oxygen consumption rate. To further investigate the role of GPR120, a receptor family involved in Gq/11 subtype, human brown precursor cells were treated with either GPR120‐specific agonist cdpA or a potent Gq/11 inhibitor (YM‐254890) during differentiation. GPR120 agonism significantly promoted EPA‐mediated brown adipogenesis, while inhibition of Gq/11 signaling completely abrogated brown adipogenesis. Consistently, the genetic deletion of GRR120 by CRISPR/Cas9 abrogated EPA‐mediated brown adipogenic effects. Collectively, these results demonstrate that EPA supplementation mediates Gq/11 signaling of GPR120 leading to augmentation of brown adipogenesis in human brown precursor cells.Support or Funding InformationNIH 1P20GM104320

Free, ethyl‐ and methyl‐ ester eicosapentaenoic acid inhibit the proliferation of human and murine colon cancer cells in vitro

In the United States colorectal cancer is the second and third leading cause of cancer related deaths in men and women, respectively. Approximately 50,000 deaths from colon cancer are expected in 2017 alone. Omega‐3 polyunsaturated fatty acids (n‐3 PUFAs) have been extensively researched and shown to have anti‐inflammatory actions. Cancer development and progression is closely related to inflammatory processes, therefore, n‐3 PUFAs are of particular interest in their role in cancer prevention and treatment. Our objectives were to determine if eicosapentaenoic acid (EPA), a common n‐3 PUFA found in fish oil, inhibited the proliferation of colon cancer cells in vitro. One murine colon cancer cell line, MC‐38, and two human colon cancer cell lines, HCT‐116 and SW620, were seeded at a density of 5 × 104 (MC38 and HCT‐116) and 1.25 × 104 (SW620) cells per well on 24 well plates. After 24 hours each plate was treated with 0, 6.25, 12.5, 25, 50, and 100 microM EPA dissolved in ethanol. All cells received the same volume of ethanol. The experimental and control groups were plated in triplicate and cell number was determined 24, 48, and 72 hours after treatment. All data are reported as mean ± SEM for n=3 except for the HCT‐116 which had a n=2. One hundred microM EPA inhibited the proliferation of MC‐38 cells after 24 and 48 hours to 66.6 ± 6.0% and 72.2 ± 15.9% of control, respectively. However, at 72 hours the 100 microM EPA treatment had no effect on the proliferation of MC‐38 cells with 98.9 ± 7.4% of cells remaining when compared to vehicle control. Similarly the HCT‐116 cell line exhibited a decrease in cellular proliferation after 24 and 48 hours of treatment with 100 microM EPA to 64.0 ± 27.3 and 56.3 ± 13.6% of control, respectively. As with the MC‐38 cells, EPA no longer inhibited the proliferation of HCT‐116 cells after 72 hours of treatment, with 115.4 ± 5.2% of cells remaining when compared to vehicle control. These data suggested the EPA was either degraded or metabolized by the cancer cells within 48 hours and therefore had no effect at 72 hours. In order to combat this issue, two more stable forms of EPA, ethyl‐ and methyl‐ester EPA were used as treatments for the SW620 cell line. The experiment was conducted in the same method as the free EPA trials. After 72 hour of treatment with 100 microM free EPA, SW620 cells exhibited a decrease in proliferation to 79.6 ± 3.5 % of control. SW620 cells treated with 100 microM ethyl‐ and methyl‐ester EPA for 72 hours exhibited a larger decrease in cell number to 30.0 ± 13.2 and 32.6 ± 5.8% of control, respectively. In conclusion, the free‐EPA inhibited cellular proliferation up to 48 hours after treatment, after which its effects were diminished. The use of ethyl‐ and methyl‐ester EPA allowed for prolonged inhibition of cellular proliferation indicating that these esterified forms of EPA may be more efficacious in vivo.Support or Funding InformationThis research was supported by donations from the Heather Custer Memorial Fund and Kiel Colon Cancer.

Antibacterial activities of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) against planktonic and biofilm growing Streptococcus mutans.

The aim of this study was to evaluate the potential antibacterial activities of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) against planktonic and biofilm modes of Streptococcus mutans (S.mutans). The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined. The effects on planktonic growth and biofilm metabolic activity were evaluated by growth curve determination and MTT assay, respectively. Then, colony forming unit (CFU) counting, scanning electron microscopy (SEM) and real-time PCR were performed to further investigate the actions of DHA and EPA on exponential phase-S.mutans. Confocal laser scanning microscopy (CLSM) was used to detect the influences on mature biofilms. The MICs of DHA and EPA against S.mutans were 100μM and 50μM, respectively; the MBC of both compounds was 100μM. In the presence of 12.5μM-100μM DHA or EPA, the planktonic growth and biofilm metabolic activity were reduced in varying degrees. For exponential-phase S.mutans, the viable counts, the bacterial membranes and the biofilm-associated gene expression were damaged by 100μM DHA or EPA treatment. For 1-day-old biofilms, the thickness was decreased and the proportion of membrane-damaged bacteria was increased in the presence of 100μM DHA or EPA. These results indicated that, DHA and EPA possessed antibacterial activities against planktonic and biofilm growing S.mutans.

Enriching Islet Phospholipids With Eicosapentaenoic Acid Reduces Prostaglandin E2 Signaling and Enhances Diabetic β-Cell Function

Prostaglandin E2 (PGE2) is derived from arachidonic acid, whereas PGE3 is derived from eicosapentaenoic acid (EPA) using the same downstream metabolic enzymes. Little is known about the impact of EPA and PGE3 on β-cell function, particularly in the diabetic state. In this work, we determined that PGE3 elicits a 10-fold weaker reduction in glucose-stimulated insulin secretion through the EP3 receptor as compared with PGE2. We tested the hypothesis that enriching pancreatic islet cell membranes with EPA, thereby reducing arachidonic acid abundance, would positively impact β-cell function in the diabetic state. EPA-enriched islets isolated from diabetic BTBR Leptinob/ob mice produced significantly less PGE2 and more PGE3 than controls, correlating with improved glucose-stimulated insulin secretion. NAD(P)H fluorescence lifetime imaging showed that EPA acts downstream and independently of mitochondrial function. EPA treatment also reduced islet interleukin-1β expression, a proinflammatory cytokine known to stimulate prostaglandin production and EP3 expression. Finally, EPA feeding improved glucose tolerance and β-cell function in a mouse model of diabetes that incorporates a strong immune phenotype: the NOD mouse. In sum, increasing pancreatic islet EPA abundance improves diabetic β-cell function through both direct and indirect mechanisms that converge on reduced EP3 signaling.

Long-Term Administration of Eicosapentaenoic Acid Improves Post-Myocardial Infarction Cardiac Remodeling in Mice by Regulating Macrophage Polarization.

BackgroundConsumption of n‐3 fatty acids reduces the incidence of cardiovascular mortality in populations that consume diets rich in fish oil. Eicosapentaenoic acid (EPA) is an n‐3 fatty acid known to reduce the frequency of nonfatal coronary events; however, the frequency of mortality after myocardial infarction (MI) is not reduced. The aims of this study were to determine whether long‐term administration of EPA regulated cardiac remodeling after MI and to elucidate the underlying therapeutic mechanisms of EPA.Methods and ResultsC57BL/6J mice were divided into control (phosphate‐buffered saline–treated) and EPA‐treated groups. After 28 days of treatment, the mice were subjected to either sham surgery or MI by left anterior descending coronary artery ligation. Mortality due to MI or heart failure was significantly lower in the EPA‐treated mice than in the phosphate‐buffered saline–treated mice. However, the incidence of cardiac rupture was comparable between the EPA‐treated mice and the phosphate‐buffered saline–treated mice after MI. Echocardiographic tests indicated that EPA treatment attenuated post‐MI cardiac remodeling by preventing issues such as left ventricular systolic dysfunction and left ventricle dilatation 28 days after MI induction. Moreover, during the chronic remodeling phase, ie, 28 days after MI, flow cytometry demonstrated that EPA treatment significantly inhibited polarization toward proinflammatory M1 macrophages, but not anti‐inflammatory M2 macrophages, in the infarcted heart. Furthermore, EPA treatment attenuated fibrosis in the noninfarcted remote areas during the chronic phase.ConclusionsLong‐term administration of EPA improved the prognosis of and attenuated chronic cardiac remodeling after MI by modulating the activation of proinflammatory M1 macrophages.

Stimulation of glycerol kinase in grass carp preadipocytes by EPA.

This study was conducted to assess the effect of eicosapentaenoic acid (EPA) on grass carp preadipocyte glycerol kinase (GyK) expression, as well as to explore the mechanism. Here, we cloned partial sequence of grass carp GyK gene and analyzed its tissue distribution. The result showed that GyK gene expressed most in the liver, followed by adipose tissue and the kidney. Besides, 400μM oleic acid (18:1n-9, OA) was used to establish a hypertrophic preadipocyte model. GyK gene expression and enzyme activity were significantly enhanced after model cells were treated with 100μM eicosapentaenoic acid (20:5n-3, EPA) for 6, 12, and 24h. Meanwhile, peroxisome proliferative-activated receptor (PPAR)γ, adipose triglyceride lipase (ATGL), and the two isoforms of grass carp HSL gene were first identified by Sun et al (2016), and they defined the two isoforms as HSLa and HSLb. Therefore, maybe HSLa and HSLb are appropriate.. The content of triglyceride was dramatically increased by EPA treatment for 24h. Further, a competitive ATGL antagonist, HY-15859, attenuated the increase in GyK induced by EPA at 12h. Surprisingly, the enhanced lipolysis and PPARγ gene expression induced by serum deprivation were paralleled by an increase in GyK gene expression, whereas a stabilization in GyK enzyme activity. Other fatty acids, including docosahexaenoic acid, alpha-linolenic acid, linoleic acid, and OA also promoted GyK gene expression. Moreover, an irreversible PPARγ antagonist, GW9662, was used to investigate the role of PPARγ in GyK induction. Data showed that GW9662 abolished the induction of GyK by EPA at 12h. Together, these data suggested that EPA elevated grass carp preadipocytes GyK expression. ATGL and PPARγ contributed to the induction of GyK. PPARγ may be a key regulator in response to GyK expression induced by EPA.

Add-on Antiplatelet Effects of Eicosapentaenoic Acid With Tailored Dose Setting in Patients on Dual Antiplatelet Therapy.

The aim of this study was to investigate the antiplatelet effects of eicosapentaenoic acid (EPA) at a sufficient dose following coronary stent implantation. Thirty-one patients on dual antiplatelet therapy with aspirin and clopidogrel were treated with highly purified EPA-E (Epadel®) for 12 weeks. Based on our previous study, patients with a high baseline EPA/arachidonic acid (AA) ratio (≥ 0.37; n = 11) were given a standard dose (1800 mg daily) of EPA-E, whereas those with a low EPA/AA ratio (< 0.37; n = 20) were given a high dose (2700 mg daily) to reach the target value of > 0.92. Platelet function was then evaluated with agonist-induced aggregation using light transmittance aggregometry and VerifyNow®. After EPA-E treatment, the EPA/AA ratio significantly increased from 0.28 to 1.31 (P < 0.001). Collagen (1, 2, and 4 μg/mL)-induced maximal platelet aggregation (MPA) was significantly suppressed after EPA-E administration (from 28.0 to 24.0, P = 0.033; from 44.0 to 40.0, P = 0.016; from 60.0 to 56.0, P = 0.010; respectively). However, there were no changes in MPA induced by adenosine diphosphate and AA and in P2Y12 reaction units (PRU) and aspirin reaction units. After EPA-E treatment, PRU was significantly suppressed in 8 patients showing high on-treatment platelet reactivity (HTPR) (baseline 305; 266-321 versus on-treatment 256; 233-261, P = 0.012), but not in those without HTPR (201; 156-220 versus 183; 159-233, P = 0.212). In conclusion, EPA treatment at a sufficient dose suppressed platelet aggregation and showed possible add-on effects in patients with clopidogrel hyporesponsiveness.

Docosapentaenoic Acid (22:5n-3) Downregulates mRNA Expression of Pro-inflammatory Factors in LPS-activated Murine Macrophage Like RAW264.7 Cells.

Docosapentaenoic acid (22:5n-3, n-3 DPA) is a n-3 polyunsaturated fatty acid (PUFA) found in fish oil, and has been reported to have health benefits. This study investigated conversion of n-3 DPA, and examined the anti-inflammatory effects of n-3 DPA on activated macrophages. Murine macrophage-like RAW264.7 cells were incubated in culture media containing n-3 DPA for 72 h. The level of n-3 DPA in the fatty acid composition of the total lipid fraction increased in a dose-dependent manner. Furthermore, the levels of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) were higher in treated cells than in control cells. In RAW264.7 cells stimulated by lipopolysaccharide (LPS), n-3 DPA significantly down-regulated mRNA expression of pro-inflammatory factors such as IL-6, IL-1β, iNOS and COX-2. Production of IL-6 was also reduced by n-3 DPA in a dose-dependent manner. We found that n-3 DPA treatment resulted in greater IL-6 mRNA down-regulation than that achieved with EPA treatment, and was similar to that of DHA treatment. Furthermore, expression levels of IL-6 and IL-1β mRNAs were measured in the presence of the delta-6 desaturase inhibitor SC26196 in the culture medium to inhibit the conversion of n-3 DPA to DHA. There was no significant difference in the down-regulation in the mRNA expression of pro-inflammatory cytokines in RAW264.7 cells by n-3 DPA with or without presence of SC26196. These results demonstrate that n-3 DPA exhibits anti-inflammatory effects in activated RAW264.7 cells, which are independent of DHA conversion.

Eicosapentaenoic acid abolishes inhibition of insulin-induced mTOR phosphorylation by LPS via PTP1B downregulation in skeletal muscle

Dietary n-3 polyunsaturated fatty acids (n-3 PUFAs) increase insulin signaling in skeletal muscle. In the current study, we investigated the effect of eicosapentaenoic acid (EPA) on insulin-induced mammalian target of rapamycin (mTOR) phosphorylation in myotubes. We showed that EPA did not affect basal and insulin-induced mTOR phosphorylation in myotubes. However, EPA abolished lipopolysaccharide (LPS) -induced deficiency in insulin signaling (P < 0.05). Pre-incubation of nuclear factor κB (NF-κΒ) and c-Jun N-terminal kinases (JNK) inhibitors prevented the decreased insulin-induced mTOR phosphorylation elicited by LPS (P < 0.05). In addition, in protein tyrosine phosphatase-1B (PTP1B) knockdown myotubes, LPS failed to decrease insulin-induced mammalian target of rapamycin (mTOR) phosphorylation in myotubes (P > 0.05). In myotubes, LPS stimulated PTP1B expression via NF-κB and activation protein-1 (AP1). Pre-incubation of 50 μM EPA prevented the LPS-induced activation of AP1 and NF-κΒ as well as PTP1B expression (P < 0.05). Interestingly, incubation of peroxisome proliferator-activated receptor γ (PPARγ) antagonist (GW9662) prior to EPA treatment, the effect of EPA on insulin-induced mTOR phosphorylation was blocked. Accordingly, EPA did not inhibit the LPS-induced activation of AP1 or NF-κΒ as well as PTP1B expression when incubation of GW9662 prior to EPA treatment. The in vivo study showed that EPA prevented LPS-induced PTPT1B expression and a decrease in insulin-induced mTOR phosphorylation in muscle of mice. In summary, EPA abolished LPS inhibition of insulin-induced mTOR phosphorylation in myotubes, and one of the key mechanisms was to inhibit AP1 and NF-κB activation and PTP1B transcription.

An LC–MS-based lipidomics approach for studying the impact of dietary eicosapentaenoic acid on modulating methylmercury toxicity in mice

Methylmercury (MeHg) exposure has been a public health problem for many decades. There is a growing interest in searching for possible dietary nutrients that may affect MeHg toxicity. The study aims to evaluate the impact of eicosapentaenoic acid (EPA) on modulating MeHg toxicity in mice. The study was based on a two-level factorial design, where the factors were presence or absence of EPA and MeHg in the feed. A liquid chromatography-mass spectrometry-based lipidomics approach was used to identify and quantify the main phospholipid species in mouse liver and plasma. The effects of EPA and MeHg on phospholipid species were evaluated by principal component analysis and statistics. Some biochemical and toxicological markers were measured and hepatic histological assay was carried out. EPA treatment significantly elevated the phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEtn) species that contain n-3 PUFA, and reduced the levels of PtdCho and PtdEtn species that contain arachidonic acid (ARA), while MeHg exhibited opposite effects on these specific PtdCho species in liver. MeHg induced higher prostaglandin E2 and lower prostaglandin E3, thus increasing pro-inflammatory factors, while EPA decreased these ARA-derived inflammatory factors. Moreover, MeHg induced chronic inflammatory symptoms in mice, including severe hepatic necrosis, higher aspartate aminotransferase and alanine aminotransferase activities in plasma, higher thiobarbituric acid reactive substances and lower glutathione in liver. These symptoms were all alleviated by EPA treatment. EPA may have protective effect against MeHg-induced toxicity due to the favorable modification of membrane phospholipid composition and inhibition of inflammatory factors.

Eicosapentaenoic acid regulates brown adipose tissue metabolism in high-fat-fed mice and in clonal brown adipocytes

Brown adipose tissue (BAT) plays a key role in energy expenditure through its specialized thermogenic function. Therefore, BAT activation may help prevent and/or treat obesity. Interestingly, subcutaneous white adipose tissue (WAT) also has the ability to differentiate into brown-like adipocytes and may potentially contribute to increased thermogenesis. We have previously reported that eicosapentaenoic acid (EPA) reduces high-fat (HF)-diet-induced obesity and insulin resistance in mice. Whether BAT mediates some of these beneficial effects of EPA has not been determined. We hypothesized that EPA activates BAT thermogenic program, contributing to its antiobesity effects. BAT and WAT were harvested from B6 male mice fed HF diets supplemented with or without EPA. HIB 1B clonal brown adipocytes treated with or without EPA were also used. Gene and protein expressions were measured in adipose tissues and H1B 1B cells by quantitative polymerase chain reaction and immunoblotting, respectively. Our results show that BAT from EPA-supplemented mice expressed significantly higher levels of thermogenic genes such as PRDM16 and PGC1α and higher levels of uncoupling protein 1 compared to HF-fed mice. By contrast, both WATs (subcutaneous and visceral) had undetectable levels of these markers with no up regulation by EPA. HIB 1B cells treated with EPA showed significantly higher mRNA expression of PGC1α and SIRT2. EPA treatment significantly increased maximum oxidative and peak glycolytic metabolism in H1B 1B cells. Our results demonstrate a novel and promising role for EPA in preventing obesity via activation of BAT, adding to its known beneficial anti-inflammatory effects.

Omega-3 fatty acids control productions of superoxide and nitrogen oxide and insulin content in INS-1E cells.

Omega-3 fatty acids have multiple effects in peripheral tissues and pancreatic beta cell function. Dietary depletion of omega-3 fatty acids is associated with pancreatic islet dysfunction and insulin resistance in rats. Herein, the effects of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on pancreatic beta cell redox state and function were investigated. INS-1E insulin-secreting cells were incubated with EPA and DHA in combination with palmitic acid, and productions of reactive oxygen species (ROS), nitric oxide (NO) and insulin were measured. The involvement of the NADPH oxidase complex in ROS production and expression of the antioxidant enzymes was also investigated. After incubation for 1 or 48h, productions of superoxide (by hydroethidine method), nitric oxide (by 4,5-diaminofluorescein diacetate-DAF-2DA assay), insulin (by radioimmunoassay), and expressions (by western blot analysis) of glutathione peroxidase (GPx-1) and gp91PHOX were measured. EPA and DHA reduced superoxide production after 1-h incubation. After 48h, palmitic acid reduced superoxide production that was normalized by EPA treatment. Palmitic acid increased NO production that was reverted by EPA and DHA. Palmitic acid increased insulin secretion after 48h, whereas both omega-3 fatty acids increased intracellular insulin content. EPA and DHA enhanced GPx-1 expression as well as gp91PHOX glycosylated form. In conclusion, EPA and DHA increased intracellular insulin content and antioxidant enzymatic defense capacity and decreased pro-oxidant generating activities that are associated with maintenance of pancreatic beta cell redox state in response to palmitic acid.