EPA Treatment Research Articles

Abstract 73: A Novel Lysosome-Mediated Degradation Mechanism for Hepatic Lipid Droplet Turnover

Dietary supplementation with fish oil, rich in n-3 fatty acids such as EPA (20:5n-3), has been shown in many studies to exert a hypotriglyceridemic effect by reducing both plasma triglycerides (TG) in the form of VLDL and intrahepatic TG that is presented in cytoplasm as lipid droplets. However, cellular or molecular mechanisms by which n-3 fatty acid treatment could reduce hepatic TG remain to be defined. We used a well-characterized cell culture system that possesses the ability to synthesize and secreted TG-rich VLDL1 to determine the effect of EPA treatment on the synthesis, storage, turnover, and secretion of TG. Robust accumulation of lipid droplets occurs when hepatic cells were cultured under lipid-rich conditions, and prolonged exposure of the cells to lipids could lead to impaired TG secretion and steatosis. Treatment of the lipid-laden cells with EPA effectively cleared the storage lipid droplets within a few hours. The enhanced turnover of lipid droplets in EPA-treated cells was not attributable to increased TG secretion, because EPA-treatment also impaired VLDL1 assembly/secretion. The droplet turnover required the activity of lysosomes and could be blocked either by NH4Cl treatment or by silencing LAMP1 or Rab7. Inactivation of Atg5, ATGL, or HSL had no effect on the droplets turnover, indicating that the process is independent of autophagosome formation or cytosolic lipase activities. Live cell imaging of cells treated with EPA showed dynamic “kiss-and-run” events between lysosomes and the droplets, which were accompanied with augmented beta-oxidation. Treatment of cells with nocodazole or silencing the Rab7 effctors FYCO1 and RILP, respectively, completely blocked the EPA-induced droplet turnover, suggesting that the movement of lysosomes was essential for the kiss-and-run process. Moreover, overexpression of Arl8b, a lysosome-associated Arf-like GTPase, markedly accelerated the EPA-induced droplet turnover. These results reveal a novel mechanism responsible for the hypolipidemic effect of n-3 fatty treatment, and suggest a therapeutic potential for n-3 fatty acids in the treatment of hepatosteatosis.

The influence of EPA and DHA on markers of inflammation in 3T3-L1 cells at different stages of cellular maturation

BackgroundEPA and DHA have been reported to have anti-obesity and anti-inflammatory properties. Recent studies revealed that these positive actions of n-3 PUFA at least partially are connected with their influence on metabolism and secretory functions of the adipose tissue. However, their impact on old adipocytes is still poorly understood. Therefore the aim of the present study was to evaluate the influence of EPA and DHA on markers of inflammation in 3T3-L1 cells at different stages of cellular maturation.MethodsYoung, mature and old differentiated 3T3-L1 adipocytes were cultured for 48 h in the presence of 100 μM EPA, or 50 μM DHA complexed to albumin, whereas in control conditions only albumin was added to the medium. The Oil Red O staining was used to confirm adipocytes differentiation, and measure triglycerides content in cells. The concentration of adipokines (interleukin 6, adiponectin and leptin) in conditioned media was measured using mouse-specific ELISA kits.ResultsThe fat accumulation in 3T3-L1 adipocytes was positively correlated with their age; however, EPA and DHA did not affect lipid accumulation on any stage of maturation. EPA and DHA increased the concentration of secreted adiponectin when compared with control, but only in the case of young adipocytes (58% and 35%, respectively). Moreover, EPA supplementation increased interleukin 6 concentration in conditioned medium, while DHA exerted an opposite effect on all stages of cellular maturation. Furthermore, EPA treatment increased leptin release from young cells, while DHA did not affect the secretion of this adipokine. In mature 3T3-L1 adipocytes both experimental factors decreased synthesis of leptin; however, in old cells no impact of these PUFA was noted.ConclusionsIn summary, age is an important determinant of fat accumulation in adipocytes and affects adipokines secretion by these cells. Moreover, the impact of investigated fatty acids: EPA and DHA on fat cells varies depending on the stage of maturation, and seems to be stronger in young cells than in mature and old ones. Docosahexaenoic acid exerts an anti-inflammatory action; however, on the basis of the obtained data it was not possible to determine whether eicosapentaenoic acid shows anti- or pro-inflammatory properties.

EPA promotes fatty acid storage and thermogenic capacity in primary differentiated mouse subcutaneous adipocytes

Adipose tissue dysfunction has been associated with lipid dysregulation and insulin resistance. Compared with visceral fat, subcutaneous adipose tissue has been recently emerged as a lipid storage and thermogenic depot, playing a beneficial role in lipid homeostasis and insulin resistance. N‐3 PUFAs are known to have a role against hyperlipidemia and insulin resistance. Herein, we determined if EPA promotes beneficial metabolic activities of subcutaneous adipocytes. Primary stromal‐vascular (SV) cells isolated from mouse inguinal adipose tissue were cultured and induced to differentiate into adipocytes. EPA was added to SV cell culture during differentiation. On day 8 of differentiation, glucose metabolism, fatty acid storage, and thermogenic capacity were assessed. When compared to controls, EPA‐treated subcutaneous adipocytes had increased levels of basal glucose uptake and GLUT4 mRNA expression, but decreased glucose and lactate levels in the cultured media, suggesting decreased glycolysis. To assess fatty acid storage capacity, lipolysis, fatty acid uptake, and TAG synthesis were determined. We observed that EPA‐treated adipocytes had significantly decreased glycerol release and downregulation of HSL and ATGL gene expression, while markedly increased expression of genes involved in fatty acid uptake, CD36 and TAG synthesis, GPAT1 and GPAT3. Moreover, EPA treatment led to a significant increase in the expression of thermogenic genes including PGC1α, COXiv, UCP1, UCP2 and UCP3 as well as adiponectin expression. We conclude that EPA enhances fatty acid storage and energy dissipation capacity in subcutaneous adipocytes, contributing in part to its metabolic benefits.Grant Funding Source: NIH

Effects of EPA, γ-linolenic acid or coenzyme Q10 on serum prostate-specific antigen levels: a randomised, double-blind trial

The main objective of the present study was to determine the potential of n-3 and n-6 fatty acids or coenzyme Q10 (CoQ10) to alter serum prostate-specific antigen (PSA) levels in normal healthy men. A total of 504 healthy men with serum PSA level ≤ 2·5 ng/ml were recruited into the study. Serum PSA values were not segregated by decade of age. Participants were randomly assigned to a daily dietary supplement containing n-3 fatty acids (1·12 g of EPA and 0·72 g of DHA per capsule) (group 1, n 126), n-6 fatty acid (600 mg γ-linolenic acid (GLA) each capsule) (group 2, n 126), CoQ10 (100 mg per capsule) (group 3, n 126) or a similar regimen of placebo (group 4, n 126) for 12 weeks. Study medication was administered as two capsules to be taken twice daily. Serum levels of PSA, EPA, DHA, GLA, lipid profile and reproductive hormones were also measured. EPA treatment significantly reduced serum PSA level by 30·0 (95 % CI 25, 36) % (P= 0·004) from baseline. In contrast, GLA therapy significantly increased serum PSA concentration by 15·0 (95 % CI 11, 20) % (P= 0·02). CoQ10 therapy also significantly reduced serum PSA level by 33·0 (95 % CI 27, 40) % (P= 0·002). In multivariable analysis, serum values of PSA were strongly correlated with duration of EPA (r - 0·62; 95 % CI - 0·42, - 0·77; P= 0·003), n-6 (r 0·42; 95 % CI 0·31, 0·58; P= 0·02) and CoQ10 use (r - 0·77; 95 % CI - 0·56, - 0·87; P= 0·001). There were also significant correlations between serum values of DHA, EPA, GLA and CoQ10 and serum PSA levels. The present study demonstrates that dietary supplements containing EPA, GLA or CoQ10 may significantly affect serum PSA levels.

Role of CTCA in Acute Coronary Syndrome—Predicting Non-Significant Coronary Artery Disease

Results: No significant differences were observed in all variables between EPA-treated (n= 12) and controls (n= 11) before EPA treatment. After three months of EPA treatment, ratio of plasma levels of EPA to arachidonic acid (AA) in the EPA-treated group were significantly higher than those in the control group (p= 0.009) (Table 1). There were no significant changes in plasma levels of glucose, triglyceride, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, or C-reactive protein in both groups. Plasma adiponectin levels increased significantly (p= 0.048) after EPA treatment. No statistically significant changes were obtained in plasma levels of visfatin (p= 0.175) or leptin concentrations (p= 0.152). Conclusion: These results indicated that EPA had an adiponectin-dependent anti-atherosclerotic effect. EPA are, therefore, thought to be beneficial in preventing vascular complications in patients with hyperlipidaemia.

N-3 and n-6 polyunsaturated fatty acids differentially regulate adipose angiotensinogen and other inflammatory adipokines in part via NF-κB-dependent mechanisms

Excessive secretion of proinflammatory adipokines has been linked to metabolic disorders. We have previously documented anti-inflammatory effects of n-3 polyunsaturated fatty acids (n-3 PUFAs) in adipose tissue; however, the mechanisms by which these fatty acids regulate adipokine secretion remain unclear. Here, we determined differential effects of eicosapentaenoic acid (EPA, n-3 PUFA) vs. arachidonic acid (AA, n-6 PUFA) on expression and secretion of angiotensinogen (Agt), interleukin 6 (IL-6) and monocyte chemotactic protein (MCP-1) in 3T3-L1 adipocytes. While both PUFAs increased intracellular Agt protein and mRNA expression, Agt secretion into culture media was increased only by AA treatment, which in turn was prevented by co-treatment with EPA. At various AA/EPA ratios, increasing AA concentrations significantly increased secretion of the above three adipokines, whereas increasing EPA dose-dependently, while lowering AA, decreased their secretion. Moreover, IL-6 and MCP-1 were more significantly reduced by EPA treatment compared to Agt (IL-6>MCP>Agt). Next, we tested whether nuclear factor-κB (NF-κB), a major proinflammatory transcription factor, was involved in regulation of these adipokines by PUFAs. EPA significantly inhibited NF-κB activation compared to control or AA treatments. Moreover, EPA attenuated tumor necrosis factor-α-induced MCP-1 and further reduced its secretion in the presence of an NF-κB inhibitor. Taken together, we reported here novel beneficial effects of EPA in adipocytes. We demonstrated direct anti-inflammatory effects of EPA, which are at least in part due to the inhibitory effects of this n-3 PUFA on the NF-κB pathway in adipocytes. In conclusion, these studies further support beneficial effects of n-3 PUFAs in adipocyte inflammation and metabolic disorders.

The Effect of dietary fatty acids on the recruitment of brown‐like adipocytes in inguinal white adipose tissue

The physiological significance of brown adipose tissue (BAT) in energy metabolism in adult humans has been recently re‐evaluated. The evidence from recent studies indicates a potential of converting white to brown adipocytes in white adipose tissue (WAT). Herein, we explored the regulatory effect of dietary fatty acids on this conversion in WAT. We found that BAT‐specific UCP1 was expressed at a significantly higher level in inguinal adipose tissue (ING) compared to epididymal adipose tissue in mice. High fat diet (HFD) modulated UCP1 expression in BAT and ING in a different manner. After one week of HFD, UCP1 was increased in BAT, but not in ING of mice. However, UCP1 was dramatically decreased after chronic HFD feeding in both tissues. Additionally, we examined the effects of saturated and polyunsaturated fatty acids on the differentiation of brown‐like adipocytes in primary culture of stromal‐vascular (SV) cells from ING of normal mice. Fatty acids were added to SV cell culture either during 8 days of differentiation or after differentiation for 24 hours. In differentiated adipocytes, 24h treatment with palmitate significantly reduced UCP1, while n‐3 fatty acid (EPA) treatment did not change UCP1 expression. However, UCP1 and PGC1α expression was significantly increased in SV cell cultures after 8‐ day EPA treatment. Our data suggests that EPA is capable of inducing the differentiation of inguinal SV cells into brown‐like adipocytes, but failed to induce UCP1 expression in mature white adipocytes. We conclude that HFD or saturated FA reduces UCP1, while EPA has a potential to increase the recruitment of brown‐like adipocytes in inguinal WAT.Grant Funding Source : General Mills Foundation, MN Obesity Consortium

Membrane Raft Organization Is More Sensitive to Disruption by (n-3) PUFA Than Nonraft Organization in EL4 and B Cells

Model membrane and cellular detergent extraction studies show (n-3) PUFA predominately incorporate into nonrafts; thus, we hypothesized (n-3) PUFA could disrupt nonraft organization. The first objective of this study was to determine whether (n-3) PUFA disrupted nonrafts of EL4 cells, an extension of our previous work in which we discovered an (n-3) PUFA diminished raft clustering. EPA or DHA treatment of EL4 cells increased plasma membrane accumulation of the nonraft probe 1,1'-dilinoleyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate by ~50–70% relative to a BSA control. Förster resonance energy transfer imaging showed EPA and DHA also disrupted EL4 nanometer scale nonraft organization by increasing the distance between nonraft molecules by ~25% compared with BSA. However, changes in nonrafts were due to an increase in cell size; under conditions where EPA or DHA did not increase cell size, nonraft organization was unaffected. We next translated findings on EL4 cells by testing if (n-3) PUFA administered to mice disrupted nonrafts and rafts. Imaging of B cells isolated from mice fed low- or high-fat (HF) (n-3) PUFA diets showed no change in nonraft organization compared with a control diet (CD). However, confocal microscopy revealed the HF (n-3) PUFA diet disrupted lipid raft clustering and size by ~40% relative to CD. Taken together, our data from 2 different model systems suggest (n-3) PUFA have limited effects on nonrafts. The ex vivo data, which confirm previous studies with EL4 cells, provide evidence that (n-3) PUFA consumed through the diet disrupt B cell lipid raft clustering.

Endocannabinoid (EC) system expression in osteoblast like cells is influenced by differentiation and eicosapentaenoic acid treatment

The cannabinoid receptor, CB2 is expressed at increasing levels with osteoblast differentiation. Anandamide (AEA) is an endogenous agonist for cannabinoid receptors. Previously, we reported that arachidonic acid (AA), the substrate for n‐acyl phosphatidylethanolamine phospholipase D (NAPE‐PLD) for the synthesis of AEA, increased NAPE‐PLD expression compared to n‐3 PUFA treatment (EPA+DHA) in osteoblast‐like cells. The aim of this study was to determine if fatty acid treatments that alter enzyme substrate concentrations modify EC system expression and osteoblast markers. MC3T3‐E1 cells were grown for 6, 10, or 20 d in osteogenic medium; 72 h prior to collection cells were treated with 10 μM of AA, EPA, DHA, oleic acid (OA), or vehicle control. Increased NAPE‐PLD, FAAH, and CB2 mRNA expression and greater alkaline phosphatase (ALP) activity were observed with longer culture time. While CB1 expression was barely detectable and did not increase with time in culture, AA and EPA treatment increased CB1 compared to the other groups. NAPE‐PLD expression was lower in osteoblast‐like cells treated with EPA compared to all other groups. CB2 also decreased with EPA treatment. These results reiterate the impact of osteoblast maturation on NAPE‐PLD and CB2 expression. EPA might hinder EC synthesis and CB2 receptor expression during osteoblast differentiation. Supported by USDA.

The pharmacotherapy of depression with EPA

This lecture considers the pharmacotherapy of depression with EPA under the following headings: • Epidemiological evidence • Biochemical evidence • First trial of pure EPA in severe treatment-resistant depression • Subsequent randomized double-blind placebo-controlled trials • Putative mechanisms. Results obtained from our own group, including structural neuroimaging and 31-phosphorus neurospectroscopy findings, showing an association of EPA treatment of depression with a marked decrease in cerebral phosphodiesters, a marked increase in cerebral phosphomonoesters and reduced lateral ventricular volume, will also be presented.

Reductions of acetylcholine release and nerve growth factor expression are correlated with memory impairment induced by interleukin‐1β administrations: effects of omega‐3 fatty acid EPA treatment

Interleukin (IL)-1beta may play an important role in Alzheimer's disease. However, the relationships between glucocorticoids and acetylcholine (ACh), and between neurotrophins and ACh in IL-1-induced memory deficits are unknown. While ethyl-eicosapentaenoate (E-EPA) has recently been reported to reduce inflammation and improve memory, cholinergic and neurotrophic mechanisms by which E-EPA improves memory is unclear. This study evaluated: (i) the correlation between ACh release and memory impairment; (ii) the effect of glucocorticoids on ACh release; (iii) the relationship between nerve growth factor (NGF) and inflammation; and (iv) the effects of E-EPA treatment on IL-1beta-induced changes. Intracerebroventricular IL-1beta administrations produced a significant reduction in hippocampal ACh release in rats fed control diet, which was partially attenuated by mifepristone (RU 486) and completely blocked by IL-1 receptor antagonist. In eight-arm radial maze, significantly less ACh release was correlated with the memory deficits after IL-1beta administrations. mRNA expression of hippocampal NGF was lower, whereas IL-1beta was higher when compared with controls. E-EPA treatment significantly improved the memory, which was correlated with normalizing ACh release, and expressions of NGF and IL-1beta. This study revealed important mechanisms by which IL-1beta impairs, while E-EPA improves memory through IL-1-glucocorticoid-ACh release and IL-1-NGF-ACh release pathways.

P-266 Effectiveness of Early EPA Treatment in Plaque Regression in Patients with Acute Coronary Syndrome

P-266 Effectiveness of Early EPA Treatment in Plaque Regression in Patients with Acute Coronary Syndrome

Fish oil targets PTEN to regulate NFκB for downregulation of anti-apoptotic genes in breast tumor growth

The molecular mechanism for the beneficial effect of fish oil on breast tumor growth is largely undefined. Using the xenograft model in nude mice, we for the first time report that the fish oil diet significantly increased the level of PTEN protein in the breast tumors. In addition, the fish oil diet attenuated the PI 3 kinase and Akt kinase activity in the tumors leading to significant inhibition of NFkappaB activation. Fish oil diet also prevented the expression of anti-apoptotic proteins Bcl-2 and Bcl-XL in the breast tumors with concomitant increase in caspase 3 activity. To extend these findings we tested the functional effects of DHA and EPA, the two active omega-3 fatty acids of fish oil, on cultured MDA MB-231 cells. In agreement with our in vivo data, DHA and EPA treatment increased PTEN mRNA and protein expression and inhibited the phosphorylation of p65 subunit of NFkappaB in MDA MB-231 cells. Furthermore, DHA and EPA reduced expression of Bcl-2 and Bcl-XL. NFkappaB DNA binding activity and NFkappaB-dependent transcription of Bcl-2 and Bcl-XL genes were also prevented by DHA and EPA treatment. Finally, we showed that PTEN expression significantly inhibited NFkappaB-dependent transcription of Bcl-2 and Bcl-XL genes. Taken together, our data reveals a novel signaling pathway linking the fish oil diet to increased PTEN expression that attenuates the growth promoting signals and augments the apoptotic signals, resulting in breast tumor regression.

Effects of eicosapentaenoic acid ethyl ester on visfatin and apelin in lean and overweight (cafeteria diet-fed) rats

Previous studies have demonstrated that the n-3 fatty acid EPA improves insulin resistance induced by high-fat diets. The aim of the present study was to investigate the potential role of visfatin and apelin in the insulin-sensitising effects of EPA ethyl ester. The effects of EPA on muscle and adipose GLUT mRNA, as well as on liver glucokinase (GK) and glucose-6-phosphatase (G6Pase) activity, were investigated. Male Wistar rats fed on a standard diet or a high-fat cafeteria diet were daily treated by oral administration with EPA ethyl ester (1 g/kg) for 5 weeks. A significant decrease (P < 0.01) in white adipose tissue (WAT) visfatin mRNA levels was found in the cafeteria-fed rats, which was reversed by EPA administration (P < 0.05). Moreover, a negative relationship was observed between homeostatic model assessment (HOMA) and the visfatin:total WAT ratio. In contrast, cafeteria-diet feeding caused a significant increase (P < 0.01) in apelin mRNA in visceral WAT. EPA increased (P < 0.01) apelin gene expression, and a negative relationship between HOMA index with visceral apelin mRNA and serum apelin:total WAT ratio was also observed. EPA treatment did not induce changes in skeletal muscle GLUT1, GLUT4 or insulin receptor mRNA levels. Neither liver GK and G6Pase activity nor the GK:G6Pase ratio was modified by EPA. These data suggest that somehow the insulin-sensitising effects of EPA could be related to its stimulatory action on both visfatin and apelin gene expression in visceral fat, while changes in skeletal muscle GLUT, as well as in hepatic glucose production, are not likely to be the main contributing factors in the improvement in insulin resistance induced by EPA.

Polyunsaturated fatty acids differentially alter PGF 2α and PGE 2 release from bovine trophoblast and endometrial tissues during short-term culture

Two studies tested the hypothesis that eicosapentaenoic (20:5ω3; EPA), docosahexaenoic acids (22:6ω3; DHA) or linoleic acid (C18:2ω6; LIN) reduced bovine endometrial and trophoblast prostaglandin F 2α (PGF 2α) and prostaglandin E 2 (PGE 2) release during short-term culture. In Study 1, endometrial tissues were collected from non-lactating, non-pregnant cows and endometrial plus trophoblast tissues from pregnant cows 16 days post-insemination. In Study 2, endometrial and trophoblast tissues were collected on day 17 of pregnancy, from cows synchronised using a double prostaglandin (PG) or Ovagen™ synchronisation. Tissues were incubated in medium only (M) or media supplemented with fatty acids: eicosapentaenoic (20:5ω3; EPA), docosahexaenoic acids (22:6ω3; DHA) or linoleic acid (C18:2ω6; LIN). In Study 1, PGE 2 release from ‘pregnant’ endometria was higher ( P = 0.094) than from ‘non-pregnant’ endometria, while PGF 2α concentrations were similar. Fatty acids treatment had no effect on PGF 2α or PGE 2 release from either pregnant or non-pregnant endometria. Individual fatty acid treatments had no effect on the ratio of PGF 2α to PGE 2 from trophoblast tissues, but when the data from the 3 fatty acid treatments were combined (EPA, DHA and LIN treatment groups) the ratio of PGF 2α to PGE 2 was reduced ( P = 0.026) when compared to medium only. In Study 2, PGE 2 concentrations were higher ( P = 0.013) from the trophoblast collected from Ovagen™ cows as compared to that of the PG synchrony group. When the data from the 3-omega fatty acids were combined (DHA and EPA treatment groups), the 3-omega treatments decreased ( P < 0.05) PGE 2 biosynthesis from both endometrial and trophoblast tissues from animals synchronised following PG synchrony but not Ovagen™ synchrony. Short-term culture with low concentrations of 3-omega fatty acids tended to reduce prostaglandin release from trophoblast collected 16 days after insemination, with the type of synchrony modifying PGE 2 production from the trophoblast tissues collected 17 days after insemination. The ability of exogenous fatty acids to modify embryonic prostaglandin release needs to be examined in the context of supplementing dairy cows with different sources of fats. Synchronisation method altered trophoblast PGE 2 release, highlighting the importance of the hormonal environment in modifying embryonic prostaglandin synthesis and release.

Eicosapentaenoic acid actions on adiposity and insulin resistance in control and high-fat-fed rats: role of apoptosis, adiponectin and tumour necrosis factor-α

n-3 PUFA have shown potential anti-obesity and insulin-sensitising properties. However, the mechanisms involved are not clearly established. The aim of the present study was to assess the effects of EPA administration, one of the n-3 PUFA, on body-weight gain and adiposity in rats fed on a standard or a high-fat (cafeteria) diet. The actions on white adipose tissue lipolysis, apoptosis and on several genes related to obesity and insulin resistance were also studied. Control and cafeteria-induced overweight male Wistar rats were assigned into two subgroups, one of them daily received EPA ethyl ester (1 g/kg) for 5 weeks by oral administration. The high-fat diet induced a very significant increase in both body weight and fat mass. Rats fed with the cafeteria diet and orally treated with EPA showed a marginally lower body-weight gain (P = 0.09), a decrease in food intake (P < 0.01) and an increase in leptin production (P < 0.05). EPA administration reduced retroperitoneal adipose tissue weight (P < 0.05) which could be secondary to the inhibition of the adipogenic transcription factor PPARgamma gene expression (P < 0.001), and also to the increase in apoptosis (P < 0.05) found in rats fed with a control diet. TNFalpha gene expression was significantly increased (P < 0.05) by the cafeteria diet, while EPA treatment was able to prevent (P < 0.01) the rise in this inflammatory cytokine. Adiposity-corrected adiponectin plasma levels were increased by EPA. These actions on both TNFalpha and adiponectin could explain the beneficial effects of EPA on insulin resistance induced by the cafeteria diet.

Eicosapentaenoic acid modifies lipid composition in caveolae and induces translocation of endothelial nitric oxide synthase

Endothelial nitric oxide synthase (eNOS) plays a crucial role in the regulation of a variety of cardiovascular functions. Many studies have shown that dietary n-3 polyunsaturated fatty acids (PUFAs) have beneficial effects on coronary atherosclerosis. However, the mechanisms of n-3 PUFAs regulation in eNOS activation remain unknown. In the present study we investigated the effects of eicosapentaenoic acid (EPA, 20:5 n-3) on subcellular distribution of eNOS and lipid composition of caveolae. We demonstrated for the first time that EPA treatment profoundly altered lipid composition and fatty acyl substitutions of phospholipids in caveolae. We found that caveolin-1 was solely located in caveolae fractions in control cells, and EPA treatment displaced caveolin-1 from caveolae. eNOS was detected in the caveolin-enriched fractions and noncaveolae fractions in control cells. EPA treatment induced the translocation of eNOS from caveolae fractions to soluble fractions. P-eNOS was also distributed in both fractions. After EPA treatment, the level of p-eNOS in each fraction was increased but the distribution of which was unaffected. Moreover, the results of immunofluorescence confirmed that EPA could redistribute caveolin-1 and eNOS in plasma membrane. eNOS activity in HUVEC cells was increased after EPA treatment, which was in a dose dependent manner. And incubation with 50 μM EPA had the maximum effect on eNOS activity. Our results suggested that eNOS translocation was paralleled by a stimulated capacity for NO production in the cells. We found that total Akt and p-Akt were primarily presented in heavy membranes in control cells, and the relative level of p-Akt increased but the distribution did not change after EPA treatment. The distribution of CaM was slightly changed after EPA treatment. Our results indicated that n-3 PUFAs profoundly altered caveolae microenvironment, thereby modifying location and function of proteins in caveolae. EPA-induced alterations of lipid and proteins in caveolae may be an important mechanism in the pathophysiologic process of atherosclerosis.

Eicosapentaenoic acid and 3,10 dithia stearic acid inhibit the desaturation oftrans-vaccenic acid intocis-9,trans-11-conjugated linoleic acid through different pathways in Caco-2 and T84 cells

Stearoyl-CoA desaturase (SCD) is a key enzyme that determines the composition and metabolic fate of ingested fatty acids, in particular the conversion of trans-vaccenic acid (TVA) to conjugated linoleic acid (CLA). The present study addressed the hypothesis that intestinal TVA absorption and biotransformation into CLA can be modulated by EPA and 3,10-dithia stearic acid (DSA) via altered SCD mRNA levels and desaturation indices (cis-9, trans-11-CLA:TVA and oleic acid:stearic acid ratios) in Caco-2 and T84 cells, two well-established in vitro models of the human intestinal epithelium. The study determined the effect of acute (3 h with 0.3 mm-EPA or 0.3 mm-DSA) and acute-on-chronic (1 week with 0.03 mm-EPA or -DSA, followed by respectively, 0.3 mm-EPA or -DSA for 3 h) treatments. In both cell lines, acute EPA treatment did not alter SCD desaturation indices, whereas the acute-on-chronic treatment affected these surrogate markers of SCD activity. This was associated with reduced sterol regulatory-element binding protein-1c and SCD mRNA levels. In contrast, acute and acute-on-chronic DSA treatments significantly reduced SCD desaturation indices without affecting SCD mRNA levels in Caco-2 cells. The present study on intestinal cells shows that the conversion rate of TVA to c9, t11-CLA is affected by other fatty acids present in the diet such as EPA, confirming previous observations in hepatic and mammary cell models.

Docosahexaenoic Acid and Eicosapentaenoic Acid Induce Changes in the Physical Properties of a Lipid Bilayer Model Membrane

We investigated the effect of fatty acids such as stearic acid (SA, 18:0), oleic acid (OA, 18:1), eicosapentaenoic acid (EPA, 20:5), and docosahexaenoic acid (DHA, 22:6) on a dipalmitoylphosphatidylcholine (DPPC) bilayer by determining the phase transition temperature, fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH), and detergent insolubility. Treatment with unsaturated fatty acid broadened and shifted the phase transitions of the DPPC bilayer to a lower temperature. The phase transition temperature and the value of fluorescence anisotropy of DPH at 37 degrees C decreased progressively with increasing treatment amounts of unsaturated fatty acid. A large amount of the DPPC bilayer treated with unsaturated fatty acid was dissolved in Triton X-100, obtaining a low level of detergent insolubility. These modifications of the bilayer physical properties were most pronounced with DHA and EPA treatment. These data show that unsaturated fatty acids, particularly DHA and EPA, induce a marked change in the lipid bilayer structure. The composition of fatty acids in the DPPC bilayer was similar after treatment with various unsaturated fatty acids, suggesting that the different actions of unsaturated fatty acids are attributed to change in the molecular structure (e.g., kinked conformation by double bonds). We further explored the change in physical properties induced by fatty acids dispersed in a water-in-oil-in-water multiple emulsion and found that unsaturated fatty acids acted efficiently on the DPPC bilayer, even when incorporated in emulsion form.

Inactivation of Ascaris suum Eggs by Ammonia

Uncharged ammonia is known to cause inactivation of a number of wastewater pathogens, but its effect on Ascaris eggs has never been isolated or quantified. The objectives of this research were to determine the conditions under which ammonia inactivates eggs of the swine Ascaris species, Ascaris suum, and to quantify the impact of ammonia on the U.S. EPA's requirements for alkaline treatment to produce Class A sludge. Eggs were incubated in controlled, laboratory solutions such that the effects of ammonia concentration and speciation, pH, and temperature could be separated. With a 24-h incubation, the inactivation at all pH levels (range 7-11) was not statistically different in the absence of ammonia. The presence of ammonia (0-1000 ppm as N) significantly increased Ascaris egg inactivation at pH 9 and 11, and the ovicidal effect was directly related to the concentration of the uncharged NH3 species. Increasing temperatures (32-52 degrees C) caused increased inactivation at all pH levels and ammonia concentrations. The current EPA treatment requirements to produce Class A biosolids by alkaline treatment have temperature, pH, and time requirements, but do not account for the effectof differences in ammonia concentration on inactivation. To illustrate the potential savings in temperature and pH that could be achieved when accounting for ammonia inactivation, the combinations of ammonia concentration, temperature, and pH neededto achieve 99% inactivation after 72 h were determined. The presence of ammonia at concentrations encountered in sludges and feces (up to 8000 ppm as N) allowed for 99% egg inactivation to be achieved at temperatures up to 14 degrees C lower than ammonia-free controls. Thus, environmentally relevant concentrations of ammonia may significantly increase the rate of Ascaris egg inactivation during alkaline stabilization.