Transcriptomic analyses of intestinal gene expression of juvenile Atlantic cod (Gadus morhua) fed diets with Camelina oil as replacement for fish oil

For aquaculture of marine species to continue to expand, dietary fish oil (FO) must be replaced with more sustainable vegetable oil (VO) alternatives. Most VO are rich in n-6 polyunsaturated fatty acids (PUFA) and few are rich in n-3 PUFA but Camelina oil (CO) is unique in that, besides high 18:3n-3 and n-3/n-6 PUFA ratio, it also contains substantial long-chain monoenes, commonly found in FO. Cod (initial mass ~ 1.4 g) were fed for 12 weeks diets in which FO was replaced with CO. Growth performance, feed efficiency and biometric indices were not affected but lipid levels in liver and intestine tended to increase and those of flesh, decrease, with increasing dietary CO although only significantly for intestine. Reflecting diet, tissue n-3 long-chain PUFA levels decreased whereas 18:3n-3 and 18:2n-6 increased with inclusion of dietary CO. Dietary replacement of FO by CO did not induce major metabolic changes in intestine, but affected genes with potential to alter cellular proliferation and death as well as change structural properties of intestinal muscle. Although the biological effects of these changes are unclear, given the important role of intestine in nutrient absorption and health, further attention should be given to this organ in future.     

Hydroxy-alkenals from the peroxidation of n-3 and n-6 fatty acids and urinary metabolites

4-Hydroxy-2E-hexenal (4-HHE) and 4-hydroxy-2E-nonenal (4-HNE) have been characterized as prominent by-products of n-3 and n-6 hydroperoxy derivatives of n-3 and n-6 fatty acids, respectively. We also have characterized the homolog 4-hydroxy-2E,6Z-dodecadienal (4-HDDE) as a specific by-product of the 12-lipoxygenase product of arachidonic acid 12-hydroperoxy-eicosatetraenoate (12-HpETE). The three hydroxy-alkenals have been found in human plasma with 4-HHE being the most prominent followed by 4-HNE. They were found increased in tissues submitted to oxidative stress, according to the fatty acid characteristic of those tissues, e.g., 4-HNE and 4-HDDE in blood platelets and 4-HHE in the retina. We have shown they covalently bind to the primary amine moiety of ethanolamine phospholipids (PE), especially the plasmalogen subclass, with the highest hydrophobic alkenal (4-HDDE) being the most reactive. Their carboxylic acid metabolites, 4-hydroxy-2E-hexenoic acid (4-HHA), 4-hydroxy-2E-nonenoic acid (4-HNA) and 4-hydroxy-2E,6Z-dodecadienoic acid (4-HDDA), respectively, were found in human urine and measured in higher amounts in situations in which oxidative stress has been reported such as aging and diabetes. As reported above with their hydroxy-alkenals precursors, 4-HHA proved to be the most prominent followed by 4-HNA. Altogether, the three hydroxy-alkenals, either in their free form or bound to membrane PE, may be considered as specific markers of lipid peroxidation able to discriminate between n-3 and n-6 fatty acids. This is corroborated by the measurement of their urinary carboxylic acid metabolites.     

Evidence for in situ ethanolamine phospholipid adducts with hydroxy-alkenals

Hydroxy-alkenals, such as 4-hydroxy-2(E)-nonenal (4-HNE; from n-6 fatty acids), are degradation products of fatty acid hydroperoxides, including those generated by free radical attack of membrane polyunsaturated fatty acyl moieties. The cytotoxic effects of hydroxy-alkenals are well known and are mainly attributable to their interaction with different molecules to form covalent adducts. Indeed, ethanolamine phospholipids (PEs) can be covalently modified in a cellular system by hydroxy-alkenals, such as 4-HNE, 4-hydroxy-2(E)-hexenal (4-HHE; from n-3 fatty acids), and 4-hydroxy-dodecadienal (4-HDDE; from the 12-lipoxygenase product of arachidonic acid), to form mainly Michael adducts. In this study, we describe the formation of PE Michael adducts in human blood platelets in response to oxidative stress and in retinas of streptozotocin-induced diabetic rats. We have successfully characterized and evaluated, for the first time, PEs coupled with 4-HHE, 4-HNE, and 4-HDDE by gas chromatography-mass spectrometry measurement of their ethanolamine moieties. We also report that aggregation of isolated human blood platelets enriched with PE-4-hydroxy-alkenal Michael adducts was altered. These data suggest that these adducts could be used as specific markers of membrane disorders occurring in pathophysiological states with associated oxidative stress and might affect cell function.     

Feeding long-chain n-3 polyunsaturated fatty acids during gestation increases intestinal glucose absorption potentially via the acute activation of AMPK

The current study utilized Ussing chambers to examine the impact of supplementing maternal gestation and/or lactation diets with n-3 polyunsaturated fatty acids (PUFA) provided via a protected fish oil (PFO) product on intestinal fatty acid profiles and ex vivo glucose uptake in the jejunum of weanling piglets. Jejunum tissues were enriched with n-3 PUFA as a result of feeding the sows the PFO during gestation and/or lactation (P<.05). Glucose uptake improved by twofold (P<.042) in intestinal preparations obtained from the offspring of sows fed PFO during gestation or throughout gestation/lactation versus lactation alone. This was also reflected in the jejunum protein expressions of glucose transporter 2 (GLUT2) and sodium-dependent glucose transporter 1 (SGLT1). Furthermore, adding docosahexaenoic acid (DHA) or an AMP-activated protein kinase (AMPK) agonist to the chamber buffer improved glucose uptake (P<.05) in intestinal preparations obtained from the offspring fed the control diet, devoid of the PFO product and containing minimal concentrations of n-3 PUFA. Collectively, these data indicate two important points. First, long-term exposure to n-3 PUFA via the maternal gestation diet effectively enhances glucose uptake in the weanling piglet, and the underlying mechanism may be associated with changes in the intestinal fatty acid profile. Secondly, there is an apparent direct and acute effect of DHA that is achieved within a time frame that precludes substantial changes in the intestinal fatty acid profile. Additionally, both mechanisms may involve activation of AMPK. Thus, n-3 PUFA delivered in utero and postnatally via the maternal diet may help the offspring adapt quickly to rapidly changing diets early in life and allow optimal nutrient uptake.     

α-Linolenic acid-enriched butter attenuated high fat diet-induced insulin resistance and inflammation by promoting bioconversion of n-3 PUFA and subsequent oxylipin formation

α-Linolenic acid (ALA) is an essential fatty acid and the precursor for long-chain n-3 PUFA. However, biosynthesis of n-3 PUFA is limited in a Western diet likely due to an overabundance of n-6 PUFA. We hypothesized that dietary reduction of n-6/n-3 PUFA ratio is sufficient to promote the biosynthesis of long-chain n-3 PUFA, leading to an attenuation of high fat (HF) diet-induced obesity and inflammation. C57BL/6 J mice were fed a HF diet from ALA-enriched butter (n3Bu, n-6/n-3=1) in comparison with isocaloric HF diets from either conventional butter lacking both ALA and LA (Bu, n-6/n-3=6), or margarine containing a similar amount of ALA and abundant LA (Ma, n-6/n-3=6). Targeted lipidomic analyses revealed that n3Bu feeding promoted the bioconversion of long-chain n-3 PUFA and their oxygenated metabolites (oxylipins) derived from ALA and EPA. The n3Bu supplementation attenuated hepatic TG accumulation and adipose tissue inflammation, resulting in improved insulin sensitivity. Decreased inflammation by n3Bu feeding was attributed to the suppression of NF-κB activation and M1 macrophage polarization. Collectively, our work suggests that dietary reduction of the n-6/n-3 PUFA ratio, as well as total n-3 PUFA consumed, is a crucial determinant that facilitates n-3 PUFA biosynthesis and subsequent lipidomic modifications, thereby conferring metabolic benefits against obesity-induced inflammation and insulin resistance.     

The effect of dietary fish oils on eicosanoid biosynthesis in peritoneal macrophages is influenced by both dietary N-6 polyunsaturated fats and total dietary fat

Recent research has implicated dietary fish oils in the reduction of eicosanoids formed from arachidonic acid and amelioration of chronic diseases such as coronary heart disease, atherosclerosis and inflammation. Feeding studies were conducted to determine if the efficacy of dietary n-3 polyunsaturated fatty acids (PUFA) from fish oils was influenced by the quantity of n-6 polyunsaturated fatty acids and the total level of fat in the diet. Groups of mice were fed diets composed of 5 and 20% total fat with varying proportions of linoleic acid as a source of n-6 PUFA. Menhaden oil as a source of n-3 PUFA was fed at two levels of n-6 at each level of total fat. Eicosanoid biosynthesis was stimulated and assayed in the mouse peritoneum using zymosan as an inflammatory stimulus. Production of LTE4 and PGE2 was enhanced by increasing n-6 PUFA in the diet at both levels of total fat. High dietary fat significantly suppressed leukotriene (LT) synthesis. Dietary menhaden oil reduced LTE4 and PGE2 synthesis at both levels of dietary n-6 in the low fat study. In animals on 20% dietary fat menhaden oil significantly reduced LT synthesis only at a relatively low dietary n-6 PUFA. On a high n-6 PUFA high fat diets, menhaden oil did not significant affect LTE4 synthesis in response to zymosan stimulation. The results suggest that the effectiveness of fish oils in reducing eicosanoids in response to specific stimulation is influenced by the level of n-6 and the total quantity of fat in the diet.     

Brain anticonvulsant protection of mice given chronic carbamazepine under various fatty acid and magnesium diet conditions

The anticonvulsant and mood stabilizer drug carbamazepine (CBZ) was evaluated for anti-seizure activity after drug pretreatment of young weaning mice given various oil-based diets. These diets had various mono-(MUFA) and poly-(PUFA) unsaturated fatty acid contents, were associated or not with magnesium deprivation, and were given over the entire experimental period (34 days). The diets included a commercial and three purified synthetic diets (n-6 PUFA, n-3 PUFA and MUFA-based chows containing 5% corn/sunflower oils 1:3, 5% rapeseed oil and 5% high oleic acid sunflower oil/sunflower oil 7:3, respectively). A 10-days CBZ treatment (50mg/kg/day fragmented in two daily intraperitoneal injections of 25mg/kg) was given 20 days after initiating diet administration and evaluations of mice was performed 4 days after arrest of CBZ in various seizure tests. In these conditions, CBZ pretreatment still exhibited anticonvulsant protection especially in magnesium-deficient animals. Ethosuximide (ESM)-like profiles under MUFA and n-3 PUFA diets and unusual GABA(A)ergic profile under n-6 PUFA diet in magnesium-deficiency dependent audiogenic seizures (MDDAS) test as well as protection against NMDA-induced seizures in all lipid (n-3 PUFA>MUFA and n-6 PUFA) diet conditions were observed in CBZ-pretreated mice. By highlighting ESM-like and anti-NMDA mechanisms previously induced by an n-3 PUFA diet, present CBZ anticonvulsant properties suggest brain protective targets common to CBZ and n-3 PUFAs.     

Inhibitory effect of dietary n-3 polyunsaturated fatty acids to intestinal IL-15 expression is associated with reduction of TCRalphabeta+CD8alpha+CD8beta-intestinal intraepithelial lymphocytes

Intestinal intraepithelial lymphocytes (IELs) and their cytokines play an important role in the regulation of gut immune response and take part in gut immune barrier function. n-3 polyunsaturated fatty acid (PUFA) is an immunoregulator that has been shown to influence the process of gut inflammation. Interleukin (IL)-15 is a T-cell growth factor that has been shown to influence the differentiation of IEL. The aim of this study was to analyze the effects of dietary n-3 PUFA on IEL. IEL phenotype and cytokine (TNF-alpha, IFN-gamma, IL-4, IL-10 and TGF-beta1) profile were measured by FACS and real-time RT-PCR in healthy adult rats fed with fish oil diet for 90 days. Rats fed with corn oil diet served as controls. Intestinal IL-15 expression was measured by immunohistochemistry and real-time RT-PCR. The results demonstrated a decrease of intestinal IL-15 expression in the fish oil group. Associated with this deduction, n-3 PUFA significantly decreased the proportion of TCRalphabeta+CD8alpha+CD8beta- cells and IEL-derived TNF-alpha, IFN-gamma, IL-4 and IL-10. In conclusion, n-3 PUFA could inhibit intestinal mucosal expression of IL-15 and may influence phenotype and function of IEL through this mechanism.     

A Metabolomic Analysis of Omega-3 Fatty Acid-Mediated Attenuation of Western Diet-Induced Nonalcoholic Steatohepatitis in LDLR -/- Mice

Background

Nonalcoholic steatohepatitis (NASH) is a progressive form of nonalcoholic fatty liver disease and a risk factor for cirrhosis, hepatocellular carcinoma and liver failure. Previously, we reported that dietary docosahexaenoic acid (DHA, 22:6,n-3) was more effective than eicosapentaenoic acid (EPA, 20:5,n-3) at reversing western diet (WD) induced NASH in LDLR^-/- mice.

Methods

Using livers from our previous study, we carried out a global non-targeted metabolomic approach to quantify diet-induced changes in hepatic metabolism.

Results

Livers from WD + olive oil (WD + O)-fed mice displayed histological and gene expression features consistent with NASH. The metabolomic analysis of 320 metabolites established that the WD and n-3 polyunsaturated fatty acid (PUFA) supplementation had broad effects on all major metabolic pathways. Livers from WD + O-fed mice were enriched in saturated (SFA) and monounsaturated fatty acids (MUFA), palmitoyl-sphingomyelin, cholesterol, n-6 PUFA, n-6 PUFA-containing phosphoglycerolipids, n-6 PUFA-derived oxidized lipids (12-HETE) and depleted of C_20-22 n-3 PUFA-containing phosphoglycerolipids, C_20-22 n-3 PUFA-derived oxidized lipids (18-HEPE, 17,18-DiHETE) and S-lactoylglutathione, a methylglyoxal detoxification product. WD + DHA was more effective than WD + EPA at attenuating WD + O-induced changes in NASH gene expression markers, n-6 PUFA and oxidized lipids, citrate and S-lactosyl glutathione. Diet-induced changes in hepatic MUFA and sphingolipid content were associated with changes in expression of enzymes involved in MUFA and sphingolipid synthesis. Changes in hepatic oxidized fatty acids and S-lactoylglutathione, however, correlated with hepatic n-3 and n-6 C_20-22 PUFA content. Hepatic C_20-22 n-3 PUFA content was inversely associated with hepatic α-tocopherol and ascorbate content and positively associated with urinary F2- and F3-isoprostanes, revealing diet effects on whole body oxidative stress.

Conclusion

DHA regulation of hepatic SFA, MUFA, PUFA, sphingomyelin, PUFA-derived oxidized lipids and S-lactoylglutathione may explain the protective effects of DHA against WD-induced NASH in LDLR^-/- mice.     

Enhanced solubilization and intestinal absorption of cholesterol by oxidized linoleic acid

Solubilization of cholesterol in the intestinal lumen by bile acids and the subsequent formation of mixed micelles is an important step in the absorption of cholesterol. We propose that oxidized fatty acids (ox-FA) may mimic bile acids and form mixed micelles with cholesterol much more efficiently, as compared with unoxidized fatty acids, thereby increasing there absorption. In an in vitro assay at concentrations of 1, 5, and 10 mM, oxidized linoleic acid (ox-18:2) increased the solubilization of cholesterol (3.06, 8.16, and 15.46 nmol/ml) in a dose dependent manner compared with a 10 mM unoxidized linoleic acid (unox-18:2 at 0.97 nmol/ml). The uptake of cholesterol solubilized in the presence of ox-18:2 by Caco-2 cells and everted rat intestinal sacs was greater (1.78 and 1.95 nmol/ml respectively) as compared with the cholesterol solubilized in the presence of unox-18:2 (0.29 and 0.61 nmol/ml; P = 0.05). In addition, when LDL receptor deficient mice were fed a high fat diet along with ox-18:2 their plasma cholesterol levels were greater than animals fed the high fat diet alone (1290 mg/dl vs. 1549 mg/dl, P = 0.013). From these results, we suggest that ox-FA, by enhancing the solubilization of luminal cholesterol, increases the uptake of cholesterol that might lead to hypercholesterolemia and atherosclerosis.     

Na+ transport in perfused intestine and in isolated enterocytes of freshwater trout deprived of essential fatty acid

1. Unidirectional Na+ fluxes obtained in perfused preparation of intestine and Na+ effluxes from enterocytes isolated by a specific method have been determined in the middle intestine of freshwater rainbow trout. 2. Fish were fed semi-purified diets containing 8% by wt of either cod liver oil as reference, grape seed oil or hydrogenated coconut oil which are both deficient in fatty acids (FA) of the (n-3) series essential for the trout. 3. Important modifications in the Na+ intestinal absorption were induced by these deficient diets when measured in perfused intestine. 4. Conversely, no changes were seen in the Na+ active or passive effluxed when measured in enterocytes isolated from trout fed the same diets. 5. Na+,K+-ATPase activities were also unmodified by any diet. 6. It is concluded that deprivation of essential fatty acids reduces Na+ absorption capabilities of trout intestine, an effect which is mainly located at the apical membrane of enterocytes. 7. These results strongly demonstrate that apical entry and active serosal transport are separately modified in trout intestine by EFA deficient diet. 8. Furthermore the FA of the (n-3) series are essential for the retention of functional properties of membranes and ionic permeabilities in particular.     

N-3 PUFA improved post-menopausal depression induced by maternal separation and chronic mild stress through serotonergic pathway in rats—effect associated with lipid mediators

Early life maternal separation (MS) increases the vulnerability to depression in rats with chronic mild stress (CMS). N-3 polyunsaturated fatty acids (PUFA) improved depressive behaviors in rats with acute stress; however, their effects on rats with MS+CMS were not apparent. The purpose of the present study was to investigate the hypothesis that lifetime n-3 PUFA supplementation improves post-menopausal depression through the serotonergic and glutamatergic pathways while modulating n-3 PUFA-derived metabolites. Female rats were fed diets of either 0% n-3 PUFA during lifetime or 1% energy n-3 PUFA during pre-weaning, post-weaning, or lifetime periods. Rats were allocated to non-MS or MS groups and underwent CMS after ovariectomy. N-3 PUFA increased brain n-3 PUFA-derived endocannabinoid/oxylipin levels, and reversed depressive behaviors. N-3 PUFA decreased blood levels of adrenocorticotropic hormone and corticosterone, and brain expressions of corticotropin-releasing factor and miRNA-218, which increased the expression of the glucocorticoid receptor. N-3 PUFA decreased the expression of tumor necrosis factor-α, interleukin (IL)-6, IL-1β, and prostaglandin E₂, while increased the expression of miRNA-155. N-3 PUFA also increased brainstem serotonin levels and hippocampal expression of the serotonin-1A receptor, cAMP response element-binding protein (CREB), phospho-CREB, and brain-derived neurotrophic factor. However, n-3 PUFA did not affect brain expression of α-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor subtype 1, N-methyl-D-aspartate receptor subtype 2B, or miRNA-132. Moreover, n-3 PUFA exposure during lifetime caused greater effects than pre- and post-weaning periods. The present study suggested that n-3 PUFA improved depressive behaviors through serotonergic pathway while modulating the metabolites of n-3 PUFA in post-menopausal depressed rats with chronic stress.     

High dose of an n-3 polyunsaturated fatty acid diet lowers activity of C57BL/6 mice

n-3 Polyunsaturated fatty acids (PUFA) are increasingly consumed as food additives and supplements; however, the side effects of these fatty acids, especially at high doses, remain unclear. We previously discovered a high fat n-3 PUFA diet made of fish/flaxseed oils promoted significant weight gain in C57BL/6 mice, relative to a control, without changes in food consumption. Therefore, here we tested the effects of feeding mice high fat (HF) and low fat (LF) n-3 PUFA diets, relative to a purified control diet (CD), on locomotor activity using metabolic cages. Relative to CD, the HF n-3 PUFA diet, but not the LF n-3 PUFA diet, dramatically reduced ambulatory, rearing, and running wheel activities. Furthermore, the HF n-3 PUFA diet lowered the respiratory exchange ratio. The data suggest mixed fish/flaxseed oil diets at high doses could exert some negative side effects and likely have limited therapeutic applications.     

Alteration of colonic stem cell gene signatures during the regenerative response to injury

Since aberrant wound healing and chronic inflammation can promote malignant transformation, we determined whether dietary bioactive fish oil (FO)-derived n-3 polyunsaturated fatty acids (n-3 PUFA) modulate stem cell kinetics in a colitis-wounding model. Lgr5-LacZ and Lgr5-EGFP-IRES-creER(T2) mice were fed diets enriched with n-3 PUFA vs n-6 PUFA (control) and exposed to dextran sodium sulfate (DSS) for 5days in order to induce crypt damage and colitis throughout the colon. Stem cell number, cell proliferation, apoptosis, expression of stem cell (Lgr5, Sox9, Bmi1, Hopx, mTert, Ascl2, and DCAMKL-1) and inflammation (STAT3) markers were quantified. DSS treatment resulted in the ablation of Lgr5(+) stem cells in the distal colon, concurrent with the loss of distal crypt structure and proliferating cells. Lgr5, Ascl2 and Hopx mRNA expression levels were decreased in damaged colonic mucosa. Lgr5(+) stem cells reappeared at day 5 of DSS recovery, with normal levels attained by day 6 of recovery. There was no effect of diet on the recovery of stem cells. FO fed animals exhibited higher levels of phospho-STAT3 at all time points, consistent with a higher wounding by DSS in FO feeding. n-3 PUFA-fed mice exhibited a reduction in stem cell associated factors, Ascl2, Axin2 and EphB3. These results indicate that rapidly cycling Lgr5(+) stem cells residing at position 1 in the colon epithelium are highly susceptible to DSS-induced damage and that dietary cues can impact stem cell regulatory networks.     

A fish oil-rich diet reduces vascular oxidative stress in apoE–/– mice

Abstract

Oxidative stress contributes to lipid peroxidation and decreases nitric oxide (NO) bioavailability in atherosclerosis. While long-chain (n-3) polyunsaturated fatty acids (PUFA) are easily oxidized in vitro, they improve endothelial function. Hence, this study postulates that long-chain (n-3) PUFA decrease atherogenic oxidative stress in vivo. To test this, apoE^–/– mice were fed a corn oil- or a fish oil (FO)-rich diet for 8, 14 or 20 weeks and parameters related to NO and superoxide (O₂^.–) plus markers of lipid peroxidation and protein oxidative damage in the aortic root were evaluated. The FO-rich diet increased NO production and endothelial NO synthase (NOS) expression and lowered inducible NOS, p22^phox expression and O₂^.–production after 14 and 20 weeks of diet. Protein lipoxidative damage (including 4-hydroxynonenal) was decreased after a long-term FO-diet. This supports the hypothesis that a FO-rich diet could counteract atherogenic oxidative stress, showing beneficial effects of long-chain (n-3) PUFA.     

Does enteral nutrition of dietary polyunsaturated fatty acids promote oxidative stress and tumour growth in ductal pancreatic cancer?: Experimental trial in Syrian Hamster

BACKGROUND: Type and composition of dietary fat intake is supposed to play an important role in carcinogenesis. Thus we investigated the effects of n-3, n-6 and n-9 polyunsaturated fatty acids (PUFA) on oxidative stress (lipidperoxidation) and tumour growth in ductal pancreatic cancer. METHODS: Ninety male hamsters were randomized into 6 groups (gr.) (n=15) and allocated to 3 main dietary categories: gr. 1 and 2 received a standard high fat diet (SHF, rich in n-6 PUFA), while gr. 3 and 4 were fed with a diet containing a mixture of n-3, n-6 and n-9 PUFA (SMOF) and gr. 5 and 6 had free access to a diet rich in n-3 PUFA (FISH-OIL). Gr. 1, 3 and 5 received weekly subcutaneous (s.c.) injections of 10 mg N-nitrosobis-2-oxypropylamine (BOP)/kg body weight in order to induce ductal pancreatic adenocarcinoma. Healthy control gr. 2, 4 and 6 were treated with 0.5 ml 0.9% sodium chloride s.c. After 32 weeks all animals were sacrificed. Removed pancreata were weighed and analysed histologically and biochemically. Activities of glutathionperoxidase (GSH-Px), superoxiddismutase (SOD) and levels of lipidperoxidation were measured in samples of pancreatic carcinoma as well as in tumour-free pancreatic tissue. RESULTS: While different diets did not significantly alter the overall incidence of histologically proven pancreatic adenocarcinoma, the number of macroscopically visible tumours was decreased in the FISH-OIL-gr. CONCLUSION: Different diets did not significantly influence the incidence of histologically proven pancreatic adenocarcinoma. However, administration of a diet rich in n-3 PUFA (FISH-OIL) resulted in a decrease of macroscopically visible tumours, thus indicating its beneficial effects in respect to attenuation of tumour growth.     

Treatment with n-3 polyunsaturated fatty acids reverses endothelial dysfunction and oxidative stress in experimental menopause

Menopause is associated with endothelial dysfunction and oxidative stress. In this condition, reduced n-3 polyunsaturated fatty acids (n-3 PUFAs) contribute to cardiovascular disease. We investigated whether treatment with n-3 PUFA reverses endothelial dysfunction and oxidative stress in experimental menopause. Thirty female rats underwent either sham-surgery or bilateral ovariectomy or bilateral ovariectomy+oral n-3 PUFA (0.8 g kg^-1 day^-1 for 2 months).Ovariectomy caused endothelial dysfunction to acetylcholine, which was reversed by superoxide scavenger Tiron. Erythrocyte membrane lipid composition was characterized by reduced n-3 PUFA total content and omega-3 index, and by concomitant increase in n-6:n-3 PUFA ratio. Ovariectomy-related oxidative stress, demonstrated by both enhanced superoxide production and 3-nitrotyrosine expression in aorta, was associated with increased nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunit NOX-4 protein expression. Endothelial nitric oxide synthase (eNOS) functional inhibition by l-NG-nitroarginine methyl ester, protein expression and activity did not change.In ovariectomized rats, treatment with n-3 PUFA increased n-3 PUFA total content and omega-3 index and decreased n-6:n-3 PUFA ratio in erythrocyte membrane, reversed vascular oxidative stress, endothelial dysfunction, aortic 3-nitrotyrosine and markedly lowered NOX-4 protein expression; eNOS protein expression also increased, paralleled by reversal of inhibitory binding to Caveolin-1, while ex-vivo functional inhibition and NOS synthesis were unchanged.These findings demonstrate in vivo a therapeutic benefit of n-3 PUFA on menopause-associated endothelial dysfunction by reversal of alterations in membrane lipid composition induced by ovariectomy and by reduction of vascular oxidative stress. In this setting they also identify NOX-4 as a potential target to reduce oxidative stress-mediated vascular complications.