Lina Cordeddu

Endocannabinoids May Mediate the Ability of (n-3) Fatty Acids to Reduce Ectopic Fat and Inflammatory Mediators in Obese Zucker Rats

Dietary (n-3) long-chain PUFA [(n-3) LCPUFA] ameliorate several metabolic risk factors for cardiovascular diseases, although the mechanisms of these beneficial effects are not fully understood. In this study, we compared the effects of dietary (n-3) LCPUFA, in the form of either fish oil (FO) or krill oil (KO) balanced for eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) content, with a control (C) diet containing no EPA and DHA and similar contents of oleic, linoleic, and alpha-linolenic acids, on ectopic fat and inflammation in Zucker rats, a model of obesity and related metabolic dysfunction. Diets were fed for 4 wk. Given the emerging evidence for an association between elevated endocannabinoid concentrations and metabolic syndrome, we also measured tissue endocannabinoid concentrations. In (n-3) LCPUFA-supplemented rats, liver triglycerides and the peritoneal macrophage response to an inflammatory stimulus were significantly lower than in rats fed the control diet, and heart triglycerides were lower, but only in KO-fed rats. These effects were associated with a lower concentration of the endocannabinoids, anandamide and 2-arachidonoylglycerol, in the visceral adipose tissue and of anandamide in the liver and heart, which, in turn, was associated with lower levels of arachidonic acid in membrane phospholipids, but not with higher activity of endocannabinoid-degrading enzymes. Our data suggest that the beneficial effects of a diet enriched with (n-3) LCPUFA are the result of changes in membrane fatty acid composition. The reduction of substrates for inflammatory molecules and endocannabinoids may account for the dampened inflammatory response and the physiological reequilibration of body fat deposition in obese rats.

Effect of dietary krill oil supplementation on the endocannabinoidome of metabolically relevant tissues from high-fat-fed mice

BackgroundOmega-3 polyunsaturated fatty acids (ω-3-PUFA) are known to ameliorate several metabolic risk factors for cardiovascular disease, and an association between elevated peripheral levels of endogenous ligands of cannabinoid receptors (endocannabinoids) and the metabolic syndrome has been reported. We investigated the dose-dependent effects of dietary ω-3-PUFA supplementation, given as krill oil (KO), on metabolic parameters in high fat diet (HFD)-fed mice and, in parallel, on the levels, in inguinal and epididymal adipose tissue (AT), liver, gastrocnemius muscle, kidneys and heart, of: 1) the endocannabinoids, anandamide and 2-arachidonoylglycerol (2-AG), 2) two anandamide congeners which activate PPARα but not cannabinoid receptors, N-oleoylethanolamine and N-palmitoylethanolamine, and 3) the direct biosynthetic precursors of these compounds.MethodsLipids were identified and quantified using liquid chromatography coupled to atmospheric pressure chemical ionization single quadrupole mass spectrometry (LC-APCI-MS) or high resolution ion trap-time of flight mass spectrometry (LC-IT-ToF-MS).ResultsEight-week HFD increased endocannabinoid levels in all tissues except the liver and epididymal AT, and KO reduced anandamide and/or 2-AG levels in all tissues but not in the liver, usually in a dose-dependent manner. Levels of endocannabinoid precursors were also generally down-regulated, indicating that KO affects levels of endocannabinoids in part by reducing the availability of their biosynthetic precursors. Usually smaller effects were found of KO on OEA and PEA levels.ConclusionsOur data suggest that KO may promote therapeutic benefit by reducing endocannabinoid precursor availability and hence endocannabinoid biosynthesis.

Sheep cheese naturally enriched in α-linolenic, conjugated linoleic and vaccenic acids improves the lipid profile and reduces anandamide in the plasma of hypercholesterolaemic subjects

Intake of dairy fat has long been considered as a risk factor for CVD. Pasture and dietary lipid supplementation have been reported to be reliable strategies in ruminant nutrition, in order to increase the content of α-linolenic acid (ALA), conjugated linoleic acid (CLA) and vaccenic acid (VA), and decrease SFA in milk fat. In the present study, we aimed at verifying whether consumption of a sheep cheese, naturally enriched in ALA, CLA and VA, would modify the plasma lipid and endocannabinoid profiles in mildly hypercholesterolaemic subjects. A total of forty-two adult volunteers (nineteen males and twenty-three females) with diagnosed mildly hypercholesterolaemia (total cholesterol 5·68-7·49 mmol/l) were randomly assigned to eat 90 g/d of a control or enriched cheese for 3 weeks, with a cross-over after 3 weeks of washout. Plasma lipids, endocannabinoids, adipokines and inflammatory markers were measured. The intake of enriched cheese significantly increased the plasma concentrations of CLA, VA, the n-3 fatty acids ALA and EPA, and more remarkably decreased that of the endocannabinoid anandamide. LDL-cholesterol decreased significantly (7%). No changes were detected in the levels of inflammatory markers; however, a significant correlation was found between the plasma levels of anandamide and leptin. The control cheese modified none of the parameters measured. The results obtained do not support the view that intake of dairy fat is detrimental to hypercholesterolaemic subjects. Indeed, they show that a naturally enriched cheese possesses beneficial properties, since it ameliorates the plasma lipid profile, and more remarkably reduces endocannabinoid biosynthesis.

Krill oil significantly decreases 2-arachidonoylglycerol plasma levels in obese subjects

We have previously shown that krill oil (KO), more efficiently than fish oil, was able to downregulate the endocannabinoid system in different tissues of obese zucker rats.We therefore aimed at investigating whether an intake of 2 g/d of either KO or menhaden oil (MO), which provides 309 mg/d of EPA/DHA 2:1 and 390 mg/d of EPA/DHA 1:1 respectively, or olive oil (OO) for four weeks, is able to modify plasma endocannabinoids in overweight and obese subjects.The results confirmed data in the literature describing increased levels of endocannabinoids in overweight and obese with respect to normo-weight subjects. KO, but not MO or OO, was able to significantly decrease 2-arachidonoylglycerol (2-AG), although only in obese subjects. In addition, the decrease of 2-AG was correlated to the plasma n-6/n-3 phospholipid long chain polyunsaturated fatty acid (LCPUFA) ratio. These data show for the first time in humans that relatively low doses of LCPUFA n-3 as KO can significantly decrease plasma 2-AG levels in obese subjects in relation to decrease of plasma phospholipid n-6/n-3 LCPUFA ratio. This effect is not linked to changes of metabolic syndrome parameters but is most likely due to a decrease of 2-AG biosynthesis caused by the replacement of 2-AG ultimate precursor, arachidonic acid, with n-3 PUFAs, as previously described in obese Zucker rats.

Conjugated linoleic acids (CLA) as precursors of a distinct family of PUFA

One of the possibilities for distinct actions of c9,t11- and the t10,c12-conjugated linoleic acid (CLA) isomers may be at the level of metabolism since the conjugated diene structure gives to CLA isomers and their metabolites a distinct pattern of incorporation into the lipid fraction and metabolism. In fact, CLA appears to undergo similar transformations as linoleic acid but with subtle isomer differences, which may account for their activity in lowering linoleic acid metabolites in those tissues rich in neutral lipids where CLA is preferentially incorporated. Furthermore, c9,t11 and t10,c12 isomers are metabolized at a different rate in the peroxisomes, where the shortened metabolite from t10,c12 is formed at a much higher proportion than the metabolite from c9,t11. This may account for the lower accumulation of t10,c12 isomer into cell lipids. CLA isomers may therefore be viewed as a “new” family of polyunsaturated fatty acids (PUFA) producing a distinct range of metabolites using the same enzymatic system as the other (i.e., n−3, n−6 and n−9) PUFA families. It is likely that perturbation of PUFA metabolism by CLA will have an impact on eicosanoid formation and metabolism, closely linked to the biological activities attributed to CLA.

Effect of acute administration of Pistacia lentiscus L. essential oil on rat cerebral cortex following transient bilateral common carotid artery occlusion

BackgroundIschemia/reperfusion leads to inflammation and oxidative stress which damages membrane highly polyunsaturated fatty acids (HPUFAs) and eventually induces neuronal death. This study evaluates the effect of the administration of Pistacia lentiscus L. essential oil (E.O.), a mixture of terpenes and sesquiterpenes, on modifications of fatty acid profile and endocannabinoid (eCB) congener concentrations induced by transient bilateral common carotid artery occlusion (BCCAO) in the rat frontal cortex and plasma.MethodsAdult Wistar rats underwent BCCAO for 20 min followed by 30 min reperfusion (BCCAO/R). 6 hours before surgery, rats, randomly assigned to four groups, were gavaged either with E.O. (200 mg/0.45 ml of sunflower oil as vehicle) or with the vehicle alone.ResultsBCCAO/R triggered in frontal cortex a decrease of docosahexaenoic acid (DHA), the membrane highly polyunsaturated fatty acid most susceptible to oxidation. Pre-treatment with E.O. prevented this change and led further to decreased levels of the enzyme cyclooxygenase-2 (COX-2), as assessed by Western Blot. In plasma, only after BCCAO/R, E.O. administration increased both the ratio of DHA-to-its precursor, eicosapentaenoic acid (EPA), and levels of palmytoylethanolamide (PEA) and oleoylethanolamide (OEA).ConclusionsAcute treatment with E.O. before BCCAO/R elicits changes both in the frontal cortex, where the BCCAO/R-induced decrease of DHA is apparently prevented and COX-2 expression decreases, and in plasma, where PEA and OEA levels and DHA biosynthesis increase. It is suggested that the increase of PEA and OEA plasma levels may induce DHA biosynthesis via peroxisome proliferator-activated receptor (PPAR) alpha activation, protecting brain tissue from ischemia/reperfusion injury.

Vagus Nerve Stimulation Reduces Body Weight and Fat Mass in Rats

Among the manifold effects of vagus nerve stimulation (VNS) delivered as an add-on treatment to patients with drug-resistant epilepsy, a moderate loss of body weight has been observed in some individuals. We have now investigated this effect in rats. Exposure of rats to VNS for 4 weeks reduced feed conversion efficiency as well as body weight gain (by ∼25%) and the amount of mesenteric adipose tissue (by ∼45%) in comparison with those in sham-operated control animals. A pair-fed experiment showed that both lower dietary intake and increase energy expenditure independently contributed to the reduction of body weight and mesenteric adipose tissue. Moreover, VNS increased the level of non-esterified fatty acids in plasma and mesenteric adipose tissue by ∼50 and 80%, respectively, without affecting that in the liver. In addition, VNS reduced the amounts of endocannabinoids and increased N-palmitoylethanolamide, an endogenous ligand of the transcription factor PPARα (peroxisome proliferator–activated receptor α) in mesenteric adipose tissue but not in the hypothalamus. These effects were accompanied by increased expression of the gene for brain-derived neurotrophic factor (BDNF) in the hypothalamus and up-regulation of the abundance of PPARα in the liver. Our results suggest that the reduction in body fat induced by VNS in rats may result from the action of both central and peripheral mediators. The reduced feed conversion efficiency associated with VNS may be mediated by hypothalamic BDNF, down-regulation of endocannabinoid tone in mesenteric adipose tissue and a PPARα-dependent increase in fatty acid oxidation in the liver, which in concerted action may account for the anorexic effect and increased energy expenditure.

Impairment of 8-iso-PGF2ALPHA isoprostane metabolism by dietary conjugated linoleic acid (CLA)

8-iso-PGF(2alpha) isoprostane (IP) is one of the most-used markers of lipid peroxidation in experimental models and humans. After its formation, it is promptly metabolized to 2,3 dinor (DIN) in peroxisomes. Conjugated linoleic acid (CLA) is preferentially beta-oxidized in peroxisomes which may compete with IP, and thereby may affect its metabolism. In order to verify whether CLA is able to influence IP formation and/or metabolism and to explain the mechanism, we challenged rats supplemented with CLA or with triolein (as a control fatty acid), with a single dose of carbon tetrachloride (CCl(4)) or of bacterial lipopolysaccharide (LPS). The results showed that IP and its precursor arachidonic acid hydroperoxide, as well as malondialdehyde (MDA), increase significantly in the liver of rats challenged with CCl(4), irrespective of the diet, while in LPS-treated rats only nitrites in liver and isoprostane in plasma increase. On the other hand, the peroxisomal beta-oxidation products of IP, the DIN, is significantly lower in the CLA group with respect to control and triolein groups. To further investigate whether this is due to competition between CLA and IP at the cellular level, we incubated human fibroblasts from healthy subjects or patients with adrenoleukodystrophy (ALD), with CLA and/or commercially available IP. The rationale of this approach is based on the deficient peroxisomal beta-oxidation of fibroblasts from ALD patients, leading to a reduced formation of DIN. In both normal and ALD cells, the presence of CLA significantly inhibits the formation of DIN from IP. We may conclude that both in vitro and in vivo studies strongly suggest that CLA may impair IP catabolism in peroxisomes. Consequently an increase of IP, as a sole result of CLA intake, cannot be considered as a marker of lipid peroxidation.

Metabolism of c9,t11-conjugated linoleic acid (CLA) in humans

The c9,t11 isomer of conjugated linoleic acid (CLA) is the most abundant CLA form present in the human diet, and is particularly prevalent in milk and dairy products, and is known to exert several health benefits in experimental animal models. A possible mechanism of action of c9,t11CLA relies on its metabolism via desaturases and elongases and partial beta oxidation in peroxisomes. In this study, we aimed to establish plasma incorporation of c9,t11CLA and its downstream metabolites in healthy volunteers after daily dietary intakes of 0.8g, 1.6g or 3.2g of c9,t11CLA in capsule form for two months. Following supplementation, the plasma concentrations of c9,t11CLA and its metabolites conjugated dienes (CD) 18:3 and the beta oxidation product CD 16:2 were incorporated in a linear fashion, while on the other hand CD 20:3 reached a plateau following intakes of 1.6g/d of dietary intake, and was not further increased following higher CLA intakes. We may conclude that supplementation of c9,t11 CLA levels result in linear responses of CLA and its main metabolites in plasma. In addition, only the highest concentration of CLA intake tested (3.2g/d) yielded plasma concentrations of CLA and metabolites close to the range found sufficient to exert nutritional effects in experimental animal models.

Hepatic retinol secretion and storage are altered by dietary CLA: common and distinct actions of CLA c9,t11 and t10,c12 isomers

Conjugated linoleic acid (CLA) is a polyunsaturated fatty acid obtained from ruminant products. Previous studies in rats and pigs showed that a dietary equimolar mixture of c9,t11 and t10,c12 CLA isomers induces changes in serum and tissue levels of retinoids (vitamin A derivatives). However, the mechanism(s) responsible for these actions remain(s) unexplored. Given the numerous crucial biological functions regulated by retinoids, it is key to establish whether the perturbations in retinoid metabolism induced by dietary CLA mediate some of the beneficial effects associated with intake of this fatty acid or, rather, have adverse consequences on health. To address this important biological question, we began to explore the mechanisms through which dietary CLA alters retinoid metabolism. By using enriched preparations of CLA c9,t11 or CLA t10,c12, we uncoupled the effects of these two CLA isomers on retinoid metabolism. Specifically, we show that both isomers induce hepatic retinyl ester accumulation. However, only CLA t10,c12 enhances hepatic retinol secretion, resulting in increased serum levels of retinol and its specific carrier, retinol-binding protein (RBP). Dietary CLA t10,c12 also redistributes retinoids from the hepatic stores toward the adipose tissue and possibly stimulates hepatic retinoid oxidation. Using mice lacking RBP, we also demonstrate that this key protein in retinoid metabolism mediates hepatic retinol secretion and its redistribution toward fat tissue induced by CLA t10,c12 supplementation.