Use of Saccharomyces boulardii CNCM I-745 as therapeutic strategy for prevention of nonsteroidal anti-inflammatory drug-induced intestinal injury.

BACKGROUND AND PURPOSE: Nonsteroidal anti-inflammatory drugs (NSAIDs) can be associated with severe adverse digestive effects. This study examined the protective effects of the probiotic Saccharomyces boulardii CNCM I-745 in a rat model of diclofenac-induced enteropathy. EXPERIMENTAL APPROACH: Enteropathy was induced in 40-week-old male rats by intragastric diclofenac (4 mg·kg-1 BID for 14 days). S. boulardii CNCM I-745 (3 g·kg-1 BID by oral gavage) was administered starting 14 days before (preventive protocol) or along with (curative protocol) diclofenac administration. Ileal damage, inflammation, barrier integrity, gut microbiota composition and toll-like receptors (TLRs)-nuclear factor κB (NF-κB) pathway were evaluated. KEY RESULTS: Diclofenac elicited intestinal damage, along with increments of myeloperoxidase, malondialdehyde, tumour necrosis factor and interleukin-1ß, overexpression of TLR2/4, myeloid differentiation primary response 88 (Myd88) and NF-κB p65, increased faecal calprotectin and butyrate levels, and decreased blood haemoglobin levels, occludin and butyrate transporter monocarboxylate transporter 1 (MCT1) expression. In addition, diclofenac provoked a shift of bacterial taxa in both faecal and ileal samples. Treatment with S. boulardii CNCM I-745, in both preventive and curative protocols, counteracted the majority of these deleterious changes. Only preventive administration of the probiotic counteracted NSAID-induced decreased expression of MCT1 and increase in faecal butyrate levels. Occludin expression, after probiotic treatment, did not significantly change. CONCLUSIONS AND IMPLICATIONS: Treatment with S. boulardii CNCM I-745 prevents diclofenac-induced enteropathy through anti-inflammatory and antioxidant activities. Such effects are likely to be related to increased tissue butyrate bioavailability, through an improvement of butyrate uptake by the enteric mucosa.

Anti-Inflammatory Effect of Beta-Caryophyllene Mediated by the Involvement of TRPV1, BDNF and trkB in the Rat Cerebral Cortex after Hypoperfusion/Reperfusion.

We have previously shown that bilateral common carotid artery occlusion followed by reperfusion (BCCAO/R) is a model to study early hypoperfusion/reperfusion-induced changes in biomarkers of the tissue physiological response to oxidative stress and inflammation. Thus in this study, we investigate with immunochemical assays if a single dose of beta-caryophyllene (BCP), administered before the BCCAO/R, can modulate the TRPV1, BDNF, and trkB receptor in the brain cortex; the glial markers GFAP and Iba1 were also examined. Frontal and temporal-occipital cortical regions were analyzed in two groups of male rats, sham-operated and submitted to BCCAO/R. Six hours before surgery, one group was gavage fed a dose of BCP (40 mg/per rat in 300 µL of sunflower oil), the other was pre-treated with the vehicle alone. Western blot analysis showed that, in the frontal cortex of vehicle-treated rats, the BCCAO/R caused a TRPV1 decrease, an increment of trkB and GFAP, no change in BDNF and Iba1. The BCP treatment caused a decrease of BDNF and an increase of trkB levels in both sham and BCCAO/R conditions while inducing opposite changes in the case of TRPV1, whose levels became higher in BCCAO/R and lower in sham conditions. Present results highlight the role of BCP in modulating early events of the cerebral inflammation triggered by the BCCAO/R through the regulation of TRPV1 and the BDNF-trkB system.

Fatty Acid Metabolism and Derived-Mediators Distinctive of PPAR-α Activation in Obese Subjects Post Bariatric Surgery.

Bariatric surger (BS) is characterized by lipid metabolic changes as a response to the massive release of non-esterified fatty acids (NEFA) from adipose depots. The study aimed at evaluating changes in polyunsaturated fatty acids (PUFA) metabolism and biosynthesis of the lipid mediators N-acylethanolamines (NAE), as indices of nuclear peroxisome proliferator-activated receptor (PPAR)-α activation. The observational study was performed on 35 subjects (27 female, 8 male) with obesity, undergoing bariatric surgery. We assessed plasma FA and NAE profiles by LC-MS/MS, clinical parameters and anthropometric measures before and 1 and 6 months after bariatric surgery. One month after bariatric surgery, as body weight and clinical parameters improved significantly, we found higher plasma levels of N-oleoylethanolamine, arachidonic and a 22:6-n3/20:5-n3 ratio as evidence of PPAR-α activation. These changes corresponded to higher circulating levels of NEFA and a steep reduction of the fat mass. After 6 months 22:6-n3/20:5-n3 remained elevated and fat mass was further reduced. Our data suggest that the massive release of NEFA from adipose tissue at 1-Post, possibly by inducing PPAR-α, may enhance FA metabolism contributing to fat depot reduction and improved metabolic parameters in the early stage. However, PUFA metabolic changes favor n6 PUFA biosynthesis, requiring a nutritional strategy aimed at reducing the n6/n3 PUFA ratio.

Decreased Metabolic Flexibility in Skeletal Muscle of Rat Fed with a High-Fat Diet Is Recovered by Individual CLA Isomer Supplementation via Converging Protective Mechanisms.

Energy balance, mitochondrial dysfunction, obesity, and insulin resistance are disrupted by metabolic inflexibility while therapeutic interventions are associated with improved glucose/lipid metabolism in skeletal muscle. Conjugated linoleic acid mixture (CLA) exhibited anti-obesity and anti-diabetic effects; however, the modulatory ability of its isomers (cis9, trans11, C9; trans10, cis12, C10) on the metabolic flexibility in skeletal muscle remains to be demonstrated. Metabolic inflexibility was induced in rat by four weeks of feeding with a high-fat diet (HFD). At the end of this period, the beneficial effects of C9 or C10 on body lipid content, energy expenditure, pro-inflammatory cytokines, glucose metabolism, and mitochondrial efficiency were examined. Moreover, oxidative stress markers, fatty acids, palmitoyletanolamide (PEA), and oleyletanolamide (OEA) contents along with peroxisome proliferator-activated receptors-alpha (PPARα), AKT, and adenosine monophosphate-activated protein kinase (AMPK) expression were evaluated in skeletal muscle to investigate the underlying biochemical mechanisms. The presented results indicate that C9 intake reduced mitochondrial efficiency and oxidative stress and increased PEA and OEA levels more efficiently than C10 while the anti-inflammatory activity of C10, and its regulatory efficacy on glucose homeostasis are associated with modulation of the PPARα/AMPK/pAKT signaling pathway. Our results support the idea that the dissimilar efficacy of C9 and C10 against the HFD-induced metabolic inflexibility may be consequential to their ability to activate different molecular pathways.

Palmitoylethanolamide counteracts hepatic metabolic inflexibility modulating mitochondrial function and efficiency in diet-induced obese mice.

Peroxisome proliferator-activated receptor (PPAR)-α activation controls hepatic lipid homeostasis, stimulating fatty acid oxidation, and adapting the metabolic response to lipid overload and storage. Here, we investigate the effect of palmitoylethanolamide (PEA), an endogenous PPAR-α ligand, in counteracting hepatic metabolic inflexibility and mitochondrial dysfunction induced by high-fat diet (HFD) in mice. Long-term PEA administration (30 mg/kg/die per os) in HFD mice limited hepatic lipid accumulation, increased energy expenditure, and markedly reduced insulin resistance. In isolated liver mitochondria, we have demonstrated PEA capability to modulate mitochondrial oxidative capacity and energy efficiency, leading to the reduction of intracellular lipid accumulation and oxidative stress. Moreover, we have evaluated the effect of PEA on mitochondrial bioenergetics of palmitate-challenged HepG2 cells, using Seahorse analyzer. In vitro data showed that PEA recovered mitochondrial dysfunction and reduced lipid accumulation in insulin-resistant HepG2 cells, increasing fatty acid oxidation. Mechanistic studies showed that PEA effect on lipid metabolism was limited by AMP-activated protein kinase (AMPK) inhibition, providing evidence for a pivotal role of AMPK in PEA-induced adaptive metabolic setting. All these findings identify PEA as a modulator of hepatic lipid and glucose homeostasis, limiting metabolic inflexibility induced by nutrient overload.

Downregulation of inflammatory markers by conjugated linoleic acid isomers in human cultured astrocytes.

OBJECTIVES: Conjugated linoleic acid (CLA) isomers have been shown to possess anti-inflammatory activity in the central nervous system. In this study, we aimed to evaluate whether modulation of the fatty acid profile by the CLA isomers c9,t11 or t10,c12CLA was associated with changes in the expression of pro-inflammatory molecules in human astrocytes. METHODS: Cultured astrocytes were treated for 6 days with 100 µM fatty acids (c9,t11CLA or t10,c12CLA or oleic acid). Following the treatment, the fatty acid profile of the cell and pro-inflammatory molecule expression were assessed. RESULTS: Only the t10,c12CLA isomer induced a significant decrease in arachidonic acid and increased the ratio of docosahexaenoic acid/eicosapentaenoic acid, which constitutes indirect evidence of peroxisome proliferator-activated receptor alpha activation. Inhibition of tumour necrosis factor-α, interleukin-1ß, and RANTES expression was observed in astrocytes treated with c9,t11CLA and t10,c12CLA. DISCUSSION: Current data demonstrate that CLA isomers, particularly t10,c12, may affect neuroinflammation by reducing the pro-inflammatory molecules in cultured astrocytes, suggesting a potential nutritional role of CLA isomers in modulating the astrocyte inflammatory response.

Inhibition of N-acylethanolamine acid amidase reduces nicotine-induced dopamine activation and reward.

Tobacco smoke is the leading preventable cause of death in the world and treatments aimed to increase success rate in smoking cessation by reducing nicotine dependence are sought. Activation of peroxisome proliferator-activated receptor-alpha (PPARα) by synthetic or endogenous agonists was shown to suppress nicotine-induced activation of mesolimbic dopamine system, one of the major neurobiological substrates of nicotine dependence, and nicotine-seeking behavior in rats and monkeys. An alternative indirect way to activate PPARα is inhibition of N-acylethanolamine acid amidase (NAAA), one of the major hydrolyzing enzyme for its endogenous agonists palmitoylethanolamide (PEA) and oleoylethanolamide (OEA). We synthetized a novel specific brain permeable NAAA inhibitor, AM11095. We administered AM11095 to rats and carried out brain lipid analysis, a functional observational battery (FOB) to assess toxicity, in vivo electrophysiological recording from dopamine cells in the ventral tegmental area, brain microdialysis in the nucleus accumbens shell and behavioral experiments to assess its effect on nicotine -induced conditioned place preference (CPP). AM11095 (5 and 25 mg/kg, i.p.) was devoid of neurotoxic and behavioral effects and did not affect motor behavior and coordination. This NAAA inhibitor (5 mg/kg i.p.) increased OEA and PEA levels in the hippocampus and cortex, prevented nicotine-induced activation of mesolimbic dopamine neurons in the ventral tegmental area, nicotine-induced elevation of dopamine levels in the nucleus accumbens shell and decreased the expression of nicotine CPP. Our results indicate that NAAA inhibitors represent a new class of pharmacological tools to modulate brain PEA/PPARα signalling and show potential in the treatment of nicotine dependence.

Acute administration of beta-caryophyllene prevents endocannabinoid system activation during transient common carotid artery occlusion and reperfusion.

BACKGROUND: The transient global cerebral hypoperfusion/reperfusion achieved by induction of Bilateral Common Carotid Artery Occlusion followed by Reperfusion (BCCAO/R) has been shown to stimulate early molecular changes that can be easily traced in brain tissue and plasma, and that are indicative of the tissue physiological response to the reperfusion-induced oxidative stress and inflammation. The aim of the present study is to probe the possibility to prevent the molecular changes induced by the BCCAO/R with dietary natural compounds known to possess anti-inflammatory activity, such as the phytocannabinoid beta-caryophyllene (BCP). METHODS: Two groups of adult Wistar rats were used, sham-operated and submitted to BCCAO/R. In both groups, 6 h before surgery, half of the rats were gavage-fed with a single dose of BCP (40 mg/per rat in 300 µl of sunflower oil as vehicle), while the second half were pre-treated with the vehicle alone. HPLC, Western Blot and immunohistochemistry were used to analyze cerebral cortex and plasma. RESULTS: After BCCAO/R, BCP prevented the increase of lipoperoxides occurring in the vehicle-treated rats in both cerebral cortex and plasma. In the frontal cortex, BCP further prevented activation of the endocannabinoid system (ECS), spared the docosahexaenoic acid (DHA), appeared to prevent the increase of cyclooxygenase-2 and increased the peroxisome-proliferator activated receptor-alpha (PPAR-alpha) protein levels, while, in plasma, BCP induced the reduction of arachidonoylethanolamide (AEA) levels as compared to vehicle-treated rats. CONCLUSIONS: Collectively, the pre-treatment with BCP, likely acting as agonist for CB2 and PPAR-alpha receptors, modulates in a beneficial way the ECS activation and the lipoperoxidation, taken as indicative of oxidative stress. Furthermore, our results support the evidence that BCP may be used as a dietary supplement to control the physiological response to the hypoperfusion/reperfusion-induced oxidative stress.

Preventive Effects of Resveratrol on Endocannabinoid System and Synaptic Protein Modifications in Rat Cerebral Cortex Challenged by Bilateral Common Carotid Artery Occlusion and Reperfusion.

This study aims to evaluate the putative roles of a single acute dose of resveratrol (RVT) in preventing cerebral oxidative stress induced by bilateral common carotid artery occlusion, followed by reperfusion (BCCAO/R) and to investigate RVT's ability to preserve the neuronal structural integrity. Frontal and temporal-occipital cortices were examined in two groups of adult Wistar rats, sham-operated and submitted to BCCAO/R. In both groups, 6 h before surgery, half the rats were gavage-fed with a single dose of RVT (40 mg/per rat in 300 µL of sunflower oil as the vehicle), while the second half received the vehicle alone. In the frontal cortex, RVT pre-treatment prevented the BCCAO/R-induced increase of lipoperoxides, augmented concentrations of palmitoylethanolamide and docosahexaenoic acid, increased relative levels of the cannabinoid receptors type 1 (CB1) and 2 (CB2), and peroxisome-proliferator-activated-receptor (PPAR)-α proteins. Increased expression of CB1/CB2 receptors mirrored that of synaptophysin and post-synaptic density-95 protein. No BCCAO/R-induced changes occurred in the temporal-occipital cortex. Collectively, our results demonstrate that, in the frontal cortex, RVT pre-treatment prevents the BCCAO/R-induced oxidative stress and modulates the endocannabinoid and PPAR-α systems. The increased expression of synaptic structural proteins further suggests the possible efficacy of RVT as a dietary supplement to preserve the nervous tissue metabolism and control the physiological response to the hypoperfusion/reperfusion challenge.

High-density lipoprotein contribute to G0-G1/S transition in Swiss NIH/3T3 fibroblasts.

High density lipoproteins (HDLs) play a crucial role in removing excess cholesterol from peripheral tissues. Although their concentration is lower during conditions of high cell growth rate (cancer and infections), their involvement during cell proliferation is not known. To this aim, we investigated the replicative cycles in synchronised Swiss 3T3 fibroblasts in different experimental conditions: i) contact-inhibited fibroblasts re-entering cell cycle after dilution; ii) scratch-wound assay; iii) serum-deprived cells induced to re-enter G1 by FCS, HDL or PDGF. Analyses were performed during each cell cycle up to quiescence. Cholesterol synthesis increased remarkably during the replicative cycles, decreasing only after cells reached confluence. In contrast, cholesteryl ester (CE) synthesis and content were high at 24 h after dilution and then decreased steeply in the successive cycles. Flow cytometry analysis of DiO-HDL, as well as radiolabeled HDL pulse, demonstrated a significant uptake of CE-HDL in 24 h. DiI-HDL uptake, lipid droplets (LDs) and SR-BI immunostaining and expression followed the same trend. Addition of HDL or PDGF partially restore the proliferation rate and significantly increase SR-BI and pAKT expression in serum-deprived cells. In conclusion, cell transition from G0 to G1/S requires CE-HDL uptake, leading to CE-HDL/SR-BI pathway activation and CEs increase into LDs.

Nutritional properties of dietary omega-3-enriched phospholipids.

Dietary fatty acids regulate several physiological functions. However, to exert their properties, they have to be present in the diet in an optimal balance. Particular attention has been focused on tissue highly polyunsaturated fatty acids (HPUFAs) n-6/n-3 ratio, influenced by the type and the esterified form of dietary fatty acids. Dietary EPA and DHA when esterified to phospholipids (PLs) are more efficiently incorporated into tissue PLs and seem to possess peculiar properties through specific mechanism(s) of action, such as the capacity to affect endocannabinoid biosynthesis at much lower doses than EPA and DHA in triglyceride form, probably because of the above mentioned higher incorporation into tissue PLs. Downregulation of the endocannabinoid system seems to mediate the positive effects exerted by omega-3-enriched PLs on several parameters of metabolic syndrome. PLs are one of the major dietary forms of EPA and DHA we are exposed to with the everyday diet; therefore, it is not surprising that it guarantees an effective EPA and DHA nutritional activity. Future studies should address whether EPA and DHA in PL form are also more effective than other formulations in ameliorating other pathological conditions where n-3 HPUFAs seem to exert beneficial activities such as cancer and psychiatric disorders.

Role of HDL in cholesteryl ester metabolism of lipopolysaccharide-activated P388D1 macrophages.

Infections share with atherosclerosis similar lipid alterations, with accumulation of cholesteryl esters (CEs) in activated macrophages and concomitant decrease of cholesterol-HDL (C-HDL). Yet the precise role of HDL during microbial infection has not been fully elucidated. Activation of P388D1 by lipopolysaccharide (LPS) triggered an increase of CEs and neutral lipid contents, along with a remarkable enhancement in 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate-HDL uptake. Similar results were found in human monocyte-derived macrophages and monocytes cocultured with phytohemagglutinin-activated lymphocytes. Inhibition of cholesterol esterification with Sandoz-58035 resulted in 80% suppression of CE biosynthesis in P388D1. However, only a 35% decrease of CE content, together with increased scavenger receptor class B member 1 (SR-B1) protein expression, was found after 72 h and thereafter up to 16 passages of continuous ACAT suppression. Chronic inhibition blunted the effect of LPS treatment on cholesterol metabolism, increased the ratio of free cholesterol/CE content and enhanced interleukin 6 secretion. These results imply that, besides de novo biosynthesis and acquisition by LDL, HDL contributes probably through SR-B1 to the increased CE content in macrophages, partly explaining the low levels of C-HDL during their activation. Our data suggest that in those conditions where more CEs are required, HDL rather than removing, may supply CEs to the cells.

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

BACKGROUND: Ischemia/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. METHODS: Adult 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. RESULTS: BCCAO/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). CONCLUSIONS: Acute 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.

A mixture of oleic, erucic and conjugated linoleic acids modulates cerebrospinal fluid inflammatory markers and improve somatosensorial evoked potential in X-linked adrenoleukodystrophy female carriers.

X-linked adrenoleukodystrophy is a rare inherited demyelinating disorder characterized by an abnormal accumulation of very long chain fatty acids, mainly hexacosanoic acid (26:0), due to a mutation of the gene encoding for a peroxisomal membrane protein. The only available, and partially effective, therapeutic treatment consists of dietary intake of a 4:1 mixture of triolein and trierucin, called Lorenzo's oil (LO), targeted to inhibit the elongation of docosanoic acid (22:0) to 26:0. In this study we tested whether, besides inhibiting elongation, an enhancement of peroxisomal beta oxidation induced by conjugated linoleic acid (CLA), will improve somatosensory evoked potentials and modify inflammatory markers in adrenoleukodystrophy females carriers. We enrolled five heterozygous women. They received a mixture of LO (40 g/day) with CLA (5 g/day) for 2 months. The therapeutic efficacy was evaluated by the means of plasma levels of 26:0, 26:0/22:0 ratio, modification of cerebrospinal fluid (CSF) inflammatory markers and somatosensory evoked potentials. Changes of fatty acid profile, and in particular CLA incorporation, were also evaluated in CSF and plasma. The results showed that CLA promptly passes the blood brain barrier and the mixture was able to lower both 26:0 and 26:0/22:0 ratio in plasma. The mixture improved somatosensory evoked potentials, which were previously found unchanged or worsened with dietary LO alone, and reduced IL-6 levels in CSF in three out of five patients. Our data suggest that the synergic activity of CLA and LO, by enhancing peroxisomal beta-oxidation and preventing 26:0 formation, improves the somatosensory evoked potentials and reduces neuroinflammation.

A lipoprotein source of cholesteryl esters is essential for proliferation of CEM-CCRF lymphoblastic cell line.

Tumour are characterised by a high content of cholesteryl esters (CEs) stored in lipid droplets purported to be due to a high rate of intracellular esterification of cholesterol. To verify whether and which pathways involved in CE accumulation are essential in tumour proliferation, the effect of CE deprivation, from both exogenous and endogenous sources, on CEM-CCRF cells was investigated. Cholesterol synthesis, esterification and content, low-density lipoprotein (LDL) binding and high-density lipoprotein (HDL)-CE uptake were evaluated in cultured in both conventional and delipidated bovine serum with or without oleic or linoleic acids, cholesteryl oleate, LDL and HDL. High content of CEs in lipid droplets in this cell line was due to esterification of both newly synthesised cholesterol and that obtained from hydrolysis of LDL; moreover, a significant amount of CE was derived from HDL-CE uptake. Cell proliferation was slightly affected by either acute or chronic treatment up to 400 µM with Sz-58035, an acyl-cholesteryl cholesterol esterification inhibitor (ACAT); although when the enzyme activity was continuously inhibited, CE content in lipid droplets was significantly higher than those in control cells. In these cells, analysis of intracellular and medium CEs revealed a profile reflecting the characteristics of bovine serum, suggesting a plasma origin of CE molecules. Cell proliferation arrest in delipidated medium was almost completely prevented in the first 72 h by LDL or HDL, although in subsequent cultures with LDL, it manifested an increasing mortality rate. This study suggests that high content of CEs in CEM-CCRF is mainly derived from plasma lipoproteins and that part of CEs stored in lipid droplets are obtained after being taken up from HDL. This route appears to be up-regulated according to cell requirements and involved in low levels of c-HDL during cancer. Moreover, the dependence of tumour cells on a source of lipoprotein provides a novel impetus in developing therapeutic strategies for use in the treatment of some tumours.

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

BACKGROUND: Omega-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. METHODS: Lipids 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). RESULTS: Eight-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. CONCLUSIONS: Our data suggest that KO may promote therapeutic benefit by reducing endocannabinoid precursor availability and hence 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.

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.

Differential effect of oleic and palmitic acid on lipid accumulation and apoptosis in cultured hepatocytes.

BACKGROUND AND AIM: Studies have shown monounsaturated oleic acid to be less toxic than palmitic acid and to prevent/attenuate palmitic acid hepatocites toxicity in steatosis models in vitro. However, to what degree these effects are mediated by steatosis extent is unknown. METHODS: We evaluated whether steatosis per se is associated with hepatocytes apoptosis and determined the role of oleic and palmitic acid, the most abundant fatty acids in western diets, on triglyceride accumulation and apoptosis in an in vitro model of steatosis induced in three hepatocytic cell lines (HepG2, HuH7, WRL68). The impact of incubation for 24 h with oleic (0.66 and 1.32 mM) and palmitic acid (0.33 and 0.66 mM), alone or combined (molar ratio 2 : 1) on steatosis, apoptosis, and insulin signalling, was evaluated. RESULTS: Concurrent with PPARgamma and SREBP-1 gene activation, steatosis extent was larger when cells were treated with oleic than with palmitic acid; the latter fatty acid was associated with increased PPARalpha expression. Cell apoptosis was inversely proportional to steatosis deposition. Moreover, palmitic, but not oleic acid, impaired insulin signalling. Despite the higher amount of fat resulting from incubation of the two fatty acids combined, the apoptosis rate and impaired insulin signalling were lower than in cells treated with palmitic acid alone, indicating a protective effect of oleic acid. CONCLUSIONS: Oleic acid is more steatogenic but less apoptotic than palmitic acid in hepatocityc cell cultures. These data may provide a biological basis for clinical findings on dietary patterns and pathogenetic models of nonalcoholic fatty liver disease.

Inhibitory effect of conjugated alpha-linolenic acid from bifidobacteria of intestinal origin on SW480 cancer cells.

In this study, we assessed the ability of six strains of bifidobacteria (previously shown by us to possess the ability to convert linoleic acid to c9, t11-conjugated linoleic acid (CLA) to grow in the presence of alpha-linolenic acid and to generate conjugated isomers of the fatty acid substrate during fermentation for 42 h. The six strains of bifidobacteria were grown in modified MRS (mMRS) containing alpha-linolenic acid for 42 h at 37 degrees C, after which the fatty acid composition of the growth medium was assessed by gas liquid chromatography (GLC). Indeed, following fermentation of one of the strains, namely Bifidobacterium breve NCIMB 702258, in the presence of 0.41 mg/ml alpha-linolenic acid, 79.1% was converted to the conjugated isomer, C18:3 c9, t11, c15 conjugated alpha-linolenic acid (CALA). To examine the inhibitory effect of the fermented oils produced, SW480 colon cancer cells were cultured in the presence of the extracted fermented oil (10-50 microg/ml) for 5 days. The data indicate an inhibitory effect on cell growth (p