Systemic Changes in Endocannabinoids and Endocannabinoid-like Molecules in Response to Partial Nephrectomy-Induced Ischemia in Humans.

Renal ischemia-reperfusion (IR), a routine feature of partial nephrectomy (PN), can contribute to the development of acute kidney injury (AKI). Rodent studies show that the endocannabinoid system (ECS) is a major regulator of renal hemodynamics and IR injury; however, its clinical relevance remains to be established. Here, we assessed the clinical changes in systemic endocannabinoid (eCB) levels induced by surgical renal IR. Sixteen patients undergoing on-clamp PN were included, with blood samples taken before renal ischemia, after 10 min of ischemia time, and 10 min following blood reperfusion. Kidney function parameters (serum creatinine (sCr), blood urea nitrogen (BUN), and serum glucose) and eCB levels were measured. Baseline levels and individual changes in response to IR were analyzed and correlation analyses were performed. The baseline levels of eCB 2-arachidonoylglycerol (2-AG) were positively correlated with kidney dysfunction biomarkers. Unilateral renal ischemia increased BUN, sCr, and glucose, which remained elevated following renal reperfusion. Renal ischemia did not induce changes in eCB levels for all patients pooled together. Nevertheless, stratifying patients according to their body mass index (BMI) revealed a significant increase in N-acylethanolamines (anandamide, AEA; N-oleoylethanolamine, OEA; and N-palmitoylethanolamine, PEA) in the non-obese patients. No significant changes were found in obese patients who had higher N-acylethanolamines baseline levels, positively correlated with BMI, and more cases of post-surgery AKI. With the inefficiency of 'traditional' IR-injury 'preventive drugs', our data support future research on the role of the ECS and its manipulation in renal IR.

Omega-3 carboxylic acids and fenofibrate differentially alter plasma lipid mediators in patients with non-alcoholic fatty liver disease.

Fibrates and omega-3 polyunsaturated acids are used for the treatment of hypertriglyceridemia but have not demonstrated consistent effects on cardiovascular (CV) risk. In this study, we investigate how these two pharmacological agents influence plasma levels of bioactive lipid mediators, aiming to explore their efficacy beyond that of lipid-lowering agents. Plasma from overweight patients with non-alcoholic fatty liver disease (NAFLD) and hypertriglyceridemia, participating in a randomized placebo-controlled study investigating the effects of 12 weeks treatment with fenofibrate or omega-3 free carboxylic acids (OM-3CA) (200 mg or 4 g per day, respectively), were analyzed for eicosanoids and related PUFA species, N-acylethanolamines (NAE) and ceramides. OM-3CA reduced plasma concentrations of proinflammatory PGE2 , as well as PGE1 , PGD1 and thromboxane B2 but increased prostacyclin, and eicosapentaenoic acid- and docosahexaenoic acid-derived lipids of lipoxygenase and cytochrome P450 monooxygenase (CYP) (e.g., 17-HDHA, 18-HEPE, 19,20-DiHDPA). Fenofibrate reduced plasma concentrations of vasoactive CYP-derived eicosanoids (DHETs). Although OM-3CA increased plasma levels of the NAE docosahexaenoyl ethanolamine and docosapentaenoyl ethanolamine, and fenofibrate increased palmitoleoyl ethanolamine, the effect of both treatments may have been masked by the placebo (olive oil). Fenofibrate was more efficacious than OM-3CA in significantly reducing plasma ceramides, pro-inflammatory lipids associated with CV disease risk. Neither treatment affected putative lipid species associated with NAFLD. Our results show that OM-3CA and fenofibrate differentially modulate the plasma mediator lipidome, with OM-3CA promoting the formation of lipid mediators with potential effects on chronic inflammation, while fenofibrate mainly reducing ceramides. These findings suggest that both treatments could ameliorate chronic inflammation with possible impact on disease outcomes, independent of triglyceride reduction.

Palmitoylethanolamide dampens neuroinflammation and anxiety-like behavior in obese mice.

High-fat diet (HFD) consumption leads to obesity and a chronic state of low-grade inflammation, named metainflammation. Notably, metainflammation contributes to neuroinflammation due to the increased levels of circulating free fatty acids and cytokines. It indicates a strict interplay between peripheral and central counterparts in the pathogenic mechanisms of obesity-related mood disorders. In this context, the impairment of internal hypothalamic circuitry runs in tandem with the alteration of other brain areas associated with emotional processing (i.e., hippocampus and amygdala). Palmitoylethanolamide (PEA), an endogenous lipid mediator belonging to the N-acylethanolamines family, has been extensively studied for its pleiotropic effects both at central and peripheral level. Our study aimed to elucidate PEA capability in limiting obesity-induced anxiety-like behavior and neuroinflammation-related features in an experimental model of HFD-fed obese mice. PEA treatment promoted an improvement in anxiety-like behavior of obese mice and the systemic inflammation, reducing serum pro-inflammatory mediators (i.e., TNF-α, IL-1ß, MCP-1, LPS). In the amygdala, PEA increased dopamine turnover, as well as GABA levels. PEA also counteracted the overactivation of HPA axis, reducing the expression of hypothalamic corticotropin-releasing hormone and its type 1 receptor. Moreover, PEA attenuated the immunoreactivity of Iba-1 and GFAP and reduced pro-inflammatory pathways and cytokine production in both the hypothalamus and hippocampus. This finding, together with the reduced transcription of mast cell markers (chymase 1 and tryptase ß2) in the hippocampus, indicated the weakening of immune cell activation underlying the neuroprotective effect of PEA. Obesity-driven neuroinflammation was also associated with the disruption of blood-brain barrier (BBB) in the hippocampus. PEA limited the albumin extravasation and restored tight junction transcription modified by HFD. To gain mechanistic insight, we designed an in vitro model of metabolic injury using human neuroblastoma SH-SY5Y cells insulted by a mix of glucosamine and glucose. Here, PEA directly counteracted inflammation and mitochondrial dysfunction in a PPAR-α-dependent manner since the pharmacological blockade of the receptor reverted its effects. Our results strengthen the therapeutic potential of PEA in obesity-related neuropsychiatric comorbidities, controlling neuroinflammation, BBB disruption, and neurotransmitter imbalance involved in behavioral dysfunctions.

Profound Modification of Fatty Acid Profile and Endocannabinoid-Related Mediators in PPARα Agonist Fenofibrate-Treated Mice.

Fenofibrate (FBR), an oral medication used to treat dyslipidemia, is a ligand of the peroxisome proliferator-activated receptor α (PPARα), a nuclear receptor that regulates the expression of metabolic genes able to control lipid metabolism and food intake. PPARα natural ligands include fatty acids (FA) and FA derivatives such as palmitoylethanolamide (PEA) and oleoylethanolamide (OEA), known to have anti-inflammatory and anorexigenic activities, respectively. We investigated changes in the FA profile and FA derivatives by HPLC and LC-MS in male C57BL/6J mice fed a standard diet with or without 0.2% fenofibrate (0.2% FBR) for 21 days. Induction of PPARα by 0.2% FBR reduced weight gain, food intake, feed efficiency, and liver lipids and induced a profound change in FA metabolism mediated by parallel enhanced mitochondrial and peroxisomal ß-oxidation. The former effects led to a steep reduction of essential FA, particularly 18:3n3, with a consequent decrease of the n3-highly unsaturated fatty acids (HUFA) score; the latter effect led to an increase of 16:1n7 and 18:1n9, suggesting enhanced hepatic de novo lipogenesis with increased levels of hepatic PEA and OEA, which may activate a positive feedback and further sustain reductions of body weight, hepatic lipids and feed efficiency.

Baobab-Fruit Shell and Fibrous Filaments Are Sources of Antioxidant Dietary Fibers.

Since 2008, baobab-fruit dried pulp is listed as an ingredient on the European Union's Novel Food Catalogue. By pulp production, 80% of the baobab fruit is discarded, forming side streams, namely, shell, fibrous filaments, and seeds. This study explored pulp and side-stream functional properties, including total dietary fiber (TDF), total antioxidant capacity (TAC), polyphenols, and water- (WHC) and oil-holding capacities (OHC), along with endocannabinoids (ECs) and N-acylethanolamines (NAEs) in pulp, seeds, and seed oil. Shell excelled in TDF (85%), followed by fibrous filaments (79%), and showed the highest soluble and direct TAC (72 ± 0.7 and 525 ± 1.0 µmol eq. Trolox/g, respectively). Pulp was the richest in polyphenols, followed by shell, fibrous filaments, and seeds. Quercetin predominated in shell (438.7 ± 2.5 µg/g); whereas epicatechin predominated in pulp (514 ± 5.7 µg/g), fibrous filaments (197.2 ± 0.1 µg/g), and seeds (120.1 ± 0.6 µg/g); followed by procyanidin B2 that accounted for 26-40% of total polyphenols in all the products. WHC and OHC ranged between 2-7 g H2O-Oil/g, with fibrous filaments showing the highest values. ECs were not found, whereas NAEs were abundant in seed oil (2408.7 ± 11.1 ng/g). Baobab shell and fibrous filaments are sources of polyphenols and antioxidant dietary fibers, which support their use as functional food ingredients.

Endocannabinoids, endocannabinoid-like molecules and their precursors in human small intestinal lumen and plasma: does diet affect them?

PURPOSE: To determine the small intestinal concentration of endocannabinoids (ECs), N-acylethanolamines (NAEs) and their precursors N-acylphosphatidylethanolamines (NAPEs) in humans. To identify relationships between those concentrations and habitual diet composition as well as individual inflammatory status. METHODS: An observational study was performed involving 35 participants with an ileostomy (18W/17M, aged 18-70 years, BMI 17-40 kg/m2). Overnight fasting samples of ileal fluid and plasma were collected and ECs, NAEs and NAPEs concentrations were determined by LC-HRMS. Dietary data were estimated from self-reported 4-day food diaries. RESULTS: Regarding ECs, N-arachidonoylethanolamide (AEA) was not detected in ileal fluids while 2-arachidonoylglycerol (2-AG) was identified in samples from two participants with a maximum concentration of 129.3 µg/mL. In contrast, mean plasma concentration of AEA was 2.1 ± 0.06 ng/mL and 2-AG was 4.9 ± 1.05 ng/mL. NAEs concentrations were in the range 0.72-17.6 µg/mL in ileal fluids and 0.014-0.039 µg/mL in plasma. NAPEs concentrations were in the range 0.3-71.5 µg/mL in ileal fluids and 0.19-1.24 µg/mL in plasma being more abundant in participants with obesity than normal weight and overweight. Significant correlations between the concentrations of AEA, OEA and LEA in biological fluids with habitual energy or fat intakes were identified. Plasma PEA positively correlated with serum C-reactive protein. CONCLUSION: We quantified ECs, NAEs and NAPEs in the intestinal lumen. Fat and energy intake may influence plasma and intestinal concentrations of these compounds. The luminal concentrations reported would allow modulation of the homeostatic control of food intake via activation of GPR119 receptors located on the gastro-intestinal mucosa. CLINICAL TRIAL REGISTRY NUMBER AND WEBSITE: NCT04143139; www.clinicaltrials.gov .

Neuroprotective and Immunomodulatory Action of the Endocannabinoid System under Neuroinflammation.

Endocannabinoids (eCBs) are lipid-based retrograde messengers with a relatively short half-life that are produced endogenously and, upon binding to the primary cannabinoid receptors CB1/2, mediate multiple mechanisms of intercellular communication within the body. Endocannabinoid signaling is implicated in brain development, memory formation, learning, mood, anxiety, depression, feeding behavior, analgesia, and drug addiction. It is now recognized that the endocannabinoid system mediates not only neuronal communications but also governs the crosstalk between neurons, glia, and immune cells, and thus represents an important player within the neuroimmune interface. Generation of primary endocannabinoids is accompanied by the production of their congeners, the N-acylethanolamines (NAEs), which together with N-acylneurotransmitters, lipoamino acids and primary fatty acid amides comprise expanded endocannabinoid/endovanilloid signaling systems. Most of these compounds do not bind CB1/2, but signal via several other pathways involving the transient receptor potential cation channel subfamily V member 1 (TRPV1), peroxisome proliferator-activated receptor (PPAR)-α and non-cannabinoid G-protein coupled receptors (GPRs) to mediate anti-inflammatory, immunomodulatory and neuroprotective activities. In vivo generation of the cannabinoid compounds is triggered by physiological and pathological stimuli and, specifically in the brain, mediates fine regulation of synaptic strength, neuroprotection, and resolution of neuroinflammation. Here, we review the role of the endocannabinoid system in intrinsic neuroprotective mechanisms and its therapeutic potential for the treatment of neuroinflammation and associated synaptopathy.

Structural analysis of a plant fatty acid amide hydrolase provides insights into the evolutionary diversity of bioactive acylethanolamides.

N-Acylethanolamines (NAEs) are fatty acid derivatives that in animal systems include the well-known bioactive metabolites of the endocannabinoid signaling pathway. Plants use NAE signaling as well, and these bioactive molecules often have oxygenated acyl moieties. Here, we report the three-dimensional crystal structures of the signal-terminating enzyme fatty acid amide hydrolase (FAAH) from Arabidopsis in its apo and ligand-bound forms at 2.1- and 3.2-Å resolutions, respectively. This plant FAAH structure revealed features distinct from those of the only other available FAAH structure (rat). The structures disclosed that although catalytic residues are conserved with the mammalian enzyme, AtFAAH has a more open substrate-binding pocket that is partially lined with polar residues. Fundamental differences in the organization of the membrane-binding "cap" and the membrane access channel also were evident. In accordance with the observed structural features of the substrate-binding pocket, kinetic analysis showed that AtFAAH efficiently uses both unsubstituted and oxygenated acylethanolamides as substrates. Moreover, comparison of the apo and ligand-bound AtFAAH structures identified three discrete sets of conformational changes that accompany ligand binding, suggesting a unique "squeeze and lock" substrate-binding mechanism. Using molecular dynamics simulations, we evaluated these conformational changes further and noted a partial unfolding of a random-coil helix within the region 531-537 in the apo structure but not in the ligand-bound form, indicating that this region likely confers plasticity to the substrate-binding pocket. We conclude that the structural divergence in bioactive acylethanolamides in plants is reflected in part in the structural and functional properties of plant FAAHs.

Intestinal NAPE-PLD contributes to short-term regulation of food intake via gut-to-brain axis.

Our objective was to explore the physiological role of the intestinal endocannabinoids in the regulation of appetite upon short-term exposure to high-fat-diet (HFD) and understand the mechanisms responsible for aberrant gut-brain signaling leading to hyperphagia in mice lacking Napepld in the intestinal epithelial cells (IECs). We generated a murine model harboring an inducible NAPE-PLD deletion in IECs (NapepldΔIEC). After an overnight fast, we exposed wild-type (WT) and NapepldΔIEC mice to different forms of lipid challenge (HFD or gavage), and we compared the modification occurring in the hypothalamus, in the vagus nerve, and at endocrine level 30 and 60 min after the stimulation. NapepldΔIEC mice displayed lower hypothalamic levels of N-oleoylethanolamine (OEA) in response to HFD. Lower mRNA expression of anorexigenic Pomc occurred in the hypothalamus of NapepldΔIEC mice after lipid challenge. This early hypothalamic alteration was not the consequence of impaired vagal signaling in NapepldΔIEC mice. Following lipid administration, WT and NapepldΔIEC mice had similar portal levels of glucagon-like peptide-1 (GLP-1) and similar rates of GLP-1 inactivation. Administration of exendin-4, a full agonist of GLP-1 receptor (GLP-1R), prevented the hyperphagia of NapepldΔIEC mice upon HFD. We conclude that in response to lipid, NapepldΔIEC mice displayed reduced OEA in brain and intestine, suggesting an impairment of the gut-brain axis in this model. We speculated that decreased levels of OEA likely contributes to reduce GLP-1R activation, explaining the observed hyperphagia in this model. Altogether, we elucidated novel physiological mechanisms regarding the gut-brain axis by which intestinal NAPE-PLD regulates appetite rapidly after lipid exposure.

Disease-Specific Derangement of Circulating Endocannabinoids and N-Acylethanolamines in Myeloproliferative Neoplasms.

Growing evidence highlights the endocannabinoid (EC) system involvement in cancer progression. Lipid mediators of this system are secreted by hematopoietic cells, including the ECs 2-arachidonoyl-glycerol (2AG) and arachidonoyl-ethanolamide (AEA), the 2AG metabolite 1AG, and members of N-acylethanolamine (NAE) family-palmitoyl-ethanolamide (PEA) and oleoyl-ethanolamide (OEA). However, the relevance of the EC system in myeloproliferative neoplasms (MPN) was never investigated. We explored the EC plasma profile in 55 MPN patients, including myelofibrosis (MF; n = 41), polycythemia vera (PV; n = 9), and essential thrombocythemia (ET; n = 5) subclasses and in 10 healthy controls (HC). AEA, PEA, OEA, 2AG, and 1AG plasma levels were measured by LC-MS/MS. Overall considered, MPN patients displayed similar EC and NAE levels compared to HC. Nonetheless, AEA levels in MPN were directly associated with the platelet count. MF patients showed higher levels of the sum of 2AG and 1AG compared to ET and PV patients, higher OEA/AEA ratios compared to HC and ET patients, and higher OEA/PEA ratios compared to HC. Furthermore, the sum of 2AG and 1AG positively correlated with JAK2V617F variant allele frequency and splenomegaly in MF and was elevated in high-risk PV patients compared to in low-risk PV patients. In conclusion, our work revealed specific alterations of ECs and NAE plasma profile in MPN subclasses and potentially relevant associations with disease severity.

Biosynthesis of N-Docosahexanoylethanolamine from Unesterified Docosahexaenoic Acid and Docosahexaenoyl-Lysophosphatidylcholine in Neuronal Cells.

We investigated the synthesis of N-docosahexaenoylethanolamine (synaptamide) in neuronal cells from unesterified docosahexaenoic acid (DHA) or DHA-lysophosphatidylcholine (DHA-lysoPC), the two major lipid forms that deliver DHA to the brain, in order to understand the formation of this neurotrophic and neuroprotective metabolite of DHA in the brain. Both substrates were taken up in Neuro2A cells and metabolized to N-docosahexaenoylphosphatidylethanolamine (NDoPE) and synaptamide in a time- and concentration-dependent manner, but unesterified DHA was 1.5 to 2.4 times more effective than DHA-lysoPC at equimolar concentrations. The plasmalogen NDoPE (pNDoPE) amounted more than 80% of NDoPE produced from DHA or DHA-lysoPC, with 16-carbon-pNDoPE being the most abundant species. Inhibition of N-acylphosphatidylethanolamine-phospholipase D (NAPE-PLD) by hexachlorophene or bithionol significantly decreased the synaptamide production, indicating that synaptamide synthesis is mediated at least in part via NDoPE hydrolysis. NDoPE formation occurred much more rapidly than synaptamide production, indicating a precursor-product relationship. Although NDoPE is an intermediate for synaptamide biosynthesis, only about 1% of newly synthesized NDoPE was converted to synaptamide, possibly suggesting additional biological function of NDoPE, particularly for pNDoPE, which is the major form of NDoPE produced.

Population genomics demystifies the defoliation phenotype in the plant pathogen Verticillium dahliae.

Verticillium dahliae is a broad host-range pathogen that causes vascular wilts in plants. Interactions between three hosts and specific V. dahliae genotypes result in severe defoliation. The underlying mechanisms of defoliation are unresolved. Genome resequencing, gene deletion and complementation, gene expression analysis, sequence divergence, defoliating phenotype identification, virulence analysis, and quantification of V. dahliae secondary metabolites were performed. Population genomics previously revealed that G-LSR2 was horizontally transferred from the fungus Fusarium oxysporum f. sp. vasinfectum to V. dahliae and is exclusively found in the genomes of defoliating (D) strains. Deletion of seven genes within G-LSR2, designated as VdDf genes, produced the nondefoliation phenotype on cotton, olive, and okra but complementation of two genes restored the defoliation phenotype. Genes VdDf5 and VdDf6 associated with defoliation shared homology with polyketide synthases involved in secondary metabolism, whereas VdDf7 shared homology with proteins involved in the biosynthesis of N-lauroylethanolamine (N-acylethanolamine (NAE) 12:0), a compound that induces defoliation. NAE overbiosynthesis by D strains also appears to disrupt NAE metabolism in cotton by inducing overexpression of fatty acid amide hydrolase. The VdDfs modulate the synthesis and overproduction of secondary metabolites, such as NAE 12:0, that cause defoliation either by altering abscisic acid sensitivity, hormone disruption, or sensitivity to the pathogen.

A double-blind randomized placebo controlled study assessing safety, tolerability and efficacy of palmitoylethanolamide for symptoms of knee osteoarthritis.

BACKGROUND: The aim of the study was to assess the safety, tolerability and efficacy of palmitoylethanolamide (PEA) when dosed at 300 mg and 600 mg per day on symptoms of knee osteoarthritis. METHODS: This was a single site, comparative, double-blind placebo controlled study in adults with mild to moderate knee osteoarthritis with 111 participants randomized to receive 300 mg PEA, 600 mg PEA or placebo each day, in divided doses b.i.d, for 8 weeks. The primary outcome was the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC). The secondary outcomes were the Numerical Rating Scales (NRS) for pain, the Depression Anxiety Stress Scale (DASS), the Perceived Stress Scale (PSS), the Pittsburg Sleep Quality Index (PSQI), the Short Form Health Survey (SF-36), the use of rescue pain medication and clinical safety assessment. RESULTS: There was a significant reduction in the total WOMAC score in the 300 mg PEA (p = 0.0372) and the 600 mg PEA (p = 0.0012) groups, the WOMAC pain score (300 mg PEA, p = 0.0074; 600 mg PEA, p = < 0.001), the WOMAC stiffness score (PEA 300 mg, p < 0.0490; 600 mg PEA, p = 0.001) and in the WOMAC function score in the 600 mg PEA group (p = 0.033) compared to placebo. The NRS pain evaluations for "worst pain" and "least pain" were significantly reduced in the 300 mg PEA group (p < 0.001, p = 0.005) and the 600 mg PEA group (p < 0.001, p < 0.001) compared to placebo. There was a significant reduction in anxiety (DASS) in both active treatment groups (300 mg PEA, p = 0.042; 600 mg PEA group (p = 0.043) compared to placebo. There were no changes in the clinical markers and the product was well tolerated. CONCLUSIONS: The study demonstrated that palmitoylethanolamide may be a novel treatment for attenuating pain and reducing other associated symptoms of knee osteoarthritis. Further studies on the pharmacological basis of this anti-inflammatory effect are now required.

Cocaine-Induced Reinstatement of Cocaine Seeking Provokes Changes in the Endocannabinoid and N-Acylethanolamine Levels in Rat Brain Structures.

There is strong support for the role of the endocannabinoid system and the noncannabinoid lipid signaling molecules, N-acylethanolamines (NAEs), in cocaine reward and withdrawal. In the latest study, we investigated the changes in the levels of the above molecules and expression of cannabinoid receptors (CB1 and CB2) in several brain regions during cocaine-induced reinstatement in rats. By using intravenous cocaine self-administration and extinction procedures linked with yoked triad controls, we found that a priming dose of cocaine (10 mg/kg, i.p.) evoked an increase of the anadamide (AEA) level in the hippocampus and prefrontal cortex only in animals that had previously self-administered cocaine. In the same animals, the level of 2-arachidonoylglycerol (2-AG) increased in the hippocampus and nucleus accumbens. Moreover, the drug-induced relapse resulted in a potent increase in NAEs levels in the cortical areas and striatum and, at the same time, a decrease in the tissue levels of oleoylethanolamide (OEA) and palmitoylethanolamide (PEA) was noted in the nucleus accumbens, cerebellum, and/or hippocampus. At the level of cannabinoid receptors, a priming dose of cocaine evoked either upregulation of the CB1 and CB2 receptors in the prefrontal cortex and lateral septal nuclei or downregulation of the CB1 receptors in the ventral tegmental area. In the medial globus pallidus we observed the upregulation of the CB2 receptor only after yoked chronic cocaine treatment. Our findings support that in the rat brain, the endocannabinoid system and NAEs are involved in cocaine induced-reinstatement where these molecules changed in a region-specific manner and may represent brain molecular signatures for the development of new treatments for cocaine addiction.

Plasma endocannabinoid levels in lean, overweight, and obese humans: relationships to intestinal permeability markers, inflammation, and incretin secretion.

Intestinal production of endocannabinoid and oleoylethanolamide (OEA) is impaired in high-fat diet/obese rodents, leading to reduced satiety. Such diets also alter the intestinal microbiome in association with enhanced intestinal permeability and inflammation; however, little is known of these effects in humans. This study aimed to 1) evaluate effects of lipid on plasma anandamide (AEA), 2-arachidonyl- sn-glycerol (2-AG), and OEA in humans; and 2) examine relationships to intestinal permeability, inflammation markers, and incretin hormone secretion. Twenty lean, 18 overweight, and 19 obese participants underwent intraduodenal Intralipid infusion (2 kcal/min) with collection of endoscopic duodenal biopsies and blood. Plasma AEA, 2-AG, and OEA (HPLC/tandem mass spectrometry), tumor necrosis factor-α (TNFα), glucagon-like peptide-1 (GLP-1), and glucose-dependent insulinotropic peptide (GIP) (multiplex), and duodenal expression of occludin, zona-occludin-1 (ZO-1), intestinal-alkaline-phosphatase (IAP), and Toll-like receptor 4 (TLR4) (by RT-PCR) were assessed. Fasting plasma AEA was increased in obese compared with lean and overweight patients ( P < 0.05), with no effect of BMI group or ID lipid infusion on plasma 2-AG or OEA. Duodenal expression of IAP and ZO-1 was reduced in obese compared with lean ( P < 0.05), and these levels related negatively to plasma AEA ( P < 0.05). The iAUC for AEA was positively related to iAUC GIP ( r = 0.384, P = 0.005). Obese individuals have increased plasma AEA and decreased duodenal expression of ZO-1 and IAP compared with lean and overweight subjects. The relationships between plasma AEA with duodenal ZO-1, IAP, and GIP suggest that altered endocannabinoid signaling may contribute to changes in intestinal permeability, inflammation, and incretin release in human obesity.

Plasma N-acylethanolamine and endocannabinoid levels in burning mouth syndrome: Potential role in disease pathogenesis.

OBJECTIVE: The objective was to measure endocannabinoid (eCB) ligands and non-cannabinoid N-acylethanolamine (NAE) molecules in plasma from individuals with burning mouth syndrome (BMS) and to determine whether plasma eCB/NAE levels correlated with pain, inflammation and depressive symptomatology in this cohort. STUDY DESIGN: Plasma content of the eCBs, anandamide (AEA) and 2-arachidonoyl-glycerol (2-AG), and the NAE molecules, palmitoylethanolamide (PEA) and oleoylethanolamide (OEA) were assessed in healthy subjects (n = 8) and in a cohort of newly diagnosed BMS patients (n = 9) using liquid chromatography-tandem mass spectrometry. Plasma eCBs and NAE profiles were correlated with self-rated oral cavity pain intensities, depressive symptomatology and plasma IL-8 levels. RESULTS: Plasma levels of PEA, but not OEA, AEA or 2-AG, were significantly elevated in patients with BMS, when compared to plasma from healthy individuals. Plasma PEA, OEA and AEA levels correlated with depressive symptomatology. CONCLUSIONS: This is the first evidence to indicate that circulating eCB/NAE levels are altered in BMS.

Fatty acid amide hydrolase (FAAH) regulates hypercapnia/ischemia-induced increases in n-acylethanolamines in mouse brain.

N-acylethanolamines (NAEs) are endogenous lipid ligands for several receptors including cannabinoid receptors and peroxisome proliferator-activated receptor-alpha (PPAR-α), which regulate numerous physiological functions. Fatty acid amide hydrolase (FAAH) is largely responsible for the degradation of NAEs. However, at high concentrations of ethanolamines and unesterified fatty acids, FAAH can also catalyze the reverse reaction, producing NAEs. Several brain insults such as ischemia and hypoxia increase brain unesterified fatty acids. Because FAAH can catalyze the synthesis of NAE, we aimed to test whether FAAH was necessary for CO2 -induced hypercapnia/ischemia increases in NAE. To test this, we examined levels of NAEs, 1- and 2-arachidonoylglycerols as well as their corresponding fatty acid precursors in wild-type and mice lacking FAAH (FAAH-KO) with three Kill methods: (i) head-focused, high-energy microwave irradiation (microwave), (ii) 5 min CO2 followed by microwave irradiation (CO2 + microwave), and (iii) 5 min CO2 only (CO2 ). Both CO2 -induced groups increased, to a similar extent, brain levels of unesterified oleic, arachidonic, and docosahexaenoic acid and 1- and 2-arachidonoylglycerols compared to the microwave group in both wild-type and FAAH-KO mice. Oleoylethanolamide (OEA), arachidonoylethanolamide (AEA), and docosahexaenoylethanolamide (DHEA) levels were about 8-, 7-, and 2.5-fold higher, respectively, in the FAAH-KO mice compared with the wild-type mice. Interestingly, the concentrations of OEA, AEA, and DHEA increased 2.5- to 4-fold in response to both CO2 -induced groups in wild-type mice, but DHEA increased only in the CO2 group in FAAH-KO mice. Our study demonstrates that FAAH is necessary for CO2 - induced increases in OEA and AEA but not DHEA. Targeting brain FAAH could impair the production of NAEs in response to brain injuries.

A novel composite formulation of palmitoylethanolamide and quercetin decreases inflammation and relieves pain in inflammatory and osteoarthritic pain models.

BACKGROUND: Osteoarthritis (OA) is a common progressive joint disease in dogs and cats. The goal of OA treatment is to reduce inflammation, minimize pain, and maintain joint function. Currently, non-steroidal anti-inflammatory drugs (e.g., meloxicam) are the cornerstone of treatment for OA pain, but side effects with long-term use pose important challenges to veterinary practitioners when dealing with OA pain. Palmitoylethanolamide (PEA) is a naturally-occurring fatty acid amide, locally produced on demand by tissues in response to stress. PEA endogenous levels change during inflammatory and painful conditions, including OA, i.e., they are typically increased during acute conditions and decreased in chronic inflammation. Systemic treatment with PEA has anti-inflammatory and pain-relieving effects in several disorders, yet data are lacking in OA. Here we tested a new composite, i.e., PEA co-ultramicronized with the natural antioxidant quercetin (PEA-Q), administered orally in two different rat models of inflammatory and OA pain, namely carrageenan paw oedema and sodium monoiodoacetate (MIA)-induced OA. Oral treatment with meloxicam was used as benchmark. RESULTS: PEA-Q decreased inflammatory and hyperalgesic responses induced by carrageenan injection, as shown by: (i) paw oedema reduction, (ii) decreased severity in histological inflammatory score, (iii) reduced activity of myeloperoxidase, i.e., a marker of inflammatory cell infiltration, and (iv) decreased thermal hyperalgesia. Overall PEA-Q showed superior effects compared to meloxicam. In MIA-treated animals, PEA-Q exerted the following effects: (i) reduced mechanical allodynia and improved locomotor function, (ii) protected cartilage against MIA-induced histological damage, and (iii) counteracted the increased serum concentration of tumor necrosis factor alpha, interleukin 1 beta, metalloproteases 1, 3, 9 and nerve growth factor. The magnitude of these effects was comparable to, or even greater than, those of meloxicam. CONCLUSION: The present findings shed new light on some of the inflammatory and nociceptive pathways and mediators targeted by PEA-Q and confirm its anti-inflammatory and pain-relieving effects in rodent OA pain models. The translatability of these observations to canine and feline OA pain is currently under investigation.

Alterations of anti-inflammatory lipids in plasma from women with chronic widespread pain - a case control study.

BACKGROUND: Chronic widespread pain conditions (CWP) such as the pain associated with fibromyalgia syndrome (FMS) are significant health problems with unclear aetiology. Although CWP and FMS can alter both central and peripheral pain mechanisms, there are no validated markers for such alterations. Pro- and anti-inflammatory components of the immune system such as cytokines and endogenous lipid mediators could serve as systemic markers of alterations in chronic pain. Lipid mediators associated with anti-inflammatory qualities - e.g., oleoylethanolamide (OEA), palmitoylethanolamide (PEA), and stearoylethanolamide (SEA) - belong to N-acylethanolamines (NAEs). Previous studies have concluded that these lipid mediators may modulate pain and inflammation via the activation of peroxisome proliferator activating receptors (PPARs) and the activation of PPARs may regulate gene transcriptional factors that control the expression of distinct cytokines. METHODS: This study investigates NAEs and cytokines in 17 women with CWP and 21 healthy controls. Plasma levels of the anti-inflammatory lipids OEA, PEA, and SEA, the pro-inflammatory cytokines TNF-α, IL-1ß, IL-6, and IL-8, and the anti-inflammatory cytokine IL-10 were investigated. T-test of independent samples was used for group comparisons. Bivariate correlation analyses, and multivariate regression analysis were performed between lipids, cytokines, and pain intensity of the participants. RESULTS: Significantly higher levels of OEA and PEA in plasma were found in CWP. No alterations in the levels of cytokines existed and no correlations between levels of lipids and cytokines were found. CONCLUSIONS: We conclude that altered levels of OEA and PEA might indicate the presence of systemic inflammation in CWP. In addition, we believe our findings contribute to the understanding of the biochemical mechanisms involved in chronic musculoskeletal pain.

Vaccenic acid suppresses intestinal inflammation by increasing anandamide and related N-acylethanolamines in the JCR:LA-cp rat.

Vaccenic acid (VA), the predominant ruminant-derivedtransfat in the food chain, ameliorates hyperlipidemia, yet mechanisms remain elusive. We investigated whether VA could influence tissue endocannabinoids (ECs) by altering the availability of their biosynthetic precursor, arachidonic acid (AA), in membrane phospholipids (PLs). JCR:LA-cprats were assigned to a control diet with or without VA (1% w/w),cis-9,trans-11 conjugated linoleic acid (CLA) (1% w/w) or VA+CLA (1% + 0.5% w/w) for 8 weeks. VA reduced the EC, 2-arachidonoylglycerol (2-AG), in the liver and visceral adipose tissue (VAT) relative to control diet (P< 0.001), but did not change AA in tissue PLs. There was no additive effect of combining VA+CLA on 2-AG relative to VA alone (P> 0.05). Interestingly, VA increased jejunal concentrations of anandamide and those of the noncannabinoid signaling molecules, oleoylethanolamide and palmitoylethanolamide, relative to control diet (P< 0.05). This was consistent with a lower jejunal protein abundance (but not activity) of their degrading enzyme, fatty acid amide hydrolase, as well as the mRNA expression of TNFα and interleukin 1ß (P< 0.05). The ability of VA to reduce 2-AG in the liver and VAT provides a potential mechanistic explanation to alleviate ectopic lipid accumulation. The opposing regulation of ECs and other noncannabinoid lipid signaling molecules by VA suggests an activation of benefit via the EC system in the intestine.