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Fatty acid amide hydrolase (FAAH) is a conserved hydrolase in eukaryotes with promiscuous activity toward a range of acylamide substrates. The native substrate repertoire for FAAH has just begun to be explored in plant systems outside the model Arabidopsis thaliana. Here, we used ex vivo lipidomics to identify potential endogenous substrates for Medicago truncatula FAAH1 (MtFAAH1). We incubated recombinant MtFAAH1 with lipid mixtures extracted from M. truncatula and resolved their profiles via gas chromatography–mass spectrometry (GC–MS). Data revealed that besides N‐acylethanolamines (NAEs), sn‐1 or sn‐2 isomers of monoacylglycerols (MAGs) were substrates for MtFAAH1. Combined with in vitro and computational approaches, our data support both amidase and esterase activities for MtFAAH1. MAG‐mediated hydrolysis via MtFAAH1 may be linked to biological roles that are yet to be discovered. [ABSTRACT FROM AUTHOR]

N-arachidonoylethanolamine (also known as anandamide) and 2-arachidonoylglycerol are activators of the cannabinoid receptors. The endocannabinoid system also includes structurally and functionally related lipid mediators that do not target cannabinoid receptors, such as oleoylethanolamide, palmitoylethanolamide, and stearoylethanolamide. These bioactive lipids are involved in various physiological processes, including regulation of pain. The primary aim of the study was to analyze associations between serum levels of these lipids and pain in participants in the Pain in Neuropathy Study, an observational, cross-sectional, multicentre, research project in which diabetic patients with painless or painful neuropathy underwent deep phenotyping. Our hypothesis was that painful neuropathy would be associated with higher levels of the 5 lipids compared with painless neuropathy. Secondary aims were to analyze other patient-reported outcome measures and clinical data in relationship to lipid levels. The lipid mediators were analyzed in serum samples using liquid chromatography tandem mass spectrometry (LC-MS/MS). Serum levels of anandamide were significantly higher in the painful group, but the effect size was small (Cohen d = 0.31). Using cluster analysis of lipid data, patients were dichotomized into a “high-level” endocannabinoid group and a “low-level” group. In the high-level group, 61% of patients had painful neuropathy, compared with 45% in the low-level group (P = 0.039). This work is of a correlative nature only, and the relevance of these findings to the search for analgesics targeting the endocannabinoid system needs to be determined in future studies. [ABSTRACT FROM AUTHOR]

Chronic kidney disease (CKD) development after acute kidney injury (AKI) involves multiple mechanisms, including inflammation, epithelial-mesenchymal transition (EMT), and extracellular matrix deposition, leading to progressive tubulointerstitial fibrosis. Recently, a central role for peroxisome-proliferator activated receptor (PPAR)-α has been addressed in preserving kidney function during AKI. Among endogenous lipid mediators, oleoylethanolamide (OEA), a PPAR-α agonist, has been studied for its metabolic and anti-inflammatory effects. Here, we have investigated OEA effects on folic acid (FA)-induced kidney injury in mice and the underlying mechanisms. OEA improved kidney function, normalized urine output, and reduced serum BUN, creatinine, and albuminuria. Moreover, OEA attenuated tubular epithelial injury, as shown by histological analysis, and decreased expression of neutrophil gelatinase-associated lipocalin and kidney injury molecule-1. Gene expression analysis of kidney tissue indicated that OEA limited immune cell infiltration and inflammation. Moreover, OEA significantly inhibited Wnt7b and Catnb1 gene transcription and α-smooth muscle actin expression, indicating suppression of EMT. Accordingly, OEA exhibited an anti-fibrotic effect, as shown by Masson staining and the reduced levels of transforming growth factor (TGF)-β1, fibronectin, and collagen IV. Mechanistically, the nephroprotective effect of OEA was related to PPAR-α activation since OEA failed to exert its beneficial activity in FA-insulted PPAR-α-/- mice. PPAR-α involvement was also confirmed in HK2 cells where GW6471, a PPAR-α antagonist, blunted OEA activity on the TGF-β1 signalling pathway and associated pro-inflammatory and fibrotic patterns. Our findings revealed that OEA counteracts kidney injury by controlling inflammation and fibrosis, making it an effective therapeutic tool for limiting AKI to CKD progression.

Background: Endocannabinoids and related N-acylethanolamines (NAEs) are bioactive lipids with important physiological functions and putative roles in mental health and addictions. Although chronic cannabis use is associated with endocannabinoid system changes, the status of circulating endocannabinoids and related NAEs in people with cannabis use disorder (CUD) is uncertain. Methods: Eleven individuals with CUD and 54 healthy non-cannabis using control participants (HC) provided plasma for measurement by high-performance liquid chromatography–mass spectrometry of endocannabinoids (2-arachidonoylglycerol (2-AG) and N-arachidonoylethanolamine (AEA)) and related NAE fatty acids (N-docosahexaenoylethanolamine (DHEA) and N-oleoylethanolamine (OEA)). Participants were genotyped for the functional gene variant of FAAH (rs324420, C385A) which may affect concentrations of AEA as well as other NAEs (OEA, DHEA). Results: In overnight abstinent CUD, AEA, OEA and DHEA concentrations were significantly higher (31–40%; p < 0.05) and concentrations of the endocannabinoid 2-AG were marginally elevated (55%, p = 0.13) relative to HC. There were no significant correlations between endocannabinoids/NAE concentrations and cannabis analytes, self-reported cannabis use frequency or withdrawal symptoms. DHEA concentration was inversely related with marijuana craving (r = −0.86; p = 0.001). Genotype had no significant effect on plasma endocannabinoids/NAE concentrations. Conclusions: Our preliminary findings, requiring replication, might suggest that activity of the endocannabinoid system is elevated in chronic cannabis users. It is unclear whether this elevation is a compensatory response or a predating state. Studies examining endocannabinoids and NAEs during prolonged abstinence as well as the potential role of DHEA in craving are warranted. [ABSTRACT FROM AUTHOR]

Obesity is associated with gastrointestinal (GI) tract and central nervous system (CNS) disorders. High-fat diet (HFD) feeding-induced obesity in mice induces dysbiosis, causing a shift toward bacteria-derived metabolites with detrimental effects on metabolism and inflammation: events often contributing to the onset and progression of both GI and CNS disorders. Palmitoylethanolamide (PEA) is an endogenous lipid mediator with beneficial effects in mouse models of GI and CNS disorders. However, the mechanisms underlining its enteroprotective and neuroprotective effects still need to be fully understood. Here, we aimed to study the effects of PEA on intestinal inflammation and microbiota alterations resulting from lipid overnutrition. Ultramicronized PEA (30 mg/kg/die per os) was administered to HFD-fed mice for 7 weeks starting at the 12th week of HFD regimen. At the termination of the study, the effects of PEA on inflammatory factors and cells, gut microbial features and tryptophan (TRP)-kynurenine metabolism were evaluated. PEA regulates the crosstalk between the host immune system and gut microbiota via rebalancing colonic TRP metabolites. PEA treatment reduced intestinal immune cell recruitment, inflammatory response triggered by HFD feeding, and corticotropin-releasing hormone levels. In particular, PEA modulated HFD-altered TRP metabolism in the colon, rebalancing serotonin (5-HT) turnover and reducing kynurenine levels. These effects were associated with a reshaping of gut microbiota composition through increased butyrate-promoting/producing bacteria, such as Bifidobacterium, Oscillospiraceae and Turicibacter sanguinis, with the latter also described as 5-HT sensor. These data indicate that the rebuilding of gut microbiota following PEA supplementation promotes host 5-HT biosynthesis, which is crucial in regulating intestinal function.

The aim of this study was to compare concentrations of endogenous N-acylethanolamine (NAE) lipid mediators—palmitoylethanolamide (PEA), oleoylethanolamide (OEA), and anandamide (AEA)—in fresh, decontaminated, cryopreserved, and freeze-dried amniotic membrane (AM) allografts, thereby determining whether AM's analgesic and anti-inflammatory efficiency related to NAEs persists during storage. The concentrations of NAEs were measured using ultra-high-performance liquid chromatography–tandem mass spectrometry. Indirect fluorescent immunohistochemistry was used to detect the PEA PPAR-α receptor. The concentrations of PEA, OEA, and AEA were significantly higher after decontamination. A significant decrease was found in cryopreserved AM compared to decontaminated tissue for PEA but not for OEA and AEA. However, significantly higher values for all NAEs were detected in cryopreserved samples compared to fresh tissue before decontamination. The freeze-dried AM had similar values to decontaminated AM with no statistically significant difference. The nuclear staining of the PPAR-α receptor was clearly visible in all specimens. The stability of NAEs in AM after cryopreservation was demonstrated under tissue bank storage conditions. However, a significant decrease, but still higher concentration of PEA compared to fresh not decontaminated tissue, was found in cryopreserved, but not freeze-dried, AM. Results indicate that NAEs persist during storage in levels sufficient for the analgesic and anti-inflammatory effects. This means that cryopreserved AM allografts released for transplant purposes before the expected expiration (usually 3–5 years) will still show a strong analgesic effect. The same situation was confirmed for AM lyophilized after one year of storage. This work thus contributed to the clarification of the analgesic effect of NAEs in AM allografts. [ABSTRACT FROM AUTHOR]

Introduction: Oleoylethanolamide (OEA), an endogenous N-acylethanolamine acting as a gut-to-brain signal to control food intake and metabolism, has been attracting attention as a target for novel therapies against obesity and eating disorders. Numerous observations suggested that the OEA effects might be peripherally mediated, although they involve central pathways including noradrenergic, histaminergic and oxytocinergic systems of the brainstem and the hypothalamus. Whether these pathways are activated directly by OEA or whether they are downstream of afferent nerves is still highly debated. Some early studies suggested vagal afferent fibers as the main route, but our previous observations have contradicted this idea and led us to consider the blood circulation as an alternative way for OEA's central actions. Methods: To test this hypothesis, we first investigated the impact of subdiaphragmatic vagal deafferentation (SDA) on the OEA-induced activation of selected brain nuclei. Then, we analyzed the pattern of OEA distribution in plasma and brain at different time points after intraperitoneal administration in addition to measuring food intake. Results: Confirming and extending our previous findings that subdiaphragmatic vagal afferents are not necessary for the eating-inhibitory effect of exogenous OEA, our present results demonstrate that vagal sensory fibers are also not necessary for the neurochemical effects of OEA. Rather, within a few minutes after intraperitoneal administration, we found an increased concentration of intact OEA in different brain areas, associated with the inhibition of food intake. Conclusion: Our results support that systemic OEA rapidly reaches the brain via the circulation and inhibits eating by acting directly on selected brain nuclei. [ABSTRACT FROM AUTHOR]

Background and Purpose: Devising novel strategies to therapeutically favour inflammation resolution and provide neuroprotection is an unmet clinical need. Enhancing endocannabinoid tone by inhibiting the catabolic enzyme fatty acid amide hydrolase (FAAH), or stimulating melatonin receptors has therapeutic potential to treat neuropathological states in which neuroinflammation plays a central role. Experimental Approach: A rodent hippocampal explant model of inflammatory injury was used to assess the effects of UCM1341, a dual‐acting compound with FAAH inhibitory action and agonist activity at melatonin receptors, against neuroinflammatory damage. FAAH activity was measured by a radiometric assay, and N‐acylethanolamine levels were assessed by HPLC–MS/MS methods. FAAH distribution, evolution of inflammation and the contribution of UCM1341 to the expression of proteins controlling macrophage behaviour were investigated by biochemical and confocal analyses. Key Results: UCM1341 exhibited greater neuroprotection against neuroinflammatory degeneration, compared with the reference compounds URB597 (FAAH inhibitor) and melatonin. During neuroinflammation, UCM1341 augmented the levels of anandamide and N‐oleoylethanolamine, but not N‐palmitoylethanolamine, up‐regulated PPAR‐α levels, attenuated demyelination and prevented the release of TNF‐α. UCM1341 modulated inflammatory responses by contributing to microglia/macrophage polarization, stimulating formation of lipid‐laden macrophages and regulating expression of proteins controlling cholesterol metabolism and efflux. The neuroprotective effects of UCM1341 were prevented by PPARα, TRPV1 and melatonin receptor antagonists. Conclusion and Implications: UCM1341, by enhancing endocannabinoid and melatoninergic signalling, benefits neuroprotection and stimulates inflammation resolution pathways. Our findings provide an encouraging prospect of therapeutically targeting endocannabinoid and melatoninergic systems in inflammatory demyelinating states in the CNS. [ABSTRACT FROM AUTHOR]

IntroductionOleoylethanolamide (OEA), an endogenous N-acylethanolamine acting as a gut-to-brain signal to control food intake and metabolism, has been attracting attention as a target for novel therapies against obesity and eating disorders. Numerous observations suggested that the OEA effects might be peripherally mediated, although they involve central pathways including noradrenergic, histaminergic and oxytocinergic systems of the brainstem and the hypothalamus. Whether these pathways are activated directly by OEA or whether they are downstream of afferent nerves is still highly debated. Some early studies suggested vagal afferent fibers as the main route, but our previous observations have contradicted this idea and led us to consider the blood circulation as an alternative way for OEA’s central actions.MethodsTo test this hypothesis, we first investigated the impact of subdiaphragmatic vagal deafferentation (SDA) on the OEA-induced activation of selected brain nuclei. Then, we analyzed the pattern of OEA distribution in plasma and brain at different time points after intraperitoneal administration in addition to measuring food intake.ResultsConfirming and extending our previous findings that subdiaphragmatic vagal afferents are not necessary for the eating-inhibitory effect of exogenous OEA, our present results demonstrate that vagal sensory fibers are also not necessary for the neurochemical effects of OEA. Rather, within a few minutes after intraperitoneal administration, we found an increased concentration of intact OEA in different brain areas, associated with the inhibition of food intake.ConclusionOur results support that systemic OEA rapidly reaches the brain via the circulation and inhibits eating by acting directly on selected brain nuclei.

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.

Background: Although neuropathic pain is a significant problem in polyneuropathy, the underlying molecular mechanisms are poorly understood. The endogenous bioactive lipids 2-arachidonoyl-glycerol (2-AG), oleoylethanolamide (OEA), palmitoylethanolamide (PEA), and stearoylethanolamide (SEA) are known to influence pain and inflammation in the peripheral nervous system. The aim of this study was to explore the plasma levels of endocannabinoids and related lipids and health-related quality of life in patients with polyneuropathy with and without pain. Methods: Patients (n = 48) with Chronic Idiopathic Axonal Neuropathy were included. Clinical data were retrieved from medical files. All patients filled out the SF-36 and EQ-5D questionnaires. In addition, blood samples were analyzed for 2-AG, OEA, PEA, and SEA. Results: Neuropathic pain was reported in 21 of the patients. There were significantly lower levels of 2-AG in patients with neuropathic pain (P = 0.03), but there were no significant differences in OEA (P = 0.61), PEA (P = 0.95), or SEA (P = 0.97) levels. The patients reporting pain in the hands had significantly lower SEA levels, 10.0 versus 15.0 (P = 0.03). The levels of 2-AG were significantly higher among patients reporting paresthesia in their feet (80.1 vs. 56.3; P = 0.02). Levels of PEA, SEA, and 2-AG were decreased in patients with loss of vibration. PEA and SEA were decreased in patients with loss of pain and temperature, and SEA decreased in patients with loss of sense of touch. However, the differences in the levels of bioactive endogenous lipids were not statistically significant when corrected for multiple comparisons. Conclusion: Alterations of 2-AG levels between polyneuropathy patients with and without neurogenic pain indicate that it could play an essential role. Further studies are warranted.

Background: Endocannabinoids and related N-acylethanolamines (NAEs) are bioactive lipids with important physiological functions and putative roles in mental health and addictions. Although chronic cannabis use is associated with endocannabinoid system changes, the status of circulating endocannabinoids and related NAEs in people with cannabis use disorder (CUD) is uncertain. Methods: Eleven individuals with CUD and 54 healthy non-cannabis using control participants (HC) provided plasma for measurement by high-performance liquid chromatography–mass spectrometry of endocannabinoids (2-arachidonoylglycerol (2-AG) and N-arachidonoylethanolamine (AEA)) and related NAE fatty acids (N-docosahexaenoylethanolamine (DHEA) and N-oleoylethanolamine (OEA)). Participants were genotyped for the functional gene variant of FAAH (rs324420, C385A) which may affect concentrations of AEA as well as other NAEs (OEA, DHEA). Results: In overnight abstinent CUD, AEA, OEA and DHEA concentrations were significantly higher (31–40%; p < 0.05) and concentrations of the endocannabinoid 2-AG were marginally elevated (55%, p = 0.13) relative to HC. There were no significant correlations between endocannabinoids/NAE concentrations and cannabis analytes, self-reported cannabis use frequency or withdrawal symptoms. DHEA concentration was inversely related with marijuana craving (r = −0.86; p = 0.001). Genotype had no significant effect on plasma endocannabinoids/NAE concentrations. Conclusions: Our preliminary findings, requiring replication, might suggest that activity of the endocannabinoid system is elevated in chronic cannabis users. It is unclear whether this elevation is a compensatory response or a predating state. Studies examining endocannabinoids and NAEs during prolonged abstinence as well as the potential role of DHEA in craving are warranted.

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.

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. [ABSTRACT FROM AUTHOR]

The aim of this study was to compare concentrations of endogenous N-acylethanolamine (NAE) lipid mediators—palmitoylethanolamide (PEA), oleoylethanolamide (OEA), and anandamide (AEA)—in fresh, decontaminated, cryopreserved, and freeze-dried amniotic membrane (AM) allografts, thereby determining whether AM’s analgesic and anti-inflammatory efficiency related to NAEs persists during storage. The concentrations of NAEs were measured using ultra-high-performance liquid chromatography–tandem mass spectrometry. Indirect fluorescent immunohistochemistry was used to detect the PEA PPAR-α receptor. The concentrations of PEA, OEA, and AEA were significantly higher after decontamination. A significant decrease was found in cryopreserved AM compared to decontaminated tissue for PEA but not for OEA and AEA. However, significantly higher values for all NAEs were detected in cryopreserved samples compared to fresh tissue before decontamination. The freeze-dried AM had similar values to decontaminated AM with no statistically significant difference. The nuclear staining of the PPAR-α receptor was clearly visible in all specimens. The stability of NAEs in AM after cryopreservation was demonstrated under tissue bank storage conditions. However, a significant decrease, but still higher concentration of PEA compared to fresh not decontaminated tissue, was found in cryopreserved, but not freeze-dried, AM. Results indicate that NAEs persist during storage in levels sufficient for the analgesic and anti-inflammatory effects. This means that cryopreserved AM allografts released for transplant purposes before the expected expiration (usually 3–5 years) will still show a strong analgesic effect. The same situation was confirmed for AM lyophilized after one year of storage. This work thus contributed to the clarification of the analgesic effect of NAEs in AM allografts.

Fatty acid amide hydrolase (FAAH) is a conserved amidase that is known to modulate the levels of endogenous N‐acylethanolamines (NAEs) in both plants and animals. The activity of FAAH is enhanced in vitro by synthetic phenoxyacylethanolamides resulting in greater hydrolysis of NAEs. Previously, 3‐n‐pentadecylphenolethanolamide (PDP‐EA) was shown to exert positive effects on the development of Arabidopsis seedlings by enhancing Arabidopsis FAAH (AtFAAH) activity. However, there is little information regarding FAAH activity and the impact of PDP‐EA in the development of seedlings of other plant species. Here, we examined the effects of PDP‐EA on growth of upland cotton (Gossypium hirsutum L. cv Coker 312) seedlings including two lines of transgenic seedlings overexpressing AtFAAH. Independent transgenic events showed accelerated true‐leaf emergence compared with non‐transgenic controls. Exogenous applications of PDP‐EA led to increases in overall seedling growth in AtFAAH transgenic lines. These enhanced‐growth phenotypes coincided with elevated FAAH activities toward NAEs and NAE oxylipins. Conversely, the endogenous contents of NAEs and NAE‐oxylipin species, especially linoleoylethanolamide and 9‐hydroxy linoleoylethanolamide, were lower in PDP‐EA treated seedlings than in controls. Further, transcripts for endogenous cotton FAAH genes were increased following PDP‐EA exposure. Collectively, our data corroborate that the enhancement of FAAH enzyme activity by PDP‐EA stimulates NAE‐hydrolysis and that this results in enhanced growth in seedlings of a perennial crop species, extending the role of NAE metabolism in seedling development beyond the model annual plant species, Arabidopsis thaliana. [ABSTRACT FROM AUTHOR]

Peroxisome proliferator-activator receptors (PPARs) regulate lipid and glucose metabolism, control inflammatory processes, and modulate several brain functions. Three PPAR isoforms have been identified, PPARα, PPARβ/δ, and PPARγ, which are expressed in different tissues and cell types. Hereinafter, we focus on PPARα involvement in the pathophysiology of neuropsychiatric and neurodegenerative disorders, which is underscored by PPARα localization in neuronal circuits involved in emotion modulation and stress response, and its role in neurodevelopment and neuroinflammation. A multiplicity of downstream pathways modulated by PPARα activation, including glutamatergic neurotransmission, upregulation of brain-derived neurotrophic factor, and neurosteroidogenic effects, encompass mechanisms underlying behavioral regulation. Modulation of dopamine neuronal firing in the ventral tegmental area likely contributes to PPARα effects in depression, anhedonia, and autism spectrum disorder (ASD). Based on robust preclinical evidence and the initial results of clinical studies, future clinical trials should assess the efficacy of PPARα agonists in the treatment of mood and neurodevelopmental disorders, such as depression, schizophrenia, and ASD. [ABSTRACT FROM AUTHOR]

[Display omitted] • Lipid overload triggers metainflammation and neuroinflammation in obese mice. • PEA, an endogenous lipid, owns well-known metabolic and central activities. • PEA lessens anxiety-like behavior modulating monoamines in the amygdala. • PEA limits systemic and central immune response induced by high-fat diet feeding. • PEA blunts high-fat diet-induced astrogliosis, microgliosis and mastocytosis. 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-1β, 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 β2) 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. [ABSTRACT FROM AUTHOR]

The increasing interest in endocannabinoid-like compounds (ECL) in food stems from their important physiological roles, including energy metabolism and satiety. In this study, the effect of fermentation or alkalisation on the formation of ECL compounds in table olives was investigated. N -acylethanolamines, monoacylglycerols, N -acylamino acids, and N -acylneurotransmitters were monitored. Results revealed that alkaline treatment led to a significant increase in the concentrations of N -oleoylethanolamide (80%), N -palmitoylethanolamide (93%), N -linoleoylethanolamide (51%), and 1-oleoylglycerol (679%) compared to control. While N -oleoylethanolamide, N -palmitoylethanolamide, N -linoleoylethanolamide, 1- and 2-oleoylglycerol, 1- and 2-linoleoylglycerol, and oleoylphenylalanine were initially absent or present in trace amounts, their levels significantly rose during fermentation. The formation rate of these compounds was higher in olives fermented in water than those in brine. The study provides detailed information on how specific ECL compounds respond to different processing methods, offering valuable information for optimising table olive production to enhance its nutritional benefits. [Display omitted] • Biologically active oleoylphenylalanine forms during olive fermentation. • N -acylethanolamine levels increase during both olive fermentation and alkalization. • N -Acylethanolamines and monoacylglycerols show higher increases in NaCl absence. [ABSTRACT FROM AUTHOR]

Abstract Fatty acid amide hydrolase (FAAH) is a conserved amidase that is known to modulate the levels of endogenous N‐acylethanolamines (NAEs) in both plants and animals. The activity of FAAH is enhanced in vitro by synthetic phenoxyacylethanolamides resulting in greater hydrolysis of NAEs. Previously, 3‐n‐pentadecylphenolethanolamide (PDP‐EA) was shown to exert positive effects on the development of Arabidopsis seedlings by enhancing Arabidopsis FAAH (AtFAAH) activity. However, there is little information regarding FAAH activity and the impact of PDP‐EA in the development of seedlings of other plant species. Here, we examined the effects of PDP‐EA on growth of upland cotton (Gossypium hirsutum L. cv Coker 312) seedlings including two lines of transgenic seedlings overexpressing AtFAAH. Independent transgenic events showed accelerated true‐leaf emergence compared with non‐transgenic controls. Exogenous applications of PDP‐EA led to increases in overall seedling growth in AtFAAH transgenic lines. These enhanced‐growth phenotypes coincided with elevated FAAH activities toward NAEs and NAE oxylipins. Conversely, the endogenous contents of NAEs and NAE‐oxylipin species, especially linoleoylethanolamide and 9‐hydroxy linoleoylethanolamide, were lower in PDP‐EA treated seedlings than in controls. Further, transcripts for endogenous cotton FAAH genes were increased following PDP‐EA exposure. Collectively, our data corroborate that the enhancement of FAAH enzyme activity by PDP‐EA stimulates NAE‐hydrolysis and that this results in enhanced growth in seedlings of a perennial crop species, extending the role of NAE metabolism in seedling development beyond the model annual plant species, Arabidopsis thaliana.

The endocannabinoid system (ECS) is dysregulated during obesity and metabolic disorders. Weight loss favours the re-establishment of ECS homeostatic conditions, but also the fatty acid composition of the diet can modulate endocannabinoid profiles. However, the combined impact of nutrient quality and energy restriction on the ECS remains unclear. In this 12 weeks randomized controlled trial, men and women (40–70 years) with obesity (BMI: 31.3 ± 3.5 kg/ m2) followed either a low nutrient quality 25% energy-restricted (ER) diet (n =39) high in saturated fats and fructose, or a high nutrient quality ER diet (n =34) amongst others enriched in n -3 polyunsaturated fatty acids (PUFAs) or kept their habitual diet (controls). Profiles of plasma- and adipose N-acylethanolamines and mono-acyl glycerol esters were quantified using LC-MS/MS. Gene expression of ECS-related enzymes and receptors was determined in adipose tissue. Measurements were performed under fasting conditions before and after 12 weeks. Our results showed that plasma level of the DHA-derived compound docosahexaenoylethanolamide (DHEA) was decreased in the low nutrient quality ER diet (P <0.001) compared with the high nutrient quality ER diet, whereas anandamide (AEA) and arachidonoylglycerol (2-AG) levels were unaltered. However, adipose tissue gene expression of the 2-AG synthesizing enzyme diacylglycerol lipase alpha (DAGL-α) was increased following the low nutrient quality ER diet (P <.009) and differed upon intervention with both other diets. Concluding, nutrient quality of the diet affects N-acylethanolamine profiles and gene expression of ECS-related enzymes and receptors even under conditions of high energy restriction in abdominally obese humans. ClinicalTrials.gov NCT02194504 [Display omitted] [ABSTRACT FROM AUTHOR]

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.

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 1β (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.

Background: Although neuropathic pain is a significant problem in polyneuropathy, the underlying molecular mechanisms are poorly understood. The endogenous bioactive lipids 2-arachidonoyl-glycerol (2-AG), oleoylethanolamide (OEA), palmitoylethanolamide (PEA), and stearoylethanolamide (SEA) are known to influence pain and inflammation in the peripheral nervous system. The aim of this study was to explore the plasma levels of endocannabinoids and related lipids and health-related quality of life in patients with polyneuropathy with and without pain. Methods: Patients (n = 48) with Chronic Idiopathic Axonal Neuropathy were included. Clinical data were retrieved from medical files. All patients filled out the SF-36 and EQ-5D questionnaires. In addition, blood samples were analyzed for 2-AG, OEA, PEA, and SEA. Results: Neuropathic pain was reported in 21 of the patients. There were significantly lower levels of 2-AG in patients with neuropathic pain (P = 0.03), but there were no significant differences in OEA (P = 0.61), PEA (P = 0.95), or SEA (P = 0.97) levels. The patients reporting pain in the hands had significantly lower SEA levels, 10.0 versus 15.0 (P = 0.03). The levels of 2-AG were significantly higher among patients reporting paresthesia in their feet (80.1 vs. 56.3; P = 0.02). Levels of PEA, SEA, and 2-AG were decreased in patients with loss of vibration. PEA and SEA were decreased in patients with loss of pain and temperature, and SEA decreased in patients with loss of sense of touch. However, the differences in the levels of bioactive endogenous lipids were not statistically significant when corrected for multiple comparisons. Conclusion: Alterations of 2-AG levels between polyneuropathy patients with and without neurogenic pain indicate that it could play an essential role. Further studies are warranted. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 β-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.

N -Acylethanolamine (NAE) signaling has received considerable attention in vertebrates as part of the endocannabinoid signaling system, where anandamide acts as a ligand for G protein–coupled cannabinoid receptors. Recent studies indicate that G proteins also are required for some types of NAE signaling in plants. The genetic ablation of the Gβγ dimer or loss of the full set of extra-large G proteins strongly attenuated NAE-induced chloroplast responses in seedlings. Intriguing parallels and distinct differences have emerged between plants and animals in NAE signaling, despite the conserved use of these lipid mediators to modulate cellular processes. Here we compare similarities and differences and identify open questions in a fundamental lipid signaling pathway in eukaryotes with components that are both conserved and diverged in plants. N -Acylethanolamines (NAEs) are lipid signaling molecules in plants and animals. Some NAE signaling pathways are facilitated by heterotrimeric G proteins in both animal and plant systems. Similarities and differences in NAE signaling offer opportunities to study the diversity and evolution of this fundamental lipid signaling pathway in eukaryotes. [ABSTRACT FROM AUTHOR]

Background: Neuropathic pain is a complex condition characterized by sensory, cognitive and affective symptoms that magnify the perception of pain. The underlying pathogenic mechanisms are largely unknown and there is an urgent need for the development of novel medications. The endocannabinoid system modulates pain perception and drugs targeting the cannabinoid receptor type 2 (CB2) devoid of psychoactive side effects could emerge as novel analgesics. An interesting model to evaluate the mechanisms underlying resistance to pain is the fragile X mental retardation protein knockout mouse (Fmr1KO), a model of fragile X syndrome that exhibits nociceptive deficits and fails to develop neuropathic pain. Methods: A partial sciatic nerve ligation was performed to wild‐type (WT) and Fmr1KO mice having (HzCB2 and Fmr1KO‐HzCB2, respectively) or not (WT and Fmr1KO mice) a partial deletion of CB2 to investigate the participation of the endocannabinoid system on the pain‐resistant phenotype of Fmr1KO mice. Results: Nerve injury induced canonical hypersensitivity in WT and HzCB2 mice, whereas this increased pain sensitivity was absent in Fmr1KO mice. Interestingly, Fmr1KO mice partially lacking CB2 lost this protection against neuropathic pain. Similarly, pain‐induced depressive‐like behaviour was observed in WT, HzCB2 and Fmr1KO‐HzCB2 mice, but not in Fmr1KO littermates. Nerve injury evoked different alterations in WT and Fmr1KO mice at spinal and supra‐spinal levels that correlated with these nociceptive and emotional alterations. Conclusions: This work shows that CB2 is necessary for the protection against neuropathic pain observed in Fmr1KO mice, raising the interest in targeting this receptor for the treatment of neuropathic pain. Significance: Neuropathic pain is a complex chronic pain condition and current treatments are limited by the lack of efficacy and the incidence of important side effects. Our findings show that the pain‐resistant phenotype of Fmr1KO mice against nociceptive and emotional manifestations triggered by persistent nerve damage requires the participation of the cannabinoid receptor CB2, raising the interest in targeting this receptor for neuropathic pain treatment. Additional multidisciplinary studies more closely related to human pain experience should be conducted to explore the potential use of cannabinoids as adequate analgesic tools. [ABSTRACT FROM AUTHOR]

Chronic kidney disease (CKD) development after acute kidney injury (AKI) involves multiple mechanisms, including inflammation, epithelial-mesenchymal transition (EMT), and extracellular matrix deposition, leading to progressive tubulointerstitial fibrosis. Recently, a central role for peroxisome-proliferator activated receptor (PPAR)-α has been addressed in preserving kidney function during AKI. Among endogenous lipid mediators, oleoylethanolamide (OEA), a PPAR-α agonist, has been studied for its metabolic and anti-inflammatory effects. Here, we have investigated OEA effects on folic acid (FA)-induced kidney injury in mice and the underlying mechanisms. OEA improved kidney function, normalized urine output, and reduced serum BUN, creatinine, and albuminuria. Moreover, OEA attenuated tubular epithelial injury, as shown by histological analysis, and decreased expression of neutrophil gelatinase-associated lipocalin and kidney injury molecule-1. Gene expression analysis of kidney tissue indicated that OEA limited immune cell infiltration and inflammation. Moreover, OEA significantly inhibited Wnt7b and Catnb1 gene transcription and α-smooth muscle actin expression, indicating suppression of EMT. Accordingly, OEA exhibited an anti-fibrotic effect, as shown by Masson staining and the reduced levels of transforming growth factor (TGF)-β1, fibronectin, and collagen IV. Mechanistically, the nephroprotective effect of OEA was related to PPAR-α activation since OEA failed to exert its beneficial activity in FA-insulted PPAR-α-/- mice. PPAR-α involvement was also confirmed in HK2 cells where GW6471, a PPAR-α antagonist, blunted OEA activity on the TGF-β1 signalling pathway and associated pro-inflammatory and fibrotic patterns. Our findings revealed that OEA counteracts kidney injury by controlling inflammation and fibrosis, making it an effective therapeutic tool for limiting AKI to CKD progression. [Display omitted] • OEA limits AKI to CKD transition in mice. • OEA reduces renal inflammation caused by FA. • OEA counteracts fibrosis progression in the kidney of FA mice. • OEA reduces the TGFβ-mediated activation of profibrotic pathways in HK-2 cells. • OEA renoprotective effects is blunted in PPARα-/- mice. [ABSTRACT FROM AUTHOR]

Peroxisome proliferator-activator receptors (PPARs) regulate lipid and glucose metabolism, control inflammatory processes, and modulate several brain functions. Three PPAR isoforms have been identified, PPARα, PPARβ/δ, and PPARγ, which are expressed in different tissues and cell types. Hereinafter, we focus on PPARα involvement in the pathophysiology of neuropsychiatric and neurodegenerative disorders, which is underscored by PPARα localization in neuronal circuits involved in emotion modulation and stress response, and its role in neurodevelopment and neuroinflammation. A multiplicity of downstream pathways modulated by PPARα activation, including glutamatergic neurotransmission, upregulation of brain-derived neurotrophic factor, and neurosteroidogenic effects, encompass mechanisms underlying behavioral regulation. Modulation of dopamine neuronal firing in the ventral tegmental area likely contributes to PPARα effects in depression, anhedonia, and autism spectrum disorder (ASD). Based on robust preclinical evidence and the initial results of clinical studies, future clinical trials should assess the efficacy of PPARα agonists in the treatment of mood and neurodevelopmental disorders, such as depression, schizophrenia, and ASD.

The family of lipid neuromodulators has been rapidly growing, as the use of different -omics techniques led to the discovery of a large number of naturally occurring N-acylethanolamines (NAEs) and N-acyl amino acids belonging to the complex lipid signaling system termed endocannabinoidome. These molecules exert a variety of biological activities in the central nervous system, as they modulate physiological processes in neurons and glial cells and are involved in the pathophysiology of neurological and psychiatric disorders. Their effects on dopamine cells have attracted attention, as dysfunctions of dopamine systems characterize a range of psychiatric disorders, i.e., schizophrenia and substance use disorders (SUD). While canonical endocannabinoids are known to regulate excitatory and inhibitory synaptic inputs impinging on dopamine cells and modulate several dopamine-mediated behaviors, such as reward and addiction, the effects of other lipid neuromodulators are far less clear. Here, we review the emerging role of endocannabinoid-like neuromodulators in dopamine signaling, with a focus on non-cannabinoid N-acylethanolamines and their receptors. Mounting evidence suggests that these neuromodulators contribute to modulate synaptic transmission in dopamine regions and might represent a target for novel medications in alcohol and nicotine use disorder.

Abstract In plants, N‐acylethanolamines (NAEs) are most abundant in desiccated seeds and their levels decline during germination and early seedling establishment. However, endogenous NAE levels rise in seedlings when ABA or environmental stress is applied, and this results in an inhibition of further seedling development. When the most abundant, polyunsaturated NAEs of linoleic acid (18:2) and linolenic acid (18:3) were exogenously applied, seedling development was affected in an organ‐specific manner. NAE 18:2 primarily affected primary root elongation and NAE 18:3 primarily affected cotyledon greening and expansion and overall seedling growth. The molecular components and signaling mechanisms involved in this pathway are not well understood. In addition, the bifurcating nature of this pathway provides a unique system in which to study the spatial aspects and interaction of these lipid‐specific and organ‐targeted signaling pathways. Using whole transcriptome sequencing (RNA‐seq) and differential expression analysis, we identified early (1–3 hr) transcriptional changes induced by the exogenous treatment of NAE 18:2 and NAE 18:3 in cotyledons, roots, and seedlings. These two treatments led to a significant enrichment in ABA‐response and chitin‐response genes in organs where the treatments led to changes in development. In Arabidopsis seedlings, NAE 18:2 treatment led to the repression of genes involved in cell wall biogenesis and organization in roots and seedlings. In addition, cotyledons, roots, and seedlings treated with NAE 18:3 also showed a decrease in transcripts that encode proteins involved in growth processes. NAE 18:3 also led to changes in the abundance of transcripts involved in the modulation of chlorophyll biosynthesis and catabolism in cotyledons. Overall, NAE 18:2 and NAE 18:3 treatment led to lipid‐type and organ‐specific gene expression changes that include overlapping and non‐overlapping gene sets. These data will provide future, rich opportunities to examine the genetic pathways involved in transducing early signals into downstream physiological changes in seedling growth.

Alterations in the expanded endocannabinoid system (eECS) and cell membrane composition have been implicated in the pathophysiology of schizophrenia spectrum disorders. We enrolled 54 antipsychotic (AP)-naïve first-episode psychosis (FEP) patients and 58 controls and applied a targeted metabolomics approach followed by multivariate data analysis to investigate the profile changes in the serum levels of endocannabinoids: 2-arachidonoylglycerol (2-AG) and anandamide, endocannabinoids-like N-acylethanolamines (NAEs: linoleoylethanolamide, oleoylethanolamide, and palmitoylethanolamide), and their dominating lipid precursor’s phosphatidylcholines. Biomolecule profiles were measured at the onset of first-episode psychosis (FEP) and 0.6 years and 5.1 years after the initiation of AP treatment. The results indicated that FEP might be characterized by elevated concentrations of NAEs and by decreased 2-AG levels. At this stage of the disease, the NAE-mediated upregulation of peroxisome proliferator-activated receptors (PPARs) manifested themselves in energy expenditure. A 5-year disease progression and AP treatment adverse effects led to a robust increase in 2-AG levels, which contributed to strengthened cannabinoid (CB1) receptor-mediated effects, which manifested in obesity. Dynamic 2-AG, NAEs, and their precursors in terms of phosphatidylcholines are relevant to the description of the metabolic shifts resulting from the altered eECS function during and after FEP.

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. [ABSTRACT FROM AUTHOR]

In plants, N‐acylethanolamines (NAEs) are most abundant in desiccated seeds and their levels decline during germination and early seedling establishment. However, endogenous NAE levels rise in seedlings when ABA or environmental stress is applied, and this results in an inhibition of further seedling development. When the most abundant, polyunsaturated NAEs of linoleic acid (18:2) and linolenic acid (18:3) were exogenously applied, seedling development was affected in an organ‐specific manner. NAE 18:2 primarily affected primary root elongation and NAE 18:3 primarily affected cotyledon greening and expansion and overall seedling growth. The molecular components and signaling mechanisms involved in this pathway are not well understood. In addition, the bifurcating nature of this pathway provides a unique system in which to study the spatial aspects and interaction of these lipid‐specific and organ‐targeted signaling pathways. Using whole transcriptome sequencing (RNA‐seq) and differential expression analysis, we identified early (1–3 hr) transcriptional changes induced by the exogenous treatment of NAE 18:2 and NAE 18:3 in cotyledons, roots, and seedlings. These two treatments led to a significant enrichment in ABA‐response and chitin‐response genes in organs where the treatments led to changes in development. In Arabidopsis seedlings, NAE 18:2 treatment led to the repression of genes involved in cell wall biogenesis and organization in roots and seedlings. In addition, cotyledons, roots, and seedlings treated with NAE 18:3 also showed a decrease in transcripts that encode proteins involved in growth processes. NAE 18:3 also led to changes in the abundance of transcripts involved in the modulation of chlorophyll biosynthesis and catabolism in cotyledons. Overall, NAE 18:2 and NAE 18:3 treatment led to lipid‐type and organ‐specific gene expression changes that include overlapping and non‐overlapping gene sets. These data will provide future, rich opportunities to examine the genetic pathways involved in transducing early signals into downstream physiological changes in seedling growth. [ABSTRACT FROM AUTHOR]

Fatty acid amide hydrolase (FAAH) is an enzyme that belongs to the amidase signature (AS) superfamily and is widely distributed in multicellular eukaryotes. FAAH hydrolyzes lipid signaling molecules – namely, N -acylethanolamines (NAEs) – which terminates their actions. Recently, the crystal structure of Arabidopsis thaliana FAAH was solved and key residues were identified for substrate-specific interactions. Here, focusing on residues surrounding the substrate-binding pocket, a comprehensive analysis of FAAH sequences from angiosperms reveals a distinctly different family of FAAH-like enzymes. We hypothesize that FAAH, in addition to its role in seedling development, also acts in an N -acyl amide communication axis to facilitate plant–microbe interactions and that structural diversity provides for the flexible use of a wide range of small lipophilic signaling molecules. Fatty acid amide hydrolase (FAAH) is the signal-terminating enzyme of the N -acylethanolamine signaling pathway with an established role in seedling development. The crystal structure of Arabidopsis thaliana FAAH was recently solved, revealing for the first time the structural features of FAAH from plants and explaining the enzyme's promiscuity toward N -acyl amide substrates. A second group of FAAH enzymes in angiosperms has been identified with conserved substitutions in the substrate-binding pocket altering the size, shape, and physicochemical properties for substrate recognition. [ABSTRACT FROM AUTHOR]

[Display omitted] • A LC-MS/MS method for determination of fatty acids and related N -Acylethanolamines in rat plasma and liver. • First assay for endogenous fatty acids and N -Acylethanolamines in the liver. • Two extraction methods were used and compared. • Extensive validation using human plasma to ensure applicability. A sensitive and specific method for the simultaneous determination of palmitoyl ethanolamide (PEA), oleoyl ethanolamide (OEA), linoleoyl ethanolamide (LEA), 1-palmitoyl glycerol (1-PG), 1-oleoyl glycerol (1-OG), 1-linoleoyl glycerol (1-LG), palmitic acid (PA), oleic Acid (OA), and linoleic acid (LA) in rat plasma and liver by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) has been developed. Multiple-reaction mode (MRM) of a triple quadrupole mass spectrometer was used for detection. A reverse phase C8 column was used for chromatographic separation, and an 11.5-minute gradient elution time was used. Deuterated internal standards included 2-AG-d 5 , OEA-d 4 , LEA-d 4 , and OA-d 9. In terms of linearity, accuracy, repeatability, intermediate precision, recovery, matrix effect, and stability, this newly designed method was approved. These nine lipids were successfully determined using this method in two sample groups: plasma from individuals with non-alcoholic fatty liver diseases (NAFLD) (n = 40) and healthy individuals (n = 40), as well as plasma and liver samples from rats with NAFLD (n = 15) and healthy ones (n = 11). [ABSTRACT FROM AUTHOR]

Common pathophysiological mechanisms have emerged for different neurological and neuropsychiatric conditions. In particular, mechanisms of oxidative stress, immuno-inflammation, and altered metabolic pathways converge and cause neuronal and non-neuronal maladaptative phenomena, which underlie multifaceted brain disorders. The peroxisome proliferator-activated receptors (PPARs) are nuclear receptors modulating, among others, anti-inflammatory and neuroprotective genes in diverse tissues. Both endogenous and synthetic PPAR agonists are approved treatments for metabolic and systemic disorders, such as diabetes, fatty liver disease, and dyslipidemia(s), showing high tolerability and safety profiles. Considering that some PPAR-acting drugs permeate through the blood–brain barrier, the possibility to extend their scope from the periphery to central nervous system has gained interest in recent years. Here, we review preclinical and clinical evidence that PPARs possibly exert a neuroprotective role, thereby providing a rationale for repurposing PPAR-targeting drugs to counteract several diseases affecting the central nervous system.

Bone is a highly complex and metabolically active tissue undergoing a continuous remodeling process, which endures throughout life. A complex cell-signaling system that plays role in regulating different physiological processes, including bone remodeling, is the endocannabinoid system (ECS). Bone mass expresses CB1 and CB2 cannabinoid receptors and enzymatic machinery responsible for the metabolism of their endogenous ligands, endocannabinoids (AEA and 2-AG). Exogenous AEA is reported to increase the early phase of human osteoblast differentiation in vitro. However, regarding this cell context little is known about how endocannabinoids and endocannabinoid-related N-acylethanolamines like PEA and OEA are modulated, in vitro, during cell differentiation and, in vivo, over time up to adulthood. Here we characterized the endocannabinoid tone during the different phases of the osteoblast differentiation process in MC3T3-E1 cells, and we measured endocannabinoid levels in mouse femurs at life cycle stages characterized by highly active bone growth (i.e., of juvenile, young adult, and mature adult bone). Endocannabinoid tone was significantly altered during osteoblast differentiation, with substantial OEA increment, decline in 2-AG and AEA, and consistent modulation of their metabolic enzymes in maturing and mineralized MC3T3-E1 cells. Similarly, in femurs, we found substantial, age-related, decline in 2-AG, OEA, and PEA. These findings can expand existing knowledge underlying physiological bone cell function and contribute to therapeutic strategies for preventing bone-related metabolic changes accruing through lifespan.

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.

Abstract 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-1β, 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.

The management of chronic pain is an integral challenge of small animal veterinary practitioners. Multiple pharmacological agents are usually employed to treat maladaptive pain including opiates, non-steroidal anti-inflammatory drugs, anticonvulsants, antidepressants, and others. In order to limit adverse effects and tolerance development, they are often combined with non-pharmacologic measures such as acupuncture and dietary interventions. Accumulating evidence suggests that non-neuronal cells such as mast cells and microglia play active roles in the pathogenesis of maladaptive pain. Accordingly, these cells are currently viewed as potential new targets for managing chronic pain. Palmitoylethanolamide is an endocannabinoid-like compound found in several food sources and considered a body’s own analgesic. The receptor-dependent control of non-neuronal cells mediates the pain-relieving effect of palmitoylethanolamide. Accumulating evidence shows the anti-hyperalgesic effect of supplemented palmitoylethanolamide, especially in the micronized and co-micronized formulations (i.e., micro-palmitoylethanolamide), which allow for higher bioavailability. In the present paper, the role of non-neuronal cells in pain signaling is discussed and a large number of studies on the effect of palmitoylethanolamide in inflammatory and neuropathic chronic pain are reviewed. Overall, available evidence suggests that there is place for micro-palmitoylethanolamide in the dietary management of chronic pain in dogs and cats.

Background: Cortical neuronal dysfunction has been proposed to underlie the psychopathology and cognitive dysfunction of schizophrenia. Previously we have reported altered sphingolipid and N-acylphosphatidylserine (NAPS) metabolism in the frontal cortex in schizophrenia. We continue to expand these investigations to define the biochemical basis for these critical neuropathologies.Methods: We undertook a targeted high resolution mass spectrometric analysis to validate our previous reports of elevated sphingolipids and NAPS in the frontal cortex of a new cohort of schizophrenia subjects. Furthermore we expanded these analyses to include ceramides, N-acylphosphatidylethanolamines (NAPE), and N-acylethanolamines (NAE). In the same tissue samples we examined N-acetylaspartylglutamate (NAAG), a modulator of excitatory amino acid transmission, hypothesized to be involved in the pathology of schizophrenia.Results: We repeated our observations of elevated sulfatides in the frontal cortex in schizophrenia. An in-depth analysis of other sphingolipids revealed decrements in ceramide levels and increased levels of lactosylceramides. NAPS also were found to be augmented in schizophrenia as we previously reported. In addition, levels of NAPES, established biomarkers of neuronal stress, were elevated while their metabolites, NAEs were decreased. With regard to excitatory amino acid neurotransmission, NAAG levels were decreased by 50% while the metabolic precursor, N-acetylaspartate was unaltered.Conclusions: Our data support the concept of cortical neuronal dysfunction in schizophrenia as indicated by altered metabolism of structural sphingolipids and NAAG, a modulator of excitatory amino acid neurotransmission. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

N-acylethanolamines (NAEs) are lipid signaling mediators, which can be synthesized from dietary fatty acids via n-acylphosphatidylethanolamine-phospholipase D (NAPE-PLD) and in turn influence physiological outcomes; however, the roles of NAPE-PLD upon dietary fatty acid modulation are not fully understood. Presently, we examine if NAPE-PLD is necessary to increase NAEs in response to dietary fatty acid manipulation. Post-weaning male wild-type (C57Bl/6), NAPE-PLD (-/+) and NAPE-PLD (-/-) mice received isocaloric fat diets containing either beef tallow, corn oil, canola oil or fish oil (10% wt/wt from fat) for 9 weeks. Brain docosahexaenoic acid (DHA) levels were higher (P<.01) in NAPE-PLD (-/+) (10.01±0.31 μmol/g) and NAPE-PLD (-/-) (10.89±0.61 μmol/g) than wild-type (7.72±0.61 μmol/g) consuming fish oil. In NAPE-PLD (-/-) mice, brain docosahexaenoylethanolamide (DHEA) levels were higher (P<.01) after fish oil feeding suggesting that NAPE-PLD was not necessary for DHEA synthesis. Liver and jejunum arachidonoylethanolamide, 1,2-arachidonoylglycerol and DHEA levels reflected their corresponding fatty acid precursors suggesting that alternate pathways are involved in NAE synthesis. NAPE-PLD (-/-) mice had lower oleoylethanolamide levels in the jejunum and a leaner phenotype compared to wild-type mice. Overall, these results demonstrate that dietary fatty acid can augment tissue NAEs in the absence of NAPE-PLD. [ABSTRACT FROM AUTHOR]

Characterized by chronic widespread pain, generalized hyperalgesia, and psychological stress, fibromyalgia (FM) is difficult to diagnose and lacks effective treatments. Endocannabinoids-arachidonoylethanolamide (AEA), 2-arachidonoylglycerol (2-AG), and the related oleoylethanolamide (OEA), palmitoylethanolamide (PEA), and stearoylethanolamide (SEA)-are endogenous lipid mediators with analgesic and anti-inflammatory characteristics, in company with psychological modulating properties (eg, stress and anxiety), and are included in a new emerging "ome," the endocannabinoidome. This case-control study compared the concentration differences of AEA, OEA, PEA, SEA, and 2-AG in 104 women with FM and 116 healthy control subjects. All participants rated their pain, anxiety, depression, and current health status. The relationships between the lipid concentrations and the clinical assessments were investigated using powerful multivariate data analysis and traditional bivariate statistics. The concentrations of OEA, PEA, SEA, and 2-AG were significantly higher in women with FM than in healthy control subjects; significance remained for OEA and SEA after controlling for body mass index and age. 2-AG correlated positively with FM duration and body mass index, and to some extent negatively with pain, anxiety, depression, and health status. In FM, AEA correlated positively with depression ratings. The elevated circulating levels of endocannabinoidome lipids suggest that these lipids play a role in the complex pathophysiology of FM and might be signs of ongoing low-grade inflammation in FM. Although the investigated lipids are significantly altered in FM, their biological roles are uncertain with respect to the clinical manifestations of FM. Thus plasma lipids alone are not good biomarkers for FM. PERSPECTIVE: This study reports about elevated plasma levels of endocannabinoidome lipid mediators in FM. The lipids' suitability to work as biomarkers for FM in the clinic were low; however, their altered levels indicate that a metabolic asymmetry is ongoing in FM, which could serve as a baseline during explorative FM pain management. [ABSTRACT FROM AUTHOR]

It has been suggested that anandamide (N-arachidonoylethanolamine), an endogenous cannabinoid substance, may be produced through Ca2+-stimulated hydrolysis of the phosphatidylethanolamine (PE) derivative N-arachidonoyl PE. The presence of N-arachidonoyl PE in adult brain tissue and the enzyme pathways that underlie its biosynthesis are, however, still undetermined. We report here that rat brain tissue contains both anandamide (11 +/- 7 pmol/gm wet tissue) and N-arachidonoyl PE (22 +/- 16 pmol/gm), as assessed by gas chromatography/mass spectrometry. We describe a N-acyltransferase activity in brain that catalyzes the biosynthesis of N-arachidonoyl PE by transferring an arachidonate group from the sn-1 carbon of phospholipids to the amino group of PE. We also show that sn-1 arachidonoyl phospholipids are present in brain, where they constitute approximately 0.5% of total phospholipids. N-acyltransferase activity is Ca2+ dependent and is enriched in brain and testis. Within the brain, N-acyltransferase activity is highest in brainstem; intermediate in cortex, striatum, hippocampus, medulla, and cerebellum; and lowest in thalamus, hypothalamus, and olfactory bulb. Pharmacological inhibition of N-acyltransferase activity in primary cultures of cortical neurons prevents Ca2+-stimulated N-arachidonoyl PE biosynthesis. Our results demonstrate, therefore, that rat brain tissue contains the complement of enzymatic activity and lipid substrates necessary for the biosynthesis of the anandamide precursor N-arachidonoyl PE. They also suggest that biosynthesis of N-arachidonoyl PE and formation of anandamide are tightly coupled processes, which may concomitantly be stimulated by elevations in intracellular Ca2+ occurring during neural activity.

It has been suggested that anandamide (N-arachidonoylethanolamine), an endogenous cannabinoid substance, may be produced through Ca2+-stimulated hydrolysis of the phosphatidylethanolamine (PE) derivative N-arachidonoyl PE. The presence of N-arachidonoyl PE in adult brain tissue and the enzyme pathways that underlie its biosynthesis are, however, still undetermined. We report here that rat brain tissue contains both anandamide (11 +/- 7 pmol/gm wet tissue) and N-arachidonoyl PE (22 +/- 16 pmol/gm), as assessed by gas chromatography/mass spectrometry. We describe a N-acyltransferase activity in brain that catalyzes the biosynthesis of N-arachidonoyl PE by transferring an arachidonate group from the sn-1 carbon of phospholipids to the amino group of PE. We also show that sn-1 arachidonoyl phospholipids are present in brain, where they constitute approximately 0.5% of total phospholipids. N-acyltransferase activity is Ca2+ dependent and is enriched in brain and testis. Within the brain, N-acyltransferase activity is highest in brainstem; intermediate in cortex, striatum, hippocampus, medulla, and cerebellum; and lowest in thalamus, hypothalamus, and olfactory bulb. Pharmacological inhibition of N-acyltransferase activity in primary cultures of cortical neurons prevents Ca2+-stimulated N-arachidonoyl PE biosynthesis. Our results demonstrate, therefore, that rat brain tissue contains the complement of enzymatic activity and lipid substrates necessary for the biosynthesis of the anandamide precursor N-arachidonoyl PE. They also suggest that biosynthesis of N-arachidonoyl PE and formation of anandamide are tightly coupled processes, which may concomitantly be stimulated by elevations in intracellular Ca2+ occurring during neural activity.

Understanding the mechanisms involved in the biogenesis of N-arachidonoylethanolamine (anandamide) and N-palmitoylethanolamine is important in view of the possible role of these lipids as endogenous cannabinoid substances. Anandamide (which activates cannabinoid CB1 receptors) and N-palmitoylethanolamine (which activates a CB2-like receptor subtype in mast cells) may both derive from cleavage of precursor phospholipid, N-acylphosphatidylethanolamine (NAPE), catalyzed by Ca(2+)-activated D-type phosphodiesterase activity. We report here that the de novo biosynthesis of NAPE is enhanced in a Ca(2+)-dependent manner when rat cortical neurons are stimulated with the Ca(2+)-ionophore ionomycin or with membrane-depolarizing agents such as veratridine and kainate. This reaction is likely to be mediated by a neuronal N-acyltransferase activity, which catalyzes the transfer of an acyl group from phosphatidylcholine to the ethanolamine moiety of phosphatidylethanolamine. In addition, we show that Ca2+-dependent NAPE biosynthesis is potentiated by agents that increase cAMP levels, including forskolin and vasoactive intestinal peptide. Our results thus indicate that NAPE levels in cortical neurons are controlled by Ca2+ ions and cAMP. Such regulatory effect may participate in maintaining a supply of cannabimimetic N-acylethanolamines during synaptic activity, and prime target neurons for release of these bioactive lipids.