BACKGROUND:Cachexia is a debilitating syndrome associated with involuntary weight loss, often occurring in cancer patients. In both humans and animal models, alterations in endocannabinoid (eCB) signaling occur in association with both metabolic disorders and several types of tumors. The wider signaling system, including the two eCBs, anandamide (AEA) and 2-arachidonoyl-glycerol (2-AG), their congeners and other long chain fatty acid amides, as well as their metabolic enzymes and receptors, is known as endocannabinoidome (eCBome). The eCBome is involved, among others, in the control of energy balance and cancer and interacts with the gut microbiome. METHODS:Using mass spectrometry-based targeted lipidomics, we measured the hepatic and intestinal concentrations of eCBome mediators in mice injected with colon carcinoma 26 (C26) cells, a model of cancer cachexia characterized, among others, by weight loss, hepatic dyslipidemia, and gut microbiome dysbiosis. RESULTS:We report that, 10 days after C26 cell injection, concomitant with >10% weight loss, eCBome lipids levels, namely 2-AG, AEA, and some of its N-acyl-ethanolamine congeners, as well as N-oleoyl-glycine, N-acyl-serotonins, and N-acyl-taurines (NATs), are altered in intestinal sections and the liver of C26 mice (e.g., hepatic 2-AG -30%, jejunal 2-AG + 30%, jejunal AEA -55%, hepatic N-oleoyl-ethanolamine (OEA) + 223%, hepatic NATs +144%, +141%, +216%). Gut dysbiosis was evident in these mice (PERMANOVA at the family level: R² = 69%, p < 0.001), with altered levels in 3 phyla (mainly the Proteobacteria, +1484%), 12 families, and 12 genera (all with adjusted p < 0.05). Additionally, 2-AG, AEA, OEA, N-arachidonoyl-serotonin, and NAT levels in the liver positively correlated with hepatic total lipids, triglycerides, and cholesterol, whereas N-docosahexaenoyl-ethanolamine and N-docosahexaenoyl-serotonin showed negative correlations. Jejunal AEA negatively, and hepatic OEA and NATs positively, correlated with weight loss. Intestinal eCBome mediators correlated with several cecal microbial taxa, including genera known to include strains beneficial in metabolic disorders, such as Bacteroides, Parabacteroides, Dysosmobacter, and Prevotella. CONCLUSIONS:These observations pinpoint eCBome mediators as new multi-functional players in the hepatic complications and gut dysbiosis accompanying cancer cachexia.

N-palmitoyl-D-glucosamine (PGA) belongs to the class of molecules known as Autacoid Local Injury Antagonism (ALIA)-amides, whose parent compound is N-palmitoyl-ethanolamine (PEA) an endocannabinoid-like mediator belonging to the expanded endocannabinoid system (the endocannabinoidome). The mechanism of action of ALIA-amides is mainly targeted at the down-regulation of the hyperactivity of peripheral mast cells and non-neuronal cells of the central nervous system. This study aimed to investigate if PGA is able to produce the typical "entourage" effect of PEA, consisting of increasing the endogenous levels of the endocannabinoids anandamide (AEA) and 2-arachidonoylglycerol (2-AG), and to also elevate endogenous PEA levels. PGA (10 µM) treatment of HaCaT cells for 40 min and 6 h resulted in higher cellular concentrations of AEA, 2-AG and PEA. The increase of endocannabinoid and PEA levels was observed both after 40 min and 6 h of PGA treatment. The unselective serine hydrolase inhibitor MAFP (10 µM) inhibited the PGA-induced increase in cellular concentrations of AEA, 2-AG and PEA. The mRNA expression of endocannabinoid and PEA anabolic (NAPEPLD, ABHD4, GDE1, DAGLA and DAGLB) and catabolic (FAAH, MAGL and NAAA) enzymes was also measured, revealing a significative reduction of GDE1, DAGLB, FAAH, MAGL and NAAA, after PGA treatment for 6 h. Finally, AEA, 2-AG and PEA were also measured in HaCaT cells after siRNA against ABHD4 and PGA treatment for 40 min, revealing an increase of their endogenous levels. In conclusion, we demonstrated for the first time that PGA increases PEA and endocannabinoid levels, thus potentially strengthening a part of endocannabinoidome signaling.

BACKGROUND:Despite the broad range of treatment options available for intestinal inflammation, the development of novel therapeutics remains essential due to the diminishing effectiveness of current therapies over time. Both the endocannabinoid system (ECS) and nociceptin/orphanin FQ peptide (NOP) receptors have been implicated in the pathogenesis of diseases associated with intestinal inflammation, highlighting their potential as therapeutic targets. OBJECTIVES:We hypothesized that an interaction exists between cannabinoid receptors 1 and 2 (CB1 and CB2) and the NOP receptor, which may hold therapeutic relevance for the treatment of colitis. MATERIAL AND METHODS:In this study, we used 3 selective ligands: a CB1 antagonist (AM6545), a CB2 antagonist (AM630) and a NOP agonist (SCH221510) in a mouse model of colitis induced by 3% dextran sulfate sodium (DSS). Quantification of several secondary messengers was conducted using western blot analysis. Real-time quantitative polymerase chain reaction (qPCR) was employed to assess CB1 expression levels in colonic tissue, while liquid chromatography-mass spectrometry (LC-MS) was used to evaluate the concentrations of endocannabinoids and related lipid mediators. RESULTS:We observed a statistically significant increase in the macroscopic score and a nonsignificant increase in the microscopic score in inflamed mice treated with both AM6545 and SCH221510 compared to those treated with SCH221510 alone. Additionally, the combination-treated group exhibited significantly lower levels of extracellular signal-regulated kinases 1/2 (ERK1/2) and significantly higher levels of phosphorylated protein kinase B (p-AKT) and β-arrestin relative to the SCH221510-only group. CONCLUSIONS:Our study offers novel insights into the interaction between the ECS and the NOP receptor, which may inform the development of new therapeutic strategies for inflammatory conditions such as colitis.

Circadian light influences brain functions in mammals. Photic non-image-forming stimuli are transduced into electrochemical signals by photosensitive retinal ganglion cells containing melanopsin, a selective blue light-responsive photopigment. The hypothalamus receives light-related information via the retinohypothalamic tract (RHT). Here, we demonstrate that, in the mouse, a lateral branch of the RHT (l-RHT) projects monosynaptically to orexin-A (OX-A) neurons in the perifornical hypothalamic area (PFH). Intravitreal injection of the anterograde tracer cholera toxin-β (CTβ) filled most of the vesicular glutamate transporter (VGluT1)/cannabinoid receptor 1 (CB1R)-positive retinal-derived inputs projecting to the OX-A neurons. Monocular injection of Fluo4-Dextran, a fluorimetric sensor of calcium mobilization, yielded fast labeling of these inputs 10 min after eye exposure to blue light, concomitantly with the enhancement of hypothalamic 2-arachidonoylglycerol (2-AG) levels, and inhibition of OX-A neuronal firing, an effect prevented by in vivo administration of the CB1R antagonist AM251. Our findings provide anatomical and functional evidence of a selective retino-hypothalamic network responsive to blue light, whose control should be suitable for therapies to counteract sleep disorders, seasonal affective disorder, or even conditions like narcolepsy or anxiety.

The endocannabinoid system (ECS) and nociceptin receptor (NOP) have been implicated in the pathology of inflammatory bowel diseases (IBD) mediating pain and alleviating inflammation. In this study we searched for the possible activation of ECS and NOP system and the correlation between CB1, CB2 and NOP receptors in IBD patients. Patients diagnosed with IBDs who underwent colonic surgical resection or biopsy at colonoscopy and control group (patients without diagnosis of IBD, which colonoscopy for the different medical indications) were recruited into the study. In surgical specimen the quantification of endocannabinoids was obtained by liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry. In biopsy specimen, the expression of genes encoding CB1, CB2, and NOP receptors was determined with real-time RT-PCR. The relative expression of CB1 and CB2 receptors in human samples with IBD compared to the unrelated controls (HC) group was reduced compared to the controls, but no differences in relative expression of NOP receptor were detected. Statistical significance difference was reached only between the level of a relative expression of CB1 receptor in ulcerative colitis (UC) and HC groups (498 (57.45-1890) and 4946 (2098--12818) (P<0.05)). A statistically significant positive correlation between the relative expression of CB1 and NOP receptors (r=0.83, P<0.05) as well as CB2 and NOP receptors in Crohn's disease (CD) (r=0.87, P<0.05) was found. Several endocannabinoids were significantly (P<0.05) increased in tissue collected from UC and CD patients in comparison to controls. CB1 and CB2 but not NOP receptors were found to be downregulated in IBD. Correlation of CB1, CB2 and NOP expression may suggest their common roles and shared molecular pathways in CD. Upregulated level of several endocannabinoids may point out to their role in IBD. Their estimation may be possibly useful in IBD diagnostics.

BACKGROUND:Despite extensive studies on the neurobiological correlates of traumatic brain injury (TBI), little is known about its molecular determinants on long-term consequences, such as dementia and Alzheimer's disease (AD). METHODS:Here, we carried out behavioural studies and an extensive biomolecular analysis, including inflammatory cytokines, gene expression and the combination of LC-HRMS and MALDI-MS Imaging to elucidate the targeted metabolomics and lipidomics spatiotemporal alterations of brains from wild-type and APP-SWE mice, a genetic model of AD, at the presymptomatic stage, subjected to mild TBI. RESULTS:We found that brain injury does not affect cognitive performance in APP-SWE mice. However, we detected an increase of key hallmarks of AD, including Aβ1-42 levels and BACE1 expression, in the cortices of traumatized transgenic mice. Moreover, significant changes in the expanded endocannabinoid (eCB) system, or endocannabinoidome (eCBome), occurred, including increased levels of the endocannabinoid 2-AG in APP-SWE mice in both the cortex and hippocampus, and N-acylserotonins, detected for the first time in the brain. The gene expression of enzymes for the biosynthesis and inactivation of eCBs and eCB-like mediators, and some of their main molecular targets, also underwent significant changes. We also identified the formation of heteromers between cannabinoid 1 (CB1) and serotonergic 2A (5HT2A) receptors, whose levels increased in the cortex of APP-SWE mTBI mice, possibly contributing to the exacerbated pathophysiology of AD induced by the trauma. CONCLUSIONS:Mild TBI induces biochemical changes in AD genetically predisposed mice and the eCBome may play a role in the pathogenetic link between brain injury and neurodegenerative disorders also by interacting with the serotonergic system.

[This corrects the article DOI: 10.3389/fphar.2018.00249.].

BACKGROUND AND PURPOSE:Preclinical studies of amyotrophic lateral sclerosis (ALS) have shown altered endocannabinoid (eCB) signalling that may contribute to the disease. Results from human studies are sparse and inconclusive. The aim of this study was to determine the association between serum levels of eCBs or their congeners, the so-called endocannabinoidome, and disease status and activity in ALS patients. METHODS:Serum concentrations of 2-arachidonoylglycerol and N-arachidonoylethanolamine (AEA), and AEA congeners palmitoylethanolamide (PEA), oleoylethanolamide (OEA), eicosapentaenoylethanolamide (EPEA), 2-docosahexaenoylglycerol (2-DHG) and docosahexaenoylethanolamide (DHEA) were measured in samples from 65 ALS patients, 32 healthy controls (HCs) and 16 neurological disease controls (NALS). A subset of 46 ALS patients underwent a longitudinal study. Disease activity and progression were correlated with eCB and congener levels. RESULTS:Most circulating mediators were higher in ALS than HCs (all p < 0.001), but not NALS. Across clinical stages, ALS patients showed increased levels of PEA, OEA and EPEA (all p < 0.02), which were confirmed by the longitudinal study (all p < 0.03). Serum PEA and OEA levels were independent predictors of survival and OEA levels were higher in patients complaining of appetite loss. Cluster analysis revealed two distinct profiles of circulating mediators associated with corresponding patterns of disease activity (severe vs. mild). Patients belonging to the 'severe' cluster showed significantly higher levels of OEA and PEA and lower levels of 2-DHG compared to NALS and HCs. CONCLUSION:Circulating endocannabinoidome profiles are indicative of disease activity, thus possibly paving the way to a personalized, rather than a 'one-fits-all', therapeutic approach targeting the endocannabinoidome.

A cutaneous mast cell tumor (cMCT) is among the most common tumors in dogs. Endocannabinoids (eCBs) belong to the endocannabinoid system (ECS), which involves also cannabinoid receptors and an enzymatic system of biosynthesis and degradation. In this study, plasma levels of N-arachidonoylethanolamine (AEA), 2-arachidonoylglycerol (2-AG), N-palmitoylethanolamine (PEA), and N-oleoylethanolamine (OEA) were evaluated in 17 dogs with MCTs of varying histological grades and clinical stages, as well as in a control group of 11 dogs. Dogs affected by cMCT had higher plasma levels of 2-AG (p = 0.0001) and lower levels of AEA (p = 0.0012) and PEA (p = 0.0075) compared to the control group, while no differences were observed at the OEA level between healthy and cMCT dogs (p = 0.9264). The ability of eCBs to help discriminate between healthy and cMCT dogs was interrogated through the area under the ROC curve (AUC). An accuracy of 0.98 (95% confidence interval [CI], 0.94–1.02) was found for 2-AG, of 0.85 (95% CI, 0.71–0.99) for AEA, and of 0.81% for PEA (95% CI, 0.64–0.69). Values > 52.75 pmol/mL for 2-AG showed 94% sensitivity and 90% specificity in distinguishing cMCT. This is the first study to demonstrate alterations in plasmatic levels of eCBs in dogs affected by MCTs, suggesting the significance of these biomarkers in the tumorigenic process and their potential use as biomarkers in the future.

Supplementary Figure S3. O2PLS regression coefficients representing correlation between polar (A-G) and lipophilic metabolites (H-I) with transcripts.

Supplementary Tables with parameters

<p>Time courses of pH and oxygen in presence of enzalutamide continuous treatment</p>

<p>Supplementary Figure S2. PCA derived scores and loadings plot displaying sample projections of (A) NMR polar spectra, (B) NMR lipid extracts and (C) PCR transcripts values.</p>

<p>Supplementary Table S2. Parameter values used in the numerical simulations.</p>

The Figure shows the long term behavior of sensitive and resistant cells (in absence of enzalutamide

<p>Enzalutamide does not induce apoptosis in TRAMP-C2 cells</p>

<p>Supplementary Materials and Methods</p>

Supplemantary informations for the model parameterization

Supplementary Table S1. Topology-based pathway analysis.

<p>Simulation and treatment of TRAMP mice treated with enzalutamide, cabazitaxel and the combination</p>