AEA Levels Research Articles

Endogenous anandamide and self-reported pain are significantly reduced after a 2-week multimodal treatment with and without radon therapy in patients with knee osteoarthritis: a pilot study

Multimodal therapies comprising spa applications are widely used as non-pharmaceutical treatment options for musculoskeletal diseases. The purpose of this randomized, controlled, open pilot study was to elucidate the involvement of the endocannabinoid system in a multimodal therapy approach. Twenty-five elderly patients with knee osteoarthritis (OA) received a 2-week spa therapy with or without combination of low-dose radon therapy in the Bad Gastein radon gallery. A 10-point numerical rating scale (pain in motion and at rest), WOMAC questionnaire, and the EuroQol-5D (EQ-5D) questionnaire were recorded at baseline, and during treatment period at weeks one and two, and at 3-month and 6-month follow-ups. Plasma levels of the endocannabinoid anandamide (AEA) were determined at baseline and at 2 weeks, and serum levels of several cartilage metabolism markers at all five time-points. A significant and sustained reduction of self-reported knee pain was observed in the study population, but no further significant effect of the additional radon therapy up and above base therapy. This pain reduction was accompanied by a significant reduction of AEA plasma levels during treatment in both groups. No significant differences were seen in serum marker concentrations between the groups treated with or without radon, but a small reduction of serum cartilage degradation markers was observed during treatment in both groups. This is the first study investigating AEA levels in the context of a non-pharmacological OA treatment. Since the endocannabinoid system represents a potential target for the development of new therapeutics, further studies will have to elucidate its involvement in OA pain.

Spinal nociceptive sensitization and plasma palmitoylethanolamide levels during experimentally induced migraine attacks.

Migraine pathophysiology has been suggested to include dysregulation of the endocannabinoid system (ES). We simultaneously evaluated plasma anandamide (AEA) and palmitoylethanolamide (PEA) levels and spinal sensitization in a validated human model of migraine based on systemic nitroglycerin (NTG) administration. Twenty-four subjects with episodic migraine (MIG) and 19 healthy controls (HC) underwent blood sampling and investigation of nociceptive withdrawal reflex thresholds (RTh: single-stimulus threshold; TST: temporal summation threshold) before and 30 (T30), 60 (T60), and 120 (T120) minutes after sublingual NTG administration (0.9 mg). At baseline, the MIG and HC groups were comparable for plasma AEA (P = 0.822) and PEA (P = 0.182) levels, and for RTh (P = 0.142) and TST values (P = 0.150). Anandamide levels increased after NTG administration (P = 0.022) in both groups, without differences between them (P = 0.779). By contrast, after NTG administration, PEA levels increased in the MIG group at T120 (P = 0.004), while remaining stable in the HC group. Nitroglycerin administration induced central sensitization in the MIG group, which was recorded as reductions in RTh (P = 0.046) at T30 and T120, and in TST (P = 0.001) at all time points. In the HC group, we observed increases in RTh (P = 0.001) and TST (P = 0.008), which suggest the occurrence of habituation. We found no significant correlations between the ES and neurophysiological parameters. Our findings suggest a role for PEA in the ictal phase of episodic migraine. The ES does not seem to be directly involved in the modulation of NTG-induced central sensitization, which suggests that the observed PEA increase and spinal sensitization are parallel, probably unrelated, phenomena.

Plasma OEA level is correlated with muscle mass and mobility in old rats

Rationale : Sarcopenia is an age-related loss of muscle mass and strength associated with changes in skeletal muscle protein homeostasis due to lipid accumulation and anabolic resistance. These two latter phenomena are also observed in obesity which is known to be associated with overactivated endocannabinoid system. The aim of this study was to evaluate the disturbances of the endocannabinoid system in ageing and their putative link to skeletal muscle mass and function in old rats. Methods : 6-month adult and 24-month old male rats were used to evaluate locomotor activity in openfield and voluntary gait by Catwalk tests. Weight of the hindlimb muscles was measured after sacrifice, and plasma concentrations of endocannabinoid (AEA, 2AG) and endocannaiboid-like metabolites (PEA and OEA) were measured by LCF-MS/MS. Comparison between groups were analyzed by student t-test and Pearson’s correlation analysis was used. Results : Compared to adult rats, old rats display higher fat mass (19±1 vs 14±1%, P<0.01) and lower lean mass (72±2 vs 80±2%, P<0.01), exemplified by a 49% decrease in hindlimb muscle mass (P<0.01), characteristics of sarcopenia. This loss of muscle mass was associated with reduced traveled distance (2720±550 vs 4804±960cm, P<0.01), mean speed (12.8±1.8 vs 10.1±0.8cm/sec, P<0.01) and duration of activity (268±49 vs 374±50sec, P<0.01) in openfield, and Catwalk analysis revealed that voluntary gait was severely impaired in old rats. Plasma levels of AEA, 2AG, PEA and OEA (pmol/ml) are described in the following table: AEA2AGPEAOEA Adult rats (n=11)2.0±0.248.9±6.249.8±6.021.4±2.6 Old rats (n=8)2.2±0.364.6±8.3 (P=0.07)37.1±2.6*12.9±1.1** * P<0.05, ** P<0,01 vs adults. Correlation analysis revealed that plasma levels of OEA, and to some extents PEA, were positively correlated with traveled distance (r²=0.31,P<0.02), mean speed (r²=0.20, P=0.06), duration of activity (r²=0.27, P<0.03) and several parameters of voluntary gait (P<0.05). Conclusion : These results demonstrate that plasma levels of endocannabinoids and endocannabinoid like metabolites are altered in sarcopenic old rats and that OEA plasma levels are associated with skeletal muscle dysfunction and loss of mobility with age.

Anandamide Level in Men with Oligoasthenoteratozoospermia

Background: Male infertility is a relatively common medical condition. Male partners are found to be solely responsible for 20–30% of infertility cases. Oligoasthenoteratozoospermia (OAT) is the most common laboratory finding in infertile men. Endocannabinoids are endogenously produced substances, that primarily act at cannabinoid receptors (CBRs), thus reproducing some of the biological actions of the natural cannabis sativa components (the “cannabinoids”). The endocannabinoid system is involved in several physiological processes, including fertility and body weight control. Objective: The aim of this study was to detect the level of anandamide (AEA) in patients with OAT and its relation to BMI. Patients and methods: This study was a case control study. It was hold between June 2018 to April 2019 in the Dermatology, Venereology and Andrology Department at Aswan University Hospital. The study included 20 patients with OAT and 9 as controls. Semen analysis using CASA was done. AEA level in seminal plasma was measured using ELISA kits. BMI was calculated. Results: AEA level in seminal plasma significantly decreased in men with ligoasthenoterat- ozoospermia than control (P < 0.009). AEA concentration on seminal fluid was significantly increased with an increase in BMI (P= 0.023). On the other hand, a significant negative relationship was present between BMI and semen volume (P= 0.03). Conclusion: AEA concentration in seminal plasma was significantly decreased in patients with OAT. AEA concentration in seminal fluid was significantly increased with an increase in BMI. On the other hand, a significant negative relationship was present between BMI and semen volume.

Serum endocannabinoid levels in suicide attempters: A pilot study

The search for a biomarker for suicide risk is a longstanding pursuit in clinical psychiatry. Literature addressing the role of endocannabinoids in suicide attempters (SA) is sparse. This cross-sectional study is aimed at comparing 8 AM serum concentrations of 4 endogenous cannabinoids (anandamide, AEA; 2-arachidonoylglycerol, 2-AG; N-palmitoiletanolamida, PEA; and oleoylethanolamide, OEA) in 30 suicide attempters (SA) and 12 psychiatric controls (PC). 8 AM AEA and PEA serum levels were higher in SA compared to PC without controlling for cannabis use (n = 42) (3.58 ± 5.77 vs. 1.62 ± 2.49, F = 3.04, P = 0.089; and 3.31 ± 4.82 vs. 1.21 ± 1.20, F = 6.22, p = 0.017, respectively). Serum ACTH was higher in PC compared to SA (32.11 ± 21.60 vs. 20.05 ± 9.96, F = 9.031, p = 0.0.005). After controlling for cannabis use in the urine test (n = 28), 8 AM AEA and PEA serum levels remained higher in SA compared to PC (4.57 ± 6.38 vs. 0.64 ± 1.11, F = 4.852, P = 0.037; and 4.35 ± 5.46 vs. 1.21 ± 1.25, F = 4.125, p = 0.053, respectively). The present study offers preliminary evidence about the role of AEA and PEA in suicidal behavior (SB). Furthermore, in the context of the mental pain model of SB, our findings suggest that some endocannabinoids may play a role in the pathophysiology of SB. Our pilot study deserves replication by other studies with bigger sample sizes.

Effect of acute physical exercise on motor sequence memory

Acute physical exercise improves memory functions by increasing neural plasticity in the hippocampus. In animals, a single session of physical exercise has been shown to boost anandamide (AEA), an endocannabinoid known to promote hippocampal plasticity. Hippocampal neuronal networks encode episodic memory representations, including the temporal organization of elements, and can thus benefit motor sequence learning. While previous work established that acute physical exercise has positive effects on declarative memory linked to hippocampal plasticity mechanisms, its influence on memory for motor sequences, and especially on neural mechanisms underlying possible effects, has been less investigated. Here we studied the impact of acute physical exercise on motor sequence learning, and its underlying neurophysiological mechanisms in humans, using a cross-over randomized within-subjects design. We measured behavior, fMRI activity, and circulating AEA levels in fifteen healthy participants while they performed a serial reaction time task before and after a short period of exercise (moderate or high intensity) or rest. We show that exercise enhanced motor sequence memory, significantly for high intensity exercise and tending towards significance for moderate intensity exercise. This enhancement correlated with AEA increase, and dovetailed with local increases in caudate nucleus and hippocampus activity. These findings demonstrate that acute physical exercise promotes sequence learning, thus attesting the overarching benefit of exercise to hippocampus-related memory functions.

Comparison of endocannabinoids levels, FAAH gene polymorphisms, and appetite regulatory substances in women with and without binge eating disorder: a cross- sectional study

Binge eating disorder (BED) is known as the most common eating disorder with both psychosocial and biological factors involved. In this regard, there is a need to recognize probable disturbances in substances involved in food intake regulation in BED. In this study, we hypothesized that the levels of endocannabinoids, fatty acid amid hydrolase (FAAH) gene polymorphisms, and appetite regulatory substances are different in overweight and obese women with and without BED. A Binge Eating Scale was used to estimate the prevalence of BED in 180 women classified as overweight or obese. The levels of anandamide (AEA), 2-arachidonoylglycerol (2-AG), leptin, insulin, and orexin-A were measured by enzyme-linked immunosorbent assay kits. The subjects were genotyped for polymorphisms of FAAH gene using amplification refractory mutation system–polymerase chain reaction. Data were analyzed using SPSS software. About 41.6% (n = 75) of the subjects were diagnosed with BED. Women with BED exhibited significantly higher levels of AEA, 2-AG, leptin, and insulin compared to non-BED women (P < .05). Binary logistic regression analysis also showed that AEA, leptin, and insulin were the predictors of having BED after adjusting for body mass index (P < .05). In addition, the frequency of A allele of FAAH gene was higher in women with BED compared to women without BED; however, there were no significant differences between these 2 groups (P = .08). These results supported our hypothesis in the cases of AEA, 2-AG, leptin, and insulin but not orexin and FAAH gene polymorphisms. The findings of the current study provide further evidence concerning the role of these substances in BED.

Differential effect of interferon-alpha treatment on AEA and 2-AG levels

The endocannabinoid (eCB) system is one of the key players in immunoregulation, and reduced activity of the eCB system has been linked with depressive-like behaviours in animal studies and depression in clinical samples. There is a well-established link between immune activation and depression, such as following the administration of the pro-inflammatory cytokine, interferon-α (IFN-α), used to treat hepatitis C viral (HCV) infection. However, the role of peripheral endocannabinoids (eCBs), anandamide (AEA) and 2-arachidonoylglycerol (2-AG), following immunotherapy with IFN-α and in IFN-α -induced depression, have not been examined yet.In this study, we investigated whether circulating AEA and 2-AG were modified by treatment with IFN-α and whether they were involved in the development of IFN-α-induced depression. We also explored whether circulating eCBs were associated with peripheral cytokines during and after IFN-α treatment. We measured serum concentrations of AEA and 2-AG using High Performance Liquid Chromatography with Tandem Mass Spectrometry, and serum concentrations of cytokines using Meso Scale Discovery electrochemiluminescence V-PLEX assay, in 70 patients with HCV infection and 41 healthy subjects. We assessed HCV patients at baseline, IFN-α-treatment weeks (TW) 4 and 24, end of treatment (END) and at six months follow-up (FU). We assessed depression using M.I.N.I. International Neuropsychiatric Interview.We found a different pattern of change in peripheral AEA and 2-AG during and after IFN-α treatment. Whilst 2-AG increased earlier in immunotherapy (TW4), remained elevated throughout treatment, and reduced at six months follow-up (FU), AEA increased later in treatment (TW24) and remained elevated six months post-treatment. We also found that baseline levels of AEA were lower in HCV patients compared with healthy controls, whereas there were no differences in 2-AG levels. Interestingly, AEA, but not 2-AG, was significantly, negatively correlated with interleukin (IL)-2 and IL-17a at six months follow-up. We did not find any difference in both eCBs between patients with and without IFN-α-induced depression, at any time point.Our findings suggest that AEA and 2-AG are involved in different stages of immunoregulation following IFN-α treatment, where AEA might be involved in chronic inflammation. Lack of association between peripheral eCBs and IFN-α-induced depression suggests that different biological mechanisms may underpin inflammation-induced depression compared with classic “psychiatric” depression, or that any changes in the eCB system in depression may not be captured by peripheral AEA and 2-AG.

The endocannabinoid system is affected by a high-fat-diet in rainbow trout

The endocannabinoid system (ECs) is a well known contributor to the hedonic regulation of food intake (FI) in mammals whereas in fish, the knowledge regarding hedonic mechanisms that control FI is limited. Previous studies reported the involvement of ECs in FI regulation in fish since anandamide (AEA) treatment induced enhanced FI and changes of mRNA abundance of appetite-related neuropeptides through cannabinoid receptor 1 (cnr1). However, no previous studies in fish evaluated the impact of palatable food like high-fat diets (HFD) on mechanisms involved in hedonic regulation of FI including the possible involvement of ECs. Therefore, we aimed to evaluate the effect of feeding a HFD on the response of ECs in rainbow trout (Oncorhynchus mykiss). First, we demonstrated a higher intake over 4 days of HFD compared with a control diet (CD). Then, we evaluated the postprandial response (1, 3 and 6 h) of components of the ECs in plasma, hypothalamus, and telencephalon after feeding fish with CD and HFD. The results obtained indicate that the increased FI of HFD occurred along with increased levels of 2-arachidonoylglycerol (2-AG) and AEA in plasma and in brain areas like hypothalamus and telencephalon putatively involved in hedonic regulation of FI in fish. Decreased mRNA abundance of EC receptors like cnr1, gpr55 and trpv1 suggest a feed-back counter-regulatory mechanism in response to the increased levels of EC. Furthermore, the results also suggest that neural activity players associated to FI regulation in mammals as cFOS, γ-Amino butyric acid (GABA) and brain derived neurotrophic factor (BDNF)/neurotrophic receptor tyrosine kinase (NTRK) systems could be involved in the hedonic eating response to a palatable diet in fish.

Colonization with the commensal fungus Candida albicans perturbs the gut-brain axis through dysregulation of endocannabinoid signaling

Anxiety disorders are the most prevalent mental health disorder worldwide, with a lifetime prevalence of 5–7 % of the human population. Although the etiology of anxiety disorders is incompletely understood, one aspect of host health that affects anxiety disorders is the gut-brain axis. Adolescence is a key developmental window in which stress and anxiety disorders are a major health concern. We used adolescent female mice in a gastrointestinal (GI) colonization model to demonstrate that the commensal fungus Candida albicans affects host health via the gut-brain axis. In mice, bacterial members of the gut microbiota can influence the host gut-brain axis, affecting anxiety-like behavior and the hypothalamus-pituitary-adrenal (HPA) axis which produces the stress hormone corticosterone (CORT). Here we showed that mice colonized with C. albicans demonstrated increased anxiety-like behavior and increased basal production of CORT as well as dysregulation of CORT production following acute stress. The HPA axis and anxiety-like behavior are negatively regulated by the endocannabinoid N-arachidonoylethanolamide (AEA). We demonstrated that C. albicans-colonized mice exhibited changes in the endocannabinoidome. Further, increasing AEA levels using the well-characterized fatty acid amide hydrolase (FAAH) inhibitor URB597 was sufficient to reverse both neuroendocrine phenotypes in C. albicans-colonized mice. Thus, a commensal fungus that is a common colonizer of humans had widespread effects on the physiology of its host. To our knowledge, this is the first report of microbial manipulation of the endocannabinoid (eCB) system that resulted in neuroendocrine changes contributing to anxiety-like behavior.

Peripheral deficiency and antiallodynic effects of 2-arachidonoyl glycerol in a mouse model of paclitaxel-induced neuropathic pain

BackgroundModulation of the endocannabinoid system has been shown to alleviate neuropathic pain. The aim of this study was to evaluate if treatment with paclitaxel, a chemotherapeutic agent that induces neuropathic pain, affects endocannabinoid levels at a time when mice develop paclitaxel-induced mechanical allodynia. We also evaluated the peripheral antiallodynic activity of the endocannabinoid 2-arachidonoyl glycerol (2-AG) and an inhibitor of monoacylglycerol lipase (MAGL), an enzyme responsible for 2-AG hydrolysis. MethodsFemale BALB/c mice were treated intraperitoneally with paclitaxel to induce mechanical allodynia. Levels of the endocannabinoids, N-arachidonoylethanolamine (anandamide, AEA), 2-AG, and the N-acylethanolamines (NAEs), N-palmitoylethanolamide (PEA) and N-oleoylethanolamide (OEA), which are structurally-related to AEA, in the brain, spinal cord and paw skin were measured using LC–MS/MS. Protein expression of MAGL in the paw skin was measured using Wes™. The effects of subcutaneous (s.c.) injection of 2-AG and JZL184 (a MAGL inhibitor) into the right hind paw of mice with paclitaxel-induced mechanical allodynia were assessed using the dynamic plantar aesthesiometer. The effects of pretreatment, s.c., into the right hind paw, with cannabinoid type 1 (CB1) receptor antagonist AM251 and CB2 receptor antagonist AM630 on the antiallodynic effects of 2-AG were also evaluated. ResultsThe levels of 2-AG were reduced only in the paw skin of paclitaxel-treated mice, whilst the levels of AEA, PEA and OEA were not significantly altered. There was no change in the expression of MAGL in the paw skin. Administration of 2-AG and JZL184 produced antiallodynic effects against paclitaxel-induced mechanical allodynia in the injected right paw, but did not affect the uninjected left paw. The antiallodynic activity of 2-AG was antagonized by both AM251 and AM630. ConclusionThese results indicate that during paclitaxel-induced mechanical allodynia there is a deficiency of 2-AG in the periphery, but not in the CNS. Increasing 2-AG in the paw by local administration of 2-AG or a MAGL inhibitor, alleviates mechanical allodynia in a CB1 and CB2 receptor-dependent manner.

The association of circulating endocannabinoids with appetite regulatory substances in obese women

BackgroundsEndocannabinoids especially anadamide (AEA) and 2‑arachidonoylglycerol (2-AG) together with appetite modulators have recently been of great importance in body weight regulation and obesity incidence. The present study was carried out to investigate AEA and 2-AG levels and their association with leptin, insulin, orexin – A, and anthropometric indices in obese women. MethodsThe demographic and anthropometric data of 180 overweight/ obese women with mean age 34.2±8.27 years old, and mean BMI 32.54±3.73kg/m2 were evaluated. The plasma levels of anadamide and 2‑arachidonoylglycerol levels and also serum levels of leptin, insulin and orexin- A concentrations were measured. Pearson and spearmen correlation tests along with hieratical regression test were used to assess the association of endocannabinoids levels with anthropometric indices and appetite modulators. ResultsSignificant correlations were revealed between AEA and 2-AG with leptin, BMI, waist circumference (WC) and body fat percent (BF%) (P<0.001). 2-AG levels correlated positively with mean insulin levels (P<0.001). Neither AEA nor 2-AG correlated significantly with serum orexin - A levels. Leptin, insulin, BMI, WC, and BF% were significant independent predictors of AEA and 2-AG in the hierarchical regression model (P<.001) and explained 65% and 68% of variance in AEA and 2-AG respectively (P<0.001). ConclusionThe findings showed that levels of AEA and 2-AG were associated with BMI, WC, BF%, and leptin and insulin levels. Also, BMI, WC, BF%, leptin and, insulin levels can have predictive value for determining AEA and 2-AG.

CSF Levels of the Endocannabinoid Anandamide are Reduced in Patients with Untreated Narcolepsy Type 1: A Pilot Study.

Endocannabinoids (ECs) modulate both excitatory and inhibitory components in the CNS. There is a growing body of evidence that shows ECs influence both hypothalamic orexinergic and histaminergic neurons involved in narcolepsy physiopathology. Therefore, ECs may influence sleep and sleep-wake cycle. To evaluate EC levels in the CSF of untreated narcoleptic patients to test whether ECs are dysregulated in Narcolepsy Type 1 (NT1) and Type 2 (NT2). We compared CSF Anandamide (AEA), 2-Arachidonoylglycerol (2-AG) and orexin in narcoleptic drug-naïve patients and in a sample of healthy subjects. We compared NT1 (n=6), NT2 (n=6), and healthy controls (n=6). We found significantly reduced AEA levels in NT1 patients compared to both NT2 and controls. No differences were found between AEA levels in NT2 versus controls and between 2-AG levels in all groups, although a trend toward a decrease in NT1 was evident. Finally, the CSF AEA level was related to CSF orexin levels in all subjects. We demonstrated that the EC system is dysregulated in NT1.

Fibronectin induces capacitation-associated events through the endocannabinoid system in bull sperm

Mammalian ejaculated spermatozoa must undergo a series of changes in the female reproductive tract, collectively called capacitation, in order to fertilize the oocyte. We reported that fibronectin (Fn), a glycoprotein from the extracellular matrix, and anandamide (AEA), one of the major members of the endocannabinoid family, are present in the bovine oviductal fluid and regulate bull sperm function. Also, AEA induces bovine sperm capacitation, through CB1 and TRPV1 receptors.In this work, we investigated if Fn induces bovine sperm capacitation thought the activation of the endocannabinoid system in this process. We incubated sperm with Fn (100 μg/ml) and/or capsazepine, a TRPV1 antagonist (0.1 μM) and some events related to sperm capacitation such as LPC-induced acrosome reaction, sperm-release from the oviduct, induction of PKA phosphorylated substrates (pPKAs) and protein tyrosine phosphorylation (pY) and nitric oxide (NO) production were assessed. Also, we studied the activity of fatty acid amide hydrolase (FAAH), the enzyme that degrades AEA. We found that Fn, via α5β1 integrin, induced capacitation-associated events. Also, Fn stimulated signaling pathways associated to capacitation as cAMP/PKA and NO/NO synthase. Moreover, Fn decreased the FAAH activity and this correlated with sperm capacitation. Capsazepine reversed fibronectin-induced capacitation, and pPKAs and NO levels. The incubation of spermatozoa with R-methanandamide (1.4 nM), a stable analogue of AEA, increased cAMP and pPKAs levels. The presence of H89 (50 μM) or KT5720 (100 nM) (PKA inhibitors) prevented AEA-induced capacitation. In addition, R-methanandamide and capsaicin (0.01 μM), a TRPV1 agonist, increased NO production via the PKA pathway. These results indicate that Fn, through α5β1, supports capacitation in bovine spermatozoa. This effect is dependent on the activation of TRPV1 through cAMP/PKA and NO signaling pathways. We propose that Fn could be considered as a new agent that promotes sperm capacitation in bull sperm. Our findings contribute to better understand the significance of Fn signaling in the capacitating events that lead to successful fertilization and embryo development in mammals including humans.

The effects of fatty acid amide hydrolase and monoacylglycerol lipase inhibitor treatments on lipopolysaccharide-induced airway inflammation in mice

Cannabinoids and the endocannabinoid system significantly contributes to the airway inflammation. Fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) are two main enzymes responsible for the metabolism of the endocannabinoids anandamide (AEA) and 2-arachydonoyl glycerol (2-AG), respectively. In the present study, we aimed to investigate the effects of local and systemic FAAH and MAGL inhibitor treatments in experimental airway inflammation and tracheal hyperreactivity in mice.Airway inflammation was induced by intranasal (i.n.) lipopolysaccharide (LPS) application (60 μl; 0,1 mg/ml in PBS) to mice and the control group received PBS. Systemic (intraperitoneal (i.p.)) or local (i.n.) FAAH inhibitor URB597 and MAGL inhibitor JZL184 treatments were administered 1h before LPS/PBS application. Fourty 8 h after LPS/PBS application, tracheas were removed to assess airway reactivity, and the lungs and bronchoalveolar lavage (BAL) fluids were isolated for histopathological evaluation, cytokine and endocannabinoid measurements.LPS application lead to an increase in 5-hydroxytryptamine (5-HT) contractions in isolated tracheal rings while carbachol contractions remained unchanged. The increased 5-HT contractions were prevented by both systemic and local URB597 and JZL184 treatments. Systemic treatment with URB597 and JZL184, and local treatment with JZL184 reduced peribronchial and paranchymal inflammation in the LPS group while i.n. application of URB597 worsened the inflammation in the lungs. Systemic URB597 treatment increased lung AEA level whereas it had no effect on 2-AG level. However, JZL184 treatment increased 2-AG level by either systemic or local application, and also elevated AEA level. Inflammation-induced increase in neutrophil numbers was only prevented by systemic URB597 treatment. However, both URB597 and JZL184 treatments abolished the increased TNF-α level either they are administered systemically or locally.These results indicate that FAAH and MAGL inhibition may have a protective effect in airway inflammation and airway hyperreactivity, and therefore their therapeutic potential for airway diseases should be further investigated.

SAT-LB100 Central and Peripheral Endocannabinoid Levels in Obese Versus Lean Women

The endocannabinoid system (ECS) is thought to be involved in obesity because its activation increases appetite and weight gain (Pagotto et al 2006). The ECS is hyperactivated in the hypothalamus of obese mice, and peripheral overactivation has been observed in humans; circulating 2-arachidonoyl glycerol (2-AG) levels positively correlated with body fat, visceral fat and fasting glucose (Osei-Hyiaman et al 2005) (Bluher et al 2006) (Motaghedi and McGraw 2008) (Cavuoto et al 2007) (Artmann et al 2008) (Bermudez-Silva 2009). The aim of this study was to evaluate whether differential activation of the peripheral versus the central ECS occurred in humans and to test the hypothesis that the ECS is hyperactivated in the human central nervous system (CNS).Cerebral spinal fluid (CSF) and blood samples were collected from 13 obese and 11 lean control women to measure 2-AG and anandamide (AEA) levels.AEA levels were higher in the plasma of obese women (obese: 4.03 ± 0.91 pmol/mL, N=13; lean: 1.84 ± 0.21 pmol/mL, N=10; p<0.05) but were lower in the CSF of obese women. The plasma/CSF ratio was 41.58 ± 5.78 (N=10) in lean women and 103.0 ± 37.36 (N=6) in obese women (p=0.054). There were no correlations between plasma and CSF AEA levels or with any biochemical parameter. The 2-AG analysis was not possible because of technical problems.Our data suggested that in human obesity, the peripheral ECS may be more active than the central ECS. Indeed, the system appeared to be suppressed in the CNS of obese women. Therefore, the peripheral activation of the ECS may be more relevant for obesity. Keywords: Anandamide, 2-arachidonoyl glycerol, obesity, endocannabinoid, endocannabinoid system, cb1 receptor.

Effect of flaxseed oil supplementation on the erythrocyte membrane fatty acid composition and endocannabinoid system modulation in patients with coronary artery disease: a double-blind randomized controlled trial

BackgroundThe endocannabinoid system (ECS) overactivation, associated with increased inflammatory process, may act as a risk factor for coronary artery disease (CAD). Dietary fat may influence the ECS tone. The aim of the present study was to investigate the effect of flaxseed oil on the erythrocyte membrane fatty acid profile and ECS activity by the measurement of serum N-arachydonoil ethanolamine (AEA) and cannabinoid receptor type-1 (CB1), cannabinoid receptor type-2 (CB2), and fatty acid amide hydrolase (FAAH) mRNA expression.MethodsThis clinical trial was performed on 44 patients with CAD. The intervention group received 1.5% fat milk supplemented with flaxseed oil (containing 2.5 g α-linolenic acid or ALA), while the placebo group received 1.5% fat milk for 10 weeks. The fatty acid profile of erythrocyte membrane phospholipids was measured by gas chromatography. The AEA level was determined using an ELISA kit, and real-time PCR was performed to measure CB1, CB2, and FAAH mRNA expression pre- and post-intervention.ResultsFlaxseed oil supplementation resulted in a significant increase in the ALA content and a significant reduction in linoleic acid (LA) content of membrane phospholipids, compared to the placebo group (MD = − 0.35 and 2.89, respectively; P < 0.05). The within group analysis showed that flaxseed oil supplementation caused a significant reduction in both LA and arachidonic acid (MD = − 4.84 and − 4.03, respectively; P < 0.05) and an elevation in the ALA (MD = 0.37, P < 0.001) content of membrane phospholipids compared with the baseline. In the intervention group, a marked reduction was observed in the serum AEA level after 10 weeks of intervention, compared with the placebo group (MD = 0.64, P = 0.016). Changes in CB2 mRNA expression in the flaxseed oil group were significant (fold change = 1.30, P = 0.003), compared with the placebo group.ConclusionFlaxseed oil supplementation could attenuate the ECS tone by decreasing the AEA level and increasing CB2 mRNA expression. Therefore, flaxseed oil may be considered a promising agent with cardioprotective properties.

Modulatory effect of methanandamide on gastric vagal afferent satiety signals depends on nutritional status.

Gastric vagal afferent responses to tension are dampened in high fat diet-induced obesity. Endocannabinoids are known to dose-dependently inhibit and excite gastric vagal afferents but their effect on gastric vagal afferents in diet-induced obesity are unknown. In individual gastric vagal afferent neurons of diet-induced obese mice the co-expression of components of the endocannabinoid system, including CB1, GHSR, TRPV1 and FAAH, was increased compared with lean mice. In high fat diet-induced obese mice, methanandamide only inhibited gastric vagal afferent responses to tension, possibly due to the observed change in the balance of receptors, hormones and breakdown enzymes in this system. Collectively, these data suggest that endocannabinoid signalling, by gastric vagal afferents, is altered in diet-induced obesity which may impact satiety and gastrointestinal function. Gastric vagal afferents (GVAs) play a role in appetite regulation. The endocannabinoid anandamide (AEA) dose-dependently inhibits and excites tension-sensitive GVAs. However, it is also known that high fat diet (HFD) feeding alters GVA responses to stretch. The aim of this study was to determine the role of AEA in GVA signalling in lean and HFD-induced obese mice. Male C57BL/6 mice were fed (12weeks) a standard laboratory diet (SLD) or HFD. Protein and mRNA expression of components of the cannabinoid system was determined in individual GVA cell bodies and the gastric mucosa. An in vitro GVA preparation was used to assess the effect of methanandamide (mAEA) on tension-sensitive GVAs and the second messenger pathways involved. In individual GVA cell bodies, cannabinoid 1 (CB1) and ghrelin (GHSR) receptor mRNA was higher in HFD mice than SLD mice. Conversely, gastric mucosal AEA and ghrelin protein levels were lower in HFD mice than SLD mice. In SLD mice, mAEA exerted dose-dependent inhibitory and excitatory effects on tension-sensitive GVAs. Only an inhibitory effect of mAEA was observed in HFD mice. The excitatory effect of mAEA was dependent on CB1, transient receptor potential vanilloid 1 (TRPV1) and the protein kinase C. Conversely, the inhibitory effect was dependent on CB1, growth hormone secretagogue receptor, TRPV1 and the protein kinase A. Endocannabinoids, acting through CB1 and TRPV1, have a pivotal role in modulating GVA satiety signals depending on the second messenger pathway utilised. In HFD mice only an inhibitory effect was observed. These changes may contribute to the development and/or maintenance of obesity.

The fundamental role of the endocannabinoid system in endometrium and placenta: implications in pathophysiological aspects of uterine and pregnancy disorders.

BACKGROUNDThe endocannabinoid system (ECS) consists of the cannabinoid receptors CB1 and CB2, the main endocannabinoids anandamide (AEA) and 2-arachidonoylglycerol (2-AG) and their metabolic enzymes N-acylphosphatidylethanolamine-specific phospholipase D, fatty acid amide hydrolase, diacylglycerol lipase and monoacylglycerol lipase. This system is involved in the modulation of essential physiological processes. Its role in the reproductive system has become significantly important in recent years, given its major role in events such as gametogenesis, decidualisation, implantation and placentation.OBJECTIVE AND RATIONALEIn this paper, we review the literature and summarize the role of the ECS elements in reproduction and their potential as early markers for diagnosis of reproductive disorders or as pharmacological targets for treatment.SEARCH METHODSOriginal research and review papers published from 1964 to June 2019 were selected in terms of relevance, reliability and quality by searching PubMed, MEDLINE and Web of Science, using the following search terms: endocannabinoid system and endometriosis; endocannabinoid system and ectopic pregnancy; endocannabinoid system and miscarriage; endocannabinoid system and pre-eclampsia; endocannabinoid system and endometrial cancer; endocannabinoid system and reproduction; endocannabinoid, endometrium; placenta; N-acylethanolamines; anandamide; 2-arachidonoylglycerol; and cannabinoids.OUTCOMESThis review demonstrates relevant information concerning ECS alterations in endometriosis, ectopic pregnancy, miscarriage, pre-eclampsia and endometrial cancer. We highlight the importance of the endocannabinoids in endometrial and placental physiology and pathophysiology, from studies in vitro and in vivo and in clinical observations. The most studied of the endogenous cannabinoids is AEA. The levels of AEA were increased in plasma of patients with endometriosis and miscarriage, as well as in the fallopian tube of women with ectopic pregnancy and in endometrial biopsies of endometrial cancer. Changes in the pattern of expression of the cannabinoid receptor CB1 were also observed in endometrial biopsies of endometriosis, fallopian tube and decidua of patients with ectopic pregnancy and pre-eclamptic placenta. Moreover, alterations in CB2 expression have been reported in association with endometrial cancer. In general, studies on the cannabinoid signalling through CB2 and on the biological activities of the other major endocannabinoid, namely 2-AG, as well as its metabolic enzymes are scarce and avidly required.WIDER IMPLICATIONSThe pathophysiological mechanisms involved in the described endometrial and placental pathologies are still unclear and lack the means for an early diagnosis. Based on current evidence, though alterations in ECS are demonstrated at tissue level, it is difficult to associate plasmatic changes in AEA with specific endometrial and placental diseases. Thus, pairing alterations in AEA levels with 2-AG and/or other endocannabinoid-like molecules may provide more accurate and early diagnoses. In addition, patients may benefit from new therapies that target the ECS and endocannabinoid signalling.

Cerebrospinal fluid endocannabinoid levels in Gilles de la Tourette syndrome

Gilles de la Tourette syndrome (TS) is a complex neurodevelopmental disorder characterized by the presence of motor and vocal tics as well as psychiatric comorbidities such as attention-deficit/hyperactivity disorder (ADHD), obsessive-compulsive disorder (OCD), depression, and anxiety. The underlying cause of the disease is still unknown, but several lines of evidence suggest a paramount role of the dopaminergic system. Based on the clinical observation that cannabis-based medicine including cannabis and delta-9-tetrahydrocannabinol (THC, dronabinol) may improve TS, alternatively, an involvement of the endocannabinoid system (ECS) has been suggested. In this study we measured cerebrospinal fluid (CSF) levels of the two most important endocannabinoids “N”-arachidonoylethanolamine (AEA, anandamide) and 2-arachidonoylglycerol (2-AG), the endocannabinoid-like molecule palmitoyl ethanolamide (PEA), and the lipid arachidonic acid (AA) in a sample of adult patients with TS (n = 20) compared with controls (n = 19) using liquid-liquid lipid extraction and simultaneous quantification by liquid chromatography multiple reaction monitoring (LC/MRM). CSF levels of AEA (p = 0.0018), 2-AG (p = 0.0003), PEA (p = 0.02), and AA (p < 0.0001) were significantly increased in TS compared with controls. Levels of 2-AG correlated with the severity of comorbid ADHD (p < 0.01). This is the first study, demonstrating alterations in the ECS suggesting an involvement of this system in the pathophysiology of TS. It can be speculated that elevated endocannabinoid levels either represent secondary changes in order to compensate for alterations in other neurotransmitter systems such as the dopaminergic system, are simply an epiphenomenon or, alternatively, represent the primary cause of TS.