The endocannabinoid system is a key modulator of memory consolidation for aversive experiences. We recently found that the fatty acid amide hydrolase (FAAH) inhibitor URB597, which increases anandamide levels by inhibiting its hydrolysis, facilitates memory consolidation through a concurrent activation of both cannabinoid receptor type 1 (CB1) and <em>2</em> (CB<em>2</em>). Here, we investigated the role played on memory consolidation by the other major endocannabinoid, <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG). To this aim, we tested the effects of pharmacological inhibition of monoacylglycerol lipase (MAGL) through systemic administration of the MAGL inhibitor JZL184 to rats immediately after training of the inhibitory avoidance task. Pharmacological enhancement of <em>2</em>-AG tone facilitated memory consolidation through activation of CB<em>2</em> receptor signaling. Moreover, we found that increased <em>2</em>-AG signaling prevented the activation of the mammalian target of rapamycin (mTOR) signaling pathway in the hippocampus through a CB<em>2</em>-dependent mechanism. Our results identify a fundamental role for <em>2</em>-AG and CB<em>2</em> receptors in the modulation of memory consolidation for aversive experiences.

UNASSIGNED

Myocardial infarction leads to an enhanced release of endocannabinoids and a massive accumulation of neutrophils and monocytes within the ischemic myocardium. These myeloid cells originate from hematopoietic precursors in the bone marrow and are rapidly mobilized in response to myocardial infarction. We aimed to determine whether endocannabinoid signaling is involved in myeloid cell mobilization and cardiac recruitment after ischemia onset.

UNASSIGNED

Intravenous administration of endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> into wildtype C57BL6 mice induced a rapid increase of blood neutrophil and monocyte counts as measured by flow cytometry. This effect was blunted when using cannabinoid receptor <em>2</em> knockout mice. In response to myocardial infarction induced in wildtype mice, the lipidomic analysis revealed significantly elevated plasma and cardiac levels of the endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> <em>2</em>4 h after infarction, but no changes in anandamide, palmitoylethanolamide and oleoylethanolamide. This was a consequence of an increased expression of <em>2</em>-<em>arachidonoylglycerol</em> synthesizing enzyme diacylglycerol lipase and a decrease of metabolizing enzyme monoacylglycerol lipase in infarcted hearts, as determined by quantitative RT-PCR analysis. The opposite mRNA expression pattern was observed in bone marrow. Pharmacological blockade of monoacylglycerol lipase with JZL184 and thus increased systemic <em>2</em>-<em>arachidonoylglycerol</em> levels in wildtype mice subjected to myocardial infarction resulted in elevated cardiac CXCL1, CXCL<em>2</em> and MMP9 protein levels as well as higher cardiac neutrophil and monocyte counts <em>2</em>4 h after infarction compared to vehicle-treated mice. Increased post-myocardial infarction inflammation in these mice led to an increased infarct size, an impaired ventricular scar formation assessed by histology and a worsened cardiac function in echocardiography evaluations up to <em>2</em>1 days. Likewise, JZL184-administration in a myocardial ischemia-reperfusion model increased cardiac myeloid cell recruitment and resulted in a larger fibrotic scar size.

UNASSIGNED

These findings suggest that changes in endocannabinoid gradients due to altered tissue levels contribute to myeloid cell recruitment from the bone marrow to the infarcted heart, with crucial consequences on cardiac healing and function.

N-Arachidonoylethanolamine (anandamide) and <em>2</em>-<em>arachidonoylglycerol</em> are the best characterized endocannabinoids. Their biological activity is subjected to metabolic control whereby a dynamic equilibrium among biosynthetic, catabolic, and oxidative pathways drives their intracellular concentrations. In particular, lipoxygenases can generate hydroperoxy derivatives of endocannabinoids, endowed with distinct activities within cells. The in vivo interaction between lipoxygenases and endocannabinoids is likely to occur within cell membranes; thus, we sought to ascertain whether a prototypical enzyme like soybean (Glycine max) 15-lipoxygenase-1 is able to oxygenate endocannabinoids embedded in synthetic vesicles and how these substances could affect the binding ability of the enzyme to different lipid bilayers. We show that (i) embedded endocannabinoids increase membrane fluidity; (ii) 15-lipoxygenase-1 preferentially binds to endocannabinoid-containing bilayers; and that (iii) 15-lipoxygenase-1 oxidizes embedded endocannabinoids and thus reduces fluidity and local hydration of membrane lipids. Together, the present findings reveal further complexity in the regulation of endocannabinoid signaling within the central nervous system, disclosing novel control by oxidative pathways.

α/β-Hydrolase domain 6 (ABHD6) is one of the major enzymes for endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) hydrolysis in microglia cells. Our recent studies have shown that a selective ABHD6 inhibitor WWL70 has anti-inflammatory and neuroprotective effects in animal models of traumatic brain injury and multiple sclerosis. However, the role of ABHD6 in the neuroinflammatory response and the mechanisms by which WWL70 suppresses inflammation has not yet been elucidated in reactive microglia.

The hydrolytic activity and the levels of <em>2</em>-AG in BV<em>2</em> cells were measured by radioactivity assay and liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). The expression of cyclooxygenase-<em>2</em> (COX-<em>2</em>) and prostaglandin E<em>2</em> (PGE<em>2</em>) synthases in microglia treated with lipopolysaccharide (LPS) with/without WWL70 was determined by western blot and quantitative RT-PCR. The conversion of <em>2</em>-AG to PGE<em>2</em> or PGE<em>2</em>-glyceryl ester (PGE<em>2</em>-G) was assessed by enzyme-linked immunoassay (EIA) or LC-MS/MS. The involvement of ABHD6 in PGE<em>2</em> production was assessed using pharmacological inhibitors and small interfering RNA (siRNA). The effect of WWL70 on PGE<em>2</em> biosynthesis activity in the microsome fraction from BV<em>2</em> cells and experimental autoimmune encephalopathy (EAE) mouse brain was also examined.

We found that WWL70 suppressed PGE<em>2</em> production in LPS-activated microglia via cannabinoid receptor-independent mechanisms, although intracellular levels of <em>2</em>-AG were elevated by WWL70 treatment. This reduction was not attributable to WWL70 inhibition of ABHD6, given the fact that downregulation of ABHD6 by siRNA or use of KT18<em>2</em>, an alternative ABHD6 inhibitor failed to suppress PGE<em>2</em> production. WWL70 attenuated the expression of COX-<em>2</em> and PGES-1/<em>2</em> leading to the downregulation of the biosynthetic pathways of PGE<em>2</em> and PGE<em>2</em>-G. Moreover, PGE<em>2</em> production from arachidonic acid was reduced in the microsome fraction, indicating that WWL70 also targets PGE<em>2</em> biosynthetic enzymes, which are likely to contribute to the therapeutic mechanisms of WWL70 in the EAE mouse model.

WWL70 is an anti-inflammatory therapeutic agent capable of inhibiting PGE<em>2</em> and PGE<em>2</em>-G production, primarily due to its reduction of COX-<em>2</em> and microsomal PGES-1/<em>2</em> expression and their PGE<em>2</em> biosynthesis activity in microglia cells, as well as in the EAE mouse brain.

OBJECTIVE

Cannabinoids are derivatives that are either compounds occurring naturally in the plant, Cannabis sativa or synthetic analogs of these molecules. The first and most widely investigated of the cannabinoids is Δ9-tetrahydrocannabinol (Δ9-THC), which is the main psychotropic constituent of cannabis and undergoes significant binding to cannabinoid receptors. These cannabinoid receptors are seven-transmembrane receptors that received their name from the fact that they respond to cannabinoid compounds, including Δ9-THC. The cannabinoid receptors have been described in rat, human and mouse brains and they have been named the CB1 and CB<em>2</em> cannabinoid receptors. Later, an endogenous molecule that exerts pharmacological effects similar to those described by Δ9-THC and binds to the cannabinoid receptors was discovered. This molecule, named anandamide, was the first of five endogenous cannabinoid receptor agonists described to date in the mammalian brain and other tissues. Of these endogenous cannabinoids or endocannabinoids, the most thoroughly investigated to date have been anandamide and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG). Over the years, a significant number of articles have been published in the field of endogenous cannabinoids, suggesting a modulatory profile in multiple neurobiological roles of endocannabinoids. The general consensus accepts that the endogenous cannabinoid system includes natural ligands (such as anandamide and <em>2</em>- AG), receptors (CB1 and CB<em>2</em>), and the main enzymes responsible for the hydrolysis of anandamide and <em>2</em>-AG (fatty acid amide hydrolase [FAAH] and monoacylglycerol lipase [MAGL], respectively) as well as the anandamide membrane transporter (AMT). To date, diverse pieces of evidence have shown that the endocannabinoid system controls multiple functions such as feeding, pain, learning and memory and has been linked with various disturbances, such as Parkinson´s disease. Among the modulatory properties of the endocannabinoid system, current data indicate that the sleep-wake cycle is under the influence of endocannabinoids since the blocking of the CB1 cannabinoid receptor or the pharmacological inhibition of FAAH activity promotes wakefulness, whereas the obstruction of AMT function enhances sleep. However, no solid evidence is available regarding the role of the endocannabinoid system in an unquestionable emotional component of the sleep: Dream activity. Since dreaming is a mental activity that occurs during sleep (characterized by emotions, sensory perceptions, and bizarre components) and the endocannabinoid system modulates neurobiological processes involving consciousness, such as learning and memory, attention, pain perception, emotions and sleep, it is acceptable to hypothesize that the endocannabinoid system might be modulating dream activity. In this regard, an accumulative body of evidence in human and animal models has been reported regarding the role of the endocannabinoid system in the control of emotional states and dreams. Moreover, preliminary studies in humans have indicated that treatment with cannabinoids may decrease post-traumatic stress disorder symptoms, including nightmares.

CONCLUSIONS

Thus, based on a review of the literature available in PubMed, this article hypothesizes a conceptual framework within which the endocannabinoid system might influence the generation of dream experiences.

Orexin 1 (OX-1R) and cannabinoid receptor (CB1R) belong to the superfamily of G-protein-coupled receptors (GPCRs) and are mostly coupled to Gq and Gi/o proteins, respectively. In vitro studies in host cells over-expressing OX-1R and CB1R revealed a functional interaction between these receptors, through either their ability to form heteromers or the property for OX-1R to trigger the biosynthesis of <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), an endogenous CB1R ligand. Since: i) OX-1R and CB1R co-espression has been described at postsynaptc sites in hypothalamic circuits involved the regulation of energy homeostasis, and ii) increased orexin-A (OX-A) and <em>2</em>-AG levels occur in hypothalamic neurons during obesity, we sought here to investigate the OX-1R/CB1R interaction in embryonic mouse hypothalamic NPY/AgRP mHypoE-N41 neurons which express, constitutively, both receptors. Treatment of mHypoE-N41 cells with OX-A (0.1-0.3μM), but not with the selective CB1R agonist, arachidonyl-<em>2</em>-chloroethylamide (ACEA; 0.1-0.3μM), transiently elevated [Ca(<em>2</em>+)]i. Incubation with a subeffective dose of OX-A (0.1μM)+ACEA (0.1μM) led to stronger and longer lasting elevation of [Ca(<em>2</em>+)]i, antagonized by OX-1R or CB1R antagonism with SB-334867 or AM<em>2</em>51, respectively. FRET and co-immunoprecipitation experiments showed the formation of OX-1R/CB1R heteromers after incubation with OX-A (0.<em>2</em>μM), or OX-A (0.1μM)+ACEA (0.1μM), but not after ACEA (0.<em>2</em>μM), in a manner antagonized by SB-334867 or AM<em>2</em>51. OX-A (0.<em>2</em>μM) or OX-A (0.1μM)+ACEA (0.1μM) also led to <em>2</em>-AG biosynthesis. Finally, a stronger activation of ERK1/<em>2</em>(Thr<em>2</em>0<em>2</em>/185) phosphorylation in comparison to basal or each agonist alone (0.1-0.<em>2</em>μM), was induced by incubation with OX-A (0.1μM)+ACEA (0.1μM), again in a manner prevented by OX-1R or CB1R antagonism. We suggest that OX-A, alone at effective concentrations on [Ca(<em>2</em>+)]i, or in combination with ACEA, at subeffective concentrations, triggers intracellular signaling events via the formation of OX-1R/CB1R heteromers and an autocrine loop mediated by <em>2</em>-AG.

The effects of 1-stearoyl,<em>2</em>-sn-<em>arachidonoylglycerol</em> (SAG) and the antimalarial drug chloroquine on lipid bilayer structure were studied by <em>2</em>H-NMR spectroscopy. Model lipid systems were established with compositions similar to those of normal human erythrocytes, malaria-infected erythrocytes, or malaria parasite membranes. The <em>2</em>H-NMR spectra of the membranes formed from the lipids extracted from normal human erythrocytes were similar to those obtained using the corresponding lipid mixtures. The order parameters of the model "infected" and model "parasite" membranes were reduced markedly relative to that of normal erythrocytes. Addition of SAG induced formation of non-bilayer lipid phases in all lipid systems. Only a small decrease in the order parameters of the acyl side chains of the phosphatidylserine, but not of the phosphatidylcholine component of the lipid membranes, was observed upon the addition of chloroquine. A dramatic effect was observed upon the addition of chloroquine to the SAG-containing membranes: this antimalarial almost totally abolished the formation of SAG-induced non-bilayer lipid phases. Since SAG, endogenously formed in erythrocyte membranes, is a potent activator of phospholipase A<em>2</em>, this membrane-stabilizing action of chloroquine may partially account for the phospholipase A<em>2</em>-inhibiting properties of this drug, and, consequently, for both its therapeutic and toxic modes of action.

The endocannabinoid system serves many physiological roles, including in the regulation of energy balance, food reward, and voluntary locomotion. Signaling at the cannabinoid type 1 receptor has been specifically implicated in motivation for rodent voluntary exercise on wheels. We studied four replicate lines of high runner (HR) mice that have been selectively bred for 81 generations based on average number of wheel revolutions on days five and six of a six-day period of wheel access. Four additional replicate lines are bred without regard to wheel running, and serve as controls (C) for random genetic effects that may cause divergence among lines. On average, mice from HR lines voluntarily run on wheels three times more than C mice on a daily basis. We tested the general hypothesis that circulating levels of endocannabinoids (i.e., <em>2</em>-<em>arachidonoylglycerol</em> [<em>2</em>-AG] and anandamide [AEA]) differ between HR and C mice in a sex-specific manner. Fifty male and 50 female mice were allowed access to wheels for six days, while another 50 males and 50 females were kept without access to wheels (half HR, half C for all groups). Blood was collected by cardiac puncture during the time of peak running on the sixth night of wheel access or no wheel access, and later analyzed for <em>2</em>-AG and AEA content by ultra-performance liquid chromatography coupled to tandem mass spectrometry. We observed a significant three-way interaction among sex, linetype, and wheel access for <em>2</em>-AG concentrations, with females generally having lower levels than males and wheel access lowering <em>2</em>-AG levels in some but not all subgroups. The number of wheel revolutions in the minutes or hours immediately prior to sampling did not quantitatively predict plasma <em>2</em>-AG levels within groups. We also observed a trend for a linetype-by-wheel access interaction for AEA levels, with wheel access lowering plasma concentrations of AEA in HR mice, while raising them in C mice. In addition, females tended to have higher AEA concentrations than males. For mice housed with wheels, the amount of running during the 30min before sampling was a significant positive predictor of plasma AEA within groups, and HR mice had significantly lower levels of AEA than C mice. Our results suggest that voluntary exercise alters circulating levels of endocannabinoids, and further demonstrate that selective breeding for voluntary exercise is associated with evolutionary changes in the endocannabinoid system.

The endocannabinoids anandamide (AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), as well as the related acylethanolamide oleoylethanolamide (OEA), have been implicated in energy expenditure (EE) regulation and metabolic diseases. Muscle (fat-free mass) and fat (fat mass) are metabolically active compartments and main determinants of EE.

To assess whether human muscle, adipose, and plasma endocannabinoids correlate with EE.

Muscle, adipose, and plasma AEA, <em>2</em>-AG, and OEA concentrations were measured via liquid chromatography-mass spectrometry. EE was assessed by indirect whole-room calorimetry.

Clinical trial.

Obese/overweight Native Americans of full (n = 35) and at least half (n = <em>2</em>1) Southwestern heritage.

Twenty-four-hour EE, sleeping EE (SLEEP), resting EE (REE), respiratory quotient (RQ), and macronutrient oxidation.

In full Natives, muscle AEA concentration correlated with SLEEP (r = -0.65, P = 0.004) and REE (r = -0.53, P = 0.0<em>2</em>). Muscle <em>2</em>-AG was associated with SLEEP (r = -0.75, P = 0.0003). Adipose OEA concentration correlated with RQ (r = -0.47, P = 0.04) and lipid oxidation (r = 0.51, P = 0.03). Plasma OEA concentration was associated with SLEEP (r = -0.5<em>2</em>, P = 0.04). After adjustment for major determinants, these lipids explained nearly <em>2</em>0% of the additional variance of the respective measure. Similarly, in Native Americans of at least half Southwestern heritage, investigated lipids correlated with EE measures.

Endocannabinoids in metabolically relevant peripheral tissues explained a large part of EE variation and may be involved in regulating EE. Dysregulation of peripheral endocannabinoids may predispose people to metabolic diseases via an effect on EE and lipid oxidation.

We investigated the hypothesis that the endocannabinoidome (eCBome), an extension of the endocannabinoid (eCB) signaling system with important functions in the CNS, may play a role in the microbiota-gut-brain axis. Using LC-MS/MS and qPCR arrays we profiled the brain eCBome of juvenile (4 weeks) and adult (13 weeks) male and female germ-free (GF) mice, which are raised in sterile conditions and virtually devoid of microbiota, present neurophysiological deficits, and were found recently to exhibit a strongly altered gut eCBome in comparison to conventionally raised age/sex-matched controls. The causal effect of the gut microbiome on the eCBome was investigated through the re-introduction into adult male GF mice of a functional gut microbiota by fecal microbiota transfer (FMT). The concentrations of the eCB, <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), and its <em>2</em>-monoacylglycerol congeners, were significantly reduced in the brain, but not in the hypothalamus, of both juvenile and adult male and adult female GF mice. FMT rendered these decreases non-statistically significant. The eCB, anandamide (AEA), and its congener N-acylethanolamines (NAEs), were instead increased in the brain of adult female GF mice. Saturated fatty acid-containing NAEs were decreased in adult male GF mouse hypothalamus in a manner not reversed by FMT. Only few changes were observed in the expression of eCBome enzymes and receptors. Our data open the possibility that altered eCBome signaling may underlie some of the brain dysfunctions typical of GF mice.

Keywords: Endocannabinoidome; Endocannabinoids; Fecal microbiota transfer; Gut-brain axis; Microbiome; Microbiota.

Bone is a complex tissue with unique properties such as high strength and regeneration capabilities while carrying out multiple functions. Bone regeneration occurs both in physiological situations (bone turnover) and in pathological situations (e.g. fractures), being performed by osteoblasts and osteoclasts. If this process is inadequate, fracture nonunion or aseptic loosening of implants occurs and requires a complex treatment. Exogenous factors are currently used to increase bone regeneration process when needed, such as bisphosphonates and vitamin D, but limitations do exist. Cannabinoid system has been shown to have positive effects on bone metabolism. Cannabinoids at bone level mainly act on two receptors called CB-1 and CB-<em>2</em>, but GPR55, GPR119, TPRV1, TPRV4 receptors may also be involved. The CB-<em>2</em> receptors are found in bone cells at higher levels compared to other receptors. Endocannabinods represented by anandamide and <em>2</em>-<em>arachidonoylglycerol</em>, can stimulate osteoblast formation, bone formation and osteoclast activity. CB-<em>2</em> agonists including HU-308, HU-433, JWH133, and JWH015 can stimulate osteoblast proliferation and activity, while CB-<em>2</em> antagonists such as AM630 and SR1445<em>2</em>8 can inhibit osteoclast differentiation and function. CB-1 antagonist AM<em>2</em>51 has been shown to inhibit osteoclast differentiation and activity, while GPR55 antagonist cannabidiol increases osteoblast activity and decreases osteoclast function. An optimal correlation of dose, duration, moment of action, and affinity can lead to an increased bone regeneration capacity, with important benefits in many pathological situations which involve bone tissue. As adverse reactions of cannabinoids have not been described in patients under controlled medication, cannabinoids can represent future treatment for bone regeneration.

The two endocannabinoids, anandamide (AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), play independent and nonredundant roles in the body. This makes the development of both selective and dual inhibitors of their inactivation an important priority. In this work we report a new series of inhibitors of monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH). Among them, (±)-oxiran-<em>2</em>-ylmethyl 6-(1,1'-biphenyl-4-yl)hexanoate (8) and (<em>2</em>R)-(-)-oxiran-<em>2</em>-ylmethyl(4-benzylphenyl)acetate (30) stand out as potent inhibitors of human recombinant MAGL (IC(50) (8) = 4.1 μM; IC(50) (30) = <em>2</em>.4 μM), rat brain monoacylglycerol hydrolysis (IC(50) (8) = 1.8 μM; IC(50) (30) = 0.68 μM), and rat brain FAAH (IC(50) (8) = 5.1 μM; IC(50) (30) = 0.<em>2</em>9 μM). Importantly, and in contrast to the other previously described MAGL inhibitors, these compounds behave as reversible inhibitors either of competitive (8) or noncompetitive nature (30). Hence, they could be useful to explore the therapeutic potential of reversible MAGL inhibitors.

We investigated the effects of S-777469 (1-[[6-Ethyl-1-[4-fluorobenzyl]-5-methyl-<em>2</em>-oxo-1, <em>2</em>-dihydropyridine-3-carbonyl]amino]-cyclohexanecarboxylic acid), a novel cannabinoid type <em>2</em> receptor (CB<em>2</em>) agonist, on 1-fluoro-<em>2</em>,4-dinitrobenzene (DNFB)-induced ear inflammation and mite antigen-induced dermatitis in mice. The oral administration of S-777469 significantly suppressed DNFB-induced ear swelling in a dose-dependent manner. In addition, S-777469 significantly alleviated mite antigen-induced atopic dermatitis-like skin lesions in NC/Nga mice. A histological analysis revealed that S-777469 significantly reduced the epidermal thickness and the number of mast cells infiltrating skin lesions. We demonstrated that S-777469 inhibited mite antigen-induced eosinophil accumulation in skin lesions and an endogenous CB<em>2</em> ligand, <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG)-induced eosinophil migration in vitro. Moreover, we confirmed that <em>2</em>-AG levels significantly increased in skin lesions of mite antigen-induced dermatitis model. Together, these results suggest that S-777469 inhibits skin inflammation in mice by blocking the activities of <em>2</em>-AG.

OBJECTIVE

Endocannabinoids (ECs) are associated with obesity and ectopic fat accumulation, both of which play a role in the development of cardiovascular disease (CVD) in type <em>2</em> diabetes (T<em>2</em>D). The effect of prolonged caloric restriction on ECs in relation to fat distribution and cardiac function is still unknown. Therefore, our aim was to investigate this relationship in obese T<em>2</em>D patients with coronary artery disease (CAD).

METHODS

In a prospective intervention study, obese T<em>2</em>D patients with CAD (n = <em>2</em>7) followed a 16 week very low calorie diet (VLCD; 450-1000 kcal/day). Cardiac function and fat accumulation were assessed with MRI and spectroscopy. Plasma levels of lipid species, including ECs, were measured using liquid chromatography-mass spectrometry.

RESULTS

VLCD decreased plasma levels of virtually all measured lipid species of the class of N-acylethanolamines including the EC anandamide (AEA; -15%, p = 0.016), without decreasing monoacylglycerols including the EC <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG). Baseline plasma AEA levels strongly correlated with the volume of subcutaneous white adipose tissue (SAT; R<em>2</em> = 0.44, p < 0.001). VLCD decreased the volume of SAT (-53%, p < 0.001), visceral white adipose tissue (VAT) (-5<em>2</em>%, p < 0.001), epicardial white adipose tissue (-15%, p < 0.001) and paracardial white adipose tissue (-<em>2</em>8%, p < 0.001). VLCD also decreased hepatic (-86%, p < 0.001) and myocardial (-33%, p < 0.001) fat content. These effects were accompanied by an increased left ventricular ejection fraction (54.8 ± 8.7-56.<em>2</em> ± 7.9%, p = 0.016).

CONCLUSIONS

Caloric restriction in T<em>2</em>D patients with CAD decreases AEA levels, but not <em>2</em>-AG levels, which is paralleled by decreased lipid accumulation in adipose tissue, liver and heart, and improved cardiovascular function. Interestingly, baseline AEA levels strongly correlated with SAT volume. We anticipate that dietary interventions are worthwhile strategies in advanced T<em>2</em>D, and that reduction in AEA may contribute to the improved cardiometabolic phenotype induced by weight loss.

Background: Acute kidney injury (AKI) is associated with a significantly increased risk of morbidity and mortality. Ischemia-reperfusion injury (IRI) is a major cause of AKI. In this study, we investigated the role of the endocannabinoid (EC) system in renal IRI using a well-established mouse model. Materials and Methods: Renal ischemia was induced in male C57BL/6 mice by clamping both kidney pedicles for 30 min followed by <em>2</em>4 h of reperfusion. To increase renal <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) levels, mice were pretreated with JZL184 (16 mg/kg), 30 min before IRI. Serum creatinine and blood urea nitrogen (BUN), renal tubular damage, renal content of ECs and renal expression of markers of inflammation and oxidative stress were measured. Results: Renal IRI was associated with significantly increased serum BUN and creatinine, increased tubular damage score, increased expression of renal markers of inflammation and oxidative stress and elevated renal <em>2</em>-AG content. Pretreatment with JZL184 was associated with a significant increase in renal <em>2</em>-AG content and there was also improved serum BUN, creatinine and tubular damage score. However, renal expression of inflammation and oxidative stress markers remained unchanged. Conclusions: This is the first report documenting that renal IRI is associated with an increase in kidney <em>2</em>-AG content. Further enhancement of <em>2</em>-AG levels using JZL184 improved indices of renal function and histology, but did not lower renal expression of markers of inflammation and oxidative stress. Further studies are needed to determine the mechanisms responsible for the effects observed and the potential value of <em>2</em>-AG as a therapeutic target in renal IRI.

BACKGROUND

The endocannabinoid (eCB) system plays a key role in the development of obesity and its comorbidities. Limited information exists on the changes in circulating eCBs following bariatric surgery.

OBJECTIVE

This study aims to (i) assess the circulating levels of eCBs and related molecules and (ii) examine the association between their levels and numerous clinical/metabolic features pre- and post-operatively.

METHODS

Sixty-five morbidly obese patients (age 4<em>2</em>.78 ± 9.<em>2</em>7 years; BMI 4<em>2</em>.00 ± 5.01 kg/m<em>2</em>) underwent laparoscopic sleeve gastrectomy (LSG) surgery, and were followed up for 1<em>2</em> months. Data collected included anthropometrics and metabolic parameters. The serum levels of the eCBs, <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), anandamide (AEA); and their related molecules, arachidonic acid (AA) and oleoylethanolamine (OEA) were measured by liquid chromatography-mass spectrometry.

RESULTS

Levels of <em>2</em>-AG, AEA, and AA were reduced post operatively with no differences in serum OEA levels. The delta changes in eCB levels between pre- and post-operation were correlated with the delta of different metabolic parameters. Positive correlations were found between delta AA and waist circumference (WC) (r = 0.<em>2</em>8, P < 0.05), free fat mass (r = 0.<em>2</em>6, P < 0.05), SteatoTest score (r = 0.45, P < 0.05), and ALT (r = 0.3<em>2</em>, P < 0.05). Delta AEA levels positively correlated with WC (r = 0.30, P < 0.05). Delta <em>2</em>-AG levels positively correlated with total cholesterol (r = 0.<em>2</em>7, P < 0.05), triglycerides (r = 0.55, P < 0.05), and SteatoTest score (r = 0.<em>2</em>7, P < 0.05). Delta OEA levels negatively correlated with fasting glucose levels (r = - 0.<em>2</em>7, P < 0.05).

CONCLUSIONS

This study provides compelling evidence that LSG surgery induces reductions in the circulating <em>2</em>-AG, AEA, and AA levels, and that these changes are associated with clinical benefits related to the surgery including reduced fat mass, hepatic steatosis, glucose, and improved lipid profile.

A balanced cytotrophoblast cell turnover is crucial for placental development and anomalies in this process associated with gestational diseases. The endocannabinoid system (ECS) has emerged as a new player in several biological processes. However, its influence during placental development is still unknown. We report here the expression of the endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) main metabolic enzymes in human cytotrophoblasts and syncytiotrophoblast. We also showed that <em>2</em>-AG induced a decrease in placental alkaline phosphatase activity, human chorionic gonadotropin secretion and Leptin mRNA levels. Moreover, <em>2</em>-AG reduced glial cell missing 1 and syncytin-<em>2</em> transcription and the number of nuclei in syncytium. These effects were mediated by cannabinoid receptors and may result from <em>2</em>-AG inhibition of the cAMP/PKA signalling pathway. Our data suggest that <em>2</em>-AG may interfere with the biochemical and morphological differentiation of human cytotrophoblasts, through a CB receptor-dependent mechanism, shedding light on a role for the ECS in placental development.

Inhibition of endocannabinoid degradation has been suggested as tool for activation of endogenous tumor defense. One of these strategies lies in blockade of fatty acid amide hydrolase (FAAH) which catalyzes the degradation of endocannabinoids (anandamide [AEA], <em>2</em>-<em>arachidonoylglycerol</em> [<em>2</em>-AG]) and endocannabinoid-like substances (N-oleoylethanolamine [OEA], N-palmitoylethanolamine [PEA]). This study addressed the impact of two FAAH inhibitors (arachidonoyl serotonin [AA-5HT], URB597) on A549 lung cancer cell metastasis and invasion. LC-MS analyses revealed increased levels of FAAH substrates (AEA, <em>2</em>-AG, OEA, PEA) in cells incubated with either FAAH inhibitor. In athymic nude mice FAAH inhibitors were shown to elicit a dose-dependent antimetastatic action yielding a 67% and 6<em>2</em>% inhibition of metastatic lung nodules following repeated administration of 15 mg/kg AA-5HT and 5 mg/kg URB597, respectively. In vitro, a concentration-dependent anti-invasive action of either FAAH inhibitor was demonstrated, accompanied with upregulation of tissue inhibitor of matrix metalloproteinases-1 (TIMP-1). Using siRNA approaches, a causal link between the TIMP-1-upregulating and anti-invasive action of FAAH inhibitors was confirmed. Moreover, knockdown of FAAH by siRNA was shown to confer decreased cancer cell invasiveness and increased TIMP-1 expression. Inhibitor experiments point toward a role of CB<em>2</em> and transient receptor potential vanilloid 1 in conferring anti-invasive effects of FAAH inhibitors and FAAH siRNA. Finally, antimetastatic and anti-invasive effects were confirmed for all FAAH substrates with AEA and OEA causing a TIMP-1-dependent anti-invasive action. Collectively, the present study provides first-time proof for an antimetastatic action of FAAH inhibitors. As mechanism of its anti-invasive properties an upregulation of TIMP-1 was identified.

The failure to undergo remyelination is a critical impediment to recovery in multiple sclerosis. Chondroitin sulfate proteoglycans (CSPGs) accumulate at demyelinating lesions creating a nonpermissive environment that impairs axon regeneration and remyelination. Here, we reveal a new role for <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), the major CNS endocannabinoid, in the modulation of CSPGs deposition in a progressive model of multiple sclerosis, the Theiler's murine encephalomyelitis virus-induced demyelinating disease. Treatment with a potent reversible inhibitor of the enzyme monoacylglycerol lipase, which accounts for 85% of the <em>2</em>-AG degradation in the mouse CNS, modulates neuroinflammation and reduces CSPGs accumulation and astrogliosis around demyelinated lesions in the spinal cord of Theiler's murine encephalomyelitis virus-infected mice. Inhibition of <em>2</em>-AG hydrolysis augments the number of mature oligodendrocytes and increases MBP, leading to remyelination and functional recovery of mice. Our findings establish a mechanism for <em>2</em>-AG promotion of remyelination with implications in axonal repair in CNS demyelinating pathologies.SIGNIFICANCE STATEMENT The deposition of chondroitin sulfate proteoglycans contributes to the failure in remyelination associated with multiple sclerosis. Here we unveil a new role for <em>2</em>-<em>arachidonoylglycerol</em>, the major CNS endocannabinoid, in the modulation of chondroitin sulfate proteoglycan accumulation in Theiler's murine encephalomyelitis virus-induced demyelinating disease. The treatment during the chronic phase with a potent reversible inhibitor of the enzyme monoacylglycerol lipase, which accounts for 85% of the <em>2</em>-<em>arachidonoylglycerol</em> degradation in the mouse CNS, modulates neuroinflammation and reduces chondroitin sulfate proteoglycan deposition around demyelinated lesions in the spinal cord of Theiler's murine encephalomyelitis virus-infected mice. The increased <em>2</em>-<em>arachidonoylglycerol</em> tone promotes remyelination in a model of progressive multiple sclerosis ameliorating motor dysfunction.

The involvement of the endocannabinoid system in drug addiction was initially studied by the use of compounds with different affinities for each cannabinoid receptor or for the proteins involved in endocannabinoids inactivation. The generation of genetically modified mice with selective mutations in these endocannabinoid system components has now provided important advances in establishing their specific contribution to drug addiction. These genetic tools have identified the particular interest of CB1 cannabinoid receptor and endogenous anandamide as potential targets for drug addiction treatment. Novel genetic tools will allow determining if the modulation of CB<em>2</em> cannabinoid receptor activity and <em>2</em>-<em>arachidonoylglycerol</em> tone can also have an important therapeutic relevance for drug addiction.

The endocannabinoid system comprises several molecular entities such as endogenous ligands [anandamide (AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG)], receptors (CB1 and CB<em>2</em>), enzymes such as [fatty acid amide hydrolase (FAHH) and monoacylglycerol lipase (MAGL)], as well as the anandamide membrane transporter. Although the role of this complex neurobiological system in the sleep-wake cycle modulation has been studied, the contribution of the blocker of FAAH/transient receptor potential cation channel subfamily V member 1 (TRPV1), N-arachidonoyl-serotonin (AA-5-HT) in sleep has not been investigated. Thus, in the present study, varying doses of AA-5-HT (5, 10, or <em>2</em>0 mg/Kg, i.p.) injected at the beginning of the lights-on period of rats, caused no statistical changes in sleep patterns. However, similar pharmacological treatment given to animals at the beginning of the dark period decreased wakefulness (W) and increased slow wave sleep (SWS) as well as rapid eye movement sleep (REMS). Power spectra analysis of states of vigilance showed that injection of AA-5-HT during the lights-off period diminished alpha spectrum across alertness in a dose-dependent fashion. In opposition, delta power spectra was enhanced as well as theta spectrum, during SWS and REMS, respectively. Moreover, the highest dose of AA-5-HT decreased wake-related contents of neurotransmitters such as dopamine (DA), norepinephrine (NE), epinephrine (EP), serotonin (5-HT) whereas the levels of adenosine (AD) were enhanced. In addition, the sleep-inducing properties of AA-5-HT were confirmed since this compound blocked the increase in W caused by stimulants such as cannabidiol (CBD) or modafinil (MOD) during the lights-on period. Additionally, administration of AA-5-HT also prevented the enhancement in contents of DA, NE, EP, 5-HT and AD after CBD of MOD injection. Lastly, the role of AA-5-HT in sleep homeostasis was tested in animals that received either CBD or MOD after total sleep deprivation (TSD). The injection of CBD or MOD increased alertness during sleep rebound period after TSD. However, AA-5-HT blocked this effect by allowing animals to display an enhancement in sleep across sleep rebound period. Overall, our findings provide evidence that AA-5-HT is an important modulator of sleep, sleep homeostasis and neurotransmitter contents.

<AbstractText>The endocannabinoid (eCB) system is involved in diverse aspects of human physiology and behavior but little is known about the impact of circadian rhythmicity on the system. The two most studied endocannabinoids, AEA (ananamide) and <em>2</em>-AG (<em>2</em>-<em>arachidonoylglycerol</em>), can be measured in peripheral blood however the functional relevance of peripheral eCB levels is not clear. Having previously detailed the <em>2</em>4-h profile of serum <em>2</em>-AG, here we report the <em>2</em>4-h serum profile of AEA to determine if these two endocannabinoids vary in parallel across the biological day including a nocturnal 8.5-h sleep period. Further, we assessed and compared the effect of a physiological challenge, in the form of sleep restriction to 4.5-h, on these two profiles.</AbstractText><AbstractText>In this randomized crossover study, we examined serum concentrations of AEA across a <em>2</em>4-h period in fourteen young adults. Congeners of AEA, the structural analogs oleoylethanolamide (OEA) and palmitoylethanolamide (PEA) were simultaneously assayed. Prior to <em>2</em>4-h blood sampling, each participant was exposed to two nights of normal (8.5 h) or restricted sleep (4.5 h). The two sleep conditions were separated by at least one month. In both sleep conditions, during the period of blood sampling, each individual ate the same high-carbohydrate meal at 0900, 1400, and 1900.</AbstractText><AbstractText>Mean <em>2</em>4-h concentrations of AEA were 0.697 ± 0.11 pmol/ml. A reproducible biphasic <em>2</em>4-h profile of AEA was observed with a first peak occurring during early sleep (0<em>2</em>00) and a second peak in the mid-afternoon (1500) while a nadir was detected in the mid-morning (1000). The <em>2</em>4-h profiles for both OEA and PEA followed a similar pattern to that observed for AEA. AEA, OEA, and PEA levels were not affected by sleep restriction at any time of day, contrasting with the elevation of early afternoon levels previously observed for <em>2</em>-AG.</AbstractText><AbstractText>The <em>2</em>4-h rhythm of AEA is markedly different from that of <em>2</em>-AG, being of lesser amplitude and biphasic, rather than monophasic. These observations suggest distinct regulatory pathways of the two eCB and indicate that time of day needs to be carefully controlled in studies attempting to delineate their relative roles. Moreover, unlike <em>2</em>-AG, AEA is not altered by sleep restriction, suggesting that physiological perturbations may affect AEA and <em>2</em>-AG differently. Similar <em>2</em>4-h profiles were observed for OEA and PEA following normal and restricted sleep, further corroborating the validity of the wave-shape and lack of response to sleep loss observed for the AEA profile. Therapeutic approaches involving agonism or antagonism of peripheral eCB signaling will likely need to be tailored according to time of day.</AbstractText>

Objectives. The purpose of this study is to investigate the relationship between plasma endocannabinoids and insulin resistance (IR) in patients with obstructive sleep apnea (OSA). Methods. A population of 64 with OSA and <em>2</em>4 control subjects was recruited. Body mass index (BMI), waist circumference, lipids, blood glucose and insulin, homeostasis model of assessment for insulin resistance index (HOMA-IR), anandamide (AEA), 1/<em>2</em>-<em>arachidonoylglycerol</em> (1/<em>2</em>-AG), and apnea-hypopnea index (AHI) were analyzed. Results. Fasting blood insulin (<em>2</em><em>2</em>.9 ± 7.8 mIU/L versus 18.5 ± 7.<em>2</em> mIU/L, P < 0.05), HOMA-IR (<em>2</em>.9 ± 1.0 versus <em>2</em>.4 ± 0.9, P < 0.01), AEA (3.<em>2</em> ± 0.7 nmol/L versus <em>2</em>.5 ± 0.6 nmol/L, P < 0.01), and 1/<em>2</em>-AG (40.8 ± 5.7 nmol/L versus 34.3 ± 7.7 nmol/L, P < 0.01) were higher in OSA group than those in control group. In OSA group, AEA, 1/<em>2</em>-AG, and HOMA-IR increase with the OSA severity. The correlation analysis showed significant positive correlation between HOMA-IR and AHI (r = 0.44, P < 0.01), AEA and AHI (r = 0.5<em>2</em>, P < 0.01), AEA and HOMA-IR (r = 0.6<em>2</em>, P < 0.01), and 1/<em>2</em>-AG and HOMA-IR (r = 0.33, P < 0.01). Further analysis showed that only AEA was significantly correlated with AHI and HOMA-IR after adjusting for confounding factors. Conclusions. The present study indicated that plasma endocannabinoids levels, especially AEA, were associated with IR and AHI in patients with OSA.

Propofol pretreatment before reperfusion, or propofol conditioning, has been shown to be cardioprotective, while its mechanism is unclear. The current study investigated the roles of endocannabinoid signaling in propofol cardioprotection in an in vivo model of myocardial ischemia/reperfusion (I/R) injury and in in vitro primary cardiomyocyte hypoxia/reoxygenation (H/R) injury. The results showed that propofol conditioning increased both serum and cell culture media concentrations of endocannabinoids including anandamide (AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) detected by LC-MS/MS. The reductions of myocardial infarct size in vivo and cardiomyocyte apoptosis and death in vitro were accompanied with attenuations of oxidative injuries manifested as decreased reactive oxygen species (ROS), malonaldehyde (MDA), and MPO (myeloperoxidase) and increased superoxide dismutase (SOD) production. These effects were mimicked by either URB597, a selective endocannabinoids degradation inhibitor, or VDM11, a selective endocannabinoids reuptake inhibitor. In vivo study further validated that the cardioprotective and antioxidative effects of propofol were reversed by selective CB<em>2</em> receptor antagonist AM630 but not CB1 receptor antagonist AM<em>2</em>51. We concluded that enhancing endogenous endocannabinoid release and subsequent activation of CB<em>2</em> receptor signaling represent a major mechanism whereby propofol conditioning confers antioxidative and cardioprotective effects against myocardial I/R injury.