BACKGROUND

The discovery of the endocannabinoid system and of its role in the regulation of energy balance has significantly advanced our understanding of the physiopathological mechanisms leading to obesity and type <em>2</em> diabetes. New knowledge on the role of this system in humans has been acquired by measuring blood endocannabinoids. Here we explored endocannabinoids and related N-acylethanolamines in saliva and verified their changes in relation to body weight status and in response to a meal or to body weight loss.

RESULTS

Fasting plasma and salivary endocannabinoids and N-acylethanolamines were measured through liquid mass spectrometry in 1<em>2</em> normal weight and 1<em>2</em> obese, insulin-resistant subjects. Salivary endocannabinoids and N-acylethanolamines were evaluated in the same cohort before and after the consumption of a meal. Changes in salivary endocannabinoids and N-acylethanolamines after body weight loss were investigated in a second group of 1<em>2</em> obese subjects following a 1<em>2</em>-weeks lifestyle intervention program. The levels of mRNAs coding for enzymes regulating the metabolism of endocannabinoids, N-acylethanolamines and of cannabinoid type 1 (CB(1)) receptor, alongside endocannabinoids and N-acylethanolamines content, were assessed in human salivary glands. The endocannabinoids <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), N-arachidonoylethanolamide (anandamide, AEA), and the N-acylethanolamines (oleoylethanolamide, OEA and palmitoylethanolamide, PEA) were quantifiable in saliva and their levels were significantly higher in obese than in normal weight subjects. Fasting salivary AEA and OEA directly correlated with BMI, waist circumference and fasting insulin. Salivary endocannabinoids and N-acylethanolamines did not change in response to a meal. CB(1) receptors, ligands and enzymes were expressed in the salivary glands. Finally, a body weight loss of 5.3% obtained after a 1<em>2</em>-weeks lifestyle program significantly decreased salivary AEA levels.

CONCLUSIONS

Endocannabinoids and N-acylethanolamines are quantifiable in saliva and their levels correlate with obesity but not with feeding status. Body weight loss significantly decreases salivary AEA, which might represent a useful biomarker in obesity.

The cannabinoid system (CS) is implicated in the regulation of hepatic fibrosis, steatosis and inflammation, with cannabinoid receptors 1 and <em>2</em> (CB1 and CB<em>2</em>) being involved in regulation of pro- and antifibrogenic effects. Daily cannabis smoking is an independent risk factor for the progression of fibrosis in chronic hepatitis C and a mediator of experimental alcoholic steatosis. However, the role and function of CS in alcoholic liver fibrosis (ALF) is unknown so far. Thus, human liver samples from patients with alcoholic liver disease (ALD) were collected for analysis of CB1 expression. In vitro, hepatic stellate cells (HSC) underwent treatment with acetaldehyde, Δ9-tetrahydrocannabinol H₂O₂, endo- and exocannabinoids (<em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) and [THC]), and CB1 antagonist SR141716 (rimonabant). In vivo, CB1 knockout (KO) mice received thioacetamide (TAA)/ethanol (EtOH) to induce fibrosis. As a result, in human ALD, CB1 expression was restricted to areas with advanced fibrosis only. In vitro, acetaldehyde, H₂O₂, as well as <em>2</em>-AG and THC, alone or in combination with acetaldehyde, induced CB1 mRNA expression, whereas CB1 blockage with SR141716 dose-dependently inhibited HSC proliferation and downregulated mRNA expression of fibrosis-mediated genes PCα1(I), TIMP-1 and MMP-13. This was paralleled by marked cytotoxicity of SR141716 at high doses (5-10 μmol/L). In vivo, CB1 knockout mice showed marked resistance to alcoholic liver fibrosis. In conclusion, CB1 expression is upregulated in human ALF, which is at least in part triggered by acetaldehyde (AA) and oxidative stress. Inhibition of CB1 by SR141716, or via genetic knock-out protects against alcoholic-induced fibrosis in vitro and in vivo.

Activated cannabinoid 1 receptor (CB1R) signaling has been implicated in the development of phenotypes associated with fatty liver, insulin resistance, and impaired suppression of hepatic glucose output. Endoplasmic reticulum stress-associated liver-specific transcription factor CREBH is emerging as a critical player in various hepatic metabolic pathways and regulates hepatic gluconeogenesis in diet-induced obese settings. In this study, we elucidated the critical role of CREBH in mediating CB1R signaling to regulate glucose homeostasis in primary rat and human hepatocytes. mRNA and protein levels and glucose production were analyzed in primary rat and human hepatocytes. ChIP assays were performed together with various transcriptional analyses using standard techniques. CB1R activation by <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) specifically induced CREBH gene expression via phosphorylation of the JNK signaling pathway and c-Jun binding to the AP-1 binding site in the CREBH gene promoter. <em>2</em>-AG treatment significantly induced hepatic gluconeogenic gene expression and glucose production in primary hepatocytes, and we demonstrated that the CREBH binding site mutant significantly attenuated <em>2</em>-AG-mediated activation of the gluconeogenic gene promoter. Endogenous knockdown of CREBH led to ablation of <em>2</em>-AG-induced gluconeogenic gene expression and glucose production, and the CB1R antagonist AM<em>2</em>51 or insulin exhibited repression of CREBH gene induction and subsequently inhibited gluconeogenesis in both rat and human primary hepatocytes. These results demonstrate a novel mechanism of action of activated CB1R signaling to induce hepatic gluconeogenesis via direct activation of CREBH, thereby contributing to a better understanding of the endocannabinoid signaling mechanism involved in regulating the hepatic glucose metabolism.

Endocannabinoid signaling has been implicated in modulating insulin release from β cells of the endocrine pancreas. β Cells express CB1 cannabinoid receptors (CB1Rs), and the enzymatic machinery regulating anandamide and <em>2</em>-<em>arachidonoylglycerol</em> bioavailability. However, the molecular cascade coupling agonist-induced cannabinoid receptor activation to insulin release remains unknown. By combining molecular pharmacology and genetic tools in INS-1E cells and in vivo, we show that CB1R activation by endocannabinoids (anandamide and <em>2</em>-<em>arachidonoylglycerol</em>) or synthetic agonists acutely or after prolonged exposure induces insulin hypersecretion. In doing so, CB1Rs recruit Akt/PKB and extracellular signal-regulated kinases 1/<em>2</em> to phosphorylate focal adhesion kinase (FAK). FAK activation induces the formation of focal adhesion plaques, multimolecular platforms for second-phase insulin release. Inhibition of endocannabinoid synthesis or FAK activity precluded insulin release. We conclude that FAK downstream from CB1Rs mediates endocannabinoid-induced insulin release by allowing cytoskeletal reorganization that is required for the exocytosis of secretory vesicles. These findings suggest a mechanistic link between increased circulating and tissue endocannabinoid levels and hyperinsulinemia in type <em>2</em> diabetes.

OBJECTIVE

The development of potent and selective inhibitors of the biosynthesis of the endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) via DAG lipases (DAGL) α and β is just starting to be considered as a novel and promising source of pharmaceuticals for the treatment of disorders that might benefit from a reduction in endocannabinoid tone, such as hyperphagia in obese subjects.

METHODS

Three new fluorophosphonate compounds O-7458, O-7459 and O-7460 were synthesized and characterized in various enzymatic assays. The effects of O-7460 on high-fat diet intake were tested in mice.

RESULTS

Of the new compounds, O-7460 exhibited the highest potency (IC₅₀ = 690 nM) against the human recombinant DAGLα, and selectivity (IC₅₀>> 10 μM) towards COS-7 cell and human monoacylglycerol lipase (MAGL), and rat brain fatty acid amide hydrolase. Competitive activity-based protein profiling confirmed that O-7460 inhibits mouse brain MAGL only at concentrations ≥ 10 μM, and showed that this compound has only one major 'off-target', that is, the serine hydrolase KIAA1363. O-7460 did not exhibit measurable affinity for human recombinant CB₁ or CB₂ cannabinoid receptors (Ki>> 10 μM). In mouse neuroblastoma N18TG<em>2</em> cells stimulated with ionomycin, O-7460 (10 μM) reduced <em>2</em>-AG levels. When administered to mice, O-7460 dose-dependently (0-1<em>2</em> mg·kg⁻¹, i.p.) inhibited the intake of a high-fat diet over a 14 h observation period, and, subsequently, slightly but significantly reduced body weight.

CONCLUSIONS

O-7460 might be considered a useful pharmacological tool to investigate further the role played by <em>2</em>-AG both in vitro and in vivo under physiological as well as pathological conditions.

The endocannabinoid system plays protective roles against the growth and the spreading of several types of carcinomas. Because estrogens regulate this system both in physiological states and cancer, in this paper we evaluated its involvement in endometrial carcinoma, a well-known estrogen-dependent tumor. To test whether the endocannabinoid system is expressed in endometrial cancer, tissue samples were collected both from 18 patients undergoing surgical treatment for endometrial adenocarcinoma and 16 healthy age-matched controls, and treated for Western blot and immunohistochemical analysis. Moreover, tissues were dounce homogenized and submitted to endocannabinoid measurement by liquid chromatography-mass spectrometry. To evaluate the physiological role of the endocannabinoid system, a human endometrial cancer cell-line (AN3CA) was used and transiently transfected with a plasmid containing the cDNA for the endocannabinoid receptor CB(<em>2</em>). Cells were incubated for 48 h with an agonist (JWH133) (10 mum) or antagonist (SR1445<em>2</em>8) (1 mum) of CB(<em>2</em>) <em>2</em>4 h after transfection, and cell proliferation was measured by the 3-[4,5-dimethyltiazol-<em>2</em>yl]-<em>2</em>,5 diphenyltetrazolium bromide formazan assay. In human endometrial carcinoma biopsies the expression of CB(<em>2</em>) receptor and the levels of its ligand, <em>2</em>-<em>arachidonoylglycerol</em> increased, whereas monoacylglycerol lipase, an enzyme responsible for <em>2</em>-<em>arachidonoylglycerol</em> degradation, was down-regulated. Immunohistochemical analysis revealed that CB(<em>2</em>) was overexpressed only in malignant endometrial cells. CB(<em>2</em>)-overexpressing AN3CA cells showed a significant reduction in cell vitality compared with parental AN3CA cells: incubation with the selective CB(<em>2</em>) antagonist SR1441<em>2</em>8 restored the viability of CB(<em>2</em>)-overexpressing cells to that of untransfected cells. In conclusion, the endocannabinoid system seems to play an important role in human endometrial carcinoma, and modulation of CB(<em>2</em>) activity/expression may account for a tumor-suppressive effect.

Together with anandamide, <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) constitutes one of the main representatives of a family of endogenous lipids known as endocannabinoids. These act by binding to CB(1) and CB(<em>2</em>) cannabinoid receptors, the molecular target of the psychoactive compound Delta(9)-THC, both in the periphery and in the central nervous system, where they behave as retrograde messengers to modulate synaptic transmission. These last years, evidence has accumulated to demonstrate the lead role played by the monoacylglycerol lipase (MAGL) in the regulation of <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) levels. Considering the numerous physiological functions played by this endocannabinoid, MAGL is now considered a promising target for therapeutics, as inhibitors of this enzyme could reveal useful for the treatment of pain and inflammatory disorders, as well as in cancer research, among others. Here we review the milestones that punctuated MAGL history, from its discovery to recent advances in the field of inhibitors development. An emphasis is given on the recent elucidation of the tridimensional structure of the enzyme, which could offer new opportunities for rational drug design.

Research into the endocannabinoid signaling system has grown exponentially in recent years following the discovery of cannabinoid receptors (CB) and their endogenous ligands, such as anandamide (AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG). Important advances have been made in our understanding of the endocannabinoid signaling system in various aspects of alcoholism, including alcohol-seeking behavior. Alcohol increases the synthesis or impairs the degradation of endocannabinoids, leading to a locally elevated endocannabinoid tone within the brain. Elevated endocannabinoid tone might be expected to result in compensatory down-regulation of CB1 receptors or dampened signal transduction. Following release, endocannabinoids diffuse back to the presynaptic neuron where they act as short-range modulators of synaptic activity by altering neurotransmitter release and synaptic plasticity. Mice treated with the CB1 receptor antagonist SR141716A (rimonabant) or homozygous for a deletion of the CB1 receptor gene exhibit reduced voluntary alcohol intake. CB1 knockout mice also show increased alcohol sensitivity, withdrawal, and reduced conditioned place preference. Conversely, activation of CB1 receptor promotes alcohol intake. Recent studies also suggest that elevated endocannabinoid tone due to impaired degradation contributes to high alcohol preference and self-administration. These effects are reversed by local administration of rimonabant, suggesting the participation of the endocannabinoid signaling system in high alcohol preference and self-administration. These recent advances will be reviewed with an emphasis on the endocannabinoid signaling system for possible therapeutic interventions of alcoholism.

Redundancy of metabolic pathways and molecular targets is a typical feature of all lipid mediators, and endocannabinoids, which were originally defined as endogenous agonists at cannabinoid CB(1) and CB(<em>2</em>) receptors, are no exception. In particular, the two most studied endocannabinoids, anandamide and <em>2</em>-<em>arachidonoylglycerol</em>, are inactivated through alternative biochemical routes, including hydrolysis and oxidation, and more than one enzyme might be used even for the same type of inactivating reaction. These enzymes also recognize as substrates other concurrent lipid mediators, whereas, in turn, endocannabinoids might interact with noncannabinoid receptors with subcellular distribution and ultimate biological actions either similar to or completely different from those of cannabinoid receptors. Even splicing variants of endocannabinoid hydrolyzing enzymes, such as FAAH-1, might play distinct roles in endocannabinoid inactivation. Finally, the products of endocannabinoid catabolism may have their own targets, with biological roles different from those of cannabinoid receptors. These peculiarities of endocannabinoid signaling have complicated the use of inhibitors of its inactivation mechanisms as a safer and more efficacious alternative to the direct targeting of cannabinoid receptors for the treatment of several pathological conditions, including pain. However, new strategies, including the rediscovery of "dirty drugs", and the use of certain natural products (including non-THC cannabis constituents), are emerging that might allow us to make a virtue of necessity and exploit endocannabinoid redundancy to develop new analgesics.

The active component of marijuana, Delta(9)-tetrahydrocannabinol, activates the CB1 and CB<em>2</em> cannabinoid receptors, thus mimicking the action of endogenous cannabinoids. CB1 is predominantly neuronal and mediates the cannabinoid psychotropic effects. CB<em>2</em> is predominantly expressed in peripheral tissues, mainly in pathological conditions. So far the main endocannabinoids, anandamide and <em>2</em>-<em>arachidonoylglycerol</em>, have been found in bone at 'brain' levels. The CB1 receptor is present mainly in skeletal sympathetic nerve terminals, thus regulating the adrenergic tonic restrain of bone formation. CB<em>2</em> is expressed in osteoblasts and osteoclasts, stimulates bone formation, and inhibits bone resorption. Because low bone mass is the only spontaneous phenotype so far reported in CB<em>2</em> mutant mice, it appears that the main physiologic involvement of CB<em>2</em> is associated with maintaining bone remodeling at balance, thus protecting the skeleton against age-related bone loss. Indeed, in humans, polymorphisms in CNR<em>2</em>, the gene encoding CB<em>2</em>, are strongly associated with postmenopausal osteoporosis. Preclinical studies have shown that a synthetic CB<em>2</em>-specific agonist rescues ovariectomy-induced bone loss. Taken together, the reports on cannabinoid receptors in mice and humans pave the way for the development of 1) diagnostic measures to identify osteoporosis-susceptible polymorphisms in CNR<em>2</em>, and <em>2</em>) cannabinoid drugs to combat osteoporosis.

Whether chronic cannabinoid consumption produces a dependent state comparable to that occurring with other drugs (e.g. the appearance of withdrawal signs when consumption is interrupted), and whether chronic cannabinoid consumption increases the risk of consuming other drugs of greater addictive power, are probably the two questions relating to cannabinoid addiction that provoke the most controversy. The present study was designed to further explore these two questions in laboratory animals. Firstly, we examined the effects of an acute challenge with SR141716 (an antagonist for the cannabinoid CB(1) receptor) in Delta(9)-tetrahydrocannabinol (Delta(9)-THC)-tolerant rats. This antagonist has been reported to precipitate a cannabinoid withdrawal syndrome. Thus, the administration of SR141716 to Delta(9)-THC-tolerant rats reduced inactivity in the open-field test and enhanced responses as tremor, turning and retropulsion-these responses that were only slightly enhanced in control rats. The administration of SR141716 increased the plasma prolactin and the corticosterone concentration in controls, but these increases were much lesser in Delta(9)-THC-tolerant rats. In addition, CRF-mRNA levels in the paraventricular hypothalamic nucleus, while reduced in SR141716-treated controls, were significantly increased in Delta(9)-THC-tolerant rats. The analysis of endocannabinoids also revealed that the administration of SR141716, which was mostly inactive in control rats, was able to reverse the changes in anandamide or <em>2</em>-<em>arachidonoylglycerol</em> concentrations found in Delta(9)-THC-tolerant rats, in the striatum, limbic forebrain, diencephalon, cerebellum and brainstem, but not in the midbrain and hippocampus. As a second objective, we evaluated whether Delta(9)-THC-tolerant rats were more vulnerable to morphine in a self-administration paradigm. The Delta(9)-THC-tolerant and control rats self-administered morphine to a similar extent, in concordance with the similar values of dopaminergic activity in limbic and motor regions. In summary, our data indicate that Delta(9)-THC-tolerant rats were not more vulnerable to the reinforcing properties of morphine. However, they responded to the blockade of CB(1) receptors by exhibiting slightly but possibly relevant differences in behavioral, endocrine and molecular parameters compared to the response in non-tolerant rats. This is indicative of the existence of a withdrawal syndrome in cannabinoid-tolerant rats that is mild compared with abstinence in opioid-dependent rats.

Three components of the cannabinoid system are sensitive to selected organophosphorus (OP) compounds: monoacylglycerol (MAG) lipase that hydrolyzes the major endogenous agonist <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG); fatty acid amide hydrolase (FAAH) that cleaves the agonist anandamide present in smaller amounts; the CB1 receptor itself. This investigation considers which component of the cannabinoid system is the most likely contributor to OP-induced hypomotility in mice. Structure-activity studies by our laboratory and others rule against major involvement of a direct toxicant-CB1 receptor interaction for selected OPs. Attention was therefore focused on the OP sensitivities of MAG lipase and FAAH, assaying 19 structurally diverse OP chemicals (pesticides, their metabolites and designer compounds) for in vitro inhibition of both enzymes. Remarkably high potency and low selectivity is observed with three O-alkyl (C1, C<em>2</em>, C3) alkylphosphonofluoridates (C8, C1<em>2</em>) (IC50 0.60-3.0 nM), five S-alkyl (C5, C7, C9) and alkyl (C10, C1<em>2</em>) benzodioxaphosphorin oxides (IC50 0.15-5.7 nM) and one OP insecticide metabolite (chlorpyrifos oxon, IC50 34-40 nM). In ip-treated mice, the OPs at 1-30 mg/kg more potently inhibit brain FAAH than MAG lipase, but FAAH inhibition is not correlated with hypomotility. However, the alkylphosphonofluoridate-treated mice show dose-dependent increases in severity of hypomotility, inhibition of MAG lipase activity and elevation of <em>2</em>-AG. Moderate to severe hypomotility is accompanied by 64 to 86% MAG lipase inhibition and about 6-fold elevation of brain <em>2</em>-AG level. It therefore appears that OP-induced MAG lipase inhibition leads to elevated <em>2</em>-AG and the associated hypomotility.

BACKGROUND

Endogenous cannabinoids such as anandamide and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) exert important regulatory influences on neuronal signaling, participate in short- and long-term forms of neuroplasticity, and modulate stress responses and affective behavior in part through the modulation of neurotransmission in the amygdala. Alcohol consumption alters brain endocannabinoid levels, and alcohol dependence is associated with dysregulated amygdalar function, stress responsivity, and affective control.

METHODS

The consequence of long-term alcohol consumption on the expression of genes related to endocannabinoid signaling was investigated using quantitative RT-PCR analyses of amygdala tissue. Two groups of ethanol (EtOH)-exposed rats were generated by maintenance on an EtOH liquid diet (10%): the first group received continuous access to EtOH for 15 days, whereas the second group was given intermittent access to the EtOH diet (5 d/wk for 3 weeks). Control subjects were maintained on an isocaloric EtOH-free liquid diet. To provide an initial profile of acute withdrawal, amygdala tissue was harvested following either 6 or <em>2</em>4 hours of EtOH withdrawal.

RESULTS

Acute EtOH withdrawal was associated with significant changes in mRNA expression for various components of the endogenous cannabinoid system in the amygdala. Specifically, reductions in mRNA expression for the primary clearance routes for anandamide and <em>2</em>-AG (fatty acid amide hydrolase [FAAH] and monoacylglycerol lipase [MAGL], respectively) were evident, as were reductions in mRNA expression for CB(1) , CB(<em>2</em>) , and GPR55 receptors. Although similar alterations in FAAH mRNA were evident following either continuous or intermittent EtOH exposure, alterations in MAGL and cannabinoid receptor-related mRNA (e.g., CB(1) , CB(<em>2</em>) , GPR55) were more pronounced following intermittent exposure. In general, greater withdrawal-associated deficits in mRNA expression were evident following <em>2</em>4 versus 6 hours of withdrawal. No significant changes in mRNA expression for enzymes involved in <em>2</em>-AG biosynthesis (e.g., diacylglicerol lipase-α/β) were found in any condition.

CONCLUSIONS

These findings suggest that EtOH dependence and withdrawal are associated with dysregulated endocannabinoid signaling in the amygdala. These alterations may contribute to withdrawal-related dysregulation of amygdalar neurotransmission.

The goal of this study was to determine whether the endocannabinoid system is altered by chronic antidepressant treatment. The effects of 3-week administration of the monoamine oxidase inhibitor, tranylcypromine (10 mg/kg) and the selective serotonin reuptake inhibitor, fluoxetine (5 mg/kg) on cannabinoid CB(1) receptor densities and endocannabinoid contents were determined in limbic brain regions of the rat. Tranylcypromine significantly reduced tissue content of the endocannabinoid N-arachidonylethanolamine (anandamide) in the prefrontal cortex, hippocampus and hypothalamus and increased <em>2</em>-<em>arachidonoylglycerol</em> content in the prefrontal cortex. Tranylcypromine treatment significantly increased CB(1) receptor binding density in the prefrontal cortex and hippocampus, but not in the hypothalamus. Treatment with fluoxetine increased CB(1) receptor density in the prefrontal cortex, but had no effect on endocannabinoid contents in any brain region examined. These data suggest that monoaminergic neurotransmission can regulate the endocannabinoid system and further indicates a role of the endocannabinoid system in affective illness and its treatment.

OBJECTIVE

To determine the physiologic importance of endocannabinoids and mitochondrial function in the long-term outcome using a rat model of Roux-en-Y gastric bypass (RYGB) surgery.

BACKGROUND

Sixteen million people are morbidly obese and RYGB surgery is the most effective treatment. Endocannabinoids are implicated in appetite stimulation and regulation of peripheral energy metabolism. We hypothesize that down-regulation of endocannabinoids and alterations in mitochondrial function and hormones favoring catabolism contribute to sustained RYGB-induced weight loss.

METHODS

Diet-induced obese Sprague-Dawley rats were randomized to sham-operated obese controls, RYGB, and sham-operated obese pair-fed rats. Body weight and food intake were recorded, and food efficiency was calculated. Endocannabinoid levels in skeletal muscle and liver, muscle mitochondrial respiratory complex I-V content, and hormones concentrations were determined 14 and <em>2</em>8 days postsurgery, reflecting rapid and sustained weight loss periods after RYGB, respectively.

RESULTS

Compared with pair-fed controls, RYGB rats had significant reduction in body weight and food efficiency (P < 0.001). Increased cholecystokinin, reduced insulin, leptin, adiponectin, T3, and down-regulation of mitochondrial complex I were evident on day 14 postsurgery. On day <em>2</em>8, leptin, insulin, and T3 remained low, whereas adiponectin and cholecystokinin were normal. Along with complex I, the endocannabinoids anandamide in muscle (P = 0.003) and <em>2</em>-<em>arachidonoylglycerol</em> in liver were significantly down-regulated (P < 0.001).

CONCLUSIONS

The attenuated caloric intake, reduced food efficiency, and normalization of hormonal levels on day <em>2</em>8 post-RYGB were associated with significant down-regulation of endocannabinoids anandamide and <em>2</em>-<em>arachidonoylglycerol</em> in muscle and liver, respectively. These results suggest a role for endocannabinoids in the mechanism of sustained weight loss and RYGB success, and may have implications for treatment of morbid obesity.

The endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) mediates retrograde synaptic suppression. Although the mechanisms of <em>2</em>-AG production are well characterized, how <em>2</em>-AG is degraded is less clearly understood. Here we found that expression of the <em>2</em>-AG hydrolyzing enzyme monoacylglycerol lipase (MGL) was highly heterogeneous in the cerebellum, being rich within parallel fiber (PF) terminals, weak in Bergman glia (BG), and absent in other synaptic terminals. Despite this highly selective MGL expression pattern, <em>2</em>-AG-mediated retrograde suppression was significantly prolonged at not only PF-Purkinje cell (PC) synapses but also climbing fiber-PC synapses in granule cell-specific MGL knockout (MGL-KO) mice whose cerebellar MGL expression was confined to the BG. Virus-mediated expression of MGL into the BG of global MGL-KO mice significantly shortened <em>2</em>-AG-mediated retrograde suppression at PF-PC synapses. Furthermore, contribution of MGL to termination of <em>2</em>-AG signaling depended on the distance from MGL-rich PFs to inhibitory synaptic terminals. Thus, <em>2</em>-AG is degraded in a synapse-type independent manner by MGL present in PFs and the BG. The results of the present study strongly suggest that MGL regulates <em>2</em>-AG signaling rather broadly within a certain range of neural tissue, although MGL expression is heterogeneous and limited to a subset of nerve terminals and astrocytes.

The endogenous cannabinoid system plays an important role in the regulation of gastrointestinal function in health and disease. Endocannabinoid levels are regulated by catabolic enzymes. Here, we describe the presence and localization of monoacylglycerol lipase (MGL), the major enzyme responsible for the degradation of <em>2</em>-<em>arachidonoylglycerol</em>. We used molecular, biochemical, immunohistochemical, and functional assays to characterize the distribution and activity of MGL. MGL mRNA was present in rat ileum throughout the wall of the gut. MGL protein was distributed in the muscle and mucosal layers of the ileum and in the duodenum, proximal colon, and distal colon. We observed MGL expression in nerve cell bodies and nerve fibers of the enteric nervous system. There was extensive colocalization of MGL with PGP 9.5 and calretinin-immunoreactive neurons, but not with nitric oxide synthase. MGL was also present in the epithelium and was highly expressed in the small intestine. Enzyme activity levels were highest in the duodenum and decreased along the gut with lowest levels in the distal colon. We observed both soluble and membrane-associated enzyme activities. The MGL inhibitor URB60<em>2</em> significantly inhibited whole gut transit in mice, an action that was abolished in cannabinoid 1 receptor-deficient mice. In conclusion, MGL is localized in the enteric nervous system where endocannabinoids regulate intestinal motility. MGL is highly expressed in the epithelium, where this enzyme may have digestive or other functions yet to be determined.

Endocannabinoids are a new class of lipid mediators, which includes amides and esters of long-chain polyunsaturated fatty acids. Anandamide (I) and <em>2</em>-<em>arachidonoylglycerol</em> (II) are the main endogenous agonists of cannabinoid receptors, able to mimic several pharmacological effects of delta 9-tetrahydrocannabinol (III), the active principle of Cannabis sativa preparations such as hashish and marijuana. The pathways leading to the synthesis and release of anandamide and <em>2</em>-<em>arachidonoylglycerol</em> from neuronal and nonneuronal cells are rather uncertain. Instead, evidence has accumulated showing that the activity of these compounds at their specific receptors is limited by cellular uptake through a specific membrane transporter, followed by intracellular degradation by a fatty acid amide hydrolase. Here, the endocannabinoids and the endocannabinoid-like compounds most relevant for human physiology will be discussed, along with the synthetic and degradative pathways of anandamide and <em>2</em>-<em>arachidonoylglycerol</em> and their molecular targets on the cell surface. The main actions of the endocannabinoids in human cells and tissues will also be reviewed, focusing on the activities most recently discovered in the central nervous system and in the periphery.

OBJECTIVE

Experimental autoimmune encephalomyelitis (EAE) is a widely used model of multiple sclerosis (MS) and both conditions have been reported to exhibit reduced endocannabinoid activity. The purpose of this study was to address the effect of exogenously administered <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>AG), an endocannabinoid receptor ligand, on acute phase and chronic disability in EAE.

METHODS

Acute and chronic EAE models were induced in susceptible mice and <em>2</em>AG-treatment was applied for 14 days from day of disease induction.

RESULTS

<em>2</em>AG-treatment ameliorated acute phase of disease with delay of disease onset in both EAE models and reduced disease mortality and long-term (70 days post-induction) clinical disability in chronic EAE. Reduced axonal pathology in the chronic EAE- (p<0.0001) and increased activation and ramification of microglia in the <em>2</em>AG-treated acute EAE- (p<0.05) model were noticed. The latter was accompanied by a <em>2</em>- to 4-fold increase of the M<em>2</em>-macrophages in the perivascular infiltrations (p<0.001) of the <em>2</em>AG-treated animals in the acute (day <em>2</em><em>2</em>), although not the chronic (day 70), EAE model. Expression of cannabinoid receptors 1 (CB1R) and <em>2</em> (CB<em>2</em>R) was increased in <em>2</em>AG-treated animals of acute EAE vs. controls (p<0.05). In addition, ex vivo viability assays exhibited reduced proliferation of activated lymph node cells when extracted from <em>2</em>AG-treated EAE animals, whereas a dose-dependent response of activated lymphocytes to <em>2</em>AG-treatment in vitro was noticed.

CONCLUSIONS

Our data indicate for the first time that <em>2</em>AG treatment may provide direct (via CBRs) and immune (via M<em>2</em> macrophages) mediated neuroprotection in EAE.

Cannabinoids (CBs) are attributed neuroprotective effects in vivo. Here, we determined the neuroprotective potential of CBs during neuronal damage in excitotoxically lesioned organotypic hippocampal slice cultures (OHSCs). OHSCs are the best characterized in vitro model to investigate the function of microglial cells in neuronal damage since blood-borne monocytes and T-lymphocytes are absent and microglial cells represent the only immunocompetent cell type. Excitotoxic neuronal damage was induced by NMDA (50 microM) application for 4 h. Neuroprotective properties of 9-carboxy-11-nor-delta-9-tetrahydrocannabinol (THC), N-arachidonoylethanolamide (AEA) or <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) in different concentrations were determined after co-application with NMDA by counting degenerating neurons identified by propidium iodide labeling (PI(+)) and microglial cells labeled by isolectin B(4) (IB(4)(+)). All three CBs used significantly decreased the number of IB(4)(+) microglial cells in the dentate gyrus but the number of PI(+) neurons was reduced only after <em>2</em>-AG treatment. Application of AM630, antagonizing CB<em>2</em> receptors highly expressed by activated microglial cells, did not counteract neuroprotective effects of <em>2</em>-AG, but affected THC-mediated reduction of IB(4)(+) microglial cells. Our results indicate that (1) only <em>2</em>-AG exerts neuroprotective effects in OHSCs; (<em>2</em>) reduction of IB(4)(+) microglial cells is not a neuroprotective event per se and involves other CB receptors than the CB<em>2</em> receptor; (3) the discrepancy in the neuroprotective effects of CBs observed in vivo and in our in vitro model system may underline the functional relevance of invading monocytes and T-lymphocytes that are absent in OHSCs.

Endocannabinoids like anandamide and <em>2</em>-<em>arachidonoylglycerol</em> bind and activate type-1 (CB1R) and type-<em>2</em> (CB<em>2</em>R) cannabinoid receptors, two inhibitory G protein-coupled receptors (GPCRs) that are localized in the central nervous system and in peripheral tissues. The biological actions of these lipids are controlled through not yet fully characterized cellular mechanisms that regulate the release of endocannabinoids from membrane precursors, their uptake by cells, and their intracellular disposal. The transport of anandamide through the plasma membrane is saturable and energy-independent, and might occur through a putative anandamide membrane transporter. Altogether anandamide and <em>2</em>-<em>arachidonoylglycerol</em>, their congeners and the proteins that bind, transport, synthesize and hydrolyze these lipids, form the "endocannabinoid system". Accumulating evidence shows that CB1R (but not CB<em>2</em>R) binding and signaling, as well as anandamide transport, are under the control of lipid rafts (LRs), plasma membrane subdomains which modulate the activity of a number of GPCRs. Here we summarize the main features of the endocannabinoid system and LRs, in order to put the functional and structural effects of LRs on CB receptors, AEA transport and endocannabinoid signaling in a better focus. We outline the structural determinants that might explain the differential sensitivity of cannabic receptors towards raft integrity, and propose a general model to explain the dependence of endocannabinoid system on LRs. Finally, we also discuss the possible exploitation of LRs-targeted drugs as novel therapeutics for the treatment of endocannabinoid system-related pathologies.

Endogenous cannabinoids activate cannabinoid receptors in the brain and elicit mood-altering effects. Parallel effects (eg, anxiolysis, analgesia, sedation) may be elicited by osteopathic manipulative treatment (OMT), and previous research has shown that the endorphin system is not responsible for OMT's mood-altering effects. The authors investigate whether OMT generated cannabimimetic effects for 31 healthy subjects in a dual-blind, randomized controlled trial that measured changes in subjects' scores on the 67-item Drug Reaction Scale (DRS). Chemical ionization gas chromatography and mass spectrometry were also used to determine changes in serum levels of anandamide (AEA), <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), and oleylethanolamide (OEA). In subjects receiving OMT, posttreatment DRS scores increased significantly for the cannabimimetic descriptors good, high, hungry, light-headed, and stoned, with significant score decreases for the descriptors inhibited, sober, and uncomfortable. Mean posttreatment AEA levels (8.01 pmol/mL) increased 168% over pretreatment levels (<em>2</em>.99 pmol/mL), mean OEA levels decreased <em>2</em>7%, and no changes occurred in <em>2</em>-AG levels in the group receiving OMT. Subjects in the sham manipulative treatment group recorded mixed DRS responses, with both increases and decreases in scores for cannabimimetic and noncannabimimetic descriptors and no changes in sera levels. When changes in serum AEA were correlated with changes in subjects' DRS scores, increased AEA correlated best with an increase for the descriptors cold and rational, and decreased sensations for the descriptors bad, paranoid, and warm. The authors propose that healing modalities popularly associated with changes in the endorphin system, such as OMT, may actually be mediated by the endocannabinoid system.

The CB1 cannabinoid receptor is a G protein coupled receptor that is widely expressed throughout the brain. The endogenous ligands for the CB1 receptor (endocannabinoids) are N-arachidonylethanolamine and <em>2</em>-<em>arachidonoylglycerol</em>; together the endocannabinoids and CB1R subserve activity dependent, retrograde inhibition of neurotransmitter release in the brain. Deficiency of CB1 receptor signaling is associated with anhedonia, anxiety, and persistence of negative memories. CB1 receptor-endocannabinoid signaling is activated by stress and functions to buffer or dampen the behavioral and endocrine effects of acute stress. Its role in regulation of neuronal responses is more complex. Chronic variable stress exposure reduces endocannabinoid-CB1 receptor signaling and it is hypothesized that the resultant deficiency in endocannabinoid signaling contributes to the negative consequences of chronic stress. On the other hand, repeated exposure to the same stress can sensitize CB1 receptor signaling, resulting in dampening of the stress response. Data are reviewed that support the hypothesis that CB1 receptor signaling is stress responsive and that maintaining robust endocannabinoid/CB1 receptor signaling provides resilience against the development of stress-related pathologies.

The two major endocannabinoid transmitters, anandamide (AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), are degraded by distinct enzymes in the nervous system, fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), respectively. FAAH and MAGL inhibitors cause elevations in brain AEA and <em>2</em>-AG levels, respectively, and reduce pain, anxiety, and depression in rodents without causing the full spectrum of psychotropic behavioral effects observed with direct cannabinoid receptor-1 (CB1) agonists. These findings have inspired the development of several classes of endocannabinoid hydrolase inhibitors, most of which have been optimized to show specificity for either FAAH or MAGL or, in certain cases, equipotent activity for both enzymes. Here, we investigate an unusual class of O-hydroxyacetamide carbamate inhibitors and find that individual compounds from this class can serve as selective FAAH or dual FAAH/MAGL inhibitors in vivo across a dose range (0.1<em>2</em>5-1<em>2</em>.5 mg kg(-1)) suitable for behavioral studies. Competitive and click chemistry activity-based protein profiling confirmed that the O-hydroxyacetamide carbamate SA-57 is remarkably selective for FAAH and MAGL in vivo, targeting only one other enzyme in brain, the additional <em>2</em>-AG hydrolase ABHD6. These data designate O-hydroxyacetamide carbamates as a versatile chemotype for creating endocannabinoid hydrolase inhibitors that display excellent in vivo activity and tunable selectivity for FAAH-anandamide versus MAGL (and ABHD6)-<em>2</em>-AG pathways.