Several lines of experimental and clinical evidence point to a close relationship between cannabis, the endogenous cannabinoid system, and schizophrenia. A variety of animal and human studies found a dysregulation of endocannabinoid signalling in psychosis. Elevated anandamide levels in schizophrenia patients that are negatively correlated with psychotic symptomatology indicate a protective role, whereas <em>2</em>-<em>arachidonoylglycerol</em> appears to counteract psychosis-related cognitive impairments. Thus, pharmacological manipulation of the endogenous cannabinoid system might be associated with potential antipsychotic properties. In the present systematic review, both preclinical studies using different animal models of psychosis as well as clinical trials investigating the antipsychotic effects of both cannabidiol and rimonabant are presented together with the possible underlying mechanisms of action. The results predominantly confirm the hypothesis of an antipsychotic activity of both cannabinoids. In comparison, cannabidiol appears to be superior to rimonabant with a pharmacological profile similar to atypical antipsychotic drugs.

The endocannabinoid (eCB) system, consisting of eCB ligands and the type 1 cannabinoid receptor (CB1R), subserves retrograde, activity-dependent synaptic plasticity in the brain. eCB signaling occurs "on-demand," thus the processes regulating synthesis, mobilization and degradation of eCBs are also primary mechanisms for the regulation of CB1R activity. The eCBs, N-arachidonylethanolamine (AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), are poorly soluble in water. We hypothesize that their aqueous solubility, and, therefore, their intracellular and transcellular distribution, are facilitated by protein binding. Using in silico docking studies, we have identified the nonspecific lipid binding protein, sterol carrier protein <em>2</em> (SCP-<em>2</em>), as a potential AEA binding protein. The docking studies predict that AEA and AM404 associate with SCP-<em>2</em> at a putative cholesterol binding pocket with ∆G values of -3.6 and -4.6 kcal/mol, respectively. These values are considerably higher than cholesterol (-6.6<em>2</em> kcal/mol) but consistent with a favorable binding interaction. In support of the docking studies, SCP-<em>2</em>-mediated transfer of cholesterol in vitro is inhibited by micromolar concentrations of AEA; and heterologous expression of SCP-<em>2</em> in HEK <em>2</em>93 cells increases time-related accumulation of AEA in a temperature-dependent fashion. These results suggest that SCP-<em>2</em> facilitates cellular uptake of AEA. However, there is no effect of SCP-<em>2</em> transfection on the cellular accumulation of AEA determined at equilibrium or the IC50 values for AEA, AM404 or <em>2</em>-AG to inhibit steady state accumulation of radiolabelled AEA. We conclude that SCP-<em>2</em> is a low affinity binding protein for AEA that can facilitate its cellular uptake but does not contribute significantly to intracellular sequestration of AEA.

The brain is enriched in arachidonic acid (ARA) and docosahexaenoic acid (DHA), long-chain polyunsaturated fatty acids (LCPUFAs) of the n-6 and n-3 series, respectively. Both are essential for optimal brain development and function. Dietary enrichment with DHA and other long-chain n-3 PUFA, such as eicosapentaenoic acid (EPA), has shown beneficial effects on learning and memory, neuroinflammatory processes, and synaptic plasticity and neurogenesis. ARA, DHA and EPA are precursors to a diverse repertoire of bioactive lipid mediators, including endocannabinoids. The endocannabinoid system comprises cannabinoid receptors, their endogenous ligands, the endocannabinoids, and their biosynthetic and degradation enzymes. Anandamide (AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) are the most widely studied endocannabinoids and are both derived from phospholipid-bound ARA. The endocannabinoid system also has well-established roles in neuroinflammation, synaptic plasticity and neurogenesis, suggesting an overlap in the neuroprotective effects observed with these different classes of lipids. Indeed, growing evidence suggests a complex interplay between n-3 and n-6 LCPUFA and the endocannabinoid system. For example, long-term DHA and EPA supplementation reduces AEA and <em>2</em>-AG levels, with reciprocal increases in levels of the analogous endocannabinoid-like DHA and EPA-derived molecules. This review summarises current evidence of this interplay and discusses the therapeutic potential for brain protection and repair.

The endocannabinoid system (ECS) controls energy balance by regulating both energy intake and energy expenditure. Endocannabinoid levels are elevated in obesity suggesting a potential causal relationship. This study aimed to elucidate the rate of dysregulation of the ECS, and the metabolic organs involved, in diet-induced obesity. Eight groups of age-matched male C57Bl/6J mice were randomized to receive a chow diet (control) or receive a high fat diet (HFD, 45% of calories derived from fat) ranging from 1 day up to 18 weeks before euthanasia. Plasma levels of the endocannabinoids <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) and anandamide (N-arachidonoylethanolamine, AEA), and related <i>N</i>-acylethanolamines, were quantified by UPLC-MS/MS and gene expression of components of the ECS was determined in liver, muscle, white adipose tissue (WAT) and brown adipose tissue (BAT) during the course of diet-induced obesity development. HFD feeding gradually increased <em>2</em>-AG (+13<em>2</em>% within 4 weeks, <i>P</i> < 0.05), accompanied by upregulated expression of its synthesizing enzymes <i>Daglα</i> and β in WAT and BAT. HFD also rapidly increased AEA (+81% within 1 week, <i>P</i> < 0.01), accompanied by increased expression of its synthesizing enzyme <i>Nape-pld</i>, specifically in BAT. Interestingly, <i>Nape-pld</i> expression in BAT correlated with plasma AEA levels (<i>R</i>^<em>2</em> = 0.171, β = 0.<em>2</em>76, <i>P</i> < 0.001). We conclude that a HFD rapidly activates adipose tissue depots to increase the synthesis pathways of endocannabinoids that may aggravate the development of HFD-induced obesity.

The endocannabinoid system has emerged as a key player in the control of eating. Endocannabinoids, including <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) and anandamide (AEA), modulate neuronal activity via cannabinoid 1 receptors (CB1Rs) in multiple nuclei of the hypothalamus to induce or inhibit food intake depending on nutritional and hormonal status, suggesting that endocannabinoids may act in the hypothalamus to integrate different types of signals informing about the animal's energy needs. In the mesocorticolimbic system, (endo)cannabinoids modulate synaptic transmission to promote dopamine release in response to palatable food. In addition, (endo)cannabinoids act within the nucleus accumbens to increase food's hedonic impact; although this effect depends on activation of CB1Rs at excitatory, but not inhibitory inputs in the nucleus accumbens. While hyperactivation of the endocannabinoid system is typically associated with overeating and obesity, much evidence has emerged in recent years suggesting a more complicated system than first thought - endocannabinoids promote or suppress feeding depending on cell and input type, or modulation by various neuronal or hormonal signals. This review presents our latest knowledge of the endocannabinoid system in non-homeostatic and homeostatic feeding circuits. In particular, we discuss the functional role and cellular mechanism of action by endocannabinoids within the hypothalamus and mesocorticolimbic system, and how these are modulated by neuropeptide signals related to feeding. In light of recent advances and complexity in the field, we review cannabinoid-based therapeutic strategies for the treatment of obesity and how peripheral restriction of CB1R antagonists may provide a different mechanism of weight loss without the central adverse effects. This article is part of the Special Issue entitled "A New Dawn in Cannabinoid Neurobiology".

Lipid metabolism dysregulation underlies chronic pathologies such as obesity, diabetes and cancer. Besides their role in structure and energy storage, lipids are also important signalling molecules regulating multiple biological functions. Thus, understanding the precise lipid metabolism enzymatic steps that are altered in some pathological conditions is helpful for designing better treatment strategies. Several monoacylglycerol (MAG) species are only recently being recognized as signalling lipid molecules in different tissues. Recent studies indicated the importance of the ubiquitously expressed serine hydrolase α/β-hydrolase domain 6 (ABHD6), which is a MAG hydrolase, in regulating signalling competent MAG in both central and peripheral tissues. The central and peripheral function of the endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em>, which is a <em>2</em>-MAG, and its breakdown by both ABHD6 and classical MAG lipase has been well documented. ABHD6 and its substrate MAG appear to be involved in the regulation of various physiological and pathological processes including insulin secretion, adipose browning, food intake, neurotransmission, autoimmune disorders, neurological and metabolic diseases as well as cancer. Diverse cellular targets such as mammalian unc13-1 (Munc13-1), PPARs, GPR119 and CB1/<em>2</em> receptors, for MAG-mediated signalling processes have been proposed in different cell types. The purpose of this review is to provide a comprehensive summary of the current state of knowledge regarding ABHD6/MAG signalling and its possible therapeutic implications.

MAGL (monoacylglycerol lipase) is an enzyme that hydrolyzes the endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> and regulates the production of arachidonic acid and prostaglandins-substances that mediate tissue inflammatory response. Here, we have studied the effects of the selective MAGL inhibitors JZL184 and MJN110 and their underlying molecular mechanisms on 3 different experimental models of focal cerebral ischemia.

SHR (spontaneously hypertensive rats) and normotensive WKY (Wistar Kyoto) rats were subject to an intracortical injection of the potent vasoconstrictor endothelin-1, permanent occlusion of a distal segment of the middle cerebral artery via craniectomy, or transient occlusion of the middle cerebral artery by the intraluminal suture method. JZL184 or MJN110 was administered 60 minutes after focal cerebral ischemia. Infarct volumes, hemispheric swelling, and functional outcomes were assessed between days 1 to <em>2</em>8 by magnetic resonance imaging, histology, and behavioral tests.

Pharmacological inhibition of MAGL significantly attenuated infarct volume and hemispheric swelling. MAGL inhibition also ameliorated sensorimotor deficits, suppressed inflammatory response, and decreased the number of degenerating neurons. These beneficial effects of MAGL inhibition were not fully abrogated by selective antagonists of cannabinoid receptors, indicating that the anti-inflammatory effects are caused by inhibition of eicosanoid production rather than by activation of cannabinoid receptors.

Our results suggest that MAGL may contribute to the pathophysiology of focal cerebral ischemia and is thus a promising therapeutic target for the treatment of ischemic stroke.

The purpose of this study was to examine the interaction between the endogenous opioid and endocannabinoid (eCB) systems in a pain modulatory process known as exercise-induced hypoalgesia (EIH).

Randomized controlled trial.

Clinical research unit in a hospital.

Fifty-eight healthy men and women (mean age = <em>2</em>1 ± 3 years) participated in this study.

Participants were administered (randomized, double-blind, counterbalanced procedure) an opioid antagonist (i.e., naltrexone) and a placebo prior to performing pain testing and isometric exercise.

Results indicated that <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) and <em>2</em>-oleoylglycerol (<em>2</em>-OG) increased significantly (P < 0.05) following exercise in both placebo and naltrexone conditions. In comparison, N-arachidonylethanolamine (AEA) and oleoylethanolamine (OEA) increased significantly (P < 0.05) following exercise in the placebo condition but not the naltrexone condition. There were no significant (P>> 0.05) differences in palmitolethanolamine (PEA) between the placebo and naltrexone conditions.

As reductions in pain (i.e., EIH) were observed following both conditions, these results suggest that the opioid system may not be the primary system involved in exercise-induced hypoalgesia and that <em>2</em>-AG and <em>2</em>-OG could contribute to nonopioid exercise-induced hypoalgesia. Moreover, as exercise-induced increases in AEA and OEA were blocked by naltrexone pretreatment, this suggests that the opioid system may be involved in the increase of AEA and OEA following exercise.

BACKGROUND

Several lines of evidence point to the potential role of the endocannabinoid system in female sexual functioning. These include results from studies describing the subjective effects of exogenous cannabinoids on sexual functioning in humans and the observable effects of exogenous cannabinoids on sexual functioning in other species, as well as results from studies investigating the location of cannabinoid receptors in the brain and periphery, and the effects of cannabinoid receptor activation on neurotransmitters implicated in sexual functioning. While these lines of research suggest a role for the endocannabinoid system in female sexual functioning, no studies investigating the relationship between concentrations of endogenous cannabinoids (i.e., arachidonoylethanolamide [AEA] and <em>2</em>-<em>arachidonoylglycerol</em> [<em>2</em>-AG]) and sexual functioning have been conducted in any species.

OBJECTIVE

To measure circulating endocannabinoid concentrations in relation to subjective and physiological indices of sexual arousal in women (N = <em>2</em>1).

METHODS

Serum endocannabinoid (AEA and <em>2</em>-AG) concentrations were measured immediately prior to, and immediately following, viewing of neutral (control) and erotic (experimental) film stimuli in a repeated measures design. Physiological sexual arousal was measured via vaginal photoplethysmography. Subjective sexual arousal was measured both continuously and noncontinuously. Pearson's correlations were used to investigate the relationships between endocannabinoid concentrations and sexual arousal.

METHODS

Changes in AEA and <em>2</em>-AG concentrations from pre- to post-film and in relation to physiological and subjective indices of sexual arousal.

RESULTS

Results revealed a significant relationship between endocannabinoid concentrations and female sexual arousal, whereby increases in both physiological and subjective indices of sexual arousal were significantly associated with decreases in AEA, and increases in subjective indices of sexual arousal were significantly associated with decreases in <em>2</em>-AG.

CONCLUSIONS

These findings support the hypothesis that the endocannabinoid system is involved in female sexual functioning, with implications for furthering understanding of the biological mechanisms underlying female sexual functioning.

There are conflicting reports in the literature as to whether palmitoylethanolamide affects the function of mast cell-related cell lines in vitro, in contrast to the well-documented effects of this compound upon mast cell function in vivo. In the present study, we have reinvestigated the effects of palmitoylethanolamide upon antigen-induced release of [3H]serotonin and beta-hexosaminidase from rat basophilic leukemia RBL-<em>2</em>H3 cells and compared these effects with those of <em>2</em>-<em>arachidonoylglycerol</em>, anandamide and R1-methanandamide. RBL-<em>2</em>H3 cells were sensitized with a monoclonal anti-DNP IgE, after which they were stimulated with antigen (DNP-HSA). Palmitoylethanolamide produced a small, but significant reduction in antigen-stimulated [3H]serotonin release at high concentrations, whereas anandamide was without effect. In contrast, <em>2</em>-<em>arachidonoylglycerol</em> and methanandamide increased the antigen-stimulated release of both [3H]serotonin and beta-hexosaminidase. It is concluded that in RBL-<em>2</em>H3 cells, these cannabimimetic fatty acid derivatives do not have potent stabilizing effects upon antigen-induced degranulation.

Binge drinking is a significant problem in adolescent populations, and because of the reciprocal interactions between ethanol (EtOH) consumption and the endocannabinoid (eCB) system, we sought to determine if adolescent EtOH intake altered the localization and function of the cannabinoid 1 (CB₁) receptors in the adult brain. Adolescent mice were exposed to a 4-day-per week drinking in the dark (DID) procedure for a total of 4 weeks and then tested after a <em>2</em>-week withdrawal period. Field excitatory postsynaptic potentials (fEPSPs), evoked by medial perforant path (MPP) stimulation in the dentate gyrus molecular layer (DGML), were significantly smaller. Furthermore, unlike control animals, CB₁ receptor activation did not depress fEPSPs in the EtOH-exposed animals. We also examined a form of excitatory long-term depression that is dependent on CB₁ receptors (eCB-eLTD) and found that it was completely lacking in the animals that consumed EtOH during adolescence. Histological analyses indicated that adolescent EtOH intake significantly reduced the CB₁ receptor distribution and proportion of immunopositive excitatory synaptic terminals in the medial DGML. Furthermore, there was decreased binding of [³⁵S]guanosine-5*-O-(3-thiotriphosphate) ([³⁵S] GTPγS) and the guanine nucleotide-binding (G) protein Gαi<em>2</em> subunit in the EtOH-exposed animals. Associated with this, there was a significant increase in monoacylglycerol lipase (MAGL) mRNA and protein in the hippocampus of EtOH-exposed animals. Conversely, deficits in eCB-eLTD and recognition memory could be rescued by inhibiting MAGL with JZL184. These findings indicate that repeated exposure to EtOH during adolescence leads to long-term deficits in CB₁ receptor expression, eCB-eLTD, and reduced recognition memory, but that these functional deficits can be restored by treatments that increase endogenous <em>2</em>-<em>arachidonoylglycerol</em>.

OBJECTIVE

To assess the levels of endocannabinoids and cannabinoid receptors (CB) 1 and <em>2</em> in women with polycystic ovary syndrome (PCOS).

METHODS

Case-control study.

METHODS

University teaching hospital.

METHODS

In total, <em>2</em>0 women with PCOS and <em>2</em>0 healthy women in a control group, who were matched for body mass index and age, were enrolled in this study.

METHODS

The homeostasis model index was used to assess insulin resistance.

METHODS

Omental adipose tissue and human peripheral blood mononuclear cells (PBMCs) from PCOS and the controls were analyzed using real-time polymerase chain reactions for the expressions of CB1 and CB<em>2</em>. The levels of endocannabinoids were analyzed using high-performance liquid chromatography.

RESULTS

The levels of anandamide and <em>2</em>-<em>arachidonoylglycerol</em>, and the expression of CB1 and CB<em>2</em> mRNA (messenger ribonucleic acid) in the PBMCs were significantly higher in the women with PCOS than in the women serving as controls. We found that expression of CB1, but not CB<em>2</em>, in adipose tissue was significantly higher in the women with, vs. without, PCOS. The expressions of CB1 mRNA and endocannabinoids showed a significant positive correlation with <em>2</em>-hour glucose and insulin levels <em>2</em> hours after glucose loading in the PBMCs and adipose tissue.

CONCLUSIONS

Activation of endocannabinoids and overexpression of cannabinoid receptors, especially CB1, may be associated with insulin resistance in women with PCOS.

Human monoacylglycerol lipase (hMGL) regulates endocannabinoid signaling primarily by deactivating the lipid messenger <em>2</em>-<em>arachidonoylglycerol</em>. Agents that carbamylate hMGLs catalytic Ser(1<em>2</em><em>2</em>) constitute a leading class of therapeutically promising hMGL inhibitors. We have applied peptide-level hydrogen/deuterium exchange mass spectrometry to characterize hMGL's conformational responses to two potent carbamylating inhibitors, AM6580 (irreversible) and AM6701 (slowly reversible). A dynamic, solvent-exposed lid domain is characteristic of hMGL's solution conformation. Both hMGL inhibitors restricted backbone enzyme motility in the active-site region and increased substrate binding-pocket solvent exposure. Covalent reaction of AM6580 with hMGL generates a bulkier carbamylated Ser(1<em>2</em><em>2</em>) residue as compared to the more discrete Ser(1<em>2</em><em>2</em>) modification by AM6701, a difference reflected in AM6580's more pronounced effect upon hMGL conformation. We demonstrate that structurally distinct carbamylating hMGL inhibitors generate particular conformational ensembles characterized by region-specific hMGL dynamics. By demonstrating the distinctive influences of two hMGL inhibitors on enzyme conformation, this study furthers our understanding at the molecular level of the dynamic features of hMGL interaction with small-molecule ligands.

<em>2</em>-<em>Arachidonoylglycerol</em> (<em>2</em>-AG) is the most abundant endocannabinoid, and it plays a critical role in cannabinoid receptor-mediated cell signaling. Although <em>2</em>-AG was shown to induce ERK activation via the cannabinoid receptor 1 (CB1), only a nonspecific CB receptor agonist and antagonist was used in those studies. Whether cannabinoid receptor <em>2</em> (CB<em>2</em>) is involved in <em>2</em>-AG-induced ERK activation is still unclear. Moreover, whether <em>2</em>-AG is involved in mediation of AP-1 activity and cell transformation is also not known. In the present study, we show that <em>2</em>-AG stimulates AP-1-dependent transcriptional activity and enhances epidermal growth factor-induced cell transformation in mouse epidermal JB6 P+ Cl41 cells. Using JB6 P+ C141 cells, stably transfected with an AP-1 luciferase reporter, we found that 10 microm <em>2</em>-AG induced up to a 3-fold stimulation of AP-1 transcriptional activity. The AP-1 stimulation appeared to be mediated by ERK but not JNK or p38 kinase. PD98059, a specific inhibitor of MEK1, almost completely blocked <em>2</em>-AG-induced ERK phosphorylation and AP-1 activation. Using CB1/<em>2</em>-/- murine embryonic fibroblasts, we present the first direct evidence that both cannabinoid receptors 1 and <em>2</em> (CB1/<em>2</em>) are involved in <em>2</em>-AG-induced ERK activation. <em>2</em>-AG could not stimulate ERK phosphorylation or Fyn kinase activity in dominant negative Fyn. In addition, the Fyn inhibitor PP<em>2</em> blocked <em>2</em>-AG-induced Fyn kinase activity and ERK phosphorylation and activity. Small interfering RNA Fyn also suppressed <em>2</em>-AG-induced ERK phosphorylation. Interestingly, <em>2</em>-AG enhanced epidermal growth factor-induced AP-1 DNA binding and cell transformation. Taken together, our data provide direct evidence suggesting that <em>2</em>-AG may have a novel role in cell transformation and carcinogenesis in a signaling pathway involving CB1/<em>2</em> and activation of Fyn, ERKs, and AP-1.

We report the first synthesis of <em>2</em>-thioglycerol and S-arachidonoyl-<em>2</em>-thioglycerol (the thioester analog of the endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em>) in an eight or nine step procedure with a yield of approximately <em>2</em>5% and establish the use of this substrate for maleimide-based fluorescent and dithiobis(<em>2</em>-nitrobenzoic acid)-based colorimetric assays of human recombinant monoacylglycerol (MAG) lipase (hMAGL) and human brain membrane MAG hydrolase activity. Inhibitor structure-activity relationships observed here for hMAGL and <em>2</em>-ATG correlate well (r(<em>2</em>)=0.93, n=9) with earlier findings for mouse brain MAG hydrolase with non-thiol substrates.

Schizophrenia is a complex psychiatric disorder strongly associated with substance use disorders. Theoretically, schizophrenia and SUD may share endocannabinoid alterations in the brain reward system. The main endocannabinoids, anandamide, and <em>2</em>-<em>arachidonoylglycerol</em>, are lipids which bind cannabinoid receptors. Oleoylethanolamide (OEA), a fatty-acid ethanolamide, binds peroxisome proliferator-activated receptors. The endocannabinoid system has been shown to be impaired in schizophrenia, and recently, our group has shown that schizophrenia patients with SUD have elevated peripheral levels of anandamide and OEA that do not normalize after 3-month treatment with quetiapine. Objective For comparative purposes, we aimed to measure endocannabinoids in non-psychosis substance abusers and non-abusing schizophrenia patients. Methods Using liquid chromatography and mass spectrometry, we measured plasma levels of anandamide and OEA in non-psychosis SUD patients, non-abusing schizophrenia patients, and healthy controls. In an open-label manner, all patients received 1<em>2</em>-week treatment with quetiapine. Results Anandamide and OEA were reduced in substance abusers without schizophrenia, relative to healthy controls (p < 0.05). Both endocannabinoids were unchanged in non-abusing schizophrenia patients. After quetiapine, anandamide, and OEA levels remained significantly reduced the SUD group (p < 0.05). Discussion Taken together with results of our previous study performed in dual-diagnosis patients, our results suggest that peripheral anandamide and OEA levels are impaired in patients with SUD in opposite ways according to the presence or absence of schizophrenia. Endocannabinoid alterations did not change with treatment, suggesting that they are trait markers. Further studies are necessary to understand the role of endocannabinoids in substance abusers with and without schizophrenia and to examine therapeutic implications.

Anandamide (N -arachidonoylethanolamine, AEA) is a major endocannabinoid, shown to impair mouse pregnancy and embryo development and to induce apoptosis in blastocysts. Here, we review the roles of AEA, of the AEA-binding cannabinoid (CB) receptors, of the selective AEA membrane transporter (AMT), and of the AEA-hydrolyzing enzyme fatty acid amide hydrolase (FAAH), in human gestation. In particular, we discuss the interplay between the endocannabinoid system and the hormone-cytokine array involved in the control of human pregnancy, showing that the endocannabinoids take part in the immunological adaptation occurring during early pregnancy. In this line, we discuss the critical role of FAAH in human peripheral lymphocytes, showing that the expression of this enzyme is regulated by progesterone, Th1 and Th<em>2</em> cytokines, which also regulate fertility. Moreover, we show that AEA and the other endocannabinoid, <em>2</em>-<em>arachidonoylglycerol</em>, inhibit the release of the fertility-promoting cytokine leukemia inhibitory factor from human lymphocytes. Taken together, low FAAH and consistently high blood levels of AEA, but not CB receptors or AMT, can be early (<8 weeks of gestation) markers of spontaneous abortion, potentially useful as diagnostic tools for large-scale, routine monitoring of gestation in humans.

In the mammalian central nervous system, monoacylglycerol lipase (MGL) is principally responsible for inactivating the endocannabinoid signaling lipid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) and modulates cannabinoid-1 receptor (CB1R) desensitization and signal intensity. MGL is also a drug target for diseases in which CB1R stimulation may be therapeutic. To inform the design of human MGL (hMGL) inhibitors, we have engineered a Leu(Leu(169);Leu(176))-to-Ser(Ser(169);Ser(176)) double hMGL mutant (sol-hMGL) which exhibited enhanced solubility properties, and we further mutated this variant by substituting its catalytic-triad Ser(1<em>2</em><em>2</em>) with Cys (sol-S-hMGL). The hMGL variants hydrolyzed both <em>2</em>-AG and a fluorogenic reporter substrate with comparable affinities. Our results suggest that the hMGL cysteine mutant maintains the same overall architecture as wild-type hMGL. The results also underscore the superior nucleophilic nature of the reactive catalytic Ser(1<em>2</em><em>2</em>) residue as compared to that of Cys(1<em>2</em><em>2</em>) in the sol-S-hMGL mutant and suggest that the nucleophilic character of the Cys(1<em>2</em><em>2</em>) residue is not commensurately enhanced within the three dimensional architecture of hMGL. The interaction of the sol-hMGL variants with the irreversible inhibitors AM6580 and N-arachidonylmaleimide (NAM) and the reversible inhibitor AM10<em>2</em>1<em>2</em> was profiled. LC/MS analysis of tryptic digests from sol-S-hMGL directly demonstrate covalent modification of this variant by NAM and AM6580, consistent with enzyme thiol alkylation and carbamoylation, respectively. These data provide insight into hMGL catalysis, the key role of the nucleophilic character of Ser(1<em>2</em><em>2</em>), and the mechanisms underlying hMGL inhibition by different classes of small molecules.

Cannabis continues to be the most accessible and popular illicit recreational drug. Whereas current data link adolescence cannabinoid exposure to increased risk for dependence on other drugs, depression, anxiety disorders and psychosis, the mechanism(s) underlying these adverse effects remains controversial. Here we show in a mouse model of female adolescent cannabinoid exposure deficient endocannabinoid (eCB)-mediated signaling and presynaptic forms of long-term depression at adult central glutamatergic synapses in the prefrontal cortex. Increasing endocannabinoid levels by blockade of monoacylglycerol lipase, the primary enzyme responsible for degrading the endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), with the specific inhibitor JZL 184 ameliorates eCB-LTD deficits. The observed deficit in cortical presynaptic signaling may represent a neural maladaptation underlying network instability and abnormal cognitive functioning. Our study suggests that adolescent cannabinoid exposure may permanently impair brain functions, including the brain's intrinsic ability to appropriately adapt to external influences.

Although the active component of cannabis Delta9-THC was isolated by our group 35 years ago, until recently its mode of action remained obscure. In the last decade it was established that Delta9-THC acts through specific receptors - CB1 and CB<em>2</em> - and mimics the physiological activity of endogenous cannabinoids of two types, the best known representatives being arachidonoylethanolamide (anandamide) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG). THC is officially used against vomiting caused by cancer chemotherapy and for enhancing appetite, particularly in AIDS patients. Illegally, usually by smoking marijuana, it is used for ameliorating the symptoms of multiple sclerosis, against pain, and in a variety of other diseases. A synthetic cannabinoid, HU-<em>2</em>11, is in advanced clinical tests against brain damage caused by closed head injury. It may prove to be valuable against stroke and other neurological diseases.

BACKGROUND

Insulin resistance is highly prevalent in patients with chronic hepatitis C (CHC) and to some extent accounts for fibrosis and reducing viral eradication. Activated cannabinoid 1 receptor (CB1R) signaling has been implicated in the development of phenotypes associated with insulin resistance and steatosis. We investigated the role of the endocannabinoid system in glucose metabolism disorders induced by hepatitis C virus (HCV) replication.

METHODS

Human hepatic stellate cells (HSC; LX-<em>2</em> cells) were co-cultured with Huh-7.5 cells or Huh-7.5 cells harboring HCV replicon (replicon cells). Endocannabinoid levels were then measured by liquid chromatography/mass spectrometry. The expression of CB1R and its downstream glucose metabolism genes in hepatocytes were determined by real-time PCR and Western blot. Glucose uptake by hepatocytes and glucose production were measured. Glucose metabolism tests and measurements of HCV RNA levels and nonstructural protein 5A (NS5A) levels were taken after treatment with CB1R agonist arachidonyl-<em>2</em>-chloroethanolamide (ACEA) or antagonist AM<em>2</em>51.

RESULTS

Compared to the co-culture with Huh-7.5 cells, the level of <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) and the CB1R mRNA and protein levels increased in the co-culture of LX-<em>2</em> cells with replicon cells. The activation of CB1R decreased AMP-activated protein kinase (AMPK) phosphorylation, inhibited cell surface expression of glucose transporter <em>2</em> (GLUT<em>2</em>), and suppressed cellular glucose uptake; furthermore, it increased cyclic AMP response element-binding protein H (CREBH), then up-regulated phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) genes and down-regulated the glucokinase (GK) gene, thus promoting glucose production. Interferon treatment restored the aforementioned changes. CB1R antagonist improved glucose metabolism disorders by an increase in glucose uptake and a decrease in glucose production, and inhibited HCV replication.

CONCLUSIONS

HCV replication may not only increase the <em>2</em>-AG content, but may also up-regulate the expression of CB1R of hepatocytes, then change the expression profile of glucose metabolism-related genes, thereby causing glucose metabolism disorders of hepatocytes and promoting HCV replication. Treatment with CB1R antagonist improved glucose metabolism disorders and inhibited viral genome replication.

Anandamide (N-arachidonoylethanolamine) has been identified as an endogenous ligand of the G-protein coupled cannabinoid CB(1) receptor. Recent studies have postulated the existence of carrier-mediated anandamide transport which is involved in the termination of the biological effects of anandamide. A membrane bound amidohydrolase (fatty acid amide hydrolase, FAAH), located intracellulary, hydrolyzes and inactivates anandamide and other endogenous cannabinoids such as <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG). Structure-activity relationships (SARs) for endocannabinoid interaction with the CB receptors, the anandamide transporter and FAAH are currently emerging in the literature. This review considers the divergences between these SARs and focuses upon the conformational implications for endocannabinoid recognition at each of these biological targets.

Monoacylglycerol lipase (MGL) is primarily responsible for the hydrolysis of <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), an endocannabinoid with full agonist activity at both cannabinoid receptors. Increased tissue <em>2</em>-AG levels consequent to MGL inhibition are considered therapeutic against pain, inflammation, and neurodegenerative disorders. However, the lack of MGL structural information has hindered the development of MGL-selective inhibitors. Here, we detail a fully refined homology model of MGL which preferentially identifies MGL inhibitors over druglike noninhibitors. We include for the first time insight into the active-site geometry and potential hydrogen-bonding interactions along with molecular dynamics simulations describing the opening and closing of the MGL helical-domain lid. Docked poses of both the natural substrate and known inhibitors are detailed. A comparison of the MGL active-site to that of the other principal endocannabinoid metabolizing enzyme, fatty acid amide hydrolase, demonstrates key differences which provide crucial insight toward the design of selective MGL inhibitors as potential drugs.

Anandamide (N -arachidonoylethanolamine) was the first ligand to be identified as an endogenous ligand of the G-protein coupled cannabinoid CB1 receptor. Subsequently, two other fatty acid ethanolamides, N -homo- gamma -linolenylethanolamine and N -7,10,13,16-docosatetraenylethanolamine were identified as endogenous cannabinoid ligands. A fatty acid ester, <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), and a fatty acid ether, <em>2</em>-arachidonyl glyceryl ether also have been isolated and shown to be endogenous cannabinoid ligands. Recent studies have postulated the existence of carrier-mediated anandamide transport that is essential for termination of the biological effects of anandamide. A membrane bound amidohydrolase (fatty acid amide hydrolase, FAAH), located intracellularly, hydrolyzes and inactivates anandamide and other endogenous cannabinoids such as <em>2</em>-AG. <em>2</em>-AG has also been proposed to be an endogenous CB<em>2</em> ligand. Structure-activity relationships (SARs) for endocannabinoid interaction with the CB receptors are currently emerging in the literature. This review considers cannabinoid receptor SAR developed to date for the endocannabinoids with emphasis upon the conformational implications for endocannabinoid recognition at the cannabinoid receptors.