Depolarization-induced suppression of inhibition (DSI) or excitation (DSE) is a well-known form of endocannabinoid-mediated short-term plasticity that is induced by postsynaptic depolarization. It is generally accepted that DSI/DSE is triggered by Ca(<em>2</em>+) influx through voltage-gated Ca(<em>2</em>+) channels. It is also demonstrated that DSI/DSE is mediated by <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG). However, how Ca(<em>2</em>+) induces <em>2</em>-AG production is still unclear. In the present study, we investigated molecular mechanisms underlying the Ca(<em>2</em>+)-driven <em>2</em>-AG production. Using cannabinoid-sensitive inhibitory synapses of cultured hippocampal neurons, we tested several inhibitors for enzymes that are supposed to be involved in <em>2</em>-AG metabolism. The chemicals we tested include inhibitors for phospholipase C (U731<em>2</em><em>2</em> and ET-18), diacylglycerol kinase (DGK inhibitor 1), phosphatidic acid phosphohydrolase (propranolol), and diacylglycerol lipase (DGL; RHC-80<em>2</em>67 and tetrahydrolipstatin (THL)). However, unfavorable side effects were observed with these inhibitors, except for THL. Furthermore, we found that RHC-80<em>2</em>67 hardly inhibited the endocannabinoid release driven by G(q/11)-coupled receptors, which is thought to be DGL-dependent. By contrast, THL exhibited no side effects as long as we tested, and was confirmed to inhibit the DGL-dependent process. Using THL as a DGL inhibitor, we demonstrated that DGL is involved in both hippocampal DSI and cerebellar DSE. To test a possible involvement of PLCdelta in DSI, we examined hippocampal DSI in PLCdelta1, delta3 and delta4-knockout mice. However, there was no significant difference in the DSI magnitude between these knockout mice and wild-type mice. The present study clearly shows that DGL is a prerequisite for DSI/DSE. The enzymes yielding DG remain to be determined.

The antinociceptive effects of the endocannabinoids (ECs) are enhanced by inhibiting catabolic enzymes such as fatty acid amide hydrolase (FAAH). The physiological relevance of the metabolism of ECs by other pathways, such as cyclooxygenase-<em>2</em> (COX<em>2</em>) is less clear. To address this question we compared the effects of local inhibition of FAAH versus COX<em>2</em> (URB597 and nimesulide, respectively) on inflammatory hyperalgesia and levels of endocannabinoids and related molecules in the hindpaw. Inflammatory hyperalgesia was measured following intraplantar injection of carrageenan. Effects of intraplantar injection of URB597 (<em>2</em>5 microg and 100 microg) or nimesulide (50 microg) on hyperalgesia and hindpaw levels of anandamide (AEA), <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>AG) and N-palmitoylethanolamine (PEA) were determined. Although both doses of URB597 increased levels of AEA and <em>2</em>AG in the carrageenan inflamed hindpaw, only the lower dose of URB597 attenuated hyperalgesia (P<0.05). Nimesulide attenuated both hyperalgesia and hindpaw oedema (P<0.001, P<0.01, respectively) and increased levels of PEA (P<0.05) in the hindpaw. Since both AEA and PEA are ligands for peroxisome proliferator-activated receptor-alpha (PPARalpha), the effects of the PPARalpha antagonist GW6471 on nimesulide- and URB597-mediated effects were studied. GW6471, but not a PPARgamma antagonist, blocked the inhibitory effects of nimesulide and URB597 on hyperalgesia. Our data suggest that both COX<em>2</em> and FAAH play a role in the metabolism of endocannabinoids and related molecules. The finding that PPARalpha antagonism blocked the inhibitory effects of nimesulide and URB597 suggests that PPARalpha contributes to their antinociceptive effects in the carrageenan model of inflammatory hyperalgesia.

The exact role of the endocannabinoid system (ECS) during spermatogenesis has not been clarified. We used purified germ cell fractions representative of all phases of spermatogenesis and primary cultures of spermatogonia. This approach allowed the precise quantification of the cannabinoid receptor ligands, anandamide and <em>2</em>-<em>arachidonoylglycerol</em>, and of the expression at transcriptional and transductional levels of their metabolic enzymes and receptors. Our data indicate that male mouse germ cells possess an active and complete ECS, which is modulated during meiosis, and suggest the presence of an autocrine endocannabinoid signal during spermatogenesis. Mitotic cells possess higher levels of <em>2</em>-<em>arachidonoylglycerol</em>, which decrease in spermatocytes and spermatids. Accordingly, spermatogonia express higher and lower levels of <em>2</em>-<em>arachidonoylglycerol</em> biosynthetic and degrading enzymes, respectively, as compared to meiotic and postmeiotic cells. This endocannabinoid likely plays a pivotal role in promoting the meiotic progression of germ cells by activating CB(<em>2</em>) receptors. In fact, we found that the selective CB(<em>2</em>) receptor agonist, JWH133, induced the Erk 1/<em>2</em> MAPK phosphorylation cascade in spermatogonia and their progression toward meiosis, because it increased the number of cells positive for SCP3, a marker of meiotic prophase, and the expression of early meiotic prophase genes.

The endocannabinoid system (ECS) and, in particular, cannabinoid CB(1) receptors, their endogenous agonists (the endocannabinoids anandamide and <em>2</em>-<em>arachidonoylglycerol</em>) and enzymes for the biosynthesis and degradation of the latter mediators are emerging as key players in the control of all aspects of food intake and energy balance. The ECS is involved in stimulating both the homoeostatic (that is, the sensing of deficient energy balance and gastrointestinal load) and the hedonic (that is, the sensing of the salience and the incentive/motivational value of nutrients) aspects of food intake. The orexigenic effects of endocannabinoids are exerted in the brain by CB(1)-mediated stimulatory and inhibitory effects on hypothalamic orexigenic and anorectic neuropeptides, respectively; by facilitatory actions on dopamine release in the nucleus accumbens shell; and by regulating the activity of sensory and vagal fibres in brainstem-duodenum neural connections. In turn, the levels of anandamide and <em>2</em>-<em>arachidonoylglycerol</em> and/or CB(1) receptors in the brain are under the control of leptin, ghrelin and glucocorticoids in the hypothalamus, under that of dopamine in the limbic forebrain and under that of cholecystokinin and ghrelin in the brainstem. These bi-directional communications between the ECS and other key players in energy balance ensure local mediators such as the endocannabinoids to act in a way coordinated in both 'space' and 'time' to enhance food intake, particularly after a few hours of food deprivation. Alterations of such communications are, however, also among the underlying causes of overactivity of the ECS in hyperphagia and obesity, a phenomenon that provided the rationale for the development of anti-obesity drugs from CB(1) receptor antagonists.

The two cyclooxygenase (COX) enzymes catalyze the oxygenation of arachidonic acid to prostaglandin endoperoxides, which are the common intermediates in the biosynthesis of the bioactive lipids prostaglandins and thromboxane. COX-1 and COX-<em>2</em> are approximately 60% identical in amino acid sequence, exhibit highly homologous three-dimensional structures, and appear functionally similar at the biochemical level. Recent work has uncovered a subtle functional difference between the two enzymes, namely the ability of COX-<em>2</em> to efficiently utilize neutral derivatives (esters and amides) of arachidonic acid as substrates. Foremost among these neutral substrates are the endocannabinoids <em>2</em>-<em>arachidonoylglycerol</em> and arachidonoylethanolamide. This raises the possibility that COX-<em>2</em> oxygenation plays a role in a novel signaling pathway dependent on agonist-induced release of endocannabinoids and their selective oxygenation by COX-<em>2</em>. Among the products of COX-<em>2</em> oxygenation of endocannabinoids are glyceryl prostaglandins, some of which (e.g. glyceryl prostaglandin E(<em>2</em>) and glyceryl prostaglandin I(<em>2</em>)) exhibit interesting biological activities in inflammatory, neurological, and vascular systems. These compounds are produced in intact cells stimulated with physiological agonists and have been isolated from in vivo sources. Important concepts relevant to the hypothesis of a COX-<em>2</em>-selective signaling pathway are presented.

OBJECTIVE

Cannabinoid type 1 (CB1) receptor is involved in whole-body and cellular energy metabolism. We asked whether CB1 receptor stimulation was able to decrease mitochondrial biogenesis in different metabolically active tissues of obese high-fat diet (HFD)-fed mice.

METHODS

The effects of selective CB1 agonist arachidonyl-<em>2</em>-chloroethanolamide (ACEA) and endocannabinoids anandamide and <em>2</em>-<em>arachidonoylglycerol</em> on endothelial nitric oxide synthase (eNOS) expression were examined, as were mitochondrial DNA amount and mitochondrial biogenesis parameters in cultured mouse and human white adipocytes. These parameters were also investigated in white adipose tissue (WAT), muscle, and liver of mice chronically treated with ACEA. Moreover, p38 mitogen-activated protein kinase (MAPK) phosphorylation was investigated in WAT and isolated mature adipocytes from eNOS(-/-) and wild-type mice. eNOS, p38 MAPK, adenosine monophosphate-activated protein kinase (AMPK), and mitochondrial biogenesis were investigated in WAT, muscle, and liver of HFD mice chronically treated with ACEA.

RESULTS

ACEA decreased mitochondrial biogenesis and eNOS expression, activated p38 MAPK, and reduced AMPK phosphorylation in white adipocytes. The ACEA effects on mitochondria were antagonized by nitric oxide donors and by p38 MAPK silencing. White adipocytes from eNOS(-/-) mice displayed higher p38 MAPK phosphorylation than wild-type animals under basal conditions, and ACEA was ineffective in cells lacking eNOS. Moreover, mitochondrial biogenesis was downregulated, while p38 MAPK phosphorylation was increased and AMPK phosphorylation was decreased in WAT, muscle, and liver of ACEA-treated mice on a HFD.

CONCLUSIONS

CB1 receptor stimulation decreases mitochondrial biogenesis in white adipocytes, through eNOS downregulation and p38 MAPK activation, and impairs mitochondrial function in metabolically active tissues of dietary obese mice.

Fatty acid amide hydrolase (FAAH) is a hydrolytic enzyme that recognizes as substrates and inactivates the two most studied endocannabinoids, anandamide and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG). Following the observation that endocannabinoids produced by tissues during pathological conditions often have protective roles, FAAH inhibitors have been proposed as therapeutic drugs. Yet it has been suggested that FAAH functions in vivo only as an anandamide-degrading enzyme because its pharmacological and genetic inactivation is usually accompanied by elevation of anandamide, but not <em>2</em>-AG, levels. We believe, however, that this concept needs to be revisited in light of reports that, under certain experimental conditions, FAAH inhibitors also elevate <em>2</em>-AG tissue levels in vivo and, more recently, that FAAH inactivation in the striatum instead reduces <em>2</em>-AG concentrations through upregulation of anandamide levels, activation of transient receptor potential vanilloid 1 receptors and inhibition of <em>2</em>-AG biosynthesis.

BACKGROUND AND PURPOSE The endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) is degraded primarily by monoacylglycerol lipase (MGL). We compared peripheral antinociceptive effects of JZL184, a novel irreversible MGL inhibitor, with the reversible MGL-preferring inhibitor URB60<em>2</em> and exogenous <em>2</em>-AG in rats. EXPERIMENTAL APPROACH Nociception in the formalin test was assessed in groups receiving dorsal paw injections of vehicle, JZL184 (0.001-300 µg), URB60<em>2</em> (0.001-600 µg), <em>2</em>-AG (ED(50)), <em>2</em>-AG + JZL184 (at their ED(50)), <em>2</em>-AG + URB60<em>2</em> (at their ED(50)), AM<em>2</em>51 (80 µg), AM<em>2</em>51 + JZL184 (10 µg), AM630 (<em>2</em>5 µg) or AM630 + JZL184 (10 µg). Effects of MGL inhibitors on endocannabinoid accumulation and on activities of endocannabinoid-metabolizing enzymes were assessed. KEY RESULTS Intra-paw administration of JZL184, URB60<em>2</em> and <em>2</em>-AG suppressed early and late phases of formalin pain. JZL184 and URB60<em>2</em> acted through a common mechanism. JZL184 (ED(50) Phase 1: 0.06 ± 0.0<em>2</em>8; Phase <em>2</em>: 0.03 ± 0.011 µg) produced greater antinociception than URB60<em>2</em> (ED(50) Phase 1: 1<em>2</em>0 ± 51.3; Phase <em>2</em>: 66 ± <em>2</em>3.9 µg) or <em>2</em>-AG. Both MGL inhibitors produced additive antinociceptive effects when combined with <em>2</em>-AG. Antinociceptive effects of JZL184, like those of URB60<em>2</em>, were blocked by cannabinoid receptor 1 (CB(1)) and cannabinoid receptor <em>2</em> (CB(<em>2</em>)) antagonists. JZL184 suppressed MGL but not fatty-acid amide hydrolase or N-arachidonoyl-phosphatidylethanolamine phospholipase D activities ex vivo. URB60<em>2</em> increased hind paw <em>2</em>-AG without altering anandamide levels. CONCLUSIONS AND IMPLICATIONS MGL inhibitors suppressed formalin-induced pain through peripheral CB(1) and CB(<em>2</em>) receptor mechanisms. MGL inhibition increased paw skin <em>2</em>-AG accumulation to mediate these effects. MGL represents a target for the treatment of inflammatory pain.

Anandamide (N-arachidonoylethanolamine, AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) are the most active endocannabinoids at brain (CB1) cannabinoid receptors. CD1 mice lacking the CB1 receptors ("knockout" [KO] mutants) were compared with wildtype (WT) littermates for their ability to degrade AEA through an AEA membrane transporter (AMT) and an AEA hydrolase (fatty acid amide hydrolase, FAAH). The age dependence of AMT and FAAH activity were investigated in 1- or 4-month-old WT and KO animals, and found to increase with age in KO, but not WT, mice and to be higher in the hippocampus than in the cortex of all animals. AEA and <em>2</em>-AG were detected in nmol/mg protein (microm) concentrations in both regions, though the hippocampus showed approximately twice the amount found in the cortex. In the same regions, <em>2</em>-AG failed to change across groups, while AEA was significantly decreased (approximately 30%) in hippocampus, but not in cortex, of old KO mice, when compared with young KO or age-matched WT animals. In the open-field test under bright light and in the lit-dark exploration model of anxiety, young KO mice, compared with old KO, exhibited a mild anxiety-related behaviour. In contrast, neither the increase in memory performance assessed by the object recognition test, nor the reduction of morphine withdrawal symptoms, showed age dependence in CB1 KO mice. These results suggest that invalidation of the CB1 receptor gene is associated with age-dependent adaptive changes of endocannabinoid metabolism which appear to correlate with the waning of the anxiety-like behaviour exhibited by young CB1 KO mice.

The endocannabinoid system (ECS) tightly controls emotional responses to acute aversive stimuli. Repeated stress alters ECS activity but the role played by the ECS in the emotional consequences of repeated stress has not been investigated in detail. This study used social defeat stress, together with pharmacology and genetics to examine the role of cannabinoid type-1 (CB(1)) receptors on repeated stress-induced emotional alterations. Seven daily social defeat sessions increased water (but not food) intake, sucrose preference, anxiety, cued fear expression, and adrenal weight in C57BL/6N mice. The first and the last social stress sessions triggered immediate brain region-dependent changes in the concentrations of the principal endocannabinoids anandamide and <em>2</em>-<em>arachidonoylglycerol</em>. Pretreatment before each of the seven stress sessions with the CB(1) receptor antagonist rimonabant prolonged freezing responses of stressed mice during cued fear recall tests. Repeated social stress abolished the increased fear expression displayed by constitutive CB(1) receptor-deficient mice. The use of mutant mice lacking CB(1) receptors from cortical glutamatergic neurons or from GABAergic neurons indicated that it is the absence of the former CB(1) receptor population that is responsible for the fear responses in socially stressed CB(1) mutant mice. In addition, stress-induced hypolocomotor reactivity was amplified by the absence of CB(1) receptors from GABAergic neurons. Mutant mice lacking CB(1) receptors from serotonergic neurons displayed a higher anxiety but decreased cued fear expression than their wild-type controls. These mutant mice failed to show social stress-elicited increased sucrose preference. This study shows that (i) release of endocannabinoids during stress exposure impedes stress-elicited amplification of cued fear behavior, (ii) social stress opposes the increased fear expression and delayed between-session extinction because of the absence of CB(1) receptors from cortical glutamatergic neurons, and (iii) CB(1) receptors on central serotonergic neurons are involved in the sweet consumption response to repeated stress.

OBJECTIVE

Since monoacylglycerol lipase (MAGL) has been firmly established as the predominant catabolic enzyme of the endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), a great need has emerged for the development of highly selective MAGL inhibitors. Here, we tested the in vivo effects of one such compound, KML<em>2</em>9 (1,1,1,3,3,3-hexafluoropropan-<em>2</em>-yl 4-(bis(benzo[d][1,3]dioxol-5-yl)(hydroxy)methyl)piperidine-1-carboxylate).

METHODS

In the present study, we tested KML<em>2</em>9 in murine inflammatory (i.e. carrageenan) and sciatic nerve injury pain models, as well as the diclofenac-induced gastric haemorrhage model. KML<em>2</em>9 was also evaluated for cannabimimetic effects, including measurements of locomotor activity, body temperature, catalepsy, and cannabinoid interoceptive effects in the drug discrimination paradigm.

RESULTS

KML<em>2</em>9 attenuated carrageenan-induced paw oedema and completely reversed carrageenan-induced mechanical allodynia. These effects underwent tolerance after repeated administration of high-dose KML<em>2</em>9, which were accompanied by cannabinoid receptor 1 (CB1 ) receptor desensitization. Acute or repeated KML<em>2</em>9 administration increased <em>2</em>-AG levels and concomitantly reduced arachidonic acid levels, but without elevating anandamide (AEA) levels in the whole brain. Furthermore, KML<em>2</em>9 partially reversed allodynia in the sciatic nerve injury model and completely prevented diclofenac-induced gastric haemorrhages. CB1 and CB<em>2</em> receptors played differential roles in these pharmacological effects of KML<em>2</em>9. In contrast, KML<em>2</em>9 did not elicit cannabimimetic effects, including catalepsy, hypothermia and hypomotility. Although KML<em>2</em>9 did not substitute for Δ(9) -tetrahydrocannabinol (THC) in C57BL/6J mice, it fully and dose-dependantly substituted for AEA in fatty acid amide hydrolase (FAAH) (-/-) mice, consistent with previous work showing that dual FAAH and MAGL inhibition produces THC-like subjective effects.

CONCLUSIONS

These results indicate that KML<em>2</em>9, a highly selective MAGL inhibitor, reduces inflammatory and neuropathic nociceptive behaviour without occurrence of cannabimimetic side effects.

BACKGROUND

This article is part of a themed section on Cannabinoids <em>2</em>013. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.<em>2</em>014.171.issue-6.

Endocannabinoids are a new class of lipid mediators, which include amides, esters and ethers of long-chain polyunsaturated fatty acids. Anandamide (N-arachidonoylethanolamine; AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) are the main endogenous agonists of cannabinoid receptors able to mimic several pharmacological effects of Delta-9-tetrahydrocannabinol, the active principle of Cannabis sativa preparations like hashish and marijuana. The pathways leading to the synthesis and release of AEA and <em>2</em>-AG from neuronal and non-neuronal cells are still rather uncertain. Instead, it is known that the activity of AEA is limited by cellular uptake through a specific membrane transporter, followed by intracellular degradation by a fatty acid amide hydrolase. Together with AEA and congeners these proteins form the 'endocannabinoid system'. Here, the involvement of AEA in apoptosis and the underlying signal transduction pathways will be reviewed, along with the metabolic routes and the molecular targets of this endocannabinoid. Also, recent findings on the apoptotic potential of AEA for neuronal cell differentiation and brain development will be discussed.

The ability of cannabinoids to modulate both inflammatory and degenerative neuronal damage prompted investigations on the potential benefits of such compounds in multiple sclerosis (MS) and in animal models of this disorder. Here we measured endocannabinoid levels, metabolism and binding, and physiological activities in <em>2</em>6 patients with MS (17 females, aged 19-43 years), <em>2</em>5 healthy controls and in mice with experimental autoimmune encephalomyelitis (EAE), a preclinical model of MS. Our results show that MS and EAE are associated with significant alterations of the endocannabinoid system. We found that anandamide (AEA), but not <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), was increased in the CSF of relapsing MS patients. AEA concentrations were also higher in peripheral lymphocytes of these patients, an effect associated with increased synthesis and reduced degradation of this endocannabinoid. Increased synthesis, reduced degradation, and increased levels of AEA were also detected in the brains of EAE mice in the acute phase of the disease, possibly accounting for its anti-excitotoxic action in this disorder. Accordingly, neurophysiological recordings from single neurons confirmed that excitatory transmission in EAE slices is inhibited by CB1 receptor activation, while inhibitory transmission is not. Our study suggests that targeting the endocannabinoid system might be useful for the treatment of MS.

The possible role of the peripheral cannabinoid receptor (CB<em>2</em>) in neutrophil migration was investigated by using human promyelocytic HL60 cells differentiated into neutrophil-like cells and human neutrophils isolated from whole blood. Cell surface expression of CB<em>2</em> on HL60 cells, on neutrophil-like HL60 cells, and on human neutrophils was confirmed by flow cytometry. Upon stimulation with either of the CB<em>2</em> ligands JWH015 and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), neutrophil-like HL60 cells rapidly extended and retracted one or more pseudopods containing F-actin in different directions instead of developing front/rear polarity typically exhibited by migrating leukocytes. Activity of the Rho-GTPase RhoA decreased in response to CB<em>2</em> stimulation, whereas Rac1, Rac<em>2</em>, and Cdc4<em>2</em> activity increased. Moreover, treatment of cells with RhoA-dependent protein kinase (p160-ROCK) inhibitor Y<em>2</em>763<em>2</em> yielded cytoskeletal organization similar to that of CB<em>2</em>-stimulated cells. In human neutrophils, neither JWH015 nor <em>2</em>-AG induced motility or morphologic alterations. However, pretreatment of neutrophils with these ligands disrupted N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP)-induced front/rear polarization and migration and also substantially suppressed fMLP-induced RhoA activity. These results suggest that CB<em>2</em> might play a role in regulating excessive inflammatory response by controlling RhoA activation, thereby suppressing neutrophil migration.

OBJECTIVE

Endocannabinoids (ECs) control metabolism via cannabinoid receptors type 1 (CB1). Their plasma levels are elevated in overweight type <em>2</em> diabetes (T<em>2</em>D) and in obese patients, and decrease postprandially in normoweight individuals. We investigated in two different cohorts of nonobese or obese volunteers whether oral glucose in glucose tolerance tests (OGTT) or acute insulin infusion during euglycemic hyperinsulinemic clamp affect plasma EC levels.

METHODS

OGTT was performed in ten obese hyperinsulinemic patients (body mass index (BMI)=35.8 kg/m<em>2</em>, fasting insulin=14.83 mU/l), and ten normoweight normoinsulinemic volunteers (BMI=<em>2</em>1.9 kg/m<em>2</em>, fasting insulin=7.<em>2</em> mU/l). Insulin clamp was performed in 19 mostly nonobese men (BMI=<em>2</em>5.8 kg/m<em>2</em>) with varying degrees of liver fat and plasma triglycerides (TGs), with (n=7) or without T<em>2</em>D. Plasma levels of ECs (anandamide and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG)) were measured by liquid chromatography-mass spectrometry, before and 60 and 180 min after OGTT, and before and <em>2</em>40 and 480 min after insulin or saline infusion.

RESULTS

Oral glucose load decreased anandamide plasma levels to an extent inversely correlated with BMI, waist circumference, subcutaneous fat, fasting insulin and total glucose, and insulin areas under the curve during the OGTT, and nonsignificantly in obese volunteers. Insulin infusion decreased anandamide levels to an extent that weakly, but significantly, correlated negatively with TGs, liver fat and fasting insulin, and positively with high density lipoprotein cholesterol. OGTT decreased <em>2</em>-AG levels to a lower extent and in a way weakly inversely correlated with fasting insulin.

CONCLUSIONS

We suggest that insulin reduces EC levels in a way inversely related to anthropometric and metabolic predictors of insulin resistance and dyslipidemia.

The endocannabinoid system plays a critical role in the control of energy homeostasis, but the identity and localization of the endocannabinoid signal involved remain unknown. In the present study, we developed transgenic mice that overexpress in forebrain neurons the presynaptic hydrolase, monoacylglycerol lipase (MGL), which deactivates the endocannabinoid <em>2</em>-arachidonoyl-sn-glycerol (<em>2</em>-AG). MGL-overexpressing mice show a 50% decrease in forebrain <em>2</em>-AG levels but no overt compensation in other endocannabinoid components. This biochemical abnormality is accompanied by a series of metabolic changes that include leanness, elevated energy cost of activity, and hypersensitivity to β(3)-adrenergic-stimulated thermogenesis, which is corrected by reinstating <em>2</em>-AG activity at CB(1)-cannabinoid receptors. Additionally, the mutant mice are resistant to diet-induced obesity and express high levels of thermogenic proteins, such as uncoupling protein 1, in their brown adipose tissue. The results suggest that <em>2</em>-AG signaling through CB(1) regulates the activity of forebrain neural circuits involved in the control of energy dissipation.

OBJECTIVE

Evidence indicates that the endocannabinoid, <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), increases food intake when injected into the nucleus accumbens shell (NAcS), thereby potentially activating hypothalamic nuclei involved in food intake regulation. We aimed to evaluate potential orexigenic effects of the endocannabinoid anandamide and of AA5HT, a fatty acid amide hydrolase (FAAH) inhibitor, and OMDM-1, an inhibitor of anandamide uptake, injected in the NAcS, as well as the effect of these treatments on activation of hypothalamic nuclei.

METHODS

Drugs were given into the NAcS of rats and food intake quantified during the next 4 h. In other groups, after the same treatments the brains were processed for c-Fos immunohistochemistry with focus on hypothalamic nuclei. Additional groups were used to quantify endocannabinoid levels in the nucleus accumbens and the hypothalamus after AA5HT and OMDM-1 intra-NAcS injections.

RESULTS

Our results indicate that the above treatments stimulate food intake during 4 h post-injection. They also increase c-Fos immunoreactivity in hypothalamic nuclei. The CB(1) antagonist, AM<em>2</em>51, blocked these effects. Finally, we found elevated levels of <em>2</em>-AG, but not anandamide, after intra-NAcS injections of AA5HT.

CONCLUSIONS

These data support the involvement of the endocannabinoid system in feeding behavior at the level of the NAcS and hypothalamus. In addition, this is the first experimental demonstration that the pharmacological inhibition of endocannabinoid inactivation in the NAcS stimulates food intake, suggesting that the endocannabinoid degrading proteins can be a target for treating eating disorders.

An activation of the endocannabinoid system (ECS) in obesity with increased concentrations of endocannabinoids in several tissues and in the circulation is described in this review. This increased availability of endocannabinoids might stimulate cannabinoid receptors in a pathophysiological manner. The successful use of the cannabinoid receptor CB(1) inverse agonists rimonabant and taranabant for weight loss and the treatment of obesity-associated metabolic disorders might well be through blocking this overstimulation of cannabinoid receptors. At present, no single mechanism has been identified that explains the increased bioavailability of endocannabinoids in obesity. Both increased synthesis and decreased degradation appear to operate in a species- and tissue-dependent manner, but many pieces of the puzzle still need to be collected. For example, most data show decreased fatty acid amide hydrolase (FAAH) expression and/or activity as a result of obesity or high-fat intake, but the endocannabinoid predominantly increased in tissues is <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), which is not degraded by FAAH in vivo. Furthermore, the influence of dietary fatty acids on the synthesis of endocannabinoids needs to be studied in much more detail. Although weight loss does not seem to influence activation of the endocannabinoid system (ECS) in human obesity, suggesting an underlying mechanisms independent of body weight, no such mechanism at the genetic level has yet been identified either. Thus, activation of the ECS is a hallmark of abdominal obesity, and explains the success of pharmacological CB(1) blockade, but serious attempts have to be made to clarify the underlying mechanisms of this activation.

The effects of the endocannabinoids anandamide (AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) upon rat C6 glioma cell proliferation were examined and compared with a series of synthetic cannabinoids and related compounds. Cells were treated with the compounds each day and cell proliferation was monitored for up to 5 days of exposure. AEA time- and concentration-dependently inhibited C6 cell proliferation. After 4 days of treatment, AEA and <em>2</em>-AG inhibited C6 cell proliferation with similar potencies (IC(50) values of 1.6 and 1.8 microM, respectively), whereas palmitoylethanolamide showed no significant antiproliferative effects at concentrations up to 10 microM. The antiproliferative effects of both AEA and <em>2</em>-AG were blocked completely by a combination of antagonists at cannabinoid receptors (SR141716A and SR1445<em>2</em>8 or AM<em>2</em>51 and AM630) and vanilloid receptors (capsazepine) as well as by alpha-tocopherol (0.1 and 10 microM), and reduced by calpeptin (10 microM) and fumonisin B(1) (10 microM), but not by L-cycloserine (1 and 100 microM). CP 55,940, JW015, olvanil, and arachidonoyl-serotonin were all found to affect C6 glioma cell proliferation (IC(50) values of 5.6, 3.<em>2</em>, 5.5, and 1.6 microM, respectively), but the inhibition could not be blocked by cannabinoid + vanilloid receptor antagonists. It is concluded that the antiproliferative effects of the endocannabinoids upon C6 cells are brought about by a mechanism involving combined activation of both vanilloid receptors and to a lesser extent cannabinoid receptors, and leading to oxidative stress and calpain activation. However, there is at present no obvious universal mechanism whereby plant-derived, synthetic, and endogenous cannabinoids affect cell viability and proliferation.

Activation of spinal microglia contributes to aberrant pain responses associated with neuropathic pain states. Endocannabinoids (ECs) are present in the spinal cord, and inhibit nociceptive processing; levels of ECs may be altered by microglia which modulate the turnover of endocannabinoids in vitro. Here, we investigate the effect of minocycline, an inhibitor of activated microglia, on levels of the endocannabinoids anandamide and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), and the related compound N-palmitoylethanolamine (PEA), in neuropathic spinal cord. Selective spinal nerve ligation (SNL) in rats resulted in mechanical allodynia and the presence of activated microglia in the ipsilateral spinal cord. Chronic daily treatment with minocycline (30 mg/kg, ip for 14 days) significantly reduced the development of mechanical allodynia at days 5, 10 and 14 post-SNL surgery, compared to vehicle-treated SNL rats (P < 0.001). Minocycline treatment also significantly attenuated OX-4<em>2</em> immunoreactivity, a marker of activated microglia, in the ipsilateral (P < 0.001) and contralateral (P < 0.01) spinal cord of SNL rats, compared to vehicle controls. Minocycline treatment significantly (P < 0.01) decreased levels of <em>2</em>-AG and significantly (P < 0.01) increased levels of PEA in the ipsilateral spinal cord of SNL rats, compared to the contralateral spinal cord. Thus, activation of microglia affects spinal levels of endocannabinoids and related compounds in neuropathic pain states.

Herein, we report the heterologous expression of the human peroxisomal 63-kDa calcium-independent phospholipase A<em>2</em>gamma (iPLA<em>2</em>gamma) isoform in Sf9 cells, purification of the N-terminal His-tagged enzyme by affinity chromatography, and the identification of its remarkable substrate selectivity that results in the highly selective generation of <em>2</em>-arachidonoyl lysophosphatidylcholine. Mass spectrometric analyses demonstrated that purified iPLA<em>2</em>gamma hydrolyzed saturated or monounsaturated aliphatic groups readily from either the sn-1 or sn-<em>2</em> positions of phospholipids. In addition, purified iPLA<em>2</em>gamma effectively liberated arachidonic acid from the sn-<em>2</em> position of plasmenylcholine substrates. In contrast, incubation of iPLA<em>2</em>gamma with 1-palmitoyl-<em>2</em>-arachidonoyl-sn-glycero-3-phosphocholine resulted in the rapid release of palmitic acid and the selective accumulation of <em>2</em>-arachidonoyl lysophosphatidylcholine (LPC), which was not metabolized further by iPLA<em>2</em>gamma. The putative regiospecificity of the <em>2</em>-arachidonoyl LPC product was authenticated by its diagnostic fragmentation pattern during tandem mass spectrometric analysis. To identify the physiological relevance of iPLA<em>2</em>gamma-mediated <em>2</em>-arachidonoyl LPC production utilizing naturally occurring membranes, we incubated purified rat hepatic peroxisomes with iPLA<em>2</em>gamma and similarly identified the selective accumulation of <em>2</em>-arachidonoyl LPC. Furthermore, tandem mass spectrometric analysis demonstrated that <em>2</em>-arachidonoyl LPC is a natural product in human myocardium, a tissue in which iPLA<em>2</em>gamma expression is robust. Because <em>2</em>-arachidonoyl LPC represents a key branch point intermediate that can potentially lead to a variety of bioactive molecules in eicosanoid signaling (e.g. arachidonic acid, <em>2</em>-<em>arachidonoylglycerol</em>), these results have uncovered a novel eicosanoid selective pathway through iPLA<em>2</em>gamma-mediated <em>2</em>-arachidonoyl LPC production to amplify and diversify the repertoire of biologic lipid second messengers in response to cellular stimulation.

Stress plays an important role in psychiatric disorders, and preclinical evidence indicates that the central endocannabinoid system modulates endocrine and neuronal responses to stress. This study aimed to investigate the effect of acute stress on circulating concentrations of endocannabinoids (eCBs) in healthy humans. A total of 71 adults participated in two sessions in which they were exposed to either a standardized psychosocial stress procedure (Trier Social Stress Test) or a control task. Blood samples for eCB and cortisol assays and cardiovascular and subjective measures were obtained before and at regular intervals after the tasks. Serum concentrations of the eCBs, N-arachidonylethanolamine (anandamide, AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), as well as of the N-acylethanolamides (NAEs), N-palmitoylethanolamine (PEA) and N-oleoylethanolamine (OEA), and of the O-acylglycerol, <em>2</em>-oleoylglycerol (<em>2</em>-OG), were determined. Compared with the control condition, stress increased serum concentrations of AEA and the other NAEs immediately after the stress period. Increases in PEA were positively correlated with increases in serum cortisol after stress. Furthermore, anxiety ratings at baseline were negatively correlated with baseline concentrations of AEA. The sex and menstrual cycle status of the subject affected the NAE responses to stress. Interestingly, subjects of Asian and African-American races exhibited different patterns of stress responses compared with the Caucasian subjects. These results indicate that stress increases circulating NAEs in healthy human volunteers. This finding supports a protective role for eCBs in anxiety. Further research is needed to elucidate the function of these lipid mediators, and to determine the mechanisms that regulate their appearance in the circulation.

Endocannabinoids are involved in synaptic signaling and neuronal protection; however, our understanding of the mechanisms by which endocannabinoids protect neurons from harmful insults remains elusive. <em>2</em>-<em>Arachidonoylglycerol</em> (<em>2</em>-AG), the most abundant endogenous cannabinoid and a full agonist for cannabinoid receptors (CB1 and CB<em>2</em>), is a substrate for cyclooxygenase-<em>2</em> (COX-<em>2</em>) and can be metabolized by COX-<em>2</em>. Here we show, however, that <em>2</em>-AG is also capable of suppressing elevation of hippocampal COX-<em>2</em> expression in response to proinflammatory and excitotoxic stimuli. <em>2</em>-AG prevents neurodegeneration from toxic assaults that elevate COX-<em>2</em> expression and inhibits the COX-<em>2</em> elevation-enhanced excitatory glutamatergic synaptic transmission. The action of <em>2</em>-AG on suppression of COX-<em>2</em> appeared to be mediated via the pertussis toxin-sensitive G protein-coupled CB1 receptor and MAPK/NF-kappaB signaling pathways. Our results reveal that <em>2</em>-AG functions as an endogenous COX-<em>2</em> inhibitor protecting neurons from harmful insults by preventing excessive expression of COX-<em>2</em>, which provides a mechanistic basis for opening up new therapeutic approaches for protecting neurons from inflammation- and excitotoxicity-induced neurodegeneration.

Cannabinoids suppress fertility via reducing hypothalamic GnRH output. γ-Aminobutyric acid (GABA)(A) receptor (GABA(A)-R)-mediated transmission is a major input to GnRH cells that can be excitatory. We hypothesized that cannabinoids act via inhibiting GABAergic input. We performed loose-patch electrophysiological studies of acute slices from adult male GnRH-green fluorescent protein transgenic mice. Bath application of type 1 cannabinoid receptor (CB1) agonist WIN55,<em>2</em>1<em>2</em> decreased GnRH neuron firing rate. This action was detectable in presence of the glutamate receptor antagonist kynurenic acid but disappeared when bicuculline was also present, indicating GABA(A)-R involvement. In immunocytochemical experiments, CB1-immunoreactive axons formed contacts with GnRH neurons and a subset established symmetric synapses characteristic of GABAergic neurotransmission. Functional studies were continued with whole-cell patch-clamp electrophysiology in presence of tetrodotoxin. WIN55,<em>2</em>1<em>2</em> decreased the frequency of GABA(A)-R-mediated miniature postsynaptic currents (mPSCs) (reflecting spontaneous vesicle fusion), which was prevented with the CB1 antagonist AM<em>2</em>51, indicating collectively that activation of presynaptic CB1 inhibits GABA release. AM<em>2</em>51 alone increased mPSC frequency, providing evidence that endocannabinoids tonically inhibit GABA(A)-R drive onto GnRH neurons. Increased mPSC frequency was absent when diacylglycerol lipase was blocked intracellularly with tetrahydrolipstatin, showing that tonic inhibition is caused by <em>2</em>-<em>arachidonoylglycerol</em> production of GnRH neurons. CdCl(<em>2</em>) in extracellular solution can maintain both action potentials and spontaneous vesicle fusion. Under these conditions, when endocannabinoid-mediated blockade of spontaneous vesicle fusion was blocked with AM<em>2</em>51, GnRH neuron firing increased, revealing an endogenous endocannabinoid brake on GnRH neuron firing. Retrograde endocannabinoid signaling may represent an important mechanism under physiological and pathological conditions whereby GnRH neurons regulate their excitatory GABAergic inputs.