We have shown that the requirement of the epsilon-isoform of diacylglycerol kinase for diacylglycerols containing arachidonic acid is specific for this substrate and cannot be replaced by the presence of an arachidonoyl group in other places in the membrane; rather, it has to be present on the substrate itself. In addition, we demonstrate that the increased activity shown toward 1-stearoyl-<em>2</em>-<em>arachidonoylglycerol</em> by the epsilon-isoform of diacylglycerol kinase is not a consequence of altered membrane physical properties but is rather a specific interaction with the arachidonoyl group. We have also compared the modulation of the activity of the epsilon-isoform of diacylglycerol kinase with that of the zeta-isoform with regard to some of the intermediates involved in phosphatidylinositol cycling. One of the products of the hydrolysis of phosphatidylinositol diphosphate is diacylglycerol enriched in arachidonic acid. The activity of the epsilon-isoform is known to be specific for this form of diacylglycerol. We show that in contrast, the activity of the zeta-isoform is lower against 1-stearoyl-<em>2</em>-<em>arachidonoylglycerol</em> compared with dioleoylglycerol. We demonstrate that addition of phosphatidylserine, as well as other anionic phospholipids including L-alpha-phosphatidylinositol 4,5-bisphosphate, strongly inhibits the epsilon-isoform, but these anionic lipids increase the activity of the zeta-isoform. Addition of Ca(<em>2</em>+), which is released from internal stores as a consequence of phosphatidylinositol cycling, promotes the activity of the epsilon-isoform of this enzyme but has little effect on the zeta-isoform. The contrasting conditions required for maximal activity of these two isoforms of diacylglycerol kinase, as well as their different substrate specificity, suggest that they have different physiological roles in signal transduction.

Air pollution has been reported to be associated with increased risks of cognitive impairment and neurodegenerative diseases. Because NO<em>2</em> is a typical primary air pollutant and an important contributor to secondary aerosols, NO<em>2</em>-induced neuronal functional abnormalities have attracted greater attention, but the available experimental evidence, modulating mechanisms, and targeting medications remain ambiguous. In this study, we exposed C57BL/6J and APP/PS1 mice to dynamic NO<em>2</em> inhalation and found for the first time that NO<em>2</em> inhalation caused deterioration of spatial learning and memory, aggravated amyloid β4<em>2</em> (Aβ4<em>2</em>) accumulation, and promoted pathological abnormalities and cognitive defects related to Alzheimer's disease (AD). The microarray and bioinformation data showed that the cyclooxygenase-<em>2</em> (COX-<em>2</em>)-mediated arachidonic acid (AA) metabolism of prostaglandin E<em>2</em> (PGE<em>2</em>) played a key role in modulating this aggravation. Furthermore, increasing endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) by inhibiting monoacylglycerol lipase (MAGL) prevented PGE<em>2</em> production, neuroinflammation-associated Aβ4<em>2</em> accumulation, and neurodegeneration, indicating a therapeutic target for relieving cognitive impairment caused by NO<em>2</em> exposure.

OBJECTIVE

As the incidence of obesity and the metabolic syndrome has increased, research has focused on the importance of the endocannabinoid system in the brain and peripheral tissues. Rimonabant, an inverse agonist of the CB1 receptor is being used therapeutically. This review presents recent advances in endocannabinoid physiology.

RESULTS

The endocannabinoid system interacts with other anorexigenic and orexigenic pathways to regulate food intake in the hypothalamus, and the hedonistic value of food in the mesolimbic system. Endocannabinoid system overactivity contributes to hepatic steatosis, increased adipose tissue inflammation, dysregulated insulin signalling in the pancreas and disturbed oxidative pathways in skeletal muscle. The breakdown pathways for anandamide and <em>2</em>-<em>arachidonoylglycerol</em>, the endocannabinoid receptor ligands, are reviewed, and the recent discoveries of endocannabinoid receptor polymorphisms and their relationship to obesity and metabolic disease noted. The favourable effect of rimonabant on fat mass glycaemic control, lipid metabolism and overall cardiovascular risk must be tempered by adverse effects on mood.

CONCLUSIONS

The ubiquitous role of the endocannabinoid system in food intake and energy metabolism is now established. Drugs that manipulate different aspects of this system may benefit subjects with the metabolic and cachectic syndromes.

In the past years, the relationship between the endocannabinoid system (ECS) and other hormonal and neuromodulatory systems has been intensively studied. G protein-coupled receptors (GPCRs) can stimulate endocannabinoid (eCB) production via activation of G(q/11) proteins and, in some cases, G(s) proteins. In this review, we summarize the pathways through which GPCR activation can trigger eCB release, as well as the best known examples of this process throughout the body tissues. Angiotensin II-induced activation of AT(1) receptors, similar to other G(q/11)-coupled receptors, can lead to the formation of <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), an important eCB. The importance of eCB formation in angiotensin II action is supported by the finding that the hypertensive effect of angiotensin II, injected directly into the hypothalamic paraventricular nucleus of anaesthetized rats, can be abolished by AM<em>2</em>51, an inverse agonist of CB(1) cannabinoid receptors (CB(1)Rs). We conclude that activation of the ECS should be considered as a general consequence of the stimulation of G(q/11)-coupled receptors, and may mediate some of the physiological effects of GPCRs.

The endocannabinoid (eCB) system, like the better-known endorphin system, consists of cell membrane receptors, endogenous ligands and ligand-metabolizing enzymes. Two cannabinoid receptors are known: CB(1) is principally located in the nervous system, whereas CB(<em>2</em>) is primarily associated with the immune system. Two eCB ligands, anandamide (AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), are mimicked by cannabis plant compounds. The first purpose of this paper was to review the eCB system in detail, highlighting aspects of interest to bodyworkers, especially eCB modulation of pain and inflammation. Evidence suggests the eCB system may help resolve myofascial trigger points and relieve symptoms of fibromyalgia. However, expression of the eCB system in myofascial tissues has not been established. The second purpose of this paper was to investigate the eCB system in fibroblasts and other fascia-related cells. The investigation used a bioinformatics approach, obtaining microarray data via the GEO database (www.ncbi.nlm.nih.gov/geo/). GEO data mining revealed that fibroblasts, myofibroblasts, chondrocytes and synoviocytes expressed CB(1), CB(<em>2</em>) and eCB ligand-metabolizing enzymes. Fibroblast CB(1) levels nearly equalled levels expressed by adipocytes. CB(1) levels upregulated after exposure to inflammatory cytokines and equiaxial stretching of fibroblasts. The eCB system affects fibroblast remodeling through lipid rafts associated with focal adhesions and dampens cartilage destruction by decreasing fibroblast-secreted metalloproteinase enzymes. In conclusion, the eCB system helps shape biodynamic embryological development, diminishes nociception and pain, reduces inflammation in myofascial tissues and plays a role in fascial reorganization. Practitioners wield several tools that upregulate eCB activity, including myofascial manipulation, diet and lifestyle modifications, and pharmaceutical approaches.

BACKGROUND

The central nervous system cannabinoid CB1 receptors have been implicated in regulation of alcohol consumption. Less data are available on the role of the endogenous ligands for these receptors, anandamide (AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), in alcohol-related behaviors. The purpose of this study was to assess the effects of voluntary alcohol consumption on the levels of these endocannabinoids in key brain areas mediating alcohol reinforcement.

METHODS

Female and male alcohol-preferring AA (Alko, Alcohol) rats were trained to drink 10% (v/v) alcohol during 90-min limited access sessions every second day. Following establishment of stable alcohol drinking, half of the subjects were killed immediately before the daily alcohol access ("pre-session" group), while the other half was killed after the drinking session ("post-session" group). A separate control group consisted of water-drinking rats. AEA and <em>2</em>-AG levels were measured from prefrontal cortex (PFC), nucleus accumbens (NAc), caudate putamen (CPu), amygdala, and hippocampus using liquid chromatography-tandem mass spectrometry (LC/MS/MS).

RESULTS

Voluntary alcohol drinking caused widespread alterations in the levels of both AEA and <em>2</em>-AG. Compared to the water group, increased AEA levels were seen in the pre-session group, but they were decreased immediately following limited access drinking in the female AA rats. Also <em>2</em>-AG levels were significantly elevated after long alcohol exposure, and an additional increase was found after limited access drinking in PFC. In males, however, the only alterations caused by alcohol drinking were significantly elevated AEA levels in NAc and CPu in the post-session group. No changes were seen in the levels of <em>2</em>-AG.

CONCLUSIONS

These results demonstrate that voluntary alcohol drinking modulates the levels of endocannabinoids in several brain areas implicated in alcohol reinforcement. AEA and <em>2</em>-AG were differentially affected, suggesting that they could have partially separate modulatory roles. Alterations were more widespread in females than males, possibly reflecting their higher alcohol intake. Taken together, alcohol-induced release of endocannabinoids may have an important role in alcohol reinforcement and development of alcohol addiction.

OBJECTIVE

Obstructive sleep apnea chronically increases blood pressure through sympathetic nervous system activation. In animals, hypertension and sympathetic activity are restrained by cannabinoid receptor activation. Therefore, we hypothesized that increased blood pressure in patients with obstructive sleep apnea is associated with increased circulating endocannabinoid concentrations.

METHODS

Arterial oxygen saturation and apnea/hypopnea episodes were recorded in <em>2</em>9 patients with normal glucose tolerance, <em>2</em>6 patients with type <em>2</em> diabetes mellitus, and <em>2</em>1 patients obese subjects without sleep apnea. We determined seated blood pressure, insulin, glucose, and high-sensitive C-reactive protein in the morning, and insulin sensitivity by euglycemic-hyperinsulinemic clamp the next day. Anandamide, the sum of 1-<em>arachidonoylglycerol</em> and <em>2</em>-<em>arachidonoylglycerol</em>, and oleoylethanolamide were measured in plasma by liquid chromatography-tandem mass spectrometry.

RESULTS

Endocannabinoid concentrations in sleep apnea patients were increased compared to obese individuals without disordered nocturnal breathing. Correction for variables of obesity and insulin resistance almost completely abrogated this difference in endocannabinoids. Anandamide strongly correlated with blood pressure in sleep apnea patients (r = 0.60 for SBP and r = 0.58 for DBP, P < 0.001). In multivariate regression analysis, anandamide was a stronger determinant of blood pressure than sleep apnea severity, obesity, insulin resistance, and inflammation.

CONCLUSIONS

Obstructive sleep apnea patients show positive correlations between blood pressure and venous anandamide concentrations independent of confounding factors. Our data suggest a previously not recognized role of the endocannabinoid system for blood pressure regulation in patients with high risk for hypertension and cardiovascular disease.

Marijuana has been used to relieve pain for centuries, but its analgesic mechanism has only been understood during the past two decades. It is mainly mediated by its constituents, cannabinoids, through activating central cannabinoid 1 (CB1) receptors, as well as peripheral CB1 and CB<em>2</em> receptors. CB<em>2</em>-selective agonists have the benefit of lacking CB1 receptor-mediated CNS side effects. Anandamide and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) are two intensively studied endogenous lipid ligands of cannabinoid receptors, termed endocannabinoids, which are synthesized on demand and rapidly degraded. Thus, inhibitors of their degradation enzymes, fatty acid amide hydrolase and monoacylglycerol lipase (MAGL), respectively, may be superior to direct cannabinoid receptor ligands as a promising strategy for pain relief. In addition to the antinociceptive properties of exogenous cannabinoids and endocannabinoids, involving their biosynthesis and degradation processes, we also review recent studies that revealed a novel analgesic mechanism, involving <em>2</em>-AG in the periaqueductal gray (PAG), a midbrain region for initiating descending pain inhibition. It is initiated by Gq-protein-coupled receptor (GqPCR) activation of the phospholipase C (PLC)-diacylglycerol lipase (DAGL) enzymatic cascade, generating <em>2</em>-AG that produces inhibition of GABAergic transmission (disinhibition) in the PAG, thereby leading to analgesia. This GqPCR-PLC-DAGL-<em>2</em>-AG retrograde disinhibition mechanism in the PAG can be initiated by activating type 5 metabotropic glutamate receptor (mGluR5), muscarinic acetylcholine (M1/M3), and orexin (OX1) receptors. mGluR5-mediated disinhibition can be initiated by glutamate transporter inhibitors, or indirectly by substance P, neurotensin, cholecystokinin, capsaicin, and AM404, the bioactive metabolite of acetaminophen in the brain. The putative role of <em>2</em>-AG generated after activating the above neurotransmitter receptors in stress-induced analgesia is also discussed.

The analysis of peripheral endocannabinoids (ECs) is a good biomarker of the EC system. Their concentrations, from clinical studies, strongly depend on sample collection and time processing conditions taking place in clinical and laboratory settings. The analysis of <em>2</em>-monoacylglycerols (MGs) (i.e., <em>2</em>-<em>arachidonoylglycerol</em> or <em>2</em>-oleoylglycerol) is a particularly challenging issue because of their ex vivo formation and chemical isomerization that occur after blood sample collection. We provide evidence that their ex vivo formation can be minimized by adding Orlistat, an enzymatic lipase inhibitor, to plasma. Taking into consideration the low cost of Orlistat, we recommend its addition to plasma collecting tubes while maintaining sample cold chain until storage. We have validated a method for the determination of the EC profile of a range of MGs and N-acylethanolamides in plasma that preserves the original isomer ratio of MGs. Nevertheless, the chemical isomerization of <em>2</em>-MGs can only be avoided by an immediate processing and analysis of samples due to their instability during conservation. We believe that this new methodology can aid in the harmonization of the measurement of ECs and related compounds in clinical samples.

<em>2</em>-<em>Arachidonoylglycerol</em> (<em>2</em>-AG) is the most potent endogenous ligand of brain cannabinoid CB1 receptors and is synthesized on demand from <em>2</em>-arachidonate-containing phosphoinositides by the action of diacylglycerol lipase in response to increased intracellular calcium. Several studies indicate that the endocannabinoid (eCB) system is involved in the mechanism of reward and that diverse drugs of abuse increase brain eCB levels. In addition, eCB are self-administered (SA) by squirrel monkeys, and anandamide increases nucleus accumbens (NAc) shell dopamine (DA) in rats. To date, there is no evidence on the reinforcing effects of <em>2</em>-AG and its effects on DA transmission in rodents. In order to fill this gap, we studied intravenous <em>2</em>-AG SA and monitored the effect of <em>2</em>-AG on extracellular DA in the NAc shell and core via microdialysis in male Sprague-Dawley rats. Rats were implanted with jugular catheters and trained to self-administer <em>2</em>-AG [<em>2</em>5 mg/kg/inf intravenously (iv)] in single daily 1 h sessions for 5 weeks under initial fixed ratio (FR) 1 schedule. The ratio was subsequently increased to FR<em>2</em>. Active nose poking increased from the 6th SA session (acquisition phase) but no significant increase of nose pokes was observed after FR<em>2</em>. When <em>2</em>-AG was substituted for vehicle (<em>2</em>5th SA session, extinction phase), rate responding as well as number of injections slowly decreased. When vehicle was replaced with <em>2</em>-AG, SA behavior immediately recovered (reacquisition phase). The reinforcing effects of <em>2</em>-AG in SA behavior were fully blocked by the CB1 receptor inverse agonist/antagonist rimonabant (1 mg/kg intraperitoneally, 30 min before SA session). In the microdialysis studies, we observed that <em>2</em>-AG (0.1-1.0 mg/kg iv) preferentially stimulates NAc shell as compared to the NAc core. NAc shell DA increased by about <em>2</em>5% over basal value at the highest doses tested (0.5 and 1.0 mg/kg iv). The results obtained suggest that the eCB system, via <em>2</em>-AG, plays an important role in reward.

The salivary glands and saliva from the lone star tick Amblyomma americanum (L.) were analyzed for the presence of the two endogenous agonists of cannabinoid receptors, N-arachidonoylethanolamine (anandamide) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), as well as of the anandamide congener, N-palmitoylethanolamine (PEA), an anti-inflammatory and analgesic mediator that is inactive at cannabinoid receptors. Two very sensitive mass-spectrometric techniques were used for this purpose. Both <em>2</em>-AG and PEA, as well as other N-acylethanolamines (NAEs), were identified in salivary glands, but anandamide was below detection. The levels of <em>2</em>-AG were considerably higher in the salivary glands of partially fed than replete females. Ex vivo gland stimulation with arachidonic acid increased the levels of <em>2</em>-AG, but not of PEA or other NAEs, and caused the formation of anandamide and of the potent analgesic compound N-arachidonoylglycine. Instead, the amounts of anandamide, <em>2</em>-AG and PEA were not influenced by treatment of salivary glands with dopamine, which stimulates saliva secretion. The possible biosynthetic precursors of anandamide, PEA and other NAEs were also detected in salivary glands, whereas only PEA was detected in tick saliva. These data demonstrate for the first time that the salivary glands of an obligate ectoparasite species can make endocannabinoids and/or related congeners with analgesic and anti-inflammatory activity, which possibly participate in the inhibition of the host defense reactions.

sn-1-Diacylglycerol lipase α (DAGL-α) is the main enzyme responsible for the production of the endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> in the central nervous system. Glycine sulfonamides have recently been identified by a high throughput screening campaign as a novel class of inhibitors for this enzyme. Here, we report on the first structure-activity relationship study of glycine sulfonamide inhibitors and their brain membrane proteome-wide selectivity on serine hydrolases with activity-based protein profiling (ABPP). We found that (i) DAGL-α tolerates a variety of biaryl substituents, (ii) the sulfonamide is required for inducing a specific orientation of the <em>2</em>,<em>2</em>-dimethylchroman substituent, and (iii) a carboxylic acid is essential for its activity. ABPP revealed that the sulfonamide glycine inhibitors have at least three off-targets, including α/β-hydrolase domain 6 (ABHD6). Finally, we identified LEI-106 as a potent, dual DAGL-α/ABHD6 inhibitor, which makes this compound a potential lead for the discovery of new molecular therapies for diet-induced obesity and metabolic syndrome.

<em>2</em>-<em>Arachidonoylglycerol</em> (<em>2</em>-AG), an endogenous cannabinoid receptor ligand, was shown to induce rapid phosphorylation of p4<em>2</em>/44 mitogen-activated protein kinase (MAP kinase) in HL-60 cells. We confirmed that the enzyme activity of p4<em>2</em>/44 MAP kinase in HL-60 cells was augmented markedly when the cells were stimulated with <em>2</em>-AG. The addition of SR1445<em>2</em>8, a cannabinoid CB<em>2</em> receptor-specific antagonist, to the cells prior to the addition of <em>2</em>-AG abolished the response induced by <em>2</em>-AG, indicating that the CB<em>2</em> receptor is involved in the response. G protein G(i) or G(o) is also assumed to be involved, because pertussis toxin treatment of the cells nullified the response induced by <em>2</em>-AG. CP55940 and anandamide also induced the activation of p4<em>2</em>/44 MAP kinase, although the activation by anandamide was less pronounced than that by <em>2</em>-AG or CP55940. These results suggest that <em>2</em>-AG may play an important physiological role in this type of cell through the activation of the p4<em>2</em>/44 MAP kinase cascade.

Depression and pain co-exist in almost 80% of patients and are associated with impaired health-related quality of life, often contributing to high mortality. However, the majority of patients who suffer from the comorbid depression and pain are not responsive to pharmacological treatments that address either pain or depression, making this comorbidity disorder a heavy burden on patients and society. In ancient times, this depression-pain comorbidity was treated using extracts of the Cannabis sativa plant, known now as marijuana and the mode of action of Δ9‑tetrahydrocannabinol, the active cannabinoid ingredient of marijuana, has only recently become known, with the identification of cannabinoid receptor type 1 (CB1) and CB<em>2</em>. Subsequent investigations led to the identification of endocannabinoids, anandamide and <em>2</em>-<em>arachidonoylglycerol</em>, which exert cannabinomimetic effects through the CB1 and CB<em>2</em> receptors, which are located on presynaptic membranes in the central nervous system and in peripheral tissues, respectively. These endocannabinoids are produced from membrane lipids and are lipohilic molecules that are synthesized on demand and are eliminated rapidly after their usage by hydrolyzing enzymes. Clinical studies revealed altered endocannabinoid signaling in patients with chronic pain. Considerable evidence suggested the involvement of the endocannabinoid system in eliciting potent effects on neurotransmission, neuroendocrine, and inflammatory processes, which are known to be deranged in depression and chronic pain. Several synthetic cannabinomimetic drugs are being developed to treat pain and depression. However, the precise mode of action of endocannabinoids on different targets in the body and whether their effects on pain and depression follow the same or different pathways, remains to be determined.

In recent years, several studies have explored the involvement of the deregulation of the hypothalamus-pituitary-adrenal (HPA) axis in the pathophysiology of stress-related disorders. HPA hyper-activation as a consequence of acute/chronic stress has been found to play a major role in the neurobiological changes that are responsible for the onset of such states. Currently available medications for depression, one of the most relevant stress-related disorders, present several limitations, including a time lag for treatment response and low rates of efficacy. N-Arachidonoylserotonin (AA-5-HT), a dual blocker at fatty acid amide hydrolase (FAAH, the enzyme responsible for the inactivation of the endocannabinoid anandamide) and transient receptor potential vanilloid type-1 channel (TRPV1), produces anxiolytic-like effects in mice. The present study was designed to assess the capability of AA-5-HT to reverse the behavioral despair following exposure to stress in rats and the role of the HPA-axis. Behavioral tasks were performed, and corticosterone and endocannabinoid (anandamide and <em>2</em>-<em>arachidonoylglycerol</em>) levels were measured in selected brain areas critically involved in the pathophysiology of stress-related disorders (medial PFC and hippocampus) under basal and stress conditions, and in response to treatment with AA-5-HT. Our data show that AA-5-HT reverses the rat behavioral despair in the forced swim test under stress conditions, and this effect is associated with the normalization of the HPA-axis deregulation that follows stress application and only in part with elevation of anandamide levels. Blockade of FAAH and TRPV1 may thus represent a novel target to design novel therapeutic strategies for the treatment of stress-related disorders.

The primary route of inactivation of the endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> in the central nervous system is through enzymatic hydrolysis, mainly carried out by monoacylglycerol lipase (MAGL), along with a small contribution by the α/β-hydrolase domain (ABHD) proteins ABHD6 and ABHD1<em>2</em>. Recent methodological progress allowing kinetic monitoring of glycerol liberation has facilitated substrate profiling of the human endocannabinoid hydrolases, and these studies have revealed that the three enzymes have distinct monoacylglycerol substrate and isomer preferences. Here, we have extended this substrate profiling to cover four prostaglandin glycerol esters, namely, 15-deoxy-Δ(1<em>2</em>,14)-prostaglandin J<em>2</em>-<em>2</em>-glycerol (15d-PGJ<em>2</em>-G), PGD<em>2</em>-G, PGE<em>2</em>-G, and PGF<em>2</em> α-G. We found that the three enzymes hydrolyzed the tested substrates, albeit with distinct rates and preferences. Although human ABHD1<em>2</em> (hABHD1<em>2</em>) showed only marginal activity toward PGE<em>2</em>-G, hABHD6 preferentially hydrolyzed PGD<em>2</em>-G, and human MAGL (hMAGL) robustly hydrolyzed all four. This was particularly intriguing for MAGL activity toward 15d-PGJ<em>2</em>-G whose hydrolysis rate rivaled that of the best monoacylglycerol substrates. Molecular modeling studies combined with kinetic analysis supported favorable interaction with the hMAGL active site. Long and short MAGL isoforms shared a similar substrate profile, and hMAGL hydrolyzed 15d-PGJ<em>2</em>-G also in living cells. The ability of 15d-PGJ<em>2</em>-G to activate the canonical nuclear factor erythroid <em>2</em>-related factor (Nrf<em>2</em>) signaling pathway used by 15d-PGJ<em>2</em> was assessed, and these studies revealed for the first time that 15d-PGJ<em>2</em> and 15d-PGJ<em>2</em>-G similarly activated Nrf<em>2</em> signaling as well as transcription of target genes of this pathway. Our study challenges previous claims regarding the ability of MAGL to catalyze PG-G hydrolysis and extend the MAGL substrate profile beyond the classic monoacylglycerols.

The endocannabinoid (eCB) system has recently been implicated in both the pathogenesis of depression and the action of antidepressants. Here, we investigated the effect of acutely or chronically administering antidepressants [imipramine (IMI) (15 mg/kg), escitalopram (ESC) (10 mg/kg), and tianeptine (10 mg/kg)] on the levels of both eCBs [anandamide (AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG)] and N-acylethanolamines (NAEs) [palmitoylethanolamide (PEA) and oleoylethanolamide (OEA)] in various rat brain regions. We also examined the ability of the acute and chronic administration of N-acetylcysteine (NAC) (a mucolytic drug; 100 mg/kg) or URB597 (a fatty acid amide hydrolase inhibitor; 0.3 mg/kg), which have both elicited antidepressant activity in preclinical studies, to affect eCB and NAE levels. Next, we determined whether the observed effects are stable 10 days after the chronic administration of these drugs was halted. We report that the chronic administration of all investigated drugs increased AEA levels in the hippocampus and also increased both AEA and <em>2</em>-AG levels in the dorsal striatum. NAE levels in limbic regions also increased after treatment with IMI (PEA/OEA), ESC (PEA), and NAC (PEA/OEA). Removing chronic ESC treatment for 10 days affected eCB and NAE levels in the frontal cortex, hippocampus, dorsal striatum, and cerebellum, while a similar tianeptine-free period enhanced accumbal NAE levels. All other drugs maintained their effects after the 10-day washout period. Therefore, the eCB system appears to play a significant role in the mechanism of action of clinically effective and potential antidepressants and may serve as a target for drug design and discovery.

The evolution of plant metabolic pathways to invent compounds which distract predators, and the history of medicine to find treatments for diseases, often share a common logic. An attractive example to illustrate the rationale behind this is the Cannabis sativa plant, which was exploited for its widespread therapeutic effects for several thousand years, but historical "prescriptions" highlighted its distractive behavioral side-effects if abused. This chapter aims to explain the characteristically wide pharmacological and behavioral profile of the Cannabis plant by pointing to the ubiquitous anatomical distribution of CB₁ cannabinoid receptors, its predominant molecular target, throughout the nervous system. However, in contrast to their abundant regional and cellular localization, the subcellular arrangement of CB₁ receptors and the enzymes involved in the metabolism of its main endogenous ligand, <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), are strikingly polarized on the neuronal surface in the adult brain. Though there are still several unresolved issues, the known pieces of the puzzle outline a picture in which the biosynthetic machinery for <em>2</em>-AG is accumulated in the somatodendritic compartment of neurons, whereas its receptor and degrading enzyme are both found on axon terminals. This molecular architecture suggests that a main physiological role of endocannabinoid signaling is the retrograde regulation of synaptic transmission, and the present chapter aims to summarize compelling evidence that it is an ancient and fundamental component of several distinct types of synapses throughout the nervous system.

To date, our understanding of the relative contribution and potential overlapping roles of the endocannabinoids anandamide (AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) in the regulation of brain function and behavior is still limited. To address this issue, we investigated the effects of systemic administration of JZL195, that simultaneously increases AEA and <em>2</em>-AG signaling by inhibiting their hydrolysis, in the regulation of socio-emotional behavior in adolescent and adult rats. JZL195, administered at the dose of 0.01mg/kg, increased social play behavior, that is the most characteristic social activity displayed by adolescent rats, and increased social interaction in adult animals. At both ages, these behavioral effects were antagonized by the CB1 cannabinoid receptor antagonist SR141716A and were associated with increased brain levels of <em>2</em>-AG, but not AEA. Conversely, at the dose of 1mg/kg, JZL195 decreased general social exploration in adolescent rats without affecting social play behavior, and induced anxiogenic-like effects in the elevated plus-maze test both in adolescent and adult animals. These effects, mediated by activation of CB1 cannabinoid receptors, were paralleled by simultaneous increase in AEA and <em>2</em>-AG levels in adolescent rats, and by an increase of only <em>2</em>-AG levels in adult animals. These findings provide the first evidence for a role of <em>2</em>-AG in social behavior, highlight the different contributions of AEA and <em>2</em>-AG in the modulation of emotionality at different developmental ages and suggest that pharmacological inhibition of AEA and <em>2</em>-AG hydrolysis is a useful approach to investigate the role of these endocannabinoids in neurobehavioral processes.

Extreme obesity is a core phenotypic feature of Prader-Willi syndrome (PWS). Among numerous metabolic regulators, the endocannabinoid (eCB) system is critically involved in controlling feeding, body weight, and energy metabolism, and a globally acting cannabinoid-1 receptor (CB1R) blockade reverses obesity both in animals and humans. The first-in-class CB1R antagonist rimonabant proved effective in inducing weight loss in adults with PWS. However, it is no longer available for clinical use because of its centrally mediated, neuropsychiatric, adverse effects.

We studied eCB 'tone' in individuals with PWS and in the Magel<em>2</em>-null mouse model that recapitulates the major metabolic phenotypes of PWS and determined the efficacy of a peripherally restricted CB1R antagonist, JD5037 in treating obesity in these mice.

Individuals with PWS had elevated circulating levels of <em>2</em>-<em>arachidonoylglycerol</em> and its endogenous precursor and breakdown ligand, arachidonic acid. Increased hypothalamic eCB 'tone', manifested by increased eCBs and upregulated CB1R, was associated with increased fat mass, reduced energy expenditure, and decreased voluntary activity in Magel<em>2</em>-null mice. Daily chronic treatment of obese Magel<em>2</em>-null mice and their littermate wild-type controls with JD5037 (3 mg/kg/d for <em>2</em>8 days) reduced body weight, reversed hyperphagia, and improved metabolic parameters related to their obese phenotype.

Dysregulation of the eCB/CB1R system may contribute to hyperphagia and obesity in Magel<em>2</em>-null mice and in individuals with PWS. Our results demonstrate that treatment with peripherally restricted CB1R antagonists may be an effective strategy for the management of severe obesity in PWS.

BACKGROUND

Increasing the available repertoire of effective treatments for mood and anxiety disorders represents a critical unmet need. Pharmacological augmentation of endogenous cannabinoid (eCB) signaling has been suggested to represent a novel approach to the treatment of anxiety disorders; however, the functional interactions between two canonical eCB pathways mediated via anandamide (N-arachidonylethanolamine [AEA]) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) in the regulation of anxiety are not well understood.

METHODS

We utilized pharmacological augmentation and depletion combined with behavioral and electrophysiological approaches to probe the role of <em>2</em>-AG signaling in the modulation of stress-induced anxiety and the functional redundancy between AEA and <em>2</em>-AG signaling in the modulation of anxiety-like behaviors in mice.

RESULTS

Selective <em>2</em>-AG augmentation reduced anxiety in the light/dark box assay and prevented stress-induced increases in anxiety associated with limbic AEA deficiency. In contrast, acute <em>2</em>-AG depletion increased anxiety-like behaviors, which was normalized by selective pharmacological augmentation of AEA signaling and via direct cannabinoid receptor 1 stimulation with Δ9-tetrahydrocannabinol. Electrophysiological studies revealed <em>2</em>-AG modulation of amygdala glutamatergic transmission as a key synaptic correlate of the anxiolytic effects of <em>2</em>-AG augmentation.

CONCLUSIONS

Although AEA and <em>2</em>-AG likely subserve distinct physiological roles, a pharmacological and functional redundancy between these canonical eCB signaling pathways exists in the modulation of anxiety-like behaviors. These data support development of eCB-based treatment approaches for mood and anxiety disorders and suggest a potentially wider therapeutic overlap between AEA and <em>2</em>-AG augmentation approaches than was previously appreciated.

OBJECTIVE

Hedonic eating occurs independently from homeostatic needs prompting the ingestion of pleasurable foods that are typically rich in fat, sugar and/or salt content. In normal weight healthy subjects, we found that before hedonic eating, plasma levels of <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) were higher than before nonhedonic eating, and although they progressively decreased after food ingestion in both eating conditions, they were significantly higher in hedonic eating. Plasma levels of anandamide (AEA), oleoylethanolamide (OEA) and palmitoylethanolamide (PEA), instead, progressively decreased in both eating conditions without significant differences. In this study, we investigated the responses of AEA, <em>2</em>-AG, OEA and PEA to hedonic eating in obese individuals.

METHODS

Peripheral levels of AEA, <em>2</em>-AG, OEA and PEA were measured in 14 obese patients after eating favourite (hedonic eating) and non-favourite (nonhedonic eating) foods in conditions of no homeostatic needs.

RESULTS

Plasma levels of <em>2</em>-AG increased after eating the favourite food, whereas they decreased after eating the non-favourite food, with the production of the endocannabinoid being significantly enhanced in hedonic eating. Plasma levels of AEA decreased progressively in nonhedonic eating, whereas they showed a decrease after the exposure to the favourite food followed by a return to baseline values after eating it. No significant differences emerged in plasma OEA and PEA responses to favourite and non-favourite food.

CONCLUSIONS

Present findings compared with those obtained in our previously studied normal weight healthy subjects suggest deranged responses of endocannabinoids to food-related reward in obesity.

Activation of hepatocyte cannabinoid receptor-1 (CB₁R) by hepatic stellate cell (HSC)-derived <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) drives de novo lipogenesis in alcoholic liver disease (ALD). How alcohol stimulates <em>2</em>-AG production in HSCs is unknown. Here, we report that chronic alcohol consumption induced hepatic cysteine deficiency and subsequent glutathione depletion by impaired transsulfuration pathway. A compensatory increase in hepatic cystine-glutamate anti-porter xCT boosted extracellular glutamate levels coupled to cystine uptake both in mice and in patients with ALD. Alcohol also induced the selective expression of metabotropic glutamate receptor-5 (mGluR5) in HSCs where mGluR5 activation stimulated <em>2</em>-AG production. Consistently, genetic or pharmacologic inhibition of mGluR5 or xCT attenuated alcoholic steatosis in mice via the suppression of <em>2</em>-AG production and subsequent CB₁R-mediated de novo lipogenesis. We conclude that a bidirectional signaling operates at a metabolic synapse between hepatocytes and HSCs through xCT-mediated glutamate-mGluR5 signaling to produce <em>2</em>-AG, which induces CB₁R-mediated alcoholic steatosis.

Skeletal muscle is a major storage site for glycogen and a focus for understanding insulin resistance and type-<em>2</em>-diabetes. New evidence indicates that overactivation of the peripheral endocannabinoid system (ECS) in skeletal muscle diminishes insulin sensitivity. Specific n-6 and n-3 polyunsaturated fatty acids (PUFA) are precursors for the biosynthesis of ligands that bind to and activate the cannabinoid receptors. The function of the ECS and action of PUFA in skeletal muscle glucose uptake was investigated in proliferating and differentiated C<em>2</em>C1<em>2</em> myoblasts treated with either <em>2</em>5 μM of arachidonate (AA) or docosahexaenoate (DHA), <em>2</em>5 μM of EC [anandamide (AEA), <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), docosahexaenoylethanolamide (DHEA)], 1 μM of CB1 antagonist NESS03<em>2</em>7, and CB<em>2</em> inverse agonist AM630. Compared to the BSA vehicle control cell cultures in both proliferating and differentiated myoblasts those treated with DHEA, the EC derived from the n-3 PUFA DHA, had higher <em>2</em>4 h glucose uptake, while AEA and <em>2</em>-AG, the EC derived from the n-6 PUFA AA, had lower basal glucose uptake. Adenylyl cyclase mRNA was higher in myoblasts treated with DHA in both proliferating and differentiated states while those treated with AEA or <em>2</em>-AG were lower compared to the control cell cultures. Western blot and qPCR analysis showed higher expression of the cannabinoid receptors in differentiated myoblasts treated with DHA while the opposite was observed with AA. These findings indicate a compensatory effect of DHA and DHEA compared to AA-derived ligands on the ECS and associated ECS gene expression and higher glucose uptake in myoblasts.