Search

2-arachidonoylglycerol (2-AG) is the most abundant endocannabinoid (EC), acting as a full agonist at both CB1 and CB2 cannabinoid receptors. It is synthesized on demand in postsynaptic membranes through the sequential action of phosphoinositide-specific phospholipase Cβ1 (PLCβ1) and diacylglycerol lipase α (DAGLα), contributing to retrograde signaling upon interaction with presynaptic CB1. However, 2-AG production might also involve various combinations of PLC and DAGL isoforms, as well as additional intracellular pathways implying other enzymes and substrates. Three other alternative pathways of 2-AG synthesis rest on the extracellular cleavage of 2-arachidonoyl-lysophospholipids by three different hydrolases: glycerophosphodiesterase 3 (GDE3), lipid phosphate phosphatases (LPPs), and two members of ecto-nucleotide pyrophosphatase/phosphodiesterases (ENPP6–7). We propose the names of AlterAG-1, -2, and -3 for three pathways sharing an ectocellular localization, allowing them to convert extracellular lysophospholipid mediators into 2-AG, thus inducing typical signaling switches between various G-protein-coupled receptors (GPCRs). This implies the critical importance of the regioisomerism of both lysophospholipid (LPLs) and 2-AG, which is the object of deep analysis within this review. The precise functional roles of AlterAGs are still poorly understood and will require gene invalidation approaches, knowing that both 2-AG and its related lysophospholipids are involved in numerous aspects of physiology and pathology, including cancer, inflammation, immune defenses, obesity, bone development, neurodegeneration, or psychiatric disorders. [ABSTRACT FROM AUTHOR]

Background: An increasing number of people around the world suffer from Crohn's disease (CD), one of the inflammatory bowel diseases (IBDs). Recent evidence suggests that the endogenous cannabinoid system plays an important role in IBD. The main endocannabinoids, anandamide (AEA) and 2-arachidonoylglycerol (2-AG), are ligands of the CB1 and CB2 receptors that are expressed on immune cells. These receptors as well as endocannabinoids are believed to mediate inflammatory activity and participate in the pathophysiology of CD. Objective: Very little is known about the secretion of endogenous cannabinoids throughout the course of CD. Therefore, the aim of this study was to determine and analyze the 2-AG and AEA concentrations in children who suffered from CD. Methods: We studied 34 adolescents with CD, mean age 13.5 years ± 2.0. Blood samples were collected three times in the active phase of the disease (during admission to hospital, before treatment), 2–4 weeks later, during the treatment and clinical improvement of the patients, and 3–6 months later, in the period of remission. The control group included 33 healthy teenagers of the same age who were examined once. In all patients, fasting blood samples were collected in the morning. 2AG and AEA concentrations in serum were measured using EIA kits (Abclonal, Woburn, MA, USA and ELK Biotechnology Co., Ltd. Wuhan, China, respectively). Results: The median 2-AG concentration in the study group was stable: acute phase 907.4 [379.3; 1300.5] pg/mL, during treatment 715.1 [416.7; 1302.5] pg/mL; remission 991.1 [381.1; 1652.5] pg/mL, and similar to the values observed in the control group 761.8 [504.3; 1497.0] pg/mL. No significant differences were found between the results obtained at all time points in the study group and compared to the control group (in all cases p > 0.44). In the case of AEA, in the study group before treatment, the median concentration was 2.63 [2.24; 2.79] ng/mL and similar to values obtained during treatment 2.56 [2.33; 3.06] ng/mL, and in remission, with 2.61 [2.46; 2.85] ng/mL. All these concentrations were lower compared to the values measured in the control group of 3.18 [2.57; 3.88] ng/mL (p = 0.023, p = 0.035, p = 0.056, respectively). Conclusions: While 2-AG levels remained stable throughout the disease course and were comparable to controls, AEA concentrations were consistently lower in CD patients. Therefore, therapeutic interventions aimed at increasing AEA-related signaling, such as administration of its analogues or fatty acid amide hydrolase inhibitors, may improve symptoms associated with Crohn's disease in children. [ABSTRACT FROM AUTHOR]

Asman Sadino,1,2,* Nyi Mekar Saptarini,3 Jutti Levita,4,* Dwi Syah Fitra Ramadhan,5 Adryan Fristiohady,6 Supat Jiranusornkul7 1Doctoral Program in Pharmacy, Faculty of Pharmacy, Universitas Padjadjaran, Sumedang, West Java, 45363, Indonesia; 2Department of Pharmacology and Clinical Pharmacy, Faculty of Mathematics and Natural Sciences, Garut University, Garut, West Java, 44151, Indonesia; 3Department of Pharmaceutical Analysis and Medicinal Chemistry, Faculty of Pharmacy, Universitas Padjadjaran, Sumedang, West Java, 45363, Indonesia; 4Department of Pharmacology and Clinical Pharmacy, Faculty of Pharmacy, Universitas Padjadjaran, Sumedang, West Java, 45363, West Java, Indonesia; 5Department of Pharmacy, Poltekkes Kemenkes Makassar, Makassar, South Sulawesi, 90222, Indonesia; 6Faculty of Pharmacy, Halu Oleo University, Kendari, Southeast Sulawesi, 93132, Indonesia; 7Department of Pharmaceutical Science, Faculty of Pharmacy, Chiang Mai University, Chiang Mai, Thailand*These authors contributed equally to this workCorrespondence: Jutti Levita, Department of Pharmacology and Clinical Pharmacy, Faculty of Pharmacy, Universitas Padjadjaran, Sumedang, West Java, 46363, Indonesia, Email jutti.levita@unpad.ac.idBackground: Human monoacylglycerol lipase (MGL) is accountable for the hydrolysis of 2-arachidonoylglycerol (2-AG), thus contributing pivotally to neuroprotection because 2-AG is the main source of arachidonic acid, the precursor of prostaglandins production. Inhibiting MGL reduces inflammatory damage in the ischemic brain and enhances cerebral blood flow. Plants have been reported for their neuroprotective effect, such as Morinda citrifolia on pentylenetetrazol (PTZ)-induced kindling seizures in mice, by reducing the seizures and restoring behavioral and biochemical changes, although the mechanism is not described.Purpose: To evaluate the binding affinity and stability of phytoconstituents in M. citrifolia fruits toward human MGL (PDB ID 3PE6), compared to the known MGL inhibitors (JZL195 and ZYH). The in silico pharmacology study was validated by an in vitro study of the phytosterols and the ethanol extract of M. citrifolia fruits (EEMC) towards MGL.Methods: Initially, nine phytoconstituents of M. citrifolia fruits were docked to the catalytic pocket of human MGL (PDB ID: 3PE6), and compounds with the best affinity were subjected to a molecular dynamic (MD) simulation. The in vitro study was performed using the MGL inhibitor screening assay kit.Results: The best binding affinity and stability toward human MGL were shown by stigmasterol and beta-sitosterol, and the MM-PBSA total binding energy of stigmasterol and beta-sitosterol to MGL is stronger than that of JZL195 and ZYH. Moreover, beta-sitosterol and EEMC inhibit MGL with an IC50 value of, respectively, 8.10 μg/mL and 196.20 μg/mL, while JZL195 shows an IC50 of 0.028 μg/mL.Conclusion: Beta-sitosterol of Morinda citrifolia fruits may have the potential to protect human neurons by occupying the catalytic site of human MGL, thus competitively inhibiting the substrate of the enzyme. However, the inhibitory activity towards human MGL is lower than JZL195.Keywords: 2-arachidonoylglycerol, beta-sitosterol, phytosterols, prostaglandin, serine hydrolase, stigmasterol

• Maternal obesity alters the neurological and behavioral phenotype in the offspring. • Treatment with cannabidiol rescues anxiety and social disturbances in the offspring. • Both perinatal maternal obesity and CBD present sexually dimorphic influences. • CBD modulates glial reactivity, oxytocin, glutamate and endocannabinoid signaling. Maternal obesity is associated with an increased risk of psychiatric disorders such as anxiety, depression, schizophrenia and autism spectrum disorder in the offspring. While numerous studies focus on preventive measures targeting the mothers, only a limited number provide practical approaches for addressing the damages once they are already established. We have recently demonstrated the interplay between maternal obesity and treatment with cannabidiol (CBD) on hypothalamic inflammation and metabolic disturbances, however, little is known about this relationship on behavioral manifestations and neurochemical imbalances in other brain regions. Therefore, here we tested whether CBD treatment could mitigate anxiety-like and social behavioral alterations, as well as neurochemical disruptions in both male and female offspring of obese dams. Female Wistar rats were fed a cafeteria diet for 12 weeks prior to mating, and during gestation and lactation. Offspring received CBD (50 mg/kg) from weaning for 3 weeks. Behavioral tests assessed anxiety-like manifestations and social behavior, while neuroinflammatory and neurochemical markers were evaluated in the prefrontal cortex (PFC) and hippocampus. CBD treatment attenuated maternal obesity-induced anxiety-like and social behavioral alterations, followed by rescuing effects on imbalanced neurotransmitter and endocannabinoid concentrations and altered expression of glial markers, CB1, oxytocin and dopamine receptors, with important differences between sexes. Overall, the findings of this study provide insight into the signaling pathways for the therapeutic benefits of CBD on neuroinflammation and neurochemical imbalances caused by perinatal maternal obesity in the PFC and the hippocampus, which translates into the behavioral manifestations, highlighting the sexual dimorphism encompassing both the transgenerational effect of obesity and the endocannabinoid system. [ABSTRACT FROM AUTHOR]

Background Methods Results Conclusions Hereditary angioedema (HAE) is a rare genetic disorder characterized by local, self‐limiting edema due to temporary increase in vascular permeability. HAE with normal C1 esterase inhibitor (C1INH) activity includes the form with mutations in the F12 gene encoding for coagulation factor XII (FXII‐HAE) causing an overproduction of bradykinin (BK) leading to angioedema attack. BK binding to B2 receptors (BK2R) leads to an activation of phospholipase C (PLC) and subsequent generation of second messengers: diacylglycerols (DAGs) and possibly the endocannabinoids (eCBs), 2‐arachidonoylglycerol (2‐AG) and anandamide (AEA), and eCB‐related N‐acylethanolamines [palmitoylethanolamide (PEA) and oleoylethanolamide (OEA)]. To date, there are no data on the role of these lipid mediators in FXII‐HAE.Here, we analyzed plasma levels of PLC, DAGs, and eCBs in 40 patients with FXII‐HAE and 40 sex‐ and age‐matched healthy individuals.Plasma PLC activity was increased in FXII‐HAE patients compared to controls. Concentrations of DAG 18:1–20:4, a lipid second messenger produced by PLC, were higher in FXII‐HAE compared to controls, and positively correlated with PLC activity and cleaved high molecular kininogen (cHK). Also the concentrations of the DAG metabolite, 2‐AG were altered in FXII‐HAE. AEA and OEA were decreased in FXII‐HAE patients compared to controls; by contrast, PEA, was increased. The levels of all tested mediators did not differ between symptomatic and asymptomatic patients. Moreover, C1INH‐HAE patients had elevated plasma levels of PLC, which correlated with cHK, but the levels of DAGs and eCBs were the same as controls.BK overproduction and BKR2 activation are linked to alteration of PLCs and their metabolites in patients with FXII‐HAE. Our results may pave way to investigations on the functions of these mediators in the pathophysiology of FXII‐HAE, and provide new potential biomarkers and therapeutic targets. [ABSTRACT FROM AUTHOR]

One of cannabis' most iconic effects is the stimulation of hedonic high-calorie eating-the "munchies"-yet habitual cannabis users are, on average, leaner than non-users. We asked whether this phenotype might result from lasting changes in energy balance established during adolescence, when use of the drug often begins. We found that daily low-dose administration of cannabis' intoxicating constituent, Δ9-tetrahydrocannabinol (THC), to adolescent male mice causes an adult metabolic phenotype characterized by reduced fat mass, increased lean mass and utilization of fat as fuel, partial resistance to diet-induced obesity and dyslipidemia, enhanced thermogenesis, and impaired cold- and β-adrenergic receptor-stimulated lipolysis. Further analyses revealed that this phenotype is associated with molecular anomalies in the adipose organ, including ectopic overexpression of muscle-associated proteins and heightened anabolic processing. Thus, adolescent exposure to THC may promote an enduring "pseudo-lean" state that superficially resembles healthy leanness but might in fact be rooted in adipose organ dysfunction.

For thousands of years, medicinal cannabis has been used for pain treatment, but its use for pain management is still controversial. Meta-analysis of the literature has shown contrasting results on the addition of cannabinoids to opioids compared with placebo/other active agents to reduce pain. Clinical studies are mainly focused on medicinal cannabis use in chronic pain management, for which the analgesic effect has been proven in many studies. This review focuses on the potential use of medical cannabis for acute pain management in preclinical studies, studies on healthy subjects and the few pioneering studies in the clinical setting.

Marijuana has been used by humans for thousands of years for both medicinal and recreational purposes. This included the treatment of pain, inflammation, seizures, and nausea. In the 1960s, the structure of the principal psychoactive ingredient Δ9-tetrahydrocannabinol was determined, and over the next few decades, two cannabinoid receptors were characterized along with the human endocannabinoid system and what it affects. This includes metabolism, the cardiovascular and reproductive systems, and it is involved in such conditions as inflammation, cancer, glaucoma, and liver and musculoskeletal disorders. In the central nervous system, the endocannabinoid system has been linked to appetite, learning, memory, and conditions such as depression, anxiety, schizophrenia, stroke, multiple sclerosis, neurodegeneration, addiction, and epilepsy. It was the profound effectiveness of cannabidiol, a non-psychoactive ingredient of marijuana, to relieve the symptoms of Dravet syndrome, a severe form of childhood epilepsy, that recently helped spur marijuana research. This has helped substantially to change society's attitude towards this potential source of useful drugs. However, research has also revealed that the actions of endocannabinoids, such as anandamide and 2-arachidonoylglycerol, and the phytocannabinoids, tetrahydrocannabinol and cannabidiol, were not just due to interactions with the two cannabinoid receptors but by acting directly on many other targets including various G-protein receptors and cation channels, such as the transient receptor potential channels for example. This mini-review attempts to survey the effects of these 4 important cannabinoids on these currently identified targets.

Marijuana has been used by humans for thousands of years for both medicinal and recreational purposes. This included the treatment of pain, inflammation, seizures, and nausea. In the 1960s, the structure of the principal psychoactive ingredient Δ9-tetrahydrocannabinol was determined, and over the next few decades, two cannabinoid receptors were characterized along with the human endocannabinoid system and what it affects. This includes metabolism, the cardiovascular and reproductive systems, and it is involved in such conditions as inflammation, cancer, glaucoma, and liver and musculoskeletal disorders. In the central nervous system, the endocannabinoid system has been linked to appetite, learning, memory, and conditions such as depression, anxiety, schizophrenia, stroke, multiple sclerosis, neurodegeneration, addiction, and epilepsy. It was the profound effectiveness of cannabidiol, a non-psychoactive ingredient of marijuana, to relieve the symptoms of Dravet syndrome, a severe form of childhood epilepsy, that recently helped spur marijuana research. This has helped substantially to change society's attitude towards this potential source of useful drugs. However, research has also revealed that the actions of endocannabinoids, such as anandamide and 2-arachidonoylglycerol, and the phytocannabinoids, tetrahydrocannabinol and cannabidiol, were not just due to interactions with the two cannabinoid receptors but by acting directly on many other targets including various G-protein receptors and cation channels, such as the transient receptor potential channels for example. This mini-review attempts to survey the effects of these 4 important cannabinoids on these currently identified targets. [ABSTRACT FROM AUTHOR]

Background: Some evidence suggests that cannabidiol (CBD) has potential to help alleviate HIV symptoms due to its antioxidant and anti-inflammatory properties. Here we examined acute CBD effects on various behaviors and the endocannabinoid system in HIV Tat transgenic mice. Methods: Tat transgenic mice (female/male) were injected with CBD (3, 10, 30 mg/kg) and assessed for antinociception, activity, coordination, anxietylike behavior, and recognition memory. Brains were taken to quantify endocannabinoids, cannabinoid receptors, and cannabinoid catabolic enzymes. Additionally, CBD and metabolite 7-hydroxy-CBD were quantified in the plasma and cortex. Results: Tat decreased supraspinal-related nociception and locomotion. CBD and sex had little to no effects on any of the behavioral measures. For the endocannabinoid system male sex was associated with elevated concentration of the proinflammatory metabolite arachidonic acid in various CNS regions, including the cerebellum that also showed higher FAAH expression levels for Tat(+) males. GPR55 expression levels in the striatum and cerebellum were higher for females compared to males. CBD metabolism was altered by sex and Tat expression. Conclusion: Findings indicate that acute CBD effects are not altered by HIV Tat, and acute CBD has no to minimal effects on behavior and the endocannabinoid system. [ABSTRACT FROM AUTHOR]

Introduction: Microglia and macrophages can influence the evolution of myelin lesions through the production of extracellular vesicles (EVs). While microglial EVs promote in vitro differentiation of oligodendrocyte precursor cells (OPCs), whether EVs derived from macrophages aid or limit OPC maturation is unknown. Methods: Immunofluorescence analysis for the myelin protein MBP was employed to evaluate the impact of EVs from primary rat macrophages on cultured OPC differentiation. Raman spectroscopy and liquid chromatographymass spectrometry was used to define the promyelinating lipid components of myelin EVs obtained in vitro and isolated from human plasma. Results and discussion: Here we show that macrophage-derived EVs do not promote OPC differentiation, and those released from macrophages polarized towards an inflammatory state inhibit OPC maturation. However, their lipid cargo promotes OPC maturation in a similar manner to microglial EVs. We identify the promyelinating endocannabinoids anandamide and 2-arachidonoylglycerol in EVs released by both macrophages and microglia in vitro and circulating in human plasma. Analysis of OPC differentiation in the presence of the endocannabinoid receptor antagonists SR141716A and AM630 reveals a key role of vesicular endocannabinoids in OPC maturation. From this study, EVassociated endocannabinoids emerge as important mediators in microglia/ macrophage-oligodendrocyte crosstalk, which may be exploited to enhance myelin repair. [ABSTRACT FROM AUTHOR]

2-arachidonoylglycerol (2-AG) is the most abundant endocannabinoid (EC), acting as a full agonist at both CB1 and CB2 cannabinoid receptors. It is synthesized on demand in postsynaptic membranes through the sequential action of phosphoinositide-specific phospholipase Cβ1 (PLCβ1) and diacylglycerol lipase α (DAGLα), contributing to retrograde signaling upon interaction with presynaptic CB1. However, 2-AG production might also involve various combinations of PLC and DAGL isoforms, as well as additional intracellular pathways implying other enzymes and substrates. Three other alternative pathways of 2-AG synthesis rest on the extracellular cleavage of 2-arachidonoyl-lysophospholipids by three different hydrolases: glycerophosphodiesterase 3 (GDE3), lipid phosphate phosphatases (LPPs), and two members of ecto-nucleotide pyrophosphatase/phosphodiesterases (ENPP6–7). We propose the names of AlterAG-1, -2, and -3 for three pathways sharing an ectocellular localization, allowing them to convert extracellular lysophospholipid mediators into 2-AG, thus inducing typical signaling switches between various G-protein-coupled receptors (GPCRs). This implies the critical importance of the regioisomerism of both lysophospholipid (LPLs) and 2-AG, which is the object of deep analysis within this review. The precise functional roles of AlterAGs are still poorly understood and will require gene invalidation approaches, knowing that both 2-AG and its related lysophospholipids are involved in numerous aspects of physiology and pathology, including cancer, inflammation, immune defenses, obesity, bone development, neurodegeneration, or psychiatric disorders.

In mouse motor synapses, the exogenous application of the endocannabinoid (EC) 2‐arachidonoylglycerol (2‐AG) increases acetylcholine (ACh) quantal size due to the activation of CB1 receptors and the stimulation of ACh vesicular uptake. In the present study, microelectrode recordings of miniature endplate potentials (MEPP) revealed that this effect of 2‐AG is independent of brain‐derived neurotrophic factor (BDNF) signaling but involves the activation of calcitonin gene–related peptide (CGRP) receptors along with CB1 receptors. Potentiation of MEPP amplitude in the presence of 2‐AG was prevented by blockers of CGRP receptors and ryanodine receptors (RyR) and by inhibitors of phospholipase C (PLC) and Ca2+/calmodulin‐dependent protein kinase II (CaMKII). Therefore, we suggest a hypothetical chain of events, which starts from the activation of presynaptic CB1 receptors, involves PLC, RyR, and CaMKII, and results in CGRP release with the subsequent activation of presynaptic CGRP receptors. Activation of CGRP receptors is probably a part of a complex molecular cascade leading to the 2‐AG‐induced increase in ACh quantal size and MEPP amplitude. We propose that the same chain of events may also take place if 2‐AG is endogenously produced in mouse motor synapses, because the increase in MEPP amplitude that follows after prolonged tetanic muscle contractions (30 Hz, 2 min) was prevented by the blocking of CB1 receptors. This work may help to unveil the previously unknown aspects of the functional interaction between ECs and peptide modulators aimed at the regulation of quantal size and synaptic transmission. [ABSTRACT FROM AUTHOR]

Background: The reasons for developing depression are not fully understood. However, it is known that the serotonergic system plays a role in the etiology, but the endocannabinoid system receives attention. Method: In this study, 161 patients with a depressive disorder and 161 healthy participants were examined for the distribution of the CNR1 rs4940353, 5‐HT2A rs6311, and 5‐HT1A rs6295 by high‐resolution melting genotyping. The concentration of arachidonoyl ethanolamide (AEA) and 2‐arachidonoylglycerol (2‐AG) in the blood was measured by liquid chromatography–tandem mass spectrometry. Additionally, depression and anxiety symptoms were evaluated based on self‐questionnaires. Fifty‐nine patients participated in a second appointment to measure the concentration of AEA, 2‐AG, and symptoms of depression and anxiety. Results: We observed higher AEA and decreased 2‐AG concentrations in patients with depression compared to healthy participants. During the treatment, the concentrations of AEA and 2‐AG did not change significantly. In patients higher symptoms of anxiety correlated with lower concentrations of 2‐AG. Gender differences were found concerning increased 2‐AG concentration in male patients and increased anxiety symptoms in female patients. Genotypic variations of 5‐HT1A rs6295 and 5‐HT2A rs6311 are associated with altered serotonergic activity and serotonin content in patients. Conclusion: In conclusion, it seems that the endocannabinoid system, especially the endocannabinoids 2‐AG and AEA, and genetic variations of the 5‐HT1A and 5‐HT2A could play a role in patients with depression and may be involved in a depressive disorder. The present study showed an altered endocannabinoid system and the genetic variations of the 5‐HT1A, 5‐HT2A, and CNR1 genes in patients with a depressive disorder compared to healthy participants. [ABSTRACT FROM AUTHOR]

The endocannabinoid system (ECS) is a new target for the development of retinal disease therapeutics, whose pathophysiology involves neurodegeneration and neuroinflammation. The endocannabinoid 2-arachidonoylglycerol (2-AG) affects neurons and microglia by activating CB1/CB2 cannabinoid receptors (Rs). The aim of this study was to investigate the effects of 2-AG on the CB1R expression/downregulation and retinal neurons/reactive microglia, when administered repeatedly (4 d), in three different paradigms. These involved the 2-AG exogenous administration (a) intraperitoneally (i.p.) and (b) topically and (c) by enhancing the 2-AG endogenous levels via the inhibition (AM11920, i.p.) of its metabolic enzymes (MAGL/ABHD6). Sprague Dawley rats were treated as mentioned above in the presence or absence of CB1/CB2R antagonists and the excitatory amino acid, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA). Immunohistochemistry, Western blot and a 2-AG level analyses were performed. The 2-AG repeated treatment (i.p.) induced the CB1R downregulation, abolishing its neuroprotective actions. However, 2-AG attenuated the AMPA-induced activation of microglia via the CB2R, as concurred by the AM630 antagonist effect. Topically administered 2-AG was efficacious as a neuroprotectant/antiapoptotic and anti-inflammatory agent. AM11920 increased the 2-AG levels providing neuroprotection against excitotoxicity and reduced microglial activation without affecting the CB1R expression. Our findings show that 2-AG, in the three paradigms studied, displays differential pharmacological profiles in terms of the downregulation of the CB1R and neuroprotection. All treatments, however, attenuated the activation of microglia via the CB2R activation, supporting the anti-inflammatory role of 2-AG in the retina. [ABSTRACT FROM AUTHOR]

Background: An increasing number of people around the world suffer from Crohn’s disease (CD), one of the inflammatory bowel diseases (IBDs). Recent evidence suggests that the endogenous cannabinoid system plays an important role in IBD. The main endocannabinoids, anandamide (AEA) and 2-arachidonoylglycerol (2-AG), are ligands of the CB1 and CB2 receptors that are expressed on immune cells. These receptors as well as endocannabinoids are believed to mediate inflammatory activity and participate in the pathophysiology of CD. Objective: Very little is known about the secretion of endogenous cannabinoids throughout the course of CD. Therefore, the aim of this study was to determine and analyze the 2-AG and AEA concentrations in children who suffered from CD. Methods: We studied 34 adolescents with CD, mean age 13.5 years ± 2.0. Blood samples were collected three times in the active phase of the disease (during admission to hospital, before treatment), 2–4 weeks later, during the treatment and clinical improvement of the patients, and 3–6 months later, in the period of remission. The control group included 33 healthy teenagers of the same age who were examined once. In all patients, fasting blood samples were collected in the morning. 2AG and AEA concentrations in serum were measured using EIA kits (Abclonal, Woburn, MA, USA and ELK Biotechnology Co., Ltd. Wuhan, China, respectively). Results: The median 2-AG concentration in the study group was stable: acute phase 907.4 [379.3; 1300.5] pg/mL, during treatment 715.1 [416.7; 1302.5] pg/mL; remission 991.1 [381.1; 1652.5] pg/mL, and similar to the values observed in the control group 761.8 [504.3; 1497.0] pg/mL. No significant differences were found between the results obtained at all time points in the study group and compared to the control group (in all cases p > 0.44). In the case of AEA, in the study group before treatment, the median concentration was 2.63 [2.24; 2.79] ng/mL and similar to values obtained during treatment 2.56 [2.33; 3.06] ng/mL, and in remission, with 2.61 [2.46; 2.85] ng/mL. All these concentrations were lower compared to the values measured in the control group of 3.18 [2.57; 3.88] ng/mL (p = 0.023, p = 0.035, p = 0.056, respectively). Conclusions: While 2-AG levels remained stable throughout the disease course and were comparable to controls, AEA concentrations were consistently lower in CD patients. Therefore, therapeutic interventions aimed at increasing AEA-related signaling, such as administration of its analogues or fatty acid amide hydrolase inhibitors, may improve symptoms associated with Crohn’s disease in children.

Abstract Background The reasons for developing depression are not fully understood. However, it is known that the serotonergic system plays a role in the etiology, but the endocannabinoid system receives attention. Method In this study, 161 patients with a depressive disorder and 161 healthy participants were examined for the distribution of the CNR1 rs4940353, 5‐HT2A rs6311, and 5‐HT1A rs6295 by high‐resolution melting genotyping. The concentration of arachidonoyl ethanolamide (AEA) and 2‐arachidonoylglycerol (2‐AG) in the blood was measured by liquid chromatography–tandem mass spectrometry. Additionally, depression and anxiety symptoms were evaluated based on self‐questionnaires. Fifty‐nine patients participated in a second appointment to measure the concentration of AEA, 2‐AG, and symptoms of depression and anxiety. Results We observed higher AEA and decreased 2‐AG concentrations in patients with depression compared to healthy participants. During the treatment, the concentrations of AEA and 2‐AG did not change significantly. In patients higher symptoms of anxiety correlated with lower concentrations of 2‐AG. Gender differences were found concerning increased 2‐AG concentration in male patients and increased anxiety symptoms in female patients. Genotypic variations of 5‐HT1A rs6295 and 5‐HT2A rs6311 are associated with altered serotonergic activity and serotonin content in patients. Conclusion In conclusion, it seems that the endocannabinoid system, especially the endocannabinoids 2‐AG and AEA, and genetic variations of the 5‐HT1A and 5‐HT2A could play a role in patients with depression and may be involved in a depressive disorder.

Purpose: Diabetic cardiomyopathy (DM) is the cause of late cardiac dysfunction in diabetic patients. Myocardial fibrosis is the main pathological mechanism, and it is associated with transforming growth factor-β1(TGF-β1) expression up-regulation. 2-Arachidonoylglycerol (2-AG) is an endogenous cannabinoid that can effectively improve myocardial cell energy metabolism and cardiac function. Here, we evaluated the protective effect of 2-AG on diabetic cardiomyopathy. Methods: Male C57BL/6 mice were injected with 2-AG intraperitoneally for 4 weeks (10 micro g/kg/day) after 12 weeks of diabetic modeling. After 4 weeks, heart function was evaluated by echocardiography. Heart structure was assessed by hematoxylin and eosin staining. Cardiac fibrosis was analyzed using immunohistochemistry, Sirius red stain, and western blot. Results: After modeling in diabetic mice, cardiac ultrasonography showed decreased cardiac function and pathological findings showed myocardial fibrosis. 2-AG could effectively inhibit the up-regulation of TGF-β1 and Smad2/3, reduce myocardial fibrosis, and ultimately improve cardiac function in diabetic mice. Conclusion: 2-AG reduces cardiac fibrosis via the TGF-β1/Smad2/3 pathway and is a potential pathway for the treatment of cardiac dysfunction in diabetic mice. [ABSTRACT FROM AUTHOR]

Abstract Background Engaging the endocannabinoid system through inhibition of monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH), degrading endocannabinoids (endoCBs) 2-arachidonoylglycerol (2-AG) and anandamide (AEA), was proposed as a promising approach to ameliorate migraine pain. However, the activity of MAGL and FAAH and action of endoCB on spiking activity of meningeal afferents, from which migraine pain originates, has not been explored thus far. Therefore, we here explored the analgesic effects of endoCB enhancement in rat and human meningeal tissues. Methods Both MAGL and FAAH activity and local 2-AG and AEA levels were measured by activity-based protein profiling (ABPP) and LC–MS/MS, respectively, in rat meninges obtained from hemiskulls of P38-P40 Wistar rats and human meninges from elderly patients undergoing non-migraine related neurosurgery. The action on endoCBs upon administration of novel dual MAGL/FAAH inhibitor AKU-005 on meningeal afferents excitability was tested by investigating paired KCl-induced spiking and validation with local (co-)application of either AEA or 2-AG. Finally, the specific TRPV1 agonist capsaicin and blocker capsazepine were tested. Results The basal level of 2-AG exceeded that of AEA in rat and human meninges. KCl-induced depolarization doubled the level of AEA. AKU-005 slightly increased spontaneous spiking activity whereas the dual MAGL/FAAH inhibitor significantly decreased excitation of nerve fibres induced by KCl. Similar inhibitory effects on meningeal afferents were observed with local applications of 2-AG or AEA. The action of AKU-005 was reversed by CB1 antagonist AM-251, implying CB1 receptor involvement in the anti-nociceptive effect. The inhibitory action of AEA was also reversed by AM-251, but not with the TRPV1 antagonist capsazepine. Data cluster analysis revealed that both AKU-005 and AEA largely increased long-term depression-like meningeal spiking activity upon paired KCl-induced spiking. Conclusions In the meninges, high anti-nociceptive 2-AG levels can tonically counteract meningeal signalling, whereas AEA can be engaged on demand by local depolarization. AEA-mediated anti-nociceptive effects through CB1 receptors have therapeutic potential. Together with previously detected MAGL activity in trigeminal ganglia, dual MAGL/FAAH inhibitor AKU-005 appears promising as migraine treatment. Graphical Abstract

Endocannabinoids are a group of endogenous mediators derived from membrane lipids, which are implicated in a wide variety of physiological functions such as blood pressure regulation, immunity, pain, memory, reward, perception, reproduction, and sleep. N-Arachidonoylethanolamine (anandamide; AEA) and 2-arachidonoylglycerol (2-AG) represent two major endocannabinoids in the human body and they exert many of their cellular and organ system effects by activating the Gi/o protein-coupled, cannabinoid type 1 (CB1) and type 2 (CB2) receptors. However, not all effects of cannabinoids are ascribable to their interaction with CB1 and CB2 receptors; indeed, macromolecules like other types of receptors, ion channels, transcription factors, enzymes, transporters, and cellular structure have been suggested to mediate the functional effects of cannabinoids. Among the proposed molecular targets of endocannabinoids, potassium channels constitute an intriguing group, because these channels not only are crucial in shaping action potentials and controlling the membrane potential and cell excitability, thereby regulating a wide array of physiological processes, but also serve as potential therapeutic targets for the treatment of cancer and metabolic, neurological and cardiovascular disorders. This review sought to survey evidence pertaining to the CB1 and CB2 receptor-independent actions of endocannabinoids on ion channels, with an emphasis on AEA and potassium channels. To better understand the functional roles as well as potential medicinal uses of cannabinoids in human health and disease, further mechanistic studies to delineate interactions between various types of cannabinoids and ion channels, including members in the potassium channel superfamily, are warranted.

BackgroundMortality in patients with end-stage renal disease (ESRD) on maintenance hemodialysis (MHD) remains exceptionally high. While traditional risk factors such as obesity are paradoxically associated with better survival, nontraditional risk factors including cachexia increase the likelihood of poor outcomes. There is accumulating evidence that the endocannabinoid (ECB) system plays a major role in energy preservation and storage, factors which can prevent the deleterious effects of cachexia. Hence, in this study, we evaluated the association of circulating ECB levels with mortality in MHD patients.MethodsSerum concentrations of anandamide (AEA) and 2-arachidonoyl-sn-glycerol (2-AG), major ECB ligands, were measured in MHD patients. Their correlation with various clinical/laboratory indices and association with 12-month all-cause mortality were examined.ResultsSerum 2-AG levels positively correlated with body mass index, serum triglycerides and body anthropometric measures. Meanwhile, serum AEA levels correlated positively with serum interleukin-6, and negatively with serum very low-density lipoprotein levels. While increased serum 2-AG levels were associated with reduced risk of all-cause mortality (hazard ratio [HR] 0.52, 95% CI 0.28-0.98), there was no clear association between serum AEA levels and mortality (HR 0.91, 95% CI 0.48-1.72).ConclusionsIn MHD patients, the circulating levels of ECB ligand, 2-AG, may play an important role in determining body mass and risk of mortality. These observations were unique to 2-AG as similar findings were not obtained with serum AEA. Future studies need to investigate the mechanisms responsible for these associations and examine the modulation of the ECB system as a potential target for therapy in ESRD.

Abstract Background The role of the Endocannabinoids (ECs) in insulin resistance, and their association with visceral obesity and metabolic profile have been studied extensively. Since the association between ECs and metabolic factors in Gestational Diabetes Mellitus (GDM) are not clear, we aimed to evaluate the levels of N-Arachidonoylethanolamide (AEA) and 2-Arachidonoylglycerol (2-AG) and their association with C-reactive protein (CRP), glycemic indices, blood pressure, and anthropometric indices in pregnant women with GDM. Methods The present case–control study was conducted among 96 singleton pregnant women aged 18–40 years, including 48 healthy pregnant women (control group) and 48 women with a positive diagnosis of GDM (case group). Odds Ratios (ORs) and 95% Confidence Intervals (CIs) for GDM were checked according to endocannabinoids and anthropometric indices using Multivariable Logistic Regression. Results AEA was significantly associated with increased risk of GDM in models 1, 2 and 3 (OR = 1.22, 95% CI: 1.06–1.41; OR = 1.54, 95% CI: 1.19–1.97; OR = 1.46, 95% CI:1.11–1.91). A positive but no significant association was found for AEA in model 4 (OR = 1.38,95% CI: 0.99–1.92). Similar to AEA, 2-AG was also positively associated with the likelihood of GDM in Models 1, 2, and 3 but the association attenuated to null in model 4 (OR = 1.25; 95% CI: 0.94- 1.65). Conclusions Our findings showed that levels of ECs were significantly higher in pregnant women with GDM compared to healthy ones. Also, ECs levels were associated with the likelihood of GDM, independent of BMI and weight gain.

Background: Engaging the endocannabinoid system through inhibition of monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH), degrading endocannabinoids (endoCBs) 2-arachidonoylglycerol (2-AG) and anandamide (AEA), was proposed as a promising approach to ameliorate migraine pain. However, the activity of MAGL and FAAH and action of endoCB on spiking activity of meningeal afferents, from which migraine pain originates, has not been explored thus far. Therefore, we here explored the analgesic effects of endoCB enhancement in rat and human meningeal tissues. Methods: Both MAGL and FAAH activity and local 2-AG and AEA levels were measured by activity-based protein profiling (ABPP) and LC–MS/MS, respectively, in rat meninges obtained from hemiskulls of P38-P40 Wistar rats and human meninges from elderly patients undergoing non-migraine related neurosurgery. The action on endoCBs upon administration of novel dual MAGL/FAAH inhibitor AKU-005 on meningeal afferents excitability was tested by investigating paired KCl-induced spiking and validation with local (co-)application of either AEA or 2-AG. Finally, the specific TRPV1 agonist capsaicin and blocker capsazepine were tested. Results: The basal level of 2-AG exceeded that of AEA in rat and human meninges. KCl-induced depolarization doubled the level of AEA. AKU-005 slightly increased spontaneous spiking activity whereas the dual MAGL/FAAH inhibitor significantly decreased excitation of nerve fibres induced by KCl. Similar inhibitory effects on meningeal afferents were observed with local applications of 2-AG or AEA. The action of AKU-005 was reversed by CB1 antagonist AM-251, implying CB1 receptor involvement in the anti-nociceptive effect. The inhibitory action of AEA was also reversed by AM-251, but not with the TRPV1 antagonist capsazepine. Data cluster analysis revealed that both AKU-005 and AEA largely increased long-term depression-like meningeal spiking activity upon paired KCl-induced spiking. Conclusions: In the meninges, high anti-nociceptive 2-AG levels can tonically counteract meningeal signalling, whereas AEA can be engaged on demand by local depolarization. AEA-mediated anti-nociceptive effects through CB1 receptors have therapeutic potential. Together with previously detected MAGL activity in trigeminal ganglia, dual MAGL/FAAH inhibitor AKU-005 appears promising as migraine treatment. [ABSTRACT FROM AUTHOR]

The endocannabinoid 2-arachidonoylglycerol (2-AG) is an inflammatory mediator and ligand for the cannabinoid receptors CB1 and CB2. We investigated the atherogenic mechanisms set in motion by 2-AG. Therefore, we created two atherosclerotic mouse models with distinct cell-specific knockouts of the CB2 receptor on either myeloid or endothelial cells. These mice were treated with JZL184, resulting in elevated plasma levels of 2-AG. After a high-fat high-cholesterol diet, atherosclerotic plaques were analyzed. The atherogenic effect of 2-AG was abrogated in mice lacking myeloid expression of the CB2 receptor but not in mice lacking endothelial expression of the CB2 receptor. In vitro, treatment of human monocytes with 2-AG led to the increased production of reactive oxygen species (ROS) and IL-1β. In conclusion, 2-AG shows an atherogenic effect in vivo, dependent on the presence of the CB2 receptor on myeloid cells. In addition, our in vitro data revealed 2-AG to promote inflammatory signalling in monocytes. 2-Arachidonoylglycerol shows an atherogenic effect that is abrogated in mice lacking myeloid expression of the CB2 receptor. [ABSTRACT FROM AUTHOR]

Introduction: Lipids and fatty acids are key components in metabolic processes of the human placenta, thereby contributing to the development of the fetus. Placental dyslipidemia and aberrant activity of lipases have been linked to diverse pregnancy associated complications, such as preeclampsia and preterm birth. The serine hydrolases, diacylglycerol lipase α and β (DAGLα, DAGLβ) catalyze the degradation of diacylglycerols, leading to the formation of monoacylglycerols (MAG), including one main endocannabinoid 2-arachidonoylglycerol (2-AG). The major role of DAGL in the biosynthesis of 2-AG is evident from various studies in mice but has not been investigated in the human placenta. Here, we report the use of the small molecule inhibitor DH376, in combination with the ex vivo placental perfusion system, activity-based protein profiling (ABPP) and lipidomics, to determine the impact of acute DAGL inhibition on placental lipid networks. Methods: DAGLα and DAGLβ mRNA expression was detected by RT-qPCR and in situ hybridization in term placentas. Immunohistochemistry staining for CK7, CD163 and VWF was applied to localize DAGLβ transcripts to different cell types of the placenta. DAGLβ activity was determined by in- gel and MS-based activitybased protein profiling (ABPP) and validated by addition of the enzyme inhibitors LEI-105 and DH376. Enzyme kinetics were measured by EnzChek™ lipase substrate assay. Ex vivo placental perfusion experiments were performed +/- DH376 [1 µM] and changes in tissue lipid and fatty acid profiles were measured by LC-MS. Additionally, free fatty acid levels of the maternal and fetal circulations were determined. Results: We demonstrate that mRNA expression of DAGLβ prevails in placental tissue, compared to DAGLα (p ≤ 0.0001) and that DAGLβ is mainly located to CK7 positive trophoblasts (p ≤ 0.0001). Although few DAGLα transcripts were identified, no active enzyme was detected applying in-gel or MS-based ABPP, which underlined that DAGLβ is the principal DAGL in the placenta. DAGLβ dependent substrate hydrolysis in placental membrane lysates was determined by the application of LEI-105 and DH376. Ex vivo pharmacological inhibition of DAGLβ by DH376 led to reduced MAG tissue levels (p ≤ 0.01), including 2-AG (p≤0.0001). We further provide an activity landscape of serine hydrolases, showing a broad spectrum of metabolically active enzymes in the human placenta. Discussion: Our results emphasize the role of DAGLβ activity in the human placenta by determining the biosynthesis of 2-AG. Thus, this study highlights the special importance of intra-cellular lipases in lipid network regulation. Together, the activity of these specific enzymes may contribute to the lipid signaling at the maternal-fetal interface, with implications for function of the placenta in normal and compromised pregnancies. [ABSTRACT FROM AUTHOR]

ContextPrevious studies have shown that the endocannabinoid system plays a major role in energy metabolism through the actions of its main mediators, 2-arachidonoyl-sn-glycerol (2-AG) and anandamide (AEA).ObjectiveWe examined serum levels of major endocannabinoid mediators and their association with clinical parameters in patients with end-stage renal disease (ESRD).Design and settingSerum concentrations of 2-AG and AEA were measured in patients on maintenance hemodialysis (MHD) and controls, and correlations with various clinical and laboratory indices were examined. 2-AG was also measured in age and sex-matched healthy subjects for comparison of levels in patients undergoing MHD.Main outcome measureSerum 2-AG.ResultsSerum 2-AG levels were significantly elevated in patients with ESRD compared with healthy controls. Higher levels of 2-AG were found in patients on MHD compared to healthy subjects, and similar findings were seen in a second set of subjects in independent analyses. Among 96 patients on MHD, 2-AG levels correlated significantly and positively with serum triglycerides (ρ = 0.43; P < 0.0001), body mass index (ρ = 0.40; P < 0.0001), and body anthropometric measures and negatively with serum high-density lipoprotein cholesterol (ρ = -0.33; P = 0.001) following adjustment for demographic and clinical variables.ConclusionsIn patients on MHD, levels of serum 2-AG, a major endocannabinoid mediator, were increased. In addition, increasing serum 2-AG levels correlated with increased serum triglycerides and markers of body mass. Future studies will need to evaluate the potential mechanisms responsible for these findings.

Abstract Background Increased circulating endocannabinoids levels are typically associated with aerobic exercise. This phenomenon is associated with a “runner’s high,” a state of euphoria and well-being experienced after a long exercise. We will provide in this review a transparent and standardized methodology following the PRISMA-P and Cochrane Handbook for Systematic Reviews of Interventions for conducting a systematic review and meta-analysis for synthesizing the available evidence about the effects of physical activity on the circulating levels of AEA and 2-AG endocannabinoids in healthy subjects. Methods A multi-disciplinary team with basic and clinical expertise in exercise science developed this protocol. PubMed, EMBASE, Web of Science, CINAHL, SPORTDiscus, and Scopus will be the databases. A health sciences librarian was consulted in the development of the research. Search strategies will combine MeSH terms and free text words, including “exercise,” “exercise, physical,” “exercise training,” “physical activity,” “endocannabinoids,” “2-arachidonoyl-glycerol,” “glyceryl 2-arachidonate,” “2-AG,” “anandamide,” “AEA,” “n-arachidonoylethanolamide,” “adult,” “young adult,” and “middle-aged.” We will select experimental or quasi-experimental studies published through December 2021. The selection of studies, data extraction, assessment of the risk of bias, and the quality of evidence will be carried out in a paired and independent manner, and the consistency will be assessed using the statistics of Cohen Kappa. Methodological quality will be assessed using the Revised Cochrane risk of bias tool for randomized trials (RoB 2) and the Risk Of Bias In Nonrandomized Studies of Interventions (ROBINS-I) risk tool. We will use the Grading of Recommendations Assessment, Development, and Evaluation to assess the quality of the evidence, χ 2 and I 2 tests for heterogeneity, funnel plots, and the Egger test for publication bias. A meta-analysis for each data comparison will be performed whenever possible to determine the effect of physical activity on endocannabinoids’ circulating levels. Discussion This systematic review and meta-analysis will provide an overview of the evidence about physical activity over AEA and 2-AG endocannabinoids, including comparability of variables between studies, critical interpretation of results, and use of accurate statistical techniques. The endocannabinoid is molecules by which muscles communicate with other tissues and organs, mediating the beneficial effects of exercise on health and performance, including increased glucose uptake, improved insulin action, and mitochondrial biogenesis. They are essential to exercise. Thus, this study will review the acute effect of physical exercise on circulating levels of endocannabinoids in healthy individuals. The results of this study will potentially be transferred to doctors, health professionals, and legislators to guide their decision making, as well as will improve future research. Systematic review registration PROSPERO CRD42020202886 .

IntroductionLipids and fatty acids are key components in metabolic processes of the human placenta, thereby contributing to the development of the fetus. Placental dyslipidemia and aberrant activity of lipases have been linked to diverse pregnancy associated complications, such as preeclampsia and preterm birth. The serine hydrolases, diacylglycerol lipase α and β (DAGLα, DAGLβ) catalyze the degradation of diacylglycerols, leading to the formation of monoacylglycerols (MAG), including one main endocannabinoid 2-arachidonoylglycerol (2-AG). The major role of DAGL in the biosynthesis of 2-AG is evident from various studies in mice but has not been investigated in the human placenta. Here, we report the use of the small molecule inhibitor DH376, in combination with the ex vivo placental perfusion system, activity-based protein profiling (ABPP) and lipidomics, to determine the impact of acute DAGL inhibition on placental lipid networks.MethodsDAGLα and DAGLβ mRNA expression was detected by RT-qPCR and in situ hybridization in term placentas. Immunohistochemistry staining for CK7, CD163 and VWF was applied to localize DAGLβ transcripts to different cell types of the placenta. DAGLβ activity was determined by in- gel and MS-based activity-based protein profiling (ABPP) and validated by addition of the enzyme inhibitors LEI-105 and DH376. Enzyme kinetics were measured by EnzChek™ lipase substrate assay. Ex vivo placental perfusion experiments were performed +/- DH376 [1 µM] and changes in tissue lipid and fatty acid profiles were measured by LC-MS. Additionally, free fatty acid levels of the maternal and fetal circulations were determined.ResultsWe demonstrate that mRNA expression of DAGLβ prevails in placental tissue, compared to DAGLα (p ≤ 0.0001) and that DAGLβ is mainly located to CK7 positive trophoblasts (p ≤ 0.0001). Although few DAGLα transcripts were identified, no active enzyme was detected applying in-gel or MS-based ABPP, which underlined that DAGLβ is the principal DAGL in the placenta. DAGLβ dependent substrate hydrolysis in placental membrane lysates was determined by the application of LEI-105 and DH376. Ex vivo pharmacological inhibition of DAGLβ by DH376 led to reduced MAG tissue levels (p ≤ 0.01), including 2-AG (p≤0.0001). We further provide an activity landscape of serine hydrolases, showing a broad spectrum of metabolically active enzymes in the human placenta.DiscussionOur results emphasize the role of DAGLβ activity in the human placenta by determining the biosynthesis of 2-AG. Thus, this study highlights the special importance of intra-cellular lipases in lipid network regulation. Together, the activity of these specific enzymes may contribute to the lipid signaling at the maternal-fetal interface, with implications for function of the placenta in normal and compromised pregnancies.

Endocannabinoids are lipid metabolites that have paracrine effects. Emerging evidence indicates a role of endocannabinoids and their cognate receptors in synaptic modulation and plasticity at a wide range of synapses throughout the central nervous sys- tem. This study aimed to analyze the dynamics of changes in the concentrations of 2 endocannabinoids (anandamide -- ANA, and 2-arachidonoylglycerol -- 2-AG) and their impact on the carbohy- drate-lipid metabolism in patients before kidney transplantation and 7 days, 1, 3, and 6 months after transplantation. Considering the pro-inflammatory action of the compound, an increase in the concentration of 2-AG causes deterioration in kidney function, which, in the case of tests performed after transplantation, is associated with a poor prognosis. In our results, the highest concentration was obtained in the group of patients before transplantation and the lowest after 6 months. In the group of patients after transplantation, ANA and 2-AG were significantly correlated with lipid metabolism parameters, adipocytokines, and inflammatory markers. As both endocannabinoids play a very important role in improving kidney function after trans-plantation, future studies are needed to examine the potential mechanisms for their action and clinical implication. [ABSTRACT FROM AUTHOR]

Background: The role of the Endocannabinoids (ECs) in insulin resistance, and their association with visceral obesity and metabolic profile have been studied extensively. Since the association between ECs and metabolic factors in Gestational Diabetes Mellitus (GDM) are not clear, we aimed to evaluate the levels of N-Arachidonoylethanolamide (AEA) and 2-Arachidonoylglycerol (2-AG) and their association with C-reactive protein (CRP), glycemic indices, blood pressure, and anthropometric indices in pregnant women with GDM. Methods: The present case–control study was conducted among 96 singleton pregnant women aged 18–40 years, including 48 healthy pregnant women (control group) and 48 women with a positive diagnosis of GDM (case group). Odds Ratios (ORs) and 95% Confidence Intervals (CIs) for GDM were checked according to endocannabinoids and anthropometric indices using Multivariable Logistic Regression. Results: AEA was significantly associated with increased risk of GDM in models 1, 2 and 3 (OR = 1.22, 95% CI: 1.06–1.41; OR = 1.54, 95% CI: 1.19–1.97; OR = 1.46, 95% CI:1.11–1.91). A positive but no significant association was found for AEA in model 4 (OR = 1.38,95% CI: 0.99–1.92). Similar to AEA, 2-AG was also positively associated with the likelihood of GDM in Models 1, 2, and 3 but the association attenuated to null in model 4 (OR = 1.25; 95% CI: 0.94- 1.65). Conclusions: Our findings showed that levels of ECs were significantly higher in pregnant women with GDM compared to healthy ones. Also, ECs levels were associated with the likelihood of GDM, independent of BMI and weight gain. [ABSTRACT FROM AUTHOR]

The endocannabinoid 2-arachidonoylglycerol (2-AG) has been reported to exhibit anticancer effects, including against colorectal cancer (CRC); however, the detailed mechanisms have not been clarified. Herein, we demonstrated that 2-AG suppressed cyclooxygenase-2 (COX-2) expression induced by prostaglandin E 2 in human colon cancer HCA-7 cells. The suppression of COX-2 expression by 2-AG was through the acceleration of processing body (P-body) formation followed by COX-2 mRNA degradation. These effects were restored by TAK-715, a specific inhibitor of p38 MAPK. Therefore, the effect of 2-AG on COX-2 may be distinct from conventional non-steroidal anti-inflammatory drugs (NSAIDs). NSAIDs inhibit the function of COX-2, whereas 2-AG suppresses the protein expression of COX-2. Recently, the cardiovascular risks of NSAIDs were reported by the Food and Drug Administration in the United States. Therefore, elucidation of the effect of 2-AG is expected to contribute to the development of an alternative and novel therapeutic option that would have no or fewer risks regarding cardiovascular events. • 2-AG suppresses PGE 2 -induced COX-2 expression in HCA-7 cells. • 2-AG does not affect COX-2 transcriptional activity. • 2-AG degrades COX-2 mRNA via accelerates P-body formation. • The effect of 2-AG is mediated by p38 MAPK activation. • Suppressing COX-2 may be more effective for the prevention and/or treatment of CRC. [ABSTRACT FROM AUTHOR]

The endocannabinoid system (ECS) regulates neurotransmission linked to synaptic plasticity, cognition, and emotion. While it has been demonstrated that dysregulation of the ECS in adulthood is relevant not only to central nervous system (CNS) disorders such as autism spectrum disorder, cognitive dysfunction, and depression but also to brain function, there are few studies on how dysregulation of the ECS in the neonatal period affects the manifestation and pathophysiology of CNS disorders later in life. In this study, DO34, a diacylglycerol lipase alpha (DAGLα) inhibitor affecting endocannabinoid 2-AG production, was injected into C57BL/6N male mice from postnatal day (PND) 7 to PND 10, inducing dysregulation of the ECS in the neonatal period. Subsequently, we examined whether it affects neuronal function in adulthood through electrophysiological and behavioral evaluation. DO34-injected mice showed significantly decreased cognitive functions, attributed to impairment of hippocampal synaptic plasticity. The findings suggest that regulation of ECS activity in the neonatal period may induce enduring effects on adult brain function. • DO34-injected mice showed significantly reduced cognitive functions, attributed to impaired hippocampal synaptic plasticity. • DO34 transiently reduced 2-AG levels, resulting in impaired LTP and evoked depression-like behaviors in mice. • Regulation of ECS activity during the neonatal period may induce enduring effects on adult brain function. [ABSTRACT FROM AUTHOR]

The endocannabinoid system (ECS) is a new target for the development of retinal disease therapeutics, whose pathophysiology involves neurodegeneration and neuroinflammation. The endocannabinoid 2-arachidonoylglycerol (2-AG) affects neurons and microglia by activating CB1/CB2 cannabinoid receptors (Rs). The aim of this study was to investigate the effects of 2-AG on the CB1R expression/downregulation and retinal neurons/reactive microglia, when administered repeatedly (4 d), in three different paradigms. These involved the 2-AG exogenous administration (a) intraperitoneally (i.p.) and (b) topically and (c) by enhancing the 2-AG endogenous levels via the inhibition (AM11920, i.p.) of its metabolic enzymes (MAGL/ABHD6). Sprague Dawley rats were treated as mentioned above in the presence or absence of CB1/CB2R antagonists and the excitatory amino acid, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA). Immunohistochemistry, Western blot and a 2-AG level analyses were performed. The 2-AG repeated treatment (i.p.) induced the CB1R downregulation, abolishing its neuroprotective actions. However, 2-AG attenuated the AMPA-induced activation of microglia via the CB2R, as concurred by the AM630 antagonist effect. Topically administered 2-AG was efficacious as a neuroprotectant/antiapoptotic and anti-inflammatory agent. AM11920 increased the 2-AG levels providing neuroprotection against excitotoxicity and reduced microglial activation without affecting the CB1R expression. Our findings show that 2-AG, in the three paradigms studied, displays differential pharmacological profiles in terms of the downregulation of the CB1R and neuroprotection. All treatments, however, attenuated the activation of microglia via the CB2R activation, supporting the anti-inflammatory role of 2-AG in the retina.

Abstract Introduction Different components of the endocannabinoid (eCB) system such as their most well-known endogenous ligands, anandamide (AEA) and 2-arachidonoylglycerol (2-AG), have been implicated in brain reward pathways. While shared neurobiological substrates have been described among addiction-related disorders, information regarding the role of this system in behavioral addictions such as gambling disorder (GD) is scarce. Aims Fasting plasma concentrations of AEA and 2-AG were analyzed in individuals with GD at baseline, compared with healthy control subjects (HC). Through structural equation modeling, we evaluated associations between endocannabinoids and GD severity, exploring the potentially mediating role of clinical and neuropsychological variables. Methods The sample included 166 adult outpatients with GD (95.8% male, mean age 39 years old) and 41 HC. Peripheral blood samples were collected after overnight fasting to assess AEA and 2-AG concentrations (ng/ml). Clinical (i.e., general psychopathology, emotion regulation, impulsivity, personality) and neuropsychological variables were evaluated through a semi-structured clinical interview and psychometric assessments. Results Plasma AEA concentrations were higher in patients with GD compared with HC (p = .002), without differences in 2-AG. AEA and 2-AG concentrations were related to GD severity, with novelty-seeking mediating relationships. Conclusions This study points to differences in fasting plasma concentrations of endocannabinoids between individuals with GD and HC. In the clinical group, the pathway defined by the association between the concentrations of endocannabinoids and novelty-seeking predicted GD severity. Although exploratory, these results could contribute to the identification of potential endophenotypic features that help optimize personalized approaches to prevent and treat GD.

Abstract Background Anandamide (AEA) and 2-arachidonoylglycerol (2-AG) play a pivotal role in stimulating motivational behavior toward food and energy metabolism. Aberrant functioning of the endocannabinoid system has been observed in extreme weight conditions (EWCs), suggesting it may influence pathophysiology. Then, we aimed to analyze fasting AEA and 2-AG plasma concentrations among individuals with EWC (i.e., anorexia nervosa [AN] and obesity with and without eating disorders [EDs]) compared with healthy controls (HCs), and its association with clinical variables and body mass index (BMI). Methods The sample included 113 adult women. Fifty-seven belonged to the obesity group, 37 without EDs (OB-ED) and 20 with ED (OB+ED classified within the binge spectrum disorders), 27 individuals from the AN group, and 29 from the HC group. Peripheral blood samples, several clinical variables, and BMI were evaluated. Results Unlike 2-AG, AEA concentrations showed significant differences between groups (p

Abstract Background Several chronic pain disorders, such as migraine and fibromyalgia, have an increased prevalence in the female population. The underlying mechanisms of this sex-biased prevalence have yet to be thoroughly documented, but could be related to endogenous differences in neuromodulators in pain networks, including the endocannabinoid system. The cellular endocannabinoid system comprises the endogenous lipid signals 2-AG (2-arachidonoylglycerol) and AEA (anandamide); the enzymes that synthesize and degrade them; and the cannabinoid receptors. The relative prevalence of different components of the endocannabinoid system in specific brain regions may alter responses to endogenous and exogenous ligands. Methods Brain tissue from naïve male and estrous staged female Sprague Dawley rats was harvested from V1M cortex, periaqueductal gray, trigeminal nerve, and trigeminal nucleus caudalis. Tissue was analyzed for relative levels of endocannabinoid enzymes, ligands, and receptors via mass spectrometry, unlabeled quantitative proteomic analysis, and immunohistochemistry. Results Mass spectrometry revealed significant differences in 2-AG and AEA concentrations between males and females, as well as between female estrous cycle stages. Specifically, 2-AG concentration was lower within female PAG as compared to male PAG (*p = 0.0077); female 2-AG concentration within the PAG did not demonstrate estrous stage dependence. Immunohistochemistry followed by proteomics confirmed the prevalence of 2-AG-endocannabinoid system enzymes in the female PAG. Conclusions Our results suggest that sex differences exist in the endocannabinoid system in two CNS regions relevant to cortical spreading depression (V1M cortex) and descending modulatory networks in pain/anxiety (PAG). These basal differences in endogenous endocannabinoid mechanisms may facilitate the development of chronic pain conditions and may also underlie sex differences in response to therapeutic intervention.

Endocannabinoid transmission is emerging as a target for treating anxiety-related disorders, given its regulation of fear extinction. Boosting anandamide levels via inhibition of its metabolism by fatty acid amide hydrolase (FAAH) can enhance extinction, whereas inhibiting monoacylglycerol lipase (MAGL) to elevate 2-arachidonoylglycerol levels can impair extinction. However, whether endocannabinoids regulate fear relapse over time or extinction resistance remains unclear. In two experiments using auditory fear conditioned rats, we examined the effects of the FAAH inhibitor URB597 and the MAGL inhibitor JZL184 administered systemically on 1) spontaneous fear recovery after delayed extinction, and 2) extinction resistance resulting from immediate extinction [the immediate extinction deficit (IED)]. In Experiment 1, URB597 or JZL184 was given immediately after delayed extinction occurring 24 h after conditioning. Extinction recall and spontaneous fear recovery were tested drug-free 1 and 21 days later, respectively. We found no effects of either drug on extinction recall or spontaneous fear recovery. In Experiment 2, URB597 or JZL184 was given before immediate extinction occurring 30 min after conditioning and extinction recall was tested drug-free the next day. We also examined the effects of propranolol, a beta-adrenoceptor antagonist that can rescue the IED, as a positive control. JZL184 enhanced fear expression and impaired extinction learning but we found no lasting effects of URB597 or JZL184 on cued extinction recall. Propranolol reduced fear expression but, unexpectedly, had no enduring effect on extinction recall. The results are discussed in relation to various methodological differences between previous studies examining endocannabinoid and adrenergic regulation of fear extinction.

In this work we strived to determine whether endocannabinoid system activity could account for the differences in acute inflammatory pain sensitivity in mouse lines selected for high (HA) and low (LA) swim-stress-induced analgesia (SSIA). Mice received intraplantar injections of 5% formalin and the intensity of nocifensive behaviours was scored. To assess the contribution of the endocannabinoid system, mice were intraperitoneally (i.p.) injected with rimonabant (0.3–3 mg/kg) prior to formalin. Minocycline (45 and 100 mg/kg, i.p.) was administered to investigate microglial activation. The possible involvement of the endogenous opioid system was investigated with naloxone (1 mg/kg, i.p.). Cannabinoid receptor types 1 and 2 (Cnr1, Cnr2) and opioid receptor subtype (Oprm1, Oprd1, Oprk1) mRNA levels were quantified by qPCR in the structures of the central nociceptive circuit. Levels of anandamide (AEA) and 2-arachidonoylglycerol (2-AG) were measured by liquid chromatography coupled with the mass spectrometry method (LC-MS/MS). In the interphase, higher pain thresholds in the HA mice correlated with increased spinal anandamide and 2-AG release and higher Cnr1 transcription. Downregulation of Oprd1 and Oprm1 mRNA was noted in HA and LA mice, respectively, however no differences in naloxone sensitivity were observed in either line. As opposed to the LA mice, inflammatory pain sensitivity in the HA mice in the tonic phase was attributed to enhanced microglial activation, as evidenced by enhanced Aif1 and Il-1β mRNA levels. To conclude, Cnr1 inhibitory signaling is one mechanism responsible for decreased pain sensitivity in HA mice in the interphase, while increased microglial activation corresponds to decreased pain thresholds in the tonic inflammatory phase. [ABSTRACT FROM AUTHOR]

In this work, 2-AG was successfully detected in human plasma samples using a new sandwich-type electrochemical immune device based on poly-β-cyclodextrin P(β-CD) functionalized with AuNPs-DDT and toluidine blue. The P(β-CD) ensured the bioactivity and stability of the immobilized 2-AG antibody by providing a broad surface for the efficient immobilization of the biotinylated antibody. To complete the top section of the immunosensor (reporter), an HRP-conjugated antibody of 2-AG (secondary antibody (Ab2)) was attached to the surface of a glassy carbon electrode (GCE) modified by P(β-CD), as well as a primarily biotinylated antibody (Ab1). The biosensor fabrication process was monitored using field-emission scanning electron microscope (FE-SEM) and EDS methods. Using the differential pulse voltammetry technique, the immunosensor was utilized for detection of 2-AG in real samples. The suggested interface increased the surface area, which allowed for the immobilization of a large quantity of anti-2-AG antibody while also improving biocompatibility, stability, and electrical conductivity. Finally, the suggested immunosensor's limit of quantitation was determined to be 0.0078 ng/L, with a linear range of 0.0078 to 1.0 ng/L. The results showed that the suggested bioassay can be utilized for diagnosis of 2-AG in clinical samples as a unique and ultrasensitive electrochemical biodevice. [ABSTRACT FROM AUTHOR]

The endocannabinoids 2-arachidonoylglycerol (2-AG) and anandamide are among the best studied lipid messengers in the brain. By activating cannabinoid receptors in the CNS, endocannabinoids tune synaptic function, thereby influencing a variety of physiological and behavioural processes. Extensive research conducted over the last few decades has considerably enhanced our understanding of the molecular mechanisms and physiological functions of the endocannabinoid system. It is now well-established that endocannabinoids are synthesized by postsynaptic neurons and serve as retrograde messengers that suppress neurotransmitter release at central synapses. While the detailed mechanisms by which endocannabinoids gate synaptic function and behavioural processes are relatively well characterized, the mechanisms governing endocannabinoid transport at central synapses remain ill defined. Recently, several studies have begun to unravel the mechanisms governing intracellular and intercellular endocannabinoid transport. In this review, we will focus on new advances in the mechanisms of intracellular and synaptic endocannabinoid transport in the CNS. LINKED ARTICLES: This article is part of a themed issue on New discoveries and perspectives in mental and pain disorders. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.17/issuetoc. [ABSTRACT FROM AUTHOR]

A simple and practical synthesis of 2-arachidonoyl glycerol (2-AG), an endogenous agonist for cannabinoid receptors, based on a two-step enzymatic process and a chemical coupling, was achieved with a good yield and negligible amount of the isomerization product 1-AG. Commercial preparation of immobilized lipase from Mucor miehei (MML) was selected as the most suitable enzyme to catalyze the efficient protection of glycerol using vinyl benzoate as an acyl transfer reagent in tetrahydrofuran. The same enzyme was used to remove the protective groups in positions 1 and 3. Owing to the mild neutral conditions and easy suitability of the method, 2-AG was obtained without any isomerization to the more stable 1-AG and air oxidation of acid chain. The synthetic method proposed here allows us to easily obtain 2-AG from the protected precursor in a one-step reaction without purification requirement. [ABSTRACT FROM AUTHOR]

Background: Dysregulation of the endocannabinoid (eCB) system is implicated in various stress-related neuropsychiatric disorders (SRDs), including anxiety, depression, and post-traumatic stress disorder (PTSD). In this systematic review and meta-analysis, our objectives were to characterize circulating anandamide (AEA) and 2-arachidonoylglycerol (2-AG) concentrations at rest and in response to acute laboratory-based psychosocial stress in individuals with SRDs and without (controls). Our primary aims were to assess the effects of acute psychosocial stress on eCB concentrations in controls (Aim 1), compare baseline (prestress) eCB concentrations between individuals with SRDs and controls (Aim 2), and explore differential eCB responses to acute psychosocial stress in individuals with SRDs compared with controls (Aim 3). Methods: On June 8, 2023, a comprehensive review of the MEDLINE (PubMed) database was conducted to identify original articles meeting inclusion criteria. A total of 1072, 1341, and 400 articles were screened for inclusion in Aims 1, 2, and 3, respectively. Results: Aim 1, comprised of seven studies in controls, revealed that most studies reported stress-related increases in AEA (86%, with 43% reporting statistical significance) and 2-AG (83%, though none were statistically significant except for one study in saliva). However, meta-analyses did not support these patterns ( p' s>0.05). Aim 2, with 20 studies, revealed that most studies reported higher baseline concentrations of both AEA (63%, with 16% reporting statistical significance) and 2-AG (60%, with 10% reporting statistical significance) in individuals with SRDs compared with controls. Meta-analyses confirmed these findings ( p' s<0.05). Aim 3, which included three studies, had only one study that reported statistically different stress-related changes in 2-AG (but not AEA) between individuals with PTSD (decrease) and controls (increase), which was supported by the meta-analysis ( p <0.001). Meta-analyses showed heterogeneity across studies and aims ( I 2 =14-97%). Conclusion: Despite substantial heterogeneity in study characteristics, samples, and methodologies, consistent patterns emerged, including elevated baseline AEA and 2-AG in individuals with SRDs compared with controls, as well as smaller stress-related increases in 2-AG in individuals with SRDs compared with controls. To consider eCBs as reliable biomarkers and potential intervention targets for SRDs, standardized research approaches are needed to clarify the complex relationships between eCBs, SRDs, and psychosocial stress.

2-Arachidonoylglycerol (2-AG) is the most prevalent endocannabinoid involved in maintaining brain homeostasis. Previous studies have demonstrated that inactivating monoacylglycerol lipase (MAGL), the primary enzyme responsible for degrading 2-AG in the brain, alleviates neuropathology and prevents synaptic and cognitive decline in animal models of neurodegenerative diseases. However, we show that selectively inhibiting 2-AG metabolism in neurons impairs cognitive function in mice. This cognitive impairment appears to result from decreased expression of synaptic proteins and synapse numbers, impaired long-term synaptic plasticity and cortical circuit functional connectivity, and diminished neurogenesis. Interestingly, the synaptic and cognitive deficits induced by neuronal MAGL inactivation can be counterbalanced by inhibiting astrocytic 2-AG metabolism. Transcriptomic analyses reveal that inhibiting neuronal 2-AG degradation leads to widespread changes in expression of genes associated with synaptic function. These findings suggest that crosstalk in 2-AG signaling between astrocytes and neurons is crucial for maintaining synaptic and cognitive functions and that excessive 2-AG in neurons alone is detrimental to cognitive function.

Cyclooxygenase-2 converts arachidonic acid to prostaglandins (PGs) and the endocannabinoid, 2-arachidonoylglycerol (2-AG), to PG glyceryl esters (PG-Gs). The physiological function of PG biosynthesis has been extensively studied, but the importance of the more recently discovered PG-G synthetic pathway remains incompletely defined. This disparity is due in part to a lack of knowledge of the physiological conditions under which PG-G biosynthesis occurs. We have discovered that RAW264.7 macrophages stimulated with Kdo2-lipid A (KLA) produce primarily PGs within the first 12 h followed by robust PG-G synthesis between 12 h and 24 h. We suggest that the amount of PG-Gs quantified is less than actually synthesized, because PG-Gs are subject to a significant level of hydrolysis during the time course of synthesis. Inhibition of cytosolic phospholipase A2 by giripladib does not accelerate PG-G synthesis, suggesting the differential time course of PG and PG-G synthesis is not due to the competition between arachidonic acid and 2-AG. The late-phase PG-G formation is accompanied by an increase in the level of 2-AG and a concomitant decrease in 18:0-20:4 diacylglycerol (DAG). Inhibition of DAG lipases by KT-172 decreases the levels of 2-AG and PG-Gs, indicating that the DAG-lipase pathway is involved in delayed 2-AG metabolism/PG-G synthesis. These results demonstrate that physiologically significant levels of PG-Gs are produced by activated RAW264.7 macrophages well after the production of PGs plateaus., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The endocannabinoid (eCB) anandamide (AEA) and 2-arachidonoylglycerol (2-AG) are endogenous lipid neurotransmitters that regulate an array of physiological functions, including pain, stress homeostasis, and reward. Fatty acid-binding protein 5 (FABP5) is a key modulator of intracellular eCB transport and inactivation. Recent evidence suggests that FABP5 controls synaptic 2-AG signaling at excitatory synapses in the dorsal raphe nucleus. However, it is currently not known whether this function extends to other brain areas. To address this, we first profiled eCB levels across several brain areas in FABP5 knockout mice and wild-type controls and report that FABP5 deletion elevates AEA levels in the striatum, prefrontal cortex, midbrain, and thalamus, as well as midbrain 2-AG levels. The expression of eCB biosynthetic and catabolic enzymes was largely unaltered in these regions, although minor sex and region-specific changes in the expression of 2-AG catabolic enzymes were observed in female FABP5 KO mice. Robust FABP5 expression was observed in the striatum, a region where both AEA and 2-AG control synaptic transmission. Deletion of FABP5 impaired tonic 2-AG and AEA signaling at striatal GABA synapses of medium spiny neurons, and blunted phasic 2-AG mediated short-term synaptic plasticity without altering CB1R expression or function. Collectively, these results support the role of FABP5 as a key regulator of eCB signaling at excitatory and inhibitory synapses in the brain. [ABSTRACT FROM AUTHOR]

Background: the endocannabinoid 2-arachidonoylglycerol (2-AG) plays a pivotal role in immune cells regulation. The plasma levels of 2-AG are increased in patients with systemic lupus erythematosus (SLE) and correlate with disease activity. Moreover, in plasmacytoid dendritic cells from SLE patients, 2-AG is able to control the production of type 1 interferon (IFN) through CB2 activation. The aim of this study was to evaluate the potential role of 2-AG on T lymphocytes from SLE patients. Methods: peripheral blood mononuclear cells (PBMCs) from SLE participants and age- and sex-matched healthy donors (HD) were isolated by Ficoll–Hypaque density-gradient centrifugation. The PBMCs were treated with increasing concentrations of 2-AG, and AM251 and AM630 were used to antagonize CB1 and CB2, respectively. Flow cytometry was used to assess the expression of CD3, CD4, CD8, CD25, IFN-ɣ, IL-4, and IL-17A. Results: 2-AG (1 μM) decreased IFN-ɣ expression (p = 0.0005) in the Th1 lymphocytes of SLE patients. 2-AG did not modulate the cytokine expression of any other T lymphocyte population from either SLE or HD. Treatment with both 2-AG and AM630 increased the IFN-ɣ expression in Th1 lymphocytes of SLE patients (p = 0.03). Discussion: 2-AG is able to modulate type 2 IFN production from CD4+ T lymphocytes from SLE patients through CB2 activation. [ABSTRACT FROM AUTHOR]