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Abstract Scope This study investigated whether AM251, a cannabinoid receptor type 1 (CB1) antagonist, ameliorates hepatic levels of metabolic abnormalities and inflammatory responses in a murine nonalcoholic steatohepatitis (NASH) model via reversal of disturbances in the immune system. Methods and Results Fifteen‐week‐old male obese db/db mice were randomly assigned to the following two groups: no treatment and treatment with AM251 at 5 mg/kg for 15 days. C57BL/6J‐Lean mice were utilized as the control group. Plasma parameters, liver histopathology, and hepatic status were measured. For the in vitro study, macrophage‐derived RAW264.7 cells were cultured with AM251 or CB1 small interfering RNA (siRNA) before challenge with arachidonyl‐2′‐chloroethylamide (ACEA) or a high concentration of fatty acids (HFFAs). The db/db mice exhibited an increase in CB1 levels, lipid droplet accumulation, mitogen‐activated protein kinase‐related inflammatory responses, and macrophage and neutrophil infiltration in the liver tissues. Flow cytometry analysis revealed an elevation in macrophages and T helper cells, plus a decrease in natural killer T cells and regulatory T cells in the liver tissues of the db/db mice; treatment with 5 mg/kg AM251 reversed these changes. Moreover, in vitro experiments revealed that administration of 3.3 μM AM251 or CB1 siRNA prevented 1 mM HFFA‐ and 1 μΜ ACEA‐induced inflammatory cytokine protein expression in the RAW264.7 cells. Conclusion These findings suggested that a blockade caused by CB1 reduced obesity‐associated NASH progression via correction of immune system dysregulations and elevated inflammatory responses in the liver tissues.

Scope: This study investigated whether AM251, a cannabinoid receptor type 1 (CB1) antagonist, ameliorates hepatic levels of metabolic abnormalities and inflammatory responses in a murine nonalcoholic steatohepatitis (NASH) model via reversal of disturbances in the immune system. Methods and Results: Fifteen‐week‐old male obese db/db mice were randomly assigned to the following two groups: no treatment and treatment with AM251 at 5 mg/kg for 15 days. C57BL/6J‐Lean mice were utilized as the control group. Plasma parameters, liver histopathology, and hepatic status were measured. For the in vitro study, macrophage‐derived RAW264.7 cells were cultured with AM251 or CB1 small interfering RNA (siRNA) before challenge with arachidonyl‐2′‐chloroethylamide (ACEA) or a high concentration of fatty acids (HFFAs). The db/db mice exhibited an increase in CB1 levels, lipid droplet accumulation, mitogen‐activated protein kinase‐related inflammatory responses, and macrophage and neutrophil infiltration in the liver tissues. Flow cytometry analysis revealed an elevation in macrophages and T helper cells, plus a decrease in natural killer T cells and regulatory T cells in the liver tissues of the db/db mice; treatment with 5 mg/kg AM251 reversed these changes. Moreover, in vitro experiments revealed that administration of 3.3 μM AM251 or CB1 siRNA prevented 1 mM HFFA‐ and 1 μΜ ACEA‐induced inflammatory cytokine protein expression in the RAW264.7 cells. Conclusion: These findings suggested that a blockade caused by CB1 reduced obesity‐associated NASH progression via correction of immune system dysregulations and elevated inflammatory responses in the liver tissues. [ABSTRACT FROM AUTHOR]

Orphan nuclear receptor estrogen-related receptor γ (ERRγ) is an important transcription factor modulating gene transcription involved in endocrine control of liver metabolism. Transferrin receptor 2 (TFR2), a carrier protein for transferrin, is involved in hepatic iron overload in alcoholic liver disease (ALD). However, TFR2 gene transcriptional regulation in hepatocytes remains largely unknown. In this study, we described a detailed molecular mechanism of hepatic TFR2 gene expression involving ERRγ in response to an endocannabinoid 2-arachidonoylglycerol (2-AG). Treatment with 2-AG and arachidonyl-2′-chloroethylamide, a selective cannabinoid receptor type 1 (CB1) receptor agonist, increased ERRγ and TFR2 expression in hepatocytes. Overexpression of ERRγ was sufficient to induce TFR2 expression in both human and mouse hepatocytes. In addition, ERRγ knockdown significantly decreased 2-AG or alcohol-mediated TFR2 gene expression in cultured hepatocytes and mouse livers. Finally, deletion and mutation analysis of the TFR2 gene promoter demonstrated that ERRγ directly modulated TFR2 gene transcription via binding to an ERR-response element. This was further confirmed by chromatin immunoprecipitation assay. Taken together, these results reveal a previously unrecognized role of ERRγ in the transcriptional regulation of TFR2 gene expression in response to alcohol.

Background The deleterious effects of cannabis consumption for fertility and pregnancy outcome are recognized for years. The main psychoactive molecule of cannabis, Δ(9)-tetrahydrocannabinol (THC) is able to cross the placenta barrier and cause alterations in fetal growth, low birth weight and preterm labor. However, the effects of THC on the human placenta amnion are still unknown. Methods The distributions of CB1R and CB2R in human amnion tissues were observed by immunohistochemistry (IHC). Human amniotic epithelial cell proliferation and migration in response to THC treatment were measured by MTS and transwell assays, respectively. The PCR array was performed to study the key regulators involved in the cell migration. The protein levels of CB1R, CB2R in amnion tissues and MMP2, MMP9 in cells were detected by western blotting. Small interfering RNAs (siRNAs) were used to knockdown MMP2 and MMP9 in WISH cells. Results Our results indicated that both CB1R and CB2R primarily identified in the epithelial layer of human placental amnion tissue. The CB1R expression in the amnion tissue was higher in the preterm group than normal control. High-dose of THC (30uM, but not 20 and 10uM) significantly inhibited (p < 0.01) human amniotic epithelial cell lines (WISH) proliferation. Meanwhile, THC at both 10uM and 20uM (p < 0.05) significantly suppressed cells migration in both WISH and primary human amniotic epithelial cells. The PCR array data and siRNA experiments demonstrated that MMP2/9 were tightly involved in the regulation of THC-inhibited cell migration in WISH cells. Conclusion These results suggested that THC inhibited the migration of human amniotic epithelial cell through the regulation of MMP2 and MMP9, which in turn altered the development of the amnion during the gestation and partially resulted in preterm labor and other adverse pregnancy outcomes. [ABSTRACT FROM AUTHOR]

The endocannabinoids are lipid-derived signaling molecules that control feeding and energy balance by activating CB1-type cannabinoid receptors in the brain and peripheral tissues. Previous studies have shown that oral exposure to dietary fat stimulates endocannabinoid signaling in the rat small intestine, which provides positive feedback that drives further food intake and preference for fat-rich foods. We now describe an unexpectedly broader role for cholinergic signaling of the vagus nerve in the production of the endocannabinoid, 2-arachidonoyl-sn-glycerol (2-AG), in the small intestine. We show that food deprivation increases levels of 2-AG and its lipid precursor, 1,2-diacylglycerol, in rat jejunum mucosa in a time-dependent manner. This response is abrogated by surgical resection of the vagus nerve or pharmacological blockade of small intestinal subtype-3 muscarinic acetylcholine (m3 mAch) receptors, but not inhibition of subtype-1 muscarinic acetylcholine (m1 mAch). We further show that blockade of peripheral CB1 receptors or intestinal m3 mAch receptors inhibits refeeding in fasted rats. The results suggest that food deprivation stimulates 2-AG-dependent CB1 receptor activation through a mechanism that requires efferent vagal activation of m3 mAch receptors in the jejunum, which, in turn, may promote feeding after a fast. [ABSTRACT FROM AUTHOR]

Abstract: Accumulating evidence from both human and preclinical studies indicates maternal substance use during pregnancy can affect fetal development, birth weight and infant outcomes. Thus, the prenatal period can be regarded as an important and potentially sensitive period of development. In this manuscript, an updated overview of studies on prenatal cannabis exposure in humans is presented, including recent studies conducted within the Generation R study. Findings on fetal growth, birth outcomes, early neonatal behavior and infant behavior and cognitive development are discussed in detail. Preclinical evidence and potential mechanisms are described as well, and recommendations for future studies are provided. It is concluded that evidence seems to suggest that fetal development is affected by prenatal maternal cannabis use, while findings on effects on infant behavior or cognition are inconsistent. Beyond infancy, subtle differences may be found in specific cognitive or behavioral outcomes, although replication studies in which pregnant women and their fetuses are exposed to current and probably higher levels of Δ9-tetrahydrocannabinol and novel designs are needed to come to firm conclusions. [Copyright &y& Elsevier]

Abstract: Tobacco produces an impressive burden of disease resulting in premature death in half of users. Despite effective smoking cessation medications (nicotine replacement therapies, bupropion and varenicline), there is a very high rate of relapse following quit attempts. The use of efficient strategies for the development of novel treatments is a necessity. A ‘bench to bedside strategy’ was initially used to develop cannabinoid CB1 receptor antagonists for the treatment of nicotine addiction. Unfortunately, after being tested on experimental animals, what seemed to be an interesting approach for the treatment of nicotine addiction resulted in serious unwanted side effects when tested in humans. Current research is focusing again on pre-clinical models in an effort to eliminate unwanted side effects while preserving the initially observed efficacy. A ‘bed side to bench strategy’ was used to study the role of the insula (part of the frontal cortex) in nicotine addiction. This line of research started based on clinical observations that patients suffering stroke-induced lesions to the insula showed a greater likelihood to report immediate smoking cessation without craving or relapse. Subsequently, animal models of addiction are used to explore the role of insula in addiction. Due to the inherent limitations existing in clinical versus preclinical studies, the possibility of close interaction between both models seems to be critical for the successful development of novel therapeutic strategies for nicotine dependence. [Copyright &y& Elsevier]

Abstract: Endogenous cannabinoids play important roles in a variety of functions in the mammalian brain, including the regulation reward-related information processing. The primary mechanism through which this is achieved is the presynaptic modulation of synaptic transmission. During reward- and reinforcement-related behavior dopamine levels increase in forebrain areas and this has recently been shown to be modulated by the endocannabinoid system. Therefore, understanding how endocannabinoids are mobilized to modulate synaptic inputs impinging on midbrain dopamine neurons is crucial to a complete understanding of the roles that these molecules play in reward behavior, drug abuse and addiction. Here we summarize the literature describing short-term and long-term regulation of afferent connections on dopamine neurons in the ventral tegmental area via endocannabinoid activation of cannabinoid CB1 receptors, and describe the mechanisms through which these molecules are released during reward-based behavior and exposure to abused drugs. [Copyright &y& Elsevier]

Highlights: [•] Intrathecal injection of (m)VD-Hpα dose-dependently produced a hypotensive effect. [•] The spinal hypotension of (m)VD-Hpα was mediated by a non-CB1 and non-CB2 mechanism. [•] The hypotension of (m)VD-Hpα at the spinal level was independent of systemic NO. [Copyright &y& Elsevier]

Highlights: [•] CB1 blockade in the PVN increases fasting-induced hyperphagia. [•] CB1 blockade in the PVN enhances the orexigenic effect of ghrelin. [•] CB1 blockade potentiates ghrelin-induced PVN hypoactivity. [Copyright &y& Elsevier]

Highlights: [•] Role for central allopregnanolone in reduced neuroendocrine stress responses. [•] Allopregnanolone and oxytocin system interactions for minimising preterm birth risk. [•] Disrupted neurosteroidogenesis in compromised pregnancy and offspring outcomes. [•] Role for progesterone or allopregnanolone in correcting adverse pregnancy outcomes. [ABSTRACT FROM AUTHOR]

Highlights: [•] IL-1β induces CB1 mRNA upregulation via androgen receptors in trigeminal ganglia. [•] Androgen receptor activates CB1 gene transcription in response to testosterone. [•] Androgen receptor binds to the promoter of CB1 gene. [Copyright &y& Elsevier]

Abstract: The cannabinoid receptor type 1 (CB1) is abundantly expressed in the central nervous system where it negatively controls the release of several neurotransmitters. CB1 activity plays a crucial role in learning and memory and in synaptic plasticity. In the present study, the role of CB1 was investigated in three different hippocampus-dependent memory tasks and in in vivo hippocampal synaptic plasticity in knockout (CB1-ko) and wildtype mice. There was no difference in short-term and long-term social and object recognition memory between CB1-ko and wildtype mice. In contrast, in background contextual fear conditioning CB1-ko mice showed enhanced freezing levels in the conditioning context and increased generalised contextual fear after a high-intensity conditioning foot shock of 1.5mA, but not after 0.7mA. In in vivo field potential recordings in the dentate gyrus, CB1-ko mice displayed a decreased paired-pulse facilitation of the populations spikes, suggesting an altered inhibitory synaptic drive onto hippocampal granule cells. Furthermore, CB1-ko mice displayed significantly higher levels of in vivo long-term potentiation (LTP) in the dentate gyrus. In conclusion, CB1 deficiency leads to enhanced contextual fear memory and altered synaptic plasticity in the hippocampus, supporting the key role of endocannabinoid signalling in learning and memory, in particular following highly aversive encounters. [Copyright &y& Elsevier]

Abstract: The role of endocannabinoid signaling in the response of the brain to injury is tantalizing but not clear. In this study, transient middle cerebral artery occlusion (MCAo) was used to produce ischemia/reperfusion injury. Brain content of N-arachidonoylethanolamine (AEA) and 2-arachidonoylglycerol were determined during MCAo. Whole brain AEA content was significantly increased after 30, 60 and 120 min MCAo compared with sham-operated brain. The increase in AEA was localized to the ischemic hemisphere after 30 min MCAo, but at 60 and 120 min, was also increased in the contralateral hemisphere. 2-Arachidonoylglycerol content was unaffected by MCAo. In a second set of studies, injury was assessed 24 h after 2 h MCAo. Rats administered a single dose (3 mg/kg) of the cannabinoid receptor type 1 (CB1) receptor antagonist SR141716 prior to MCAo exhibited a 50% reduction in infarct volume and a 40% improvement in neurological function compared with vehicle control. A second CB1 receptor antagonist, LY320135 (6 mg/kg), also significantly improved neurological function. The CB1 receptor agonist, WIN 55212-2 (0.1–1 mg/kg) did not affect either infarct volume or neurological score. [Copyright &y& Elsevier]

Cannabinoids have been shown to impair cognition in vivo and block long-term potentiation (LTP), a candidate experimental model of learning and memory in vitro, via cannabinoid receptor (CB1) activation. cis-Oleamide (cOA) is an endogenous sleep-inducing lipid with putative cannabinomimetic properties. We hypothesise that cOA is cannabinomimetic and perform a comparative study with synthetic and endogenous cannabinoids on their effects on synaptic conditioning via two different patterns of stimulation in the hippocampal slice. CB1 agonists, R(+)-WIN55212-2 and anandamide, but not cOA blocked high frequency stimulation (HFS)-LTP. R(+)-WIN55212-2 and cOA (stereoselectively) attenuated responses to θ-burst-LTP, while anandamide did not. The anandamide transport inhibitor, AM404, attenuated HFS-LTP, an effect reversed by the CB1 receptor antagonist SR141716A but not mimicked by the vanilloid receptor agonist capsaicin. TFNO, an inhibitor of fatty acid amide hydrolase (FAAH), the enzyme responsible for degrading anandamide, failed to block HFS-LTP alone or in combination with cOA. On the contrary, this combination was as effective as cOA on its own in attenuating θ-burst-LTP. cOA effects on θ-burst-LTP were prevented in the presence of the GABAA receptor blocker picrotoxin, but not by pretreatment with SR141716A. These findings suggest that cOA neither directly activates CB1 receptors nor acts via the proposed “entourage” effect [Nature 389 (1997) 25] to increase titres of anandamide through FAAH inhibition. The selective effects of cOA on θ-burst-conditioning may reflect modulation of GABAergic transmission. Anandamide uptake inhibition, but not blockade of FAAH, effectively increases synaptic concentrations of endocannabinoids. [Copyright &y& Elsevier]

Cannabinoid compounds have been shown to produce antinociception and antihyperalgesia by acting upon cannabinoid receptors located in both the CNS and the periphery. A potential mechanism by which cannabinoids could inhibit nociception in the periphery is the activation of cannabinoid receptors located on one or more classes of primary nociceptive neurons. To address this hypothesis, we evaluated the neuronal distribution of cannabinoid receptor type 1 (CB1) in the trigeminal ganglion (TG) of the adult rat through combined in situ hybridization (ISH) and immunohistochemistry (IHC). CB1 receptor mRNA was localized mainly to medium and large diameter neurons of the maxillary and mandibular branches of the TG. Consistent with this distribution, in a de facto nociceptive sensory neuron population that exhibited vanilloid receptor type 1 immunoreactivity, colocalization with CB1 mRNA was also sparse (<5%). Furthermore, very few neurons (approximately 5%) in the peptidergic (defined as calcitonin gene-related peptide- or substance P-immunoreactive) or the isolectin B4-binding sensory neuron populations contained CB1 mRNA. In contrast, and consistent with the neuron-size distribution for CB1, nearly 75% of CB1-positive neurons exhibited N52-immunoreactivity, a marker of myelinated axons. These results indicate that in the rat TG, CB1 receptors are expressed predominantly in neurons that are not thought to subserve nociceptive neurotransmission in the noninjured animal. Taken together with the absence of an above background in situ signal for CB2 mRNA in TG neurons, these findings suggest that the peripherally mediated antinociceptive effects of cannabinoids may involve either as yet unidentified receptors or interaction with afferent neuron populations that normally subserve non-nociceptive functions. [Copyright &y& Elsevier]

A recombinant Semliki Forest virus (SFV) RNA construct, SFV1-mCB2 RNA, was employed for the high-level expression of the murine CB2 (mCB2) cannabinoid receptor in baby hamster kidney cells. Biosynthetic radiolabel incorporation studies in concert with urea-sodium dodecylsulfate–polyacrylamide gel electrophoresis (urea-SDS–PAGE) and western immunoblotting revealed that two major proteins of approximately 26 and 40 kDa were produced by the construct. The 40 kDa product, but not the 26 kDa product, was glycosylated as determined by 2-deoxy-d-glucose incorporation and peptide-N-glycosidase F digestion analysis. Assessment of [3H]CP55940 ([3H]-(−)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol) binding data for membranes of cells transfected with SFV1-mCB2 RNA indicated a Kd of 0.35±0.04 nM and a Bmax of 24.4±2.7 pmol/mg. A rank order of binding affinities for cannabinoids, which paralleled that reported for native mCB2 receptors, was observed. The CB2 receptor-specific antagonist SR144528 (N-[(1S)-endo-1,3,3-trimethyl bicyclo[2.2.1]heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide) blocked binding of [3H]CP55940, while the CB1 receptor-specific antagonist SR141716A [N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride] had a minimal effect. These results indicate that the recombinant receptor expressed from SFV1-mCB2 RNA exhibits properties, including ligand binding features, that are consistent with those for the native mCB2 receptor. However, the presence of both 26 and 40 kDa receptor species is consistent with alternative translation from two AUG start sites using the SFV1-mCB2 RNA expression system. [Copyright &y& Elsevier]

Novel aromatic analogues of N-oleoylethanolamine and N-arachidonoylethanolamine (anandamide, AEA) were synthesized and, based on the capability of similar compounds to interact with proteins of the endocannabinoid and endovanilloid signaling systems, were tested on: (i) cannabinoid CB1 and CB2 receptors; (ii) vanilloid VR1 receptors; (iii) anandamide cellular uptake (ACU); and (iv) the fatty acid amide hydrolase (FAAH). The (R)- and, particularly, the (S)-1′-(4-hydroxybenzyl) derivatives of N-oleoylethanolamine and AEA (OMDM-1, OMDM-2, OMDM-3, and OMDM-4) inhibited to a varied extent ACU in RBL-2H3 cells (Ki ranging between 2.4 and 17.7 μM), the oleoyl analogues (OMDM-1 and OMDM-2, Ki 2.4 and 3.0 μM, respectively) being 6- to 7-fold more potent than the arachidonoyl analogues (OMDM-3 and OMDM-4). These four compounds exhibited: (i) poor affinity for either CB1 (Ki≥5 μM) or CB2 (Ki>10 μM) receptors in rat brain and spleen membranes, respectively; (ii) almost no activity at vanilloid receptors in the intracellular calcium assay carried out with intact cells over-expressing the human VR1 (ec50≥10 μM); and (iii) no activity as inhibitors of FAAH in N18TG2 cell membranes (Ki>50 μM). The oleoyl- and arachidonoyl-N′-(4-hydroxy-3-methoxybenzyl)hydrazines (OMDM-5 and OMDM-6), inhibited ACU (Ki 4.8 and 7.0 μM, respectively), and were more potent as VR1 agonists (ec50 75 and 50 nM, respectively), weakly active as CB1 receptor ligands (Ki 4.9 and 3.2 μM, respectively), and inactive as CB2 ligands (Ki>5 μM) as well as on FAAH (Ki≥40 μM). In conclusion, we report two novel potent and selective inhibitors of ACU (OMDM-1 and OMDM-2) and one “hybrid” agonist of CB1 and VR1 receptors (OMDM-6). Unlike other compounds of the same type, OMDM-1, OMDM-2, and OMDM-6 were very stable to enzymatic hydrolysis by rat brain homogenates. [Copyright &y& Elsevier]

Orphan nuclear receptor estrogen-related receptor γ (ERRγ) is an important transcription factor modulating gene transcription involved in endocrine control of liver metabolism. Transferrin receptor 2 (TFR2), a carrier protein for transferrin, is involved in hepatic iron overload in alcoholic liver disease (ALD). However, TFR2 gene transcriptional regulation in hepatocytes remains largely unknown. In this study, we described a detailed molecular mechanism of hepatic TFR2 gene expression involving ERRγ in response to an endocannabinoid 2-arachidonoylglycerol (2-AG). Treatment with 2-AG and arachidonyl-2′-chloroethylamide, a selective cannabinoid receptor type 1 (CB1) receptor agonist, increased ERRγ and TFR2 expression in hepatocytes. Overexpression of ERRγ was sufficient to induce TFR2 expression in both human and mouse hepatocytes. In addition, ERRγ knockdown significantly decreased 2-AG or alcohol-mediated TFR2 gene expression in cultured hepatocytes and mouse livers. Finally, deletion and mutation analysis of the TFR2 gene promoter demonstrated that ERRγ directly modulated TFR2 gene transcription via binding to an ERR-response element. This was further confirmed by chromatin immunoprecipitation assay. Taken together, these results reveal a previously unrecognized role of ERRγ in the transcriptional regulation of TFR2 gene expression in response to alcohol. [ABSTRACT FROM AUTHOR]

Membrane proteins can associate into larger complexes. Examples include receptor tyrosine complexes, ion channels, transporters, and G protein-coupled receptors (GPCRs). For the latter, there is abundant evidence indicating that GPCRs assemble into complexes, through both homo- and heterodimerization. However, the tools for studying and disrupting these complexes, GPCR or otherwise, are limited. Here, we have developed stabilized interference peptides for this purpose. We have previously reported that tetrahydrocannabinol-mediated cognitive impairment arises from homo- or heterooligomerization between the GPCRs cannabinoid receptor type 1 (CB 1 R) and 5-hydroxytryptamine 2A (5-HT 2A R) receptors. Here, to disrupt this interaction through targeting CB 1 -5-HT 2A receptor heteromers in HEK293 cells and using an array of biochemical techniques, including calcium and cAMP measurements, bimolecular fluorescence complementation assays, and CD-based helicity assessments, we developed a NanoLuc binary technology (NanoBiT)-based reporter assay to screen a small library of aryl-carbon-stapled transmembrane-mimicking peptides produced by solid-phase peptide synthesis. We found that these stapling peptides have increased α-helicity and improved proteolytic resistance without any loss of disrupting activity in vitro , suggesting that this approach may also have utility in vivo In summary, our results provide proof of concept for using NanoBiT to study membrane protein complexes and for stabilizing disrupting peptides to target such membrane complexes through hydrocarbon-mediated stapling. We propose that these peptides could be developed to target previously undruggable GPCR heteromers., (© 2019 Botta et al.)

Although cannabinoid receptor 1 (CB1) antagonists have been shown to attenuate diet-induced obesity (DIO) and associated inflammation, the precise molecular mechanisms involved are not clear. In the current study, we investigated the role of microRNA (miR) in the regulation of adipose tissue macrophage (ATM) phenotype following treatment of DIO mice with the CB1 antagonist SR141716A. DIO mice were fed high-fat diet (HFD) for 12 weeks and then treated daily with SR141716A (10 mg/kg) for 4 weeks while continuing HFD. Treated mice experienced weight loss, persistent reduction in fat mass, improvements in metabolic profile, and decreased adipose inflammation. CB1 blockade resulted in down-regulation of several miRs in ATMs, including the miR-466 family and miR-762. Reduced expression of the miR-466 family led to induction of anti-inflammatory M2 transcription factors KLF4 and STAT6, whereas down-regulation of miR-762 promoted induction of AGAP-2, a negative regulator of the neuroimmune retention cues, Netrin-1 and its coreceptor UNC5B. Furthermore, treatment of primary macrophages with SR141716A up-regulated KLF4 and STAT6, reduced secretion of Netrin-1, and increased migration toward the lymph node chemoattractant CCL19. These studies demonstrate for the first time that CB1 receptor blockade attenuates DIO-associated inflammation through alterations in ATM miR expression that promote M2 ATM polarization and macrophage egress from adipose tissue. The current study also identifies additional novel therapeutic targets for diet-induced obesity and metabolic disorder., (© 2019 Mehrpouya-Bahrami et al.)

Rimonabant is a potent and selective cannabinoid CB1 receptor antagonist widely used in animal and clinical studies. Besides its antagonistic properties, numerous studies have shown that, at micromolar concentrations rimonabant behaves as an inverse agonist at CB1 receptors. The mechanism underpinning this activity is unclear. Here we show that micromolar concentrations of rimonabant inhibited Gα i/o -type G proteins, resulting in a receptor-independent block of G protein signaling. Accordingly, rimonabant decreased basal and agonist stimulated [ 35 S]GTPγS binding to cortical membranes of CB1- and GABA B -receptor KO mice and Chinese Hamster Ovary (CHO) cell membranes stably transfected with GABA B or D2 dopamine receptors. The structural analog of rimonabant, AM251, decreased basal and baclofen-stimulated GTPγS binding to rat cortical and CHO cell membranes expressing GABA B receptors. Rimonabant prevented G protein-mediated GABA B and D2 dopamine receptor signaling to adenylyl cyclase in Human Embryonic Kidney 293 cells and to G protein-coupled inwardly rectifying K + channels (GIRK) in midbrain dopamine neurons of CB1 KO mice. Rimonabant suppressed GIRK gating induced by GTPγS in CHO cells transfected with GIRK, consistent with a receptor-independent action. Bioluminescent resonance energy transfer (BRET) measurements in living CHO cells showed that, in presence or absence of co-expressed GABA B receptors, rimonabant stabilized the heterotrimeric Gαi/o-protein complex and prevented conformational rearrangements induced by GABA B receptor activation. Rimonabant failed to inhibit Gαs-mediated signaling, supporting its specificity for Gα i/o -type G proteins. The inhibition of Gα i/o protein provides a new site of rimonabant action that may help to understand its pharmacological and toxicological effects occurring at high concentrations., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The G i/o protein family transduces signals from a diverse group of G protein-coupled receptors (GPCRs). The observed specificity of G i/o -GPCR coupling and the high rate of G i/o signal transduction have been hypothesized to be enabled by the existence of stable associates between G i/o proteins and their cognate GPCRs in the inactive state (G i/o -GPCR preassembly). To test this hypothesis, we applied the recently developed technique of two-photon polarization microscopy (2PPM) to Gα i1 subunits labeled with fluorescent proteins and four GPCRs: the α 2A -adrenergic receptor, GABA B , cannabinoid receptor type 1 (CB 1 R), and dopamine receptor type 2. Our experiments with non-dissociating mutants of fluorescently labeled Gα i1 subunits (exhibiting impaired dissociation from activated GPCRs) showed that 2PPM is capable of detecting GPCR-G protein interactions. 2PPM experiments with non-mutated fluorescently labeled Gα i1 subunits and α 2A -adrenergic receptor, GABA B , or dopamine receptor type 2 receptors did not reveal any interaction between the G i1 protein and the non-stimulated GPCRs. In contrast, non-stimulated CB 1 R exhibited an interaction with the G i1 protein. Further experiments revealed that this interaction is caused solely by CB 1 R basal activity; no preassembly between CB 1 R and the G i1 protein could be observed. Our results demonstrate that four diverse GPCRs do not preassemble with non-active G i1 However, we also show that basal GPCR activity allows interactions between non-stimulated GPCRs and G i1 (basal coupling). These findings suggest that G i1 interacts only with active GPCRs and that the well known high speed of GPCR signal transduction does not require preassembly between G proteins and GPCRs., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Recent studies provide evidence that premature maternal decidual senescence resulting from heightened mTORC1 signaling is a cause of preterm birth (PTB). We show here that mice devoid of fatty acid amide hydrolase (FAAH) with elevated levels ofN-arachidonyl ethanolamide (anandamide), a major endocannabinoid lipid mediator, were more susceptible to PTB upon lipopolysaccharide (LPS) challenge. Anandamide is degraded by FAAH and primarily works by activating two G-protein-coupled receptors CB1 and CB2, encoded by Cnr1 and Cnr2, respectively. We found thatFaah(-/-)decidual cells progressively underwent premature senescence as marked by increased senescence-associated β-galactosidase (SA-β-Gal) staining and γH2AX-positive decidual cells. Interestingly, increased endocannabinoid signaling activated MAPK p38, but not p42/44 or mTORC1 signaling, inFaah(-/-)deciduae, and inhibition of p38 halted premature decidual senescence. We further showed that treatment of a long-acting anandamide in wild-type mice at midgestation triggered premature decidual senescence utilizing CB1, since administration of a CB1 antagonist greatly reduced the rate of PTB inFaah(-/-)females exposed to LPS. These results provide evidence that endocannabinoid signaling is critical in regulating decidual senescence and parturition timing. This study identifies a previously unidentified pathway in decidual senescence, which is independent of mTORC1 signaling., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Cannabis has been used medicinally for centuries to treat a variety of disorders, including those associated with the gastrointestinal tract. The discovery of our bodies' own "cannabis-like molecules" and associated receptors and metabolic machinery - collectively called the endocannabinoid system - enabled investigations into the physiological relevance for the system, and provided the field with evidence of a critical function for this endogenous signaling pathway in health and disease. Recent investigations yield insight into a significant participation for the endocannabinoid system in the normal physiology of gastrointestinal function, and its possible dysfunction in gastrointestinal pathology. Many gaps, however, remain in our understanding of the precise neural and molecular mechanisms across tissue departments that are under the regulatory control of the endocannabinoid system. This review highlights research that reveals an important - and at times surprising - role for the endocannabinoid system in the control of a variety of gastrointestinal functions, including motility, gut-brain mediated fat intake and hunger signaling, inflammation and gut permeability, and dynamic interactions with gut microbiota.

The adipocyte-derived, anorectic hormone leptin was recently shown to owe part of its regulatory effects on appetite-regulating hypothalamic neuropeptides to the elevation of reactive oxygen species (ROS) levels in arcuate nucleus (ARC) neurons. Leptin is also known to exert a negative regulation on hypothalamic endocannabinoid levels and hence on cannabinoid CB1 receptor activity. Here we investigated the possibility of a negative regulation by CB1 receptors of leptin-mediated ROS formation in the ARC. Through pharmacological and molecular biology experiments we report data showing that leptin-induced ROS accumulation is 1) blunted by arachidonyl-2'-chloroethylamide (ACEA) in a CB1-dependent manner in both the mouse hypothalamic cell line mHypoE-N41 and ARC neuron primary cultures, 2) likewise blocked by a peroxisome proliferator-activated receptor-γ (PPAR-γ) agonist, troglitazone, in a manner inhibited by T0070907, a PPAR-γ antagonist that also inhibited the ACEA effect on leptin, 3) blunted under conditions of increased endocannabinoid tone due to either pharmacological or genetic inhibition of endocannabinoid degradation in mHypoE-N41 and primary ARC neuronal cultures from MAGL(-/-) mice, respectively, and 4) associated with reduction of both PPAR-γ and catalase activity, which are reversed by both ACEA and troglitazone. We conclude that CB1 activation reverses leptin-induced ROS formation and hence possibly some of the ROS-mediated effects of the hormone by preventing PPAR-γ inhibition by leptin, with subsequent increase of catalase activity. This mechanism might underlie in part CB1 orexigenic actions under physiopathological conditions accompanied by elevated hypothalamic endocannabinoid levels., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)