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Pathological remodeling of atrial tissue renders the atria more prone to arrhythmia upon arrival of electrical triggers. Activation of the renin-angiotensin system is an important factor that contributes to atrial remodeling, which may result in atrial hypertrophy and prolongation of P-wave duration. In addition, atrial cardiomyocytes are electrically coupled via gap junctions, and electrical remodeling of connexins may result in dysfunction of coordinated wave propagation within the atria. Currently, there is a lack of effective therapeutic strategies that target atrial remodeling. We previously proposed that cannabinoid receptors (CBR) may have cardioprotective qualities. CB13 is a dual cannabinoid receptor agonist that activates AMPK signaling in ventricular cardiomyocytes. We reported that CB13 attenuates tachypacing-induced shortening of atrial refractoriness and inhibition of AMPK signaling in the rat atria. Here, we evaluated the effects of CB13 on neonatal atrial rat cardiomyocytes (NRAM) stimulated by angiotensin II (AngII) in terms of atrial myocyte enlargement and mitochondrial function. CB13 inhibited AngII-induced enhancement of atrial myocyte surface area in an AMPK-dependent manner. CB13 also inhibited mitochondrial membrane potential deterioration in the same context. However, AngII and CB13 did not affect mitochondrial permeability transition pore opening. We further demonstrate that CB13 increased Cx43 compared to AngII-treated neonatal rat atrial myocytes. Overall, our results support the notion that CBR activation promotes atrial AMPK activation, and prevents myocyte enlargement (an indicator that suggests pathological hypertrophy), mitochondrial depolarization and Cx43 destabilization. Therefore, peripheral CBR activation should be further tested as a novel treatment strategy in the context of atrial remodeling.

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Cannabinoids produce their characteristic effects mainly by binding to two types of G-protein coupled receptors (GPCRs), the CB1 and CB2 cannabinoid receptors. The CB1 receptor is the main cannabinoid receptor in the central nervous system, and it participates in many brain functions. Recent studies showed that membrane potential may serve as a novel modulatory modality of many GPCRs. Here, we used Xenopus oocytes as an expression system to examine whether membrane potential modulates the activity of the CB1 receptor. We found that the potencies of the endocannabinoid 2-AG and the phytocannabinoid THC in activating the receptor are voltage dependent; depolarization enhanced the potency of these agonists and decreased their dissociation from the receptor. This voltage dependence appears to be agonist dependent as the potency of the endocannabinoid anandamide (AEA) was voltage independent. The finding of this agonist-specific modulatory factor for the CB1 receptor may contribute to our future understanding of various physiological functions mediated by the endocannabinoid system.

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Scleroderma, or systemic sclerosis, is a multi-organ connective tissue disease resulting in fibrosis of the skin, heart, and lungs with no effective treatment. Endocannabinoids acting via cannabinoid-1 receptors (CB1R) and increased activity of inducible NO synthase (iNOS) promote tissue fibrosis including skin fibrosis, and joint targeting of these pathways may improve therapeutic efficacy. Recently, we showed that in mouse models of liver, lung and kidney fibrosis, treatment with a peripherally restricted hybrid CB1R/iNOS inhibitor (MRI-1867) yields greater anti-fibrotic efficacy than inhibiting either target alone. Here, we evaluated the therapeutic efficacy of MRI-1867 in bleomycin-induced skin fibrosis. Skin fibrosis was induced in C57BL/6J (B6) and Mdr1a/b-Bcrp triple knock-out (KO) mice by daily subcutaneous injections of bleomycin (2 IU/100 µL) for 28 days. Starting on day 15, mice were treated for 2 weeks with daily oral gavage of vehicle or MRI-1867. Skin levels of MRI-1867 and endocannabinoids were measured by mass spectrometry to assess target exposure and engagement by MRI-1867. Fibrosis was characterized histologically by dermal thickening and biochemically by hydroxyproline content. We also evaluated the potential increase of drug-efflux associated ABC transporters by bleomycin in skin fibrosis, which could affect target exposure to test compounds, as reported in bleomycin-induced lung fibrosis. Bleomycin-induced skin fibrosis was comparable in B6 and Mdr1a/b-Bcrp KO mice. However, the skin level of MRI-1867, an MDR1 substrate, was dramatically lower in B6 mice (0.023 µM) than in Mdr1a/b-Bcrp KO mice (8.8 µM) due to a bleomycin-induced increase in efflux activity of MDR1 in fibrotic skin. Furthermore, the endocannabinoids anandamide and 2-arachidonylglycerol were elevated 2-4-fold in the fibrotic vs. control skin in both mouse strains. MRI-1867 treatment attenuated bleomycin-induced established skin fibrosis and the associated increase in endocannabinoids in Mdr1a/b-Bcrp KO mice but not in B6 mice. We conclude that combined inhibition of CB1R and iNOS is an effective anti-fibrotic strategy for scleroderma. As bleomycin induces an artifact in testing antifibrotic drug candidates that are substrates of drug-efflux transporters, using Mdr1a/b-Bcrp KO mice for preclinical testing of such compounds avoids this pitfall.

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White adipose tissue (WAT) possesses the endocannabinoid system (ECS) machinery and produces the two major endocannabinoids (ECs), arachidonoylethanolamide (AEA) and 2-arachidonoylglycerol (2-AG). Accumulating evidence indicates that WAT cannabinoid 1 receptors (CB1R) are involved in the regulation of fat storage, tissue remodeling and secretory functions but their role in controlling lipid mobilization is unclear. In the present study, we used different strategies to acutely increase ECS activity in WAT and tested the consequences on glycerol production as a marker of lipolysis. Treating lean mice or rat WAT explants with JLZ195, which inhibits ECs degrading enzymes, induced an increase in 2-AG tissue contents that was associated with a CB1R-dependent decrease in lipolysis. Direct treatment of rat WAT explants with AEA also inhibited glycerol production while mechanistic studies revealed it could result from the stimulation of Akt-signaling pathway. Interestingly, AEA treatment decreased lipolysis both in visceral and subcutaneous WAT collected on lean subjects suggesting that ECS also reduces fat store mobilization in Human. In obese mice, WAT content and secretion rate of ECs were higher than in control while glycerol production was reduced suggesting that over-produced ECs may inhibit lipolysis activating local CB1R. Strikingly, our data also reveal that acute CB1R blockade with Rimonabant did not modify lipolysis in vitro in obese mice and human explants nor in vivo in obese mice. Taken together, these data provide physiological evidence that activation of ECS in WAT, by limiting fat mobilization, may participate in the progressive tissue remodeling that could finally lead to organ dysfunction. The present findings also indicate that acute CB1R blockade is inefficient in regulating lipolysis in obese WAT and raise the possibility of an alteration of CB1R signaling in conditions of obesity.

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Asperuloside is an iridoid glycoside found in many medicinal plants that has produced promising anti-obesity results in animal models. In previous studies, three months of asperuloside administration reduced food intake, body weight, and adipose masses in rats consuming a high fat diet (HFD). However, the mechanisms by which asperuloside exerts its anti-obesity properties were not clarified. Here, we investigated homeostatic and nutrient-sensing mechanisms regulating food intake in mice consuming HFD. We confirmed the anti-obesity properties of asperuloside and, importantly, we identified some mechanisms that could be responsible for its therapeutic effect. Asperuloside reduced body weight and food intake in mice consuming HFD by 10.5 and 12.8% respectively, with no effect on mice eating a standard chow diet. Fasting glucose and plasma insulin were also significantly reduced. Mechanistically, asperuloside significantly reduced hypothalamic mRNA ghrelin, leptin, and pro-opiomelanocortin in mice consuming HFD. The expression of fat lingual receptors (CD36, FFAR1-4), CB1R and sweet lingual receptors (TAS1R2-3) was increased almost 2-fold by the administration of asperuloside. Our findings suggest that asperuloside might exert its therapeutic effects by altering nutrient-sensing receptors in the oral cavity as well as hypothalamic receptors involved in food intake when mice are exposed to obesogenic diets. This signaling pathway is known to influence the subtle hypothalamic equilibrium between energy homeostasis and reward-induced overeating responses. The present pre-clinical study demonstrated that targeting the gustatory system through asperuloside administration could represent a promising and effective new anti-obesity strategy.

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The transient receptor potential vanilloid 1 (TRPV1) is a non-selective ligand-gated cation channel involved in synaptic transmission, plasticity, and brain pathology. In the hippocampal dentate gyrus, TRPV1 localizes to dendritic spines and dendrites postsynaptic to excitatory synapses in the molecular layer (ML). At these same synapses, the cannabinoid CB1 receptor (CB1R) activated by exogenous and endogenous cannabinoids localizes to the presynaptic terminals. Hence, as both receptors are activated by endogenous anandamide, co-localize, and mediate long-term depression of the excitatory synaptic transmission at the medial perforant path (MPP) excitatory synapses though by different mechanisms, it is plausible that they might be exerting a reciprocal influence from their opposite synaptic sites. In this anatomical scenario, we tested whether the absence of TRPV1 affects the endocannabinoid system. The results obtained using biochemical techniques and immunoelectron microscopy in a mouse with the genetic deletion of TRPV1 show that the expression and localization of components of the endocannabinoid system, included CB1R, change upon the constitutive absence of TRPV1. Thus, the expression of fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) drastically increased in TRPV1-/- whole homogenates. Furthermore, CB1R and MAGL decreased and the cannabinoid receptor interacting protein 1a (CRIP1a) increased in TRPV1-/- synaptosomes. Also, CB1R positive excitatory terminals increased, the number of excitatory terminals decreased, and CB1R particles dropped significantly in inhibitory terminals in the dentate ML of TRPV1-/- mice. In the outer 2/3 ML of the TRPV1-/- mutants, the proportion of CB1R particles decreased in dendrites, and increased in excitatory terminals and astrocytes. In the inner 1/3 ML, the proportion of labeling increased in excitatory terminals, neuronal mitochondria, and dendrites. Altogether, these observations indicate the existence of compensatory changes in the endocannabinoid system upon TRPV1 removal, and endorse the importance of the potential functional adaptations derived from the lack of TRPV1 in the mouse brain.