Endocannabinoids And Energy Homeostasis

National Institute On Alcohol Abuse And Alcoholism

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Abstract

In previous studies we demonstrated that both the endocannabinoid/CB1 receptor (CB1R) system and inducible nitric oxide synthase (iNOS) are upregulated in various fibrotic tissues and contribute to the initiation and progression of fibrosis. This has led us to design and synthesize hybrid CB1R/iNOS inhibitors that can simultaneously engage these 2 targets resulting in an increase in their anti-fibrotic efficacy compared to engaging only one of these targets. We were able to prove this concept in different preclinical models of liver and lung fibrosis, using the hybrid CB1R/iNOS inhibitor MRI-1867 (JCI Insight 2016; 2017). We then set out to test MRI-1867 in a well established mouse model of scleroderma, induced by daily intradermal injection of bleomycin. In contrast to its strong efficacy in mitigating liver and lung fibrosis, MRI-1867 was ineffective in modifying bleo-induced skin fibrosis. A solution to this paradox was offered by the finding of extremely low levels of MRI-1867 in the fibrotic skin, compared to much higher levels in healthy skin areas of the same animals. We found that bleomycin strongly induced the expression of two abc drug transporters, P-gp and Bcrg, locally in the fibrotic skin patch, which minimized target exposure due to the reverse transport of MRI-1867, a substrate of abc transporters, from the affected skin. To overcome this problem unique to the animal model (there is no evidence for the induction of abc transporters in human scleroderma), we repeated these experiments using mice with triple genetic knockout of P-gp (Mdr1a/b) and Bcrg. In these animals, exposure of the skin to MRI-1867 was similarly high in the bleomycin-injected fibrotic patch and normal healthy skin areas. Furthermore, fibrosis, as measured by hydroxyproline content and visualized histologically by Masson trichrome and sirius red staining was significantly reduced by daily administration of MRI-1867, 10 mg/kg/day by oral gavage. We have finalized a manuscript and submitted it for publication in Frontiers in Endocrinology. Seven-transmembrane receptors signal via G protein- and beta-arrestin-dependent pathways. We describe a peripheral CB1R antagonist (MRI-1891) highly biased toward inhibiting CB1R-induced beta-arrestin-2 (BArr2) recruitment over G-protein activation. In obese wild-type and BArr2-knockout (KO) mice, MRI-1891 treatment reduces food intake and body weight without eliciting anxiety even at a high dose causing partial brain CB1R occupancy. By contrast, the unbiased global CB1R antagonist rimonabant elicits anxiety in both strains, indicating no BArr2 involvement. Interestingly, obesity-induced muscle insulin resistance is improved by MRI-1891 in wild-type but not in BArr2-KO mice. In C2C12 myoblasts, CB1R activation suppresses insulin-induced akt-2 phosphorylation, preventable by MRI-1891, BArr2 knockdown or overexpression of CB1R-interacting protein-1a. MRI-1891, but not rimonabant, interacts with non-polar residues on the N-terminal loop of the CB1R, including F108, and on transmembrane helix-1, including S123, a combination that facilitates BArr2 bias. Thus, CB1R activation promotes muscle insulin resistance via BArr2 signaling, which is selectively mitigated by a biased CB1R antagonist at reduced risk of CNS side effects. This study has now been published in ACS Pharmacology & Translational Science, 2021.