Endocannabinoids (ECs) acting via CB1 receptors (CB1R) play an important role in the control of body weight and energy homeostasis. In clinical trials, the CB1R blocking drug rimonabant had been found effective in reducing body weight and improving cardiometabolic abnormalities in patients with the metabolic syndrome, but was withdrawn from the market in 2008 due to neuropsychiatric side effects. Several studies, including our own, indicate that CB1R in peripheral tissues contribute to the metabolic benefit of CB1R blockade (JCI 115:1298, 2005;JCI 118:3160-9, 2008), raising the possibility that peripheral CB1R may be selectively targeted in the treatment of the metabolic syndrome in order to minimize neuropsychiatric side effects. Last year, we published the first evidence in support of the validity of this approach, using a novel, peripherally restricted CB1R antagonist, AM6545. AM6545 is an orally bioavailable CB1R neutral antagonist with high CB1R binding affinity and selectivity and markedly reduced brain penetrance relative to its parent compound, the CB1R inverse agonist rimonabant. Unlike rimonabant, AM6545 failed to inhibit central CB1R-mediated behavioral effects in mice, but had metabolic effects similar to rimonabant in mice with diet-induced obesity (DIO), providing proof of concept for the therapeutic potential of peripheral CB1R blockade (JCI 120:2953-66, 2010). AM6545 was equieffective with rimonabant in reversing steatosis and dyspidemias, but was less effective than rimonabant in reducing food intake, body weight and adiposity, as well as insulin and leptin resistance. The greater efficacy of rimonabant could be due to its blockade of CB1R in the brain or to its inverse agonist properties. To distinguish between these two possibilities, we have tested a novel, peripherally restricted CB1R inverse agonist, JD5037, developed at Jenrin Discoveries Inc. JD5037 is a highly potent (Kd: 0.3 nM) peripherally restricted CB1R antagonist with no central CB1R occupancy, documented by CB1R PET, at therapeutically effective doses. JD5037 is devoid of CB1R-mediated behavioral effects, but is fully equieffective with its brain-penetrant parent in reducing food intake, body weight, steatosis, insulin and leptin resistance and dyslipidemia. In leptin-deficient ob/ob mice, JD5037 is simlarly effective in reversiong steatosis and insulin resistance, but fails to affect food intake and body weight, suggesting the role of endogenous leptin in these latter effects. Indeed, JD5037 treatment of DIO mice reverses their leptin resistance by rapidly reversing their hyperleptinemia, through decreasing leptin expression and secretion by adipocytes and increasing megalin-mediated leptin clearance via the kidney. These findings indicate that targeting peripheral CB1R by inverse agonists has great promise in the treatment of obesity and its cardiometabolic complications. This study has been submitted for publication. We have previously shown that hepatic CB1R are necessary for diet-induced steatosis, insulin and leptin resistance to develop in mice (JCI 2008). To address the question whether activation of hepatic CB1R is sufficient for these effect, we have developed a rescue model, transgenic mice that express CB1R only in hepatocytes and analyzed glycemic control using a hyperinsulinemic clamp. High fat diet induces hepatic insulin resistance in wild-type mice but not in mice with global or hepatocyte-specific knockout of CB1R. CB1R-/- mice with transgenic re-expression of CB1R in liver are hyperinsulinemic as a result of reduced insulin clearance due to downregulation of the insulin degrading enzyme, yet have increased hepatic glucose production due to increased glycogenolysis, indicating hepatic insulin resistance. In mice with CB1R present in hepatocytes, high fat diet or CB1R activation results in ER stress, via activation of the Bip/PERK/eIF2alpha protein translation pathway. In human and murine isolated hepatocytes, CB1R activation causes ER-stres-dependent suppression phosphorylation of akt-2 by insulin, through stimulation of the serine/threomnine phosphatase Phlpp1. In human liver, CB1R expression is upregulated in non-salcoholic fatty liver disease. In conclusion, endocannabinoids contribute to diet-induced insulin resistance via hepatic CB1-mediated inhibition of insulin signaling and clearance. These findings have been submitted for publication. The mammalian liver regenerates upon tissue loss, which induces quiescent hepatocytes to enter the cell cycle and undergo limited replication under the control of multiple hormones, growth factors and cytokines. ECs acting via CB1R promote neural progenitor cell proliferation , and in the liver they promote lipogenesis. Based on these findings, we tested whether CB1R are involved in the control of liver regeneration. We found that mice lacking CB1R globally or in hepatocytes only have a delayed proliferative response following 2/3 partial hepatectomy (PHX). In wild-type mice, PHX leads to increased hepatic expression of CB1R and hyperactivation of the biosynthesis of the endocannabinoid anandamide in the liver via an in vivo pathway involving conjugation of arachidonic acid and ethanolamine by fatty-acid amide hydrolase (FAAH). In wild-type but not in CB1R ko mice, PHX induces robust upregulation of key cell-cycle proteins involved in mitotic progression, including cyclin-dependent kinase 1 (Cdk1), cyclin B2, and their transcriptional regulator, forkhead box protein M1 (FoxM1), as revealed by ultrahigh throughput RNA sequencing and pathway analysis and confirmed by real-time PCR and Western blot analyses. Treatment of wild-type mice with anandamide induced similar changes mediated via the PI3K/akt pathway. We conclude that activation of hepatic CB1r by newly synthesized anandamide promotes liver regeneration by controlling the expression of cell-cycle regulators that drive M phase progression. These findings have been published in PNAS.

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Project End
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11
Fiscal Year
2011
Total Cost
$2,457,470
Indirect Cost
Name
National Institute on Alcohol Abuse and Alcoholism
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Iyer, Malliga R; Cinar, Resat; Katz, Alexis et al. (2017) Design, Synthesis, and Biological Evaluation of Novel, Non-Brain-Penetrant, Hybrid Cannabinoid CB1R Inverse Agonist/Inducible Nitric Oxide Synthase (iNOS) Inhibitors for the Treatment of Liver Fibrosis. J Med Chem 60:1126-1141
Cinar, Resat; Gochuico, Bernadette R; Iyer, Malliga R et al. (2017) Cannabinoid CB1 receptor overactivity contributes to the pathogenesis of idiopathic pulmonary fibrosis. JCI Insight 2:
Zhu, Lu; Rossi, Mario; Cui, Yinghong et al. (2017) Hepatic ?-arrestin 2 is essential for maintaining euglycemia. J Clin Invest 127:2941-2945
Jourdan, Tony; Szanda, Gerg?; Cinar, Resat et al. (2017) Developmental Role of Macrophage Cannabinoid-1 Receptor Signaling in Type 2 Diabetes. Diabetes 66:994-1007
Iyer, Malliga R; Cinar, Resat; Coffey, Nathan J et al. (2017) Synthesis of S-2-((S)-3-(4-chlorophenyl)-N'-((4-chlorophenyl)sulfonyl)-4-phenyl-4,5-dihydro-1H-pyrazole-1-carboximidamido)-3-(methyl-d3 )butanamide-d5 , octadeuterated JD5037. J Labelled Comp Radiopharm 60:460-465
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Mukhopadhyay, Partha; Baggelaar, Marc; Erdelyi, Katalin et al. (2016) The novel, orally available and peripherally restricted selective cannabinoid CB2 receptor agonist LEI-101 prevents cisplatin-induced nephrotoxicity. Br J Pharmacol 173:446-58
Cinar, Resat; Iyer, Malliga R; Liu, Ziyi et al. (2016) Hybrid inhibitor of peripheral cannabinoid-1 receptors and inducible nitric oxide synthase mitigates liver fibrosis. JCI Insight 1:
Jourdan, T; Godlewski, G; Kunos, G (2016) Endocannabinoid regulation of ?-cell functions: implications for glycaemic control and diabetes. Diabetes Obes Metab 18:549-57
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