We analyzed the molecular mechanisms involved in the catabolic effects of peripheral cannabinoid-1 receptor (CB1R) blockade in the liver in diet-induced obesity/metabolic syndrome. Endocannabinoids promote energy conservation in obesity, whereas CB1R blockade reverses body weight gain and insulin resistance and increases energy expenditure. Here we investigated the molecular mechanisms of the catabolic effects of CB1 R blockade in the liver. Exposure of primary mouse hepatocytes and HepG2 cells to the CB1 R agonist arachidonyl-2'-chloroethylamide inhibited the expression of Sirtuin-1 (Sirt1) and Rictor, a component of mechanistic target of rapamycin complex 2 (mTORC2) and suppressed insulin-induced Akt phosphorylation at serine 473. These effects were reversed by peripheral CB1 R antagonist JD5037 in control hepatocytes but not in hepatocytes deficient in Sirt1 and/or Rictor, indicating that these two proteins are required for the CB1R-mediated inhibition of insulin signaling. Feeding C57BL/6J mice a high-fat diet (HFD) inhibited hepatic Sirt1/mTORC2/Akt signaling, and the inhibition was reversed by rimonabant or JD5037 in wild-type but not liver-specific Sirt1-/- (Sirt1-LKO) mice, to levels observed in hepatocyte-specific CB1R-/- mice. A similar attenuation of hyperglycemia and hyperinsulinemia in wild-type mice with obesity but not in Sirt1-LKO mice could be attributed to insufficient reversal of HFD-induced mitochondrial reactive oxygen species generation in peripheral tissues in the latter. In contrast, JD5037 treatment was equally effective in HFD-fed wild-type and Sirt1-LKO mice in reducing hepatic steatosis, increasing fatty acid -oxidation, and activating 5'adenosine monophosphate-activated protein kinase (AMPK) through liver kinase B1 (LKB1), resulting in a similar increase in total energy expenditure in the two strains. We conclude that peripheral CB1R blockade in mice with obesity improves glycemic control through the hepatic Sirt1/mTORC2/Akt pathway, whereas it increases fatty acid oxidation through LKB1/AMPK signaling. This study has been published in Hepatology. In another study, we have generated novel, high affinity ligands for the cannabinoid-2 receptor (CB2R) that served as key tools for resolving the mechanism of activation of the CB2R at the molecular level. The CB2R is predominantly expressed in the immune system, and selective modulation of CB2R which lack the psychoactivity of CB1R has therapeutic potential in inflammatory, fibrotic, and neurodegenerative diseases. In a multi-group collaborative study with Ray Stevens' laboratory, we reported the crystal structure of human CB2R in complex with a rationally designed antagonist, AM10257, at 2.8 Angstrom resolution. The CB2R/AM10257 structure reveals a distinctively different binding pose compared with CB1R. However, the extracellular portion of the antagonist-bound CB2R shares a high degree of conformational similarity with the agonist-bound CB1R, which led to the discovery of AM10257's unexpected opposing functional profile of CB2R antagonism versus CB1R agonism. Further structural analysis using mutational studies and molecular docking revealed the molecular basis of their function and selectivity for CB2R and CB1R. Additional analyses of our designed antagonist and agonist pairs provide important insight into the activation mechanism of CB2R. The present findings should facilitate rational drug design toward precise modulation of the endocannabinoid system. This study has been published in Cell. In a third study related to the therapeutic development of our novel, hybrid CB1R/iNOS inhibitors for the treatment of fibrotic disorders, we explored a potential pitfall in the use of animal models in which bleomycin is used to induce tissue fibrosis, including fibrosis of the lungs or the skin. ATP-binding cassette (ABC) transporters are evolutionarily conserved membrane proteins that pump a variety of endogenous substrates across cell membranes. Certain subfamilies are known to interact with pharmaceutical compounds, potentially influencing drug delivery and treatment efficacy. However, the role of drug resistance-associated ABC transporters has not been examined in idiopathic pulmonary fibrosis (IPF) or its animal model: the bleomycin-induced murine model. We investigate the expression of two ABC transporters, P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP), in human IPF lung tissue and two different bleomycin-induced mouse models of pulmonary fibrosis. We obtained human IPF specimens from patients during lung transplantation and administered bleomycin to male C57BL/6J mice either by oropharyngeal aspiration (1 U/kg) or subcutaneous osmotic infusion (100 U/kg over 7 days). We found that P-gp and BCRP expression in IPF patient lungs was comparable to controls. However, murine lungs expressed increased levels of P-gp and BCRP after oropharyngeal and subcutaneous bleomycin administration. We localized this upregulation to multiple pulmonary cell types, including alveolar fibroblasts, endothelial cells, and type-2 epithelial cells. Functionally, this effect reduced murine lung exposure to nintedanib, an FDA-approved IPF therapy known to be a P-gp substrate. The study reveals a discrepancy between IPF pathophysiology and the common animal model of lung fibrosis. Bleomycin-induced drug efflux in the murine lungs may present an uncontrolled confounding variable in the preclinical study of IPF drug candidates, and these findings will facilitate disease model validation, enhance new drug discoveries that ultimately improve patient outcomes. This work has just been published American Journal of Respiratory Cell and Molecular Biology.

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National Institute on Alcohol Abuse and Alcoholism
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Iyer, Malliga R; Cinar, Resat; Coffey, Nathan J et al. (2018) Synthesis of 13 C6 -labeled, dual-target inhibitor of cannabinoid-1 receptor (CB1 R) and inducible nitric oxide synthase (iNOS). J Labelled Comp Radiopharm :
Jourdan, Tony; Park, Joshua K; Varga, Zoltán V et al. (2018) Cannabinoid-1 receptor deletion in podocytes mitigates both glomerular and tubular dysfunction in a mouse model of diabetic nephropathy. Diabetes Obes Metab 20:698-708
Valenta, Ines; Varga, Zoltan V; Valentine, Heather et al. (2018) Feasibility Evaluation of Myocardial Cannabinoid Type 1 Receptor Imaging in Obesity: A Translational Approach. JACC Cardiovasc Imaging 11:320-332
Varga, Zoltan V; Erdelyi, Katalin; Paloczi, Janos et al. (2018) Disruption of Renal Arginine Metabolism Promotes Kidney Injury in Hepatorenal Syndrome in Mice. Hepatology 68:1519-1533
Zhou, Zhou; Xu, Ming-Jiang; Cai, Yan et al. (2018) Neutrophil-Hepatic Stellate Cell Interactions Promote Fibrosis in Experimental Steatohepatitis. Cell Mol Gastroenterol Hepatol 5:399-413
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Jourdan, Tony; Nicoloro, Sarah M; Zhou, Zhou et al. (2017) Decreasing CB1 receptor signaling in Kupffer cells improves insulin sensitivity in obese mice. Mol Metab 6:1517-1528
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

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