Reversal of diet-induced hepatic steatosis by peripheral CB1 receptor blockade in mice is p53/ miRNA-22/SIRT1/PPARa dependent Shahar Azar 1 , Shiran Udi 1 , Adi Drori 1 , Rivka Hadar 1 , Alina Nemirovski 1 , Kiran V. Vemuri 2 , Maya Miller 3 , Dana Sherill-Rofe 3 , Yhara Arad 3 , Devorah Gur-Wahnon 4 , Xiaoling Li 5 , Alexandros Makriyannis 2 , Danny Ben-Zvi 3 , Yuval Tabach 3 , Iddo Z. Ben-Dov 5 , Joseph Tam 1, * ABSTRACT Objective: The endocannabinoid (eCB) system is increasingly recognized as being crucially important in obesity-related hepatic steatosis. By activating the hepatic cannabinoid-1 receptor (CB 1 R), eCBs modulate lipogenesis and fatty acid oxidation. However, the underlying molecular mechanisms are largely unknown. Methods: We combined unbiased bioinformatics techniques, mouse genetic manipulations, multiple pharmacological, molecular, and cellular biology approaches, and genomic sequencing to systematically decipher the role of the hepatic CB 1 R in modulating fat utilization in the liver and explored the downstream molecular mechanisms. Results: Using an unbiased normalized phylogenetic profiling analysis, we found that the CB 1 R evolutionarily coevolves with peroxisome proliferator-activated receptor-alpha (PPARa), a key regulator of hepatic lipid metabolism. In diet-induced obese (DIO) mice, peripheral CB 1 R blockade (using AM6545) induced the reversal of hepatic steatosis and improved liver injury in WT, but not in PPARa / mice. The antisteatotic effect mediated by AM6545 in WT DIO mice was accompanied by increased hepatic expression and activity of PPARa as well as elevated hepatic levels of the PPARa-activating eCB-like molecules oleoylethanolamide and palmitoylethanolamide. Moreover, AM6545 was unable to rescue hepatic steatosis in DIO mice lacking liver sirtuin 1 (SIRT1), an upstream regulator of PPARa. Both of these signaling molecules were modulated by the CB 1 R as measured in hepatocytes exposed to lipotoxic conditions or treated with CB 1 R agonists in the absence/presence of AM6545. Furthermore, using microRNA transcriptomic profiling, we found that the CB 1 R regulated the hepatic expression, acetylation, and transcriptional activity of p53, resulting in the enhanced expression of miR-22, which was found to specifically target SIRT1 and PPARa. Conclusions: We provide strong evidence for a functional role of the p53/miR-22/SIRT1/PPARa signaling pathway in potentially mediating the antisteatotic effect of peripherally restricted CB 1 R blockade. Ó 2020 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Keywords Obesity; Fatty liver; Endocannabinoids; microRNAs; Nuclear receptor 1. INTRODUCTION Non-alcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease worldwide, affecting more than 25% of the hu- man population, and a leading cause of morbidity and mortality [1]. NAFLD, considered the hepatic manifestation of metabolic syndrome, and its earliest stage, hepatic steatosis, are characterized by fat accumulation that exceeds 5% of hepatocytes in the absence of sig- nificant alcohol intake or secondary causes of lipid accumulation [1]. NAFLD is based on imbalances between lipid acquisition and removal that are regulated by de novo lipogenesis, fatty acid b-oxidation, up- take of circulating lipids, and their export to extrahepatic tissues. To date, various cellular, hormonal, metabolic, and genetic factors have been linked to its development [1], yet no effective medications have been approved to treat it. Endocannabinoids (eCBs) are lipid ligands produced from membrane phospholipids; they activate the cannabinoid receptors to modulate food intake, body mass, and energy expenditure, mainly by activating the cannabinoid-1 receptor (CB 1 R) abundantly expressed in the central nervous system and also present in peripheral tissues, such as the liver [2]. In fact, hepatic steatosis induced by a high-fat diet (HFD) depends on the activation of the peripheral CB 1 Rs, including those in the liver. Global or hepatic nullification of the CB 1 R protects mice from devel- oping HFD-induced fatty liver [3,4]. Hepatocyte-selective 1 Obesity and Metabolism Laboratory, Institute for Drug Research, School of Pharmacy, Faculty of Medicine, Hebrew University of Jerusalem, Jerusalem, Israel 2 Center for Drug Discovery, Northeastern University, Boston, MA, USA 3 Department of Developmental Biology and Cancer Research, Institute for Medical Research Israel-Canada, Hadassah Medical School, Hebrew University of Jerusalem, Jerusalem, Israel 4 Laboratory of Medical Transcriptomics, Department of Nephrology, Hadassah-Hebrew University Medical Center, Jerusalem, Israel 5 Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC, USA *Corresponding author. Joseph Tam, Obesity and Metabolism Laboratory, Institute for Drug Research, School of Pharmacy, Faculty of Medicine, P.O. Box 12065, Je- rusalem, 9112001, Israel. Tel.: þ972 2 675 7645; Fax: þ972 2 675 7015. E-mail: yossi.tam@mail.huji.ac.il (J. Tam). Received May 19, 2020  Revision received September 3, 2020  Accepted September 17, 2020  Available online 26 September 2020 https://doi.org/10.1016/j.molmet.2020.101087 Original Article MOLECULAR METABOLISM 42 (2020) 101087 Ó 2020 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). www.molecularmetabolism.com 1