Basic Study
Copyright ©The Author(s) 2020. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Sep 14, 2020; 26(34): 5101-5117
Published online Sep 14, 2020. doi: 10.3748/wjg.v26.i34.5101
Arachidyl amido cholanoic acid improves liver glucose and lipid homeostasis in nonalcoholic steatohepatitis via AMPK and mTOR regulation
David Fernández-Ramos, Fernando Lopitz-Otsoa, Laura Delacruz-Villar, Jon Bilbao, Martina Pagano, Laura Mosca, Maider Bizkarguenaga, Marina Serrano-Macia, Mikel Azkargorta, Marta Iruarrizaga-Lejarreta, Jesús Sot, Darya Tsvirkun, Sebastiaan Martijn van Liempd, Felix M Goni, Cristina Alonso, María Luz Martínez-Chantar, Felix Elortza, Liat Hayardeny, Shelly C Lu, José M Mato
David Fernández-Ramos, Fernando Lopitz-Otsoa, Laura Delacruz-Villar, Jon Bilbao, Maider Bizkarguenaga, José M Mato, Precision Medicine and Metabolism Laboratory, Centro de Investigación Cooperativa en Biociencias (CIC bioGUNE), Derio 48160, Bizkaia, Spain
David Fernández-Ramos, María Luz Martínez-Chantar, José M Mato, CIBERehd - Centro de Investigación Biomédica en Red de enfermedades hepáticas y digestivas, Madrid 28029, Spain
Martina Pagano, Laura Mosca, Department of Precision Medicine, University of Campania "Luigi Vanvitelli", Naples 80138, Italy
Marina Serrano-Macia, María Luz Martínez-Chantar, Liver Disease Laboratory, Centro de Investigación Cooperativa en Biociencias (CIC bioGUNE), Derio 48160, Spain
Mikel Azkargorta, Felix Elortza, Proteomics Platform, Centro de Investigación Cooperativa en Biociencias (CIC bioGUNE), Derio 48160, Spain
Marta Iruarrizaga-Lejarreta, Cristina Alonso, OWL Metabolomics, Derio 48160, Spain
Jesús Sot, Felix M Goni, Instituto Biofisika (UPV/EHU, CSIC), Leioa 48940, Spain; Departamento de Bioquímica y Biología Molecular, Universidad del País Vasco, Leioa 48940, Spain
Darya Tsvirkun, Liat Hayardeny, Pre-clinical and Chemistry, Manufacturing and Controls, Galmed Pharmaceuticals, Tel Aviv 6578317, Israel
Sebastiaan Martijn van Liempd, Metabolomics Platform, Centro de Investigación Cooperativa en Biociencias (CIC bioGUNE), Derio 48160, Spain
Shelly C Lu, Division of Digestive and Liver Diseases, Cedars-Sinai Medical Center, Los Angeles, CA 90048, United States
Author contributions: Fernández-Ramos D, Lopitz-Otsoa F, Delacruz-Villar L, Bilbao J, Pagano M, Mosca L, Bizkarguenaga M, Serrano-Macia M, Azkargorta M, Iruarrizaga-Lejarreta M, and vanLiempd SM performed the experiments, and acquired, analyzed, and interpreted the data; Tsvirkun D, Goni FM, Alonso C, Elortza F, Hayardeny L, Martínez-Chantar ML, and Lu SC critically revised the manuscript; Fernández-Ramos D, Lopitz-Otsoa F, and Mato JM drafted the manuscript; Goni FM, Martínez-Chantar ML, Lu SC, and Mato JM obtained funding; Mato JM designed, coordinated, and supervised the study; all authors approved the final version of the article.
Supported by the National Institutes of Health Grant, No. R01CA172086;Plan Nacional of I+D, No. SAF2017-88041-R;Ministerio de Economía y Competitividad de España, No. SAF2017-87301-R;Asociación Española contra el Cáncer, No. AECC17/302;Ayudas Fundación BBVA a equipos de Investigación Científica 2018;Fondo Europeo de Desarrollo Regional, Ministerio de Economia y Competitividad de España, No. PGC2018-099857-B-I00;Basque Government Grants, No. IT1264-19;Ministerio de Economia y Competitividad de España for the Severo Ochoa Excellence Accreditation, No. SEV-2016-0644. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Institutional review board statement: The study was reviewed and approved by the Food and Drug Agency at United States (Form Approved: OMS No. 0910-0014, IND No. 079200), and Ministry of Health at Israel (Institutional Committee application number: 0488-14). More information is available at ClinicalTrials.gov NCT02279524.
Institutional animal care and use committee statement: All work performed with animals was approved by an Órgano Habilitado (Comité de Bioética y Bienestar Animal, CBBA/IACUC, at CIC bioGUNE) and the competent authority (Diputación de Bizkaia) following European and Spanish directives, under the following protocol: P-CBG-CBBA-0515. CIC bioGUNE’s Animal Facility is accredited by AAALAC Intl., The Institutional Animal Care and Use Committee at CIC bioGUNE, Derio, Bizkaia, Spain).
Conflict-of-interest statement: Dr. Mato is a Galmed Pharmaceuticals and OWL Metabolomics consultant and/or speaker. Dr. Hayardeny and Dr. Tzivirkun are Galmed Pharmaceuticals employees. Dr. Iruarrizaga-Lejarreta and Dr. Alonso are OWL Metabolomics employees. All other authors have nothing to disclose.
Data sharing statement: No additional data are available.
ARRIVE guidelines statement: The authors have read the ARRIVE guidelines, and the manuscript was prepared and revised according to the ARRIVE guidelines.
Open-Access: This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Corresponding author: José M Mato, PhD, Director, Professor, Precision Medicine and Metabolism Laboratory, Centro de Investigación Cooperativa en Biociencias (CIC bioGUNE), Parque Tecnológico de Bizkaia, Derio 48160, Bizkaia, Spain. director@cicbiogune.es
Received: May 19, 2020
Peer-review started: May 19, 2020
First decision: June 4, 2020
Revised: June 19, 2020
Accepted: August 13, 2020
Article in press: August 13, 2020
Published online: September 14, 2020
Processing time: 113 Days and 4.2 Hours
Abstract
BACKGROUND

Arachidyl amido cholanoic acid (Aramchol) is a potent downregulator of hepatic stearoyl-CoA desaturase 1 (SCD1) protein expression that reduces liver triglycerides and fibrosis in animal models of steatohepatitis. In a phase IIb clinical trial in patients with nonalcoholic steatohepatitis (NASH), 52 wk of treatment with Aramchol reduced blood levels of glycated hemoglobin A1c, an indicator of glycemic control.

AIM

To assess lipid and glucose metabolism in mouse hepatocytes and in a NASH mouse model [induced with a 0.1% methionine and choline deficient diet (0.1MCD)] after treatment with Aramchol.

METHODS

Isolated primary mouse hepatocytes were incubated with 20 μmol/L Aramchol or vehicle for 48 h. Subsequently, analyses were performed including Western blot, proteomics by mass spectrometry, and fluxomic analysis with 13C-uniformly labeled glucose. For the in vivo part of the study, male C57BL/6J mice were randomly fed a control or 0.1MCD for 4 wk and received 1 or 5 mg/kg/d Aramchol or vehicle by intragastric gavage for the last 2 wk. Liver metabolomics were assessed using ultra-high-performance liquid chromatography-time of flight-MS for the determination of glucose metabolism-related metabolites.

RESULTS

Combination of proteomics and Western blot analyses showed increased AMPK activity while the activity of nutrient sensor mTORC1 was decreased by Aramchol in hepatocytes. This translated into changes in the content of their downstream targets including proteins involved in fatty acid (FA) synthesis and oxidation [P-ACCα/β(S79), SCD1, CPT1A/B, HADHA, and HADHB], oxidative phosphorylation (NDUFA9, NDUFB11, NDUFS1, NDUFV1, ETFDH, and UQCRC2), tricarboxylic acid (TCA) cycle (MDH2, SUCLA2, and SUCLG2), and ribosome (P-p70S6K[T389] and P-S6[S235/S236]). Flux experiments with 13C-uniformely labeled glucose showed that TCA cycle cataplerosis was reduced by Aramchol in hepatocytes, as indicated by the increase in the number of rounds that malate remained in the TCA cycle. Finally, liver metabolomic analysis showed that glucose homeostasis was improved by Aramchol in 0.1MCD fed mice in a dose-dependent manner, showing normalization of glucose, G6P, F6P, UDP-glucose, and Rbl5P/Xyl5P.

CONCLUSION

Aramchol exerts its effect on glucose and lipid metabolism in NASH through activation of AMPK and inhibition of mTORC1, which in turn activate FA β-oxidation and oxidative phosphorylation.

Keywords: Nonalcoholic fatty liver disease; Steatohepatitis; Methionine and choline deficient diet; Tricarboxylic acid cycle; Hemoglobin A1c; Stearoyl-CoA desaturase 1

Core Tip: Arachidyl amido cholanoic acid (Aramchol), a phase III investigational drug for the treatment of nonalcoholic steatohepatitis (NASH), has been demonstrated to be able to reduce liver steatosis, inflammation, and fibrosis in various animal models of steatohepatitis. In a phase IIb clinical trial, it reduced blood levels of glycated hemoglobin A1c, an indicator of glycemic control. In this study, we showed that Aramchol-treated hepatocytes had activated AMPK and inhibited mTORC1, which in turn activated fatty acid β-oxidation and oxidative phosphorylation, inhibiting de novo lipogenesis, gluconeogenesis, and cataplerosis. These results explain the mechanism by which Aramchol exerts its effect on glucose and lipid metabolism in NASH.