Targeting hepatic Glutaminase 1 ameliorates N o n - A l c o h o l i c Steatohepatitis b y r e s t o r i n g V e r y - L o w Density Lipoproteins triglyceride assembly J. SIMON1, M. NUÑEZ-GARCIA2, P. FERNÁNDEZ-TUSSY1, L. BARBIER-TORRES1, D. FERNÁNDEZ-RAMOS1, B. GÓMEZ-SANTOS2, X. BUQUE2,3, F. LOPITZ 1 1 1 1 1 1 OTSOA , N. GOIKOETXEA-USANDIZAGA , M. SERRANO-MACIÁ , R. RODRÍGUEZ-AGUDO , M. BIZKARGUENAGA , I. ZUBIETE-FRANCO , V. GUTIÉRREZ- DE JUAN1, D. CABRERA4, C. ALONSO5, P. IRUZUBIETA6,7, M. ROMERO-GOMEZ8, S. VAN LIEMPD4, A. CASTRO5, R. NOGUEIRAS9, M. VARELA-REY1, JUAN MANUEL FALCON-PEREZ10, E. VILLA11, J. CRESPO6,7, SC. LU12, JM. MATO1, P. ASPICHUETA2,3, T. CARDOSO-DELGADO1, ML. MARTINEZ-CHANTAR

1Liver Disease Laboratory, Liver Metabolism Laboratory, CIC bioGUNE, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), 48160 , Bizkaia,.; 2Department of Physiology, Faculty of Medicine and Nursing, University of the Basque Country, 48940 , Bizkaia, Spain; 3Biocruces Health Research Institute, 48903 , Bizkaia, Spain; 4Metabolomics Platform, CIC bioGUNE, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), 48160 Derio, Bizkaia, Spain.; 5Owl Metabolomics, 48610, Derio, Spain; 6Gastroenterology and Hepatology Department, Marqués de Valdecilla University Hospital, 39008 Santander, Spain; 7Infection, Immunity and Digestive Pathology Group, Research Institute Marqués de Valdecilla (IDIVAL), 39011 Santander, Spain; 8Unit for the Clinical Management of Digestive Diseases, Hospital Universitario Virgen del Rocío, CIBERehd, University of , 41013 Seville, Spain; 9Department of Physiology, CIMUS, University of - Instituto de Investigación Sanitaria, CIBER Fisiopatología de la Obesidad y Nutrición (CIBERobn), Santiago de Compostela 15782, Spain; 10Ikerbasque, Basque Foundation for Science, 48013 , Bizkaia, Spain.; 11Department of Gastroenterology, Azienda Ospedaliero- Universitaria & University of Modena and Reggio Emilia, Modena, Italy. 12Division of Digestive and Liver Diseases, Cedars-Sinai Medical Center, Los Angeles, CA, USA.

INTRODUCTION GLS1 UPREGULATION IN NASH Glutamine/glutamate ratio in serum from NASH patients from a previous study (Barr et al., 2012) showed a decrease compared to healthy patients. We have determined GLS1 Glucose GLUTAMINE (Gln) and GLS2 content by IHC and qPCR observing an upregulation of GLS1 in NASH both at Gln is the most abundant aminoacid (20% in blood, 40% in muscle) and Pyruvate protein and mRNA levels (data not shown). GLS2 expression was decreased the main proton donnor in the organism α-KG NH3

TCA Glutaminase (GLS) (EC 3.5.1.2) catabolizes Gln and releasing ammonia at: presented Poster Glu Gln Acetyl-CoA cycle o There are 2 different issoforms: glutaminase 2 (GLS2) and the GLS1 higher affinity glutaminase 1 (GLS1) o GLS1 expression has been shown to be upregulated in liver cancer (Yuneva et al., 2012)

IN THE PRESENT WORK

Herein, we have investigated the role of GLS1 in NASH In the present work we have characterized the GLS1 expression in our animal models too, 0.1%MCDD and CD-HFD too. Similarly as in NASH We have used 0.1% methionine and choline deficient diet (MCDD)-fed mice, an animal model where NASH is mainly caused by a patients, GLS1 is increased at protein and mRNA levels. disruption in VLDL assembly, and choline-deficient high-fat diet (CD-HFD), an animal model in which NASH is followed by weight gain, dyslipidemia and insulin resistance

TARGETING GLS1 RESOLVES NASH OXIDATIVE RATE IS DECREASED

The activity of several oxidative pathways was measured: acid soluble metabolites (ASM) production for fatty acid oxidation, labelled citrate from glutamine as an indicator of the TCA cycle activity and oxygen consumption rate (OCR) for ETC activity. In all indicators the NASH-induced increase is reverted as a consequence of the A precilinical study was carried out where GLS1 In 0.1%MCDD and CD-HFD the increased lipid GLS1-specific silencing. These results was specifically sillencing through tail vein acccumulation was partially reverted when GLS1 is suggest that the reduced oxidative stress is injection. Both lipid accumulation and downregulated. Oxidative stress was also reduced a consequence of the oxidative activity. malonilaldehyde production as indicator of so NASH phenotype is ameliorated as a oxidative stress were measured. consequence of the treatment.

VLDLVLDL EXPORT EXPORT IS IS IMPROVED IMPROVED CONCLUDING REMARKS CONTACT INFORMATION - GLS1 is upregulated at protein and mRNA levels in NASH patients, in vivo animal models and in vitro primary hepatocytes. Address: Liver Disease Laboratory,Metabolomics Unit, - Liver-specific GLS1 silencing reduces lipid accumulation and CIC bioGUNE oxidative stress in in vitro and in vivo pre-clinical assay. - Reduced lipid accumulation is not due to an increased lipid Phone number: 944 061 304

oxidation. Decreased oxidative activiy may be the cause of the Metabolism, alcohol and toxicity Teresa Cardoso Delgado As a consequence of GLS1 silencing, liver phospholipid observed oxidative stress reduction. E-mail: [email protected] content is restored (data not shown). An in vivo approach LinkedIn: Jorge Simón inhibiting VLDL uptake by poloxamer-407 allowed to isolate - As a consequence of GLS1 silencing secreted VLDL content is and analyze secreted lipoprotein particles. As observed, enriched in phospholipids and intracelular lipids. This might the the VLDL phospholipid and intracelular lipid content is restored. mechanism by which lipid accumulation is reduced in liver. This suggest an enrichment of secreted VLDL.

P01-17

NAFLD2019