Boosting mitochondria activity by silencing MCJ overcomes cholestasis-induced liver injury

Background & Aims: Mitochondria are the major organelles for the formation of reactive oxygen species (ROS) in the cell, and mitochondrial dysfunction has been described as a key factor in the pathogenesis of cholestatic liver disease. The methylation-controlled J-protein (MCJ) is a mitochondrial protein that interacts with and represses the function of complex I of the electron transport chain. The relevance of MCJ in the pathology of cholestasis has not yet been explored. Methods: We studied the relationship between MCJ and cholestasis-induced liver injury in liver biopsies from patients with chronic cholestatic liver diseases, and in livers and primary hepatocytes obtained from WT and MCJ-KO mice. Bile duct ligation (BDL) was used as an animal model of cholestasis, and primary hepatocytes were treated with toxic doses of bile acids. We evaluated the effect of MCJ silencing for the treatment of cholestasis-induced liver injury. Results: Elevated levels of MCJ were detected in the liver tissue of patients with chronic cholestatic liver disease when compared with normal liver tissue. Likewise, in mouse models, the hepatic levels of MCJ were increased. After BDL, MCJ-KO animals showed significantly decreased inflammation and apoptosis. In an in vitro model of bile-acid induced toxicity, we observed that the loss of MCJ protected mouse primary hepatocytes from bile acid-induced mitochondrial ROS over-production and ATP depletion, enabling higher cell viability. Finally, the in vivo inhibition of the MCJ expression, following BDL, showed reduced liver injury and a mitigation of the main cholestatic characteristics. Conclusions: We demonstrated that MCJ is involved in the progression of cholestatic liver injury, and our results identified MCJ as a potential therapeutic target to mitigate the liver injury caused by cholestasis. Lay summary: In this study, we examine the effect of mitochondrial respiratory chain inhibition by MCJ on bile acid-induced liver toxicity. The loss of MCJ protects hepatocytes against apoptosis, mitochondrial ROS overproduction, and ATP depletion as a result of bile acid toxicity. Our results identify MCJ as a potential therapeutic target to mitigate liver injury in cholestatic liver diseases.Acknowledgements: We thank MINECO for the Severo Ochoa Excellence Accreditation of CIC bioGUNE [SEV-2016-0644]

Arachidyl Amido Cholanoic Acid Improves Liver Glucose and Lipid Homeostasis in Nonalcoholic Steatohepatitis Via AMPK and mTOR Regulation

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 mu mol/L Aramchol or vehicle for 48 h. Subsequently, analyses were performed including Western blot, proteomics by mass spectrometry, and fluxomic analysis with(13)C-uniformly labeled glucose. For thein vivopart 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-ACC alpha/beta(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(13)C-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 beta-oxidation and oxidative phosphorylation.Supported by the National Institutes of Health Grant, No. R01CA172086; Plan Nacional of I+D, No. SAF2017-88041-R; Ministerio de Economia y Competitividad de Espana, No. SAF2017-87301-R; Asociacion Espanola contra el Cancer, No. AECC17/302; Ayudas Fundacion BBVA a equipos de Investigacion Cientifica 2018; Fondo Europeo de Desarrollo Regional, Ministerio de Economia y Competitividad de Espana, No. PGC2018-099857-BI00; Basque Government Grants, No. IT1264-19; Ministerio de Economia y Competitividad de Espana for the Severo Ochoa Excellence Accreditation, No. SEV2016-0644. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Stabilization of LKB1 and Akt by neddylation regulates energy metabolism in liver cancer

The current view of cancer progression highlights that cancer cells must undergo through a post-translational regulation and metabolic reprogramming to progress in an unfriendly environment. In here, the importance of neddylation modification in liver cancer was investigated. We found that hepatic neddylation was specifically enriched in liver cancer patients with bad prognosis. In addition, the treatment with the neddylation inhibitor MLN4924 in Phb1-KO mice, an animal model of hepatocellular carcinoma showing elevated neddylation, reverted the malignant phenotype. Tumor cell death in vivo translating into liver tumor regression was associated with augmented phosphatidylcholine synthesis by the PEMT pathway, known as a liver-specific tumor suppressor, and restored mitochondrial function and TCA cycle flux. Otherwise, in protumoral hepatocytes, neddylation inhibition resulted in metabolic reprogramming rendering a decrease in oxidative phosphorylation and concomitant tumor cell apoptosis. Moreover, Akt and LKB1, hallmarks of proliferative metabolism, were altered in liver cancer being new targets of neddylation. Importantly, we show that neddylation-induced metabolic reprogramming and apoptosis were dependent on LKB1 and Akt stabilization. Overall, our results implicate neddylation/signaling/metabolism, partly mediated by LKB1 and Akt, in the development of liver cancer, paving the way for novel therapeutic approaches targeting neddylation in hepatocellular carcinoma

Stabilization of LKB1 and Akt by neddylation regulates energy metabolism in liver cancer

The current view of cancer progression highlights that cancer cells must undergo through a post-translational regulation and metabolic reprogramming to progress in an unfriendly environment. In here, the importance of neddylation modification in liver cancer was investigated. We found that hepatic neddylation was specifically enriched in liver cancer patients with bad prognosis. In addition, the treatment with the neddylation inhibitor MLN4924 in Phb1-KO mice, an animal model of hepatocellular carcinoma showing elevated neddylation, reverted the malignant phenotype. Tumor cell death in vivo translating into liver tumor regression was associated with augmented phosphatidylcholine synthesis by the PEMT pathway, known as a liver-specific tumor suppressor, and restored mitochondrial function and TCA cycle flux. Otherwise, in protumoral hepatocytes, neddylation inhibition resulted in metabolic reprogramming rendering a decrease in oxidative phosphorylation and concomitant tumor cell apoptosis. Moreover, Akt and LKB1, hallmarks of proliferative metabolism, were altered in liver cancer being new targets of neddylation. Importantly, we show that neddylation-induced metabolic reprogramming and apoptosis were dependent on LKB1 and Akt stabilization. Overall, our results implicate neddylation/signaling/metabolism, partly mediated by LKB1 and Akt, in the development of liver cancer, paving the way for novel therapeutic approaches targeting neddylation in hepatocellular carcinoma

miR-873-5p targets mitochondrial GNMT-Complex II interface contributing to non-alcoholic fatty liver disease

Objective: Non-alcoholic fatly liver disease (NAFLD) is a complex pathology in which several dysfunctions, including alterations in metabolic pathways, mitochondrial functionality and unbalanced lipid import/export, lead to lipid accumulation and progression to inflammation and fibrosis. The enzyme glycine N-methyltransferase (GNMT), the most important enzyme implicated in S-adenosylmethionine catabolism in the liver, is downregulated during NAFLD progression. We have studied the mechanism involved in GNMT downregulation by its repressor microRNA miR-873-5p and the metabolic pathways affected in NAFLD as well as the benefit of recovery GNMT expression. Methods: miR-873-5p and GNMT expression were evaluated in liver biopsies of NAFLD/NASH patients. Different in vitro and in vivo NAFLD murine models were used to assess miR-873-5p/GNMT involvement in fatty liver progression through targeting of the miR-873-5p as NAFLD therapy. Results: We describe a new function of GNMT as an essential regulator of Complex II activity in the electron transport chain in the mitochondria. In NAFLD, GNMT expression is controlled by miR-873-5p in the hepatocytes, leading to disruptions in mitochondria! functionality in a preclinical murine non-alcoholic steatohepatitis (NASH) model. Upregulation of miR-873-5p is shown in the liver of NAFLD/NASH patients, correlating with hepatic GNMT depletion. Importantly, NASH therapies based on anti-miR-873-5p resolve lipid accumulation, inflammation and fibrosis by enhancing fatty acid beta-oxidation in the mitochondria. Therefore, miR-873-5p inhibitor emerges as a potential tool for NASH treatment. Conclusion: GNMT participates in the regulation of metabolic pathways and mitochondria! functionality through the regulation of Complex II activity in the electron transport chain. In NAFLD, GNMT is repressed by miR-873-5p and its targeting arises as a valuable therapeutic option for treatment. (C) 2019 The Authors. Published by Elsevier GmbH.This work was supported by grants from NIH (US Department of Health and Human services)-R01AT001576 (to S.C.L., J.M.M., and M.L.M.-C.), Ministerio de Economia, Industria y Competitividad: SAF2017-87301-R (to M.L.M.-C.), SAF2015-64352-R (to P.A.), Gobierno Vasco-Departamento de Salud 2013111114 (to M.L.M.-C.), Gobierno Vasco-Departamento de Educacion IT-336-10 (to PA), BIOEF (Basque Foundation for Innovation and Health Research: EiTB Maratoia BIO15/CA/016/BD (M.L.M.-C.), ELKARTEK 2016, Departamento de Industria del Gobierno Vasco (to M.L.M.-C), Asociacion Espanola contra el Cancer (to T.C.D., P.F.-T. and M.L.M.-C.), Mitotherapeutix (to M.L.M.-C.), Daniel Alagille award from EASL (to T.C.D), Fundacion Cientifica de la Asociacion Espanola Contra el Cancer (AECC Scientific Foundation) Rare Tumor Calls 2017 (to M.L.M.-C.), La Caixa Foundation Program (to M.L.M.-C.), Ayudas Fundacion BBVA a Equipos de Investigacion Cientifica 2019 (to M.L.M.-C.). Ciberehd_ISCIII_MINECO is funded by the Instituto de Salud Carlos III. We thank this work produced with the support of a 2017 Leonardo Grant for Researchers and Cultural Creators, BBVA Foundation (to M.V.R.). This work was supported by Fonds National de la Recherche Luxembourg and the Deutsche Forschungsgemeinschaft (C12/BM/3975937, FL/997/7-1, Inter "HepmiRSTAT", to I.B. and F.L.). We thank MINECO for the Severo Ochoa Excellence Accreditation (SEV2016-0644)

miR-873-5p targets mitochondrialGNMT-Complex II interface contributing tonon-alcoholic fatty liver disease

Objective:Non-alcoholic fatty liver disease (NAFLD) is a complex pathology in which several dysfunctions, including alterations in metabolicpathways, mitochondrial functionality and unbalanced lipid import/export, lead to lipid accumulation and progression to inflammation andfibrosis.The enzyme glycine N-methyltransferase (GNMT), the most important enzyme implicated in S-adenosylmethionine catabolism in the liver, isdownregulated during NAFLD progression. We have studied the mechanism involved in GNMT downregulation by its repressor microRNA miR-873-5p and the metabolic pathways affected in NAFLD as well as the benefit of recovery GNMT expression.Methods:miR-873-5p and GNMT expression were evaluated in liver biopsies of NAFLD/NASH patients. Differentin vitroandin vivoNAFLD murinemodels were used to assess miR-873-5p/GNMT involvement in fatty liver progression through targeting of the miR-873-5p as NAFLD therapy.Results:We describe a new function of GNMT as an essential regulator of Complex II activity in the electron transport chain in the mitochondria.In NAFLD, GNMT expression is controlled by miR-873-5p in the hepatocytes, leading to disruptions in mitochondrial functionality in a preclinicalmurine non-alcoholic steatohepatitis (NASH) model. Upregulation of miR-873-5p is shown in the liver of NAFLD/NASH patients, correlating withhepatic GNMT depletion. Importantly, NASH therapies based on anti-miR-873-5p resolve lipid accumulation, inflammation andfibrosis byenhancing fatty acidb-oxidation in the mitochondria. Therefore, miR-873-5p inhibitor emerges as a potential tool for NASH treatment.Conclusion:GNMT participates in the regulation of metabolic pathways and mitochondrial functionality through the regulation of Complex II activityin the electron transport chain. In NAFLD, GNMT is repressed by miR-873-5p and its targeting arises as a valuable therapeutic option for treatment

Neddylation inhibition ameliorates steatosis in NAFLD by boosting hepatic fatty acid oxidation via the DEPTOR-mTOR axis

Objective: Neddylation is a druggable and reversible ubiquitin-like post-translational modification upregulated in many diseases, including liver fibrosis, hepatocellular carcinoma, and more recently, non-alcoholic fatty liver disease (NAFLD). Herein, we propose to address the effects of neddylation inhibition and the underlying mechanisms in pre-clinical models of NAFLD. Methods: Hepatic neddylation measured by immunohistochemical analysis and NEDD8 serum levels measured by ELISA assay were evaluated in NAFLD clinical and pre-clinical samples. The effects of neddylation inhibition by using a pharmacological small inhibitor, MLN4924, or molecular approaches were assessed in isolated mouse hepatocytes and pre-clinical mouse models of diet-induced NAFLD, male adult C57BL/6 mice, and the AlfpCre transgenic mice infected with AAV-DIO-shNedd8. Results: Neddylation inhibition reduced lipid accumulation in oleic acid-stimulated mouse primary hepatocytes and ameliorated liver steatosis, preventing lipid peroxidation and inflammation in the mouse models of diet-induced NAFLD. Under these conditions, increased Deptor levels and the concomitant repression of mTOR signaling were associated with augmented fatty acid oxidation and reduced lipid content. Moreover, Deptor silencing in isolated mouse hepatocytes abolished the anti-steatotic effects mediated by neddylation inhibition. Finally, serum NEDD8 levels correlated with hepatic neddylation during the disease progression in the clinical and pre-clinical models. Conclusions: Overall, the upregulation of Deptor, driven by neddylation inhibition, is proposed as a novel effective target and therapeutic approach to tackle NAFLDThis work was supported by grants from Gobierno Vasco-Departamento de Salud 2013111114 (to M.L.M.-C), ELKARTEK 2016, Departamento de Industria del Gobierno Vasco (to M.L.M−C), Ministerio de Ciencia, Innovación y Universidades MICINN: SAF2017-87301-R, and RTI2018-096759-A-100 integrado en el Plan Estatal de Investigación Científica y Técnica y Innovación, cofinanciado con Fondos FEDER (to M.L.M− T.C.D respectively); MCIU/AEI/FEDER, UE (RTI2018-095134-B-100) (to P.A.), AECC Bizkaia (M.S-M); Asociación Española contra el Cáncer (T.C.D), Fundación Científica de la Asociación Española Contra el Cáncer (AECC Scientific Foundation) Rare Tumor Calls 2017 (to M.L.M, J.M.B., M.A.A., J.J.G.M.), La Caixa Foundation Program (to M.L.M and J.M.B.), 2018 BBVA Foundation Grants for Scientific Research Teams (to M.L.M.-C.), Ayudas para apoyar grupos de investigación del sistema Universitario Vasco IT971-16 (P.A.). MyFirst Grant AIRC n.16888, Ricerca Finalizzata Ministero della Salute RF-2016-02364358, Ricerca corrente Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico (to LV), the European Union (EU) Programme Horizon 2020 (under grant agreement No. 777377) for the project LITMUS- “Liver Investigation: Testing Marker Utility in Steatohepatitis” (to LV), Fondazione IRCCS Ca’ Granda “Liver BIBLE” PR-0391, Fondazione IRCCS Ca’ Granda core COVID-19 Biobank (RC100017A) (to LV). This research was funded by the CIBERehd (EHD15PI05/2016) and “Fondo de Investigaciones Sanitarias, Instituto de Salud Carlos III”, Spain (PI16/00598 and PI19/00819, co-funded by European Regional Development Fund/European Social Fund, “Investing in your future”); Spanish Ministry of Economy, Industry and Competitiveness (SAF2016-75197-R); “Junta de Castilla y Leon” (SA063P17); AECC Scientific Foundation (2017/2020), Spain; “Centro Internacional sobre el Envejecimiento” (OLD-HEPAMARKER, 0348_CIE_6_E), Spain; University of Salamanca Foundation, Spain (PC-TCUE18-20_051), and Fundació Marato TV3 (Ref. 201916–31), Spain. RB acknowledges BFU2017-84653-P (MINECO/FEDER, EU), SEV-2016-0644 (Severo Ochoa Excellence Program), 765445-EU (UbiCODE Program), SAF2017-90900-REDT (UBIRed Program), and IT1165-19 (Basque Country Government). Ciberehd_ISCIII_MINECO is funded by the Instituto de Salud Carlos III. We thank MINECO for the Severo Ochoa Excellence Accreditation to CIC bioGUNE (SEV-2016-0644)S

Methionine Cycle Rewiring by Targeting miR-873-5p Modulates Ammonia Metabolism to Protect the Liver from Acetaminophen

Drug-induced liver injury (DILI) development is commonly associated with acetaminophen (APAP) overdose, where glutathione scavenging leads to mitochondrial dysfunction and hepatocyte death. DILI is a severe disorder without effective late-stage treatment, since N-acetyl cysteine must be administered 8 h after overdose to be efficient. Ammonia homeostasis is altered during liver diseases and, during DILI, it is accompanied by decreased glycine N-methyltransferase (GNMT) expression and S-adenosylmethionine (AdoMet) levels that suggest a reduced methionine cycle. Anti-miR-873-5p treatment prevents cell death in primary hepatocytes and the appearance of necrotic areas in liver from APAP-administered mice. In our study, we demonstrate a GNMT and methionine cycle activity restoration by the anti-miR-873-5p that reduces mitochondrial dysfunction and oxidative stress. The lack of hyperammoniemia caused by the therapy results in a decreased urea cycle, enhancing the synthesis of polyamines from ornithine and AdoMet and thus impacting the observed recovery of mitochondria and hepatocyte proliferation for regeneration. In summary, anti-miR-873-5p appears to be an effective therapy against APAP-induced liver injury, where the restoration of GNMT and the methionine cycle may prevent mitochondrial dysfunction while activating hepatocyte proliferative response.We thank Ministerio de Ciencia e Innovación, Programa Retos-Colaboración RTC2019- 007125-1 (for J.S. and M.L.M.-C.); Instituto de Salud Carlos III: Proyectos de Investigación en Salud DTS20/00138 (for J.S. and M.L.M.-C.), PI20/00690 (for R.J.) and PT20/000127 (for M.I.L.); CIBERehd: EHD21TRF01/2022 (to M.L.M.-C.); Departamento de Industria del Gobierno Vasco (for M.L.M.-C.); Ministerio de Ciencia, Innovación y Universidades MICINN: PID2020-117116RB-I00 and RTI2018- 096759-1-100 integrado en el Plan Estatal de Investigación Cientifica y Técnica y Innovación, cofinanciado con Fondos FEDER (for M.L.M.-C. and T.C.D., respectively); BIOEF (Basque Foundation for Innovation and Health Research); Asociación Española contra el Cáncer (AECC) (to M.L.M.-C., T.C.D.); AECC: GCTRA18006CARR (to A.C.); Fundación Científica de la Asociación Española Contra el Cancer (AECC Scientific Foundation) Rare Tumor Calls 2017 (for M.L.M.); La Caixa Foundation Program (for M.L.M.); BFU2015-70067-REDC, BFU2016-77408-R and BES-2017-080435 (MINECO/FEDER, UE); Ministerio de Ciencia, Innovación y universidades PID2019-108787RB-100 (to A.C.), PID2019- 109055RB-I00 (L.A.M.-C.), PID2020-117941RB-100 (to F.J.C.); Spanish Ministry of Economy and Competitiveness Grants BFU2013-47531-R and BFU2016-77408-R (L.A.M.-C.) and the FIGHT-CNNM2 project from the EJP RD Joint Transnational Call (JTC2019) (Ref. AC19/00073) (for L.A.M.-C.); Comunidad de Madrid: EXOHEP-CM S2017/BMD-3727 and NanoLiver-CM Y2018/NMT-4949 co-funded by European Structural and Investment Fund and COST Action CA17112 (to F.J.C.); Vencer el Cáncer Foundation (to A.C.); European Research Council: Consolidator Grant 819242 (to A.C.); CIBERONC and CIBERehd were funded by the Instituto de Salud Carlos III and Cofunded by FEDER funds. Partial funding for open access charge: Universidad de Málag