Restoring cellular magnesium balance through Cyclin M4 protects against acetaminophen-induced liver damage

Acetaminophen overdose is one of the leading causes of acute liver failure and liver transplantation in the Western world. Magnesium is essential in several cellular processess. The Cyclin M family is involved in magnesium transport across cell membranes. Herein, we identify that among all magnesium transporters, only Cyclin M4 expression is upregulated in the liver of patients with acetaminophen overdose, with disturbances in magnesium serum levels. In the liver, acetaminophen interferes with the mitochondrial magnesium reservoir via Cyclin M4, affecting ATP production and reactive oxygen species generation, further boosting endoplasmic reticulum stress. Importantly, Cyclin M4 mutant T495I, which impairs magnesium flux, shows no effect. Finally, an accumulation of Cyclin M4 in endoplasmic reticulum is shown under hepatoxicity. Based on our studies in mice, silencing hepatic Cyclin M4 within the window of 6 to 24 h following acetaminophen overdose ingestion may represent a therapeutic target for acetaminophen overdose induced liver injury.Acknowledgements: This work was supported by Ministerio de Ciencia, Innovación y Universidades MICINN: PID2020-117116RB-I00 integrado en el Plan Estatal de Investigación Cientifica y Técnica y Innovación, cofinanciado con Fondos FEDER (to MLM-C), Ministerio de Ciencia e Innovación CONSOLIDER-INGENIO 2010 Program Grant CSD2008-00005 (to LAMC); Spanish Ministry of Economy and Competitiveness Grant BFU2013-47531-R, BFU2016-77408-R, PID2019-109055RB-100 (to L.A.M.-C.) (MINECO/FEDER, UE); Asociación Española contra el Cáncer (MLM-C, TC-D), Fundación Científica de la Asociación Española Contra el Cáncer (AECC Scientific Foundation) Rare Tumor Calls 2017 (to M.L.M.-C.), La Caixa Foundation Program (to M.L.M.-C.), Fundacion BBVA UMBRELLA project (to M.L.M.-C.), Ayuda RYC2020-029316-I financiada por MICIN/AEI/10.13039/501100011033 (to TC-D), Plataforma de Investigación Clínica-SCReN (PT17 0017 0020) (to M.I.-L.), programa retos RTC2019-007125-1 (to M.L.M.-C, J.S.), Proyectos Investigacion en Salud DTS20/00138 (to M.L.M.-C., J.S), ERA-Net E-Rare EJP RD Joint Translational Call for Rare Diseases FIGHT-CNNM2 (EJPRD19-040) and from Instituto Carlos III, Spain (REF G95229142) (to L.A.M.-C.), US National Institutes of Health under grant CA217817 (to D.B.), Ciberehd_ISCIII_MINECO is funded by the Instituto de Salud Carlos III. We thankMINECO for the Severo Ochoa Excellence Accreditation to CIC bioGUNE (SEV-2016-0644) and PhD fellowship fromMINECO (REF BES-2017-080435) awarded to I.G.-R. The collection and storage of patients tissues was supported by the Newcastle Biomedicine Biobank and the European Community’s Seventh Framework Programme (FP7/2001–2013) and Cancer Research UK awards Cancer Research UK grants C18342/A23390; C9380/A18084 and C9380/A26813. Finally, we would like to acknowledge Begoña Rodríguez Iruretagoyena for the technical support provided

The lipopolysaccharide-TLR4 axis regulates hepatic glutaminase 1 expression promoting liver ammonia build-up as steatotic liver disease progresses to steatohepatitis

Introduction Ammonia is a pathogenic factor implicated in the progression of metabolic-associated steatotic liver disease (MASLD). The contribution of the glutaminase 1 (GLS) isoform, an enzyme converting glutamine to glutamate and ammonia, to hepatic ammonia build-up and the mechanisms underlying its upregulation in metabolic-associated steatohepatitis (MASH) remain elusive. Methods Multiplex transcriptomics and targeted metabolomics analysis of liver biopsies in dietary mouse models representing the whole spectra of MASLD were carried out to characterize the relevance of hepatic GLS during disease pathological progression. In addition, the acute effect of liver-specific GLS inhibition in hepatic ammonia content was evaluated in cultured hepatocytes and in in vivo mouse models of diet-induced MASLD. Finally, the regulatory mechanisms of hepatic GLS overexpression related to the lipopolysaccharide (LPS)/Toll-like receptor 4 (TLR4) axis were explored in the context of MASH. Results In mouse models of diet-induced MASLD, we found that augmented liver GLS expression is closely associated with the build-up of hepatic ammonia as the disease progresses from steatosis to steatohepatitis. Importantly, the acute silencing/pharmacological inhibition of GLS diminishes the ammonia burden in cultured primary mouse hepatocytes undergoing dedifferentiation, in steatotic hepatocytes, and in a mouse model of diet-induced steatohepatitis, irrespective of changes in ureagenesis and gut permeability. Under these conditions, GLS upregulation in the liver correlates positively with the hepatic expression of TLR4 that recognizes LPS. In agreement, the pharmacological inhibition of TLR4 reduces GLS and hepatic ammonia content in LPS-stimulated mouse hepatocytes and hyperammonemia animal models of endotoxemia. Conclusions Overall, our results suggest that the LPS/TLR4 axis regulates hepatic GLS expression promoting liver ammonia build-up as steatotic liver disease progresses to steatohepatitis.TC Delgado is funded by “Ayuda RYC2020-029316-I financiada por MCIN/AEI/10.13039/501100011033 y por El FSE invierte en tu future”. This work was supported by the Gilead Sciences Research Scholars Program in Global Liver (to TCD), Nanostring® grant (to TCD); grant from Ministerio de Ciencia, Innovación y Universidades (MICINN: PID2022-139395OB-100 integrado en el Plan Estatal de Investigación Científica y Técnica e Innovación, con Fondos FEDER); grants from Ministerio de Ciencia, Innovación y Universidades MICINN: PID2020-117116RB-I00 CEX2021-001136-S integrado en el Plan Estatal de Investigación Científica y Técnica e Innovación, cofinanciado con Fondos FEDER for (MLM-C); Project funded by CIBEREHD; La Caixa Scientific Foundation (HR17-00601) (for MLM-C); ERA-Net E-Rare EJP RD Joint Translational Call for Rare Diseases FIGHT-CNNM2 (EJPRD19-040), and from Instituto Carlos III, Spain (for MLM-C, JH); the Basque Department of Education (IT1739-22) (for JH). JH and TCD are members of the European Reference Network for Rare Hereditary Metabolic Disorders (MetabERN) Project ID No. 739543

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

Restoring cellular magnesium balance through Cyclin M4 protects against acetaminophen-induced liver damage

Acetaminophen overdose is one of the leading causes of acute liver failure and liver transplantation in the Western world. Magnesium is essential in several cellular processess. The Cyclin M family is involved in magnesium transport across cell membranes. Herein, we identify that among all magnesium transporters, only Cyclin M4 expression is upregulated in the liver of patients with acetaminophen overdose, with disturbances in magnesium serum levels. In the liver, acetaminophen interferes with the mitochondrial magnesium reservoir via Cyclin M4, affecting ATP production and reactive oxygen species generation, further boosting endoplasmic reticulum stress. Importantly, Cyclin M4 mutant T495I, which impairs magnesium flux, shows no effect. Finally, an accumulation of Cyclin M4 in endoplasmic reticulum is shown under hepatoxicity. Based on our studies in mice, silencing hepatic Cyclin M4 within the window of 6 to 24 h following acetaminophen overdose ingestion may represent a therapeutic target for acetaminophen overdose induced liver injury

The outcome of boosting mitochondrial activity in alcohol-associated liver disease is organ-dependent.

BACKGROUND AND AIMS Alcohol-associated liver disease (ALD) accounts for 70% of liver-related deaths in Europe, with no effective approved therapies. Although mitochondrial dysfunction is one of the earliest manifestations of alcohol-induced injury, restoring mitochondrial activity remains a problematic strategy due to oxidative stress. Here, we identify methylation-controlled J protein (MCJ) as a mediator for ALD progression and hypothesize that targeting MCJ may help in recovering mitochondrial fitness without collateral oxidative damage. APPROACH AND RESULTS C57BL/6 mice [wild-type (Wt)] Mcj knockout and Mcj liver-specific silencing (MCJ-LSS) underwent the NIAAA dietary protocol (Lieber-DeCarli diet containing 5% (vol/vol) ethanol for 10 days, plus a single binge ethanol feeding at day 11). To evaluate the impact of a restored mitochondrial activity in ALD, the liver, gut, and pancreas were characterized, focusing on lipid metabolism, glucose homeostasis, intestinal permeability, and microbiota composition. MCJ, a protein acting as an endogenous negative regulator of mitochondrial respiration, is downregulated in the early stages of ALD and increases with the severity of the disease. Whole-body deficiency of MCJ is detrimental during ALD because it exacerbates the systemic effects of alcohol abuse through altered intestinal permeability, increased endotoxemia, and dysregulation of pancreatic function, which overall worsens liver injury. On the other hand, liver-specific Mcj silencing prevents main ALD hallmarks, that is, mitochondrial dysfunction, steatosis, inflammation, and oxidative stress, as it restores the NAD + /NADH ratio and SIRT1 function, hence preventing de novo lipogenesis and improving lipid oxidation. CONCLUSIONS Improving mitochondrial respiration by liver-specific Mcj silencing might become a novel therapeutic approach for treating ALD.This work was supported by grants from Ministerio de Ciencia e Innovación, Programa Retos-Colaboración RTC2019-007125-1 (for Jorge Simon and Maria Luz Martinez-Chantar); Ministerio de Economía, Industria y Competitividad, Retos a la Sociedad AGL2017- 86927R (for F.M.); Instituto de Salud Carlos III, Proyectos de Investigación en Salud DTS20/00138 and DTS21/00094 (for Jorge Simon and Maria Luz Martinez-Chantar, and Asis Palazon. respectively); Instituto de Salud Carlos III, Fondo de Investigaciones Sanitarias co-founded by European Regional Development Fund/European Social Fund, “Investing in your future” PI19/00819, “Una manera de hacer Europa” FIS PI20/00765, and PI21/01067 (for Jose J. G. Marin., Pau Sancho-Bru,. and Mario F. Fraga respectively); Departamento de Industria del Gobierno Vasco (for Maria Luz Martinez-Chantar); Asturias Government (PCTI) co-funding 2018-2023/ FEDER IDI/2021/000077 (for Mario F. Fraga.); Ministerio de Ciencia, Innovación y Universidades MICINN: PID2020-117116RB-I00, CEX2021-001136-S PID2020-117941RB-I00, PID2020-11827RB-I00 and PID2019-107956RA-100 integrado en el Plan Estatal de Investigación Científica y Técnica y Innovación, cofinanciado con Fondos FEDER (for Maria Luz Martinez-Chantar, Francisco J Cubero., Yulia A Nevzorova and Asis Palazon); Ayudas Ramón y Cajal de la Agencia Estatal de Investigación RY2013-13666 and RYC2018- 024183-I (for Leticia Abecia and Asis Palazon); European Research Council Starting Grant 804236 NEXTGEN-IO (for Asis Palazon); The German Research Foundation SFB/TRR57/P04, SFB1382-403224013/ A02 and DFG NE 2128/2-1 (for Francisco J Cubero and Yulia A Nevzorova); National Institute of Health (NIH)/National Institute of Alcohol Abuse and Alcoholism (NIAAA) 1U01AA026972-01 (For Pau Sancho-Bru); Junta de Castilla y León SA074P20 (for Jose J. G. Marin); Junta de Andalucía, Grupo PAIDI BIO311 (for Franz Martin); CIBERER Acciones Cooperativas y Complementarias Intramurales ACCI20-35 (for Mario F. Fraga); Ministerio de Educación, Cultura y Deporte FPU17/04992 (for Silvia Ariño); Fundació Marato TV3 201916-31 (for Jose J. G. Marin.); Ainize Pena-Cearra is a fellow of the University of the Basque Country (UPV/ EHU); BIOEF (Basque Foundation for Innovation and Health Research); Asociación Española contra el Cáncer (Maria Luz Martinez-Chantar and Teresa C. Delgado.); Fundación Científica de la Asociación Española Contra el Cáncer (AECC Scientific Foundation) Rare Tumor Calls 2017 (for Maria Luz Martinez-Chantar); La Caixa Foundation Program (for Maria Luz Martinez-Chantar); Proyecto Desarrollo Tecnologico CIBERehd (for Maria Luz Martinez-Chantar); Ciberehd_ISCIII_MINECO is funded by the Instituto de Salud Carlos III.S

Effect of Gracilaria vermiculophylla Macroalga on Non-Alcoholic Fatty Liver Disease in Obese Rats

Marine algae are valuable sources of bioactive compounds that have the potential to be used in the management of various pathologies. Despite the increasing prevalence of NAFLD, the absence of an approved effective pharmacological treatment with demonstrable effectiveness persists. In this context, the aim of the present study is to assess the effect of Gracilaria vermiculophylla red seaweed dietary supplementation on hepatic lipid accumulation, as well as on oxidative stress, inflammation and fibrosis- related markers on obese fa/fa Zucker rats fed with a standard diet, supplemented or not with 2.5% or 5% dehydrated Gracilaria vermiculophylla. After a six-week supplementation with the macroalga, no significant reduction in hepatic total lipid content or hepatic triglyceride content was observed. However, both doses were able to diminish hepatic NEFA concentration by reducing de novo lipogenesis and increasing mitochondrial biogenesis. Moreover, supplementation with the dose of 2.5% improved some oxidative stress and inflammation-related markers. Supplementation with the dose of 5% did not exert these clear beneficial effects. Thus, this study demonstrates that while Gracilaria vermiculophylla may not mitigate hepatic steatosis, it could exert protective effects on the liver by reducing NEFA content and enhancing oxidative stress and inflammation parameters.This study was supported by Instituto de Salud Carlos III (CIBERobn) under Grant CB12/03/30007, the Government of the Basque Country under Grants KK-2019-00031 and IT1482-22

Anti-miR-873-5p improves alcohol-related liver disease by enhancing hepatic deacetylation via SIRT1

Background & Aims: Current therapies for the treatment of alcohol-related liver disease (ALD) have proven largely ineffective. Patients relapse and the disease progresses even after liver transplantation. Altered epigenetic mechanisms are characteristic of alcohol metabolism given excessive acetate and NAD depletion and play an important role in liver injury. In this regard, novel therapeutic approaches based on epigenetic modulators are increasingly proposed. MicroRNAs, epigenetic modulators acting at the post-transcriptional level, appear to be promising new targets for the treatment of ALD. Methods: MiR-873-5p levels were measured in 23 liver tissue from Patients with ALD, and GNMT levels during ALD were confirmed using expression databases (transcriptome n = 62, proteome n = 68). High-resolution proteomics and metabolomics in mice following the Gao-binge model were used to investigate miR-873-5p expression in ALD. Hepatocytes exposed to 50 mM alcohol for 12 h were used to study toxicity. The effect of anti-miR-873-5p in the treatment outcomes of ALD was investigated. Results: The analysis of human and preclinical ALD samples revealed increased expression of miR-873-5p in the liver. Interestingly, there was an inverse correlation with NNMT, suggesting a novel mechanism for NAD depletion and aberrant acetylation during ALD progression. High-resolution proteomics and metabolomics identified miR-873-5p as a key regulator of NAD metabolism and SIRT1 deacetylase activity. Anti-miR-873-5p reduced NNMT activity, fuelled the NAD salvage pathway, restored the acetylome, and modulated the levels of NF-κB and FXR, two known SIRT1 substrates, thereby protecting the liver from apoptotic and inflammatory processes, and improving bile acid homeostasis. Conclusions: These data indicate that targeting miR-873-5p, a repressor of GNMT previously associated with NAFLD and acetaminophen-induced liver failure. is a novel and attractive approach to treating alcohol-induced hepatoxicity. Impact and implications: The role of miR-873-5p has not been explicitly examined in the progression of ALD, a pathology with no therapeutic options. In this study, inhibiting miR-873-5p exerted hepatoprotective effects against ALD through rescued SIRT1 activity and consequently restored bile acid homeostasis and attenuated the inflammatory response. Targeting hepatic miR-873-5p may represent a novel therapeutic approach for the treatment of ALD

Mitochondrial bioenergetics boost macrophage activation, promoting liver regeneration in metabolically compromised animals

17 páginas, 6 figurasBackground and aims: Hepatic ischemia-reperfusion injury (IRI) is the leading cause of early posttransplantation organ failure as mitochondrial respiration and ATP production are affected. A shortage of donors has extended liver donor criteria, including aged or steatotic livers, which are more susceptible to IRI. Given the lack of an effective treatment and the extensive transplantation waitlist, we aimed at characterizing the effects of an accelerated mitochondrial activity by silencing methylation-controlled J protein (MCJ) in three preclinical models of IRI and liver regeneration, focusing on metabolically compromised animal models. Approach and results: Wild-type (WT), MCJ knockout (KO), and Mcj silenced WT mice were subjected to 70% partial hepatectomy (Phx), prolonged IRI, and 70% Phx with IRI. Old and young mice with metabolic syndrome were also subjected to these procedures. Expression of MCJ, an endogenous negative regulator of mitochondrial respiration, increases in preclinical models of Phx with or without vascular occlusion and in donor livers. Mice lacking MCJ initiate liver regeneration 12 h faster than WT and show reduced ischemic injury and increased survival. MCJ knockdown enables a mitochondrial adaptation that restores the bioenergetic supply for enhanced regeneration and prevents cell death after IRI. Mechanistically, increased ATP secretion facilitates the early activation of Kupffer cells and production of TNF, IL-6, and heparin-binding EGF, accelerating the priming phase and the progression through G1 /S transition during liver regeneration. Therapeutic silencing of MCJ in 15-month-old mice and in mice fed a high-fat/high-fructose diet for 12 weeks improves mitochondrial respiration, reduces steatosis, and overcomes regenerative limitations. Conclusions: Boosting mitochondrial activity by silencing MCJ could pave the way for a protective approach after major liver resection or IRI, especially in metabolically compromised, IRI-susceptible organs.Peer reviewe