Nutraceutical Properties of Polyphenols against Liver Diseases

Current food tendencies, suboptimal dietary habits and a sedentary lifestyle are spreading metabolic disorders worldwide. Consequently, the prevalence of liver pathologies is increasing, as it is the main metabolic organ in the body. Chronic liver diseases, with non-alcoholic fatty liver disease (NAFLD) as the main cause, have an alarming prevalence of around 25% worldwide. Otherwise, the consumption of certain drugs leads to an acute liver failure (ALF), with drug-induced liver injury (DILI) as its main cause, or alcoholic liver disease (ALD). Although programs carried out by authorities are focused on improving dietary habits and lifestyle, the long-term compliance of the patient makes them difficult to follow. Thus, the supplementation with certain substances may represent a more easy-to-follow approach for patients. In this context, the consumption of polyphenol-rich food represents an attractive alternative as these compounds have been characterized to be effective in ameliorating liver pathologies. Despite of their structural diversity, certain similar characteristics allow to classify polyphenols in 5 groups: stilbenes, flavonoids, phenolic acids, lignans and curcuminoids. Herein, we have identified the most relevant compounds in each group and characterized their main sources. By this, authorities should encourage the consumption of polyphenol-rich products, as most of them are available in quotidian life, which might reduce the socioeconomical burden of liver diseases.We thank University of Basque Country (UPV/EHU), Basque Government and Asociacion Espanola Contra el Cancer (AECC) for the Pre-doctoral grants to M.C.-A., N.G.-U. and M.S.-M., respectively. 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)

Understanding gut-liver axis nitrogen metabolism in Fatty Liver Disease

The homeostasis of the most important nitrogen-containing intermediates, ammonia and glutamine, is a tightly regulated process in which the gut-liver axis plays a central role. Several studies revealed that nitrogen metabolism is altered in Metabolic Dysfunction-Associated Fatty Liver Disease (MAFLD), a consensus-driven novel nomenclature for Non-Alcoholic Fatty Liver Disease (NAFLD), the most common chronic liver disease worldwide. Both increased ammonia production by gut microbiota and decreased ammonia hepatic removal due to impaired hepatic urea cycle activity or disrupted glutamine synthetase activity may contribute to hepatic ammonia accumulation underlying steatosis, which can eventually progress to hyperammonemia in more advanced stages of steatohepatitis and overt liver fibrosis. Furthermore, our group recently showed that augmented hepatic ammoniagenesis via increased glutaminase activity and overexpression of the high activity glutaminase 1 isoenzyme occurs in Fatty Liver Disease. Overall, the improved knowledge of disrupted nitrogen metabolism and metabolic miscommunication between the gut and the liver suggests that the reestablishment of altered gut-liver axis nitrogenous balance is an appealing and attractive therapeutic approach to tackle Fatty Liver Disease, a growing and unmet health problem.This work was supported by grants from Ministerio de Ciencia, Innovacion y Universidades (MICINN) PID2020-117116RB-I00 integrated in Plan Estatal de Investigacion Cientifica y Tecnica y Innovacion (to MLM-C), and co-funded with Fondos FEDER (to MLM-C), Subprograma Retos Colaboracion RTC2019-007125-1 (to MLM-C), La Caixa Foundation Program HR17-00601 (to MLM-C), Proyectos Investigacion en Salud DTS20/00138 (to MLM-C), Departamento de Industria del Gobierno Vasco (to MLM-C), and Ciberehd_ISCIII_MINECO, which is funded by the Instituto de Salud Carlos III. This work was partially funded by the Basque Department of Education (IT1281-19) (JdH), "Investigador AECC" (INVES18050CARD) and Ayuda RYC2020-029316-I financiada por MCIN/AEI/10.13039/501100011033 y por El FSE invierte en tu futuro (to TCD)

Hu Antigen R (HuR) Protein Structure, Function and Regulation in Hepatobiliary Tumors

Hepatobiliary tumors are a group of primary malignancies encompassing the liver, the intra- and extra-hepatic biliary tracts, and the gall bladder. Within the liver, hepatocellular carcinoma (HCC) is the most common type of primary cancer, which is, also, representing the third-most recurrent cause of cancer-associated death and the sixth-most prevalent type of tumor worldwide, nowadays. Although less frequent, cholangiocarcinoma (CCA) is, currently, a fatal cancer with limited therapeutic options. Here, we review the regulatory role of Hu antigen R (HuR), a ubiquitous member of the ELAV/Hu family of RNA-binding proteins (RBPs), in the pathogenesis, progression, and treatment of HCC and CCA. Overall, HuR is proposed as a valuable diagnostic and prognostic marker, as well as a therapeutic target in hepatobiliary cancers. Therefore, novel therapeutic approaches that can selectively modulate HuR function appear to be highly attractive for the clinical management of these types of tumors.España Ministerio de Ciencia, Innovación y Universidades (MICINN), integrated in the Plan Estatal de Investigación Científica y Técnica e Innovación, grant numbers PID2020-117116RB-I00 (to M.L.M.-C.) and PGC2018-096049-B-I00Gobierno de Andalucía, grant numbers BIO-198, US-1254317, US-1257019, P18-FR-3487, and P18-HO-4091Fondo Europeo de Desarrollo Regional (FEDER) (to MLM-C and I.D.-M.); and the La Caixa Foundation Program, grant number HR17-00601 (to M.L.M.-C.).España Ministerio de Educación, Cultura y Deporte, grant number FPU016/0151

Atorvastatin Provides a New Lipidome Improving Early Regeneration After Partial Hepatectomy in Osteopontin Deficient Mice

Osteopontin (OPN), a multifunctional cytokine that controls liver glycerolipid metabolism, is involved in activation and proliferation of several liver cell types during regeneration, a condition of high metabolic demands. Here we investigated the role of OPN in modulating the liver lipidome during regeneration after partial-hepatectomy (PH) and the impact that atorvastatin treatment has over regeneration in OPN knockout (KO) mice. The results showed that OPN deficiency leads to remodeling of phosphatidylcholine and triacylglycerol (TG) species primarily during the first 24 h after PH, with minimal effects on regeneration. Changes in the quiescent liver lipidome in OPN-KO mice included TG enrichment with linoleic acid and were associated with higher lysosome TG-hydrolase activity that maintained 24 h after PH but increased in WT mice. OPN-KO mice showed increased beta-oxidation 24 h after PH with less body weight loss. In OPN-KO mice, atorvastatin treatment induced changes in the lipidome 24 h after PH and improved liver regeneration while no effect was observed 48 h post-PH. These results suggest that increased dietary-lipid uptake in OPN-KO mice provides the metabolic precursors required for regeneration 24 h and 48 h after PH. However, atorvastatin treatment offers a new metabolic program that improves early regeneration when OPN is deficient.This work was supported by IT-336-10 (Gobierno Vasco) (to PA) and SAF2015-64352-R (to PA), SAF2017-87301-R (to MLM-C) and EITB Maratoia BIO15/CA/014 (to MLM-C). MNG and DM were recipients of a predoctoral fellowship from the University of Basque Country UPV/EHU and BG-S and DS were recipients for predoctoral fellowships from the Basque Goverment. We thank technical support from Jose Antonio Lopez Gomez

Inhibition of NAE-dependent protein hyper-NEDDylation in cystic cholangiocytes halts cystogenesis in experimental models of polycystic liver disease

Background Polycystic liver diseases (PLDs) are genetic inherited disorders characterized by the progressive growth of numerous intrahepatic biliary cysts, which are the main cause of morbidity. Previous studies revealed that cystic cholangiocytes are characterized by endoplasmic reticulum stress and aberrant posttranslational modification (PTM) of proteins, in particular hyper-SUMOylation, that promote PLD pathobiology. Protein NEDDylation is a newly characterized PTM that modulates a plethora of biological processes and its dysregulation is associated with the development and progression of several human diseases. However, the role of NEDDylation in PLD remains elusive. Objective To explore the role of protein NEDDylation in PLD and its potential therapeutic regulatory value. Methods Levels and functional effects of NEDDylation, including response to Pevonedistat (first-in-class selective inhibitor of the NEDDylation E1 enzyme NAE), were assessed in vitro, in vivo, and/or in patients with PLD. NEDDylated protein levels in normal and cystic human cholangiocytes were assessed by immunoprecipitation, and the proteomic profile was further analyzed by mass spectrometry. Results and Conclusion The genes involved in the NEDDylation pathway were found overexpressed (mRNA) in polycystic human and rat liver tissue, as well as in cystic cholangiocytes in culture, compared to controls. Elevated levels of NEDDylated proteins were further confirmed in cystic cholangiocytes in vitro, which diminished under Pevonedistat incubation. Pevonedistat promoted apoptotic cell death and reduced proliferation in cystic cholangiocytes in vitro. Comparative proteomic profiling of NEDD8-immunoprecipitated proteins between normal and cystic cholangiocytes in culture reported candidate proteins involved in cystogenesis, mostly associated with protein biogenesis and quality control. All these data indicate that cystic cholangiocytes display increased protein NEDDylation, contributing to cell survival and proliferation, ultimately supporting hepatic cystogenesis. Targeting of protein hyper-NEDDylation in cystic cholangiocytes inhibits cystogenesis in experimental models, representing a novel therapeutic opportunity in PLD.Spanish Carlos III Health Institute (ISCIII), Grant/Award Numbers: CON14/00129, CPII19/00008, FIS PI12/00380, FIS PI14/ 00399, FIS PI15/01132, FIS PI17/00022, FIS PI18/01075, FIS PI20/00186, Sara Borrell CD19/00254; Diputacion Foral de Gipuzkoa, Grant/Award Numbers: DFG15/010, DFG16/004; Department of Health of the Basque Country, Grant/Award Numbers: 2015111100, 2017111010, 2019111024; Euskadi RIS3, Grant/Award Numbers: 2016222001, 2017222014, 2018222029, 2019222054, 2020333010; Department of Industry of the Basque Country, Grant/Award Number: KK-2020/00008; Spanish Ministry of Economy and Competitiveness, Grant/Award Number: RYC-2015-17755; Ministerio de Ciencia, Innovacion y Universidades, Grant/ Award Number: SAF2017-87301-R; Ayudas para apoyar grupos de investigacion del Sistema Universitario Vasco, Grant/Award Number: IT971-16; Universita Politecnica delle Marche, Grant/Award Number: PSA2017_UNIVPM; European Association for the Study of the Liver, Grant/Award Number: Sheila Sherlock Award 2017; Spanish Ministry of Science and Innovation, Grant/Award Number: BES-2014-069148; Basque Government, Grant/Award Number: PRE_2016_1_0269; Basque Foundation for Innovation and Health Research, Grant/Award Number: BIO15/CA/016/BD; Fundacion Cientifica de la Asociacion Espanola Contra el Cancer; La Caixa Scientific Foundation, Grant/ Award Number: HR17-00601; CIBERehd; Fondo Europeo de Desarrollo Regional Documen

Enhanced mitochondrial activity reshapes a gut microbiota profile that delays NASH progression

[EN] Background and Aims: Recent studies suggest that mitochondrial dysfunction promotes progression to NASH by aggravating the gut-liver status. However, the underlying mechanism remains unclear. Herein, we hypothesized that enhanced mitochondrial activity might reshape a specific microbiota signature that, when transferred to germ-free (GF) mice, could delay NASH progression. Approach and Results: Wild-type and methylation-controlled J protein knockout (MCJ-KO) mice were fed for 6 weeks with either control or a choline-deficient, L-amino acid–defined, high-fat diet (CDA-HFD). One mouse of each group acted as a donor of cecal microbiota to GF mice, who also underwent the CDA-HFD model for 3 weeks. Hepatic injury, intestinal barrier, gut microbiome, and the associated fecal metabolome were then studied. Following 6 weeks of CDA-HFD, the absence of methylation-controlled J protein, an inhibitor of mitochondrial complex I activity, reduced hepatic injury and improved gut-liver axis in an aggressive NASH dietary model. This effect was transferred to GF mice through cecal microbiota transplantation. We suggest that the specific microbiota profile of MCJ-KO, characterized by an increase in the fecal relative abundance of Dorea and Oscillospira genera and a reduction in AF12, Allboaculum, and [Ruminococcus], exerted protective actions through enhancing short-chain fatty acids, nicotinamide adenine dinucleotide (NAD+) metabolism, and sirtuin activity, subsequently increasing fatty acid oxidation in GF mice. Importantly, we identified Dorea genus as one of the main modulators of this microbiota-dependent protective phenotype. Conclusions: Overall, we provide evidence for the relevance of mitochondria–microbiota interplay during NASH and that targeting it could be a valuable therapeutic approach.S

Exogenous aralar/slc25a12 can replace citrin/slc25a13 as malate aspartate shuttle component in liver

The deficiency of CITRIN, the liver mitochondrial aspartate–glutamate carrier (AGC), is the cause of four human clinical phenotypes, neonatal intrahepatic cholestasis caused by CITRIN deficiency (NICCD), silent period, failure to thrive and dyslipidemia caused by CITRIN deficiency (FTTDCD), and citrullinemia type II (CTLN2). Clinical symptoms can be traced back to disruption of the malate-aspartate shuttle due to the lack of citrin. A potential therapy for this condition is the expression of aralar, the AGC present in brain, to replace citrin. To explore this possibility we have first verified that the NADH/NAD+ ratio increases in hepatocytes from citrin(−/−) mice, and then found that exogenous aralar expression reversed the increase in NADH/NAD+ observed in these cells. Liver mitochondria from citrin (−/−) mice expressing liver specific transgenic aralar had a small (~ 4–6 nmoles x mg prot−1 x min−1) but consistent increase in malate aspartate shuttle (MAS) activity over that of citrin(−/−) mice. These results support the functional replacement between AGCs in the liver. To explore the significance of AGC replacement in human therapy we studied the relative levels of citrin and aralar in mouse and human liver through absolute quantification proteomics. We report that mouse liver has relatively high aralar levels (citrin/aralar molar ratio of 7.8), whereas human liver is virtually devoid of aralar (CITRIN/ARALAR ratio of 397). This large difference in endogenous aralar levels partly explains the high residual MAS activity in liver of citrin(−/−) mice and why they fail to recapitulate the human disease, but supports the benefit of increasing aralar expression to improve the redox balance capacity of human liver, as an effective therapy for CITRIN deficienc

Targeting UBC9-Mediated Protein Hyper-SUMOylation in Cystic Cholangiocytes Halts Polycystic Liver Disease in Experimental Models

BACKGROUND & AIMS: Polycystic liver diseases (PLDs) are genetic disorders characterized by progressive development of multiple fluid-filled biliary cysts. Most PLD-causative genes participate in protein biogenesis and/or transport. Post-translational modifications (PTMs) are implicated in protein stability, localization and activity, contributing to human pathobiology; however, their role in PLD is unknown. Herein, we aimed to unveil the role of protein SUMOylation in PLD and its potential therapeutic targeting. METHODS: Levels and functional effects of SUMOylation, along with response to S-adenosylmethionine (SAMe, inhibitor of the SUMOylation enzyme UBC9) and/or short-hairpin RNAs (shRNAs) against UBE2I (UBC9), were evaluated invitro, invivo and/or in patients with PLD. SUMOylated proteins were determined by immunoprecipitation and proteomic analyses by mass spectrometry. RESULTS: Most SUMOylation-related genes were found overexpressed (mRNA) in polycystic human and rat liver tissue, as well as in cystic cholangiocytes in culture compared to controls. Increased SUMOylated protein levels were also observed in cystic human cholangiocytes in culture, which decreased after SAMe administration. Chronic treatment of polycystic (PCK: Pkdh1-mut) rats with SAMe halted hepatic cystogenesis and fibrosis, and reduced liver/body weight ratio and liver volume. Invitro, both SAMe and shRNA-mediated UBE2I knockdown increased apoptosis and reduced cell proliferation of cystic cholangiocytes. High-throughput proteomic analysis of SUMO1-immunoprecipitated proteins in cystic cholangiocytes identified candidates involved in protein biogenesis, ciliogenesis and proteasome degradation. Accordingly, SAMe hampered proteasome hyperactivity in cystic cholangiocytes, leading to activation of the unfolded protein response and stress-related apoptosis. CONCLUSIONS: Cystic cholangiocytes exhibit increased SUMOylation of proteins involved in cell survival and proliferation, thus promoting hepatic cystogenesis. Inhibition of protein SUMOylation with SAMe halts PLD, representing a novel therapeutic strategy. LAY SUMMARY: Protein SUMOylation is a dynamic post-translational event implicated in numerous cellular processes. This study revealed dysregulated protein SUMOylation in polycystic liver disease, which promotes hepatic cystogenesis. Administration of S-adenosylmethionine (SAMe), a natural UBC9-dependent SUMOylation inhibitor, halted polycystic liver disease in experimental models, thus representing a potential therapeutic agent for patients.Spanish Carlos III Health Institute (ISCIII) [J.M. Banales (FIS PI12/00380, PI15/01132, PI18/01075 and Miguel Servet Program CON14/00129 and CPII19/00008); M.J. Perugorria (FIS PI14/00399, PI17/00022 and PI20/00186); P.M. Rodrigues (Sara Borrell CD19/00254)] cofinanced by “Fondo Europeo de Desarrollo Regional” (FEDER); Ministerio de Ciencia, Innovación y Universidades (MICINN; M.L. Martinez-Chantar: SAF2017-87301-R); “Instituto de Salud Carlos III” [CIBERehd: J.M. Banales, M.J. Perugorria, M.L. Martinez-Chantar and L. Bujanda], Spain; “Diputación Foral Gipuzkoa” (J.M. Banales: DFG15/010, DFG16/004), Department of Health of the Basque Country (M.J. Perugorria: 2019111024, 2015111100 and J.M. Banales: 2017111010), “Euskadi RIS3” (J.M. Banales: 2016222001, 2017222014, 2018222029, 2019222054, 2020333010), BIOEF (Basque Foundation for Innovation and Health Research: EiTB Maratoia BIO15/CA/016/BD to J.M. Banales and M.L. Martinez-Chantar) and Department of Industry of the Basque Country (J.M. Banales: Elkartek: KK-2020/00008). La Caixa Scientific Foundation (J.M. Banales and M.L. Martinez-Chantar: HR17-00601). “Fundación Científica de la Asociación Española Contra el Cáncer” (AECC Scientific Foundation, to J.M. Banales and M.L. Martinez-Chantar). “Ayudas para apoyar grupos de investigación del Sistema Universitario Vasco” (IT971-16 to P.A.). Università Politecnica delle Marche PSA2017_UNIVPM grant (to M. Marzioni). National Institutes of Health (NIH) of United States of America (DK24031 to N.F. LaRusso). MJ Perugorria was funded by the Spanish Ministry of Economy and Competitiveness (MINECO: “Ramón y Cajal” Program RYC-2015-17755), P.Y. Lee-Law by the European Association for the Study of the Liver (EASL; Sheila Sherlock Award 2017), F.J. Caballero-Camino by the Spanish Ministry of Science and Innovation (BES-2014-069148), and P. Olaizola and A. Santos-Laso by the Basque Government (PRE_2016_1_0269, PRE_2015_1_0126). We thank MINECO for the Severo Ochoa Excellence Accreditation to CIC bioGUNE (SEV-2016-0644). The funding sources had no involvement in study design, data collection and analysis, decision to publish, or preparation of the article

MO38-2 Metallothionein-3: Potential therapeutic target for sorafenib resistance in hepatocellular carcinoma

1 p. JSMO2021 Virtual Congress. 2021 the Japanese Society of Medical Oncology Annual Meeting. February 18 - 21, 2021Background: Metallothionein-3 (M-3) has poorly characterized functions in hepatocellular carcinoma (HCC). HCC is a significant health problem. Globally is the second most common cause of cancer-associated death. Sorafenib was originally identified as an inhibitor of multiple oncogenic kinases and remains the only approved systemic therapy for advanced HCC. However, acquired resistance to sorafenib has been found in HCC patients, which results in poor prognosis. Overexpression of MT-3 decreased the sensitivity of HCC cells to sorefenib. Here, we investigated the impact of MT-3 up-regulation in HCC cells and the mechanisms underlying the sorafenib-resistance.Methods: To increase the expression of MT-3 HCC cells were transiently transfected with a plasmid containing MT-3 gene or with empty vector. The cDNA microarrays were accomplished using the ElectraSenseTM Reader. MS analysis was performed using a Q-Exactive MS. We used chick chorioallantoic membrane assay as in vivo model.Results: A cDNA profiling revealed that sorafenib resistance has a specific transcriptomic signature involving genes responsible for ion transport, trafficking and DNA repair. Also, The MS analysis data strongly suggest that resistance HCC cells acquired a complex regulatory network that significantly affects the ability of HCC cells to remove the ROS and activation of glycolysis. We provide the first evidence that up-regulation of MT3 resulted in increased dissociation, invasion, and intravasation from the primary tumours to the veins. In addition, MT3 profoundly impacted blood migration of Nbl cells and their extravasation to chicken organs.Conclusion: From a perspective of future utilization of our data, we anticipate that several identified genes and proteins could serve as prognostic biomarkers of outcome of sorafenib therapy. The increased expression of MT-3 within tumour mass should inform about worse prognosis and also decreased efficiency of sorafenib-based chemotherapy in HCC.Peer reviewe

Sensitive detection of SARS-CoV-2 seroconversion by flow cytometry reveals the presence of nucleoprotein-reactive antibodies in unexposed individuals

There is an ongoing need of developing sensitive and specific methods for the determination of SARS-CoV-2 seroconversion. For this purpose, we have developed a multiplexed flow cytometric bead array (C19BA) that allows the identification of IgG and IgM antibodies against three immunogenic proteins simultaneously: the spike receptor-binding domain (RBD), the spike protein subunit 1 (S1) and the nucleoprotein (N). Using different cohorts of samples collected before and after the pandemic, we show that this assay is more sensitive than ELISAs performed in our laboratory. The combination of three viral antigens allows for the interrogation of full seroconversion. Importantly, we have detected N-reactive antibodies in COVID-19-negative individuals. Here we present an immunoassay that can be easily implemented and has superior potential to detect low antibody titers compared to current gold standard serology methods.Acknowledgements: We thank Petros Tyrakis and Iván Martínez-Forero for critical reading and editing of the manuscript. Support was provided by the Severo Ochoa Excellence Accreditation from MCIU (SEV-2016-0644) and the SPRI I+D COVID-19 fund (Gobierno Vasco). Personal fellowships: A.A.-V. (La Caixa Inphinit LCF/BQ/DR20/11790022), A.B. (AECC Bizkaia), A.G.d.R (Bikaintek), A.P. (Ramón y Cajal), B.J.-L. (Gob. Vasco), and E.P.-F. (Juan de la Cierva-Formación). M.L.M.-C. acknowledges RTC2019-007125-1, DTS20/00138, SAF2017-87301-R, and BBVA UMBRELLA project. M.L.-H. acknowledges the ISCIII for grant COV20-0170 and the Government of Cantabria for grant 2020UIC22-PUB-0019. O.M., J.-M.M., and N.G.A.A. acknowledge the Agencia Estatal de Investigación (Spain) for grants CTQ2015-68756-R, RTI2018-101269-BI00, and RTI2018-095700-B-I00, respectively. A.P. has received grant funding from the European Research Council (ERC), grant agreement number 804236 (Horizon 2020), and the FERO Foundation