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203 p., El estilo de vida occidental ha provocado un aumento de la prevalencia de varias enfermedades que afectan al hígado. Por un lado, la obesidad, que se ha convertido en una pandemia mundial, aumenta el riesgo de desarrollar la enfermedad del hígado graso no alcohólico (EHGNA) además de otros tipos de cáncer como el carcinoma hepatocelular (CHC). Por otro lado, la amplia disponibilidad y el abuso del paracetamol (APAP) han provocado que la lesión hepática inducida por este fármaco se convierta en la causa más común de fallo hepático aguda. Ambas enfermedades tienen como eje principal la alteración del metabolismo de lípidos, la respuesta inmune y la activación del ciclo celular. Las opciones terapéuticas actuales son escasas y muy limitadas por lo que es necesario encontrar nuevas dianas terapéuticas. La familia de factores de transcripción E2F está compuesta por proteínas reguladoras del ciclo celular. Sin embargo, los miembros de la familia E2F ejercen otras funciones; en concreto, E2F1 participa en procesos metabólicos y tanto E2F1 como E2F2 han sido relacionados con la regeneración hepática y la maduración de determinadas células del sistema inmune. Por lo tanto, en este trabajo se propuso investigar el papel y el valor potencial como dianas terapéuticas de E2F1 y E2F2 en el CHC relacionado con NAFLD y en la lesión hepática inducida por APAP.

26 p., Los avances producidos durante los últimos años en el área de la espectrometría de masas y la sofisticación de los equipos utilizados han permitido el análisis de biomoléculas con elevada precisión. La técnica MALDI-IMS (Matrix Assisted Laser Desorption Ionization – Imaging Mass Spectropetry) permite combinar el análisis molecular con información espacial, generando imágenes de la distribución de los metabolitos estudiados en tejido. El cáncer es una de las principales causas de muerte en el mundo, y su estudio ha revelado que la reprogramación metabólica es un factor crítico en su desarrollo y progresión. En particular, se ha investigado el metabolismo de los lípidos como fuente de energía, adaptación al microambiente y señalización celular en las células neoplásicas. En el caso del hígado existen varias patologías y factores de riesgo que pueden derivar en cáncer hepático, que es de los tumores más frecuentes en los pacientes. Por este motivo, el objetivo de este trabajo fue comparar el lipidoma de muestras control con 2 tipos de tumor hepático con el fin de observar cambios en la huella lipídica de los diferentes grupos. Se han podido observar diferencias significativas para algunas familias de lípidos, lo que abre la posibilidad del uso de dichas moléculas como biomarcadores tumorales y la técnica MALDI-IMS como una herramienta de diagnóstico.

[EN]Background & Aims: Autophagy-related gene 3 (ATG3) is an enzyme mainly known for its actions in the LC3 lipidation process, which is essential for autophagy. Whether ATG3 plays a role in lipid metabolism or contributes to non-alcoholic fatty liver disease (NAFLD) remains unknown. Methods: By performing proteomic analysis on livers from mice with genetic manipulation of hepatic p63, a regulator of fatty acid metabolism, we identified ATG3 as a new target downstream of p63. ATG3 was evaluated in liver samples from patients with NAFLD. Further, genetic manipulation of ATG3 was performed in human hepatocyte cell lines, primary hepatocytes and in the livers of mice. Results: ATG3 expression is induced in the liver of animal models and patients with NAFLD (both steatosis and nonalcoholic steatohepatitis) compared with those without liver disease. Moreover, genetic knockdown of ATG3 in mice and human hepatocytes ameliorates p63-and diet-induced steatosis, while its overexpression increases the lipid load in hepatocytes. The inhibition of hepatic ATG3 improves fatty acid metabolism by reducing c-Jun N-terminal protein kinase 1 (JNK1), which increases sirtuin 1 (SIRT1), carnitine palmitoyltransferase 1a (CPT1a), and mitochondrial function. Hepatic knockdown of SIRT1 and CPT1a blunts the effects of ATG3 on mitochondrial activity. Unexpectedly, these effects are independent of an autophagic action. Conclusions: Collectively, these findings indicate that ATG3 is a novel protein implicated in the development of steatosis. Lay summary: We show that autophagy-related gene 3 (ATG3) contributes to the progression of non-alcoholic fatty liver disease

[EN] BACKGROUND & AIMS: Cholangiocarcinoma (CCA) comprises a heterogeneous group of malignant tumors associated with dismal prognosis. Alterations in post-translational modifications (PTMs), including NEDDylation, result in abnormal protein dynamics, cell disturbances and disease. Herein, we investigate the role of NEDDylation in CCA development and progression. METHODS: Levels and functions of NEDDylation, together with response to pevonedistat (NEDDylation inhibitor) or CRISPR/Cas9 against NAE1 were evaluated invitro, invivo and/or in patients with CCA. The development of preneoplastic lesions in Nae1+/- mice was investigated using an oncogene-driven CCA model. The impact of NEDDylation in CCA cells on tumor-stroma crosstalk was assessed using CCA-derived cancer-associated fibroblasts (CAFs). Proteomic analyses were carried out by mass-spectrometry. RESULTS: The NEDDylation machinery was found overexpressed and overactivated in human CCA cells and tumors. Most NEDDylated proteins found upregulated in CCA cells, after NEDD8-immunoprecipitation and further proteomics, participate in the cell cycle, proliferation or survival. Genetic (CRISPR/Cas9-NAE1) and pharmacological (pevonedistat) inhibition of NEDDylation reduced CCA cell proliferation and impeded colony formation invitro. NEDDylation depletion (pevonedistat or Nae1+/- mice) halted tumorigenesis in subcutaneous, orthotopic, and oncogene-driven models of CCA invivo. Moreover, pevonedistat potentiated chemotherapy-induced cell death in CCA cells invitro. Mechanistically, impaired NEDDylation triggered the accumulation of both cullin RING ligase and NEDD8 substrates, inducing DNA damage and cell cycle arrest. Furthermore, impaired NEDDylation in CCA cells reduced the secretion of proteins involved in fibroblast activation, angiogenesis, and oncogenic pathways, ultimately hampering CAF proliferation and migration. CONCLUSION: Aberrant protein NEDDylation contributes to cholangiocarcinogenesis by promoting cell sur

Altered methionine metabolism is associated with weight gain in obesity. The methionine adenosyltransferase (MAT), catalyzing the first reaction of the methionine cycle, plays an important role regulating lipid metabolism. However, its role in obesity, when a plethora of metabolic diseases occurs, is still unknown. By using antisense oligonucleotides (ASO) and genetic depletion of Mat1a, here, we demonstrate that Mat1a deficiency in diet-induce obese or genetically obese mice prevented and reversed obesity and obesity-associated insulin resistance and hepatosteatosis by increasing energy expenditure in a hepatocyte FGF21 dependent fashion. The increased NRF2-mediated FGF21 secretion induced by targeting Mat1a, mobilized plasma lipids towards the BAT to be catabolized, induced thermogenesis and reduced body weight, inhibiting hepatic de novo lipogenesis. The beneficial effects of Mat1a ASO were abolished following FGF21 depletion in hepatocytes. Thus, targeting Mat1a activates the liver-BAT axis by increasing NRF2-mediated FGF21 secretion, which prevents obesity, insulin resistance and hepatosteatosis. High methionine and S-adenosylmethionine serum levels are related with obesity. Here the authors show that knockdown of methionine adenosyltransferase by using antisense oligonucleotides provides beneficial effects in obesity and comorbidities.

Background & Aims: Inflammation, particularly that mediated by bacterial components translocating from the gut to the liver and binding to toll-like receptors (TLRs), is central to cholestatic liver injury. The triggering receptor expressed on myeloid cells-2 (TREM-2) inhibits TLR-mediated signaling and exerts a protective role in hepatocellular injury and carcinogenesis. This study aims to evaluate the role of TREM-2 in cholestasis.Methods: TREM-2 expression was analyzed in the livers of pa-tients with primary biliary cholangitis (PBC) or primary scle-rosing cholangitis (PSC), and in mouse models of cholestasis. Wild-type (WT) and Trem-2 deficient (Trem-2-/-) mice were subjected to experimental cholestasis and gut sterilization. Pri-mary cultured Kupffer cells were incubated with lipopolysac-charide and/or ursodeoxycholic acid (UDCA) and inflammatory responses were analyzed.Results: TREM-2 expression was upregulated in the livers of patients with PBC or PSC, and in murine models of cholestasis. Compared to WT, the response to bile duct ligation (BDL)-induced obstructive cholestasis or alpha-naphtylisothiocyanate (ANIT)-induced cholestasis was exacerbated in Trem-2-/-mice. This was characterized by enhanced necroptotic cell death, in-flammatory responses and biliary expansion. Antibiotic treat-ment partially abrogated the effects observed in Trem-2-/-mice after BDL. Experimental overexpression of TREM-2 in the liver of WT mice downregulated ANIT-induced IL-33 expression and neutrophil recruitment. UDCA regulated Trem-1 and Trem-2 expression in primary cultured mouse Kupffer cells and damp-ened inflammatory gene transcription via a TREM-2-dependent mechanism.Conclusions: TREM-2 acts as a negative regulator of inflamma-tion during cholestasis, representing a novel potential thera-peutic target.Lay summary: Cholestasis (the reduction or cessation of bile flow) causes liver injury. This injury is exacerbated when gut-derived bacterial components interact with receptors

E2F1/E2F2 expression correlates with malignancy in prostate cancer (PCa), but its functional significance remains unresolved. To define the mechanisms governed by E2F in PCa, we analyzed the contribution of E2F target genes to the control of genome integrity, and the impact of modulating E2F activity on PCa progression. We show that silencing or inhibiting E2F1/E2F2 induces DNA damage during S phase and potentiates 5-FU-induced replication stress and cellular toxicity. Inhibition of E2F downregulates the expression of E2F targets involved in nucleotide biosynthesis (TK1, DCK, TYMS), whose expression is upregulated by 5-FU. However, their enzymatic products failed to rescue DNA damage of E2F1/E2F2 knockdown cells, suggesting additional mechanisms for E2F function. Interestingly, targeting E2F1/E2F2 in PCa cells reduced WEE1 expression and resulted in premature CDK1 activation during S phase. Inhibition of CDK1/CDK2 prevented DNA damage induced by E2F loss, suggesting that E2F1/E2F2 safeguard genome integrity by restraining CDK1/CDK2 activity. Importantly, combined inhibition of E2F and ATR boosted replication stress and dramatically reduced tumorigenic capacity of PCa cells in xenografts. Collectively, inhibition of E2F in combination with drugs targeting nucleotide biosynthesis or DNA repair is a promising strategy to provoke catastrophic levels of replication stress that could be applied to PCa treatment.

Melanoma is the deadliest form of skin cancer due to its ability to colonize distant sites and initiate metastasis. Although these processes largely depend on the lipid-based cell membrane scaffold, our understanding of the melanoma lipid phenotype lags behind most other aspects of this tumor cell. Here, we examined a panel of normal human epidermal and nevus melanocytes and primary and metastatic melanoma cell lines to determine whether distinctive cell-intrinsic lipidomes can discern non-neoplastic from neoplastic melanocytes and define their metastatic potential. Lipidome profiles were obtained by UHPLC-ESI mass-spectrometry, and differences in the signatures were analyzed by multivariate statistical analyses. Significant and highly specific changes in more than 30 lipid species were annotated in the initiation of melanoma, whereas less numerous changes were associated with melanoma progression and the non-malignant transformation of nevus melanocytes. Notably, the “malignancy lipid signature” features marked drops in pivotal membrane lipids, like sphingomyelins, and aberrant elevation of ether-type lipids and phosphatidylglycerol and phosphatidylinositol variants, suggesting a previously undefined remodeling of sphingolipid and glycerophospholipid metabolism. Besides broadening the molecular definition of this neoplasm, the different lipid profiles identified may help improve the clinical diagnosis/prognosis and facilitate therapeutic interventions for cutaneous melanoma.

Objectives: Lately, many countries have restricted or even banned transfat, and palm oil has become a preferred replacement for food manufacturers. Whether palm oil is potentially an unhealthy food mainly due to its high content of saturated Palmitic Acid (PA) is a matter of debate. The aim of this study was to test whether qualitative aspects of diet such as levels of PA and the fat source are risk factors for Metabolic Syndrome (MS) and Metabolic Associated Fatty Liver Disease (MAFLD). Methods: C57BL/6 male mice were fed for 14 weeks with three types of Western diet (WD): 1. LP-WD—low concentration of PA (main fat source—corn and soybean oils); 2. HP-WD—high concentration of PA (main fat source—palm oil); 3. HP-Trans-WD—high concentration of PA (mainly transfat). Results: All types of WD caused weight gain, adipocyte enlargement, hepatomegaly, lipid metabolism alterations, and steatohepatitis. Feeding with HP diets led to more prominent obesity, hypercholesterolemia, stronger hepatic injury, and fibrosis. Only the feeding with HP-Trans-WD resulted in glucose intolerance and elevation of serum transaminases. Brief withdrawal of WDs reversed MS and signs of MAFLD. However, mild hepatic inflammation was still detectable in HP groups. Conclusions: HP and HP-Trans-WD play a crucial role in the genesis of MS and MAFLD.

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 sur

[EN] Objective Hepatocellular carcinoma (HCC) is a prevalent and aggressive cancer usually arising on a background of chronic liver injury involving inflammatory and hepatic regenerative processes. The triggering receptor expressed on myeloid cells 2 (TREM-2) is predominantly expressed in hepatic non-parenchymal cells and inhibits Toll-like receptor signalling, protecting the liver from various hepatotoxic injuries, yet its role in liver cancer is poorly defined. Here, we investigated the impact of TREM-2 on liver regeneration and hepatocarcinogenesis. Design TREM-2 expression was analysed in liver tissues of two independent cohorts of patients with HCC and compared with control liver samples. Experimental HCC and liver regeneration models in wild type and Trem-2-/- mice, and in vitro studies with hepatic stellate cells (HSCs) and HCC spheroids were conducted. Results TREM-2 expression was upregulated in human HCC tissue, in mouse models of liver regeneration and HCC. Trem-2-/- mice developed more liver tumours irrespective of size after diethylnitrosamine (DEN) administration, displayed exacerbated liver damage, inflammation, oxidative stress and hepatocyte proliferation. Administering an antioxidant diet blocked DEN-induced hepatocarcinogenesis in both genotypes. Similarly, Trem-2-/- animals developed more and larger tumours in fibrosis-associated HCC models. Trem-2-/- livers showed increased hepatocyte proliferation and inflammation after partial hepatectomy. Conditioned media from human HSCs overexpressing TREM-2 inhibited human HCC spheroid growth in vitro through attenuated Wnt ligand secretion. Conclusion TREM-2 plays a protective role in hepatocarcinogenesis via different pleiotropic effects, suggesting that TREM-2 agonism should be investigated as it might beneficially impact HCC pathogenesis in a multifactorial manner.

[EN] 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

Background & Aims: Perturbations of intracellular magnesium (Mg2+) homeostasis have implications for cell physiology. The cyclin M family, CNNM, perform key functions in the transport of Mg2+ across cell membranes. Herein, we aimed to elucidate the role of CNNM4 in the development of non-alcoholic steatohepatitis (NASH). Methods: Serum Mg2+ levels and hepatic CNNM4 expression were characterised in clinical samples. Primary hepatocytes were cultured under methionine and choline deprivation. A 0.1% methionine and choline-deficient diet, or a choline-deficient high-fat diet were used to induce NASH in our in vivo rodent models. Cnnm4 was silenced using siRNA, in vitro with DharmaFECT and in vivo with Invivofectamine (R) or conjugated to N-acetylgalactosamine. Results: Patients with NASH showed hepatic CNNM4 over-expression and dysregulated Mg2+ levels in the serum. Cnnm4 silencing ameliorated hepatic lipid accumulation, inflammation and fibrosis in the rodent NASH models. Mechanistically, CNNM4 knockdown in hepatocytes induced cellular Mg2+ accumulation, reduced endoplasmic reticulum stress, and increased microsomal triglyceride transfer activity, which promoted hepatic lipid clearance by increasing the secretion of VLDLs. Conclusions: CNNM4 is overexpressed in patients with NASH and is responsible for dysregulated Mg2+ transport. Hepatic CNNM4 is a promising therapeutic target for the treatment of NASH. Lay summary: Cyclin M4 (CNNM4) is overexpressed in nonalcoholic steatohepatitis (NASH) and promotes the export of magnesium from the liver. The liver-specific silencing of Cnnm4 ameliorates NASH by reducing endoplasmic reticulum stress and promoting the activity of microsomal triglyceride transfer protein.

Introduction and Objectives Nonalcoholic-fatty-liver disease (NAFLD) is considered the hepatic manifestation of metabolic syndrome (MetS). Mineralocorticoid receptor (MR) activation is associated with increased risk of MetS but few studies have assessed the role of liver MR on NAFLD. We aimed to evaluate the effect of MR modulation by sodium intake in liver injury in experimental models of NAFLD. Materials and Methods C57BL/6J mice were fed either a high-fat-diet (HFD) or a choline/methionine deficient (MCD) diet with different sodium concentrations. Hepatic concentration of lipid species, serum aldosterone levels, expression of MR, proinflammatory and profibrotic markers and liver histology were assessed. Results Mice fed with High-Na+/HFD showed a lower MR expression in liver (p = 0.01) and less steatosis on histology (p = 0.04). Consistently, animals from this group exhibited lower levels of serum aldosterone (p = 0.028) and lower hepatic triglyceride content (p = 0.008). This associated to a reduced expression of lipogenic genes, significant changes in lipid subspecies, lower HOMA-IR (p < 0.05), and lower expression of pro-inflammatory and profibrotic markers compared to those mice fed a Low-Na+/HFD. Additionally, mice fed a High-Na+/HFD showed higher expression of salt-inducible kinase (SIK)-1 and lower expression of serum-and-glucocorticoid-inducible kinase (SGK)-1. Similar results were observed with the MCD diet model. Conclusion We identified in two experimental models of NAFLD that High-Na+ diet content is associated to lower serum aldosterone levels and hepatic MR downregulation, associated to decreased steatosis and reduced de novo hepatic lipogenesis, proinflammatory and profibrotic markers. Decreased activation of hepatic MR seems to generate beneficial downstream inhibition of lipogenesis in experimental NAFLD.

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 hepati

288 p., Munduko Osasun Erakundearen (OME) arabera, obesitatea osasun-egoeraren kontrako arriskua da, zeinak gorputzean gehiegizko gantz-pilaketa duen bereizgarritzat eta zeinaren munduko prebalentzia ia hirukoiztu egin den azken 40 urteetan, bai garatutako bai garatu-gabekoherrialdeetan. Gibeleko metabolismoaren asaldurekin lotuta dago, zeinak gibel lipidoen metaketan eta gibel koipetsuaren gaixotasun ez-alkoholikoaren (NAFLD) progresioa ahalbidetzen duten.NAFLDaren ezaugarria esteatosia edo gibel koipetsu ez-alkoholikoa da, egoera bat zeinean gibeleko TG eduki altua lipidoen pilaketan (batez ere, serumeko gantz azidoen (GA) barneratzea eta de novo lipogenesia edo DNL) eta deuseztatze bideen (batik bat, lipidoen oxidazioa edo GAOa eta dentsitate oso baxuko lipoproteinen edo VLDLren kanporaketa) arteko konpentsazio ezak bideratzen duen. Egun, NAFLDa kartzinoma hepatozelularraren (HCC) arrisku-faktore garrantzitsua da. Esteatosiaren baitan garatzen den HCCak NAFLDarekin loturiko HCCa izena du eta ezaugarri bereizgarriak ditu (lipido metaketa eta hantura, esaterako) beste etiologia batzuetatik garatutako HCCekin alderatuz gero. HCCan gene-gidari asko daude, tumore-histologiari, zelularen seinalizazio-bidezidorrei eta berprogramazio metabolikoari dagokion ezaugarri hain heterogeneoak azaltzen dituena. NAFLDarekin loturiko HCCaren fenotipoari dagokionez, nabarmentzekoa da obesitateak, berez, asaldura metabolikoak ahalbidetzen dituela, metabolikoki osasuntsua (MHO) den eta ez-osasuntsua den paziente obesoen (MUO) arteko bereizmena egiten duen sinadura metaboliko batekin erlazionatu baita.E2F trankripzio faktoreen familia, tipikoki ziklo zelularraren erregulatzailea dena, aldaketa metabolikoen arduraduna da ere, zeinak gibeleko egoera patologikoetan eta horri loturiko konplikazio metabolikoetan (obesitatea eta diabetesa, adibidez) gertatzen diren. E2F faktoreetako nahasmenduak deskribatu dira HCCan, gibeleko minbizian duten garrantzia nabarmentzen duena. Ikerketa gehienak E2F1ean

Microsomal triglyceride transfer protein (MTTP) deficiency results in a syndrome of hypolipidemia and accelerated NAFLD. Animal models of decreased hepatic MTTP activity have revealed an unexplained dissociation between hepatic steatosis and hepatic insulin resistance. Here, we performed comprehensive metabolic phenotyping of liver-specific MTTP knockout (L-Mttp(-/-)) mice and age-weight matched wild-type control mice. Young (10-12-week-old) L-Mttp(-/-) mice exhibited hepatic steatosis and increased DAG content; however, the increase in hepatic DAG content was partitioned to the lipid droplet and was not increased in the plasma membrane. Young L-Mttp(-/-) mice also manifested normal hepatic insulin sensitivity, as assessed by hyperinsulinemic-euglycemic clamps, no PKC epsilon activation, and normal hepatic insulin signaling from the insulin receptor through AKT Ser/Thr kinase. In contrast, aged (10-month-old) L-Mttp(-/-) mice exhibited glucose intolerance and hepatic insulin resistance along with an increase in hepatic plasma membrane sn-1,2-DAG content and PKC epsilon activation. Treatment with a functionally liver-targeted mitochondrial uncoupler protected the aged L-Mttp(-/-) mice against the development of hepatic steatosis, increased plasma membrane sn-1,2-DAG content, PKC epsilon activation, and hepatic insulin resistance. Furthermore, increased hepatic insulin sensitivity in the aged controlled-release mitochondrial protonophore-treated L-Mttp(-/-) mice was not associated with any reductions in hepatic ceramide content. Taken together, these data demonstrate that differences in the intracellular compartmentation of sn-1,2-DAGs in the lipid droplet versus plasma membrane explains the dissociation of NAFLD/lipid-induced hepatic insulin resistance in young L-Mttp(-/-) mice as well as the development of lipid-induced hepatic insulin resistance in aged L-Mttp(-/-) mice

Background and Aims G protein-coupled receptor (GPR) 55 is a putative cannabinoid receptor, and l-alpha-lysophosphatidylinositol (LPI) is its only known endogenous ligand. Although GPR55 has been linked to energy homeostasis in different organs, its specific role in lipid metabolism in the liver and its contribution to the pathophysiology of nonalcoholic fatty liver disease (NAFLD) remains unknown. Approach and Results We measured (1) GPR55 expression in the liver of patients with NAFLD compared with individuals without obesity and without liver disease, as well as animal models with steatosis and nonalcoholic steatohepatitis (NASH), and (2) the effects of LPI and genetic disruption of GPR55 in mice, human hepatocytes, and human hepatic stellate cells. Notably, we found that circulating LPI and liver expression of GPR55 were up-regulated in patients with NASH. LPI induced adenosine monophosphate-activated protein kinase activation of acetyl-coenzyme A carboxylase (ACC) and increased lipid content in human hepatocytes and in the liver of treated mice by inducing de novo lipogenesis and decreasing beta-oxidation. The inhibition of GPR55 and ACC alpha blocked the effects of LPI, and the in vivo knockdown of GPR55 was sufficient to improve liver damage in mice fed a high-fat diet and in mice fed a methionine-choline-deficient diet. Finally, LPI promoted the initiation of hepatic stellate cell activation by stimulating GPR55 and activation of ACC. Conclusions The LPI/GPR55 system plays a role in the development of NAFLD and NASH by activating ACC.

Carotid atherosclerotic plaque rupture can lead to cerebrovascular accident (CVA). By comparing RNA-Seq data from vascular smooth muscle cells (VSMC) extracted from carotid atheroma surgically excised from a group of asymptomatic and symptomatic subjects, we identified more than 700 genomic variants associated with symptomatology (p < 0.05). From these, twelve single nucleotide polymorphisms (SNPs) were selected for further validation. Comparing genotypes of a hospital-based cohort of asymptomatic with symptomatic patients, an exonic SNP in the BIRC6 (BRUCE/Apollon) gene, rs35286811, emerged as significantly associated with CVA symptomatology (p = 0.002; OR = 2.24). Moreover, BIRC6 mRNA levels were significantly higher in symptomatic than asymptomatic subjects upon measurement by qPCR in excised carotid atherosclerotic tissue (p < 0.0001), and significantly higher in carriers of the rs35286811 risk allele (p < 0.0001). rs35286811 is a proxy of a GWAS SNP reported to be associated with red cell distribution width (RDW); RDW was increased in symptomatic patients (p < 0.03), but was not influenced by the rs35286811 genotype in our cohort. BIRC6 is a negative regulator of both apoptosis and autophagy. This work introduces BIRC6 as a novel genetic risk factor for stroke, and identifies autophagy as a genetically regulated mechanism of carotid plaque vulnerability.

Insulin resistance (IR) is one of the key contributing factors in the development of type 2 diabetes mellitus (T2DM). However, the molecular mechanisms leading to IR are still unclear. The implication of microRNAs (miRNAs) in the pathophysiology of multiple cardiometabolic pathologies, including obesity, atherosclerotic heart failure and IR, has emerged as a major focus of interest in recent years. Indeed, upregulation of several miRNAs has been associated with obesity and IR. Among them, miR-27b is overexpressed in the liver in patients with obesity, but its role in IR has not yet been thoroughly explored. In this study, we investigated the role of miR-27b in regulating insulin signaling in hepatocytes, both in vitro and in vivo. Therefore, assessment of the impact of miR-27b on insulin resistance through the hepatic tissue is of special importance due to the high expression of miR-27b in the liver together with its known role in regulating lipid metabolism. Notably, we found that miR-27b controls post-transcriptional expression of numerous components of the insulin signaling pathway including the insulin receptor (INSR) and insulin receptor substrate 1 (IRS1) in human hepatoma cells. These results were further confirmed in vivo showing that overexpression and inhibition of hepatic miR-27 enhances and suppresses hepatic INSR expression and insulin sensitivity, respectively. This study identified a novel role for miR-27 in regulating insulin signaling, and this finding suggests that elevated miR-27 levels may contribute to early development of hepatic insulin resistance.

Los capítulos 5.2 y 6 están sujetos a confidencialidad por el autor. 207 p., La presente tesis doctoral trata sobre la modulación del metabolismo para el tratamiento de laesteatohepatitis no alcohólica (EHNA). Esta patología se caracteriza por una acumulación de grasa en elhígado que da lugar a complicaciones derivadas como la muerte celular, la inflamación, hinchamientoo ydesarrollo de fibrosis. Para identificar las dianas terapéuticas presentadas nos hemos centrado enperturbaciones descritas en el transcurso de la EHNA o patologías más severas: el metabolismo delamonio y nitrógeno y la homeostasis de magnesio. En base a ello hemos identificado la glutaminasa 1(GLS1) y la ciclina M4 (CNNM4) como dianas potenciales a la hora de tratar la enfermedad. Se hacaracterizado una sobre-expresión de ambas proteínas en muestras de pacientes diagnosticados de EHNA,así como en modelos animales de ratón de la enfermedad. Se han realizado estudios pre-clínicos en losque se ha inducido la patología por dos dietas distintas y se ha silenciado específicamente la proteínamediante inyección por vena de la cola, inhibiendo así la expresión del enzima en el hígado. En ambosensayos pre-clínicos se ha observado una menor acumulación de lípidos intrahepáticos y una disminucióndel estrés oxidativo derivado. Además, a la hora de elucidar el mecanismo de acción mediante el cual laacumulación de grasa o esteatosis es reducida, se ha identificado que el silenciamiento específico de Gls1y Cnnm4 promueve la secreción de lípidos en forma de partículas de muy baja densidad (VLDL).

[EN] Osteopontin (OPN), a senescence-associated secretory phenotype factor, is increased in patients with nonalcoholic fatty liver disease (NAFLD). Cellular senescence has been associated with age-dependent hepatosteatosis. Thus, we investigated the role of OPN in the age-related hepatosteatosis. For this, human serum samples, animal models of aging, and cell lines in which senescence was induced were used. Metabolic fluxes, lipid, and protein concentration were determined. Among individuals with a normal liver, we observed a positive correlation between serum OPN levels and increasing age. This correlation with age, however, was absent in patients with NAFLD. In wild-type (WT) mice, serum and liver OPN were increased at 10 months old (m) along with liver p53 levels and remained elevated at 20m. Markers of liver senescence increased in association with synthesis and concentration of triglycerides (TG) in 10m OPN-deficient (KO) hepatocytes when compared to WT hepatocytes. These changes in senescence and lipid metabolism in 10m OPN-KO mice liver were associated with the decrease of 78 kDa glucose-regulated protein (GRP78), induction of ER stress, and the increase in fatty acid synthase and CD36 levels. OPN deficiency in senescent cells also diminished GRP78, the accumulation of intracellular TG, and the increase in CD36 levels. In 20m mice, OPN loss led to increased liver fibrosis. Finally, we showed that OPN expression in vitro and in vivo was regulated by p53. In conclusion, OPN deficiency leads to earlier cellular senescence, ER stress, and TG accumulation during aging. The p53-OPN axis is required to inhibit the onset of agerelated hepatosteatosis.

Nonalcoholic fatty liver disease (NAFLD) is considered the next major health epidemic with an estimated 25% worldwide prevalence. No drugs have yet been approved and NAFLD remains a major unmet need. Here, we identify MCJ (Methylation-Controlled J protein) as a target for non-alcoholic steatohepatitis (NASH), an advanced phase of NAFLD. MCJ is an endogenous negative regulator of the respiratory chain Complex I that acts to restrain mitochondrial respiration. We show that therapeutic targeting of MCJ in the liver with nanoparticle- and GalNAc-formulated siRNA efficiently reduces liver lipid accumulation and fibrosis in multiple NASH mouse models. Decreasing MCJ expression enhances the capacity of hepatocytes to mediate beta -oxidation of fatty acids and minimizes lipid accumulation, which results in reduced hepatocyte damage and fibrosis. Moreover, MCJ levels in the liver of NAFLD patients are elevated relative to healthy subjects. Thus, inhibition of MCJ emerges as an alternative approach to treat NAFLD. Non-alcoholic fatty liver (NAFLD) disease causes degeneration of the liver, affects about 25% of people globally, and has no approved treatment. Here, the authors show that the therapeutic siRNA-driven silencing of MCJ in the liver is an effective and safe treatment for NAFLD in multiple mouse models.

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.

Los capítulos 5 y 6 están sujetos a confidencialidad por el autor. 235 p., Los estudios de identificación de nuevos biomarcadores reciben una cantidad notable de financiación, pero su éxito es reducido, con solo un 0.1% de los nuevos biomarcadores identificados llegando a implantarse en la práctica clínica rutinaria. Las tecnologías de alto rendimiento (ómicas) permiten la medición de una cantidad muy elevada de variables, facilitando así la posible identificación de nuevos biomarcadores. Los desarrollos tecnológicos más recientes han permitido abaratar sus costes, facilitando así la implantación y globalización de este tipo de estudios. A su vez, esto ha supuesto un incremento en el número de candidatos a biomarcador que no superan las fases de validación, en parte por la falta de protocolos y métodos de análisis estandarizados y robustos para este tipo de datos. En esta tesis, hemos estudiado qué métodos son más eficientes en la identificación de biomarcadores derivados de la combinación de distintas ómicas, principalmente microbioma y metabolómica. Nos hemos centrado en la identificación de nuevos biomarcadores diagnósticos para tres enfermedades con un elevado impacto socioeconómico: el cáncer de próstata, el cáncer colorectal y la fibromialgia. Para todos los potenciales biomarcadores identificados hemos estudiado su robustez y capacidad predictiva mediante una combinación de métodos bioinformáticos y experimentales, para tratar de aportar biomarcadores capaces de superar la fase de validación correspondiente con éxito.

176 p., La osteopontina es una proteína multifuncional que se expresa en numerosos tipos celulares y tejidos. Está implicada en numerosos procesos fisiológicos y patológicos. La OPN se ha asociado a diferentes trastornos hepáticos, de hecho, su expresión se encuentra incrementada en diferentes etapas de la enfermedad del hígado graso no alcohólico (EHGNA). Además, juega un papel regulado el metabolismo lipídico hepático. Por lo tanto, en base a la evidencia de que la OPN está relacionado con trastornos hepáticos, cuya prevalencia aumenta con la edad; y que OPN desempeña un papel en el metabolismo de lípidos hepáticos, que se encuentra desregulado en el envejecimiento, proponemos que OPN podría tener un papel en la susceptibilidad del hígado a la progresión del EHGNA asociada al envejecimiento. Para este estudio se empleó una cohorte de pacientes con hígado sano y otra con EHGNA, además emplearon animales control y deficientes en OPN (OPN-KO) de 3, 10 y 20 meses de edad, así como líneas celulares hepáticas para estudios in-vitro. Los resultados mostraron que la OPN está implicada en el envejecimiento del hígado y que ejerce un papel protector. En suero, OPN se correlaciona positivamente con la edad en individuos y modelos animales con hígado normal. En los modelos animales, el aumento de los niveles de OPN en suero está relacionado con el aumento de los niveles en el hígado. La inducción de senescencia en células HepG2, a través de la modulación de p53, aumenta los niveles de OPN celular y su secreción. OPN está regulada para mantener el equilibrio del metabolismo lipídico del hígado durante el envejecimiento. La deficiencia de OPN en ratones hace que el hígado sea más vulnerable a la hepatoteatosis asociada a la edad, siendo el mecanismo subyacente el aumento de la lipogénesis de novo asociada a la senescencia y a una alteración de la respuesta al estrés de retículo debido a la disminución de los niveles de la chaperona GRP78.

BACKGROUND: Even though liver kinase B1 (LKB1) is usually described as a tumor suppressor in a wide variety of tissues, it has been shown that LKB1 aberrant expression is associated with bad prognosis in Hepatocellular Carcinoma (HCC). METHODS: Herein we have overexpressed LKB1 in human hepatoma cells and by using histidine pull-down assay we have investigated the role of the hypoxia-related post-translational modification of Small Ubiquitin-related Modifier (SUMO)ylation in the regulation of LKB1 oncogenic role. Molecular modelling between LKB1 and its interactors, involved in regulation of LKB1 nucleocytoplasmic shuttling and LKB1 activity, was performed. Finally, high affinity SUMO binding entities-based technology were used to validate our findings in a pre-clinical mouse model and in clinical HCC. FINDINGS: We found that in human hepatoma cells under hypoxic stress, LKB1 overexpression increases cell viability and aggressiveness in association with changes in LKB1 cellular localization. Moreover, by using site-directed mutagenesis, we have shown that LKB1 is SUMOylated by SUMO-2 at Lys178 hampering LKB1 nucleocytoplasmic shuttling and fueling hepatoma cell growth. Molecular modelling of SUMO modified LKB1 further confirmed steric impedance between SUMOylated LKB1 and the STe20-Related ADaptor cofactor (STRADalpha), involved in LKB1 export from the nucleus. Finally, we provide evidence that endogenous LKB1 is modified by SUMO in pre-clinical mouse models of HCC and clinical HCC, where LKB1 SUMOylation is higher in fast growing tumors. INTERPRETATION: Overall, SUMO-2 modification of LKB1 at Lys178 mediates LKB1 cellular localization and its oncogenic role in liver cancer. FUND: This work was supported by grants from NIH (US Department of Health and Human services)-R01AR001576-11A1 (J.M.M and M.L.M-C.), Gobierno Vasco-Departamento de Salud 2013111114 (to M.L.M.-C), ELKARTEK 2016, Departamento de Industria del Gobierno Vasco (to M.L.M.-C), MINECO: SAF2017-87301-R a

El contenido de los capítulos IV, V y VI está sujeto a confidencialidad por el autor. 117 p., Chronic liver disease refers to a wide group of pathologies caused by several damaging agents and characterized by a slow progression and final development of cirrhosis and hepatocellular carcinoma (HCC). It represents one of the most frequent causes of mortality in Western countries and its increase in last years and the lack of effective treatment highlight the necessity of the better characterization of the disease. Dysregulation of methionine and S-adenosylmethionine (SAMe) metabolism and enzymes participating in the methionine cycle has been implicated in chronic liver disease. Glycine N-methyltransferase (GNMT), the most important SAMe-catabolizing enzyme, is downregulated in liver pathologies such as NAFLD, cholestasis, fibrosis, cirrhosis and HCC. Nevertheless, there are few studies describing its downregulation in liver disease, which may be of interest to develop targeted therapies. We describe novel microRNAs (small non-coding RNAs that regulate gene expression at the posttranscriptional level by mRNA repression) repressing GNMT in chronic liver diseases such as NAFLD, cholestasis and fibrosis and HCC.MiR-873-5p regulates GNMT in NAFLD. Moreover, we describe new functions of mitochondrial GNMT essential for OXPHOS and ß-oxidation processes. Inhibiting this microRNA results in increased cytoplasmic and mitochondrial GNMT levels, enhanced Complex II activity in the ETC and sustained ß-oxidation, avoiding lipid accumulation, inflammation and fibrosis. This microRNA also participates in cholestatic fibrosis, targeting GNMT in hepatocytes, cholangiocytes, inflammatory and fibrogenic cells. Inhibition of miR-873-5p recovers GNMT expression in the different hepatic cell types, avoiding SAMe accumulation and aberrant DNA methylation in the liver, which finally results in an appropriate response and defense against bile acid induced liver damage.Finally, we identify miR-518d-5p is upregulated in HCC acting as an oncomiRs and conferring drug resistance to sorafenib

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.

Tesis restringida por decisión de la autora 151 p., La ácido graso translocasa CD36 es un transportador de ácidos grasos sin presencia y/o función destacables en el hígado sano. Sin embargo, en el hígado de pacientes con esteatohepatitis su expresión se correlaciona con el desarrollo de hepatoesteatosis, resistencia periférica a insulina y con el grado de inflamación y apoptosis. Perturbaciones en la homeostasis del retículo endoplásmico celular conducen a la activación de la respuesta a proteínas mal plegadas (UPR). Respuesta que se entrecruza con vías de señalización involucradas en inflamación y estrés oxidativo, habiéndose encontrado vinculada a diversas patologías hepáticas, como la enfermedad de hígado graso no alcohólica. En este trabajo demostramos el papel modulador de CD36 en la vía de UPR IRE1¿, su implicación en la activación de la ß-oxidación mitocondrial en el hepatocito y cómo, a pesar de que su ausencia en hígado protege frente a la inflamación y estrés oxidativo inducidos por tunicamicina, su presencia es necesaria para amortiguar la esteatosis derivada de este tratamiento. Entre los mecanismos implicados en la generación de esta hepatoesteatosis, CD36 regula la función lisosomal implicada en el aporte lipídico para la formación y secreción de VLDL.

108 p. El contenido de los capítulos 5, 6 y 7 están sujetos a confidencialidad, En esta tesis doctoral hemos corroborado que tanto una correcta función mitocondrial como la homeostasis de proteínas son fundamentales para el correcto funcionamiento del hígado y que alteraciones en ambos procesos están asociados con la lesión hepática. Hemos descubierto nuevos mecanismos subyacentes de la enfermedad hepática, a nivel mitocondrial y de modificaciones post-traduccionales que podrían ayudar al desarrollo de nuevos fármacos y terapias para su tratamiento. Concretamente, hemos descrito que MCJ es un regulador negativo del metabolismo mitocondrial en hígado y que su expresión está altamente implicada en el daño hepático por fármacos y en hígado graso no alcohólico. De manera importante, hemos demostrado que la deficiencia en MCJ podría ser una terapia prometedora para estas enfermedades, ya que su silenciamiento en modelos experimentales animales ha resultado muy efectivo, manteniendo las mitocondrias activas y evitando el daño hepático y la progresión de la enfermedad. Por otro lado hemos descrito que la proteína mitocondrial PHB1 tiene un papel en colestasis, ya que sus niveles se encuentran disminuidos específicamente en pacientes con enfermedades colestásicas y que su deficiencia predispone al daño por ligadura del conducto biliar. Hemos visto que en colestasis, a parte de su función mitocondrial, bajos niveles de PHB1 regulan la actividad del proteosoma e inducen cambios transcripcionales a través de la sobreexpresión y localización nuclear de HDAC4. En este respecto, hemos observado que la disminución de la expresión de HDAC4 mejora el daño hepático asociado a la deficiencia de PHB1. Finalmente, hemos presentado evidencias sobre el importante papel de la nedilización en el desarrollo del CHC y hemos identificado LKB1 y Akt, conocidas oncoquinasas, como nuevas dianas de Nedd8. Tanto el grado de nedilización como los niveles de LKB1 y Akt se asocian a un peor pronóstico. Además, hemos demostrado que inhibir la nedilización bloquea el crecimiento tu

154 p., La enfermedad hepática crónica es un término que incluye una larga lista de patologías hepáticas y que suele finalizar con la pérdida de integridad del parénquima hepático degenerando en cirrosis y carcinoma hepatocelular (CHC). La enfermedad hepática crónica puede estar causada por distintas etiologías, como la infección por los virus de la hepatitis B y C, toxinas, consumo de alcohol y drogas, enfermedades autoinmunes y hereditarias y la enfermedad del hígado graso no alcohólica (EHGNA). Se han investigado múltiples mecanismos moleculares que dan lugar a la enfermedad hepática crónica, aunque hoy en dia la contribución de las modificaciones postraduccionales a esta enfermedad es un área aún por estudiar. Las modificaciones postraduccionales (PTM) se producen por la conjugación reversible de una proteína o un grupo funcional a determinada proteína diana. Las PTM regulan tanto la activación como la inactivación de sus proteínas dianas, e inducen cambios en la localización celular, degradación/estabilidad, formación de complejos, etc. Dentro de las modificaciones postransduccionales, nuestros estudios se han dirigido a las denominadas ¿Ubiquitin like¿ (UBL), que incluyen todas las modificaciones ocasionadas por proteínas cuya función sea similar a la ubiquitina. Se estima que hay alrededor de 16 UBL con funciones y actividades distintas. Todas ellas comparten un ciclo de vida parecido con similitudes en su activación y procesamiento. Hoy en día, hay tres UBL que concentran la atención de la mayoría de las investigaciones: la ubiquitina, SUMO y NEDD8. NEDD8 (Neural precursor cell expressed developmentally downregulated-8) es una de las UBL más similares a la ubiquitina (59%) pero tiene dianas y funciones completamente distintas. La principal función de NEDD8 consiste en estabilizar las proteínas a las que modifica alargando su vida media. La proteína diana mejor conocida de esta UBL son las conocidas como ¿cullins¿ que forman parte de los complejos cullin-RING-ligases

186 p., La enfermedad del hígado graso no alcohólica, EHGNA o NAFLD, es la causa más frecuente en el mundo de enfermedad crónica hepática. Su desarrollo se ha asociado en algunos casos a un metabolismo errático de S-Adenosilmetionina (SAMe). SAMe es una molécula donadora de metilos que participa en múltiples procesos celulares. En éste trabajo demostramos que el exceso hepático de SAMe,a través de la vía PEMT, de la que es sustrato, es capaz de inducir el acúmulo de triglicérido en el hígado al mismo tiempo que induce la secreción de éste al torrente circulatorio en forma de VLDLs. Además, lasVLDLs que se secretan en el contexto de elevados niveles de SAMe presentan unas características específicas que contribuyen a que su aclaramiento del plasma sanguíneo sea mayor. Las características yla secreción hepática de VLDL no sólo se ve afectada en contextos con elevado SAMe, sino también cuando los niveles de SAMe son bajos. SAMe es por tanto capaz de modular el metabolismo lipídico hepático al mismo tiempo que el metabolismo sistémico de VLDL.

150 p., Osteopontin (OPN) is a cytokine and matrix molecule that is upregulated in human and mouse models of chronic liver injury and directly promotes liver fibrogenesis by activating hepatic stellate cells (HSC) and liver progenitors (LPC). OPN is secreted by multiple cell types including HSC, LPC, as well as recruited immune cells such as natural killer T (NKT) cells, which amplify the fibrogenic response. In vivo, OPN neutralization abrogates the LPC response, inhibits HSC activation, and reduces liver fibrosis, thus demonstrating that targeting OPN is a potentially attractive anti-fibrotic strategy.

El contenido de los capítulos C,D,E,F,G e I están sujetos a confidencialidad 78 p. [168 p.], Osteopontin deficiency protects from obesity-related hepatosteatosis and liver fibrosis. Here we have investigated the role of osteopontin as a direct regulator of liver metabolism and whether osteopontin could be a serum-biomarker of liver metabolic dysfunction in non-alcoholic fatty-liver (NAFL) disease patients. We demonstrate that osteopontin regulates the crosstalk between liver cholesterol and phosphatidylcholine metabolism, being the key process the conversion of cholesterol into bile acids, frequently decreased in NAFL. In osteopontin-deficient mice enhanced cholesterogenesis in hepatocytes leads to decreased de novo lipogenesis and in vivo inhibition of cholesterogenesis normalizes liver phosphatidylcholine content. Finally, serum osteopontin levels correlate with liver phosphatidylcholine and cholesterol concentration in non-obese NAFL patients, in whom adipose tissue metabolic deregulation is not evident and conversion of cholesterol into bile acids is decreased. In conclusion, osteopontin is a key regulator of liver lipid metabolism and may be an early serum-biomarker of metabolic de regulation in non-obese NAFL patients.

Los factores de transcripción E2F son cruciales en la transición G1/S del ciclo celular. El factor de transcripción E2F1 regula el metabolismo oxidativo y su falta genera resistencia a obesidad. Se desconoce si E2F1 y E2F2 están implicados en la regulación de la homeostasis metabólica hepática. Por ello, el objetivo fue investigar el papel de E2F1 y E2F2 en la modulación del metabolismo lipídico hepático y su repercusión a nivel orgánico. Se utilizaron ratones macho de 3 meses E2F1-/-, E2F2-/- y sus controles que serán alimentados con dieta control o rica en grasa (HFD) durante 10 semanas. Se analizaron parámetros séricos en estado de alimento y tras 13 horas de ayuno. Se investigaron in vivo los flujos metabólicos hepáticos implicados en la disponibilidad de fosfolípidos, diglicéridos y triglicéridos (TG) tras administración de sustratos radiactivos.