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1.
Liver Transpl ; 25(4): 627-639, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30663275

RESUMO

Liver ischemia/reperfusion injury (IRI) is an important cause of liver damage especially early after liver transplantation, following liver resection, and in other clinical situations. Using rat experimental models, we identified oxaloacetate (OAA) as a key metabolite able to protect hepatocytes from hypoxia and IRI. In vitro screening of metabolic intermediates beneficial for hepatocyte survival under hypoxia was performed by measures of cell death and injury. In vivo, the effect of OAA was evaluated using the left portal vein ligation (LPVL) model of liver ischemia and a model of warm IRI. Liver injury was evaluated in vivo by serum transaminase levels, liver histology, and liver weight (edema). Levels and activity of caspase 3 were also measured. In vitro, the addition of OAA to hepatocytes kept in a hypoxic environment significantly improved cell viability (P < 0.01), decreased cell injury (P < 0.01), and improved energy metabolism (P < 0.01). Administration of OAA significantly reduced the extent of liver injury in the LPVL model with lower levels of alanine aminotransferase (ALT; P < 0.01), aspartate aminotransferase (AST; P < 0.01), and reduced liver necrosis (P < 0.05). When tested in a warm IRI model, OAA significantly decreased ALT (P < 0.001) and AST levels (P < 0.001), prevented liver edema (P < 0.001), significantly decreased caspase 3 expression (P < 0.01), as well as histological signs of cellular vesiculation and vacuolation (P < 0.05). This was associated with higher adenosine triphosphate (P < 0.05) and energy charge levels (P < 0.01). In conclusion, OAA can significantly improve survival of ischemic hepatocytes. The hepatoprotective effect of OAA was associated with increased levels of liver bioenergetics both in vitro and in vivo. These results suggest that it is possible to support mitochondrial activity despite the presence of ischemia and that OAA can effectively reduce ischemia-induced injury in the liver.


Assuntos
Transplante de Fígado/efeitos adversos , Ácido Oxaloacético/administração & dosagem , Substâncias Protetoras/administração & dosagem , Traumatismo por Reperfusão/prevenção & controle , Isquemia Quente/efeitos adversos , Alanina Transaminase/sangue , Alanina Transaminase/metabolismo , Animais , Aspartato Aminotransferases/sangue , Aspartato Aminotransferases/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Metabolismo Energético/efeitos dos fármacos , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Fígado/citologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Masculino , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Cultura Primária de Células , Ratos , Traumatismo por Reperfusão/sangue , Traumatismo por Reperfusão/etiologia
2.
PLoS Pathog ; 9(6): e1003416, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23785285

RESUMO

To identify new regulators of antiviral innate immunity, we completed the first genome-wide gene silencing screen assessing the transcriptional response at the interferon-ß (IFNB1) promoter following Sendai virus (SeV) infection. We now report a novel link between WNT signaling pathway and the modulation of retinoic acid-inducible gene I (RIG-I)-like receptor (RLR)-dependent innate immune responses. Here we show that secretion of WNT2B and WNT9B and stabilization of ß-catenin (CTNNB1) upon virus infection negatively regulate expression of representative inducible genes IFNB1, IFIT1 and TNF in a CTNNB1-dependent effector mechanism. The antiviral response is drastically reduced by glycogen synthase kinase 3 (GSK3) inhibitors but restored in CTNNB1 knockdown cells. The findings confirm a novel regulation of antiviral innate immunity by a canonical-like WNT/CTNNB1 signaling pathway. The study identifies novel avenues for broad-spectrum antiviral targets and preventing immune-mediated diseases upon viral infection.


Assuntos
Glicoproteínas/imunologia , Imunidade Inata , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Infecções por Respirovirus/imunologia , Vírus Sendai/imunologia , Proteínas Wnt/imunologia , Via de Sinalização Wnt/imunologia , Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Transporte/imunologia , Proteínas de Transporte/metabolismo , Linhagem Celular , Proteína DEAD-box 58 , RNA Helicases DEAD-box/imunologia , RNA Helicases DEAD-box/metabolismo , Feminino , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/imunologia , Estudo de Associação Genômica Ampla , Glicoproteínas/metabolismo , Humanos , Interferon beta/imunologia , Interferon beta/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Masculino , Interferência de RNA , Proteínas de Ligação a RNA , Receptores Imunológicos , Infecções por Respirovirus/metabolismo , Infecções por Respirovirus/patologia , Vírus Sendai/metabolismo , Proteínas Wnt/metabolismo
3.
PLoS One ; 19(6): e0304405, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38857235

RESUMO

The liver is a highly specialized organ involved in regulating systemic metabolism. Understanding metabolic reprogramming of liver disease is key in discovering clinical biomarkers, which relies on robust tissue biobanks. However, sample collection and storage procedures pose a threat to obtaining reliable results, as metabolic alterations may occur during sample handling. This study aimed to elucidate the impact of pre-analytical delay during liver resection surgery on liver tissue metabolomics. Patients were enrolled for liver resection during which normal tissue was collected and snap-frozen at three timepoints: before transection, after transection, and after analysis in Pathology. Metabolomics analyses were performed using 1H Nuclear Magnetic Resonance (NMR) and Liquid Chromatography-Mass Spectrometry (LC-MS). Time at cryopreservation was the principal variable contributing to differences between liver specimen metabolomes, which superseded even interindividual variability. NMR revealed global changes in the abundance of an array of metabolites, namely a decrease in most metabolites and an increase in ß-glucose and lactate. LC-MS revealed that succinate, alanine, glutamine, arginine, leucine, glycerol-3-phosphate, lactate, AMP, glutathione, and NADP were enhanced during cryopreservation delay (all p<0.05), whereas aspartate, iso(citrate), ADP, and ATP, decreased (all p<0.05). Cryopreservation delays occurring during liver tissue biobanking significantly alter an array of metabolites. Indeed, such alterations compromise the integrity of metabolomic data from liver specimens, underlining the importance of standardized protocols for tissue biobanking in hepatology.


Assuntos
Bancos de Espécimes Biológicos , Criopreservação , Fígado , Metabolômica , Humanos , Criopreservação/métodos , Fígado/metabolismo , Metabolômica/métodos , Masculino , Pessoa de Meia-Idade , Feminino , Adulto , Idoso , Metaboloma , Fatores de Tempo , Cromatografia Líquida/métodos , Espectroscopia de Ressonância Magnética/métodos , Espectrometria de Massas/métodos , Bancos de Tecidos
4.
Gastroenterology ; 142(1): 130-139.e4, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21945831

RESUMO

BACKGROUND & AIMS: Development of fibrosis is part of the pathophysiologic process of chronic liver disease. Although it is considered deleterious, it also represents a form of tissue repair. Deposition of extracellular matrix changes the cellular environment of the liver; we investigated whether it increases resistance to noxious stimuli and the role of changes in intracellular signaling to hepatocytes in mediating this effect. METHODS: Primary cultures of mouse hepatocytes were exposed to type I collagen (COL1); cell injury was assessed by morphologic and biochemical criteria. The expression of Bcl-2 family members was evaluated by immunoblot analyses. Activation of extracellular signal-regulated kinase (ERK) was assessed using phospho-specific antibodies. Liver fibrosis was induced by repeated administration of thioacetamide or carbon tetrachloride to mice; mice were then exposed to Fas antibodies. RESULTS: Hepatocytes exposed to COL1 were more resistant to a variety of hepatotoxins, in a dose-dependent manner, and had lower levels of Bad, Bid, and Bax proapoptotic proteins compared with control hepatocytes. Activation of ERK1/2 was stronger and quicker in hepatocytes exposed to COL1. The MEK1/2 inhibitors U0126 and PD98059 reversed the protective effects of COL1 and the decrease in proapoptotic proteins. Hepatocytes isolated from ERK1(-/-) mice were insensitive to the protective effect of COL1. Fibrotic livers from wild-type mice had high levels of phospho-ERK1 and were resistant to Fas-induced cell death. ERK1(-/-) mice lost this effect. CONCLUSIONS: Production of COL1 during liver fibrosis induces a hepatoprotective response that is mediated by activation of ERK1 signaling.


Assuntos
Doença Hepática Induzida por Substâncias e Drogas/prevenção & controle , Cirrose Hepática Experimental/patologia , Fígado/patologia , Doença Aguda , Animais , Apoptose , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Western Blotting , Tetracloreto de Carbono , Células Cultivadas , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/patologia , Colágeno Tipo I/metabolismo , Citoproteção , Ativação Enzimática , Fígado/efeitos dos fármacos , Fígado/metabolismo , Cirrose Hepática Experimental/induzido quimicamente , Cirrose Hepática Experimental/metabolismo , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 1/metabolismo , MAP Quinase Quinase 2/antagonistas & inibidores , MAP Quinase Quinase 2/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais , Tioacetamida , Fatores de Tempo , Proteína X Associada a bcl-2/metabolismo , Proteína de Morte Celular Associada a bcl/metabolismo , Receptor fas/metabolismo
5.
Cancers (Basel) ; 15(12)2023 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-37370840

RESUMO

BACKGROUND: Hepatocellular carcinoma (HCC) is a major contributor to cancer-related morbidity and mortality burdens globally. Given the fundamental metabolic activity of hepatocytes within the liver, hepatocarcinogenesis is bound to be characterized by alterations in metabolite profiles as a manifestation of metabolic reprogramming. METHODS: HCC and adjacent non-tumoral liver specimens were obtained from patients after HCC resection. Global patterns in tissue metabolites were identified using non-targeted 1H Nuclear Magnetic Resonance (1H-NMR) spectroscopy whereas specific metabolites were quantified using targeted liquid chromatography-mass spectrometry (LC/MS). RESULTS: Principal component analysis (PCA) within our 1H-NMR dataset identified a principal component (PC) one of 53.3%, along which the two sample groups were distinctively clustered. Univariate analysis of tissue specimens identified more than 150 metabolites significantly altered in HCC compared to non-tumoral liver. For LC/MS, PCA identified a PC1 of 45.2%, along which samples from HCC tissues and non-tumoral tissues were clearly separated. Supervised analysis (PLS-DA) identified decreases in tissue glutathione, succinate, glycerol-3-phosphate, alanine, malate, and AMP as the most important contributors to the metabolomic signature of HCC by LC/MS. CONCLUSIONS: Together, 1H-NMR and LC/MS metabolomics have the capacity to distinguish HCC from non-tumoral liver. The characterization of such distinct profiles of metabolite abundances underscores the major metabolic alterations that result from hepatocarcinogenesis.

6.
J Hepatol ; 56(1): 70-7, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21835140

RESUMO

BACKGROUND & AIMS: Innate sensing of viral infection activates a global defense response including type I interferon (IFN) and IFN-stimulated genes (ISGs) expression. We previously reported that HCV NS3/4A protease, an essential protein in viral polyprotein processing, can abrogate antiviral signaling pathways and effectors' response when ectopically expressed in human hepatocytes by cleaving antiviral adaptor CARDIF. However, whether HCV mediates evasion of innate immunity in patients with chronic infection remains unclear. METHODS: In this study, paired liver biopsies and corresponding purified hepatocytes of chronic hepatitis C patients and controls were subjected to transcriptional analysis of selected innate immune genes and to CARDIF protein detection. RESULTS: We report that an antiviral response is largely supported by infected hepatocytes as demonstrated by upregulation of the representative antiviral genes ISG15, ISG56, and OASL as well as chemokines genes CXCL9, CXCL10, and CXCL11 measured in both HCV-derived liver biopsies and hepatocytes; that the mRNA levels of these indicator ISGs correlate inversely with HCV RNA level; and more importantly that expression of the early responsive IRF3-dependent genes type I IFNß, type III IL28A/IL29, and chemokine CCL5 are severely compromised and associated to a global decrease of CARDIF adaptor in infected hepatocytes. CONCLUSIONS: Altogether the data argue for a strong viral strategy that counteracts the host's early antiviral response of hepatocytes from chronic patients without impairing ISGs induced via classical IFN pathway.


Assuntos
Hepatite C Crônica/imunologia , Imunidade Inata , Fígado/imunologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adulto , Idoso , Estudos de Casos e Controles , Quimiocinas/genética , Feminino , Hepatite C Crônica/genética , Hepatite C Crônica/metabolismo , Hepatócitos/imunologia , Hepatócitos/metabolismo , Hepatócitos/virologia , Humanos , Imunidade Inata/genética , Fatores Reguladores de Interferon/genética , Interferons/genética , Fígado/metabolismo , Fígado/virologia , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , Regulação para Cima
7.
Apoptosis ; 17(2): 143-53, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22109881

RESUMO

Hepatocyte death due to apoptosis is a hallmark of almost every liver disease. Manipulation of cell death regulatory steps during the apoptotic process is therefore an obvious goal of biomedical research. To clarify whether metabolic changes occur prior to the characteristic apoptotic events, we used ex vivo multinuclear NMR-spectroscopy to study metabolic pathways of [U-(13)C]glucose in mouse liver during Fas-induced apoptosis. We addressed whether these changes could be associated with protection against apoptosis afforded by Epidermal Growth Factor (EGF). Our results show that serum alanine and aspartate aminotransferase levels, caspase-3 activity, BID cleavage and changes in cellular energy stores were not observed before 3 h following anti-Fas injection. However, as early as 45 min after anti-Fas treatment, we observed upregulation of carbon entry (i.e. flux) from glucose into the Krebs-cycle via pyruvate dehydrogenase (PDH) and pyruvate carboxylase (PC) (up to 139% and 123% of controls, respectively, P < 0.001). This was associated with increased glutathione synthesis. EGF treatment significantly attenuated Fas-induced apoptosis, liver injury and the late decrease in energy stores, as well as the early fluxes through PDH and PC which were comparable to untreated controls. Using ex vivo multinuclear NMR-spectroscopic analysis, we have shown that Fas receptor activation in mouse liver time-dependently affects specific metabolic pathways of glucose. These early upregulations in glucose metabolic pathways occur prior to any visible signs of apoptosis and may have the potential to contribute to the initiation of apoptosis by maintaining mitochondrial energy production and cellular glutathione stores.


Assuntos
Apoptose , Glucose/metabolismo , Hepatócitos/metabolismo , Mitocôndrias Hepáticas/metabolismo , Receptor fas/antagonistas & inibidores , Receptor fas/metabolismo , Animais , Anticorpos/administração & dosagem , Anticorpos/imunologia , Apoptose/efeitos dos fármacos , Fator de Crescimento Epidérmico/administração & dosagem , Glutationa/metabolismo , Hepatócitos/patologia , Hepatócitos/ultraestrutura , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Oxirredução , Regulação para Cima , Receptor fas/imunologia
8.
Intervirology ; 54(1): 1-9, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-20689310

RESUMO

OBJECTIVE: To determine whether liver-derived hepatitis C RNA-containing particles express the E1E2 discontinuous antigenic determinant defined by unique monoclonal antibody (mAb) D32.10 which recognizes three highly conserved segments in E1 (aa297-306) and E2 (aa480-494 and aa613-621) envelope glycoproteins. METHODS: Human hepatocytes were isolated from HCV-infected cirrhotic explanted livers. The liver-derived hepatitis C virus (HCV) particles released from three distinct cultures (genotypes 1b and 2b) were characterized. HCV RNA+ was quantified by real-time RT-PCR. The E1E2 antigenic activity was assessed by indirect ELISA and immunoblotting using D32.10. The density distributions of HCV RNA and E1E2 antigen were determined by isopycnic sucrose density gradients. HCV E1E2, E2 and core antigens were detected in the cells by immunochemical staining. RESULTS: Liver-derived HCV particles contained HCV RNA (106-107 copies/mg of protein) and core proteins and expressed the E1E2/D32.10 epitope. HCV RNA and E1E2 cosedimented between 1.15 and 1.25 g/ml in sucrose gradients. Moreover, the mAb D32.10 detected E1E2 by immunostaining in HCV-infected hepatocytes in parallel with E2 and core antigens. CONCLUSION: Our results provide evidence that the mAb D32.10 recognizes E1E2 envelope complexes expressed in the cell cytoplasm and on the surface of HCV RNA-containing particles released from short-term cultures of in vivo infected hepatocytes.


Assuntos
Regulação Viral da Expressão Gênica , Hepacivirus/genética , Hepatócitos/metabolismo , Peptídeos/imunologia , Proteínas do Envelope Viral/genética , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Células Cultivadas , Centrifugação Isopícnica , Epitopos/imunologia , Epitopos/metabolismo , Hepacivirus/metabolismo , Hepatite C/genética , Hepatite C/virologia , Hepatócitos/virologia , Humanos , Cirrose Hepática/fisiopatologia , Peptídeos/genética , Peptídeos/metabolismo , RNA Viral , Proteínas do Envelope Viral/imunologia , Proteínas do Envelope Viral/metabolismo
9.
Cancers (Basel) ; 13(17)2021 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-34503295

RESUMO

Metabolic reprogramming and deregulated cellular energetics are hallmarks of cancer. The aberrant metabolism of cancer cells is thought to be the product of differential oncogene activation and tumor suppressor gene inactivation. MYC is one of the most important oncogenic drivers, its activation being reported in a variety of cancer types and sub-types, among which are the most prevalent and aggressive of all malignancies. This review aims to offer a comprehensive overview and highlight the importance of the c-Myc transcription factor on the regulation of metabolic pathways, in particular that of glutamine and glutaminolysis. Glutamine can be extensively metabolized into a variety of substrates and be integrated in a complex metabolic network inside the cell, from energy metabolism to nucleotide and non-essential amino acid synthesis. Together, understanding metabolic reprogramming and its underlying genetic makeup, such as MYC activation, allows for a better understanding of the cancer cell phenotype and thus of the potential vulnerabilities of cancers from a metabolic standpoint.

10.
J Hepatol ; 52(2): 167-75, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-20006398

RESUMO

BACKGROUND & AIMS: Molecular sensors recognize viral nucleic acids and initiate events that subsequently enable cells to control and clear infection. Hepatitis C Virus (HCV) can interfere with the innate host response and the NS3/4A protease was reported to specifically block antiviral signaling pathways, a finding that had yet to be studied in human primary hepatocytes. METHODS: Freshly isolated human primary hepatocytes, transduced with a lentiviral vector expressing HCV NS3/4A were stimulated with extracellular and intracellular double-stranded RNA (dsRNA) and the innate immune antiviral genes were quantified by quantitative PCR and microarrays analysis. RESULTS: We demonstrate that sensing receptors of human hepatocytes in primary cultures are stimulated following recognition of either mode of dsRNA delivery, inducing transcriptional up-regulation (over 100-fold) of multiple immune genes, either selectively or independently of recognition pathways. We also report that the intracellular dsRNA-activated innate response is severely compromised upon ectopic expression of the HCV NS3/4A protease gene in normal human primary hepatocytes, and completely restored by treatment with the NS3/4A protease specific inhibitor BILN2061. CONCLUSIONS: The present study indicates that NS3/4A has a wider protease-dependent effect on the intracellular Pathogen Recognition Receptor (PRR)-mediated immune response than on its extracellular counterpart, which underlies the major role of cytosolic dsRNA receptors in HCV recognition by primary human hepatocytes.


Assuntos
Hepatócitos/metabolismo , Hepatócitos/virologia , Proteínas não Estruturais Virais/metabolismo , Células Cultivadas , Perfilação da Expressão Gênica , Hepacivirus/imunologia , Hepacivirus/metabolismo , Hepatócitos/imunologia , Humanos , Imunidade Inata , Técnicas In Vitro , Análise de Sequência com Séries de Oligonucleotídeos , RNA/genética , RNA/metabolismo , Transdução de Sinais
11.
Liver Int ; 29(6): 942-9, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19302183

RESUMO

BACKGROUND/AIM: Since the discovery of hepatitis C virus (HCV), researchers have encountered difficulties with in vitro models. The aim of this study was to determine whether HCV-infected human primary hepatocytes, isolated from cirrhotic livers at liver transplantation, can be used as a model to study HCV infection. METHODS: Hepatocytes were isolated with collagenase and cultured over a 20-day period on different matrices. Viral kinetics was monitored with/without treatment by real-time polymerase chain reaction. RESULTS: Cell yield and viability were higher with uninfected/non-cirrhotic livers (77.2+/-1.8%) in comparison with HCV-infected cirrhotic livers (68.8+/-12%). HCV-infected hepatocytes behaved similar to non-infected cells and expressed albumin and cytochrome P4502E1. HCV-positive strand was identified in supernatants and cell lysates. HCV-negative strand was only found inside cells and correlated with viral RNA recovery in the medium. Improvement in the degree of hepatocyte differentiation was associated with better HCV recovery. Antiviral treatment with interferon-alpha, EX4 and cyclosporine A induced significant reductions in HCV RNA. CONCLUSION: Primary cultures of HCV-infected human hepatocytes from end-stage cirrhotic livers is feasible, represents an excellent model to study specific virus-host interactions and can be used to assess viral replication.


Assuntos
Hepacivirus/genética , Hepatite C/virologia , Hepatócitos/virologia , Interações Hospedeiro-Patógeno , Fígado/citologia , Fígado/virologia , Análise de Variância , Western Blotting , Técnicas de Cultura de Células , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Ciclosporina/farmacologia , Primers do DNA/genética , Hepacivirus/fisiologia , Hepatócitos/efeitos dos fármacos , Humanos , Interferon-alfa/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Replicação Viral/efeitos dos fármacos
12.
Oncotarget ; 9(42): 26868-26883, 2018 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-29928490

RESUMO

HCC (Hepatocellular carcinoma) cells exhibit greater metabolic plasticity than normal hepatocytes since they must survive in a dynamic microenvironment where nutrients and oxygen are often scarce. Using a metabolomic approach combined with functional in vitro and in vivo assays, we aimed to identify an HCC metabolic signature associated with increased tumorigenicity and patient mortality. Metabolite profiling of HCC Dt81Hepa1-6 cells revealed that their increased tumorigenicity was associated with elevated levels of glycolytic metabolites. Tumorigenic Dt81Hepa1-6 also had an increased ability to uptake glucose leading to a higher glycolytic flux that stemmed from an increased expression of glucose transporter GLUT-1. Dt81Hepa1-6-derived tumors displayed increased mRNA expressions of glycolytic genes, Hypoxia-inducible factor-1alpha and of Cyclin D1. HCC tumors also displayed increased energy charge, reduced antioxidative metabolites and similar fatty acid biosynthesis compared to healthy liver. Increased tumoral expression of glycolytic and hypoxia signaling pathway mRNAs was associated with decreased survival in HCC patients. In conclusion, HCC cells can rapidly alter their metabolism according to their environment and switch to the use of glucose through aerobic glycolysis to sustain their tumorigenicity and proliferative ability. Therefore, cancer metabolic reprogramming could be essential for the tumorigenicity of HCC cells during cancer initiation and invasion.

13.
Cell Cycle ; 17(7): 903-916, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29633904

RESUMO

Hepatocellular carcinoma (HCC) is a metabolically heterogeneous cancer and the use of glucose by HCC cells could impact their tumorigenicity. Dt81Hepa1-6 cells display enhanced tumorigenicity compared to parental Hepa1-6 cells. This increased tumorigenicity could be explained by a metabolic adaptation to more restrictive microenvironments. When cultured at high glucose concentrations, Dt81Hepa1-6 displayed an increased ability to uptake glucose (P<0.001), increased expression of 9 glycolytic genes, greater GTP and ATP (P<0.001), increased expression of 7 fatty acid synthesis-related genes (P<0.01) and higher levels of Acetyl-CoA, Citrate and Malonyl-CoA (P<0.05). Under glucose-restricted conditions, Dt81Hepa1-6 used their stored fatty acids with increased expression of fatty acid oxidation-related genes (P<0.01), decreased triglyceride content (P<0.05) and higher levels of GTP and ATP (P<0.01) leading to improved proliferation (P<0.05). Inhibition of lactate dehydrogenase and aerobic glycolysis with sodium oxamate led to decreased expression of glycolytic genes, reduced lactate, GTP and ATP levels (P<0.01), increased cell doubling time (P<0.001) and reduced fatty acid synthesis. When combined with cisplatin, this inhibition led to lower cell viability and proliferation (P<0.05). This metabolic-induced tumorigenicity was also reflected in human Huh7 cells by a higher glucose uptake and proliferative capacity compared to HepG2 cells (P<0.05). In HCC patients, increased tumoral expression of Glut-1, Hexokinase II and Lactate dehydrogenase correlated with poor survival (P = 2.47E-5, P = 0.016 and P = 6.58E-5). In conclusion, HCC tumorigenicity can stem from a metabolic plasticity allowing them to thrive in a broader range of glucose concentrations. In HCC, combining glycolytic inhibitors with conventional chemotherapy could lead to improved treatment efficacy.


Assuntos
Carcinoma Hepatocelular/metabolismo , Regulação Neoplásica da Expressão Gênica , Glucose/metabolismo , Glicólise/genética , Metabolismo dos Lipídeos/genética , Neoplasias Hepáticas/metabolismo , Acetilcoenzima A/metabolismo , Adaptação Fisiológica , Animais , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/mortalidade , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ácido Cítrico/metabolismo , Ciclo do Ácido Cítrico/efeitos dos fármacos , Ciclo do Ácido Cítrico/genética , Ácidos Graxos/biossíntese , Glucose/farmacologia , Transportador de Glucose Tipo 1/genética , Transportador de Glucose Tipo 1/metabolismo , Glicólise/efeitos dos fármacos , Células Hep G2 , Hexoquinase/genética , Hexoquinase/metabolismo , Humanos , L-Lactato Desidrogenase/genética , L-Lactato Desidrogenase/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/mortalidade , Neoplasias Hepáticas/patologia , Malonil Coenzima A/metabolismo , Camundongos , Transdução de Sinais , Análise de Sobrevida , Triglicerídeos/metabolismo
14.
PLoS One ; 13(6): e0199177, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29902244

RESUMO

The liver is a highly vascularized organ receiving a dual input of oxygenated blood from the hepatic artery and portal vein. The impact of decreased blood flow on glucose metabolism and how hepatocytes could adapt to this restrictive environment are still unclear. Using the left portal vein ligation (LPVL) rat model, we found that cellular injury was delayed after the onset of liver ischemia. We hypothesized that a metabolic adaptation by hepatocytes to maintain energy homeostasis could account for this lag phase. Liver glucose metabolism was characterized by 13C- and 1H-NMR spectroscopy and analysis of high-energy metabolites. ALT levels and caspase 3 activity in LPVL animals remained normal during the first 12 h following surgery (P<0.05). Ischemia rapidly led to decreased intrahepatic tissue oxygen tension and blood flow (P<0.05) and increased expression of Hypoxia-inducible factor 1-alpha. Intrahepatic glucose uptake, ATP/ADP ratio and energy charge level remained stable for up to 12 h after ligation. Entry of glucose in the Krebs cycle was impaired with lowered incorporation of 13C from [U-13C]glucose into glutamate and succinate from 0.25 to 12 h after LPVL. However, total hepatic succinate and glutamate increased 6 and 12 h after ischemia (P<0.05). Glycolysis was initially reduced (P<0.05) but reached maximum 13C-lactate (P<0.001) and 13C-alanine (P<0.01) enrichments 12 h after LPVL. In conclusion, early liver homeostasis stems from an inherent ability of ischemic hepatocytes to metabolically adapt through increased Krebs cycle and glycolysis activity to preserve bioenergetics and cell viability. This metabolic plasticity of hepatocytes could be harnessed to develop novel metabolic strategies to prevent ischemic liver damage.


Assuntos
Ciclo do Ácido Cítrico , Glicólise , Isquemia/metabolismo , Fígado/irrigação sanguínea , Regulação para Cima , Anaerobiose , Animais , Morte Celular , Hipóxia Celular , Metabolismo Energético , Hepatócitos/patologia , Homeostase , Fígado/patologia , Masculino , Mitocôndrias/metabolismo , Ratos , Ratos Sprague-Dawley , Fatores de Tempo
15.
PLoS One ; 12(12): e0190366, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29284039

RESUMO

The liver plays a key role in maintaining physiological homeostasis and hepatocytes are largely responsible for this. The use of isolated primary hepatocytes has become an essential tool for the study of nutrient physiology, xenobiotic metabolism and several liver pathologies. Since hepatocytes are removed from their normal environment, the isolation procedure and in vitro culture of primary hepatocytes is partially known to induce undesired metabolic changes. We aimed to perform a thorough metabolic profiling of primary cells before, during and after isolation using state-of-the-art techniques. Extensive metabolite measurements using HPLC were performed in situ in the liver, during hepatocyte isolation using the two-step collagenase perfusion method and during in vitro cell culture for up to 48 hours. Assessment of mitochondrial respiratory capacity and ATP-linked respiration of isolated primary hepatocytes was performed using extracellular flux analysis. Primary hepatocytes displayed a drastic decrease in antioxidative-related metabolites (NADPH, NADP, GSH and GSSG) during the isolation procedure when compared to the in situ liver (P<0.001). Parallel assessment of citric acid cycle activity showed a significant decrease of up to 95% in Acetyl-CoA, Isocitrate/Citrate ratio, Succinate, Fumarate and Malate in comparison to the in situ liver (P<0.001). While the levels of several cellular energetic metabolites such as Adenosine, AMP, ADP and ATP were found to be progressively reduced during the isolation procedure and cell culture (P<0.001), higher ATP/ADP ratio and energy charge level were observed when primary cells were cultured in vitro compared to the in situ liver (P<0.05). In addition, a significant decrease in the respiratory capacity occurred after 24 hours in culture. Interestingly, this was not associated with a significant modification of ATP-linked respiration. In conclusion, major metabolic alterations occur immediately after hepatocytes are removed from the liver. These changes persist or increase during in vitro culture. These observations need to be taken into account when using primary hepatocytes for the study of metabolism or liver physiopathology.


Assuntos
Hepatócitos/metabolismo , Animais , Antioxidantes/metabolismo , Cromatografia Líquida de Alta Pressão , Ciclo do Ácido Cítrico , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias Hepáticas/metabolismo , Estresse Oxidativo
16.
PLoS One ; 12(2): e0171215, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28152020

RESUMO

There are limited numbers of models to study hepatocellular carcinoma (HCC) in vivo in immunocompetent hosts. In an effort to develop a cell line with improved tumorigenicity, we derived a new cell line from Hepa1-6 cells through an in vivo passage in C57BL/6 mice. The resulting Dt81Hepa1-6 cell line showed enhanced tumorigenicity compared to Hepa1-6 with more frequent (28±12 vs. 0±0 lesions at 21 days) and more rapid tumor development (21 (100%) vs. 70 days (10%)) in C57BL/6 mice. The minimal Dt81Hepa1-6 cell number required to obtain visible tumors was 100,000 cells. The Dt81Hepa1-6 cell line showed high hepatotropism with subcutaneous injection leading to liver tumors without development of tumors in lungs or spleen. In vitro, Dt81Hepa1-6 cells showed increased anchorage-independent growth (34.7±6.8 vs. 12.3±3.3 colonies; P<0.05) and increased EpCAM (8.7±1.1 folds; P<0.01) and ß-catenin (5.4±1.0 folds; P<0.01) expression. A significant proportion of Dt81Hepa1-6 cells expressed EpCAM compared to Hepa1-6 (34.8±1.1% vs 0.9±0.13%; P<0.001). Enriched EpCAM+ Dt81Hepa1-6 cells led to higher tumor load than EpCAM- Dt81Hepa1-6 cells (1093±74 vs 473±100 tumors; P<0.01). The in vivo selected Dt81Hepa1-6 cell line shows high liver specificity and increased tumorigenicity compared to Hepa1-6 cells. These properties are associated with increased expression of EpCAM and ß-catenin confirming that EpCAM+ HCC cells comprise a subset with characteristics of tumor-initiating cells with stem/progenitor cell features. The Dt81Hepa1-6 cell line with its cancer stem cell-like properties will be a useful tool for the study of hepatocellular carcinoma in vivo.


Assuntos
Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/patologia , Células-Tronco Neoplásicas/patologia , Animais , Agregação Celular , Linhagem Celular Tumoral , Proliferação de Células , Citometria de Fluxo , Humanos , Masculino , Camundongos Endogâmicos C57BL , Invasividade Neoplásica , Transplante de Neoplasias , Ensaio Tumoral de Célula-Tronco
17.
PLoS One ; 11(10): e0165360, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27792745

RESUMO

Collagen produced during the process of liver fibrosis can induce a hepatocellular protective response through ERK1 signalling. However, the influence of T cells and associated cytokine production on this protection is unknown. In addition, athymic mice are frequently used in hepatocellular carcinoma xenograft experiments but current methods limit our ability to study the impact of liver fibrosis in this setting due to high mortality. Therefore, a mouse model of liver fibrosis lacking T cells was developed using Foxn1 nu/nu mice and progressive oral administration of thioacetamide (TAA) [0.01-0.02%] in drinking water. Fibrosis developed over a period of 16 weeks (alpha-SMA positive area: 20.0 ± 2.2%, preCol1a1 mRNA expression: 11.7 ± 4.1 fold changes, hydroxyproline content: 1041.2 ± 77µg/g of liver) at levels comparable to that of BALB/c mice that received intraperitoneal TAA injections [200 µg/g of body weight (bw)] (alpha-SMA positive area: 20.9 ± 2.9%, preCol1a1 mRNA expression: 13.1 ± 2.3 fold changes, hydroxyproline content: 931.6 ± 14.8µg/g of liver). No mortality was observed. Athymic mice showed phosphorylation of ERK1/2 during fibrogenesis (control 0.03 ± 0.01 vs 16 weeks 0.22 ± 0.06AU; P<0.05). The fibrosis-induced hepatoprotection against cytotoxic agents, as assessed histologically and by serum AST levels, was not affected by the absence of circulating T cells (anti-Fas JO2 [0.5µg/g bw] for 6h (fibrotic 4665 ± 2596 vs non-fibrotic 13953 ± 2260 U/L; P<0.05), APAP [750 mg/kg bw] for 6 hours (fibrotic 292 ± 66 U/L vs non-fibrotic 4086 ± 2205; P<0.01) and CCl4 [0.5mL/Kg bw] for 24h (fibrotic 888 ± 268 vs non-fibrotic 15673 ± 2782 U/L; P<0.001)). In conclusion, liver fibrosis can be induced in athymic Foxn1 nu/nu mice without early mortality. Liver fibrosis leads to ERK1/2 phosphorylation. Finally, circulating T lymphocytes and associated cytokines are not involved in the hepatocellular protection afforded by liver fibrosis.


Assuntos
Doença Hepática Induzida por Substâncias e Drogas/prevenção & controle , Cirrose Hepática/fisiopatologia , Linfócitos T/fisiologia , Animais , Western Blotting , Carcinógenos/farmacologia , Modelos Animais de Doenças , Imunofluorescência , Fígado/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Tioacetamida/farmacologia , Transcriptoma
18.
Am J Pathol ; 170(2): 497-504, 2007 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-17255318

RESUMO

Genetic iron overload, or hemochromatosis, can be caused by mutations in HFE, hemojuvelin, and hepcidin genes. Hepcidin, a negative regulator of intestinal iron absorption, is found to be inappropriately low in both patients and in animal models, indicating that proper control of basal hepcidin levels requires both hemojuvelin and HFE. In mice, repulsive guidance molecule c (Rgmc, the hemojuvelin mouse ortholog) and hepcidin levels are transcriptionally regulated during inflammation. Here, we report that basal Rgmc levels in Hfe-deficient mice are normal and that these mice retain the ability to suppress Rgmc expression after lipopolysaccharide (LPS) challenge. Thus, Rgmc regulation by LPS is Hfe-independent. The response of Rgmc to LPS involves signaling through toll-like receptor 4 (Tlr4), because Tlr4-deficient mice do not show altered Rgmc expression after LPS administration. We further show that tumor necrosis factor-alpha, but not interleukin-6, is sufficient to cause Rgmc down-regulation by LPS. These results contrast with previous data demonstrating that hepcidin levels are directly regulated by interleukin-6 but not by tumor necrosis factor-alpha. The regulation of iron-related genes by different cytokines may allow for time-dependent control of iron metabolism changes during inflammation and may be relevant to chronic inflammation, infections, and cancer settings, leading to the development of anemia of chronic disease.


Assuntos
Regulação para Baixo , Antígenos de Histocompatibilidade Classe I/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Musculares/biossíntese , Transdução de Sinais , Receptor 4 Toll-Like/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/metabolismo , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Proteínas Ligadas por GPI , Hemocromatose/genética , Hemocromatose/metabolismo , Proteína da Hemocromatose , Hepcidinas , Humanos , Inflamação/genética , Inflamação/metabolismo , Interleucina-6/metabolismo , Ferro/metabolismo , Lipopolissacarídeos/farmacologia , Proteínas de Membrana/deficiência , Camundongos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Receptor 4 Toll-Like/deficiência
19.
Am J Physiol Gastrointest Liver Physiol ; 291(2): G229-37, 2006 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16565419

RESUMO

Hepcidin is a negative regulator of iron absorption produced mainly by the liver in response to changes in iron stores and inflammation, and its levels have been shown to regulate the intestinal basolateral iron transporter ferroportin1 (Fp1). Hereditary hemochromatosis patients and Hfe-deficient mice show inappropriate expression of hepcidin but, in apparent contradiction, still retain the ability to regulate iron absorption in response to alterations of iron metabolism. To further understand the molecular relationships among Hfe, hepcidin, and Fp1, we investigated hepcidin and Fp1 regulation in Hfe-deficient mice (Hfe-/- and beta2m-/-) in response to iron deprivation, iron loading, and acute inflammation. We found that whereas basal hepcidin levels were manifestly dependent on the presence of Hfe and on the mouse background, all Hfe-deficient mice were still able to regulate hepcidin in situations of altered iron homeostasis. In the liver, Fp1 was modulated in opposite directions by iron and LPS, and its regulation in Hfe-deficient mice was similar to that observed in wild-type mice. In addition, we found that iron-deprived mice were able to mount a robust response after LPS challenge and that Toll-like receptor 4 (TLR-4)-deficient mice fail to regulate hepcidin expression in response to LPS. In conclusion, these results suggest that although Hfe is necessary for the establishment of hepcidin basal levels, it is dispensable for hepcidin regulation through both the iron-sensing and inflammatory pathways, and hepatic Fp1 regulation is largely independent of hepcidin and Hfe. The inflammatory pathway overrides the iron-sensing pathway and is TLR-4 dependent.


Assuntos
Peptídeos Catiônicos Antimicrobianos/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Inflamação/metabolismo , Sobrecarga de Ferro/metabolismo , Ferro/metabolismo , Fígado/metabolismo , Proteínas de Membrana/metabolismo , Baço/metabolismo , Animais , Células Cultivadas , Proteína da Hemocromatose , Hepcidinas , Camundongos , Camundongos Endogâmicos C3H , Camundongos Knockout
20.
Am J Physiol Gastrointest Liver Physiol ; 285(2): G298-308, 2003 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-12702491

RESUMO

Growth factors have been shown to protect cells from a variety of apoptotic stimuli. In the liver, the Fas system is thought to be very important in the genesis of hepatocyte apoptosis. Others have already shown the importance of the phosphatidylinositol 3-kinase (PI3-kinase) pathway and of increased Bcl-xl expression in the antiapoptotic effect of growth factors on hepatocytes. We investigated the effect of EGF on Bid, a BH3-only member of the Bcl-2 family and a major player in the transduction of the Fas apoptotic signal. Hepatocyte apoptosis was induced in vitro with a purified anti-mouse Fas antibody. The effect of EGF on Bid protein expression was studied on those cultures. EGF dose dependently reduced the expression of Bid protein in primary mouse hepatocyte cultures independently of Fas stimulation. This decrease was not the result of the degradation of Bid into its active p15 fragment. Treating cells with a specific inhibitor of the EGF receptor autophosphorylation completely abolished the decrease in Bid expression afforded by EGF. Treatment with LY-294002, a PI3-kinase blocker, partly reverted the effect of EGF. When apoptosis was induced in Bid-deficient hepatocytes, EGF lost its capacity to protect cells against this type of cell death. These results show that EGF decreases the expression of Bid protein and suggest that the effect of EGF on Bid is one of the mechanisms of the antiapoptotic effect of EGF.


Assuntos
Apoptose/efeitos dos fármacos , Proteínas de Transporte/genética , Fator de Crescimento Epidérmico/farmacologia , Regulação da Expressão Gênica , Hepatócitos/efeitos dos fármacos , Animais , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3 , Western Blotting , Cromonas/farmacologia , Inibidores Enzimáticos/farmacologia , Hepatócitos/citologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Morfolinas/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Reação em Cadeia da Polimerase , Proteínas Tirosina Quinases/antagonistas & inibidores
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