Background Hedgehog signaling plays an important role in embryonic development, organogenesis and cancer. In the adult liver, Hedgehog signaling in non-parenchymal cells has been found to play a role in certain disease states such as fibrosis and cirrhosis. However, whether the Hedgehog pathway is active in mature healthy hepatocytes and is of significance to liver function are controversial. Findings Two types of mice with distinct conditional hepatic deletion of the Smoothened gene, an essential co-receptor protein of the Hedgehog pathway, were generated for investigating the role of Hedgehog signaling in mature hepatocytes. The knockout animals (KO) were inconspicuous and healthy with no changes in serum transaminases, but showed a slower weight gain. The liver was smaller, but presented a normal architecture and cellular composition. By quantitative RT-PCR the downregulation of the expression of Indian hedgehog (Ihh) and the Gli3 transcription factor could be demonstrated in healthy mature hepatocytes from these mice, whereas Patched1 was upregulated. Strong alterations in gene expression were also observed for the IGF axis. While expression of Igf1 was downregulated, that of Igfbp1 was upregulated in the livers of both genders. Corresponding changes in the serum levels of both proteins could be detected by ELISA. By activating and inhibiting the transcriptional output of Hedgehog signaling in cultured hepatocytes through siRNAs against Ptch1 and Gli3, respectively, in combination with a ChIP assay evidence was collected indicating that Igf1 expression is directly dependent on the activator function of Gli3. In contrast, the mRNA level of Igfbp1 appears to be controlled through the repressor function of Gli3, while that of Igfbp2 and Igfbp3 did not change. Interestingly, body weight of the transgenic mice correlated well with IGF-I levels in both genders and also with IGFBP-1 levels in females, whereas it did not correlate with serum growth hormone levels. Conclusions Our results demonstrate for the first time that Hedgehog signaling is active in healthy mature mouse hepatocytes and that it has considerable importance for IGF-I homeostasis in the circulation. These findings may have various implications for mouse physiology including the regulation of body weight and size, glucose homeostasis and reproductive capacity.

Resveratrol is reported to possess chemotherapeutic properties in several cancers. In this study, we wanted to investigate the molecular mechanisms of resveratrol-induced cell cycle arrest and apoptosis as well as the impact of resveratrol on NAMPT and SIRT1 protein function and asked whether there are differences in hepatocarcinoma cells (HepG2, Hep3B cells) and non-cancerous primary human hepatocytes. We found a lower basal NAMPT mRNA and protein expression in hepatocarcinoma cells compared to primary hepatocytes. In contrast, SIRT1 was significantly higher expressed in hepatocarcinoma cells than in primary hepatocytes. Resveratrol induced cell cycle arrest in the S- and G2/M- phase and apoptosis was mediated by activation of p53 and caspase-3 in HepG2 cells. In contrast to primary hepatocytes, resveratrol treated HepG2 cells showed a reduction of NAMPT enzymatic activity and increased p53 acetylation (K382). Resveratrol induced NAMPT release from HepG2 cells which was associated with increased NAMPT mRNA expression. This effect was absent in primary hepatocytes where resveratrol was shown to function as NAMPT and SIRT1 activator. SIRT1 inhibition by EX527 resembled resveratrol effects on HepG2 cells. Furthermore, a SIRT1 overexpression significantly decreased both p53 hyperacetylation and resveratrol-induced NAMPT release as well as S-phase arrest in HepG2 cells. We could show that NAMPT and SIRT1 are differentially regulated by resveratrol in hepatocarcinoma cells and primary hepatocytes and that resveratrol did not act as a SIRT1 activator in hepatocarcinoma cells.

Proefschrift Rijksuniversiteit Groningen. / Datum laatste controle: 28-11-1997. Met lit. opg. - Met een samenvatting in het Nederlands.

Introdução e objetivos: Tem sido demonstrado que um moderado desacoplamento mitocondrial em células hepáticas pode reverter a hipertrigliceridemia, a doença de fígado gorduroso e a resistência à insulina. A dissipação da energia conservada no espaço intermembranas mitocontrial, como ocorre no desacoplamento, aumenta o uso de substratos energéticos e também podem reduzir a geração mitocondrial de espécies reativas de O2 (EROs). Duas estratégias de desacoplamento mitocondrial foram estudadas neste trabalho: a primeira consistiu em reduzir a velocidade da via de síntese de triacilgliceróis por meio da triacsin C (inibidor da Acil CoA Sintetase - ACS), e dessa forma aumentar os ácidos graxos livres (AGL) como substratos das proteínas desacopladoras; a segunda foi verificar se clusianona (composto natural das raízes de Clusia congestiflora), análogo estrutural do desacoplador químico nemorosoma, é capaz de promover o desacoplamento químico. Resutados e discussão: A triacsin C, em concentrações até 1 ?M, não apresentou efeito tóxico em mitocôndrias isoladas de fígado e nem em hepatócitos primários. Nesses últimos, aumentou o consumo de oxigênio nos estados de respiração basal e de máxima velocidade respiratória. Além disso, foi verificado um aumento da expressão do fator de transcrição PGC1- alfa e de ?-hidroxiacil CoA desidrogenase (HAD), uma enzima da beta-oxidação de ácidos graxos. A clusianona aumentou o consumo de O2 no estado de repouso, diminuiu o potencial de membrana, reduziu a produção de EROs e preveniu o inchamento mitocondrial induzido por Ca2+ de forma dose dependente, porém menos potente que a nemorosona. Conclusões: Nossos resultados indicam que a triacsin C acelerou o metabolismo mitocondrial, a oxidação de ácidos graxos e a biogênese mitocondrial; a clusianona foi caracterizada como um desacoplador eficaz da fosforilação oxidativa mitocondrial, provavelmente envolvendo um mecanismo protonofórico devido as suas propriedades químicas. Dessa forma, ambas as estratégias estudadas se mostram com potencial terapêutico no tratamento de doenças como esteatose hepática, hipertrigliceridemia e obesidade. / Introduction and Objectives: It has been shown that a mild mitochondrial uncoupling in livers can reverse hypertriglyceridemia, fatty liver disease and insulin resistance. The dissipation of energy stored in mitochondrial intermembrane space as heat, as in uncoupling, increases the use of energy substrates and may also reduce the mitochondrial generation of reactive O2 species (ROS). Two strategies to induce mitochondrial uncoupling were studied in this work: the first consisted of slowing down the route of triacylglycerols synthesis by triacsin C (acyl CoA synthetase inhibitor), and thus increasing the free fatty acids (FFA) as substrates for uncoupling proteins; the second was to determine whether clusianone (natural compound from the roots of Clusia congestiflora), a structural analogue of chemical uncoupler nemorosoma, is capable of promoting the chemical uncoupling. Results and discussion: The triacsin C, in concentrations up to 1 ?M, showed no toxic effect on liver mitochondria and primary hepatocytes. In hepatocytes triacsin C increased oxygen consumption in the states of basal respiration and maximum respiratory rate. In addition, there was an increase in the expression of the transcription factor PGC1 - ? and ? - hydroxyacyl CoA dehydrogenase (ADH), an enzyme of ?- oxidation of fatty acids. The clusianone increased O2 consumption in resting state, decreased membrane potential, reduced the production of ROS and prevented the mitochondrial swelling induced by Ca2+ in a dose dependent manner, but less potent than nemorosone. Conclusions: Our results indicate that triacsin C accelerated mitochondrial metabolism, fatty acid oxidation and mitochondrial biogenesis; the clusianone was characterized as an effective uncoupler of mitochondrial oxidative phosphorylation, probably involving a protonoforic mechanism due to its chemical properties. Therefore, both strategies have therapeutic potential in the treatment of diseases such as liver steatosis, hypertriglyceridemia and obesity.

O transplante de hepatócitos é uma modalidade terapêutica atrativa para as doenças hepáticas, assim como uma alternativa para o transplante hepático. O objetivo do presente estudo é investigar a efetividade do transplante de hepatócitos de ratos nos modelos de hepatotoxicidade aguda induzida por paracetamol (1g/kg e 1,5g/kg). Os hepatócitos foram isolados de ratos Wistar machos e transplantados 24 horas após em receptoras fêmeas com hepatotoxicidade de 1g/kg. Os ratos fêmeas receberam 1x107 hepatócitos (grupo 1, n=20) ou PBS (grupo 2, n=24) através da veia porta ou no baço. A análise de sobrevida em 3 dias demonstrou que todos os animais do grupo 1 sobreviveram, enquanto 5 animais do grupo 2 morreram (P=0,03), todas as mortes ocorreram nos ratos que receberam PBS através da veia porta (P=0,001). Os níveis de alanina aminotransferase e fator V que foram medidos no experimento não mostraram diferença entre o grupo 1 e o grupo 2 em nenhum momento. A análise molecular e a histologia mostraram a presença de hepatócitos no fígado de animais transplantados através da veia porta ou pelo baço. O modelo de hepatotoxicidade aguda induzida por paracetamol (1g/kg) demonstrou que o transplante de hepatócitos de ratos aumenta a sobrevida quando o local de injeção é na veia porta. No modelo de hepatotoxicidade aguda induzida por paracetamol (1,5g/kg), os hepatócitos foram isolados de ratos Wistar machos, e transplantados 6 horas após em receptoras fêmeas. Os ratos fêmeas receberam 1x107 hepatócitos (grupo 1, n=33) ou PBS (grupo 2, n=24) no baço. A análise de sobrevida em 3 dias demonstrou que 9 animais do grupo 1, e 9 animais do grupo 2 morreram no dia 2. Não houve diferença estatística significativa na análise de sobrevida entre o grupo 1 e o grupo 2. Nossos dados demonstram que o isolamento de hepatócitos é um procedimento factível. O transplante de hepatócitos é uma técnica que pode ser aplicada em modelos animais de IHA levando ao aumento da sobrevida. Entretanto, o modelo utilizado no presente estudo apresentou um alto grau de variabilidade, tornando necessária a avaliação do transplante de hepatócitos em um modelo mais reprodutível. / Hepatocyte transplantation is an attractive therapeutic modality for liver disease as an alternative for liver transplantation. The aim of the current study was to investigate the effectiveness of rat hepatocyte transplantation in the acetaminophen-induced acute hepatotoxicity models (1g/kg and 1,5g/kg). Hepatocytes were isolated from male Wistar rats and transplanted 24 hours later in female recipients with hepatotoxicity of 1g/kg of acetaminophen. Female rats received either 1x107 hepatocytes (group1, n=20) or PBS (group 2, n=24) through the portal vein or into the spleen. Survival analyses in 3 days showed that all animals from group 1 survived, whereas 5 rats from group 2 died (P=0.03), all deaths occurred in rats that received PBS into the portal vein (P=0.001). Alanine aminotransferase and factor V levels measured within the experiment did not differ between groups 1 and 2 at any time point. Molecular analysis and histology showed presence of hepatocytes in liver of transplanted animals injected either through portal vein or spleen. Our data in acetaminophen-induced hepatotoxicity model (1g/kg) demonstrate that rat hepatocyte transplantation increases survival when the site of injection is into portal vein in this hepatotoxicity model. In the acetaminophen-induced acute hepatotoxicity model (1,5g/kg), hepatocytes were isolated from male Wistar rats and transplanted 6 hours later in female recipients. Female rats received either 1x107 hepatocytes (group1, n=33) or PBS (group 2, n=24) into the spleen. Survival analyses in 3 days showed that 9 rats from group 1 and 9 rats from group 2 died at day 2. There was no statistical significance in survival between group 1 and group 2. Our data demonstrate that hepatocyte isolation is a feasible procedure. Hepatocyte transplantation can be used in animal models of acute liver failure increasing survival. The model of the present study show a higher variability, therefore it´s necessary to evaluate hepatocyte transplantation in a more reproducible model.

O transplante de hepatócitos é uma modalidade terapêutica atrativa para as doenças hepáticas, assim como uma alternativa para o transplante hepático. O objetivo do presente estudo é investigar a efetividade do transplante de hepatócitos de ratos nos modelos de hepatotoxicidade aguda induzida por paracetamol (1g/kg e 1,5g/kg). Os hepatócitos foram isolados de ratos Wistar machos e transplantados 24 horas após em receptoras fêmeas com hepatotoxicidade de 1g/kg. Os ratos fêmeas receberam 1x107 hepatócitos (grupo 1, n=20) ou PBS (grupo 2, n=24) através da veia porta ou no baço. A análise de sobrevida em 3 dias demonstrou que todos os animais do grupo 1 sobreviveram, enquanto 5 animais do grupo 2 morreram (P=0,03), todas as mortes ocorreram nos ratos que receberam PBS através da veia porta (P=0,001). Os níveis de alanina aminotransferase e fator V que foram medidos no experimento não mostraram diferença entre o grupo 1 e o grupo 2 em nenhum momento. A análise molecular e a histologia mostraram a presença de hepatócitos no fígado de animais transplantados através da veia porta ou pelo baço. O modelo de hepatotoxicidade aguda induzida por paracetamol (1g/kg) demonstrou que o transplante de hepatócitos de ratos aumenta a sobrevida quando o local de injeção é na veia porta. No modelo de hepatotoxicidade aguda induzida por paracetamol (1,5g/kg), os hepatócitos foram isolados de ratos Wistar machos, e transplantados 6 horas após em receptoras fêmeas. Os ratos fêmeas receberam 1x107 hepatócitos (grupo 1, n=33) ou PBS (grupo 2, n=24) no baço. A análise de sobrevida em 3 dias demonstrou que 9 animais do grupo 1, e 9 animais do grupo 2 morreram no dia 2. Não houve diferença estatística significativa na análise de sobrevida entre o grupo 1 e o grupo 2. Nossos dados demonstram que o isolamento de hepatócitos é um procedimento factível. O transplante de hepatócitos é uma técnica que pode ser aplicada em modelos animais de IHA levando ao aumento da sobrevida. Entretanto, o modelo utilizado no presente estudo apresentou um alto grau de variabilidade, tornando necessária a avaliação do transplante de hepatócitos em um modelo mais reprodutível. / Hepatocyte transplantation is an attractive therapeutic modality for liver disease as an alternative for liver transplantation. The aim of the current study was to investigate the effectiveness of rat hepatocyte transplantation in the acetaminophen-induced acute hepatotoxicity models (1g/kg and 1,5g/kg). Hepatocytes were isolated from male Wistar rats and transplanted 24 hours later in female recipients with hepatotoxicity of 1g/kg of acetaminophen. Female rats received either 1x107 hepatocytes (group1, n=20) or PBS (group 2, n=24) through the portal vein or into the spleen. Survival analyses in 3 days showed that all animals from group 1 survived, whereas 5 rats from group 2 died (P=0.03), all deaths occurred in rats that received PBS into the portal vein (P=0.001). Alanine aminotransferase and factor V levels measured within the experiment did not differ between groups 1 and 2 at any time point. Molecular analysis and histology showed presence of hepatocytes in liver of transplanted animals injected either through portal vein or spleen. Our data in acetaminophen-induced hepatotoxicity model (1g/kg) demonstrate that rat hepatocyte transplantation increases survival when the site of injection is into portal vein in this hepatotoxicity model. In the acetaminophen-induced acute hepatotoxicity model (1,5g/kg), hepatocytes were isolated from male Wistar rats and transplanted 6 hours later in female recipients. Female rats received either 1x107 hepatocytes (group1, n=33) or PBS (group 2, n=24) into the spleen. Survival analyses in 3 days showed that 9 rats from group 1 and 9 rats from group 2 died at day 2. There was no statistical significance in survival between group 1 and group 2. Our data demonstrate that hepatocyte isolation is a feasible procedure. Hepatocyte transplantation can be used in animal models of acute liver failure increasing survival. The model of the present study show a higher variability, therefore it´s necessary to evaluate hepatocyte transplantation in a more reproducible model.

O transplante de hepatócitos é uma modalidade terapêutica atrativa para as doenças hepáticas, assim como uma alternativa para o transplante hepático. O objetivo do presente estudo é investigar a efetividade do transplante de hepatócitos de ratos nos modelos de hepatotoxicidade aguda induzida por paracetamol (1g/kg e 1,5g/kg). Os hepatócitos foram isolados de ratos Wistar machos e transplantados 24 horas após em receptoras fêmeas com hepatotoxicidade de 1g/kg. Os ratos fêmeas receberam 1x107 hepatócitos (grupo 1, n=20) ou PBS (grupo 2, n=24) através da veia porta ou no baço. A análise de sobrevida em 3 dias demonstrou que todos os animais do grupo 1 sobreviveram, enquanto 5 animais do grupo 2 morreram (P=0,03), todas as mortes ocorreram nos ratos que receberam PBS através da veia porta (P=0,001). Os níveis de alanina aminotransferase e fator V que foram medidos no experimento não mostraram diferença entre o grupo 1 e o grupo 2 em nenhum momento. A análise molecular e a histologia mostraram a presença de hepatócitos no fígado de animais transplantados através da veia porta ou pelo baço. O modelo de hepatotoxicidade aguda induzida por paracetamol (1g/kg) demonstrou que o transplante de hepatócitos de ratos aumenta a sobrevida quando o local de injeção é na veia porta. No modelo de hepatotoxicidade aguda induzida por paracetamol (1,5g/kg), os hepatócitos foram isolados de ratos Wistar machos, e transplantados 6 horas após em receptoras fêmeas. Os ratos fêmeas receberam 1x107 hepatócitos (grupo 1, n=33) ou PBS (grupo 2, n=24) no baço. A análise de sobrevida em 3 dias demonstrou que 9 animais do grupo 1, e 9 animais do grupo 2 morreram no dia 2. Não houve diferença estatística significativa na análise de sobrevida entre o grupo 1 e o grupo 2. Nossos dados demonstram que o isolamento de hepatócitos é um procedimento factível. O transplante de hepatócitos é uma técnica que pode ser aplicada em modelos animais de IHA levando ao aumento da sobrevida. Entretanto, o modelo utilizado no presente estudo apresentou um alto grau de variabilidade, tornando necessária a avaliação do transplante de hepatócitos em um modelo mais reprodutível. / Hepatocyte transplantation is an attractive therapeutic modality for liver disease as an alternative for liver transplantation. The aim of the current study was to investigate the effectiveness of rat hepatocyte transplantation in the acetaminophen-induced acute hepatotoxicity models (1g/kg and 1,5g/kg). Hepatocytes were isolated from male Wistar rats and transplanted 24 hours later in female recipients with hepatotoxicity of 1g/kg of acetaminophen. Female rats received either 1x107 hepatocytes (group1, n=20) or PBS (group 2, n=24) through the portal vein or into the spleen. Survival analyses in 3 days showed that all animals from group 1 survived, whereas 5 rats from group 2 died (P=0.03), all deaths occurred in rats that received PBS into the portal vein (P=0.001). Alanine aminotransferase and factor V levels measured within the experiment did not differ between groups 1 and 2 at any time point. Molecular analysis and histology showed presence of hepatocytes in liver of transplanted animals injected either through portal vein or spleen. Our data in acetaminophen-induced hepatotoxicity model (1g/kg) demonstrate that rat hepatocyte transplantation increases survival when the site of injection is into portal vein in this hepatotoxicity model. In the acetaminophen-induced acute hepatotoxicity model (1,5g/kg), hepatocytes were isolated from male Wistar rats and transplanted 6 hours later in female recipients. Female rats received either 1x107 hepatocytes (group1, n=33) or PBS (group 2, n=24) into the spleen. Survival analyses in 3 days showed that 9 rats from group 1 and 9 rats from group 2 died at day 2. There was no statistical significance in survival between group 1 and group 2. Our data demonstrate that hepatocyte isolation is a feasible procedure. Hepatocyte transplantation can be used in animal models of acute liver failure increasing survival. The model of the present study show a higher variability, therefore it´s necessary to evaluate hepatocyte transplantation in a more reproducible model.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22302号 / 医博第4543号 / 新制||医||1040(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 川口 義弥, 教授 妹尾 浩, 教授 濵﨑 洋子 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23409号 / 医博第4754号 / 新制||医||1052(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 妹尾 浩, 教授 武藤 学, 教授 小川 誠司 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19580号 / 医博第4087号 / 新制||医||1013(附属図書館) / 32616 / 京都大学大学院医学研究科医学専攻 / (主査)教授 妹尾 浩, 教授 木村 剛, 教授 柳田 素子 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM