京都大学 / 新制・課程博士 / 博士(医学) / 甲第24516号 / 医博第4958号 / 新制||医||1064(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 伊藤 貴浩, 教授 岩田 想, 教授 武藤 学 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Circadian rhythms are daily molecular oscillations within cells ranging from prokaryotes to humans. This rhythm is self sustaining, and receives external cues in order to synchronize an organism's behavior and physiology with the environment. Many metabolites utilized in metabolic processes seem to follow a pattern of circadian oscillation. Iron, an essential component in cellular processes such as respiration and DNA synthesis, is obtained almost exclusively through diet, yet little is known about how the clock governs iron metabolism. The regulation of iron within the cell is very tightly controlled, as iron is highly reactive in the generation of oxidative stress and the excretion of excess iron is very limited. There are limited findings indicating that there are molecular ties between the circadian clock and the regulation of iron metabolism. The first half of my dissertation focuses on the role of the circadian clock in modulating expression of iron metabolic components. We found that key components of iron import, in TFRC, and export, in SLC40A1, show altered expression in response to changes in the expression of clock transcription components. Furthermore, in circadian synchronized HepG2 hepatocytes TFRC and SLC40A1 showed rhythms in their mRNA expression, although expression of these genes was highly altered in conditions of high iron availability. We also examined IREB2, which expresses a master regulator of iron concentration in IRP2. IRP2 showed rhythms in phase with circadian component PER2, and IRP2's rhythmicity was lost under iron overload conditions. We observed that the ability of these three critical iron metabolic components to respond to sudden increases in available iron was mitigated in cells with clock impairment. Whole cistrome and transcriptome analysis was used to determine that rhythmicity in TFRC and SLC40A1 are not equal in their recruitment of circadian protein binding or in the stage of transcription in which circadian rhythms are generated. The cumulative effect of all of this regulation is that rhythmic variation in intracellular hepatic ferrous iron is clock controlled. The second half of my dissertation focuses on understanding how iron uptake influences clock resetting. Initially, iron was added to the cells in the form of ferrous sulfate, or chelated out of the cells using 2-2'-dipyridyl and clock gene expression was monitored. Altered rhythmicity of these components was seen at both the mRNA and protein level in cells with disrupted iron homeostasis. Then, we measured changes in period, phase, and amplitude of these rhythms, ultimately using a luciferase reporter cell line to demonstrate that even slight changes in cellular iron produce an effect on rhythmic period. We find that the circadian clock and iron metabolism pathway are intimately related, and that the intracellular iron concentration plays a role in circadian clock behavior. Overall, our research illustrates the importance of the circadian clock in liver metabolism and physiology. Improper iron metabolism due to genetic or dietary shortcomings is common in humans, and our work builds on the importance of chronotherapy in treatment of these conditions. Conversely, our research into the effect intracellular iron has on the clock contributes to the growing body of research into how circadian clocks, especially the peripheral clock of the liver, receive input from a range of metabolites in conjunction with signals from the master oscillator of the suprachiasmatic nucleus. / Ph. D. / The circadian clock is the system allowing the body to stay in synchrony with its environment. Clocks are found in organisms ranging from bacteria to humans, and use environmental cues such as light and temperature to coordinate important processes inside the cell. Many of these processes require enzymes which contain iron in order to function. Iron is obtained almost exclusively through feeding, and high iron levels are toxic to the cell. In this work, we looked at how the circadian clock helps maintain the amount of iron within the cell at healthy levels. We showed that the genes which are involved in managing iron are expressed in different amounts depending on the time of day, and that this causes the amount of iron within the cell to vary over time. We also examined how the amount of iron in the cells goes on to alter the circadian clock. The way the circadian rhythm oscillates is altered when either too much or too little iron is available to the cells. These findings have health impacts, especially in the context of the liver where poor management of the circadian clock or iron metabolism have been linked to the development of various forms of liver cancer.

Fundação Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Neste trabalho, abordamos um problema de otimização de parâmetros da biofísica em que o objetivo é a obtenção da taxa média de concentração de substrato no fígado. Este problema é altamente não-linear, multimodal e com função-objetivo não-diferenciável. Resolvemos o mesmo através de métodos de otimização da literatura e introduzimos três métodos de otimização. Os métodos introduzidos neste trabalho são baseados na hibridização de um método estocástico, que explora o espaço de busca, com um método determinístico de busca direta, que faz uma busca local mais refinada nas áreas mais promissoras deste espaço. Os novos métodos são comparados aos da literatura e é verificado que o desempenho dos primeiros é superior. / In this work, we attack a parameter optimization problem from Biophysics, where the aim is to obtain the substrate concentration rate of a liver. This problem is highly non-linear, multimodal, and with non-differentiable objective-function. We solve it using optimization methods from the literature and three methods introduced in this work. The latter methods are based on the hybridization of a stochastic technique which explores the search space, with a direct search deterministic technique which exploits the most promising areas. Our results show that the new optimization methods perform better than those from the literature.

Fundação Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Neste trabalho, abordamos um problema de otimização de parâmetros da biofísica em que o objetivo é a obtenção da taxa média de concentração de substrato no fígado. Este problema é altamente não-linear, multimodal e com função-objetivo não-diferenciável. Resolvemos o mesmo através de métodos de otimização da literatura e introduzimos três métodos de otimização. Os métodos introduzidos neste trabalho são baseados na hibridização de um método estocástico, que explora o espaço de busca, com um método determinístico de busca direta, que faz uma busca local mais refinada nas áreas mais promissoras deste espaço. Os novos métodos são comparados aos da literatura e é verificado que o desempenho dos primeiros é superior. / In this work, we attack a parameter optimization problem from Biophysics, where the aim is to obtain the substrate concentration rate of a liver. This problem is highly non-linear, multimodal, and with non-differentiable objective-function. We solve it using optimization methods from the literature and three methods introduced in this work. The latter methods are based on the hybridization of a stochastic technique which explores the search space, with a direct search deterministic technique which exploits the most promising areas. Our results show that the new optimization methods perform better than those from the literature.

The liver is the central metabolic hub in organisms and a complex, intertwining regulatory network guarantees efficient liver processes. The morphogenic Hedgehog pathway was recently shown to play a role in regulating the underlying genetic program. Transgenic mouse models with hepatocyte-specific inactivation of Hedgehog signaling showed alterations in insulin-like growth factor homeostasis and in energy metabolism associated with increased lipid accumulation in the liver. In this thesis, it was possible to connect the observed infertility of female knockout mice with an unexpected activation of sex steroid synthesis in the liver. Associated with increased steroidogenic gene expression exclusively in hepatocytes, the plasma testosterone level was significantly elevated, which led to androgenization and an anovulatory phenotype. With these characteristics, the mouse model mimicked the human polycystic ovarian syndrome and suggested an influence of liver and hepatic Hedgehog signaling on reproduction under disease conditions. Further, murine liver metabolism was challenged with starvation starting at different times of day. The transcriptomic results were analyzed with a self-organizing map approach, allowing an intuitive interpretation of data and a thus far unknown diurnally different response of hepatic regulatory mechanisms due to starvation was revealed. In contrast to the manifoldly published and observed switch from energy-consuming to energy-providing processes due to starvation started in the morning, evening starvation led to a novel hepatic expression signature with decreased gluconeogenic gene expression and increased levels of lipid and steroid metabolism-related genes. These differences can be explained by the equally diurnally regulated expression of the corresponding regulatory transcription factors and hormones. Additionally, lipidome analysis confirmed the diurnal differences after starvation. Thus, this study emphasized the immense impact of circadian regulation on liver metabolism and suggests high accuracy when starvation is the focus of research to avoid varying results.:BIBLIOGRAPHISCHE DARSTELLUNG ................................................................................ II LIST OF ABBREVIATIONS .................................................................................................. III TABLE OF CONTENTS ....................................................................................................... IV SUMMARY ............................................................................................................................ 1 ZUSAMMENFASSUNG ......................................................................................................... 5 INTRODUCTION ................................................................................................................... 9 Liver architecture and metabolism ..................................................................................... 9 Diverse possibilities of liver metabolism regulation .......................................................... 10 Connection of Hedgehog signaling to hepatic metabolism ............................................... 10 Impact of feeding schemes on hepatic metabolism .......................................................... 13 Aims of the thesis ............................................................................................................ 14 References ...................................................................................................................... 15 CHAPTER 1 ........................................................................................................................ 18 CHAPTER 2 ........................................................................................................................ 39 PERSPECTIVE ................................................................................................................... 64 CURRICULUM VITAE ........................................................................................................... V PUBLICATIONS AND PRESENTATIONS ............................................................................ VI Publications ...................................................................................................................... VI Oral presentations ............................................................................................................ VI Poster presentations ........................................................................................................ VII AUTHOR CONTRIBUTION STATEMENT .......................................................................... VIII SELBSTSTÄNDIGKEITSERKLÄRUNG .............................................................................. XII DANKSAGUNG .................................................................................................................. XIII

Thesis / Doctor of Philosophy (PhD)

Le carcinome hépatocellulaire (CHC) est une des principales causes de mortalité par cancer dans le monde. Dans environ 50% des tumeurs, on observe les signes d’une activation de la voie Wnt/β-caténine, causée par une mutation de l’exon 3 du gène CTNNB1 ou par stimulation du récepteur FRZD. Des études transcriptomiques du CHCs ont montré que ces deux modes d’activation étaient associés à des sous-types de tumeurs différents. Nous avons cherché à mieux comprendre les caractéristiques cliniques et le phénotype moléculaire de ces deux sous-types de CHCs. Dans un premier temps, nous avons fait le lien entre l'activation Wnt extracellulaire, un phénotype de cellules cancéreuses souches ou progénitrices et la présence de foyers de fibrose discrète intra-tumorale, observable par examen histopathologique, que nous avons appelés "nids fibreux". Nous avons également mis en évidence HAPLN1, une protéine de la matrice extracellulaire dont l’expression est stimulée par Wnt3a dans un modèle de cellules hépatiques progénitrices HepaRG, comme un nouveau marqueur d’agressivité du CHC. Ces résultats montrent une association entre l’activation Wnt extracellulaire et une agressivité tumorale passant par un remodelage matriciel. Dans un second temps, une Méta-analyse de données publiques de transcriptomique a permis de mettre évidence 4 sous-types de CHCs. La mutation CTNNB1, prédite par l’expression de 5 marqueurs par une méthode développée durant la thèse, était associée à un de ces sous-types et à un bon pronostic clinique. Nous avons également isolé un nouveau sous-type de CHC de bon pronostic exprimant un phénotype de tumeur différenciée et des signatures de métabolisme hépatique périportales. Ce sous-type a probablement été un facteur confondant dans les études précédentes mesurant l’association de la mutation CTNNB1 avec un bon pronostic. Enfin, nous avons mis en évidence une forte association négative entre la mutation CTNNB1 et l’inflammation ainsi que la fibrose tumorale dans trois cohortes indépendantes. Cet effet pourrait être provoqué par une inhibition de NF-κB par la β-caténine mutée, comme suggérée par des résultats préliminaires issue d’un modèle in vitro d’HepaRG mutés T41 stimulés par LPS. Nos résultats suggèrent donc que les deux modes d’activation de la voie Wnt/β-caténine sont associés à des mécanismes moléculaires, des profils d’expression, des phénotypes et des pronostics cliniques très différents. / Hepatocellular carcinoma (HCC) is the third cause of cancer-related death worldwide. Half of them show activation of Wnt/β-catenin pathway, caused by activating CTNNB1 exon 3 mutation of by stimulation of FRZD receptor. Transcriptomic based HCC classifications showed that this two types of activation were associated with distinct tumor subtypes. We tried to better understand the molecular phenotypes and the clinical features associated with these subtypes. In a first part, we linked extracellular Wnt activation with a stem/progenitor phenotype and with fibrous hotspot in HCC. Fibrous hotspot, which were called “fibrous nest”, can be detected by routine anatomic pathology analyses. We also showed that HAPLN1, an extracellular matrix protein induced by Wnt3a in progenitor HepaRG cells, was a new marker of stemness and bad outcome in HCC. Those results shows the associations between extracellular Wnt activation, extracellular matrix remodeling and tumor aggressiveness. In a second part, a transcriptome meta-analysis of 1133 HCCs highlighted 4 robust subclasses. CTNNB1 mutation, predicted by a 5-genes score based method, was associated with one of these subclasses and with good clinical features. We also highlighted a new subclass of CTNNB1 wild type HCCs associated with tumor differentiation, signatures of periportal metabolism and good outcome. This subclass was probably a confounding factor in survival studies comparing HCCs carrying mutant versus those carrying wild-type CTNNB1. Finally, we highlighted strong negative associations between CTNNB1 mutation and inflammation as well as tumor fibrosis in three independent cohorts. Preliminary results of in vitro HepaRG cells mutated for CTNNB1 in T41 and stimulated by LPS suggest an inhibitory effect of β-caténine on NF-κB. In conclusion, our results show that the two types of Wnt activation in HCC are associated with very distinct molecular phenotypes and clinical features.

Indiana University-Purdue University Indianapolis (IUPUI) / Sterol Regulatory Element Binding Protein-1 (SREBP-1) is a conserved transcription factor of the basic helix-loop-helix leucine zipper family (bHLH-Zip) that primarily regulates glycolytic and lipogenic enzymes such as L-pyruvate kinase, acetyl-CoA carboxylase, fatty acid synthase, stearoyl-CoA desaturase 1, and mitochondrial glycerol-3-phosphate acyltransferase 1. SREBP-1c activity is higher in the liver of human obese patients, as well as ob/ob and db/db mouse models of obesity and type 2 diabetes, underscoring the role of this transcription factor as a contributor to hepatic steatosis and insulin resistance. Nonetheless, SREBP-1 deficient ob/ob mice, do not display improved glycemia despite a significant decrease in hepatic lipid accumulation, suggesting that SREBP-1 might play a role at regulating carbohydrate metabolism. By silencing SREBP-1 in the liver of normal and type 2 diabetes db/db mice, we showed that indeed, SREBP-1 is needed for appropriate regulation of glycogen synthesis and gluconeogenesis enzyme gene expression. Depleting SREBP-1 activity more than 90%, resulted in a significant loss of glycogen deposition and increased expression of Pck1 and G6pc. Hence, the benefits of reducing de novo lipogenesis in db/db mice were offset by the negative impact on gluconeogenesis and glycogen synthesis. Some studies had also indicated that SREBP-1 regulates the insulin signaling pathway, through regulation of IRS2 and a subunit of the PI3K complex, p55g. To gain insight on the consequences of silencing SREBP-1 on insulin sensitivity, we analyzed the insulin signaling and mTOR pathways, as both are interconnected through feedback mechanisms. These studies suggest that SREBP-1 regulates S6K1, a downstream effector of mTORC1, and a key molecule to activate the synthesis of protein. Furthermore, these analyses revealed that depletion of SREBP-1 leads to reduced insulin sensitivity. Overall, our data indicates that SREBP-1 regulates pathways important for the fed state, including lipogenesis, glycogen and protein synthesis, while inhibiting gluconeogenesis. Therefore, SREBP-1 coordinates multiple aspects of the anabolic response in response to nutrient abundance. These results are in agreement with emerging studies showing that SREBP-1 regulates a complex network of genes to coordinate metabolic responses needed for cell survival and growth, including fatty acid metabolism; phagocytosis and membrane biosynthesis; insulin signaling; and cell proliferation.

Vitronectin (VTN) is a glycoprotein enriched in the blood and activates integrin receptors. VTN blood levels increase only in female mice 24 h after an ischemic stroke and exacerbate brain injury through IL-6-driven inflammation, but the VTN induction mechanism is unknown. Here, a 30 min middle cerebral artery occlusion (MCAO) in female mice induced VTN protein in the liver (normally the main source) in concert with plasma VTN. Male mice were excluded as VTN is not induced after stroke. MCAO also increased plasma VTN levels after de novo expression of VTN in the liver of VTN female mice, using a hepatocyte-specific (SERPINA1) promoter. MCAO did not affect SERPINA1 or VTN mRNA in the liver, brain, or several peripheral organs, or platelet VTN, compared to sham mice. Thus, hepatocytes are the source of stroke-induced increases in plasma VTN, which is independent of transcription. The cholinergic innervation by the parasympathetic vagus nerve is a potential source of brain-liver signaling after stroke. Right-sided vagotomy at the cervical level led to increased plasma VTN levels, suggesting that VTN release is inhibited by vagal tone. Co-culture of hepatocytes with cholinergic neurons or treatment with acetylcholine, but not noradrenaline (sympathetic transmitter), suppressed VTN expression. Hepatocytes have muscarinic receptors and the M1/M3 agonist bethanechol decreased VTN mRNA and protein release in vitro via M1 receptors. Finally, systemic bethanechol treatment blocked stroke-induced plasma VTN. Thus, VTN translation and release are inhibited by muscarinic signaling from the vagus nerve and presents a novel target for lessening detrimental VTN expression.

Recent evidence suggests that oxidatively modified forms of low-density lipoprotein (LDL) may be particularly atherogenic. In this investigation, the catabolism of human LDL modified by lipid peroxidation in vitro was studied with a recirculating rat liver perfusion system. A dual-labelling technique was used that permitted native LDL and modified LDL to be studied simultaneously in the liver perfusion system. Native human LDL was found to have a fractional catabolic rate (FCR) of 1.00 +/- 0.21%/h, in agreement with other investigators. Subjecting LDL to oxidation for 12 h in the presence of 30 microM FeEDTA did not significantly affect its FCR. LDL treated with a superoxide-generating system (xanthine oxidase, hypoxanthine, O2) in the presence of 30 microM FeEDTA did, however, show a significant increase in FCR (3.23 +/- 0.19%/h). The hepatic uptakes of native LDL and LDL oxidized with FeEDTA+O2 were similar, but both were significantly lower than the hepatic uptake of LDL treated with the superoxide-radical-generating system. The proteolysis of LDL with pancreatin did not influence either its susceptibility to oxidation or its FCR. LDL oxidation resulted in the preferential loss of alpha-tocopherol rather than gamma-tocopherol. These data indicate that the rat liver effectively catabolizes LDL oxidatively modified by treatment with the superoxide-generating system. Furthermore, our results suggest that only very low plasma levels of highly oxidized LDL could be found under conditions in vivo. The liver may therefore play a major role in protecting the arterial vasculature from highly atherogenic forms of LDL.