La prévalence de l’obésité et du diabète de type 2 est en constante augmentation dans les pays industrialisés. Ces pathologies sont associées à des troubles de l’équilibre énergétique de l’organisme, ce qui se traduit au niveau hépatique par le développement de NAFLD (Non‐alcoholic Fatty Liver Disease). La NAFLD est engendrée par l’accumulation excessive de lipides dans les hépatocytes ou stéatose et est associée à la résistance à l’insuline. L’insulinorésistance hépatique est dite sélective car l’insuline n’est plus capable d’inhiber la production hépatique de glucose, contribuant à l’hyperglycémie, alors que la synthèse lipidique est exacerbée, induisant une stéatose hépatique. La stéatose peut évoluer en pathologies plus graves, telles que la fibrose, la cirrhose ou le cancer hépatocellulaire (CHC). L’objectif de ma thèse a été d’étudier les mécanismes moléculaires pouvant contribuer à la sélectivité de la résistance à l’insuline hépatique et à l’évolution de la stéatose simple vers le CHC. Dans un premier temps, nous avons étudié le rôle de l’adaptateur moléculaire Grb14, un inhibiteur de l’activité kinase du récepteur de l’insuline, dans la régulation du métabolisme hépatique chez la souris. L’invalidation de Grb14 dans le foie conduit d’une part à une activation de la signalisation de l’insuline et une amélioration de tolérance au glucose, et d’autre part à une diminution de la lipogenèse consécutive à l’inhibition du facteur lipogénique LXR via la voie Nrf2. L’invalidation de Grb14 dans le foie de souris obèses et diabétiques permet de ramener la glycémie et la stéatose hépatique à des valeurs similaires aux témoins. Ces données suggèrent que Grb14 est un nouvel acteur impliqué dans la sélectivité de la résistance à l’insuline du foie. La seconde étude a permis de montrer l’implication de la stéatose hépatique induite par un régime hypercalorique sur le développement de CHC. En effet, sur un fond génétique favorisant la carcinogenèse, un régime riche en graisse et en sucre contribuant à l’insulinorésistance hépatique accélère la cinétique d’apparition des tumeurs et augmente leur nombre. / The prevalence of metabolic diseases, including obesity and type 2 diabetes, are expanding in a worldwide epidemic way. These diseases are associated with metabolic disorders, resulting in the development of NAFLD (Non‐alcoholic Fatty Liver Disease) in the liver. NAFLD is generated by excessive accumulation of lipids in hepatocytes, and is associated with insulin resistance. In liver, insulin resistance leads to a blunted inhibitory action on hepatic glucose production, inducing hyperglycemia, whereas de novo lipogenesis, which is positively regulated by insulin, is paradoxically exacerbated, contributing to hepatic steatosis. Steatosis may also evolve into more serious diseases such as fibrosis, cirrhosis and hepatocellular carcinoma (HCC). The aim of my thesis was to study the molecular mechanisms that may contribute to the selectivity of hepatic insulin resistance and the development of HCC. Initially, we studied the role of the molecular adapter Grb14, an inhibitor of the insulin receptor kinase activity, in the regulation of hepatic metabolism in mice. Invalidation of Grb14 in the liver leads on the one hand to an activation of the insulin signaling and improved glucose tolerance, and on the other hand to a decrease of LXR activity resulting in lipogenesis inhibition. Invalidation of Grb14 in the liver of obese and diabetic mice restores blood glucose levels and hepatic steatosis similar to control values. These data suggest that Grb14 is a new player involved in the selectivity of the insulin resistance in the liver. The second study demonstrated the involvement of hepatic steatosis induced by a high‐calorie diet on the development of HCC. Indeed, on a genetic background favoring carcinogenesis, a diet rich in fat and sugar contributing to hepatic insulin resistance accelerates appearance of tumors and increases their number.

Le diabète de type 2 se caractérise par une résistance à l’insuline dans les tissus périphériques, une déficience de sécrétion d’insuline et une augmentation de la production endogène de glucose. Notre but a été de démontrer le rôle spécifique de la production hépatique de glucose dans le développement du diabète. Nous avons développé un modèle de souris invalidées pour le gène codant pour la sous-unité catalytique de la G6Pase, enzyme clé de la production de glucose, spécifiquement dans le foie. Sous alimentation diabétogène, les souris transgéniques résistent au développement de l’hyperglycémie et l’hyperinsulinémie, et présentent une amélioration de la sensibilité à l’insuline et une augmentation du captage périphérique de glucose. Ces souris résistent également à l’obésité induite par ce régime déséquilibré, en liaison avec l’augmentation de la dépense énergétique, associée à l’induction des médiateurs de la thermogenèse dans les tissus adipeux brun et blanc, et au remodelage du muscle squelettique vers un phénotype oxydative. La délétion de la G6PC hépatique chez des souris rendues obèses et diabétiques résulte en une amélioration spectaculaire et rapide du métabolisme glucidique, et une stabilisation de la masse corporelle des souris obèses, associée à une induction des gènes du métabolisme oxydative dans les tissus périphériques.Ces effets bénéfiques pourraient être dus à l’augmentation de la sécrétion de facteurs hépatiques circulants connus pour réguler le métabolisme énergétique et glucidique dans les tissus périphériques. Ces travaux démontrent le rôle délétère de la production hépatique de glucose dans le développement du diabète et de l’obésité / Type 2 diabetes is characterized by insulin resistance of glucose uptake by peripheral tissues, insulin secretion deficiency and increased endogenous glucose production. Our aim is to demonstrate the specific role of hepatic glucose production in triggering insulin resistance and diabetes. For that, we developed an inducible and liver-specific knock-out mouse model for the gene encoding the catalytic subunit of G6Pase, a key enzyme of glucose production. When fed a high fat/high sucrose diet, transgenic mice resisted to the development of fasting hyperglycemia and hyperinsulinemia, and even showed enhanced insulin sensitivity and glucose uptake in peripheral tissues. These mice are also resistant to diet induced obesity, due to the induction of basal metabolism, associated with increased brown and white adipose tissue thermogenesis machinery and remodeling of skeletal muscle towards a more oxidative phenotype. When liver G6pc deletion was realized in obese and diabetic mice, this resulted in a spectacular and early amelioration of glucose metabolism compared to that before liver G6pc deletion, and to stabilization of body mass of obese mice, which was associated with induction of oxidative genes in peripheral tissues. These beneficial effects could be explained by the secretion of hepatic circulating hormones known to control glucose and energy metabolism in peripheral tissues. This work underlines the deleterious role of hepatic glucose production in the development of obesity and diabetes, and sets the liver as a master-switch in the regulation of whole-body glucose and energy metabolism

Les résultats fondamentaux et biocliniques que nous présentons au travers de cette thèse illustrent la difficulté d’évaluer la part liée au virus et celle liée à des facteurs de l’hôte dans l’induction d’une stéatose hépatique chez les patients chroniquement infectés par le HCV. Les données in vitro suggèrent que le virus joue un rôle direct dans l’induction d’une stéatose, notamment par les propriétés de sa protéine de capside, et que la variabilité du virus peut avoir un impact sur l’intensité de cette stéatose. Notre étude bioclinique suggère que la variabilité du virus semble avoir un rôle beaucoup plus modéré in vivo. Ainsi, chez les patients chroniquement infectés par le HCV, les facteurs de l’hôte joueraient un rôle majeur pour moduler le degré de la stéatose associée au virus et de prochaines études seront nécessaires pour établir la nature de ces facteurs. / The results presented in this thesis illustrate the difficulty of assessing the part related to the virus and that related to host factors in the induction of hepatic steatosis in patients chronically infected with HCV. In vitro data suggest that the virus plays a direct role in the induction of steatosis, due to the properties of its capsid protein, and that the variability of the virus can affect the intensity of the steatosis. Our bio-clinical study suggests that this variability seems to have a much more moderate impact in vivo. Thus, in patients chronically infected with HCV, host factors seem to play a major role to modulate the degree of steatosis associated with the virus. Further studies are needed to establish the nature of these factors.

A incidência da obesidade tomou proporções epidêmicas nos últimos anos, atingindo bilhões de indivíduos mundialmente. A DHGNA é uma manifestação hepática das alterações metabólicas causadas pela obesidade e os casos desta doença vêm crescendo cada vez mais. Alternativas capazes de reduzir estas alterações são fundamentais para minimizar o impacto na qualidade de vida da população e na economia do país. Diversos estudos têm mostrado que os compostos bioativos de alimentos possuem efeitos benéficos à saúde. O camu-camu (Myrciaria dubia (H. B. K). Mc Vaugh) é um fruto nativo da região amazônica com potencial agroeconômico ainda inexplorado, que contém um grande número de compostos fitoquímicos que podem atuar sobre o metabolismo corporal. Desta forma, o objetivo deste estudo foi avaliar o efeito dos compostos fenólicos do camu-camu no desenvolvimento da DHGNA em camundongos C57BL/6 que receberam dieta rica em lipídios e sacarose (HFS). O extrato rico em compostos fenólicos da polpa comercial deste fruto foi obtido através de extração em fase sólida e caracterizado por cromatografia líquida de alta eficiência (CLAE/DAD). Os extratos obtidos foram testados em doses de 7 mg e de 14 mg equivalentes de ácido gálico/Kg de peso corporal. Foram investigados os efeitos destes compostos sobre as homeostases glicídica e lipídica através de análises séricas, testes de tolerância à insulina e à glicose e conteúdo de glicogênio e triacilglicerol intra-hepático. O extrato do camu-camu apresentou flavonóis, ácido elágico e elagitaninos em sua composição. A suplementação com extrato fenólico de camu-camu diminuiu a intolerância à glicose, independente da dose administrada, e melhorou a sensibilidade à insulina e regulou o conteúdo de glicogênio intra-hepático na maior dose. Não foi observado efeito sobre os lipídios plasmáticos. Entretanto, nota-se que houve uma melhora na função hepática em decorrência da redução da atividade da alanina aminotransferase (ALT), indicadora de dano celular, independente da dose. Além disso, a suplementação com extratos fenólicos do camu-camu na maior dose reduziu o conteúdo de triacilglicerol intra-hepático (p = 0,0001) e de biomarcadores inflamatórios, como Proteína - C Reativa (PCR) (p = 0,0359) e prostaglandina E2 (PGE2) (p = 0,004). Estes efeitos foram associados, principalmente, à menor ingestão alimentar. Portanto, neste estudo, os compostos fenólicos do camu-camu foram eficientes em prevenir a progressão da DHGNA em camundongos alimentados com dieta HFS. / Obesity has reached epidemic proportions in recent years, affecting billions of people worldwide. Nonalcoholic fatty liver disease (NAFLD) is a hepatic disorder induced by the metabolic changes caused by obesity and its incidence has been growing increasingly. Alternatives designed to reduce such changes are crucial to minimize their impact on the population´s quality of life and countries economy. Several studies have shown that food bioactive compounds have beneficial effects on health. Camu-camu (Myrciaria dubia (H. B. K.) Mc Vaugh) is a native fruit from Amazonan, with unexplored agroeconomic potential, which contains a large number of phytochemical compounds that can act on body metabolism. Therefore, this study was designed to assess the effect of the phenolic compounds of camu-camu in the development of NAFLD in C57BL/6 mice fed with a lipid and saccharose-rich diet (HFS). The phenolic compound-rich extract was obtained from the commercial pulp of this fruit using solid-phase extraction and high-performance liquid chromatography with diode array detector (HPLC/DAD). The resulting extracts were tested at 7 mg and 14 mg gallic acid equivalents/kg body weight. The effects of these compounds on glucose and lipid homeostasis were investigated by serum analyses, insulin and glucose tolerance tests and intrahepatic content of glycogen and triacylglycerol. Camu-camu extract presented flavonols, ellagic acid and ellagitannins in its composition. Supplementation with camu-camu phenolic extract decreased glucose intolerance, regardless the dose, improved insulin sensitivity and normalized the intra-hepatic glycogen content at the highest dose. No effects on plasma lipid were found. However, an improvement in liver function due to the decrease in alanine aminotransferase (ALT) was observed, suggestive of cell damage, regardless the dose. Moreover, the supplementation with phenolic extracts of camu-camu at the highest dose decreased the intrahepatic content of triacylglycerol (p = 0.0001) and inflammatory biomarkers, such as C-reactive protein (CRP) (p = 0.0359) and prostaglandin E2 (PGE2) (p = 0.004). Such effects were primarily associated with lower food intake. Therefore, in this study, the phenolic compounds of camu-camu have shown to be effective in preventing the progression of NAFLD in mice fed with HFS (high-fat/sucrose) diet.

L’obésité et les maladies métaboliques associées, telles que les stéatopathies hépatiques (ou NAFLD, pour non-alcoholic fatty liver disease), sont en constante augmentation dans la population mondiale. La stéatose hépatique, correspondant au premier stade de la NAFLD et caractérisée par une simple accumulation de lipides dans le foie est considérée comme bénigne. Cependant, cette pathologie est susceptible d'évoluer en stéatohépatite (ou NASH, pour non-alcoholic steatohepatitis), bien plus grave puisqu’elle est additionnée d’une nécrose et d’une inflammation. L’origine de cette évolution est multifactorielle : l’alcool ou les médicaments peuvent en être à l’origine, mais de plus en plus d’études font part du rôle probable que pourraient avoir les contaminants de l’environnement. Ainsi dans cette étude, nous nous sommes intéressés à l’impact de la co-exposition à l’éthanol et au benzo[a]pyrène (B[a]P), un xénobiotique de la famille des hydrocarbures aromatiques polycycliques, sur la progression de la NAFLD vers la NASH. Pour cela, nous avons utilisé la lignée cellulaire hépatique humaine HepaRG. Nous avons établi, sur cette lignée, un modèle de NAFLD en incubant les cellules avec un mélange d’acides gras pendant 14 jours, nous permettant de réaliser des investigations chroniques sur les effets du B[a]P et de l’éthanol. Au terme de ces traitements, nous avons constaté que l’alcool potentialisait la cytotoxicité du B[a]P uniquement sur les cellules HepaRG stéatosées. Cette cytotoxicité était également accompagnée d’une production de cytokines pro-inflammatoires, témoignant d’un état pouvant être apparenté à une NASH. Nous avons également mis en évidence une modification du profil de métabolisation du B[a]P, accompagnée d’une sur production de ROS, un dysfonctionnement global de la chaîne respiratoire mitochondriale et une induction de la voie apoptotique. Enfin, ces observations étaient absentes ou minorées lorsque l’exposition était individuelle et lorsque les cellules n’étaient pas stéatosées. Ainsi, ces résultats suggèrent l’importance de la prise en compte d’une exposition multifactorielle à des xénobiotiques dans la progression de la NAFLD, mais également du fait que la présence de NAFLD pourrait à elle seule aggraver la toxicité de xénobiotiques. / Obesity and associated metabolic diseases such as non-alcoholic fatty liver diseases (NAFLD) are steadily increasing in the global population. Fatty liver (also called steatosis) which is the first stage of NAFLD described by a retention of lipids in the liver is considered to be benign. However, this pathology is likely to evolve into non-alcoholic steatohepatitis (NASH), much more severe since it is supplemented with necrosis and inflammation. This evolution is multifactorial in origin: alcohol or drugs may be involved, but more and more studies have mentioned the likely role of environmental contaminants. Thus, in this study we were interested in the impact of an alcohol/benzo[a]pyrene (B[a]P, a xenobiotic of the family of polycyclic aromatic hydrocarbons) co-exposure on the progression of NAFLD towards NASH. To this end, we used the human hepatic cell line called HepaRG. In this cell line, we established an in vitro model of NAFLD by incubating the cells with a mixture of fatty acids for 14 days, whereby we would carry out some chronic investigations about the effects of B[a]P and ethanol. At the end of these treatments, we found that alcohol potentiated B[a]P cytotoxicity on steatotic HepaRG cells. This cytotoxicity was also combined with pro-inflammatory cytokine production, suggesting the presence of a NASH condition. We also demonstrated a change in the metabolic profile of B[a]P, in addition to a ROS production, a global dysfunction of the mitochondrial respiratory chain and an induction of apoptosis. Finally, these observations were absent or decreased when the exposure was individual and when the cells were not steatotic. Thus, these results suggest the importance to consider multifactorial exposure to xenobiotics in the progression of NAFLD as well as the fact that the presence of NAFLD alone could exacerbate xenobiotic toxicity.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23055号 / 医博第4682号 / 新制||医||1048(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 伊達 洋至, 教授 長船 健二 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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Indiana University-Purdue University Indianapolis (IUPUI) / Regulation of the pyruvate dehydrogenase complex (PDC) is important for glucose homeostasis and control of fuel selection by tissues. Knocking out pyruvate dehydrogenase kinase 4 (PDK4), one of four kinases responsible for regulation of PDC activity, lowers blood glucose levels by limiting the supply of three carbon compounds for gluconeogenesis. Down regulation of PDK4 expression is also important for control of blood glucose by insulin. The primary goal was to determine whether PDK4 should be considered a target for the treatment of diabetes. A major concern is that inhibition of fatty acid oxidation by PDK4 deficiency may promote fat accumulation in tissues and worsen insulin sensitivity. This was examined by feeding wild type and PDK4 knockout mice a diet rich in saturated fat. Fasting blood glucose levels were lower, glucose tolerance was better, insulin sensitivity was greater, and liver fat was reduced in PDK4 knockout mice. The reduction in liver fat is contradictory to the finding that fibrate drugs increase PDK4 expression but ameliorate hepatic steatosis in rodents. To investigate this phenomenon, wild type and PDK4 knockout mice were fed the high saturated fat diet with and without clofibric acid. The beneficial effect of clofibric acid on hepatic steatosis was greater in the PDK4 knockout mice, indicating up regulation of PDK4 is not necessary and likely opposes the effect of clofibric acid on hepatic steatosis. Clofibric acid dramatically lowered the amount of hepatic CD36, a plasma membrane translocase required for fatty acid import, suggesting a novel mechanism for prevention of hepatic steatosis by fibrates. PDK4 deficiency had no effect on CD36 expression but reduced the enzymatic capacity for fatty acid synthesis, suggesting clofibric acid and PDK4 deficiency ameliorate hepatic steatosis by independent mechanisms. Investigation of the mechanism by which insulin regulates PDK4 expression revealed a novel binding site for hepatic nuclear factor 4α (HNF4α) in the PDK4 promoter. The stimulatory effect of HNF4α was sensitive to inhibition by Akt which is activated by insulin. The findings suggest PDK4 is a viable target for the treatment of hepatic steatosis and type 2 diabetes.

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