A Doença Hepática Gordurosa Não Alcoólica (DHGNA) é uma condição clínicopatológica comum, caracterizada por depósito de lipídeos no hepatócito do parênquima hepático. A esteatose hepática (EH) é um dos componentes da DHGNA e caracteriza-se pela presença de vacúolos de lipídeos, principalmente triacilgliceróis (triglicerídeos), dentro dos hepatócitos. Alterações na oxidação das gorduras no fígado ou redução na exportação de lipoproteínas de muito baixa densidade (VLDL) a partir do órgão são os principais mecanismos etiopatogênicos envolvidos com a EH. A patogênese da DHGNA é multifatorial e diversos fatores ou condições têm sido relacionados à predisposição para o seu desenvolvimento. Atualmente, diferentes tratamentos farmacológicos para DHGNA estão sendo propostos, mas ainda não há nenhum estudo comprobatório da sua eficácia. Objetiva-se avaliar os efeitos da suplementação da colina e do frutooligossacarídeo (FOS) na dieta de ratos Wistar, no modelo de esteatose hepática, induzido por dieta hiperglicídica. Foram utilizados 46 ratos machos, da raça Wistar adultos com peso variando entre 250 - 320 g, vindos do Biotério Central do Campus da USP Ribeirão Preto. Do lote inicial de animais foram distribuídos de forma aleatória nos diferentes grupos de estudo de I a IV, dependendo da indução ou não da esteatose. Considerou-se fase I o período correspondente a indução de esteatose e fase II quando se submeteu os animais a suplementação com nutrientes (Grupos III e IV), ou quando os animais receberam dieta padrão pós fase I (Grupo II) . Os animais do Grupo I (controle) receberam ração padrão do biotério que foi igual para todos os animais, sendo separado um lote da mesma ração para todo o experimento. Foi analisado as seguintes variáveis: Ingestão alimentar semanal, evolução do peso dos animais, nitrogênio urinário, amônia urinária, colesterol total e triacilgliceróis séricos, peso úmido de fígado e coração, nitrogênio e gordura tecidual, dosagem de vitamina E, malondialdeído (MDA) e glutationa no tecido hepático e análise histopatológica. Observamos que nenhum dos nutrientes empregados (colina e FOS) foi eficaz na redução da quantidade de gordura do fígado pela análise histológica. Nenhum dos nutrientes adicionados foi capaz de proteger o fígado da ação dos radicais livres, já que o MDA, um marcador indireto da geração do estresse oxidativo, manteve-se com valores elevados mesmo na fase de tratamento. Ocorreu diminuição dos níveis de triacilgliceróis em todos os grupos submetidos à indução de esteatose, do início ao final do experimento. O frutooligossacarídeo foi capaz de reduzir os níveis de colesterol sérico, em relação aos seus níveis basais, quando suplementado após indução de esteatose. / Non-alcoholic fatty liver disease (NAFLD) is a common clinical pathological condition characterized by fat accumulation in the the hepatic parenchyma hepatocyte. Liver steatosis (HS) is one of the components of NAFLD and is characterized by the presence of lipids vacuoles, mainly triacylglycerol, within the hepatocites. Alterations in fat oxidation in the liver or very low density proteins lipoproteins tranport from the organ are the main etiopatogenic mechanisms involved in HS. NAFLD patogeny is multifactor, thus several factors have been associated the propensity to develop it. Presently, many drug treatments for NAFLD are being suggested, however, there have been no studies that prove their efficacy so far. The aim of this study was to assess the effects of choline and fructooligosaccharide (FOS) suplementation in Wistar rats with HS induced by high glicid diet. Forty six adult male Wistar rats weighing between 250g and 320g from the USP (Ribeirão Preto-USP) central vivarium were used. They were divided randomly into different study groups from I to IV depending on whether steatosis would be induced or not. Phase I of the study was the period corresponding to steatosis induction and phase II was when the animals received nutrient suplementation (Groups III and IV) or when they received standard diet (Group II). Group I animals (control) received the usual vivarium food, which was the same for all of them. A certain amount of that same food was kept aside for the duration of the experiment. The following variables were analyzed: weekly food intake, weight gain, urine ammonia, urine nitrogen, total cholesterol and serum triacylglycerol, liver and heart humid weight, nitrogen and tissue fat, vitamin E, malondialdehyde (MDA) and glutathione content in the liver tissue and hitopathological analysis. As observed, neither of the nutrients (choline and FOS) was efficient in reducing the amount of fat in the liver. Neither of the nutrients added was able to protec the liver from free radicals, once the MDA, a indirect marker for oxidative stress generation, showed high levels even during the treatment phase. There was a reduction in triacylglycerol levels in all steatosis induced groups, from the beginning to the end of the experiment. Fructooligosaccharide was able to reduce the levels of serum cholesterol, in relation to its basal levels, when suplemented after steatosis induction.

L’objectif général de ce travail était de mieux définir à travers des études cliniques le rôle de l’inflammation hépatique dans l’histoire naturelle de la NAFLD. La première étude a montré que les lésions d’inflammation lobulaire ou de fibrose, même minimes, sont associées avec un risque de progression de la maladie à moyen terme. Souvent cette progression s’accompagnait d’une aggravation des facteurs de risque métabolique. La deuxième étude a démontré que les facteurs de risque métaboliques sont fréquents chez les patients avec une maladie alcoolique du foie et augmentent significativement le risque de carcinome hépatocellulaire au stade de cirrhose. Ces résultats permettent d’identifier un groupe des patients buveurs excessifs ayant un risque élevé de carcinome hépatocellulaire. La troisième étude a porté sur une cohorte transversale de plus de 5000 patients. La stéatose était un facteur associé avec la présence des lésions d’athérosclérose indépendamment des facteurs de risque cardiovasculaire classiques. Dans une cohorte de 1800 patients suivis en moyenne 8 ans, nous avons montré que la stéatose était associée à la survenue des lésions d’athérosclérose carotidienne. Ces résultats, suggèrent que la stéatose est non seulement un marqueur de risque mais un facteur qui intervient dans la pathogenèse de l’athérosclérose carotidienne. En conclusion, nos résultats suggèrent que l'inflammation hépatique, dans un contexte de stéatose contribue à la progression des lésions hépatiques, favorise l'expression de médiateurs pro-athérogènes et l’activation des voies de carcinogenèse ce qui aurait pour effet l'apparition des complications extrahépatiques chez les patients avec NAFLD. / The aim of this work was to analyze the role of chronic systemic inflammation into the natural history of NAFLD. We first undertook a study of NAFLD patients with repeat liver biopsies and demonstrated that mild lobular or portal inflammation or fibrosis in any location substantially increases the risk of progression to steatohepatitis or advanced fibrosis. Disease progression occurred concomitant with worsening of the metabolic conditions during follow-up. In the second study, we analyzed the prevalence and the impact of steatosis and metabolic risk factors on the risk of developing hepatocellular carcinoma in patients with alcoholic cirrhosis undergoing liver transplantation. The main finding of this study was that patients with advanced ALD have a high prevalence of NAFLD, and that this comorbid association confers a significantly increased risk of hepatocellular carcinoma. These findings are important for risk stratification of HCC in patients with ALD. In the third study we demonstrated that steatosis predicted carotid atherosclerosis independently of the association with classical cardiovascular risk factors. Second, in a subset of patients with longitudinal follow-up we demonstrated that baseline NAFLD was an independent predictor for incident carotid plaques. These results suggest that NAFLD is not only a marker but also an “active player” in the pathogenesis of atherosclerosis. In conclusion, our results suggests that low-grade chronic inflammation responsible for the production of pro-atherogenic cytokines and the activation of pro-oncogenic signaling pathways might be the link between liver fibrosis progression, hepatocellular carcinoma and cardiovascular risk.

La préservation statique à froid (SCS) est l’étalon-or de la préservation des organes après une greffe. En raison de la pénurie d’organes et de l’augmentation du nombre de patients figurant sur la liste d’attente, le recours aux organes provenant des donneurs à critères élargis, lesquels sont très sensibles au syndrome d’ischémie-reperfusion (IRS), ce qui entraîne une non-fonction primaire (PNF) ou un dysfonctionnement précoce (EAD), est de plus en plus fréquent.Cette recherche avait pour but d’étudier et d’identifier de nouvelles stratégies pour améliorer la qualité de la préservation des organes - d'atténuer les séquelles de l'IRS en utilisant la machine de perfusion hépatique à différentes températures et à différentes périodes d'utilisation après le prélèvement de l'organe ou en ajoutant une hémoglobine extracellulaire en tant que transporteur d'oxygène pendant le SCS.Deux modèles différents ex-vivo ont été analysés : L’un chez le petit animal avec des foies de rats normaux et stéatosiques, pour la perfusion hypothermique (HMP) et SCS avec le transporteur d'oxygène et au niveau préclinique, des foies humains stéatosiques récusés, pour la perfusion normothermique (NMP).Les résultats ont confirmé de manière significative l'intérêt de l’HMP dans la phase pré-ischémique du SCS et celui de l'utilisation de l'hémoglobine extracellulaire en améliorant la fonction hépatique, le maintien de l'anatomie des hépatocytes et en réduisant des marqueurs du stress oxydatif, de l'apoptose et de l'inflammation. Egalement, l'utilisation de NMP a permis d'analyser les foies sévèrement stéatosiques pouvant être récupérés pour une transplantation dans un avenir très proche.Cette recherche met en évidence de nouvelles approches en matière de préservation d'organes susceptibles d'augmenter le pool d'organes et d'améliorer les résultats en transplantation hépatique.Mots-clés : greffe de foie, stockage froid dans le froid, perfusion dans une machine à foie, lésion de reperfusion par ischémie, transporteur d'oxygène. / Static cold storage (SCS) is the gold standard of organ preservation after being procured for transplantation. Due to the organ shortage and the increase of number of patients in the waiting list have pushed the use organs from extended criteria donors which are very susceptible to the ischemia reperfusion syndrome (IRS) leading to primary non-function (PNF) or to early allograft dysfunction (EAD).This research was aimed to study and identify new strategies to improve the quality of organ preservation -liver, to attenuate the IRS sequels by using the liver perfusion machine (LPM) at different temperatures and times of usage after the organ procurement or by adding an extracellular hemoglobin as an oxygen carrier during SCS.Two different ex-vivo models were analyzed: small animal -normal and steatotic rat livers, for hypothermic perfusion (HMP) and SCS with the oxygen carrier and preclinical -steatotic discarded human livers, for normothermic perfusion (NMP).The results significantly confirmed the benefit of the HMP in the preischemic phase of SCS and that of the use of the extracellular hemoglobin by improving the liver function, maintenance of the hepatocytes anatomy and by a reduction of the oxidative stress, apoptosis and inflammation markers. Also, the use of NMP permitted to analyze the severely steatotic livers that can be rescued for transplantation in the very near future.This investigation unveils new approaches in organ preservation that could increase the pool of organs and improve the results in liver transplantation. Key words: liver transplantation, static cold storage, liver machine perfusion, ischemia reperfusion injury, oxygen carrier.

This study tested the ability of a novel adipose tissue derived cytokine, C1q TNF-related protein-3 (CTRP3), to prevent alcohol-induced hepatic lipid accumulation, or alcoholic fatty liver disease (ALD). Previous work has demonstrated that CTRP3 is effective at preventing high-fat diet-induced fatty liver; however, the potential of CTRP3 to inhibit ALD has not been explored. To test the potential protective effects of CTRP3, transgenic mice overexpressing CTRP3 (Tg) or wild-type littermates (WT) were subjected to one of two different models of ALD. In the first model, known as the NIAAA model, mice were fed control or alcohol-containing liquid diets (5% vol/vol) for 10 days followed by a single gavage of ethanol (5 g/kg). In the second model, the chronic model, mice were fed control or alcohol-containing diets for 6 wk with no gavage. This study found that CTRP3 reduced triglyceride accumulation in the chronic model of alcohol consumption by ~50%, whereas no reduction was observed in the NIAAA model. Further analysis of isolated primary hepatocytes from WT and Tg mice demonstrated that CTRP3 increased oxygen consumption in the presence of fatty acids, indicating that CTRP3 increases hepatic fatty acid utilization. In conclusion, this study indicates that CTRP3 attenuates hepatic triglyceride accumulation in response to long-term chronic, but not short-term, alcohol consumption.

The goal of elastography is to map the mechanical properties of soft tissues associated with health and disease. The mechanical property of interest in this work is the complex shear modulus, composed of a real part, the storage modulus, which is a measure of elasticity, and an imaginary part, the loss modulus, which is a measure of viscosity. Together, they determine the speed and attenuation of shear waves in the medium. Elastography techniques based on either ultrasound imaging or MRI can image shear wave propagation and thus are capable of measuring shear wave speed and attenuation. Dispersion, or the frequency-dependence of material parameters, is a primary confounding factor when comparing measurements between different shear wave elastography implementations. Prior attempts at quantifying this frequency-dependence suffered from inaccurate modeling assumptions and low signal-to-noise ratios (SNR). To overcome these limitations, a high-fidelity forward model of shear wave propagation in homogeneous media was developed. The model is an exact semi-analytical solution of Navier's equation and is well-suited for acoustic radiation force impulse shear wave elastography (ARFI-SWE) because it does not require precise knowledge of the strength of the source, nor its spatial or temporal distribution. Unlike models used in ARFI-SWE heretofore, it accounts for the vector polarization of shear waves and exactly represents geometric spreading of the shear wavefield, whether spherical, cylindrical, or neither. Furthermore, it is material-model independent, i.e. it makes no assumption about the frequency-dependence of material parameters. It overcomes the problem of low SNR through spatial averaging and enables estimation of the frequency-dependent complex shear modulus over a wider frequency range than has hitherto been possible. This improved ARFI-SWE was named Shear Wave Rheometry (SWR). By combining SWR with a novel torsional vibration rheometry, dispersion in tissue-mimicking gels was quantified from 1--1800 Hz. The measurements show sizable frequency-dependent variation in the shear modulus of gelatin, a material often assumed to be non-dispersive based on narrow-band measurements. SWR measurements in ex vivo bovine liver tissue yielded complex shear modulus estimates from 25--250 Hz and showed that liver tissue exhibits significant dispersion in this frequency range: a factor of 4 increase in the storage modulus and a factor of 10 increase in the loss modulus. Quality metrics showed that liver tissue can be reasonably approximated as homogeneous and isotropic for ARFI-SWE measurements in this frequency range. Results demonstrate that accounting for dispersion is essential for meaningful comparisons of measurements between systems. Moreover, improved tissue characterization enabled by SWR may have clinical relevance, for example, in the diagnosis and monitoring of chronic liver disease.

Many individuals presumed to have non-alcoholic fatty liver disease (NAFLD) consume moderate amounts of alcohol; however, little is known regarding patterns of alcohol use and how drinking behaviors may influence liver fat. We conducted a cross-sectional study of 2,475 participants of the Framingham Heart Study who underwent computed tomography (CT) to define liver fat. We performed multivariable-adjusted logistic regression models for the association between different alcohol drinking patterns, including the average alcoholic drinks/week, frequency of alcohol use, usual quantity of alcohol consumed, maximum drinks consumed in 24 hours, and binge drinking behavior, and CT-defined hepatic steatosis. We excluded heavy alcohol users defined as women who drink > 14 drinks/week and men who drink > 21 drinks/week. We also performed an analysis specific to beverage type (beer, wine, or liquor/spirit drinks).The prevalence of hepatic steatosis in our study sample (mean age ± standard deviation (SD) 49.8±10.2, 50.3% women) was 17.5%. Among individuals with presumed NAFLD, binge drinking occurred in 25.4% of individuals. In adjusted models, the odds of hepatic steatosis increased by 20% for each SD increase in the number of alcoholic drinks consumed per week (OR 1.20; 95% confidence interval (CI) 1.08, 1.36). Frequency of alcohol use (drinking days/week) was also associated with hepatic steatosis (OR 1.09; 95% CI 1.03, 1.15). The odds of hepatic steatosis increased by 15% for each SD increase in the maximum drinks per week (OR 1.15; 95% CI 1.02, 1.30). In the beverage specific analysis, alcohol use patterns were associated with hepatic steatosis among beer drinkers, but no significant associations were observed among wine drinkers. Conclusions: Even after excluding heavy alcohol users from our sample, alcohol use contributed to liver fat, which suggests alcohol-related liver fat may be present among individuals presumed to have NAFLD. Additional prospective studies are needed to validate our findings and to determine if more comprehensive alcohol use screening tools should be used in practice or clinical trial settings. / 2020-06-03T00:00:00Z

Le récepteur Sigma 1 (SigR1) est une protéine transmembranaire du RE, enrichie dans les MAMs, qui agirait comme une chaperonne. Ubiquitaire, SigR1 est très exprimé dans le système nerveux central (SNC) et le foie. Dans le SNC, SigR1 est impliqué dans un grand nombre de maladies neurodégénératives mais aussi dans le mécanisme de la douleur et dans la dépression. SigR1 est aussi impliqué dans les cancers. Il est surexprimé dans de nombreuses tumeurs cancéreuses et particulièrement dans les tumeurs hormonodépendantes dans lesquelles son expression est corrélée au statut hormonal de la tumeur. Malgré sa très forte expression dans le foie, son rôle y est inconnu. Dans l’objectif de déterminer le rôle de SigR1 dans le foie nous étudions son expression dans les différents types de tumeurs hépatiques. SigR1 est significativement surexprimé dans les adénomes hépatocellulaires (HCA) et particulièrement le sous-type muté pour le gène HNF1α, les H-HCA. Les H-HCA sont des tumeurs hépatiques bénignes stéatosées majoritairement observées chez des femmes jeunes prenant des contraceptifs oraux (œstrogènes). Quelles sont les causes et les conséquences de cette surexpression dans les hépatocytes ? En utilisant des modèles cellulaires hépatocytaires (HepG2 et Huh7) et des souris KO pour le gène HNF1α nous mettons en évidence les résultats suivants. Les œstrogènes induisent l’expression de SigR1 via son récepteur nucléaire ERα. De même, l’inhibition d’HNF1α induit une surexpression de SigR1. Cette surexpression entraine une prolifération et une stéatose des hépatocytes, correspondant au phénotype des patientes atteintes de H-HCAs. / Sigma 1 receptor (SigR1) is a transmembrane protein of the RE, enriched in the MAMs, which would act like a chaperone. Although ubiquitous, SigR1 is especially expressed in the central nervous system (CNS) and the liver. In the CNS, SigR1 has been linked to neurodegenerative diseases and also pain and depression. SigR1 is also involved in cancer. SigR1 is overexpressed in many cancer tumors and especially in hormone dependent tumors where its expression is correlated to the hormonal status of the tumor. Although SigR1 is highly expressed in the liver, its role in this organ is unknown. Aiming at finding the role of this protein in the liver we analyze its expression in several liver tumors. SigR1 is significantly overexpressed in hepatocellular adenomas mutated for HNF1α gene, H-HCA. H-HCA are benign liver tumors with marked steatosis. They are mostly found in women taking oral contraceptives (estrogens). Why is SigR1 overexpressed in H-HCA ans what are the consequences of this overexpression? Using hepatocyte cellular models (HepG2 and Huh7) and mice that are KO for HNF1α gene, we found the following results. Estrogens induce the expression of SigR1 through its nuclear receptor ERα. HNF1α inhibition also induces its expression. This overexpression leads to an increase of the cell proliferation rate and steatosis. These effects resume H-HCA patients’ phenotype.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18147号 / 医博第3867号 / 新制||医||1002(附属図書館) / 31005 / 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 坂井 義治, 教授 千葉 勉 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DGAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19964号 / 医博第4154号 / 新制||医||1017(附属図書館) / 33060 / 京都大学大学院医学研究科医学専攻 / (主査)教授 柳田 素子, 教授 横出 正之, 教授 渡邊 直樹 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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