Liver fatty acid binding protein has been reported to possess antioxidant properties in the liver. The aim of this study was to investigate the effect of this protein in a nonalcoholic fatty liver disease (NAFLD) cell culture model. Rat hepatoma cells were treated with an oleate:palmitate (2:1) mixture for either 1 and 2 days, or further treated with 500 µM clofibrate to induce L-FABP expression. Intracellular lipid accumulation was quantitated by Nile Red. Lipotoxicity was determined using the WST-1 assay. Dichlorofluorescein (DCF) was utilized to assess intracellular reactive oxidative species (ROS) level. Measurement of lipotoxicity showed statistical decreases in cell viability as lipid concentrations increased in a dose-dependent manner. NAFLD cell cultures showed characteristic cellular damage from increased ROS levels in fatty acid treated cells. All groups treated with clofibrate showed statistically increased intracellular L-FABP levels and reduced ROS levels. The results lead to the conclusion that clofibrate induces L-FABP expression and in this manner suppresses hepatocellular ROS generation.

Population health data suggests that the development of metabolic disease is influenced by early life events. Gestational diabetes (GDM) is a common complication of pregnancy, but its effects on the offspring are poorly understood. It is hypothesized that a diet high in fat and sucrose will cause excessive weight gain and obesity during pregnancy accompanied by hyperglycemia and hyperinsulinemia that are characteristic of GDM. It is also hypothesized that gestational exposure to GDM will cause obesity, hepatic steatosis and insulin resistance in the offspring when compared to the offspring from metabolically healthy, lean mothers.

Liver fatty acid binding protein has been reported to possess antioxidant properties in the liver. The aim of this study was to investigate the effect of this protein in a nonalcoholic fatty liver disease (NAFLD) cell culture model. Rat hepatoma cells were treated with an oleate:palmitate (2:1) mixture for either 1 and 2 days, or further treated with 500 µM clofibrate to induce L-FABP expression. Intracellular lipid accumulation was quantitated by Nile Red. Lipotoxicity was determined using the WST-1 assay. Dichlorofluorescein (DCF) was utilized to assess intracellular reactive oxidative species (ROS) level. Measurement of lipotoxicity showed statistical decreases in cell viability as lipid concentrations increased in a dose-dependent manner. NAFLD cell cultures showed characteristic cellular damage from increased ROS levels in fatty acid treated cells. All groups treated with clofibrate showed statistically increased intracellular L-FABP levels and reduced ROS levels. The results lead to the conclusion that clofibrate induces L-FABP expression and in this manner suppresses hepatocellular ROS generation.

Obesity and metabolic dysfunction are worldwide health epidemics and they have grown to unprecedented levels. Human NAFLD is directly linked to obesity and metabolic dysfunction, so attention was given to elucidating a more complete understanding of the liver's role in mediating the metabolically healthy obese phenotype and to better characterizing the potential contribution of dietary fat and fatty acids as a therapeutic supplement to obesogenic diets. Specifically, flaxseed is high in α-linolenic acid (ALA; 18:3 n-3) and low in linoleic acid (LA; 18:2 n-6), and contains multiple other components such as fiber and lignans, and was investigated for its high potential to modify obesity phenotype and fatty liver disease. Additionally, we explored the temporal effect of initiating high-fat diets in various phases of adulthood. However, work in this field is complicated by an ongoing search for appropriate preclinical animal models of NAFLD as they have not been able to replicate the full spectrum of human NAFLD. As such, this dissertation sought to explore fatty liver disease in popular murine models of overnutrition, as well as a novel hen model. Major findings from this work showed that (1) exposure to a high-fat diet during early adulthood preserves metabolic homeostasis, modifies liver morphology, and protects against obesity-related disease, (2) dietary enrichment with flaxseed is capable of increasing tissue n3PUFA content, but this appeared to be only weakly related to metabolic and histological outcomes, and (3) there are limitations to the laying hen as a model of NAFLD as the pathogenic changes may not adequately match the human condition.

Obesity is increasing worldwide, and the associated health-risks are also on the rise. Eventually, obesity related tissue damage leads to complications such as chronic inflammation, diabetes, cardiovascular disease, and non-alcoholic fatty liver disease. Adipose tissue expansion in obesity triggers specific mechanisms that cause tissue damage. The immune system is especially agitated with excessive fat accumulation, which triggers inflammation and subsequent immune cell infiltration of tissue. Neutrophils are a major immune cell that cause damage in obesity, and the protease neutrophil elastase (NE) is a major neutrophil released factor of tissue damage. The goal of this study is to use tissue extracted from neutrophil elastase knockout (NEKO) mice that have been fed a high-fat high-fructose diet (HFHFD), and compare them to wild-type (WT) mice fed a normal chow diet (NCD), high-fat diet (HFD), and HFHFD to understand the effect of neutrophil elastase damage in obesity. Tissue from aged (NEKO) mice will also be examined to evaluate the role of neutrophils and NE in tissue damage in aging and obesity. Mice in these experimental groups were sacrificed and had their tissue extracted for various staining protocols to discover the extent of tissue damage and immune cell infiltration between mice with and mice without NE. One experiment had 4 different diets fed to mice. The other experiment had mice aged for 2 years, and mice aged for 3 months and 4 months. Mice from the first experiment were fed for 4 months and separated into 4 groups based on diet, WT-NCD, WT-HFD, WT-HFHFD, and NEKO-HFHFD. Our data indicates that, in comparison with WT-HFHF mice, NEKO-HFHFD mice had less steatosis, fibrosis, immune cell infiltration, and apoptosis within the liver. Neutrophil infiltration into the liver is increased by the HFHFD diet. HFHFD diet also stimulates fibrosis, as indicated by collagen deposition in the liver. Neutrophil accumulation is also associated with the increase of macrophages and CD4 Th Cells in the liver, particularly in WT mice fed the HFHFD. Interestingly, the liver from NEKO-HFHFD mice had dramatically reduced infiltration of neutrophils, macrophages, and CD4+ Th cells. Our data suggests that NE is required for HFHFD induced inflammation and fibrosis in the liver. Mice from the second experiment were split into 3 groups based on age, WT-Young (3 months and 4 months), WT-Old, and NEKO-Old. All groups were fed the same normal chow diet, but WT Old and NE KO Old were both aged to 2 years old. Our data revealed that NE deletion in aged mice reduced fibrosis, elastin fragmentation, calcification, and presence of NE within the aorta. While part of the mechanism for neutrophil elastase related tissue damage has been explored through this one-year master degree research project, more work is needed to fully understand how NE is stimulated and causes tissue damage. Future work should examine the potential interaction between neutrophils and other immune cells in obesity and aging. / 2022-06-04T00:00:00Z

Die Beurteilung von Fibrose und Entzündungsaktivität ist essenziell zur Feststellung eines progredienten Krankheitsverlaufes bei Patienten mit nicht-alkoholischer Fettlebererkrankung (NAFLD). Während zur Bestimmung des Fibrosegrades ultraschall-gestützte Methoden und Serum-Marker die invasive Leberbiopsie für verschiedenen Fragestellungen bereits ersetzen können, bleibt die nicht-invasive Charakterisierung der Entzündungsaktivität eine Herausforderung. Frei zirkulierende DNA (cfDNA) als neuer nicht-invasiv zu bestimmender Biomarker für zelluläre Entzündung und Zelltod wurde in Patienten mit NAFLD bisher noch nicht evaluiert. Für die vorliegenden Dissertation wurde die Konzentration von cfDNA (90 und 222bp-Fragmente) im Plasma von 58 NAFLD-Patienten sowie 13 gesunden Kontroll-Probanden, die an zwei prospektiven Diagnostikstudien teilgenommen hatten, mittels quantitativer real-time PCR gemessen und ausgewertet. Die Ergebnisse wurden mit der Gewebesteifigkeit (Transiente Elastographie) und dem Leberfettgehalt (Controlled attenuation parameter; Magnetresonanzspektroskopie / 1H-MRS) sowie den Serumspiegeln der Aminotransferasen und des Ferritins verglichen. Der Gehalt an 90bp-cfDNA im Plasma war bei NAFLD-Patienten signifikant höher als bei den gesunden Probanden. In der NAFLD-Kohorte korrelierte die cfDNA-Konzentration signifikant mit der Krankheitsaktivität und dem Krankheitsgrad, insbesondere bei Patienten mit erhöhter Lebersteifigkeit. Die weitere Untersuchung von cfDNA als Biomarker für den Krankheitsverlauf von Patienten mit NAFLD ist daher empfehlenswert, insbesondere im Hinblick auf die Identifikation von Patienten mit einem erhöhten Risiko für progrediente NAFLD.:1 Vorbemerkung 2 Bibliographische Beschreibung 3 Abkürzungsverzeichnis 4 Einführung 4.1 Nicht-alkoholische Fettlebererkrankung 4.1.1 Definition und Epidemiologie 4.1.1.1 Herausforderung im Umgang mit der NAFLD 4.1.2 Pathogenese 4.1.3 Nichtinvasive Diagnostik 4.1.3.1 Bildgebende Verfahren 4.1.3.2 Serum-basierte Steatose- und Fibrose-Indices 4.1.4 Histopathologie 4.1.5 Genetische Prädisposition 4.1.6 Therapie 4.2 Frei zirkulierende DNA 4.2.1 Entdeckung und Ursprung frei zirkulierender DNA 4.2.2 Klinische Schwerpunkte in der bisherigen cfDNA-Forschung 4.2.3 Potenzielle Bedeutung der cfDNA bei NAFLD 4.3 Wissenschaftliche Zielsetzung 5 Publikation 6 Zusammenfassung und Ausblick 7 Literaturverzeichnis 8 Anhang 8.1 Erklärung über die eigenständige Abfassung der Arbeit 8.2 Erklärung über den wissenschaftlichen Beitrag der Promovendin an der ausgewählten Publikation 8.3 Teilnahmebescheinigung: Vorlesung zur „Guten wissenschaftlichen Praxis“ an der Medizinischen Fakultät der Universität Leipzig 8.4 Lebenslauf 8.5 Veröffentlichungen im Rahmen dieser Arbeit

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.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20560号 / 医博第4245号 / 新制||医||1022(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 柳田 素子, 教授 西渕 光昭 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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Obesity is a growing problem that impacts both adults and children. Obesity is linked to the development of unfavorable health outcomes like excess fat accumulation in the liver, a problem known as non-alcoholic fatty liver disease (NAFLD). Brown adipose tissue (BAT), a thermogenic body fat that can be turned on by cold, produces heat by consuming circulating lipids and glucose in a futile cycle. Less active cold-stimulated BAT is linked to obesity and type 2 diabetes in adults but whether this relationship exists in children is unknown. In rodents, increases in BAT activity are associated with reductions in NAFLD, effects that may be mediated through changes in the gut microbiome and reductions in peripheral serotonin. Whether the gut microbiome and serotonin play a role in regulating BAT activity in adults and children is not known. In this thesis, we have utilized magnetic resonance imaging (MRI) proton density fat fraction (PDFF) to assess BAT in the supraclavicular (SCV) region after three hours and one hour of whole-body cold exposure in adults and children, respectively. In 60 adults (aged 18-57 years), we examined whether there is a relationship between cold-stimulated BAT activity and liver fat (assessed via MRI) and whether gut microbiota plays a role in connecting these two tissues. In children, we investigated, BAT activity after whole-body cold exposure in 26 boys (aged 8-10 years). We also explored if BAT activity was different between boys with and without overweight/obesity. Finally, in young boys, we measured the levels of serotonin in platelet-poor plasma and its metabolic end product 5-hydroxyindole acetic acid (5-HIAA) in the urine. We explored if these measures of circulating serotonin were related to cold-stimulated BAT activity and if they were different between boys with and without overweight/obesity. The findings from this research indicate that higher cold-stimulated BAT activity is associated with lower liver fat in adults, but that this relationship is unlikely mediated through changes in the gut microbiota. Additionally, boys with overweight/obesity have lower cold-stimulated BAT activity and lower 5-HIAA in their urine compared to those with normal weight. Moreover, circulatory serotonin is negatively related to total adiposity. However, circulating serotonin is not related to cold-stimulated BAT activity in this cohort. These findings are important as they indicate for the first time that increasing BAT activity in adults and children could potentially be a new avenue for the treatment of NAFLD and obesity. / Thesis / Doctor of Philosophy (PhD) / Obesity is a risk factor for the accumulation of extra liver fat, a problem known as non-alcoholic fatty liver disease (NAFLD). Brown adipose tissue (BAT) is a kind of body fat that rather than storing calories like white fat burns calories when switched on by cold. Studies in adults have shown that people with obesity and type 2 diabetes have less active BAT suggesting switching it on may be helpful to promote weight loss and lower glucose. However, whether this relationship exists in children is not known. In rodents, increased BAT activity has also been linked to reductions in NAFLD, effects that might involve a hormone called serotonin, or changes in the gut microbiome but whether this is important in children and adults is also not understood. In this thesis, we utilized magnetic resonance imaging (MRI) to examine BAT activity after whole-body cold exposure in adults (3 hours) and children (1 hour). In 60 adults (aged 18-57 years), we report that higher cold-stimulated BAT activity is linked to NAFLD, but gut microbiota does not seem to play a role in this relationship. In 26 boys (aged 8-10 years), BAT is less responsive to cold in boys with overweight/obesity compared to boys with normal weight. Additionally, serotonin is lower in boys with overweight/obesity compared to boys with normal weight. These findings suggest that increasing BAT activity in adults and children could potentially be a new avenue for the treatment of NAFLD and obesity.