Orientador: Helena Coutinho Franco de Oliveira / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-27T08:22:09Z (GMT). No. of bitstreams: 1 Paiva_AdrieneAlexandra_D.pdf: 2285275 bytes, checksum: e9d86e12d4ac3d041953799c130a314a (MD5) Previous issue date: 2015 / Resumo: A doença hepática gordurosa não alcoólica (Nonalcoholic fatty liver disease - NAFLD) é a principal manifestação hepática na obesidade e síndrome metabólica. A história natural da doença envolve a esteatose, estresse oxidativo, inflamação, fibrose e morte celular. Níveis plasmáticos elevados de lipoproteínas ricas em triglicérides são fatores de risco independentes para doenças cardiovasculares. Estudos clínicos e experimentais mostram forte correlação entre triglicérides plasmáticos (TG) e os níveis de apolipoproteína CIII. A apolipoproteína CIII também é aumentada no plasma de pacientes diabéticos. Ao comparar camundongos apoCIII transgênicos com controles não transgênicos irmãos (NTg), mostramos aqui que a super-expressão de apoCIII, independentemente da dieta rica em gordura (High fat diet - HFD), resulta em características NAFLD, ou seja, aumento do conteúdo de lípidos no fígado, diminuição poder antioxidante, aumento da expressão de TNF'alfa', receptor de TNF'alfa', caspase-1 clivada e interleucina-1'beta', diminuição do receptor de adiponectina-2 e aumento da morte celular. Além disso, os indicadores de inflamação sistêmica de TNF'alfa' e a proteína C-reactiva estão também elevados em camundongos apoCIII. Esse quadro é agravado e características adicionais da NAFLD são diferencialmente induzidas por HFD em camundongos apoCIII. A HFD induziu intolerância à glicose, juntamente com aumento da gliconeogênese, evidenciando a resistência hepática à insulina, induziu um aumento significativo da TNF'alfa' (8 vezes) e IL-6 (60%) no plasma em camundongos apoCIII comparados aos NTg. Os outros indicadores de inflamação (proteína C reativa plasmática e TNF?, TNFr, caspase-1 e IL-1'beta' hepática) mantiveram-se superiores em camundongos apoCIII em HFD. A ativação da via intrínseca (Bax / Bcl-2) e a proteina efetora (caspase-3) de apoptose foram aumentados em ambos os camundongos apoCIII com low fat diet (LFD) e HFD. Como esperado, o tratamento com fenofibrato reverteu vários dos efeitos da dieta e da apoCIII. No entanto, o fibrato não normalizou o padrão inflamatório nos animais apoCIII, tais como o aumento do TNF'alfa', TNFr, IL1'beta' e redução do adiponectina-R2, mesmo com total correção do teor de lipídios no fígado. Estes resultados indicam que a superexpressão apoCIII desempenha um papel importante na inflamação hepática e na morte celular e aumenta a suscetibilidade e a gravidade da esteatose hepática induzida pela dieta / Abstract: Nonalcoholic fatty liver disease (NAFLD) is the principal liver manifestation in obesity and metabolic syndrome. The natural history of the disease involves steatosis, oxidative stress, inflammation, fibrosis and cell death. Elevated plasma levels of triglyceride-rich remnant lipoproteins are independent cardiovascular disease risk factors. Clinical and experimental studies show strong correlation and causal links between plasma triglycerides (TG) and apolipoprotein CIII levels. Apolipoprotein CIII is also increased in the plasma of diabetic patients. By comparing apolipoprotein (apo) CIII transgenic mice with control non-transgenic (NTg) littermates, we show here that the overexpression of apoCIII, independently of high fat diet (HFD), results in NAFLD features, namely, increased liver lipid content, decreased antioxidant power, increased expression of TNF'alfa', TNF'alfa' receptor, cleaved caspase-1 and interleukin-1'beta', decreased adiponectin receptor-2 and increased cell death. In addition, systemic indicators of inflammation TNF? and C-reactive protein are also elevated in apoCIII mice. This picture is aggravated and additional NAFLD features are differentially induced by HFD in apoCIII mice. HFD induced glucose intolerance together with increased gluconeogenesis, evidencing hepatic insulin resistance. Marked increases in plasma TNF'alfa' (8-fold) and IL-6 (60%) were induced by HFD in apoCIII mice compared to NTg mice. The other inflammatory indicators (plasma C reactive protein, liver TNF'alfa', TNFr, caspase-1 and IL1 'beta') remained higher in HFD apoCIII mice. Cell death signaling (Bax/Bcl2), effector (caspase-3) and apoptosis were augmented in both low and HFD apoCIII mice. As expected, fenofibrate treatment reversed several of the diet and apoCIII effects. However, fibrate did not normalize apoCIII inflammatory traits, such as increased TNF'alfa1, TNFr, IL1'beta' and reduced adiponectin-R2, even under fully corrected liver lipid content. These results indicate that apoCIII overexpression play a major role in liver inflammation and cell death increasing susceptibility to and the severity of diet induced NAFLD / Doutorado / Fisiologia / Doutora em Biologia Funcional e Molecular

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The worldwide prevalence of obesity and obesity-associated liver diseases have attracted great attention in the past decades. Obesity is an increasing health problem, which can induce a series of metabolic syndrome associated diseases, such as fatty liver disease, type 2 diabetes. The conventional causes for obesity, such as over-eating, sedentary life-style, and genetic factors, cannot fully explain the global rapid increase of obese population in the last few decades. It was found that the production of persistent organic pollutants (POPs) in the industry was closely correlated with the prevalence of obesity. POPs are organic chemicals that are resisted to degrade by various processes and widely applied in daily products to improve the quality of our life. 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) is the most abundant and toxic congener in the family of polybrominated diphenyl ethers (PBDEs), which are the commonly used flame retardants and listed as POPs in 2009. High concentration of BDE-47 has been found in indoor dust and marine fish in Hong Kong. Owing to their high lipophilic and persistent characters, BDE-47 is mainly accumulated in adipose tissue. Epidemiological data indicates that exposure to BDE-47 is associated with obesity and obesity-associated liver diseases. Therefore, based on published research, we hypothesize that BDE-47 exposure may increase the occurrence of obesity and aggravate the progression of obesity-associated fatty liver disease through promoting adipocyte differentiation and impairing lipid metabolism. To verify this hypothesis, mouse preadipocytes (3T3-L1 cells) were exposed to BDE-47 and differentiated into adipocytes. Excitedly, with BDE-47 exposure, more lipid droplets were formed and accumulated in the treated cells than that in untreated adipocytes (without BDE-47 exposure). Along with the increased content of triglyceride accumulation, augmented gene and protein levels of transcription factors (PPARγ and PGC-1α), and related genes (FABP4 and C/EBPα) were also detected in BDE-47 treated cells. In addition, the total production of reactive oxygen species (ROS), contents of lipid peroxidation and DNA oxidation were obviously increased in adipocytes treated with BDE-47 (10 μM). To explore how BDE-47 regulated the oxidative stress signal pathways, antioxidants of ROS sources were employed with BDE-47 exposure during adipocyte differentiation. Notably, mitochondrial respiration, xanthine oxidase and NADPH pathway were significantly influenced by BDE-47 exposure to generate ROS in the treated adipocytes. The effects of BDE-47 on mitochondrial respiration were also determined for further exploring the relationship between mitochondrial ROS and adipocyte differentiation. Significant elevation of mitochondrial ROS was detected in adipocytes exposed with BDE-47 (10 μM). Furthermore, to support the energy requirements for the growth of adipocytes during differentiation process, BDE-47 improved the mitochondrial metabolism for ATP production via increasing the spare mitochondrial respiration capacity. Inhibiting the mitochondrial ROS generation in BDE-47-treated adipocytes with antioxidant attenuated the generation of ROS and reduced the accumulation of lipid droplets as well. This phenomenon indicated that the ROS-induced by BDE-47 through mitochondrial chain was critical for adipocyte differentiation. Global metabolomic profiling based on high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) was performed on differentiated 3T3-L1 cells to reveal the metabolic changes induced by BDE-47. Twenty three significantly changed metabolites were identified in the adipocytes after BDE-47 exposure. The results of pathway analysis showed that purine and glutathione metabolism were the main impacted pathways and upregulated by BDE-47 treatment. In purine metabolism, increasing levels of adenosine monophosphate (AMP) and guanosine monophosphate (GMP) induced by BDE-47 led to the increment of inosine 5'-monophosphate (IMP) in adipocytes. These increases forwarded the pathway and caused high production of uric acid along with hydrogen peroxide, which contributed to the elevation of ROS after exposure to BDE-47. Inhibiting the synthesis of uric acid with antioxidant could significantly decrease the production of ROS, the levels of adipogenesis-related genes, and the accumulation of lipid droplets in BDE-47 exposed adipocytes. These results further demonstrated that exposure to BDE-47 promoted adipocyte differentiation via causing oxidative stress, upregulating purine metabolism, and increasing production of uric acid. Subsequently, C57BL/6J mouse model with diet interaction was employed to explore the obesogenic effects of BDE-47. Male C57BL/6J mice were fed with either a low-fat diet (LFD, 10% fat) or high-fat diet (HFD, 60% fat) for 15 weeks and subcutaneously injected with BDE-47 (7mg/kg [Low dose, L] or 70mg/kg [High dose, H]) or the vehicle weekly. It was found that exposure to BDE-47 (H) significantly led to the elevation of body weight and serum triglyceride content in HFD fed mice. Besides, the combination of BDE-47 and HFD also significantly increased the weight of white adipose tissue (WAT) and augmented the size of adipocytes in WAT. These have confirmed the obesogenic effects of BDE-47 in vivo. Additionally, BDE-47 (H) exposure significantly increased the accumulation of hepatic triglyceride content and lipid droplets accompanying with elevated inflammation in HFD fed mice, indicating the deterioration of hepatic steatosis in BDE-47 treated mice. Moreover, the integration analysis of lipidomic and gene expression revealed that BDE-47 up-regulated triglyceride synthesis but suppressed lipid exportation and β oxidation to impair the lipid metabolism and worsen the accumulation of hepatic lipid in HFD fed mice. In addition, the increase of liver fibrosis scars (the protein level of αSMA and collagens), serum transaminase levels, as well as lipid peroxidation have been detected in the mice with co-treatment of BDE-47 and HFD. BDE-47 exposure also increased the production of ROS and the levels of fibrotic genes in hepatocytes. However, in LFD with BDE-47 exposed mouse liver, we cannot observe such changes compared with the control (LFD-DMSO). Interestingly, the application of antioxidants reversed the BDE-47-induced fibrotic responses (the expression of αSMA and col3) in hepatocytes, which indicated that the increase of liver fibrosis scars was tightly associated with the level of oxidative stress. In conclusion, these results offered a new insight of lipid toxicities and underlying mechanism of BDE-47 induced obesity-related liver fibrosis. As far as we know, this is the first systematic study of the obesogenic effects and underlying mechanisms of BDE-47 in diet-induced mouse model. These results have showed the pathological roles of BDE-47 in the development of obesity and related liver diseases by an integration analysis of omics study and biological analysis in vivo and in vitro. Meanwhile, inhibitors were applied to investigate the mechanism of BDE-47-induced toxicity. Taken together, our results indicated that BDE-47 exposure could accelerate the development of obesity and aggravate the progression of fatty liver in obese mice via causing oxidative stress. This study may shed a light for an explanation for the worldwide prevalence of obesity and related liver diseases. Furthermore, this work reflects the potential of omics study and biological methods for toxicity assessment of environmental pollutants on human health. It would be helpful for the clinical diagnose and treatment.

The burden of liver diseases is increasing worldwide, accounting for two million deaths annually. In the past decade, tremendous progress has been made in the basic and translational research of liver tissue engineering, which seeks to build physiologically relevant liver models to better understand liver diseases, accelerate drug development, and advance regenerative medicine. Liver microtissues are small, three-dimensional (3D) hepatocyte cultures that recapitulate liver physiology and have been used in many biomedical applications. However, sourcing of high-quality human hepatocytes for microtissue fabrication poses a significant challenge. Since the inception of induced pluripotent stem cell (iPSC) technology, iPSC-derived hepatocyte-like cells (HLCs) have demonstrated significant improvement over other hepatocyte cell sources in many studies. Despite their promising potential, HLCs face certain challenges: they resemble fetal hepatocytes rather than adult hepatocytes; they undergo dedifferentiation quickly after reaching maturity; they are produced on a small scale; and they exhibit large donor-to-donor and batch-to-batch variability. This doctoral thesis focuses on engineering patient-specific liver microtissues with prolonged phenotypic maintenance and disease modeling potential. Chapter 1 provides a review of recent advances, challenges, and future directions in liver microtissue research. 3D microtissues can be generated by scaffold-free assembly or scaffold-assisted methods using macroencapsulation, droplet microfluidics, and bioprinting. Optimization of the hepatic microenvironment entails incorporating the appropriate cell composition for enhanced cell-cell interactions and niche-specific signals, and creating scaffolds with desired chemical, mechanical and physical properties. Perfusion-based culture systems such as bioreactors and microfluidic systems are used to achieve efficient exchange of nutrients and soluble factors in the microtissues. Chapter 2 describes our efforts in optimizing methods of generating human HLCs from the peripheral blood of selected donors. Peripheral blood mononuclear cells (PBMCs) were first reprogrammed to iPSCs using Sendai viruses carrying the four Yamanaka factors. We developed an optimized protocol for hepatocyte differentiation from iPSCs, and obtained HLCs that exhibited hepatocyte-specific phenotypes and functions that were comparable to other reports. We then demonstrated the one-step generation of homogeneous, microencapsulated liver microtissues in Chapter 3. Droplet microfluidics was used to produce double emulsion droplets that served as individual microenvironments where HLCs were encapsulated in methylated collagen and alginate. The cells self-assembled in <16 hours through dynamic interactions with methylated collagen, and individual spheroids were encapsulated in polymerized alginate gel to prevent cell fusion and attachment. HLC spheroids remained viable and functional for >24 days, whereas 2D HLCs underwent dedifferentiation within 7 days of reaching maturity. The spheroids showed further maturation compared to the 2D HLCs at peak maturity. Co-culture of HLCs with human endothelial cells was also investigated in the 3D system, but no improvement was observed over monoculture spheroids with our current methods. To our knowledge, this is the first study to utilize droplet microfluidics to generate homogeneous, compartmentalized droplets that serve as optimized 3D microenvironments for HLC aggregation and maturation. It demonstrated the potential of using high-throughput droplet microfluidics to produce and encapsulate mature, functional human HLCs for long-term applications. In Chapter 4, we developed a TM6SF2 knockout and overexpression model in iPSCs to investigate its molecular function and potential role in nonalcoholic fatty liver disease (NAFLD). Transmembrane 6 superfamily member 2 (TM6SF2) is a protein of unknown function, and analysis from our model suggested that TM6SF2 dysregulation has a biphasic response. Our data showed that both knockout and overexpression can result in the upregulation of cholesterol biosynthesis and a defect in the proper processing of lipid droplets. Additionally, high expression of the TM6SF2 rs58542926 variant has an increased risk for cholesterol upregulation, compared to the major allele. Future works will focus on generating liver microtissues from the TM6SF2 knockout and transgene-expressing cells using droplet microfluidics, and validating our hypotheses with established biochemical and functional assays.

Background/objectives The relationship between nonalcoholic fatty liver disease (NAFLD) and albuminuria has been shown in many epidemiologic studies, although the results were inconsistent. This meta-analysis was conducted to summarize all available data and to estimate the risk of albuminuria among patients with NAFLD. Methods Comprehensive literature review was conducted utilizing Medline and Embase database through January 2018 to identify studies that compared the risk of albuminuria among patients with NAFLD versus those without NAFLD. Effect estimates from each study were extracted and combined using the random-effect, generic inverse variance method of DerSimonian and Laird. Results Nineteen studies (17 cross-sectional studies and two cohort studies) with 24 804 participants fulfilled the eligibility criteria and were included in this meta-analysis. The risk of albuminuria among patients with NAFLD was significantly higher than those without NAFLD with the pooled odds ratio (OR) of 1.67 [95% confidence interval (CI): 1.32-2.11]. Subgroup analysis demonstrated the significantly increased risk of albuminuria among patients with NAFLD without diabetes with pooled OR of 2.25 (95% CI: 1.65-3.06). However, we found no significant association between albuminuria and NAFLD among diabetic patients [pooled OR 1.28 (95% CI: 0.94-1.75)]. Conclusion A significantly increased risk of albuminuria among patients with NAFLD was observed in this meta-analysis. Physicians should pay more attention to the early detection and subsequent treatment of individuals with microalbuminuria especially in patients with NAFLD.

Background & aims Cytochrome P450 2A5 (CYP2A5) is induced by ethanol, and the ethanol induction of CYP2A5 is regulated by nuclear factor-erythroid 2-related factor 2 (NRF2). Cyp2a5 knockout (Cyp2a5−/−) mice develop more severe alcoholic fatty liver than Cyp2a5+/+ mice. Fibroblast growth factor 21 (FGF21), a PPARα-regulated liver hormone, is involved in hepatic lipid metabolism. Alcoholic and non-alcoholic fatty liver are enhanced in Pparα knockout (Pparα−/−) mice. This study investigates the relationship between the PPARα-FGF21 axis and the enhanced alcoholic fatty liver in Cyp2a5−/− mice. Methods Mice were fed the Lieber-Decarli ethanol diet to induce alcoholic fatty liver. Results More severe alcoholic fatty liver disease was developed in Cyp2a5−/− mice than in Cyp2a5+/+ mice. Basal FGF21 levels were higher in Cyp2a5−/− mice than in Cyp2a5+/+ mice, but ethanol did not further increase the elevated FGF21 levels in Cyp2a5−/− mice while FGF21 was induced by ethanol in Cyp2a5+/+ mice. Basal levels of serum FGF21 were lower in Pparα−/− mice than in Pparα+/+ mice; ethanol induced FGF21 in Pparα+/+ mice but not in Pparα−/− mice, whereas ethanol induced hypertriglyceridemia in Pparα−/− mice but not in Pparα+/+ mice. Administration of recombinant FGF21 normalized serum FGF21 and triglyceride in Pparα−/− mice. Alcoholic fatty liver was enhanced in liver-specific Fgf21 knockout mice. Pparα and Cyp2a5 double knockout (Pparα−/−/Cyp2a5−/−) mice developed more severe alcoholic fatty liver than Pparα+/+/Cyp2a5−/− mice. Conclusions These results suggest that CYP2A5 protects against the development of alcoholic fatty liver disease, and the PPARα-FGF21 axis contributes to the protective effects of CYP2A5 on alcoholic fatty 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

京都大学 / 0048 / 新制・論文博士 / 博士(農学) / 乙第13019号 / 論農博第2829号 / 新制||農||1042(附属図書館) / 学位論文||H28||N4965(農学部図書室) / 32947 / (主査)教授 久米 新一, 教授 松井 徹, 教授 祝前 博明 / 学位規則第4条第2項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

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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