The non-alcoholic fatty liver disease (NAFLD) is the most common liver pathology in developed countries with an estimated prevalence of 20 to 30% in the American population. A typically benign and asymptomatic pathology, NAFLD is characterized by hepatic steatosis and abnormal levels of hepatic enzymes stemming from an increase in circulating free fatty acids originating from white adipose tissue lipolysis, an increased de novo lipogenesis, reduced fatty acid oxidation and decreased hepatic triglycerides secretion, all within an insulin resistance context. NAFLD has the potential to progress to the non-alcoholic steatohepatitis (NASH), a condition marked by inflammation, advanced oxidative stress and fibrosis. NASH is expected to be the leading cause of liver transplant by 2020 due to its complications (i.e.: cirrhosis, hepatocellular carcinoma and liver failure). Various xenobiotics such as pesticides have been shown to promote the apparition and development of NAFLD. Of interest to this study is the neonicotinoid imidacloprid, more contemporarily known for its suspected role in the colony collapse disorder of various anthophilae species. Imidacloprid has been shown to induce hepatic oxidative stress in rats, a significant factor in the development of NAFLD and its progression to NASH. Lifestyle modifications, namely physical exercise, is a current treatment which has been proven beneficial to prevent and treat NAFLD by reducing hepatic steatosis, oxidative stress and improving insulin sensitivity. The role of any neonicotinoid on the development of NAFLD has yet to be examine and few have looked at the role of exercise in the treatment of NAFLD brought about by pesticide contamination.

研究背景及實驗目的: 非酒精性脂肪性肝炎(NASH)使得肥胖和2 型糖尿病變得複雜，肝臟炎症的持續產生是其主要的發病機理。CXCL10 是一種促進炎症的細胞因數，其在肥胖和2 型糖尿病中的表達顯著升高。CXCL10 以及其受體CXCR3 是否在NASH 的發生發展中起作用尚不清楚。在本研究中，我們探索了CXCL10 以及其受體CXCR3 在脂肪性肝炎中的功能， 並評估了CXCL10 在NASH 中的臨床價值。 / 實驗方法：CXCL10 基因敲除鼠，CXCR3 敲除鼠以及野生型C57BL/6 小鼠給予蛋氨酸膽鹼缺乏食(MCD)4 周或者8 周。CXCL10 的信號通路以及下游靶點通過細胞因數分析，cDNA array, 蛋白DNA 結合實驗，自噬溶酶體系統分析進行檢測。為了闡明CXCL10 抑制對NASH 的預防治療作用，我們給MCD 餵養的小鼠注射抗CXCL10 抗體。用不同濃度的CXCL10 抗體以及CXCR3 抑制劑NIBR2130 幹預MCD 培養的肝細胞株AML-12。臨床研究中，我們收集了147個非酒精性脂肪肝患者以及73 個健康對照的血清，用酶聯免疫吸附試驗檢測血清中CXCL10 的水準。 / 結果：野生型小鼠給予MCD 餵養後，CXCL10 以及CXCR3 的表達升高，並出現脂肪性肝炎的表現。然而，MCD 飼養的CXCL10 以及CXCR3 基因敲除鼠中，脂肪性肝炎明顯減輕。CXCL10 通過促炎細胞因數的產生以及NK-κB 信號通路促進MCD 飼養的小鼠NASH 的發生。CXCL10 通過促進脂質合成的基因SREBP-1c, ChREBP 和 SCD-1 引起脂肪變性，並通過CYP2E1 以及 C/EBPβ 的上調引起氧化應激。值得注意的是，自噬的損傷在CXCL10 以及CXCR3 導致的脂肪性肝炎的進展中起重要作用。 MCD 飼養的野生型小鼠中p62 以及LC3-II 表達明顯高於CXCL10 以及CXCR3 基因敲除鼠。通過抗CXCL10 抗體中和CXCL10 可以減輕MCD 食引起的小鼠脂肪性肝炎以及MCD 培養液引起的AML-12 細胞損傷。高選擇性的CXCR3 抑制劑NIBR2130 也可以抑制MCD 引起的肝細胞損傷。我們進一步研究了CXCL10 的臨床應用價值，發現NASH 患者血清以及肝臟中CXCL10 的水準明顯升高。更重要的是，血液中CXCL10 的水準與肝小葉炎症程度有關，是NASH 的獨立危險因素。 / 結論：我們的研究首次發現CXCL10 以及其受體CXCR3 通過促進炎症，脂質聚集，氧化應激以及自噬缺乏在NASH 的發病中起重要作用。抑制CXCL10 或者CXCR3 為NASH 患者的治療提供了新的方法。CXCL10 可作為NASH 患者非侵入性診斷的標誌物。 / Background and aims: Non-alcoholic steatoheaptitis (NASH) complicates obesity and type 2 diabetes, while recruitment and perpetuation of liver inflammation is central to its pathogenesis. Expression of C-X-C motif chemokine 10 (CXCL10), a proinflammatory cytokine, correlates positively with obesity and type 2 diabetes. Whether CXCL10 and its receptor CXCR3 play a role in NASH is unknown. In this study, we investigated the functional significance of CXCL10 and its receptor CXCR3 in steatoheaptitis. Moreover, the clinical impact of CXCL10 in NASH was examined. / Methods: Gene-deleted CXCL10 (CXCL10-/-), CXCL10 receptor CXCR3 (CXCR3-/-) and C57BL/6 wildtype (WT) mice were fed methionine and choline-deficient (MCD) diet for 4 or 8 weeks. Cytokine profiling assay, cDNA array, protein-DNA binding activity assay and autophagosome-lysosome system analysis of CXCL10 signaling and downstream targets were performed. In other experiments, we injected neutralizing anti-CXCL10 monoclonal antibodies (mAb) into MCD diet-fed WT mice, while AML-12 cells were cultured in MCD medium in the presence of anti-CXCL10 mAb or CXCR3 inhibitor (NIBR2130) for 24 hours. Human serum was obtained from 147 patients with biopsy-proven non-alcoholic fatty liver disease and 73 controls. Circulating CXCL10 levels were determined by enzyme-linked immunosorbent assay. / Results: MCD-fed WT mice developed steatohepatitis with higher hepatic CXCL10 and CXCR3 expression. CXCL10-/- and CXCR3-/- mice were refractory to MCDinduced steatohepatitis. In WT mice with steatohepatitis, but not in CXCL10-/- mice, CXCL10 was associated with the induction of pro-inflammatory chemokines and cytokines, as well as activation of nuclear factor-κB (NF-κB) signaling. CXCL10 expression was linked to steatosis through lipogenic factors, including liver X receptors and its downstream targets (SREBP-1c, ChREBP and SCD-1), and also to oxidative stress (up-regulation of CYP2E1 and C/EBPβ). In particular, autophagy deficiency was involved in CXCL10- and CXCR3-induced steatohepatitis as indicated by p62 and LC3-I/II protein accumulation in MCD-fed WT mice than in CXCL10-/- and CXCR3-/- mice. Moreover, the impaired autophagic function was related to the reduction of lysosomal function in CXCL10- or CXCR3-induced NASH. Blockade of CXCL10 by anti-CXCL10 mAb protected against MCD-induced steatohepatitis in vivo and against MCD-mediated injury to AML-12 cells in vitro. The highly selective CXCR3 antagonist NIBR2130 also inhibited MCD-induced injury in AML-12 hepatocytes. We further investigated the clinical impact of CXCL10 and found circulating and hepatic CXCL10 levels were significantly higher in human NASH. Importantly, circulating CXCL10 level was correlated with the degree of lobular inflammation and was an independent risk factor for NASH patients. / Conclusions: We demonstrate for the first time that CXCL10 and its receptor CXCR3 plays a pivotal role in the pathogenesis of NASH by promoting inflammation, fatty acid accumulation, oxidative stress and autophagy deficiency. Blockade of CXCL10 or CXCR3 is a potential novel approach for NASH intervention. CXCL10 is a noninvasive biomarker for NASH patients. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Zhang, Xiang. / Thesis (Ph.D.) Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 145-167). / Abstracts also in Chinese.

Obesity can lead to a range of health problems including type 2 diabetes (T2DM), cardiovascular disease and non-alcoholic fatty liver disease (NAFLD), and causes an estimated 2.8 million deaths annually (2016). It is a growing epidemic affecting over 600 million people worldwide (in 2014), with 26.8% of the adult population in England alone being obese, an increase of 10% in the last decade, and 62.9% overweight or obese. This trend is predicted to continue, and is attributed to an increasingly sedentary lifestyle, coupled with a high calorie “western diet”, which is estimated to cost >£25billion/year in the UK (2015), which is predicted to rise to £49.9 billion by 2050. It is clear that both sex and genetics affect the extent to which individuals exposed to a high fat diet develop adiposity and its associated morbidities, although the mechanisms underlying these differences are not well understood. Here we explore this aetiology, focusing on poly(A)-binding protein 4 (PABP4), an RNA-binding protein in which polymorphisms associated with altered cholesterol levels and cardiovascular disease risk were identified in human GWAS studies. To this end, I take advantage of an unpublished Pabp4 knock-out mouse, maintained on either normal (ND) or high fat diet (HFD), to explore the role of PABP4 in determining the response to high fat diet. PABP4 is a poorly characterised member of the PABP family, which are multifunctional central regulators of global and mRNA-specific translation, and stability. In cell lines, PABP4 is predominantly cytoplasmic, consistent with such functions. However, analogously to PABP1, the prototypical PABP family member, PABP4 is relocalised to stress granules or the nucleus in response to specific cellular stresses and/or viral infections, suggesting a role in altering gene expression programs in responses to changing cellular conditions. Whilst the expression pattern of PABP4 within tissues has not been previously characterised, western blotting of adult mouse tissues revealed that PABP4 is highly expressed in tissues relevant to obesity, T2DM and NAFLD, such as adipose, pancreas, liver and muscle, consistent with the idea that it may play a role in regulating gene expression programs in response to HFD. Immunohistochemistry of tissue sections provided additional insight, revealing a distinct cellular distribution of PABP4 in some tissues, when compared to the well characterised PABP1. Birth weight and post-birth growth can affect adult metabolism. In particular, low birth weight and catch-up growth, characterised by preferentially putting down adipose over lean mass, increases the risk of metabolic conditions in adulthood, such as obesity, T2DM and cardiovascular disease. Therefore, Pabp4-/- and wildtype mice were weighed at birth and daily until weaning. Interestingly this revealed that Pabp4-/- mice have a reduced weight at birth that is exacerbated to weaning (21days (P21)) (5.7% and 18.3% reductions respectively). This analysis also uncovered a reduced survival to weaning, with both male and female Pabp4-/- mice being present at sub-Mendelian ratios by P21 (p=0.0056). Whilst most death occurred neonatally, Pabp4-/- mice showed an increased rate of attrition until weaning, preceded in some cases by an arrest of weight gain. Weight gain was also tracked from 4 weeks to 12 weeks of age on normal diet showing that Pabp4-/- mice had reduced weight into adulthood (12% reduction at 12wks). Analysis of weight gain by sex uncovered a sexually dimorphic effect of Pabp4-deficiency, with female, but not male, Pabp4-/- mice remaining reduced in weight compared to wildtype after 8 weeks on ND (13.4% reduction in female weight). Body composition analysis showed that fat mass was equivalent to wildtype at 12 weeks of age in both sexes but that female Pabp4-/- mice had a 14.3% reduction in lean mass. Neither the catch-up growth in males nor the reduced lean mass in females was sufficient to result in a change in glucose homeostasis. As the risk of developing metabolic disorders in adult life is a consequence of both genetic and environmental factors, such as diet, Pabp4-/- were placed on a HFD at 4 weeks of age for 8 weeks. HFD models the ‘western’ diet, and has been shown to induce obesity, insulin resistance and glucose intolerance in wildtype mice. Whereas Pabp4-/- mice were only distinguishable from wild-type in terms of female lean mass on normal diet, pronounced sexually dimorphic differences were observed in HFD fed mice. Male Pabp4-/- mice appeared to be partially protected from the negative effects of an 8 week HFD regimen, with a 44% decrease in adipose mass gain compared to wildtype despite equal lean mass. Pabp4-/- male mice also had significantly reduced ectopic lipid stores, with an 81% decrease in hepatic triglyceride concentration compared to wildtype, meaning that NAFLD has not developed. Furthermore, Pabp4- /- male mice did not develop hyperinsulinemia on HFD and retained insulin sensitisation (assessed via glucose tolerance test (GTT) and insulin tolerance test (ITT)), although they displayed wildtype-like elevated plasma glucose concentrations (compared to ND). Western blotting had detected high PABP4 levels in the pancreas, indicating a possible pancreatic origin of these alterations. However, immunofluorescence revealed that PABP4 was confined to the exocrine portion of the pancreas, and was undetectable in the insulin producing pancreatic beta cells, suggesting this phenotype may not be beta cell in origin. This is consistent with the fact that the Pabp4-/- male mice retained an appropriate glucose-induced burst of insulin secretion, and therefore insulin production appears unimpaired. Thus, the primary defect may reside in the exocrine pancreas, which aids digestion, or in other key metabolism related tissues (e.g. muscle, liver, adipose and brain), or a combination thereof. In HFD fed wildtype mice, insulin resistance is caused by increased adiposity and ectopic lipid depots, which blunt insulin stimulated signalling cascades, meaning that the normal responses to insulin (e.g. cellular up take of glucose in muscle and arrested glucose production in liver, to decrease plasma glucose concentrations), are impaired. Therefore, the absence of insulin resistance in HFD fed Pabp4-/- male mice may be a consequence of the reduced increase in adipose mass and ectopic lipid deposits detected in these mice, and their consequent lack of inhibition on insulin signalling pathways. The reduced adiposity was not a result of reduced food intake or dietary fat absorption as male Pabp4-/- mice did not eat less nor exhibit apparent steatorrhea (fatty stools). These results highlight that the Pabp4-/- male mice appear to have an alteration in energy use/storage, and the investigation of this will form the basis of future work. When fed HFD, female Pabp4-/- mice revealed a divergent phenotype to that of wildtype female mice and Pabp4-/- male mice. HFD fed Pabp4-/- female mice showed no difference to HFD-fed wildtype mice in terms of weight, but still exhibited the reduction in lean mass seen on ND, but now with a 22.8% increase in volume of adipose tissue. Together, this means that HFD fed Pabp4-/- females have a higher body fat percentage (32.6% compared to 25.9 % for wildtype females). In contrast to the males, there was no difference in terms of hepatic triglycerides in HFD fed Pabp4-/- female mice and they showed greater hyperglycaemia than wildtype (GTT), although like males they retained insulin sensitisation (ITT). These potentially conflicting results in terms of insulin sensitivity and plasma glucose concentrations may result from the alterations in body composition, which can confound results when lean mass is altered and total body weight is used for calculating doses for GTT/ITT. Interestingly, adiponectin, an adipokine normally found in inverse proportion to adipose mass, was increased in plasma from HFD fed Pabp4-/- female mice (21% increase from HFD fed wildtype mice). Whilst surprising given the increase fat mass of Pabp4-/- females, the insulin sensitising properties of adiponectin may help to explain the retained insulin sensitivity detected in the female Pabp4-/- mice. / The finding that HFD revealed metabolic differences in the Pabp4-/- mice lead to the question of whether Pabp4-/- mice have issues adapting to other situations which require modulation of energy storage and glucose homeostasis. One such event is pregnancy, when maternal regulation of insulin resistance is tightly modulated throughout gestation. We therefore characterised the maternal Pabp4-/- environment in late pregnancy (E18.5), when insulin sensitivity decreases to 40-60% lower than pre-pregnancy which results reduced maternal glucose uptake, freeing the glucose up for the rapidly developing foetus. Pregnant Pabp4-/- mice had elevated plasma insulin concentration post fasting (63.7% increase), however glucose homeostasis was wildtype-like, both in terms of plasma glucose and insulin concentrations, throughout a GTT. However, plasma glucose and insulin concentrations in E18.5 Pabp4-/- foetuses were significantly decreased (9% and 44.3% respectively). Pabp4-/- foetuses also had reduced foetal and placental weight/length parameters. This establishes that the differences in weight observed at birth were present by late gestation and secondly, that the reductions in both foetal glucose and insulin concentrations which may contribute to or underlie the reduced growth. It also suggests that the differences seen in adulthood on HFD may be a consequence of metabolic differences present during pregnancy. Taken together, these data support the hypothesis that PABP4 plays a key role in the regulation of mRNAs which are important in growth, post-natal survival and metabolic adaption to high fat diet.

Submitted by Natalia Vieira (natalia.vieira@famerp.br) on 2016-05-20T17:39:42Z No. of bitstreams: 1 giovannazanellisilva_dissert.pdf: 2457622 bytes, checksum: 3faf263a6839bd66d93d3bcefc887385 (MD5) / Made available in DSpace on 2016-05-20T17:39:42Z (GMT). No. of bitstreams: 1 giovannazanellisilva_dissert.pdf: 2457622 bytes, checksum: 3faf263a6839bd66d93d3bcefc887385 (MD5) Previous issue date: 2015-11-27 / Introduction: Nonalcoholic Fatty Liver Disease (NAFLD) is characterized by an increase in intracellular content of triglycerides; its prevalence worldwidely is nearly 20-30% of the population. This disease has spectral nature that includes steatosis and nonalcoholic steatohepatitis (NASH) in the absence of significant alcohol consumption. Although NAFLD may remain as a stable disease for longer periods, this condition may progress to advanced stages of cirrhosis and liver cancer. Diabetes Mellitus Type 2, insulin resistance and obesity are important risk factors, among others, for development of NAFLD, and are directly related to sedentary lifestyle and inappropriate eating habits. Thus, alteration in lifestyle, changes in eating habits and regular physical activity play a fundamental role in treating this disease. Aim: To evaluate the effectiveness of hypocaloric diet for the treatment of NASH as well as adherence to treatment. Methods: This is a prospective longitudinal open cohort study, in which 26 NASH patients were divided into 2 group: 15 patients in the control group and 11 patients in the treatment group which were followed up for 6 months. Both groups were diagnosed by liver biopsy. The treatment group was a given lifestyle change program with supervised low-calorie diet (20-25 kcal / kg actual weight / day), monitored exercise, standard treatment of metabolic syndrome and drug maintenance treatment with metformin and N- acetylcysteine. The control group received general guidelines on diet and weight loss, encouragement to practice physical exercise, standard treatment of metabolic syndrome and maintenance of drug treatment with metformin. Criteria for inclusion: patients with at least one of the component of metabolic syndrome; BMI ≥ 25 and ≤ 40 kg /m² and sedentary for at least three months. Criteria for exclusion: other concomitant liver diseases, alcohol intake greater than or equal to 21 drinks / week, or 140 g / day for men and 14 drinks / week or 70g / day for women, medication known to be associated with NAFLD, untreated hypothyroidism or hyperthyroidism, previous bariatric surgery, or uncontrolled psychiatric disorder. Diagnostic criteria: the diagnosis of NASH was done by liver biopsy in patients with steatosis on ultrasound or MRI and at least one risk factor for advanced fibrosis into the period up to one year before entering the study. The lifestyle change program in the treatment group had a weight loss goal of 5% or more of their initial weight within six months. Evaluation criteria: a monthly basis applied the clinical evaluation protocol and on a quarterly basis the laboratorial, according to the following variables: weight, height, body mass index, waist circumference, hip circumference, aminotransferase levels, gamma GT, total cholesterol and fractions, triglycerides. Statistical analysis: the descriptive variables were expressed as frequency, mean or median, standard deviation and variation as applicable. The Student t test and Mann-Whitney test were used for comparative analysis. It was admitted confidence interval of 95% and a significance level of P <0.05. Results: Of the 15 patients enrolled with a diagnosis of NASH who were submitted to nutritional treatment, 12 patients completed the six month follow-up of the study. The average age was 51.42 years ± 8.50, being 08 (66%) women and 04 (33%) men. Of this total, only one patient refused to carry out physical activity. Two (17%) among the 12 participants who completed the six month follow-up reached the percentage of expected weight loss. The average percentage of adaptation to the proposed diet was 82.93% ± 13.51%. Conclusion: The lifestyle change program tested for six months associated with NASH treatment, was not effective for clinical and biochemical improvement even with satisfactory adherence by most patients. Our data point out to the potential role of more restrictive diets and intensive supervision in this context combined with multidisciplinary team for the treatment of NASH. This real-life study produced crucial information for readjustment of the multidisciplinary treatment protocol for patients followed up in the service. / Introducao: A doenca hepatica gordurosa nao alcoolica (DHGNA) e caracterizada pelo aumento do conteudo intracelular de triglicerideos, com prevalencia mundial de aproximadamente 20 a 30% da populacao. Esta doenca tem natureza espectral que engloba esteatose e esteatohepatite nao-alcoolica (NASH), na ausencia de consumo significante de alcool. Embora DHGNA possa permanecer como uma doenca estavel por longos periodos, esta condicao pode progredir para estagios avancados de cirrose e cancer de figado. O Diabetes Mellitus Tipo 2, resistencia a insulina e obesidade sao importantes fatores de risco, dentre outros, para desenvolvimento da DHGNA, e estao diretamente relacionadas ao estilo de vida sedentario e habitos alimentares inapropriados. Dessa forma, alteracao no estilo de vida, mudancas nos habitos alimentares e atividade fisica regular tem papel fundamental no tratamento desta doenca. Objetivo: Avaliar a eficacia da dieta hipocalorica durante o tratamento do NASH, assim como, a adesao ao tratamento instituido. Metodo e Casuistica: Trata-se de um estudo de coorte aberto prospectivo, longitudinal, no qual foram incluidos consecutivamente 26 pacientes, divididos em dois grupos: 15 pacientes no grupo tratamento e 11 pacientes no grupo controle acompanhados durante 6 meses. Ambos tinham diagnostico de NASH por biopsia. O grupo tratamento recebeu programa de mudanca no estilo de vida com dieta hipocalorica supervisionada (20 - 25 kcal/kg de peso atual/dia), exercicio fisico supervisionado, tratamento padrao dos componentes da sindrome metabolica e manutencao de tratamento medicamentoso com metformina e N-acetilcisteina. O grupo controle recebeu orientacoes gerais de dieta e perda de peso, estimulo a pratica de exercicio fisico, tratamento padrao dos componentes da sindrome metabolica e manutencao do tratamento medicamentoso com metformina. Criterios de inclusao: portadores de pelo menos uma caracteristica de sindrome metabolica, IMC . 25 e . 40kg/m2, e sedentarios por no minimo tres meses. Criterios de exclusao: outras doencas hepaticas concomitantes, ingestao alcoolica igual ou superior a 21 doses/semana ou 140g/dia para homens e 14 doses/semana ou 70g/dia para mulheres, medicacao conhecidamente relacionada com DHGNA, hipotireoidismo ou hipertireoidismo nao tratados, pos operatorio de cirurgia bariatrica, compulsao alimentar ou outro disturbio psiquiatrico nao controlado. Criterios diagnosticos: o diagnostico de NASH foi feito por biopsia de figado em pacientes portadores de esteatose ao ultrassom ou ressonancia magnetica e pelo menos um fator de risco para fibrose avancada, no periodo de ate 1 ano antes da entrada no estudo. Programa de mudanca no estilo de vida no grupo tratado teve como meta a reducao minima ou superior a 5% de seu peso inicial no periodo de seis meses. Criterios de avaliacao: aplicado mensalmente o protocolo de avaliacao clinica e trimestralmente o laboratorial, de acordo com as seguintes variaveis: peso, altura, indice de massa corporal, circunferencia abdominal, circunferencia do quadril, niveis de aminotransferases, gama GT, colesterol total e fracoes, triglicerideos. Analise estatistica: as variaveis descritivas foram expressas em frequencia, media ou mediana, desvio padrao e variacao conforme aplicaveis. Foram utilizados os testes t de Student e teste de Mann-Whitney para analises comparativas. Foi admitido intervalo de confianca de 95% e nivel de significancia para P<0,05. Resultados: Dos 15 pacientes incluidos com diagnostico de NASH que foram submetidos ao programa de mudanca no estilo de vida, 12 pacientes concluiram o acompanhamento de 6 meses do estudo. A idade média foi de 51,42 anos ± 8,50, sendo 08 (66%) pessoas do gênero feminino e 04(33%) do gênero masculino. Deste total, apenas um paciente negou a realização de atividade física. Dois (17%) participantes dentre os 12 que concluíram os seis meses de acompanhamento atingiram a porcentagem de perda de peso esperada. A média da porcentagem de adequação à dieta proposta dos participantes do estudo encontrada foi de 82,93% ± 13,51%. Conclusão: O programa de mudança no estilo de vida testado durante seis meses associado ao tratamento do NASH, não foi eficaz para a melhora clínica e bioquímica mesmo com adesão satisfatória pela maioria dos pacientes. Este estudo de vida real produziu informações de fundamental importância para readequação do protocolo de atendimento multidisciplinar dos pacientes em acompanhamento no serviço.

Submitted by Fabíola Silva (fabiola.silva@famerp.br) on 2018-01-08T16:01:25Z No. of bitstreams: 1 viniciusdelimafreitas_tese.pdf: 8494219 bytes, checksum: 35b61ce25b380b2ebf2433cf44ff18e5 (MD5) / Made available in DSpace on 2018-01-08T16:01:25Z (GMT). No. of bitstreams: 1 viniciusdelimafreitas_tese.pdf: 8494219 bytes, checksum: 35b61ce25b380b2ebf2433cf44ff18e5 (MD5) Previous issue date: 2017-05-05 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Introduction: The prevalence of hepatic steatosis and non-alcoholic fatty liver disease (NAFLD) in the Brazilian population is not known, and there are few studies about this disease in the country. Lifestyle modification, including physical activity and exercise are first line recommendation for the tratment of patients with NAFLD. Aim: To evaluate the efficacy of the Supervised physical activity and exercise program on-site and distance-supervised with duration of 12-month for patients with non-alcoholic steatohepatitis (NASH). Methods: This is a prospective, longitudinal, open cohort study including consecutive patients who had a histological diagnosis of NASH in the last 12 months and who were followed up at the outpatient NAFLD clinic. Exclusion criteria were: patients with concomitant liver diseases who could lead to steatosis; history or active significant alcohol intake such as equal or greater than 210g / week for men and 140g / week for women; drugs known to be related to NAFLD; untreated hypo or hyperthyroidism; pevious bariatric surgery; obesity equal or greater than grade III; binge eating or other uncontrolled psychiatric disorder. The patients were studied withing a pre-stated protocol study including clinical and laboratory evaluation, as well as the Baecke questionnaire and the six minute walk test (6MWT), before and after participation in the physical conditioning program. Descriptive statistics, Student's tes and the Mann-Whitney test, were performed for parametric and non-parametric variables as apropriated. The significance level adopted was p-value >0.05. Results: From the 15 included patients, three of them did not complete the multidisciplinary program during the 12-month study period. Thus, the total sample analyzed was 11 patients, that is, 73.33% of included patients. The 5% goal for body weight loss was not reached, however low density lipoprotein (LDL) presented significant reduction at the end of the study (p = 0.0130). The distance-supervised program was chosen by all patients and walking was the main physical activity (66.67%), followed by soccer. The 6-min walk distance (6MWD) was sgnificantly higher at trhee and six month when compared with basal distance at the entry of the study. Conclusion: The distancesupervised physical activity and exercise program had high adherence and was effective in improving the functional capacity for patitients with NASH. On the other hand, there was partial improvement for biochemical and antropometric variables. Aditionally, this is a distance-supervised life-style modification program with low cost and high potential cost-benefit for patients with DHGNA and NASH attended on the National Health System. / Introdução: A prevalência da Esteatose Hepática (EH) e da Doença Hepática Gordurosa Não Alcoólica (DHGNA) na população brasileira não é conhecida, e são poucos os estudos sobre esta doença no país. A mudança no estilo de vida representa a principal recomendação para o tratamento da DHGNA, assim, a atividade física e o exercício físico são ferramentas eficientes no combate à dislipidemia e acúmulo de gordura no fígado. Objetivo: Avaliar os efeitos do programa de condicionamento físico supervisionado in loco e supervisionado à distância com duração de 12 meses em pacientes com Esteatohepatite não alcoólica (NASH). Casuística e Método: Trata-se de um estudo de coorte aberto prospectivo, longitudinal, no qual foram estudados, pacientes em acompanhamento nos ambulatórios de DHGNA do Hospital de Base de São José do Rio Preto, que tiveram o diagnóstico histológico de NASH nos últimos 12 meses. Os critérios de exclusão apresentados foram: pacientes com outras doenças hepáticas concomitantes que possam cursar com esteatose; história prévia de ingestão alcoólica igual ou superior a 210g/semana para homens e 140g/semana para mulheres; medicação conhecidamente relacionada com a etiologia de DHGNA; hipotireoidismo ou hipertireoidismo não tratado; pós-operatório de cirurgia bariátrica; obesidade maior ou igual ao grau III; compulsão alimentar ou outro distúrbio psiquiátrico não controlado. Os pacientes foram analisados em protocolo de avaliação clínica e laboratorial, como o questionário de Baecke e o Teste de Caminhada de 6 minutos (TC6), antes e após a participação no programa de condicionamento físico em estudo. A estatística descritiva foi composta pelas variáveis paramétricas e não paramétricas (média, desvio padrão). As comparações entre os valores basais e, após a intervenção do programa de condicionamento físico foram efetuadas pelo teste t de Student (dados pareados) e teste não paramétrico de Mann-Whitney, com nível de significância (valores de p) inferior a 0,05. Resultados: Dos 15 pacientes incluídos no estudo, três pacientes não concluíram o programa multidisciplinar no período de 12 meses. Assim, a amostra total analisada foi de 11 pacientes, isto é, 73,33% dos incluídos no estudo. O programa supervisionado a distância foi escolhido por todos os pacientes avaliados tendo a caminhada como atividade física mais praticada (66,67%), seguido do futebol. A meta de perda de 5% do peso corporal não foi atingida ao final do estudo, e a lipoproteína plasmática de baixa densidade (LDL) apresentou redução significante (Tempo 4, p=0,0130) durante o estudo. A Distância Percorrida no Teste (DTC6) foi maior nos Tempos 1 e 2 quando comparado ao Tempo 0, com diferença significante (p < 0,05). Conclusão: O programa de condicionamento físico supervisionado à distância teve alta adesão e foi eficaz para a melhora da capacidade funcional de pacientes com NASH. A melhora foi parcial para os parâmetros bioquímicos e antropométricos. Adicionalmente, este programa de condicionamento físico, monitorado à distância, tem baixo custo e é de facil implantação no Sistema Único de Saúde, com alto potencial de custo-benefício para pacientes com DHGNA e NASH, que poderão ser maiores a longo prazo.

Non-alcoholic fatty liver disease (NAFLD) results from an unequal uptake/storage and export/oxidation of lipids within the liver and is often a secondary disease to type II diabetes (22). NAFLD causes this imbalance of lipids by altering glucose and lipid metabolism, which corresponds to a decrease in mitochondrial function leading to failure of the liver. One established treatment for type II diabetes and NAFLD is the drug metformin, which has similar properties to the newly discovered CTRP 3 protein which is part of a group of bioactive molecules secreted by adipose tissue, collectively termed adipokines (2-4). Both have similar effects on hepatic glucose and lipid metabolism and both specifically suppress hepatic gluconeogenesis (11, 17, 27, 29). The revolutionary Seahorse extracellular flux analyzer was used to measure the metabolism of H4IIE hepatocytes without use of radiolabeling (1). By detecting the Oxygen Consumption Rate (OCR) of hepatocytes the level of metabolic function within mitochondria can be measured. Once an effective protocol was established using this new technology, hepatocytes treated with metformin had a significantly lower OCR compared to control treated hepatocytes treated. However, H4IIE hepatocytes treated with metformin and palmitate had a significant increase in OCR and eventually equilibrated with the lower OCR of hepatocytes solely treated with metformin. With similar effect, hepatocytes treated with CTRP3 and palmitate caused a drastic increase in OCR while hepatocytes treated with only CTRP3 had a decrease in OCR. This suggests that CTRP3 increases fatty acid oxidation which decreases lipid concentrations within hepatocytes which could mean future protection of liver against NAFLD. In conclusion, our Seahorse XF analyzer models compare metformin and CTRP3’s similarities and suggest the possible liver protective functions of CTRP3. Our results will aid in future research of CTRP3 to further determine its possible uses as a treatment for liver-associated diseases.

Alcoholic fatty liver disease (AFLD) is a significant public health concern. Excessive alcohol (ethanol) consumption causes liver cell damage and death, which results in eventual failure of the liver and death. AFLD is the number one cause of liver-related mortality in the United States. Our lab works with the novel protein C1q TNF Related Protein 3 (CTRP3), which inhibits non-alcoholic fatty liver disease, however the effects on AFLD are unknown. Therefore, the purpose of this experiment is to determine if CTRP3 prevents ethanol-induced liver cell death. The H4IIE rat hepatoma cell line was chosen for experimentation as a cell culture model of liver tissue. To determine a suitable alcohol level H4IIE cells were treated with 50, 100, and 200 mmol of ethanol for 18-24 hours. Trypan Blue was used to identify the dead/damaged cells, as only dead/damaged cells will be stained blue with this protocol. We observed that 100 mmol of ethanol consistently induced ~10% mortality rate in these cells. Next, we tested the ability of CTRP3 to reduce ethanol-induced mortality. We added purified CTRP3 protein to the cell media along with the 100 mmol ethanol treatment. The addition of CTRP3 reduced the amount of alcohol-induced cell death/damage in the H4IIE cell line by approximately 60%. Our next goal was to determine how CTRP3 reduces ethanol-induced death. The Akt signaling pathway is a well-known inhibitor of cell death. Therefore, to determine if CTRP3 attenuated ethanol-induced cell damage/death through activation of the Akt signaling pathway, another set of cells was treated with 100 mmol of ethanol and CTRP3 (with or without insulin). Western blots were used to compare the amount of active Akt (phosphorylated) in the CTRP3-treated and non-treated cells. A Western blot utilizes an electric current to separate denatured protein samples on a SDS-page gel, separating the proteins based on size. The smaller the protein the faster it migrates across the gel. The proteins are then transferred to a membrane for analysis, through exposure to commercial antigens and chemiluminescence imaging. There was no change in the amount of total or active Akt between the samples treated with or without CTRP3. We conclude that CTRP3 protects liver cells from ethanol-induced damage/death, but not through activation of the Akt pathway.

Alcohol abuse is the third leading cause of preventable death in the United States. Excessive intake of alcohol can result to alcoholic fatty liver disease, the number one cause of live related mortalities in the US. The outlining purpose for this project is to determine the alcohol-induced changes in the liver cell protein signaling. For this project, we treated H4IIE rat hepatoma cells (with 100 and 200 mM ethanol overnight). H4IIE cells were chosen because they are a commonly used liver cell culture line that maintains characteristics of intact liver cells. After treatment we collected and prepared the cells for protein signaling analysis, using standard western blotting procedure. A western blot detects relative quantity of proteins in a sample. Briefly, protein samples are separated by size through electrophoresis, smaller proteins move faster through the gel so that the larger proteins are toward the top and smaller towards the bottom. The proteins are then transferred to a nitrocellulose membrane and protein concentration is detected by chemiluminescence. We chose to examine the effects of ethanol on the activation of the key regulator of metabolic signaling, Protein Kinase B/Akt (Akt). Based on our results, ethanol has no effect on the total amount of Akt in the H4IIE liver cells. However, ethanol significantly attenuates insulin-induced activation of Akt in a dose-dependent manner, as seen by a reduction in the amount of phosphorylated Akt. Therefore, we conclude that treatments that increase Akt activation may be a viable option for the treatment of alcoholic fatty liver disease.

Over the past five decades, per capita caloric intake has increased by approximately 28% in the United States. A hallmark of the current standard American diet is an excess of energy sources from saturated fat and refined carbohydrates. High energy diets such as the “Western” diet cause numerous pathologies, including non-alcoholic fatty liver disease (NAFLD), high blood pressure, dyslipidemia, and peripheral insulin resistance. High energy diets also negatively impact the hippocampus, a brain area important for learning and memory. It is not surprising, then, that high energy diets impair hippocampal-dependent memory. The experiments in this dissertation investigate possible diet-induced consequences that may contribute to the impairing effects of high energy diets on hippocampal-dependent memory. Our initial experiments found that diet-induced NAFLD impairs hippocampal-dependent memory, but these cognitive deficits were not due to decreases in insulin-like growth factor-1 (IGF-1) or hippocampal insulin signaling. Next, we found that a high energy diet increased the ability of epinephrine to increase blood glucose concentrations, indicating a novel way in which high energy diets impair liver function. The final set of experiments found that high energy diets do not necessarily impair memory but instead may prevent the memory-enhancing effects of acute stress. Taken together, these results indicate that high energy diets interact with acute stress to negatively impact hippocampal-dependent memory, and that hippocampal insulin resistance and IGF-1are not likely involved.

Nonalcoholic fatty liver disease (NAFLD) has become a worldwide, chronic liver disease of increasing clinical significance. It is closely associated with the rising epidemics of obesity and insulin resistance. Up to 17% of the United States population may progress from the disease stage characterized as simple, benign steatosis to the more severe, inflammatory stage of nonalcoholic steatohepatitis (NASH). This progression occurs through 2nd 'hits' of increased oxidative stress and inflammation to a liver that has been sensitized by lipotoxic stress. NASH is also characterized by increased collagen deposition resulting in fibrosis and architectural rearrangement of the liver. Progressive NAFLD is currently recognized as an important contributor to the development of cryptogenic cirrhosis and subsequent liver-related mortalities (estimated at 30-40% in these patients).The pathological progression of NAFLD, as described by the 'two hit' hypothesis, characterizes the different stages of liver injury. However, the mechanism(s) responsible for the progression to NASH are unknown. Profiling global gene expression and metabolite patterns in human liver samples representing the full spectrum of progressive human NAFLD may reveal potential mechanisms of progressive disease. Human liver samples representing each stage of NAFLD progression were analyzed by methodologies such as high-throughput microarrays, high resolution mass spectrometry, and protein immunoblot techniques. Bioinformatics tools and gene expression/regulation database software were utilized in several studies to characterize the altered hepatic profiles of these patients. Hepatic transcriptomic profiles of ADME (absorption, distribution, metabolism and elimination) and ER (endoplasmic reticulum) stress response genes exhibited initiated hepatoprotective responses in patients with NASH. The endogenous pathways of BA (bile acid) synthesis and BCAA (branched chain amino acid) metabolism also showed evidence of coordinately regulated alterations in response to disease-induced stress in NASH. The transcriptional regulation of the investigated pathways was confirmed by transcription factor binding sites enrichment analysis. The collective response to hepatic stress in human NAFLD, demonstrates a coordinated, hepatoprotective intent that may be utilized for future therapeutics in the battle against progressive liver disease.