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 To this end, I take advantage of an unpublished Pabp4 knock-out mouse, maintained on either normal (ND) or high fat diet (HFD), to explore the role of PABP4 in determining the response to high fat diet.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.

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.

No description available.

Coffee consumption has consistently been associated with a reduction in risk of type 2 diabetes mellitus (T2DM), although the mechanism for this association remains unknown. Sub-clinical inflammation, non-alcoholic fatty liver disease (NAFLD), and lipoprotein abnormalities characterize and predict T2DM. Limited evidence suggests that coffee may have a beneficial role in these disorders but further investigation is warranted. Our aim therefore was to investigate the associations of caffeinated and decaffeinated coffee with markers of inflammation, liver injury, and lipoproteins, in a non-diabetic cohort. No significant associations of caffeinated or decaffeinated coffee with inflammatory markers or lipoproteins were identified. Caffeinated coffee consumption however was inversely associated with alanine aminotransferase (β= -0.09, p= 0.0107) and aspartate aminotransferase (β= -0.05, p= 0.0161) in multivariate analysis. Decaffeinated coffee was not associated with liver enzymes. These analyses suggest that caffeinated coffee’s beneficial impact on NAFLD may be a potential mechanism for its inverse association with T2DM.

Coffee consumption has consistently been associated with a reduction in risk of type 2 diabetes mellitus (T2DM), although the mechanism for this association remains unknown. Sub-clinical inflammation, non-alcoholic fatty liver disease (NAFLD), and lipoprotein abnormalities characterize and predict T2DM. Limited evidence suggests that coffee may have a beneficial role in these disorders but further investigation is warranted. Our aim therefore was to investigate the associations of caffeinated and decaffeinated coffee with markers of inflammation, liver injury, and lipoproteins, in a non-diabetic cohort. No significant associations of caffeinated or decaffeinated coffee with inflammatory markers or lipoproteins were identified. Caffeinated coffee consumption however was inversely associated with alanine aminotransferase (β= -0.09, p= 0.0107) and aspartate aminotransferase (β= -0.05, p= 0.0161) in multivariate analysis. Decaffeinated coffee was not associated with liver enzymes. These analyses suggest that caffeinated coffee’s beneficial impact on NAFLD may be a potential mechanism for its inverse association with T2DM.

Orientador: César Tadeu Spadella / Banca: José Guilherme Minossi / Banca: Silvio Fernando Guideti Marques / Resumo: O diabetes mellitus (DM) é tido como um problema de saúde pública mundial. No Brasil ele atinge mais de 14 milhões de pessoas, sendo acompanhado de altos índices de morbidade e mortalidade. Entretanto, os mecanismos primariamente responsáveis pela agressão dos tecidos e órgãos pelo DM ainda não são completamente conhecidos, o que explica a dificuldade em se estabelecer um tratamento eficaz para prevenir ou controlar a progressão das lesões diabéticas crônicas. O estresse oxidativo celular é tido como um dos mecanismos importantes na gênese do dano tecidual relacionado à hiperglicemia. Através deste mecanismo, o DM poderia aumentar a produção de espécies reativas do oxigênio (EROs) ao nível celular, que pela sua toxicidade, seria capaz de promover o desenvolvimento das lesões diabéticas crônicas. Evidências clínicas sugerem que o fígado de indivíduos diabéticos também poderia sofrer a ação das EROs, no longo prazo, levando a uma seqüência de eventos capaz de determinar a doença gordurosa do fígado de etiologia não-alcoólica (DGFNA), com progressão para esteato-hepatite e cirrose. Todavia, a presença de estresse oxidativo no tecido hepático de portadores de DM, ainda não está bem estabelecida na literatura, o que justifica a realização de novas investigações em modelos-animais de diabetes, no intuito de melhor esclarecer a real participação deste mecanismo na gênese e evolução das lesões hepáticas diabéticas crônicas. Neste estudo foram utilizados 60 ratos machos Lewis, distribuídos em 2 grupos experimentais, com 30 animais cada um, assim designados: GN - Grupo Controle: constituído de ratos normais, não-diabéticos; GD - Grupo Diabético: constituído por animais diabéticos induzidos pela aloxana, sem qualquer tratamento. Cada um dos grupos experimentais foi dividido em 3 subgrupos de ratos, com 10 animais cada um, para serem... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Diabetes mellitus (DM) is considered to be a public-health problem worldwide. In Brazil, it affects 14 million people, and it is accompanied by high morbidity and mortality rates. However, the mechanisms primarily responsible for tissue and organ aggression by DM are not yet fully known, which explains the difficulty in establishing effective treatment to prevent or control the progression of chronic diabetic lesions. Cellular oxidative stress is considered to be one of the important mechanisms in the genesis of hyperglycemia-related tissue damage. Through this mechanism, DM could increase the production of reactive oxygen species (ROS) in the cellular level, which, due to their toxicity, could promote the development of chronic diabetic lesions. Clinical evidence suggests that the liver of diabetic individuals could also suffer the action of ROS in the long term, thus leading to a sequence of events that can determine non-alcoholic fatty liver disease (NAFLD), with progression to steatohepatitis and cirrhosis. However, the presence of oxidative stress in the hepatic tissue of individuals with DM has not been yet well established in the literature, which justifies the performance of new investigations in diabetes animal models with the purpose to clarify the actual participation of such mechanisms in the genesis and development of chronic diabetic hepatic lesions. In this study, 60 males Lewis rats were used. They were distributed into 2 experimental groups, each containing 30 animals and designated as follows: GN - Control Group: consisting of non-diabetic control rats; GD - Diabetic Group: consisting of alloxan-induced diabetic rats without any treatment. Each experimental group was divided into 3 subgroups of rats with 10 animals each to be evaluated and sacrificed respectively at 4 experimental moments, namely: M1- animals from the 3 subgroups, at the initial moment... (Complete abstract click electronic access below) / Mestre