Thesis (MScMedSc)--Stellenbosch University, 2011. / ENGLISH ABSTRACT: Non-Alcoholic Fatty Liver Disease (NAFLD) is the most prevalent chronic liver disease in Western countries and is considered the hepatic manifestation of the Metabolic Syndrome (MetS). Its heterogeneous nature ranges from hepatic steatosis through steatohepatitis to advanced fibrosis and cirrhosis where the ingestion of significant amounts of alcohol has been excluded. The disease profile of NAFLD and its necro-inflammatory subset Nonalcoholic Steatohepatitis (NASH) were described in the parent study, which provided a clinically well-characterised patient cohort for the present investigation. South African patients with NASH had significantly higher mean serum cholesterol and triglyceride levels than those with fatty liver only. The objective of this study was to implement a high-throughput real-time polymerase chain reaction (PCR) method in our laboratory to enable the assessment of cardiovascular genetic risk factors in NAFLD patients. The specific aims were to determine the clinical utility and perform analytical validation of each mutation included in the multi-gene cardiovascular disease (CVD) screening assay. The Pathology Supported Genetic Testing (PSGT) concept developed at our department provides a practical approach to personalized medicine. The CVD multi-gene screen analyses key metabolic pathways relating to atherogenic dyslipidaemia, chronic inflammation, hypercoagulation and iron dysregulation implicated in insulin resistance, which is known to be a universal factor in the pathogenesis of NAFLD. Deleterious low-penetrance mutations in the APOE (APOE2 and E4 alleles), MTHFR (677C>T and 1298A>C), F2 (20210G>A), FV (1691G>A, Leiden) and HFE (C282Y and H63D) genes were included for analysis due to their important role as genetic contributors to these biological processes. A total of 178 patients diagnosed with NAFLD and 75 controls were studied using direct DNA sequencing and a RT-PCR system for mutation detection. In addition, two patients with high ferritin levels were included as case studies. A significant association was found between HFE mutations and elevated Alanine Transaminase (ALT) levels in the NAFLD population (p = 0.04). This discovery is interpreted as the identification of a subset of patients at greater risk of developing progressive liver damage who would benefit most from genetic testing to direct more aggressive therapy at an earlier stage. The necessity of an integrative, systems-based network approach was demonstrated to more accurately distinguish between Hereditary Haemochromatosis (HH) and Insulin Resistance-associated Hepatic Iron Overload (IR-HIO) syndrome in obese patients. The PSGT approach to personalized medicine facilitates diagnosis of CVD subtypes, prevention of cumulative risk and the formulation of gene-based intervention programs tailored to the needs of the patient. These findings support the clinical utility of the CVD multi-gene test to guide chronic disease risk management in patients with NAFLD. The HFE mutation detection component of this test is of particular relevance in directing an effective treatment strategy in patients with a medical history of CVD and/or high iron stores. / AFRIKAANSE OPSOMMING: Nie-Alkoholiese Vettige Lewer Siekte (NAFLD) is die mees algemene kroniese lewer siekte in Westerse lande en word bestempel as die hepatiese manifestasie van die Metaboliese Sindroom (MetS). Die heterogene natuur van NAFLD strek van hepatiese steatose deur steatohepatietis tot gevorderde fibrose en sirrose waar grootskaalse alkohol inname uitgesluit is. Die siekte-profiel van NAFLD en sy nekro-inflammatoriese subtipe Nie-Alkoholiese Steatohepatietis (NASH) is reeds beskryf in die ouer studie, wat ‗n klinies goed-gekarakteriseerde pasiënt groep vir die huidige ondersoek daar gestel het. Suid-Afrikaanse pasiënte met NASH het beduidend hoër gemiddelde serum cholesterol en trigliseried vlakke in vergelyking met slegs vettige lewer. Die doel van hierdie studie was om ‗n hoë deurvoer rieëltyd polimerase kettingreaksie (RT-PCR) metode in ons laboratorium te implimenteer om kardiovaskulêre genetiese risiko faktore in NAFLD pasiënte te ondersoek. Die spesifieke mikpunte was om die kliniese nut en analitiese geldigheid van elke mutasie wat ingesluit is in die multi-geen kardiovaskulêre siekte (KVS) siftings toets vas te stel. Die Patologie Ondersteunde Genetiese Toetsing (PSGT) konsep wat by ons departement ontwikkel is, verskaf ‗n praktiese benadering tot persoonlike medisyne. Die KVS multi-geen toets analiseer belangrike metaboliese weë verwant aan atherogene dyslipidemie, kroniese inflammasie, oormatige bloedstolling en yster disregulering wat betrokke is by insulien weerstand wat bekend is as ‗n universele factor in the patogenese van NAFLD. Nadelige lae-penetrasie mutasies in die APOE (APOE2 en E4 allele), MTHFR (677C>T en 1298A>C) F2 (20210G>A), FV (1691G>A, Leiden) en HFE (C282Y en H63D) gene was ingesluit vir analise as gevolg van hul belangrike rol as genetiese bydraers tot die bogenoemde biologiese prosesse. ‗n Totaal van 178 pasiënte gediagnoseer met NAFLD en 75 kontroles is bestudeer deur gebruik te maak van direkte DNA volgordebepaling en ‗n RT-PCR metode vir mutasie opsporing. Twee pasiënte met verhoogde ferritien vlakke is ook as gevalle studies ingesluit. ‗n Beduidende assosiasie is gevind tussen HFE mutasies en verhoogde Alanien Transaminase (ALT) vlakke in die NAFLD studiepopulasie (p = 0.04) wat aanduidend is van ‗n subgroup van pasiënte wat die meeste baat sal vind uit genetiese toetsing om meer aggressiewe behandeling te rig op' n vroeër stadium. Die noodsaaklikheid van 'n geïntegreerde, stelsels-gebaseerde netwerk benadering is gewys om meer akkuraat te onderskei tussen Oorerflike Hemochromatose (HH) en Insulien Weerstand-geassosieerde Hepatiese Yster Oorlading (IR-HIO) sindroom in vetsugtige pasiënte. Die PSGT benadering tot persoonlike medisyne formuleer geen-gebaseerde intervensie programme aangepas tot die behoeftes van die pasiënt ek maak diagnose van KVS-subtipes en voorkoming van kumulatiewe risiko moontlik. Hierdie bevindinge ondersteun die kliniese nut van die KVS multi-geen toets om riglyne vir die risikobestuur van kroniese siektes soos NAFLD daar te stel. Die HFE mutasie opsporings komponent van hierdie toets is van besondere belang om 'n effektiewe strategie vir die behandeling van pasiënte met 'n mediese geskiedenis van KVS en/of hoë yster vlakke daar te stel.

Introdução: A obesidade é uma doença crônica que aumenta sua prevalência progressivamente no mundo todo. Uma das suas principais complicações é a doença hepática gordurosa não alcoólica (DHGNA), que pode apresentar-se de forma assintomática, com esteatose hepática, esteato-hepatite, cirrose e hepatocarcinoma. Está associada à dislipidemia, hipertensão, diabetes, síndrome metabólica e resistência insulínica. O padrão ouro para seu diagnóstico é a biopsia hepática, considerado método invasivo. Medidas indiretas são usadas para auxiliar na sua detecção, como dados do exame físico, exames de bioquímica e de imagem. Objetivo: Diagnosticar DHGNA usando a ressonância nuclear magnética (RNM) como método não invasivo, correlacionando-a com achados clínicos e laboratoriais. Metodologia: Estudo transversal de 50 crianças e adolescentes seguidas no Ambulatório de Obesidade do Hospital das Clínicas da FMRP-USP. Foram submetidas à questionário sobre histórico pessoal e familiar, à exame físico, exames laboratoriais (lipidograma, transaminases, glicemia e insulina basal) e RNM de abdome para cálculo da de gordura hepática, visceral e subcutânea. Resultados: Diagnosticado esteatose hepática em 14 (28%) dos participantes, sendo 8 com esteatose grave (porcentagem de gordura >18%) e 4 não grave (porcentagem gordura hepática entre 9 e 18%). Houve diferença estatisticamente significante entre a gordura hepática detectada pela RNM e o gênero masculino, triglicérides, TGO, TGP, relação TGO/TGP e acantose nigricans. O cálculo do Homeostasis Model Assessment Insulin Resistance (HOMA-IR) e da síndrome metabólica não apresentaram relação positiva com a porcentagem de gordura hepática. Conclusão: A frequência de esteatose hepática, utilizando a RNM como ferramenta diagnóstica, foi inferior à encontrada na literatura. Dos parâmetros estudados, os melhores preditores de esteatose hepática foram triglicérides, TGO, TGP, relação TGO/TGP, gênero masculino, acantose nigricans e Z escore para IMC elevado / Introduction: Obesity is a chronic disease whose prevalence progressively increasing worldwide. One of its main complications is non-alcoholic fatty liver disease (NAFLD), which may occur in an asymptomatic form, as simple fatty liver, steatohepatites (NASH), cirrhosis and hepatocellular carcinoma. This disease is associated with dyslipidemia, hypertension, diabetes, metabolic syndrome and insulin resistance. The gold standard for diagnosis is a liver biopsy, considered to be an invasive method. Indirect measures are used to aid its diagnosis, such as physical, biochemical and imaging tests. Objective: Diagnosing NAFLD using MRI as non-invasive method and correlating it with clinical and laboratory findings. Methodology: A cross-sectional study of 50 children and adolescents followed at the Obesity clinic of the University Hospital, FMRP-USP. A questionnaire about personal and family history was aplplied and each subject was submitted to physical examination, laboratory tests (lipip profile, transaminases, glycemia and basal insulin) and magnetic resonance imaging (MRI) of the abdomen for calculation of hepatic, visceral and subcutaneous fat. Results: Fatty liver disease was diagnosed in 14 (28%) of the participants, 8 of them with severe steatosis (fat percentage > 18%) and 4 with milder steatosis (fat percentage between 9 and 18%). There was a statistically significant difference between the hepatic fat detected by MRI and male gender, triglycerides, AST, ALT, AST/ALT ratio and acanthosis nigricans. The calculation of the Homeostasis Model Assessment Insulin Resistance (HOMA-IR) and of the metabolic syndrome did not show a positive relationship with the percentage of hepatic fat. Conclusion: The frequency of hepatic steatosis using MRI as a diagnostic tool, was lower than the values reported in the literature. Among the parameters studied, the best predictors of liver steatosis were triglycerides, AST, ALT, AST/ALT ratio, male gender, acanthosis nigricans and Z score for high body mass index

Introdução: A obesidade é uma doença crônica que aumenta sua prevalência progressivamente no mundo todo. Uma das suas principais complicações é a doença hepática gordurosa não alcoólica (DHGNA), que pode apresentar-se de forma assintomática, com esteatose hepática, esteato-hepatite, cirrose e hepatocarcinoma. Está associada à dislipidemia, hipertensão, diabetes, síndrome metabólica e resistência insulínica. O padrão ouro para seu diagnóstico é a biopsia hepática, considerado método invasivo. Medidas indiretas são usadas para auxiliar na sua detecção, como dados do exame físico, exames de bioquímica e de imagem. Objetivo: Diagnosticar DHGNA usando a ressonância nuclear magnética (RNM) como método não invasivo, correlacionando-a com achados clínicos e laboratoriais. Metodologia: Estudo transversal de 50 crianças e adolescentes seguidas no Ambulatório de Obesidade do Hospital das Clínicas da FMRP-USP. Foram submetidas à questionário sobre histórico pessoal e familiar, à exame físico, exames laboratoriais (lipidograma, transaminases, glicemia e insulina basal) e RNM de abdome para cálculo da de gordura hepática, visceral e subcutânea. Resultados: Diagnosticado esteatose hepática em 14 (28%) dos participantes, sendo 8 com esteatose grave (porcentagem de gordura >18%) e 4 não grave (porcentagem gordura hepática entre 9 e 18%). Houve diferença estatisticamente significante entre a gordura hepática detectada pela RNM e o gênero masculino, triglicérides, TGO, TGP, relação TGO/TGP e acantose nigricans. O cálculo do Homeostasis Model Assessment Insulin Resistance (HOMA-IR) e da síndrome metabólica não apresentaram relação positiva com a porcentagem de gordura hepática. Conclusão: A frequência de esteatose hepática, utilizando a RNM como ferramenta diagnóstica, foi inferior à encontrada na literatura. Dos parâmetros estudados, os melhores preditores de esteatose hepática foram triglicérides, TGO, TGP, relação TGO/TGP, gênero masculino, acantose nigricans e Z escore para IMC elevado / Introduction: Obesity is a chronic disease whose prevalence progressively increasing worldwide. One of its main complications is non-alcoholic fatty liver disease (NAFLD), which may occur in an asymptomatic form, as simple fatty liver, steatohepatites (NASH), cirrhosis and hepatocellular carcinoma. This disease is associated with dyslipidemia, hypertension, diabetes, metabolic syndrome and insulin resistance. The gold standard for diagnosis is a liver biopsy, considered to be an invasive method. Indirect measures are used to aid its diagnosis, such as physical, biochemical and imaging tests. Objective: Diagnosing NAFLD using MRI as non-invasive method and correlating it with clinical and laboratory findings. Methodology: A cross-sectional study of 50 children and adolescents followed at the Obesity clinic of the University Hospital, FMRP-USP. A questionnaire about personal and family history was aplplied and each subject was submitted to physical examination, laboratory tests (lipip profile, transaminases, glycemia and basal insulin) and magnetic resonance imaging (MRI) of the abdomen for calculation of hepatic, visceral and subcutaneous fat. Results: Fatty liver disease was diagnosed in 14 (28%) of the participants, 8 of them with severe steatosis (fat percentage > 18%) and 4 with milder steatosis (fat percentage between 9 and 18%). There was a statistically significant difference between the hepatic fat detected by MRI and male gender, triglycerides, AST, ALT, AST/ALT ratio and acanthosis nigricans. The calculation of the Homeostasis Model Assessment Insulin Resistance (HOMA-IR) and of the metabolic syndrome did not show a positive relationship with the percentage of hepatic fat. Conclusion: The frequency of hepatic steatosis using MRI as a diagnostic tool, was lower than the values reported in the literature. Among the parameters studied, the best predictors of liver steatosis were triglycerides, AST, ALT, AST/ALT ratio, male gender, acanthosis nigricans and Z score for high body mass index

CTRP3 is a secreted plasma protein of the C1q family that helps regulate hepatic gluconeogenesis and is downregulated in a diet-induced obese state. However, the role of CTRP3 in regulating lipid metabolism has not been established. Here, we used a transgenic mouse model to address the potential function of CTRP3 in ameliorating high-fat diet-induced metabolic stress. Both transgenic and wild-type mice fed a high-fat diet showed similar body weight gain, food intake, and energy expenditure. Despite similar adiposity to wild-type mice upon diet-induced obesity (DIO), CTRP3 transgenic mice were strikingly resistant to the development of hepatic steatosis, had reduced serum TNF-α levels, and demonstrated a modest improvement in systemic insulin sensitivity. Additionally, reduced hepatic triglyceride levels were due to decreased expression of enzymes (GPAT, AGPAT, and DGAT) involved in triglyceride synthesis. Importantly, short-term daily administration of recombinant CTRP3 to DIO mice for 5 days was sufficient to improve the fatty liver phenotype, evident as reduced hepatic triglyceride content and expression of triglyceride synthesis genes. Consistent with a direct effect on liver cells, recombinant CTRP3 treatment reduced fatty acid synthesis and neutral lipid accumulation in cultured rat H4IIE hepatocytes. Together, these results establish a novel role for CTRP3 hormone in regulating hepatic lipid metabolism and highlight its protective function and therapeutic potential in attenuating hepatic steatosis.

Made available in DSpace on 2015-07-22T20:50:53Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-08-31 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Associação Fundo de Incentivo à Psicofarmacologia (AFIP) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Introdução: A complexidade da fisiopatologia na Esteatose Hepática Não Alcóolica (EHNA) envolve uma interface entre as adipocinas e a regulação neuroendócrina do balanço energético, incluindo o papel do sistema Neuropeptídeo Y/Proteína agouti relacionada. Objetivo: estabelecer a relação entre a razão NeuropeptídeoY/Proteína relacionada Agouti (AgRP) e a adiponectinemia, bem como avaliar a influência de neuropeptídeos orexígenos nos aspectos nutricionais de adolescentes obesos com EHNA submetidos a uma intervenção interdisciplinar de longo prazo. Métodos: Recrutou-se adolescentes obesos pós-púberes para participarem de uma intervenção interdisciplinar. O grupo foi analisado de acordo com a presença ou não de EHNA pela ultrassonografia (US). As amostras de sangue foram coletadas para a mensuração da glicemia, transaminases hepáticas e perfil lipídico, resistência e sensibilidade insulínica. As concentrações de Adiponectina, NPY e AgRP foram avaliadas por ELISA e a mensuração da gordura visceral e subcutânea pela US. A ingestão alimentar foi aferida por meio de registro alimentar de 3 dias. Resultados: No início da terapia, observou-se que parâmetros clínicos importantes como massa corporal, IMC, gordura visceral, HOMA-IR, QUICKI, triglicérides, VLDL-colesterol e transaminases hepáticas apresentaram-se mais alterados em pacientes com EHNA, os quais melhoram após tratamento. Além disso, a energia total e ingestão de macronutrientes reduziram significativamente em ambos os grupos. Observou-se correlação positiva entre AgRP e a gordura visceral em todos os pacientes, e correlação negativa entre NPY/AgRP com a concentração de adiponectina apenas em adolescentes obesos com EHNA. Ao analisar a influência da dieta nos neuropeptídeos orexígenos, apenas os pacientes com EHNA apresentaram correlação positiva entre a ingestão de ácidos graxos saturados com os neuropeptídeos orexígenos (NPY e AgRP); e carboidrato com NPY. Foi observada correlação positiva entre ingestão de energia, (%) de lipídio e ácidos graxos saturados com o acúmulo de gordura visceral. Conclusão: Os pacientes com EHNA apresentaram parâmetros clínicos mais alterados ao compará-los àqueles que não possuíam esta doença, incluindo a correlação negativa entre adiponectina e NPY/AgRP. Estes resultados sugeriram que adolescentes obesos com EHNA apresentaram perfil inflamatório alterado, o qual pode influenciar na regulação neuroendócrina do balanço energético indicando uma barreira adicional na terapia de redução de peso. Além disso, nossos achados revelaram uma importante influência da composição da dieta no sistema orexígeno, sendo essencial considerar a ingestão excessiva de gordura saturada como um fator determinante para o desenvolvimento de EHNA. / BACKGROUND: The complexity physiophatology in the Non Alcoholic Fatty Liver Disease (NAFLD) involves interplay between adipokines and neuroendocrine regulation of energy balance, including the role of Neuropeptide Y/Agouti Related Protein system. AIM: establishing the relationship between Neuropeptide Y/Agouti Related Protein (NPY/AgRP) ratio and adiponectinemia, as well as to assess the influence of orexigenic neuropeptides in the nutritional aspects of NAFLD obese adolescents submitted to a long-term interdisciplinary approach. METHODS: A group of post-pubescent obese adolescents were recruited and included in the interdisciplinary intervention. The group was analyzed according to the presence or ausence of NAFLD by ultrassonography (US). Blood samples were collected to measure glycemia, hepatic transaminases and lipid profile, insulin resistance and sensitivity. Adiponectin, NPY and AgRP concentrations were measured by ELISA and the measurement of visceral ans subcutaneous fat by US. Food intake was measured using 3-day diet records. RESULTS: It was observed at baseline that important clinical parameters including body weight, BMI, visceral fat, HOMA-IR, QUICKI, triglycerides, VLDL-cholesterol and hepatic transaminases were more altered in NAFLD patients. After the intervention, these parameters, total energy and macronutrient intake were reduced significantly in both groups. The most important finding was the positive correlation between AgRP with visceral fat in all patients, and negative correlation between NPY/AgRP with adiponectin only in NAFLD obese adolescents. Analyzing the influence of the diet in the orexigenic neuropeptides, only the NAFLD patients presented a positive correlation between the saturated fatty acids intake and the orexigenic neuropeptides (NPY and AgRP); and carbohydrate with NPY. Indeed, it was observed a positive correlation between energy intake, lipid (%) and saturated fatty acids with visceral fat accumulation. CONCLUSION: NAFLD patients presented more altered clinical parameters than non-NAFLD, including the negative correlation between adiponectin and NPY/AgRP. These results suggested that NAFLD obese adolescents presented an altered inflammatory profile that can influence the neuroendocrine regulation of energy balance suggesting and additional impairment in the weigth loss therapy. Moreover, our findings showed an important influence of diet composition in the orexigenic system, being essential consider that the excessive saturated fatty acids intake could be a determinant factor to increase nonalcoholic fatty liver disease. / FAPESP: 2008/53069-0 / FAPESP: 2006/00684-3 / FAPESP (CEPID/Sono): 9814303-3 / TEDE

Made available in DSpace on 2016-06-02T20:19:18Z (GMT). No. of bitstreams: 1 4384.pdf: 6022457 bytes, checksum: 1327718b29745e1447375271aa448490 (MD5) Previous issue date: 2012-04-10 / Universidade Federal de Sao Carlos / Nonalcoholic Fatty Liver disease (NAFLD) is characterized by fat accumulation in the liver and associated with obesity and insulin resistance. Activin A is a member of the transforming growth factor (TGF)-β superfamily which inhibits hepatocyte growth and induces apoptosis. Follistatin is a glycoprotein which antagonizes the biological actions of activin. Exercise is an important therapeutic strategy to decrease the metabolic effects of obesity. In this study, we evaluated the pattern of Activin A and follistatin liver expression in obese rats submitted to swimming exercise. Adult male Wistar rats were allowed free access to standard rodent chow (control group, C) or HF diet (58% Kcal from fat, high-fat group, HF) during 12 weeks. Glucose tolerance test (GTT) and insulin tolerance test (ITT) were performed at 12 weeks. After 12 weeks, C and HF rats were randomly assigned to a swimming training group (C-Ex and HF-Ex) or a sedentary group (C-Sed and HF-Sed). C-Ex and HF-Ex swam for 45 minutes at 0900h and 1700h, 5 day week-1, for 4 weeks. After this period, rats were submitted again to GTT and then killed by decapitation. Plasma was collected for liver enzymes, alanine transaminase (ALT), aspartate transaminase (AST) and gammaglutamyl transaminase (GGT) determination. Liver was dissected and immediately frozen in liquid nitrogen and stored at -700 C for subsequent analysis. Actvin βA subunit and follistatin mRNA was quantified by real time RT-PCR. HF developed insulin resistance according to GTT and ITT tests. ALT levels were significantly higher in HF-Sed than in C-Sed and significantly lower in HF-Ex than in HF-Sed. AST and GGT did not vary among the groups. Actvin βA subunit mRNA was significantly higher in HF-Ex than in HF-Sed. Follistatin mRNA was significantly lower in C-Ex and HF-Ex compared to C-Sed and HF-Sed, respectively. There was no evidence of steatosis or inflammation (grade 0) in C. In contrast, in HF the severity of steatosis varied from grade 1 to grade 3. This was not associated with inflammation or fibrosis. After the training period, HF-Ex showed improvement in the extent of liver parenchyma damage, the severity of steatosis varying from grade 0 to grade 1. These data suggest that exercise may reduce the deleterious effects of high-fat diet on liver and the local expression of activin-follistatin may be involved. / A doença hepática gordurosa não alcoólica (DHGNA) é caracterizada pelo acúmulo de gordura no fígado e está associada à obesidade e à resistência insulínica. A activina A, membro da superfamília do fator de crescimento e transformação β (TGF-β), inibe o crescimento de hepatócitos e induz a apoptose. A folistatina é uma glicoproteína que antagoniza as ações biológicas da activina. O exercício é uma importante ferramenta terapêutica na diminuição dos efeitos metabólicos da obesidade. Neste estudo, foi avaliado o padrão de expressão de activina A e folistatina no fígado de ratos obesos submetidos ao exercício de natação. Ratos Wistar, machos e adultos tiveram acesso livre à dieta padrão (grupo controle, C) ou dieta hiperlipídica (58% Kcal de gordura, grupo dieta hiperlipídica, DH) durante 12 semanas. O teste de tolerância à glicose (TTG) e o teste de tolerância à insulina (TTI) foram realizados antes e após 3, 4, 8 e 12 semanas. Após 12 semanas, os animais C e DH foram distribuídos aleatoriamente em grupos exercício (CE e DHE) ou grupos sedentários (CS e DHS). Os grupos CE e DHE nadaram por 45 minutos às 9:00h e 17:00h, 5 vezes por semana, durante 4 semanas. Após este período, os animais foram submetidos novamente ao TTG e foram sacrificados por decapitação. O plasma foi coletado para analisar as enzimas hepáticas alanina transaminase (ALT), aspartato transaminase (AST) e gama glutamil transferase (GGT). O fígado foi dissecado e imediatamente congelado em nitrogênio líquido e armazenado a -80ºC para posterior análise. O RNAm da subunidade βA de activina e folistatina foi quantificado por PCR-RT em tempo real. O grupo DH desenvolveu resistência à insulina de acordo com os testes TTG e TTI. Os níveis de ALT foram significativamente maiores no grupo DHS comparados com o grupo CS e significativamente menores no grupo DHE comparados com o grupo DHS. Os níveis de AST e GGT não variaram entre os grupos. A expressão da subunidade βA de activina foi significativamente maior no grupo DHE comparada com o grupo DHS. A expressão de folistatina foi significativamente menor nos grupos CE e DHE comparado aos grupos CS e DHS, respectivamente. Não houve evidências de esteatose ou inflamação (grau 0) no grupo C. Porém, no grupo DH a severidade da esteatose variou do grau 1 ao grau 3, mas não foi associada com inflamação ou fibrose. Após o período de treinamento, o grupo DHE apresentou melhora na extensão da lesão do parênquima hepático, com severidade da esteatose variando do grau 0 ao grau 1. Estes dados sugerem que o exercício reduziu os efeitos deletérios da dieta hiperlipídica no fígado e a expressão local de activina-folistatina pode está envolvida.

Non-Alcoholic Fatty Liver Disease (NAFLD) is characterized by accumulation of lipids in hepatocytes, which accounts for at least 5% of the weight of this tissue. It affects patients in different age groups; these tend to present hepatic alterations characterized not only by the accumulation of fat but, in some cases, also by the presence of inflammation and fibrosis, even evolving to cirrhosis. It is considered the hepatic component of Metabolic Syndrome (MS) and this, in turn, is also an inducer of NAFLD. The present study proposes to evaluate the degree of steatosis in patients with NAFLD and their relationship with cardiovascular risk, the prevalence of OSAS and quality of life. This is a cross-sectional study, where 173 patients were interviewed at the outpatient clinic. Hepatology, which is a reference service of the State, University Hospital of the Federal University of Sergipe. Socio-demographic data and anthropometric measurements, results of biochemical laboratory tests and image of medical records were collected, and these patients were submitted to quality of life (QOL) questionnaires (WHOQOL bref), CVD risk (Framinghan score), individual risk of OSAS (Berlin scale) and bioimpedance test. We obtained as results a prevalence in the low risk population to develop CVD (58.4%), a prevalence of the sample for high risk of OSAS (64%), the QV assessment showed better results in the psychological and social relations domains . We conclude that there is an association between the degree of NAFLD and the evolution of OSA in the patients studied. There was no association with the increase in the risk of CVD, and when we assessed the association with the QOL, there was no significant difference between the total score quality of life in patients with mild steatosis for individuals who presented them in marked form. Thus, we emphasize the importance of the global evaluation of these patients, from the beginning of the detection of NAFLD. / A Doença Hepática Gordurosa Não Alcoólica (DHGNA) é caracterizada por acúmulo de lipídios em hepatócitos, que representa, ao menos, 5% do peso deste tecido. Acomete pacientes em diferentes faixas etárias; estes tendem a apresentar alterações hepáticas caracterizadas não apenas pelo acúmulo de gordura, mas, em alguns casos, também pela presença de inflamação e fibrose, inclusive evoluindo para cirrose. É considerada o componente hepático da Síndrome Metabólica (SM) e esta, por sua vez, também um indutor da DHGNA. O presente estudo propõe avaliar o grau de esteatose em pacientes com DHGNA e a relação destes com o risco cardiovascular, a prevalência de SAOS e a qualidade de vida. Trata-se de um estudo transversal, onde foram entrevistados 173 pacientes atendidos no ambulatório de Hepatologia, um serviço de referência no Estado, do Hospital Universitário da Universidade Federal de Sergipe. Foram coletados dados sociodemográficos e medidas antropométricas, resultados de exames laboratoriais bioquímicos e de imagem dos prontuários, e estes pacientes foram submetidos a questionários de qualidade de vida (QV) (WHOQOL bref), risco de doenças cardiovasculares (DCV’s) (Escore de Framinghan), a avaliação do risco individual de Síndrome da Apneia Obstrutiva do Sono (SAOS) (Escala de Berlim) e ao teste de bioimpedância. Obtivemos como resultados uma prevalência na população estudada de baixo risco para desenvolver DCV’s (58,4%), houve uma prevalência da amostra para alto risco de SAOS (64%), a avaliação da QV demonstrou melhores resultados nos domínios psicológicos e das relações sociais. Concluímos que há uma associação do grau de DHGNA com a evolução do SAOS nos pacientes estudados, não havendo a mesma associação em relação ao aumento do risco de DCV’s e quando avaliamos a associação com a QV não observou-se diferença significativa entre o escore total de qualidade de vida em pacientes com esteatose leve para os indivíduos que as apresentavam na forma acentuada. Sendo assim, enfatizamos a importância da avaliação global desses pacientes, desde o início da detecção da DHGNA. / Aracaju

Le diabète et/ou l’obésité sont associés à la stéatose hépatique non-alcoolique (SHNA). Cette maladie du foie affecte environ un tiers de la population nord-américaine. Elle peut progresser vers un stade d’inflammation, de stress oxydatif et de fibrose appelé la stéatohépatite pouvant éventuellement entraîner le développement d’un cancer primitif du foie comme le carcinome hépatocellulaire (CHC). Cependant, les mécanismes reliant la diète, les maladies métaboliques et le développement du cancer sont complexes et peu connus. Le coactivateur transcriptionnel PGC-1α est un important régulateur du métabolisme énergétique de la cellule et la perte de ce dernier mène à un métabolisme nutritionnel inefficace, ainsi qu’à des défauts mitochondriaux importants. Fait intéressant, une réduction de PGC-1α est retrouvée chez les patients atteints de la maladie du foie gras non-alcoolique (SHNA) et du carcinome hépatocellulaire (HCC). Nous avons précédemment démontré qu’une réduction de PGC- 1α dans le foie murin en combinaison avec une diète obésogène peut provoquer l’apparition de la stéatohépatite. Cependant, le rôle causal de PGC-1α dans le cancer du foie associé à la diète demeure inconnu. Ensuite, un variant génétique de PGC-1α (SNP rs8192678) modifie un résidu glycine en sérine à la position 482 (PGC-1α G482S) chez l’humain et mène à une perte de stabilité protéique dans des cellules hépatiques humaines. Ce polymorphisme est associé au développement de maladies métaboliques, mais son impact sur le cancer demeure inconnu. Enfin, le gène de PGC-1α (PPARGC1A) est régulé par deux promoteurs (proximal et alternatif) donnant naissance à différents isoformes (PGC-1α1-4) de fonctions inconnues. L’action indépendante de ces variants pourrait fournir des indices quant au paradoxe entourant les recherches sur PGC-1α. Nous posons l’hypothèse principale que la perte d’expression de PGC-1α dans le foie favorise le développement du cancer hépatique en réponse à une diète riche en gras/fructose et à l’agent carcinogène diéthylnitrosamine. Dans cette thèse, nous montrons que la perte de PGC-1α favorise le développement du cancer du foie dans un modèle murin combinant une diète obésogène et un carcinogène hépatique. En effet, PGC-1α est nécessaire au maintien de l’expression du marqueur épithélial E-cadhérine et à la réponse cellulaire (apoptose, yH2AX) face aux dommages hépatiques. Nous montrons également que le variant G481 stabilise PGC-1α au niveau protéique et a un effet protecteur contre le cancer du foie chez la souris. Enfin, à l’aide d’expériences in vivo et in vitro nous montrons que la forme canonique PGC-1α1 et le variant PGC-1α4 exercent des rôles distincts sur la mort des cellules hépatiques en réponse à l’inflammation. En conclusion, cette thèse apporte de nouvelles connaissances sur les fonctions de PGC-1α au sein des complications hépatiques associées aux maladies métaboliques et inflammatoires. / Diabetes and obesity are associated to nonalcoholic fatty liver disease (NAFLD). This pathology affects approximately 30% of the population in North America. It ranges from simple steatosis to a more severe necro-inflammatory form called nonalcoholic steatohepatitis (NASH) that can ultimately lead to cirrhosis and primary liver cancer, such as hepatocellular carcinoma (HCC). However, the relationship between diet, metabolic disorders, and cancer development is poorly understood. PGC-1α is a transcriptional coactivator that regulates cellular energy metabolism. Loss of PGC-1α can lead to inefficient nutrient metabolism and severe mitochondrial defects. Interestingly, patients with NAFLD/NASH and HCC exhibit reduced levels of hepatic PGC-1α. We have previously shown that low hepatic PGC-1α combined with an obesogenic diet leads to hallmarks of NASH in mice. However, whether low hepatic PGC-1α reflects a cause or a consequence of liver cancer remains to be determined. Furthermore, a single nucleotide polymorphism within the PPARGC1A coding sequence (SNP rs8192678) leads to a switch between glycine to serine residue at position 482 (PGC-1α G482S) in humans and is associated with reduced protein stability in human liver cells. This SNP is associated with metabolic disorders, but its impact on liver cancer remains un- known. Lastly, the PGC-1α gene (PPARGC1A) is regulated by two promoters (proximal and alternative) that give rise to different isoforms (PGC-1α1-4) of unknown functions. Independent actions of these isoforms could provide a plausible explanation for the paradox observed in previous studies covering the role of PGC-1α. We proposed the general hypothesis that loss of hepatic PGC-1α promotes diet-associated liver cancer development in mice through increased susceptibility to hepatotoxicity. In this thesis, we show that loss of hepatic PGC-1α promotes diet-associated liver cancer in mice. Indeed, PGC-1α is essential to maintain E-cadherin expression and liver cell response (apoptosis, yH2AX) to damage. We also show that G481 variant stabilizes hepatic PGC-1α protein and protects against liver cancer development in mice. Finally, using in vivo and in vitro experiments we show that canonical PGC-1α1 and the PGC-1α4 variant differentially regulate liver cell apoptosis in response to inflammatory signaling. In conclusion, this thesis sheds new light on the role of PGC-1α in liver complications associated with metabolic disorders and inflammation.

La stéatose hépatique non-alcoolique (NAFLD) est une altération hépatique fréquente dans le diabète de type 2 (DT2) et est associée à diverses complications telles que la mortalité. L’établissement d’outils de prédiction non-invasifs de NAFLD est primordial. Mon projet de maîtrise avait pour objectif d’établir des marqueurs génétiques de NAFLD dans le DT2 via deux stratégies : 1) une sélection non-ciblée des marqueurs génétiques (SNPs) via la méthode LASSO et 2) une sélection ciblée de SNPs rapportés comme liés à la maladie ou à des altérations associées. Une population de 4098 patients avec DT2 d’origine caucasienne (ADVANCE) a été utilisée. Des données statistiques sommaires d’études pangénomiques ont été exploitées pour sélectionner, via LASSO, les marqueurs génétiques (SNPs) à inclure dans le score de risque polygénique (PRS). J’ai également développé un modèle de 3210 SNPs ajusté par des covariables capable de prédire les taux élevés de ALT (AUC=0,69) et la mortalité non-cardiovasculaire (AUC=0,66). Le criblage du locus candidat PNPLA3-SAMM50 a mis en avant une diversité des associations génétiques aux différentes altérations métaboliques comme les taux de ALT (substitut du diagnostic de NAFLD) (rs2294915, P = 1,83x10-7), à la mortalité non-cardiovasculaire (rs2294917, P = 3,9x10-4) et à l’efficacité de la thérapie intensive antidiabétique chez certains patients de la population (porteurs GG de rs16991236, P=0,007). Mes travaux ont permis de mieux comprendre le fond génétique de NAFLD dans le DT2 et laissent envisager l’établissement d’outils de diagnostic et de suivi de la maladie plus adéquats. / Non-alcoholic fatty liver disease (NAFLD) is a liver disorder more frequent in type 2 diabetes (T2D) and is associated with complications such as mortality. For this reason, establishing non-invasive tools for predicting NAFLD is crucial. My master’s project aimed to establish genetic markers for NAFLD in T2D using two strategies: 1) a non-targeted selection of genetic markers (SNPs) by the LASSO method and 2) a targeted selection of SNPs reported as associated with the disease or its related abnormalities. A population involving 4098 patients with T2D and Caucasian ancestry was used. Summary statistics data of pangenomic studies were exploited for the selection of SNPs to be involved in the polygenic risk score (PRS). I also designed a model of 3210 SNPs adjusted by covariates and able to predict the high rates of ALT (AUC=0.69) and non-cardiovascular death (AUC=0.66). Mapping of the candidate locus PNPLA3-SAMM50 allowed the observation of diversity in terms of genetic association with the metabolic abnormalities such as ALT (surrogate of NAFLD) (rs2294915, P = 1.83x10-7), non-cardiovascular death (rs2294917, P = 3.9x10-4) and the efficiency of the intensive antidiabetic therapy within a subgroup in the population (individuals with GG of rs16991236, P = 0.007). My studies allowed for a better understanding of the genetic background of NAFLD in T2D and open perspectives for establishing more adequate tools for diagnosis and follow-up of the disease.

Nonalcoholic fatty liver disease (NAFLD) has become one of the most common hepatic diseases worldwide, making the diagnosis and management of NAFLD an emerging public health issue. Theories associated with NAFLD surmise that inflammation may be the root cause, along with the complex interplay of other chronic conditions such as obesity, metabolic syndrome, diabetes, dyslipidemia, and cardiovascular disease (CVD). It is unknown if other inflammatory conditions such as rheumatoid arthritis (RA), along with the use of methotrexate (MTX), might confer increased risk for NAFLD. Longitudinal data collected from a retrospective cohort of 17,481 adult RA patients in the United States were used to determine the incidence and factors associated with the development of NAFLD using a noninvasive tool (Fibrosis-4 score). Results of the Kaplan Meier analysis showed that 31% of this cohort developed NAFLD, in about 7 years from baseline, with most having mild to moderate disease and only 1.4% with advanced disease. RA patients also had a prevalence of chronic conditions associated with NAFLD, as seen in the general population. In the Cox proportional hazard multivariate analysis, age (middle and elderly), hypertension, CVD, dyslipidemia, metabolic syndrome, exercise, use of MTX, and non-MTX antirheumatic drugs were independent predictors for the development of NAFLD. This research could improve early diagnosis of NAFLD using a novel noninvasive tool. Increase awareness of the prevalence and causes of NALFD inform clinical practice and management of the disease and influence policy about this chronic condition in patients with RA.