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Diese Arbeit beinhaltet Untersuchungen zu den unterschiedlichen Isoformen des Leptinrezeptors und dessen Regulation in primären humanen Hepatozyten. Leptin und der Leptinrezeptor nehmen in der Physiologie des menschlichen Energiehaushaltes eine wesentliche Funktion ein und sind an der Pathogenese der Adipositas mit Folgeerkrankungen wie der Entwicklung einer Fettleber beteiligt. Es wird erstmalig geprüft inwieweit das Modellsystem primärer humaner Hepatozyten für Analysen der Leptinrezeptor-Expression und der Abspaltung von löslichem Leptinrezeptor geeignet ist. Weiterhin werden untersucht, welchen Einfluss endokrine Regulatoren wie Dexamethason, Leptin und Glucagon auf die isoformspezifischen Rezeptormengen in primären Hepatozyten haben und wie der Rezeptor unter Apoptose reguliert ist, welche durch die lipotoxischen Effekte der freien Fettsäure Palmitat und den Apoptoseinduktor Staurosporin induziert wird. Hierdurch können Rückschlüsse auf eine möglicherweise veränderte Wirksamkeit des Leptins in der Leber gezogen werden.

L’athérosclérose est un problème de santé publique majeur puisque elle représente aujourd’hui la principale cause de décès dans les pays occidentalisés. Il est donc important de comprendre les mécanismes participant à la progression et aux complications de cette entité anatomoclinique. Nous nous sommes attachés dans ce travail de thèse à démontrer la place et l’apport potentiel de l’imagerie non invasive non expérimentale dans la mise en exergue des particularités de l’athérosclérose carotidienne et/ou coronarienne, et dans la stratégie de dépistage de ses complications chez le sujet non diabétique et diabétique de type 2, en fonction de l’existence ou non d’une stéatose hépatique non alcoolique. Nous présentons notamment dans ce travail, issu en partie de l’exploitation des données cliniques, biologiques et radiologiques de trois protocoles hospitaliers de recherche clinique, les différentes publications scientifiques internationales auxquelles il a donné lieu. / Atherosclerosis is a major public health problem and is one of the major causes of death in the developed western world today. It is therefore of utmost importance that we understand the mechanisms involved in the evolution and progression of this disease and its associated complications. With the work done for this thesis, we tried to bring forth the importance of non invasive clinical imaging to study the pattern of evolution of atherosclerosis involving the carotid and/or coronary arteries. We also present the role played by imaging in prevention and early diagnosis of associated complications in non diabetic and type 2 diabetic patients, presenting with or without non alcoholic hepatic steatosis. In this study, we evaluated three different clinical research protocols used involving the clinical findings, biochemical as well as radiological examination results. The results of these protocols have been the basis for several peer reviewed international publications till date.

Introdução - A soja e outras leguminosas são consideradas como alimentos funcionais por apresentarem propriedades hipocolesterlemizantes. Esta propriedade, porém, ainda não foi relatada para o feijão caupi. Um possível componente deste grão responsável pelo feito redutor de colesterol é sua proteína. Objetivo - Produzir isolado protéico de feijão caupi e verificar a influência do grão integral e de sua proteína isolada no metabolismo lipídico de hamsters hipercolesterolemizados pela dieta. Métodos - O isolado protéico de feijão caupi foi produzido por precipitação isoelétrica, utilizando-se pH 8,5 para solubilização da proteína e 4,5 para a sua precipitação, obtendo-se um isolado com 92 % de proteína. O isolado protéico (IP) e o feijão caupi integral (FCI) foram utilizados como fonte protéica em dietas experimentais para hamsters que tiveram hipercolesterolemia induzida por dieta contendo 20 % de caseína, 13,5 % de gordura saturada e 0,1 % de colesterol, por 3 semanas. Os animais foram distribuídos em três grupos, recebendo cada grupo dieta com 20 % de caseína (controle), dieta com 20 % de proteína de isolado de feijão caupi e dieta com 20% de proteína de feijão caupi integral, por 4 semanas. Resultados - Comparando-se à dieta controle, a dieta com FCI e IP provocaram reduções significativas no colesterol total (49 % e 20 %, respectivamente) e colesterol não-HDL (54 % e 22 %, respectivamente). Análises histológicas do fígado foram realizadas e observou-se que IP e o FCI proporcionaram efeito hepatoprotetor comparado à caseína, pois o grupo que recebeu caseína apresentou em média esteatose difusa e intensa, enquanto grupo com feijão e isolado apresentaram esteatose focal e, em alguns casos, ausentes. Alguns possíveis mecanismos envolvidos para o efeito benéfico no metabolismo lipídico foram investigados. A digestibilidade verdadeira do IP foi igual à da caseína, enquanto a do feijão integral foi menor. A excreção de ácidos biliares e colesterol foi inversamente proporcional aos níveis plasmáticos do colesterol dos animais submetidos às diferentes dietas. Os animais com a dieta de feijão integral apresentaram a maior excreção de ácidos biliares e colesterol nas fezes; valores estatisticamente diferentes aos dos animais da dieta com caseína. Conclusões - O feijão caupi e sua proteína isolada reduzem o colesterol plasmático e proporcionam efeito hepatoprotetor. A digestibilidade, excreção de ácidos biliares e colesterol não estão relacionados com a redução do colesterol provocada pelo IP. O mecanismo envolvido na redução do colesterol nestes experimentos ainda não está totalmente elucidado, sugerindo que a proteína isolada do feijão caupi atue na síntese de colesterol, pois não foi observado aumento na sua excreção. / Introduction - Soya and other legume seeds are considered functional food because of their hypocholesterolemic properties. This property, however, is not reported yet for cowpea. A possible component present in this grain that could respond for the hypocholesterolemic effect is the protein fraction. Objective - To produce protein isolate of cowpea and to verify the influence of the whole seed and its isolated protein on the lipid metabolism of diet hypercholesterolemized hamsters. Methods - The cowpea protein isolate was prepared by isoelectric precipitation, using pH 8.5 for solubilization and pH 4.5 for protein precipitation. The isolate obtained presented protein content of about 92%. The protein isolate (PI) and cowpea whole seed (CWS) were used as protein source in experimental diets fed to hamsters that previously had their blood cholesterol increased by a diet containing 20 % of casein, 13,5 % of saturated fat and 0,1 % of cholesterol during 3 weeks. Animals were divided into 3 groups and fed on diets containing: 20 % casein (control), 20 % isolate protein of cowpea and 20 % of cowpea whole seed for 4 wks. Results - The results showed that for hamster that fed on diets containing PI and CWS occurred a significant decrease in total cholesterol (49 % and 20 %, respectively) and non-HDL cholesterol (54 % and 22 %, respectively), as compared to casein group. Histological analysis of hepatic tissue was performed and showed that PI and CWS presented reduction in hepatic lipotoxicity as compared to the casein group. Therefore, the group that received casein presented steatosis widely distributed throughout the hepatic lobule, while whole seed cowpea and protein isolate meal groups produced only focal steatosis and, in some cases, it was absent. Some mechanisms involved in lowering plasma cholesterol were investigated. Digestibility for PI was equal for casein group, while it was significantly lower for CWS group. The bile acids and cholesterol excretion in the feces were inversely proportional to plasma cholesterol levels. Animals on CWS presented higher levels of feces bile acids and cholesterol; these values were statistically different from animals on casein diet. Conclusions - Whole seed cowpea and its protein isolate reduce plasma cholesterol and hepatic lipotoxicity. Digestibility, the bile acids and cholesterol excretion are not related to hypocholesterolemic effect of protein isolate of cowpea. The mechanisms involved cholesterol reduction in these experiments is not yet fully elucidated. It is suggested that protein isolate of cowpea is related to the cholesterol syntheses, as it was not verified any increase in cholesterol excretion in the animals on protein isolate diet.

A esteatose hepática, que se caracteriza pelo acúmulo excessivo de gordura nas células do fígado, é um problema que vem preocupando a comunidade médico-científica, pois sua incidência vem aumentando a nível global, com expectativa de se tornar a doença crônica hepática de maior predominância em várias partes do mundo. Apesar de ser considerada uma doença benigna, a esteatose pode evoluir para doenças mais graves como cirrose, fibrose avançada, esteato hepatite (com ou sem fibrose) ou carcinoma. Entretanto, é potencialmente reversível, mesmo em quadros mais graves, o que reforça a urgência de se desenvolver métodos confiáveis para detecção e avaliação, inclusive ao longo de tratamento. Os métodos atuais para diagnóstico e quantificação da gordura hepática ainda são falhos: com a ultrassonografia não se é capaz de realizar quantificação; a tomografia computadorizada faz uso de radiação ionizante; a punção (biópsia), considerada o padrão ouro, é precisa, mas invasiva e pontual. A Ressonância Magnética (RM), tanto com espectroscopia (MRS) como com imagem (MRI), são alternativas completamente não invasivas, capazes de fornecer o diagnóstico e quantificação da gordura infiltrada no fígado. Entretanto, os trabalhos encontrados na literatura utilizam sequências de pulsos desenvolvidas especialmente para esse fim, com métodos de pós-processamento extremamente rebuscados, o que não é compatível com o estado atual dos equipamentos encontrados em ambientes clínicos nem mesmo ao nível de experiência e conhecimento das equipes técnicas que atuam em clínicas de radiodiagnóstico. Assim, o objetivo central do presente trabalho foi avaliar o potencial da RM como candidato a método de diagnóstico e de quantificação de gordura em ambientes clínicos, utilizando, para isso, sequências de pulsos convencionais, disponíveis em qualquer sistema comercial de RM, com protocolos de aquisição e processamento compatíveis com àqueles realizados em exames clínicos, tanto no que se refere à simplicidade como ao tempo total de aquisição. Foram avaliadas diferentes abordagens de MRS e MRI utilizando a biópsia hepática como padrão de referência. Foram avaliados pacientes portadores de diabetes tipo II, que apresentam alta prevalência de esteatose hepática não alcoólica, além de grande variabilidade nos percentuais de gordura. Foram realizadas medidas de correlação, acurácia, sensibilidade e especificidade de cada uma das abordagens utilizadas. Todos os métodos avaliados apresentaram alto grau de correlação positiva (> 87%) com os dados obtidos de maneira invasiva, o que revela que os valores obtidos utilizando RM estão de acordo com aquilo observado pela biópsia hepática. Muito embora os métodos de processamento utilizados não sejam tão complexos quanto seriam necessários caso uma quantificação absoluta fosse desejada, nossas análises mostraram alta acurácia, especificidade e sensibilidade da RM na avaliação da esteatose. Em conclusão, a RM se apresenta, de fato, como uma excelente candidata para avaliar, de forma não invasiva, a fração de gordura hepática, mesmo quando se considera as limitações impostas por um ambiente clínico convencional. Isso sugere que essas novas metodologias podem começar a migrar para ambientes clínicos sem depender das sequências complexas e dos processamentos exóticos que estão descritos na literatura mais atual. / Hepatic steatosis is a disease characterized by excessive accumulation of fat in liver cells. Its incidence is increasing and it is expected to become the chronic liver disease with the highest prevalence worldwide, which became a major concern to medical and scientific community. Although considered a benign disease, fatty liver can evolve to more serious diseases such as cirrhosis, advanced fibrosis, steatohepatitis (with or without fibrosis) or carcinoma. However, it is potentially reversible, even in the most severe cases, which reinforces the urgent need to develop reliable methods for detection and evaluation, including throughout treatment. Current methods for diagnosis and measurement of liver fat are still flawed: ultrasound is not able to perform quantification; CT uses ionizing radiation; biopsy, considered the gold standard, is accurate but invasive and focal. Magnetic resonance imaging (MRI) and spectroscopy (MRS) are completely non-invasive alternatives capable of providing diagnosis and quantification of infiltrated fat in the liver. However, studies in the literature frequently employ pulse sequences especially developed for this purpose, with extremely elaborate post-processing methods, which are not compatible with the current state of the art of typical clinical scanners or even with the level of experience and knowledge of technical staff working in radiology clinics. Thus, the main goal of this study was to evaluate the potential of MRI as a candidate for diagnosing and quantifying fat liver in clinical settings, using conventional pulse sequences, acquisition and post-processing protocol, compatible with those performed in clinical examinations in both simplicity and total acquisition time. We investigated different approaches of MRI and MRS using liver biopsy as reference standard. We evaluated patients with type II diabetes, which have a high prevalence of non-alcoholic fat liver disease, and great variability in the percentage of fat. Correlation, accuracy, sensitivity and specificity measurements of each of the approaches used were performed. All evaluated methods highly correlated (> 87%) with the data obtained from invasive measurement, which shows that the values obtained using MRI are consistent with that observed for liver biopsy. Although the processing methods used are not as complex as would be required if an absolute quantification was desired, our analyzes showed high accuracy, sensitivity and specificity of MRI in the evaluation of steatosis. In conclusion, MRI is an excellent candidate to assess liver fat fraction in a non-invasive way, even when considering the limitations imposed by conventional clinical setting. This suggests that these new methods may begin to migrate to clinical environments without relying on complex sequences and exotic post-processing techniques that are described in the current literature.

Au cours du développement, les modifications du métabolisme des monocarbones liées à une malnutrition peuvent être délétères autant pour la mère que pour le nouveau-né. De plus, les conséquences à long terme d'une carence en période gestationnelle et périnatale sont mal connues, notamment en ce qui concerne les pathologies cardiaques et hépatiques. Nous avons mis en oeuvre un modèle nutritionnel de rates adultes carencées en donneurs de groupements méthyles (vitamines B12, folates et choline) avant la gestation. Ces micronutriments participent à la régulation de différentes enzymes impliquées dans le métabolisme de l'homocystéine. Afin de se placer dans un contexte de physiopathologie, proche de la situation clinique évaluée, nous avons choisi d'alimenter les rates avec un régime carencé un mois avant la mise en accouplement et de poursuivre ce régime pendant la période d'allaitement. Nous avons évalué les répercussions métaboliques et fonctionnelles du régime sur les tissus myocardique et hépatique, chez le nouveau né à 21 jours. Nous avons étudié l'effet de cette carence en groupements méthyles sur le métabolisme énergétique lipidique et sur la carnitine. Conséquences de la carence au niveau myocardique : Le régime carencé en donneurs de méthyles induit une hypertrophie cardiaque avec une augmentation de l'épaisseur du myocarde et un agrandissement des cardiomyocytes. L'étude protéomique du myocarde et l'analyse des données par bioinformatique identifient PGC-1[alpha], PPAR[alpha] et ERR[alpha] comme principaux déterminants des variations d'expression des protéines du métabolisme oxydatif mitochondrial. Nous avons observé une diminution d'expression de PPAR[alpha] et ERR[alpha] et une inactivation de PGC1[alpha] par hypométhylation et hyperacétylation, en lien avec une diminution d'expression de PRMT1 et de SIRT1 et une augmentation de SAH. Conséquences de la carence au niveau du foie : La carence s'accompagne de l'apparition d'une stéatose hépatique microvésiculaire, avec une élévation des taux tissulaires de lipides et de triglycérides. De plus, nous avons observé une augmentation des marqueurs pro inflammatoires sans augmentation des marqueurs de fibrose. A cet égard, nos résultats ont montré qu'un déficit de synthèse de carnitine, impliquée dans la bêta-oxydation et le stockage des acides gras, jouerait un rôle déterminant dans la pathogenèse de la stéatose chez le nouveau-né. Il existe également une dérégulation du métabolisme oxydatif des acides gras, avec diminution d'activité des complexes I et II de la chaîne respiratoire, qui résulte d'une hypométhylation de PGC1 et d'une diminution d'expression de PPAR[alpha], ER[alpha] et ERR[alpha]. En conclusion, nos résultats montrent que la carence maternelle en donneurs de méthyles, induit des modifications sur la fonction de PGC-1[alpha]. Ces modifications sont associées à des altérations de l'oxydation des acides gras et sur la fonction mitochondriale pendant la période néonatale, ce qui entraîne l'accumulation de lipides dans les tissus myocardique et hépatique. Le lien entre la carence en donneurs de méthyles et l'altération de la méthylation de PGC-1[alpha] modifie les activités des enzymes impliquées dans la méthylation et l'acétylation de PGC-1. Ces enzymes sont aussi liées à des modifications épigénomiques qui modulent la fonction et l'expression des protéines. Nos résultats sont en accord avec les études de population de Barker et al, qui suggèrent que la nutrition maternelle pendant les étapes précoces de la vie est corrélée avec le risque de maladies cardio-vasculaires dans la vie adulte indépendamment des autres facteurs de risque / During development, changes in carbon metabolism related to malnutrition may be deleterious for both the mother and the newborn. In addition, long-term consequences of a methyl deficiency gestational and prenatal are poorly understood. We are particularly interested in studying these effects on the heart and liver. We have used a nutritional model of adult rats deficient in methyl donors (vitamin B12, folate and choline) before pregnancy. To be placed in a context of pathophysiology, close to the clinical situation, we chose to feed the rats with a methyl deficient diet one month before mating and continue this diet during the suckling period. We evaluated the metabolic and functional effects of this diet on myocardial and hepatic tissues, in the newborn pups in 21 days old. We studied the effects of methyl deficient diet on lipid and energy metabolism and carnitine. Consequences of the deficiency at the myocardial: The diet deficient in methyl donors induces cardiac hypertrophy with an increase in myocardial thickness and enlargement of cardiomyocytes. The proteomics analysis of the bioinformatics data identifies PGC-1[alpha], ERR[alpha] and PPAR[alpha] as major determinants of changes in protein expression of mitochondrial oxidative metabolism. We observed a decreased expression of PPAR[alpha] and ERR[alpha] and an inactivation of PGC1[alpha] by hypomethylation and hyperacetylation, in conjunction with a decrease in the expression of PRMT1 and SIRT1 and increased the level of SAH. Consequences of the deficiency at liver: Deficiency is accompanied by the appearance of microvesicular hepatic steatosis, with elevated tissue levels of lipids and triglycerides. Increase of inflammation was observed in this model with no changes in fibrosis score. In this respect, our results showed a deficiency of carnitine synthesis, involved in the beta-oxidation and storage of fatty acids, play a role in the pathogenesis of hepatic steatosis in the newborn. There is also a deregulation of the oxidative metabolism of fatty acids, with decreased activity of complex I and II of the respiratory chain, resulting in hypomethylation of PGC1 and decreased expression of PPAR[alpha], ER[alpha] and ERR[alpha]. In conclusion, our results show that maternal deprivation in methyl donors impaired the function of PGC-1[alpha]. These changes are associated with alterations in fatty acid oxidation and mitochondrial function during the neonatal period, with lipid accumulation in myocardial tissue and liver. The link between the methyl donor deficiency and impaired methylation of PGC-1[alpha] alters the activities of enzymes involved in methylation and acetylation of PGC-1[alpha]. These enzymes are also associated with epigenetic changes and the function and gene expression. Our results are consistent with population studies by Barker and colleagues, who suggest that maternal nutrition during early stages of life is correlated with the risk of cardiovascular disease in adult, independent of other risk factors

A aterosclerose é um processo inflamatório que se inicia na infância e progride com a idade, sendo o principal processo patológico que culmina nas doenças cardiovasculares. Ácidos graxos ômega 3 (N-3 FA) reduzem triacilgliceróis plasmáticos e inflamação, enquanto fitosteróis possuem ação hipocolesterolêmica. Portanto, o objetivo deste estudo foi de aplicar a combinação dessas duas classes de lipídios bioativos na infância, visando reduzir a aterosclerose na fase adulta. O estudo foi dividido em duas etapas. Inicialmente, o efeito de três ingredientes contendo N-3 FA foi avaliado em termos de perfil lipídico e biomarcadores inflamatórios. Camundongos adultos LDLr Knockout receberam uma dieta hiperlipídica e foram simultaneamente suplementados com água (CON), óleo de algas (ALG), óleo de peixe (FIS) e óleo de Echium (ECH) através de gavagem durante 4 semanas. Os animais suplementados com óleo de Echium apresentaram os maiores índices de redução de triacilgliceróis e VLDL, além de inibição da esteatose causada pelo dieta hiperlipídica. Entretanto, diferente dos óleos marinhos, tais efeitos não envolveram ativação de fatores de transcrição envolvidos no metabolismo lipídico, como PPARα e LXRα hepáticos. Na segunda etapa deste estudo, camundongos Knockout LDLr recém-desmamados foram suplementados durante 2 meses com emulsões preparadas com óleo de soja (CON), óleo de de Echium (ECH), óleo de alga (ALG), fitosteróis isolados (PHY), óleo de alga + fitosteróis (ALG+PHY) e óleo de Echium + fitosteróis (ECH+PHY). A seguir, dislipidemia e estresse oxidativo foram induzidos através de uma dieta hiperlipídica por mais 2 meses. Todos os animais apresentaram estrias gordurosas na aorta, sendo que a área de lesão foi maior naqueles suplementados com fitosteróis isoladamente (PHY). Entretanto, esse efeito negativo foi totalmente revertido pela co-suplementação com N-3 FA. Observou-se que os fitosteróis isolados agiram como agonistas de LXRα, e que a reversão proporcionada pelos N-3 FA envolveu aumento da expressão do fator de transcrição PPARα e redução do aumento de LXRα. Além disso, o óleo de Echium reduziu o estresse oxidativo no fígado através de mecanismos associados à modulação da atividade e expressão de enzimas antioxidantes. Nossos resultados sugerem que a melhor alternativa no desenvolvimento de alimentos funcionais para crianças, visando prevenção de aterosclerose na fase adulta, foi conferida pelos N-3 FA provenientes do óleo de Echium. / Atherosclerosis is an inflammatory process that begins in childhood and progresses with age, being the main pathological process that culminates in cardiovascular disease. Omega 3 fatty acids (n-3 FA) reduces triacylglycerol (TG) and inflammation, while phytosterols present hypocholesterolemic action. Therefore, the aim of this study was to apply the combination of these two classes of bioactive lipids in childhood to reduce atherosclerosis in adulthood. The study was divided into two steps. Initially, the effect of three ingredients containing n-3 FA was evaluated in terms of lipid and inflammatory biomarkers. LDLr knockout adult mice received a high-fat diet and were simultaneously supplemented with water (CON), algae oil (ALG), fish oil (FIS) and Echium oil (ECH) by gavage for 4 weeks. The animals supplemented with Echium oil presented higher reduction of TG and VLDL. Besides, Echium oil inhibited hepatic steatosis caused by high-fat diet. However, unlike of marine oils, this hypotriglyceridemic effect did not involve activation of transcription factors associated to lipid metabolism, such as PPARα and LXRα. At the second step of this study, weaning LDLr knockout mice were supplemented with emulsions prepared with soybean oil (CON), Echium oil (ECH), algae oil (ALG), isolated phytosterols (PHY), algae oil + phytosterols (ALG + PHY) and Echium oil + phytosterols (PHY + ECH) for 2 months. Thereafter, dyslipidemia and oxidative stress were induced by a high fat diet for 2 months more. All animals showed fatty streaks in the aorta artery. The lesion area was greatest in mice supplemented with isolated phytosterol (PHY). However, this negative effect was completely reversed by co-supplementation with n-3 FA. It was observed that isolated phytosterols acted as LXRα agonists, and that the reversal provided by the N-3 FA involved the increase of PPARα and reduction of LXRα expression. Furthermore, the Echium oil reduced oxidative stress in liver through mechanisms associated with modulation of antioxidant enzymes activity and expression. Our results suggest that the best alternative to develop functional foods for children, aimed at preventing atherosclerosis in adulthood, was conferred by the N-3 FA from Echium oil.

Introdução - A soja e outras leguminosas são consideradas como alimentos funcionais por apresentarem propriedades hipocolesterlemizantes. Esta propriedade, porém, ainda não foi relatada para o feijão caupi. Um possível componente deste grão responsável pelo feito redutor de colesterol é sua proteína. Objetivo - Produzir isolado protéico de feijão caupi e verificar a influência do grão integral e de sua proteína isolada no metabolismo lipídico de hamsters hipercolesterolemizados pela dieta. Métodos - O isolado protéico de feijão caupi foi produzido por precipitação isoelétrica, utilizando-se pH 8,5 para solubilização da proteína e 4,5 para a sua precipitação, obtendo-se um isolado com 92 % de proteína. O isolado protéico (IP) e o feijão caupi integral (FCI) foram utilizados como fonte protéica em dietas experimentais para hamsters que tiveram hipercolesterolemia induzida por dieta contendo 20 % de caseína, 13,5 % de gordura saturada e 0,1 % de colesterol, por 3 semanas. Os animais foram distribuídos em três grupos, recebendo cada grupo dieta com 20 % de caseína (controle), dieta com 20 % de proteína de isolado de feijão caupi e dieta com 20% de proteína de feijão caupi integral, por 4 semanas. Resultados - Comparando-se à dieta controle, a dieta com FCI e IP provocaram reduções significativas no colesterol total (49 % e 20 %, respectivamente) e colesterol não-HDL (54 % e 22 %, respectivamente). Análises histológicas do fígado foram realizadas e observou-se que IP e o FCI proporcionaram efeito hepatoprotetor comparado à caseína, pois o grupo que recebeu caseína apresentou em média esteatose difusa e intensa, enquanto grupo com feijão e isolado apresentaram esteatose focal e, em alguns casos, ausentes. Alguns possíveis mecanismos envolvidos para o efeito benéfico no metabolismo lipídico foram investigados. A digestibilidade verdadeira do IP foi igual à da caseína, enquanto a do feijão integral foi menor. A excreção de ácidos biliares e colesterol foi inversamente proporcional aos níveis plasmáticos do colesterol dos animais submetidos às diferentes dietas. Os animais com a dieta de feijão integral apresentaram a maior excreção de ácidos biliares e colesterol nas fezes; valores estatisticamente diferentes aos dos animais da dieta com caseína. Conclusões - O feijão caupi e sua proteína isolada reduzem o colesterol plasmático e proporcionam efeito hepatoprotetor. A digestibilidade, excreção de ácidos biliares e colesterol não estão relacionados com a redução do colesterol provocada pelo IP. O mecanismo envolvido na redução do colesterol nestes experimentos ainda não está totalmente elucidado, sugerindo que a proteína isolada do feijão caupi atue na síntese de colesterol, pois não foi observado aumento na sua excreção. / Introduction - Soya and other legume seeds are considered functional food because of their hypocholesterolemic properties. This property, however, is not reported yet for cowpea. A possible component present in this grain that could respond for the hypocholesterolemic effect is the protein fraction. Objective - To produce protein isolate of cowpea and to verify the influence of the whole seed and its isolated protein on the lipid metabolism of diet hypercholesterolemized hamsters. Methods - The cowpea protein isolate was prepared by isoelectric precipitation, using pH 8.5 for solubilization and pH 4.5 for protein precipitation. The isolate obtained presented protein content of about 92%. The protein isolate (PI) and cowpea whole seed (CWS) were used as protein source in experimental diets fed to hamsters that previously had their blood cholesterol increased by a diet containing 20 % of casein, 13,5 % of saturated fat and 0,1 % of cholesterol during 3 weeks. Animals were divided into 3 groups and fed on diets containing: 20 % casein (control), 20 % isolate protein of cowpea and 20 % of cowpea whole seed for 4 wks. Results - The results showed that for hamster that fed on diets containing PI and CWS occurred a significant decrease in total cholesterol (49 % and 20 %, respectively) and non-HDL cholesterol (54 % and 22 %, respectively), as compared to casein group. Histological analysis of hepatic tissue was performed and showed that PI and CWS presented reduction in hepatic lipotoxicity as compared to the casein group. Therefore, the group that received casein presented steatosis widely distributed throughout the hepatic lobule, while whole seed cowpea and protein isolate meal groups produced only focal steatosis and, in some cases, it was absent. Some mechanisms involved in lowering plasma cholesterol were investigated. Digestibility for PI was equal for casein group, while it was significantly lower for CWS group. The bile acids and cholesterol excretion in the feces were inversely proportional to plasma cholesterol levels. Animals on CWS presented higher levels of feces bile acids and cholesterol; these values were statistically different from animals on casein diet. Conclusions - Whole seed cowpea and its protein isolate reduce plasma cholesterol and hepatic lipotoxicity. Digestibility, the bile acids and cholesterol excretion are not related to hypocholesterolemic effect of protein isolate of cowpea. The mechanisms involved cholesterol reduction in these experiments is not yet fully elucidated. It is suggested that protein isolate of cowpea is related to the cholesterol syntheses, as it was not verified any increase in cholesterol excretion in the animals on protein isolate diet.

Abstract Metabolic Syndrome (MetSyn) is a medical condition prevalent in Australia. MetSyn is diagnosed with a varying combination of visceral obesity, insulin resistance/ impaired glucose tolerance/ Type 2 diabetes, dyslipidaemia and hypertension. Obesity is a central feature of this syndrome that is characterised by abnormalities in glucose and lipid metabolism. An understanding of the cause of the metabolic derangement that occurs in obesity, and that contributes to MetSyn, would allow effective treatment and prevention strategies to be formulated. This is a priority in the current environment of highly prevalent overweight and obesity in Australian children and adults. Lipotoxicity of insulin-dependent tissues and ectopic fat depots are emerging as fundamental processes in the pathogenesis of MetSyn. Lifestyle intervention, such as increased physical activity, show great promise as agents for disrupting the disease progression and may act via direct or indirect mechanisms on the underlying pathology of MetSyn. This study aimed to determine if diagnostic markers of MetSyn exist in obese, prepubertal, Australian children and to assess the contribution of lifestyle factors on components of MetSyn. Further, this study sought to investigate the relationship between body fat patterning (total body fat, abdominal adipose depots, skeletal intramyocellular lipids, intrahepatocellular lipids) and markers of MetSyn. An experimental intervention was then employed to examine the effect of physical activity on body fat distribution, insulin sensitivity, and haemodynamic and biochemical markers of MetSyn, and additionally to determine if the effect of exercise on parameters of MetSyn was mediated by a change in body fat patterning. Data were collected in a group of 15 obese (mean BMI Z-score 2.51 ± 0.49), prepubertal children (6 male, 9 female) aged 5.1 – 11.4 years (mean age 7.82 yrs ± 1.83). Measures included insulin sensitivity, blood biochemistry (lipid, haemostatic and adipocyte activity markers), blood pressure, two-compartment body composition by hydrometry, and nuclear magnetic resonance scanning for abdominal adipose depots, intrahepatic lipids and skeletal intramyocellular lipids. Each child’s habitual nutrition and physical activity were also ascertained using multiple-pass 24-hr diet recalls and accelerometry respectively. Data collection was conducted pre and post a 12-week physical activity intervention which consisted of cardiorespiratory activity during instructor led sessions (60 mins, twice weekly) and family led sessions (>10 mins, 4 days/wk). There is no universally accepted definition of MetSyn in childhood. The International Diabetes Federation suggests that MetSyn should not be diagnosed in children aged 6 to < 10 years. Children can be identified to be at risk of MetSyn, however, based on waist circumference ≥90th percentile and family history1,2; all subjects in this study were at risk according to these criteria. Four definitions of paediatric MetSyn previously applied to a group of young, overweight Australian children3 were used to calculate the prevalence of MetSyn in the current sample and it was found to be 27-89% at baseline and 13-80% after the experimental intervention depending upon the definition used. Acanthosis nigricans and impaired glucose tolerance (IGT) were present in one female child. Post-intervention, IGT had resolved and the child was glucose tolerant. Habitual dietary intake (energy intake and macronutrients) measured over a 3-day period pre-intervention displayed a significant positive association between fasting glucose and energy intake, as well as a significant negative association between fasting glucose and the protein component of the diet. Following the physical activity programme, energy intake was significantly positively correlated with body fat percentage (% BF). There was no difference found in dietary intake assessed prior to and following cessation of the physical activity intervention, in terms of energy or % energy from macronutrients. Habitual physical activity was not related to MetSyn diagnostic indicators. A higher level of physical fitness, estimated by predicted O2max (ml•kg-1•min-1), was significantly correlated with a lower level of diastolic blood pressure at baseline. A greater fitness level ( O2max) was moderately correlated with a lower BMI Z-score following the 12-week intervention. There was no difference between pre- and post-intervention habitual physical activity. A trend towards less sedentary time and increased light intensity activity was found, but these did not reach significance. Physical fitness level showed a trend for improvement following the intervention (P = 0.060). Anthropometrically determined body composition and body fat distribution did not change following the intervention. Radiologically determined abdominal adipose tissue depots were not significantly different post-intervention. % BF was not different when assessed with bioelectrical impedance analysis. However, % BF did reduce significantly over the 12-week intervention period when quantified by hydrometry (42.3% ± 5.0 vs 36.9% ± 8.6, P = 0.022). Adipokines, the secretory products of adipocytes displaying pleiotropic metabolic action, were investigated for their relation to lipid depots and additionally for change post-intervention. Cardiovascular (CV) disease risk was investigated by proatherogenic and protective blood lipids. When examined at baseline, fasting blood triacylglycerols (TAG) were inversely associated with basal and stimulated insulin sensitivity. Post-intervention, a higher level of HDL-C was found to be associated with greater insulin sensitivity, although this was not apparent at baseline. The relation between TAG and insulin sensitivity discovered pre-intervention was no longer evident. All other biomarkers of CV risk were not associated with body composition, glucose homeostasis, and lifestyle factors pre- and post-intervention. The effect of the physical activity intervention on indicators of haemostasis, physical fitness, blood lipids and lipoproteins, systemic inflammation, and fibrinolytic activity were analysed for change. Both systolic and diastolic blood pressure were significantly reduced following the physical activity programme. There was no significant difference found in any other measured parameter of CV risk. Log[HOMA], a surrogate index of insulin resistance, was significantly decreased post-intervention indicating reduced insulin resistance. QUICKI, a surrogate index of insulin sensitivity, was significantly improved post-intervention. The remaining indicators of insulin resistance, insulin sensitivity and β-cell function based on fasting surrogates did not significantly change over the 12-week experimental period. Dynamic insulin sensitivity and β-cell function were investigated pre- and post-intervention using paired samples t-tests. Glucose and insulin area under the curve of the OGTT were significantly reduced and whole-body insulin sensitivity index (WBISI) was significantly increased hence showing an improvement in stimulated insulin sensitivity. AUCCP/AUCglu significantly declined also indicating an improved response to oral glucose stimulation. IGI and ΔCP30/ΔG30, as markers of β-cell insulin secretion, did not change. Disposition index, the interrelationship of insulin secretion (IGI) and insulin sensitivity (WBISI), was not changed pre- and post-intervention. Hepatic insulin extraction was increased post-intervention (4.3 ± 1.2 vs 4.8 ± 1.1, P = 0.022) possibly due to greater hepatic and/or peripheral insulin sensitivity. General linear modeling (GLM) showed the improvement in whole-body insulin sensitivity discovered following the intervention was independent of % BF, abdominal adipose tissue depots, and ectopic lipid depots. Intrahepatocellular lipids (IHCL) significantly decreased after the 12-week intervention (6.99% ± 9.41 vs 5.83% ± 8.54) whilst there was no significant change in the serum markers of liver inflammation. IHCL was positively and strongly associated with total abdominal adipose tissue, intra-abdominal adipose tissue and subcutaneous abdominal adipose tissue both before and after the intervention. IHCL was positively associated with %BF measured post-intervention; this relationship almost reached significance when measured pre-intervention (P = 0.060). IHCL was not associated with insulin sensitivity either pre- or post-intervention nor with circulating lipids at either timepoint. The change in IHCL was independent of % BF and abdominal adipose tissue tested by GLM. However, there was no significant difference found in IHCL post-intervention after adjustment for insulin sensitivity (WBISI) by GLM. Prior to intervention, 10 of 15 subjects had hepatic steatosis diagnostic of non-alcoholic fatty liver disease. Eight of the 10 subjects with clinically significant hepatic steatosis had reduction of fatty infiltrate following the exercise intervention. In the whole group it was demonstrated that physical activity attenuates lipid infiltration of the liver independent of body fat. To further investigate the pathophysiology of ectopic lipid depots, biomarkers of oxidative stress and anti-oxidant status were examined in relation to IHCL. Pre-intervention, there was no association found between pro-oxidative or anti-oxidative activity and IHCL. Post-intervention, an inverse association of plasma carotenoid:cholesterol ratio with IHCL was found. Skeletal intramyocellular lipids (IMCL) measured in the right soleus were significantly increased post-intervention (2.4 ± 1.1 vs 2.6 ± 1.2, P = 0.035). There was no association between IMCL and % BF when measured pre- or post-intervention. Abdominal adipose depots were associated with IMCL at baseline and following the intervention. IMCL was not related to IHCL at either timepoint. Pre-intervention, there was a trend for a relationship between IMCL and insulin. Post-intervention, IMCL was tightly and inversely correlated with insulin sensitivity (r = -0.85 P = 0.000). Linear regression between IMCL and WBISI run pre-intervention and post-intervention found the slopes were not significantly different whereas the intercepts were highly significantly different (P = 0.001), thus, as IMCL increased there was a corresponding decrease in insulin sensitivity. GLM found the increase in IMCL was independent of % BF and abdominal adipose tissue, but was not independent of WBISI. These data indicate the greater IMCL level found post-intervention was a non-pathologic training adaptation. To further investigate the pathophysiology of ectopic lipid depots, biomarkers of oxidative stress and anti-oxidant status were examined in relation to IMCL. Pre-intervention, there was a positive association between malondialdehyde and IMCL. Post-intervention, an inverse association was found between IMCL and both plasma total carotenoids and total carotenoid:free cholesterol ratio. In summation, this study found improved metabolic health in obese, prepubertal children following a 12-week physical activity intervention without dietary intervention or intentional weight loss. Body fat and fat distribution were not prime mediators for the effect of the intervention on parameters of the Metabolic Syndrome; whereas insulin sensitivity was discovered to be a mediator of the change shown in ectopic fat depots. Causality and directionality of these fascinating relationships cannot be determined from the present study, and further research is encouraged. This thesis offers an insight into the pathogenesis of MetSyn and the use of physical activity to improve MetSyn in the setting of paediatric obesity.