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.

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.

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.

Hepatic steatosis is characterised by accumulation of triacylglycerols in the cytoplasm of hepatocytes at a level that reaches 5 to 40% of the weight of the liver. Its prevalence has increased worldwide, having a strong relationship with diabetes mellitus type 2 and obesity, diseases that constitute a global epidemic. Despite having been considered for a considerable time as a benign entity, it is known that a synergic combination with other factors, such as oxidative stress, can trigger a cascade of inflammatory reactions, leading to fibrogenesis, cirrhosis and hepatocellular carcinoma. Its etiology, despite not being totally elucidated, shows a strong correlation with insulin resistence, and is actually considered a hepatic manifestation of metabolic syndrome. However, it can be triggered by other factors; such as prolonged total parenteral nutrition, bariatric surgery, proteincalory malnutrition and the use of drugs. The actual prevalence is unknown but it affects adults and children, and is emerging as the most common chronic condition of the liver in the western world. Though an effective pharmacological treatment does not exist, all patients are encouraged to reduce their ingestion of fat and to practise regular physical exercise, with the objective of increasing their daily energy expenditure. Strategies of nutritional intervention and control of associated metabolic risk factors need to be evaluated. Aiming at contributing to the discussion of the problem, this dissertation presents two articles: a literature review of the main strategies of dietetic therapy in the treatment of steatosis, dealing with energy intake and the intake of macro- and micronutrients that exert effects on the prevention, treatment and/or retard the progression of the disease; the second article refers to an experimental study of weaned Wistar rats that received a diet with substitution of soy oil with an experimental oil rich in 9 triheptanoin, a triacylglycerol of heptanoic acid (7:0), that was being used in disorders of mitochondrial oxidative metabolism. The animals that received AIN-93 diets with 0, 30 and 50% of substitution of soy oil with experimental oil developed hepatic steatosis. The histological analysis revealed half the number of severe cases of hepatic steatosis in the animals with 50% substitution in relation to those with 0% substitution. Other variables demonstrated a potential protector effect of the experimental oil against hepatic steatosis as absolute and relative liver weight. Such an effect, associated with the clinical evidence of the success of triheptanoin with several mitochondrial disorders, awakes interest in the future of its application for other disorders. / Fundação de Amparo a Pesquisa do Estado de Alagoas / A esteatose hepática é caracterizada pelo acúmulo de triacilgliceróis no citoplasma dos hepatócitos a um nível que atinge 5 a 40% do peso do fígado. Sua prevalência tem aumentado em todo o mundo devido a forte relação com o diabetes mellitus tipo 2 e a obesidade, doenças que se constituem epidemias globais. Apesar de ter sido considerada por muito tempo como uma entidade benigna, sabe-se que a combinação sinérgica com outros fatores, como o estresse oxidativo, pode desencadear reações inflamatórias em cascata, levando à fibrogênese, cirrose e ao carcinoma hepatocelular. Sua etiologia, apesar de não estar totalmente elucidada, mostra forte correlação com a resistência à insulina, sendo, atualmente, considerada a manifestação hepática da síndrome metabólica. Entretanto, pode ser desencadeada por outros fatores, como nutrição parenteral total prolongada, cirurgia bariátrica, desnutrição calórico-protéica e o uso de drogas. Embora a prevalência verdadeira seja desconhecida, atinge adultos e crianças e está emergindo como a condição crônica do fígado mais comum no mundo ocidental. Apesar de ainda não existir tratamento farmacológico eficaz, todos os pacientes são encorajados a reduzir a ingestão de gorduras e praticar exercícios físicos regulares, com o objetivo de aumentar o gasto energético diário. Estratégias de intervenção nutricional e controle dos fatores de risco metabólico associados devem ser avaliadas. Visando contribuir com a discussão do problema, esta dissertação apresenta dois artigos: uma revisão da literatura com as principais estratégias da terapêutica dietética no tratamento da esteatose, tratando de oferta energética e aporte de macro e micronutrientes que exercem efeitos na prevenção, tratamento e/ou retardo da progressão da doença; o 7 segundo artigo refere-se a um estudo experimental com ratos Wistar recém desmamados, que receberam dieta com substituição do óleo de soja por um óleo experimental rico em trieptanoína, um triacilglicerol do ácido heptanóico (7:0), que vem sendo utilizado em desordens do metabolismo oxidativo mitocondrial. Os animais que receberam dietas AIN-93 com 0, 30 e 50% de substituição do óleo de soja pelo óleo experimental desenvolveram esteatose hepática. A análise histológica revelou metade do número de casos graves de esteatose hepática nos animais com 50% de substituição em relação àqueles submetidos a 0% de substituição. Outras variáveis evidenciaram potencial efeito protetor do óleo experimental contra a esteatose, como peso absoluto e relativo do fígado. Tal efeito, associado às evidências clínicas de sucesso do uso da trieptanoína em algumas desordens mitocondriais, desperta o interesse em investigações futuras de sua aplicação terapêutica em outras desordens.

Introdução: Em relação ao fígado, a esteatose é a consequência mais comum do consumo abusivo do etanol e predispõe à doença hepática mais grave. Os mecanismos da doença hepática alcoólica não são plenamente conhecidos e as terapias são escassas. Os objetivos desta tese foram: 1) averiguar os efeitos do etanol no fígado, utilizando o zebrafish como modelo experimental; 2) avaliar o tratamento com o Lactobacillus rhamnosus GG (LGG) na esteatose hepática; 3) observar os efeitos do etanol e do tratamento com o LGG no comportamento do zebrafish. Métodos: Foram realizados três experimentos utilizando peixes zebrafish, adultos, wildtype. O primeiro experimento foi formado por dois grupos, Controle (C) e Etanol (E), com 52 animais em cada um. O grupo E foi exposto a 0,5% de etanol por quatro semanas. Foram conduzidas análises histológicas e moleculares dos genes il-1b, tnf-α, il-10, sirt1, adiponectina e adipor2 nos fígados dos animais. No 2°experimento foram avaliados quatro grupos: Controle (C), Probiótico (P), Etanol (E) e Probiótico + Etanol (P + E), com 220 animais respectivamente. Durante quatro semanas os grupos P + E e P foram alimentados com ração com o probiótico LGG e os grupos E e C com ração sem probiótico. Foram realizadas análises histológicas e morfométricas no tecido hepático, quantificações de lipídeos séricos e hepáticos. No 3° experimento, os grupos C, E, P e P+E foram formados (n=15 animais por grupo) e, após duas semanas, o comportamento dos animais foi analisado no teste open-tank com o programa ANYmaze ®. Resultados: No 1° experimento os animais do grupo E apresentaram intensa esteatose hepática, aumento de glicogênio plasmático associado às gotículas lipídicas, alterações no retículo endoplasmático rugoso e degeneração de canalículos biliares. Houve acentuação na expressão hepática de il-1b, tnf-α, sirt1 e do adipor2, indicando que o etanol desencadeou resposta inflamatória e de proteção hepática. No 2° experimento, o grupo E apresentou intensa esteatose após quatro semanas, ao contrário do grupo P + E. A morfometria celular mostrou um aumento de 14,8 vezes no tamanho dos hepatócitos do grupo E (4° semana) quando comparado com C (p <0,0001). Os triglicerídeos séricos diminuíram no grupo P + E em comparação com C, P (p <0,001) e E (p = 0,004). O colesterol sérico do grupo P diminuiu comparado aos grupos C e E na segunda semana (p = 0,002 e p = 0,007) e do grupo P + E diminuiu comparado aos grupos E e C (p<0,0001), na quarta semana. As concentrações de triglicerídeos hepáticos reduziram no grupo P + E na quarta semana em comparação com E (p = 0,006). No 3° experimento, os animais expostos ao etanol apresentaram menor ansiedade em relação ao novo ambiente, evidenciada pela maior exploração da área superior do aquário. O efeito desibinidor do etanol não foi significativamente atenuado pelo tratamento com o LGG. Conclusões: Os resultados do primeiro estudo indicaram que o etanol desencadeia uma série de eventos celulares e moleculares e que a inflamação desempenha papel significativo na esteatose hepática. No segundo, foi demonstrado que o tratamento com LGG diminuiu os níveis séricos de triglicerídeos e de colesterol, atenuando a esteatose hepática. O terceiro estudo mostrou que o etanol teve efeito significativo no comportamento do zebrafish, que não foi modificado pelo LGG. / Introduction: Regarding to the liver, hepatic steatosis is the most common consequence of abusive alcohol consumption and predisposes to more severe liver disease. The mechanisms of alcoholic liver disease are not fully known and therapies are scarce. The objectives of this thesis were: 1) to verify the effects of ethanol in the liver using the zebrafish as an experimental model; 2) to evaluate a treatment with the probiotic Lactobacillus rhamnosus GG (LGG) in hepatic steatosis; 3) to observe the effects of ethanol and treatment with LGG in zebrafish behavior. Methods: Three experiments were performed using zebrafish, adult, wildtype. For the 1st trial, two groups were formed: Control (C) and Ethanol (E), with 52 animals in each group. E group was exposed to 0.5% ethanol during four weeks. Histological and molecular analysis of genes il-1b, tnf-α, il-10, sirt1, adiponectin, and adipor2 were conducted in zebrafish livers. For the 2nd trial, four groups were evaluated: Control (C), Probiotic (P), Ethanol (E) and Probiotic + Ethanol (P + E). During four weeks, the P + E and P groups were fed with food supplemented with LGG and E and C groups received food without probiotic. Histological and morphometric analysis in liver tissue, measurements of serum and hepatic lipids were performed. In the 3rd trial, C, E, P and P + E groups were formed and after two weeks, the animals' behavior was analyzed in opentank test with ANYmaze ® program. Results: In the 1st trial, animals in E group developed severe liver steatosis and cell abnormalities were observed: increase of glycogen associated to lipid droplets, alterations in the rough endoplasmic reticulum, degeneration of biliary canaliculi with presence of myelin figures inside. Increased hepatic expression of il-1b, tnf-α, sirt1 and adipor2 possibly indicates that ethanol triggered both inflammatory and hepatic protection responses. In the 2nd trial, E group presented severe steatosis after four weeks, in contrast to the E + P group. Cell morphometry showed a 14.8 fold in hepatocytes size of E (4th week) compared to C group (p <0.0001). Serum triglycerides decreased in the P + E group compared with C, P (p <0.001) and E groups (p = 0.004). Serum cholesterol decreased in P group compared to C and E groups at second week (p = 0.002 and p = 0.007) and in E + P group decreased compared with E and C groups (p<0.0001) at fourth week. Liver triglycerides were reduced in the P + E group at the fourth week compared to E group (p = 0.006). In the 3rd trial, there was an alteration in the behavior of animals exposed to ethanol compared to that nonexposed, an effect not significantly attenuated by treatment with LGG. Conclusions: Results of the first study indicate that ethanol triggers a series of cellular and molecular events and inflammation plays a significant role in hepatic steatosis. Then, it was shown that treatment with LGG decreased serum levels of triglycerides and cholesterol, attenuating hepatic steatosis. The third study showed that the ethanol, but not LGG has a significant effect on zebrafish behavior.

Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro / A restrição protéica neonatal promove alterações metabólicas, estruturais e morfológicas em diversos órgãos. O tipo de dieta pós-natal pode agravar esse quadro. Este trabalho teve como objetivo avaliar os efeitos da dieta com alta densidade energética (ADE) pós-natal sobre os parâmetros morfológicos hepáticos de ratos Wistar submetidos à restrição protéica neonatal. Fêmeas Wistar foram divididas em dois grupos nutricionais: normoprotéico (NP-19% proteínas) e com restrição protéica durante a gestação e primeira metade da lactação (LP-5% de proteínas). Ambas as dietas seguiram ecomendações da AIN-93 (Rhoster_). Ao desmame, os filhotes foram subdivididos em oito grupos, de acordo com o tipo de dieta pós-natal (SC - ração padrão ou HF dieta ADE): a) macho NP-SC; b) fêmea NP-SC; c) macho NP-HF; d) fêmea NP-HF; e) macho LPSC; f) fêmea LP-SC; g) macho LP-HF e h) fêmea LP-HF. A aferição da massa corporal e da pressão arterial foram feitas semanalmente. Aos 6 meses de idade, os animais sofreram eutanásia. O fígado foi retirado após perfusão e teve seu volume aferido pelo método de Scherle. Para fins estatísticos utilizou-se análise de variância e teste de comparações múltiplas de Neuman-Keuls (p<0,05). A dieta ADE foi eficaz ao induzir sobrepeso nos grupos NP-HF de ambos os sexos aos 77 dias (p<0,001). Aos 112 dias as fêmeas LP-HF apresentaram sobrepeso em relação às fêmeas LP (p<0,05) demonstrando que os fatores ambientais podem maximizar os efeitos da restrição protéica neonatal. Entretanto, em machos essa interação não foi observada. No que concerne às alterações hepáticas, a restrição protéica resultou em redução do número de hepatócitos em ambos os gêneros (p<0,01), com efeito adicional da dieta ADE apenas nos machos (p<0,01). Ademais, o insulto sofrido no período neonatal promoveu uma predisposição ao acúmulo de triglicerídeos hepáticos nos grupos RP (Vv = 15%), configurando um quadro de esteatose (p<0,01). A dieta ADE acentuou essa alteração, com os grupos RP-ADE alcançando níveis superiores a 33% de esteatose (p<0,001). Tanto a restrição protéica neonatal quanto a dieta ADE pós-desmame de forma isolada promoveram HAS leve aos 3 meses, redução do número de hepatócitos e esteatose grau 1 aos 6 meses de idade. Quando os dois estímulos foram aplicados simultaneamente, foi observada uma exacerbação do quadro hipertensivo, do déficit de hepatócitos e esteatose grau 2. Essas constatações ratificam a importância das condições intra-uterinas e da qualidade da dieta pós-natal na gênese de doenças crônicas. / Neonatal protein restriction has been associated with metabolic, structural and morphological adaptations in diverse organs. The type of postnatal diet can further exacerbate these outcomes. The present work aimed at evaluating the effects a postnatal hyperlipidic diet exerts on hepatic morphological parameters of rats subjected to perinatal protein restriction. Virgin female Wistar were divided into 2 groups: normal protein (NP) and low protein (LP). NP dams received standard diet for pregnant rats (AIN-93/Rhoster_- 19% protein) during the whole pregnancy and first half lactation, whereas LP dams had free access to na isocaloric diet lacking the recommended amount of protein (5% protein) for the same period. At weaning, offspring were assigned to one of the 8 nutritional groups according to the diet they will receive (SC standard chow or HF high fat): a) male NP-SC; b) female NP-SC; c) male NP-HF; d) female NP-HF; e) male LPSC; f) female LP-SC; g) male LP-HF e h) fêmea RP-ADE. Body mass and blood pressure were measured weekly until 6 months, when euthanasia occurred. Liver was carefully dissected and had its volume determined through Scherles method. Analysis of variance (ANOVA) and post-hoc test of Bonferroni were used to test differences among the groups (p<0,05). The HF diet provoked overweight in NP-HF groups from both genders on day 77 (p<0,001). However, LP-HFC groups showed a delayed response and sexual dimorphism, as only females developed overweight on day 112 (p<0,05). As for hepatic alterations, protein restriction resulted in hepatocyte deficit in both genders (p<0.01), existing an additional shift from HF diet exclusively in males (p<0.01). Furthermore, higher steatosis rates were found in protein restricted animals (Vv = 15%, p<0.01), having the post-weaning HF diet an additional effect on this outcome in LP-HF (Vv > 33%, p<0.001). Fetal programming and HF diet as single stimulus caused mild hypertension at 3 months, an important reduction in hepatocyte number as well as stage 1 steatosis at 6 months. However, when both stimuli were applied simultaneously, hypertension and hepatocyte number deficit were worsened and grade 2 steatosis occurred. All of these serve to highlight the paramount importance of intrauterine conditions and postnatal diet quality when it comes to the pathogenesis of chronic diseases.

Made available in DSpace on 2016-06-02T19:22:56Z (GMT). No. of bitstreams: 1 4481.pdf: 1042099 bytes, checksum: 3878e16d5c02d2ef0ac1ce99a2abeb33 (MD5) Previous issue date: 2011-09-16 / Financiadora de Estudos e Projetos / Estrogen deficiency is associated with increased oxidative stress (OS), which in turn is involved in the physiopathology of diseases such as fatty liver disease (NAFLD). Resistance training (RT) may reduce the oxidative damage by promoting an up-regulation in the antioxidant defence system. Therefore, this study aimed to assess the effects of RT on OS markers in the liver of ovariectomized rats (Ovx). Adult Sprague-Dawley rats were divided into four groups (n = 8 per group): sham-operated sedentary (Sham-Sed), ovariectomized sedentary (Sed-Ovx), sham-operated resistance training (Sham-Rt) and ovariectomized resistance training (Ovx-Rt). During a 10-week RT period, the animals climbed a 1.1 m vertical ladder with weights attached to their tails; the sessions were performed three times a week, with 4-9 climbs and 8-12 dynamic movements per climb. The OS was measured by levels of reduced glutathione (GSH) and oxidized glutathione (GSSG), the enzymatic activity of catalase (CAT) and superoxide dismutase (SOD), the lipid peroxidation (LP), the concentration of vitamin E and gene expression of glutathione peroxidase (GSH-Px). The estrogen deficiency associated with Ovx decreased in the GSH/GSSG ratio (28%), vitamin E concentration (45%), and gene expression of GSH-Px (49%). Moreover, the training induced negative changes in the hepatic anti-oxidative/oxidative balance, as evidenced by the increased TBARS levels (30%), as well as decreased GSH/GSSG ratio (19%) and vitamin E concentration (35%). These data indicate that the RT program adopted did not reverse the hepatic oxidative damage caused by Ovx; indeed, it increased hepatic OS. / A deficiência de estrogênio está associada ao aumento do estresse oxidativo (EO), que por sua vez, está envolvido na fisiopatologia de doenças como a esteatose hepática (EH). O treinamento resistido (TR) pode diminuir o dano oxidativo ao promover uma regulação ascendente no sistema de defesa antioxidante. Sendo assim, o objetivo do presente estudo foi verificar os efeitos do TR em marcadores do EO no fígado de ratas ovariectomizadas (Ovx). Ratas Sprague-Dawley adultas foram divididas em 4 grupos (n = 8 por grupo): sham operado sedentário (Sham-Sed), Ovx sedentário (Ovx-Sed), Sham-Tr e Ovx-Tr. Durante o período de 10 semanas, os animais escalaram uma escada vertical de 1,1 m com pesos atados em suas caudas. As sessões foram realizadas três vezes na semana, com 4-9 escaladas por sessão de treino. O EO foi avaliado por meio dos níveis de glutationa reduzida (GSH) e oxidada (GSSG), atividade enzimática da catalase (CAT) e superóxido dismutase (SOD), peroxidação lipídica (PL), concentração de vitamina E e expressão gênica da glutationa peroxidase (GSH-Px). A deficiência de estrogênio associada à Ovx diminuiu a relação (GSH/GSSG) (28%), concentração de vitamina E (44,7%), expressão gênica da GSH-Px (49%). Por sua vez, o TR induziu mudanças negativas no balanço antioxidativo/oxidativo hepático como evidenciado pelo aumento dos níveis de TBARS (30%), bem como diminuição da razão GSH/GSSG (19%) e concentração de vitamina E (35%). Estes dados indicaram que o treinamento resistido adotado não reverteu o dano oxidativo hepático causado pela OVx, e além disso, aumentou o estresse oxidativo hepático.

Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro / A restrição protéica neonatal promove alterações metabólicas, estruturais e morfológicas em diversos órgãos. O tipo de dieta pós-natal pode agravar esse quadro. Este trabalho teve como objetivo avaliar os efeitos da dieta com alta densidade energética (ADE) pós-natal sobre os parâmetros morfológicos hepáticos de ratos Wistar submetidos à restrição protéica neonatal. Fêmeas Wistar foram divididas em dois grupos nutricionais: normoprotéico (NP-19% proteínas) e com restrição protéica durante a gestação e primeira metade da lactação (LP-5% de proteínas). Ambas as dietas seguiram ecomendações da AIN-93 (Rhoster_). Ao desmame, os filhotes foram subdivididos em oito grupos, de acordo com o tipo de dieta pós-natal (SC - ração padrão ou HF dieta ADE): a) macho NP-SC; b) fêmea NP-SC; c) macho NP-HF; d) fêmea NP-HF; e) macho LPSC; f) fêmea LP-SC; g) macho LP-HF e h) fêmea LP-HF. A aferição da massa corporal e da pressão arterial foram feitas semanalmente. Aos 6 meses de idade, os animais sofreram eutanásia. O fígado foi retirado após perfusão e teve seu volume aferido pelo método de Scherle. Para fins estatísticos utilizou-se análise de variância e teste de comparações múltiplas de Neuman-Keuls (p<0,05). A dieta ADE foi eficaz ao induzir sobrepeso nos grupos NP-HF de ambos os sexos aos 77 dias (p<0,001). Aos 112 dias as fêmeas LP-HF apresentaram sobrepeso em relação às fêmeas LP (p<0,05) demonstrando que os fatores ambientais podem maximizar os efeitos da restrição protéica neonatal. Entretanto, em machos essa interação não foi observada. No que concerne às alterações hepáticas, a restrição protéica resultou em redução do número de hepatócitos em ambos os gêneros (p<0,01), com efeito adicional da dieta ADE apenas nos machos (p<0,01). Ademais, o insulto sofrido no período neonatal promoveu uma predisposição ao acúmulo de triglicerídeos hepáticos nos grupos RP (Vv = 15%), configurando um quadro de esteatose (p<0,01). A dieta ADE acentuou essa alteração, com os grupos RP-ADE alcançando níveis superiores a 33% de esteatose (p<0,001). Tanto a restrição protéica neonatal quanto a dieta ADE pós-desmame de forma isolada promoveram HAS leve aos 3 meses, redução do número de hepatócitos e esteatose grau 1 aos 6 meses de idade. Quando os dois estímulos foram aplicados simultaneamente, foi observada uma exacerbação do quadro hipertensivo, do déficit de hepatócitos e esteatose grau 2. Essas constatações ratificam a importância das condições intra-uterinas e da qualidade da dieta pós-natal na gênese de doenças crônicas. / Neonatal protein restriction has been associated with metabolic, structural and morphological adaptations in diverse organs. The type of postnatal diet can further exacerbate these outcomes. The present work aimed at evaluating the effects a postnatal hyperlipidic diet exerts on hepatic morphological parameters of rats subjected to perinatal protein restriction. Virgin female Wistar were divided into 2 groups: normal protein (NP) and low protein (LP). NP dams received standard diet for pregnant rats (AIN-93/Rhoster_- 19% protein) during the whole pregnancy and first half lactation, whereas LP dams had free access to na isocaloric diet lacking the recommended amount of protein (5% protein) for the same period. At weaning, offspring were assigned to one of the 8 nutritional groups according to the diet they will receive (SC standard chow or HF high fat): a) male NP-SC; b) female NP-SC; c) male NP-HF; d) female NP-HF; e) male LPSC; f) female LP-SC; g) male LP-HF e h) fêmea RP-ADE. Body mass and blood pressure were measured weekly until 6 months, when euthanasia occurred. Liver was carefully dissected and had its volume determined through Scherles method. Analysis of variance (ANOVA) and post-hoc test of Bonferroni were used to test differences among the groups (p<0,05). The HF diet provoked overweight in NP-HF groups from both genders on day 77 (p<0,001). However, LP-HFC groups showed a delayed response and sexual dimorphism, as only females developed overweight on day 112 (p<0,05). As for hepatic alterations, protein restriction resulted in hepatocyte deficit in both genders (p<0.01), existing an additional shift from HF diet exclusively in males (p<0.01). Furthermore, higher steatosis rates were found in protein restricted animals (Vv = 15%, p<0.01), having the post-weaning HF diet an additional effect on this outcome in LP-HF (Vv > 33%, p<0.001). Fetal programming and HF diet as single stimulus caused mild hypertension at 3 months, an important reduction in hepatocyte number as well as stage 1 steatosis at 6 months. However, when both stimuli were applied simultaneously, hypertension and hepatocyte number deficit were worsened and grade 2 steatosis occurred. All of these serve to highlight the paramount importance of intrauterine conditions and postnatal diet quality when it comes to the pathogenesis of chronic diseases.

Introdução: Em relação ao fígado, a esteatose é a consequência mais comum do consumo abusivo do etanol e predispõe à doença hepática mais grave. Os mecanismos da doença hepática alcoólica não são plenamente conhecidos e as terapias são escassas. Os objetivos desta tese foram: 1) averiguar os efeitos do etanol no fígado, utilizando o zebrafish como modelo experimental; 2) avaliar o tratamento com o Lactobacillus rhamnosus GG (LGG) na esteatose hepática; 3) observar os efeitos do etanol e do tratamento com o LGG no comportamento do zebrafish. Métodos: Foram realizados três experimentos utilizando peixes zebrafish, adultos, wildtype. O primeiro experimento foi formado por dois grupos, Controle (C) e Etanol (E), com 52 animais em cada um. O grupo E foi exposto a 0,5% de etanol por quatro semanas. Foram conduzidas análises histológicas e moleculares dos genes il-1b, tnf-α, il-10, sirt1, adiponectina e adipor2 nos fígados dos animais. No 2°experimento foram avaliados quatro grupos: Controle (C), Probiótico (P), Etanol (E) e Probiótico + Etanol (P + E), com 220 animais respectivamente. Durante quatro semanas os grupos P + E e P foram alimentados com ração com o probiótico LGG e os grupos E e C com ração sem probiótico. Foram realizadas análises histológicas e morfométricas no tecido hepático, quantificações de lipídeos séricos e hepáticos. No 3° experimento, os grupos C, E, P e P+E foram formados (n=15 animais por grupo) e, após duas semanas, o comportamento dos animais foi analisado no teste open-tank com o programa ANYmaze ®. Resultados: No 1° experimento os animais do grupo E apresentaram intensa esteatose hepática, aumento de glicogênio plasmático associado às gotículas lipídicas, alterações no retículo endoplasmático rugoso e degeneração de canalículos biliares. Houve acentuação na expressão hepática de il-1b, tnf-α, sirt1 e do adipor2, indicando que o etanol desencadeou resposta inflamatória e de proteção hepática. No 2° experimento, o grupo E apresentou intensa esteatose após quatro semanas, ao contrário do grupo P + E. A morfometria celular mostrou um aumento de 14,8 vezes no tamanho dos hepatócitos do grupo E (4° semana) quando comparado com C (p <0,0001). Os triglicerídeos séricos diminuíram no grupo P + E em comparação com C, P (p <0,001) e E (p = 0,004). O colesterol sérico do grupo P diminuiu comparado aos grupos C e E na segunda semana (p = 0,002 e p = 0,007) e do grupo P + E diminuiu comparado aos grupos E e C (p<0,0001), na quarta semana. As concentrações de triglicerídeos hepáticos reduziram no grupo P + E na quarta semana em comparação com E (p = 0,006). No 3° experimento, os animais expostos ao etanol apresentaram menor ansiedade em relação ao novo ambiente, evidenciada pela maior exploração da área superior do aquário. O efeito desibinidor do etanol não foi significativamente atenuado pelo tratamento com o LGG. Conclusões: Os resultados do primeiro estudo indicaram que o etanol desencadeia uma série de eventos celulares e moleculares e que a inflamação desempenha papel significativo na esteatose hepática. No segundo, foi demonstrado que o tratamento com LGG diminuiu os níveis séricos de triglicerídeos e de colesterol, atenuando a esteatose hepática. O terceiro estudo mostrou que o etanol teve efeito significativo no comportamento do zebrafish, que não foi modificado pelo LGG. / Introduction: Regarding to the liver, hepatic steatosis is the most common consequence of abusive alcohol consumption and predisposes to more severe liver disease. The mechanisms of alcoholic liver disease are not fully known and therapies are scarce. The objectives of this thesis were: 1) to verify the effects of ethanol in the liver using the zebrafish as an experimental model; 2) to evaluate a treatment with the probiotic Lactobacillus rhamnosus GG (LGG) in hepatic steatosis; 3) to observe the effects of ethanol and treatment with LGG in zebrafish behavior. Methods: Three experiments were performed using zebrafish, adult, wildtype. For the 1st trial, two groups were formed: Control (C) and Ethanol (E), with 52 animals in each group. E group was exposed to 0.5% ethanol during four weeks. Histological and molecular analysis of genes il-1b, tnf-α, il-10, sirt1, adiponectin, and adipor2 were conducted in zebrafish livers. For the 2nd trial, four groups were evaluated: Control (C), Probiotic (P), Ethanol (E) and Probiotic + Ethanol (P + E). During four weeks, the P + E and P groups were fed with food supplemented with LGG and E and C groups received food without probiotic. Histological and morphometric analysis in liver tissue, measurements of serum and hepatic lipids were performed. In the 3rd trial, C, E, P and P + E groups were formed and after two weeks, the animals' behavior was analyzed in opentank test with ANYmaze ® program. Results: In the 1st trial, animals in E group developed severe liver steatosis and cell abnormalities were observed: increase of glycogen associated to lipid droplets, alterations in the rough endoplasmic reticulum, degeneration of biliary canaliculi with presence of myelin figures inside. Increased hepatic expression of il-1b, tnf-α, sirt1 and adipor2 possibly indicates that ethanol triggered both inflammatory and hepatic protection responses. In the 2nd trial, E group presented severe steatosis after four weeks, in contrast to the E + P group. Cell morphometry showed a 14.8 fold in hepatocytes size of E (4th week) compared to C group (p <0.0001). Serum triglycerides decreased in the P + E group compared with C, P (p <0.001) and E groups (p = 0.004). Serum cholesterol decreased in P group compared to C and E groups at second week (p = 0.002 and p = 0.007) and in E + P group decreased compared with E and C groups (p<0.0001) at fourth week. Liver triglycerides were reduced in the P + E group at the fourth week compared to E group (p = 0.006). In the 3rd trial, there was an alteration in the behavior of animals exposed to ethanol compared to that nonexposed, an effect not significantly attenuated by treatment with LGG. Conclusions: Results of the first study indicate that ethanol triggers a series of cellular and molecular events and inflammation plays a significant role in hepatic steatosis. Then, it was shown that treatment with LGG decreased serum levels of triglycerides and cholesterol, attenuating hepatic steatosis. The third study showed that the ethanol, but not LGG has a significant effect on zebrafish behavior.

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.