Orientadores: Mariana Porto Zambon, Bruno Geloneze Neto / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-23T12:05:33Z (GMT). No. of bitstreams: 1 Silva_ClelianideCassiada_M.pdf: 3445763 bytes, checksum: 0e769f408904fd12add084d02186f8fd (MD5) Previous issue date: 2013 / Resumo: Objetivo: Avaliar a relação do índice TyG com componentes da síndrome metabólica e outros marcadores antropométricos e bioquímicos relacionados a risco cardiometabólico, e como indicador de resistência à insulina pelo clamp hiperglicêmico em adolescentes com diferentes níveis de adiposidade e estadios puberais. Materiais e métodos: Estudo transversal multicêntrico. Avaliou-se 835 adolescentes de 10-19 anos (489 meninas, média de 15,1 ± 2,4 anos e 346 meninos, média de 14,7 ± 2,4 anos). Realizou-se avaliação antropométrica, de composição corporal e bioquímica. O estadio puberal foi obtido por meio de autoavaliação, e a pressão arterial pelo método auscultatório. A resistência à insulina foi avaliada pelos índices TyG e HOMA2-IR, e pelo clamp hiperglicêmico (n = 42). A correlação entre duas variáveis foi avaliada pelo coeficiente de correlação parcial com ajuste para o estadio puberal. Na análise entre o índice TyG e o clamp hiperglicêmico foram analisados: qui-quadrado (X2), Kappa (k), curva ROC (Receiver Operating Characteristic), áreas abaixo das curvas (AUC), intervalos de confiança de 95% (IC95%) e determinado o ponto de corte para o índice TyG diagnosticar resistência à insulina. Resultados: Em ambos os sexos, o índice TyG correlacionou-se de forma positiva com circunferência da cintura (r = 0,30 feminino e 0,41 masculino; p<0,001), pressão arterial sistólica (r = 0,18; p<0,001 feminino e r = 0,13; p = 0,03 masculino) e diastólica (r = 0,20; p<0,001 feminino e r = 0,16; p = 0,005 masculino) e com o índice HOMA2-IR (r = 0,36 feminino e r = 0,45 masculino; p<0,001), e negativa com HDL-colesterol (r = -0,21 feminino e r = -0,41 masculino; p<0,001). Observou-se correlação inversa entre o clamp com o índice TyG (r = -0,55; p<0,001) e com o índice HOMA2-IR (r = -0,33; p = 0,03). Observou-se associação significante entre a presença de sensibilidade à insulina diminuída avaliada pelo clamp e a resistência à insulina aumentada segundo o índice TyG (X2 = 6,7; p = 0,009). O teste Kappa mostrou concordância satisfatória entre o índice TyG e a sensibilidade à insulina obtida a partir do clamp (k = 0,4; p = 0,009). O índice TyG mostrou bom desempenho para identificar resistência à insulina (AUC = 0,72; p = 0,02). Considerando os pacientes com índice TyG acima do percentil 90 como indivíduos resistentes à insulina, encontrou-se um valor limite para resistência à insulina de 4,47. Conclusão: O índice TyG é um instrumento válido na estimativa do grau da resistência à insulina e alterações nos componentes da síndrome metabólica e nos indicadores de risco cardiometabólico em adolescentes, tendo sido validado diante do clamp hiperglicêmico / Abstract: Objective: To evaluate the relationship of the TyG index with the metabolic syndrome components and other anthropometric and biochemical markers related to cardiometabolic risk, and as an indicator of insulin resistance by hyperglycemic clamp in adolescents with different levels of adiposity and pubertal stages. Materials and methods: A cross-sectional multicenter study. There were evaluated 835 adolescents aged 10-19 years (489 girls, mean 15.1 ± 2.4 years and 346 boys, mean 14.7 ± 2.4 years). There were performed anthropometric, body composition and biochemistry measurements. The pubertal stage was obtained through self-assessment, and blood pressure by auscultation method. The insulin resistance was assessed by TyG index, HOMA2-IR and hyperglycemic clamp (n = 42). The correlation between two variables was assessed by partial correlation coefficient adjusted for pubertal stage. In the analysis between the TyG index and the hyperglycemic clamp, there were analyzed: chi-square (X2), Kappa (k), ROC (Receiver Operating Characteristic) curve, areas under the curve (AUC), confidence intervals of 95% (CI95%) and determined the cut-off point for the TyG index to diagnose insulin resistance. Results: In both genders, the TyG index correlated positively with waist circumference (r = 0.30 female and r = 0.41 male; p<0.001), systolic blood pressure (r = 0.18, p<0.001 female and r = 0.13, p = 0.03 male) and diastolic (r = 0.20, p<0.001 female and r = 0.16, p = 0.005 male) and with the HOMA2-IR index (r = 0.36 female and r = 0.45 male, p<0.001), and negatively with HDL-cholesterol (r = -0.21 female and r = -0.41 male, p<0.001). There were observed inverse correlation between the clamp with TyG index (r = -0.55, p<0.001) and with the HOMA2-IR index (r = -0.33, p = 0.03). There was observed significant association between the presence of decreased insulin sensitivity measured by clamp and increased insulin resistance according to the TyG index (X2 = 6.7, p = 0.009). The Kappa test showed satisfactory agreement between the TyG index and insulin sensitivity obtained from the clamp (k = 0.4, p = 0.009). The TyG index showed good performance to identify insulin resistance (AUC = 0.72, p = 0.02). Considering patients with TyG index above the 90th percentile as insulin-resistant subjects, there was found a threshold value for insulin resistance of 4.47. Conclusion: The TyG index is a valid tool to estimate the degree of insulin resistance and changes in the metabolic syndrome components and cardiometabolic risk factors in adolescents, having been validated with the hyperglycemic clamp / Mestrado / Saude da Criança e do Adolescente / Mestra em Ciências

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22362号 / 医博第4603号 / 新制||医||1043(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 平井 豊博, 教授 稲垣 暢也, 教授 竹内 理 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Introduction: Diabetes Mellitus is a metabolic disorder that affects a person’s ability to either produce insulin (Type I diabetes mellitus) or properly use insulin (Type II diabetes mellitus) in order to maintain adequate blood glucose levels. The most severe diabetic complications arise due to hyperglycemia – a state of extremely high blood glucose levels – such as, coronary artery disease (CAD), in which coronary stent therapy is a popular method of treatment. However, research has shown a high rate of in-stent restenosis in diabetic patients with CAD, most likely due to activation of cellular adhesion molecules on endothelial cells exposed to the hyperglycemic environment. Blood vessel mimics (BVMs) have been researched as viable options for in vitro studies on vascular stents; thus, it would be beneficial to create an in vitro diabetic BVM for stent manufactures to evaluate and determine the root cause of the high failure rate of stents in the diabetic population. In addition, a diabetic BVM would help manufactures optimize coatings or stent configurations for diabetic patients. Methods: The purpose of this thesis was to take the initial steps towards the goal of a diabetic BVM. The first aim was to establish a procedure of developing glycemic cell media solutions of various glucose concentrations, and to establish a feasible method of monitoring the glucose concentration of the solutions. Glycemic cell media solutions were developed and their glucose concentrations were evaluated with a blood glucose meter (specifically, the Aviva Accu-Chek blood glucose meter) or visual blood glucose test strips (Glucoflex R visual blood glucose test strips). The second aim was to ensure that the developed glycemic cell media solutions could be monitored in a cell culture environment over time, and to determine if the hyperglycemic conditions induced any change to endothelial cells. Bovine aortic endothelial cells (BAECs) and human umbilical vein endothelial cells (HUVECs) were used to evaluate glucose consumption and cell morphology. Glucose concentration of the cell media was recorded to evaluate glucose consumption, and the cells were evaluated under a microscope in order to determine cell morphology and an increase in cell death. Results & Conclusions: Data accumulated from the first set of experiments confirmed that glycemic cell media solutions can be developed by adding Sigma G6512 D-(+)-glucose to base cell media. Additionally, the Aviva Accu-Chek blood glucose meter recorded the most accurate and precise glucose concentrations of the various glycemic cell media solutions compared to the Glucoflex-R blood glucose visual test strips. Lastly, the series of experiments with BAECs and HUVECs confirmed that the glycemic cell media solutions could be effectively monitored over time, and that these conditions evoked higher glucose consumption by the endothelial cells compared to the normal glycemic cell media solutions. Additionally, neither glycemic environment evoked significant cell death. These results met the aims of this thesis, and therefore provide the foundation for further development of a diabetic BVM.

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Résumé : La néphropathie diabétique (ND) est une complication microvasculaire du diabète évoluant ultimement en insuffisance rénale et l’hyperglycémie est connue comme étant l’un des facteurs de risques. De larges études cliniques, tel que le DCCT et l’UKPDS, ont montré que si le contrôle intensif de la glycémie se faisait de façon précoce, il serait possible de retarder le développement de la ND. Cependant, les résultats de l'EDIC montrent que si ce contrôle intensif se faisait plus tardivement, suite à une période d’hyperglycémie, il n’empêcherait plus sa progression. Les podocytes ont un rôle critique dans le maintien des fonctions rénales et leur apoptose corrèle de façon très spécifique avec la progression de la ND. Récemment, nous avons rapporté que SHP-1, une protéine tyrosine phosphatase, était augmentée en concentrations élevées de glucose (HG), menant à une inhibition des voies de signalisation de l'insuline. Notre hypothèse est que l’augmentation de l’expression de SHP-1 causée par l’hyperglycémie persiste même après réduction des niveaux de glucose, phénomène de mémoire hyperglycémique, causant une résistance à l'insuline, la mort des podocytes et une absence de réversibilité liée à la progression de ND. Les résultats in vivo montrent que la fonction et la pathologie rénale continuent de progresser et ce en dépit de la normalisation des niveaux de glucose avec implants d’insuline de 5 à 7 mois d’âge La progression de la pathologie corrèle avec le maintien de l’augmentation de l’expression de SHP-1, contribuant au maintien de l’inhibition des voies de l’insuline. En culture, des podocytes murins exposés en HG pendant 96 h et ensuite exposés en condition normale de glucose(NG) pour les dernières 24 h montrent une persistance de l’inhibition des voies de signalisation de l’insuline qui corrèle avec l’augmentation persistante de l’expression et l’activité phosphatase de SHP-1. L’activité des caspases 3/7 dans les podocytes est plus élevée lorsque ceux-ci sont exposés en HG qu’en NG. Le retour en NG pour les dernières 24 h n’a aucun effet bénéfique à réduire l’activité des caspases 3/7. Finalement, l’analyse épigénétique a été suggérée comme étant une explication du phénomène de mémoire hyperglycémique. La monométhylation de la lysine 4 de l’histone 3 (H3K4me1), un marqueur d’activation génique, est augmentée sur le promoteur de SHP-1 en HG et demeure élevée malgré le retour en NG pendant les dernières 24 h. En conclusion, l’hyperglycémie engendre une augmentation persistante de SHP-1 due possiblement à des modifications épigénétiques, causant le maintien de l’inhibition les voies de signalisation de l’insuline même après un retour à des niveaux normaux de glucose, contribuant à la progression de la ND. / Abstract : Diabetic nephropathy (DN) is the leading cause of end-stage renal disease. Renal podocytes apoptosis induced by hyperglycemia is an early event of DN. Clinical studies have shown that intensive blood glucose control reduced the development of DN but is not sufficient, if started late, to prevent its progression, introducing the concept of “hyperglycemic memory”. We have recently published that the tyrosine phosphatase SHP-1 is elevated in renal cortex of type 1 diabetic mice (Akita), contributing to insulin unresponsiveness and DN. We hypothesized that SHP-1 expression remains elevated regardless of systemic blood glucose normalization, and is responsible for hyperglycemic memory in podocytes leading to DN progression. In vivo contribution of SHP-1 in hyperglycemic memory was evaluated using Akita mice treated with insulin implants after 4 months of diabetes. Both urinary albuminuria and glomerular filtration rate were significantly increased in diabetic mice compared to non-diabetic mice and remained elevated despite normalization of blood glucose levels. Renal dysfunction was associated with a persistent increase of SHP-1 expression in renal cortex and inhibition of insulin action that were not normalized following insulin implants. Mouse podocytes were cultured in normal (5.6mM; NG), high glucose concentrations (25mM; HG) for 120 h or HG (96 h) followed by NG for an additional 24 h (HG+NG). We observed that Akt and ERK phosphorylation induced by insulin was inhibited in HG and were not restored despite returning glucose level to 5.6 mM after the HG period. This inhibition was associated with persistent increase of SHP-1 expression and phosphatase activity, leading to insulin signaling pathway inhibition. Moreover, caspase 3/7 activity in podocytes exposed to HG was higher than in podocytes cultured in NG and returning glucose concentrations to normal range for the last 24 h after the 96 h HG exposure had no effect on reducing caspase 3/7 activity. Epigenetic changes were studied to explain the hyperglycemic memory effect. On SHP-1 promoter, H3K4me1 levels, an activation mark, tended to be more elevated in podocytes exposed to HG and were maintained despite returning to NG levels after the HG conditions. In conclusion, hyperglycemia induces persistent and epigenetic changes of SHP-1 causing insulin unresponsiveness in the podocytes contributing to DN progression.

Contextualização: As complicações agudas do diabetes, embora em grande parte evitáveis, apresentavam considerável mortalidade em diversas localidades do mundo no século passado. No Brasil, a organização do Sistema Único de Saúde pode ter resultado em importante queda na mortalidade por esta causa. Objetivos: Descrever a mortalidade por complicações agudas do diabetes no Brasil entre 1991 e 2010. Métodos: Os óbitos declarados no Sistema de Informações sobre Mortalidade por complicações agudas do diabetes (CID-9 249 e 250, seguidos pelos dígitos 1, 2 ou 3, e CID-10 E10 a E14, seguidos pelos dígitos 0 ou 1) foram corrigidos para causas mal definidas e sub-registro. A partir da população obtida do Instituto Brasileiro de Geografia e Estatística, foram calculadas taxas de mortalidade padronizadas de acordo com a população mundial. Correlações lineares foram realizadas para descrever a relação entre mortalidade e idade, e regressões Joinpoint foram utilizadas para descrever tendências. Resultados: Houve queda de 70,9% na mortalidade por complicações agudas do diabetes no Brasil entre 1991 e 2010, de 8,42 para 2,45 óbitos por 100.000 habitantes. A redução ocorreu em ambos os sexos, todas as faixas etárias, todas as regiões e quase todas as unidades federativas. O declínio foi menor nos últimos anos, quando as taxas já estavam bem mais baixas. A mortalidade aumentou exponencialmente com a idade e foi maior nas regiões Norte e Nordeste. Conclusões: A marcante redução na mortalidade por complicações agudas do diabetes no Brasil nas últimas duas décadas indica que a cobertura ampla e gratuita adotada pelo sistema nacional de saúde do Brasil, com disponibilização de insulina e organização do cuidado, foi capaz de reduzir substancialmente as complicações agudas dessa doença. Entretanto, considerando especialmente as iniquidades regionais existentes, ainda há espaço para redução na mortalidade por essas complicações no Brasil. / Background: Acute complications of diabetes, though largely preventable, presented considerable mortality in various locations around the world in the 20th Century. In Brazil, the organization of the national health system may have resulted in an important decline in this cause of mortality. Objectives: To describe mortality rates from acute complications of diabetes in Brazil from 1991 to 2010. Methods: The deaths reported in the Mortality Information System for acute complications of diabetes (ICD-9 249 and 250, followed by the digits 1, 2 or 3, and ICD-10 E10 to E14, followed by the digits 0 or 1) were corrected for ill-defined and under-reporting. Using the population obtained from national censuses, we calculated mortality rates standardized to the world population. Linear correlations were performed to describe the relationship between mortality and age, and Joinpoint regressions were used to characterize trends. Results: Mortality from acute complications of diabetes decreased 70.9%, from 8.42 to 2.45 deaths / 100000 inhabitants, in Brazil from 1991 to 2010. The reduction occurred in both sexes, all ages, all regions and almost all states. The decline was less marked in recent years. Mortality rates increased exponentially with age and were higher in the North and Northeast regions. Conclusions: The marked reduction in mortality from acute complications of diabetes in Brazil over the last two decades suggests that the universal coverage adopted by the national health system of Brazil, provided without charge and in an increasingly organized fashion, coupled with greater availability of insulin, was able to substantially reduce deaths due to the acute complications of diabetes. However, especially considering regional inequities, much room still exists for further reduction in mortality from these complications in Brazil.

Glycemic intake influences the rise in blood glucose concentration following consumption of a carbohydrate containing meal, known as the postprandial glycemic response. The glycemic response is a result of both the type and amount of carbohydrate foods consumed and is commonly measured as the glycemic index (GI) or glycemic load (GL), where the GI is a ranking in comparison to glucose and the GL is an absolute value encompassing both the GI and amount of carbohydrate consumed. Evidence from controlled trials in rat models suggests that glycemic intake has a role in development of insulin resistance, however trials and observational studies of humans have produced conflicting results. As insulin resistance is a precursor to type 2 diabetes mellitus, lifestyle factors that could prevent development of this condition have important public health implications. Previous observational studies have used food frequency questionnaires to assess usual diet, which could have resulted in a lack of precision in assessment of individual serve sizes, and have been limited to daily measures of glycemic intake. Daily measures do not take fluctuations in glycemic intake on a per meal basis into account, which may be a more relevant measure for investigation in relation to disease outcomes. This PhD research was conducted in a group of Brisbane women aged 42 to 81 years participating in the multidisciplinary Brisbane Longitudinal Assessment of Ageing in Women (LAW study). Older women may be at particular risk of insulin resistance due to age, hormonal changes, and increases in abdominal obesity associated with menopause, and the LAW study provided an ideal opportunity to study the relationship between diet and insulin resistance. Using the diet history tool, we aimed to assess the glycemic intake of the population and hypothesised that daily GI and daily GL would be significantly positively associated with increased odds of insulin resistant status. We also hypothesised that a new glycemic measure representing peaks in GL at different meals would be a stronger predictor of insulin resistant status than daily measures, and that a specially designed questionnaire would be an accurate and repeatable dietary tool for assessment of glycemic intake. To address these hypotheses, we conducted a series of studies. To assess glycemic intake, information on usual diet was obtained by detailed diet history interview and analysed using Foodworks and the Australian Food and Nutrient (AUSNUT) database, combined with a customised GI database. Mean ± SD intakes were 55.6 ± 4.4% for daily GI and 115 ± 25 for daily GL (n=470), with intake higher amoung younger participants. Bread was the largest contributor to intakes of daily GI and GL (17.1% and 20.8%, respectively), followed by fruit (15.5% and 14.2%, respectively). To determine whether daily GI and GL were significantly associated with insulin resistance, the homeostasis model assessment of insulin resistance (HOMA) was used to assess insulin resistant status. Daily GL was significantly higher in subjects who were insulin resistant compared to those who were not (134 ± 33 versus 114 ± 24 respectively, P<0.001) (n=329); the odds of subjects in the highest tertile of GL intake being insulin resistant were 12.7 times higher when compared with the lowest tertile of GL (95% CI 1.6-100.1, P=0.02). Daily GI was not significantly different in subjects who were insulin resistant compared to those who were not (56.0 ± 3.3% versus 55.7 ± 4.5%, P=0.69). To evaluate whether a new glycemic measure representing fluctuations in daily glycemic intake would be a stronger predictor of insulin resistant status than other glycemic intake measures, the GL peak score was developed to express in a single value the magnitude of GL peaks during an average day. Although a significant relationship was seen between insulin resistant status and GL peak score (Nagelkerke’s R2=0.568, P=0.039), other glycemic intake measures of daily GL (R2=0.671, P<0.001) and daily GL per megajoule (R2=0.674, P<0.001) were stronger predictors of insulin resistant status. To develop an accurate and repeatable self-administered tool for assessment of glycemic intake, two sub-samples of women (n=44 for the validation study and n=52 for the reproducibility study) completed a semi-quantitative questionnaire that contained 23 food groupings selected to include the top 100 carbohydrate foods consumed by the study population. While there were significant correlations between the glycemic intake questionnaire and the diet history for GL (r=0.54, P<0.01), carbohydrate (r=0.57, P<0.01) and GI (r=0.40, P<0.01), Bland-Altman plots showed an unacceptable difference between individual intakes in 34% of subjects for daily GL and carbohydrate, and 41% for daily GI. Reproducibility results showed significant correlations for daily GL (r=0.73, P<0.001), carbohydrate (r=0.76, P<0.001) and daily GI (r=0.64, P<0.001), but an unacceptable difference between individual intakes in 25% of subjects for daily GL and carbohydrate, and 27% for daily GI. In summary, our findings show that a significant association was observed between daily glycemic load and insulin resistant status in a group of older women, using a diet history interview to obtain precise estimation of individual carbohydrate intake. Both the type and quantity of carbohydrate are important to consider when investigating relationships between diet and insulin resistance, although our results suggest the association is more closely related to overall daily glycemic intake than individual meal intake variations. A dietary tool that permits precise estimation of carbohydrate intake is essential when evaluating possible associations between glycemic intake and individual risk of chronic diseases such as insulin resistance. Our results also suggest that studies using questionnaires to estimate glycemic intake should state degree of agreement as well as correlation coefficients when evaluating validity, as imprecise estimates of carbohydrate at an individual level may have contributed to the conflicting findings reported in previous studies.

Contextualização: As complicações agudas do diabetes, embora em grande parte evitáveis, apresentavam considerável mortalidade em diversas localidades do mundo no século passado. No Brasil, a organização do Sistema Único de Saúde pode ter resultado em importante queda na mortalidade por esta causa. Objetivos: Descrever a mortalidade por complicações agudas do diabetes no Brasil entre 1991 e 2010. Métodos: Os óbitos declarados no Sistema de Informações sobre Mortalidade por complicações agudas do diabetes (CID-9 249 e 250, seguidos pelos dígitos 1, 2 ou 3, e CID-10 E10 a E14, seguidos pelos dígitos 0 ou 1) foram corrigidos para causas mal definidas e sub-registro. A partir da população obtida do Instituto Brasileiro de Geografia e Estatística, foram calculadas taxas de mortalidade padronizadas de acordo com a população mundial. Correlações lineares foram realizadas para descrever a relação entre mortalidade e idade, e regressões Joinpoint foram utilizadas para descrever tendências. Resultados: Houve queda de 70,9% na mortalidade por complicações agudas do diabetes no Brasil entre 1991 e 2010, de 8,42 para 2,45 óbitos por 100.000 habitantes. A redução ocorreu em ambos os sexos, todas as faixas etárias, todas as regiões e quase todas as unidades federativas. O declínio foi menor nos últimos anos, quando as taxas já estavam bem mais baixas. A mortalidade aumentou exponencialmente com a idade e foi maior nas regiões Norte e Nordeste. Conclusões: A marcante redução na mortalidade por complicações agudas do diabetes no Brasil nas últimas duas décadas indica que a cobertura ampla e gratuita adotada pelo sistema nacional de saúde do Brasil, com disponibilização de insulina e organização do cuidado, foi capaz de reduzir substancialmente as complicações agudas dessa doença. Entretanto, considerando especialmente as iniquidades regionais existentes, ainda há espaço para redução na mortalidade por essas complicações no Brasil. / Background: Acute complications of diabetes, though largely preventable, presented considerable mortality in various locations around the world in the 20th Century. In Brazil, the organization of the national health system may have resulted in an important decline in this cause of mortality. Objectives: To describe mortality rates from acute complications of diabetes in Brazil from 1991 to 2010. Methods: The deaths reported in the Mortality Information System for acute complications of diabetes (ICD-9 249 and 250, followed by the digits 1, 2 or 3, and ICD-10 E10 to E14, followed by the digits 0 or 1) were corrected for ill-defined and under-reporting. Using the population obtained from national censuses, we calculated mortality rates standardized to the world population. Linear correlations were performed to describe the relationship between mortality and age, and Joinpoint regressions were used to characterize trends. Results: Mortality from acute complications of diabetes decreased 70.9%, from 8.42 to 2.45 deaths / 100000 inhabitants, in Brazil from 1991 to 2010. The reduction occurred in both sexes, all ages, all regions and almost all states. The decline was less marked in recent years. Mortality rates increased exponentially with age and were higher in the North and Northeast regions. Conclusions: The marked reduction in mortality from acute complications of diabetes in Brazil over the last two decades suggests that the universal coverage adopted by the national health system of Brazil, provided without charge and in an increasingly organized fashion, coupled with greater availability of insulin, was able to substantially reduce deaths due to the acute complications of diabetes. However, especially considering regional inequities, much room still exists for further reduction in mortality from these complications in Brazil.

Submitted by Simone Souza (simonecgsouza@hotmail.com) on 2017-09-15T15:03:15Z No. of bitstreams: 1 DISS_2014_Larissa Finger.pdf: 952368 bytes, checksum: ce15137e3eda929260a105c871adbf0a (MD5) / Approved for entry into archive by Jordan (jordanbiblio@gmail.com) on 2017-09-19T13:39:00Z (GMT) No. of bitstreams: 1 DISS_2014_Larissa Finger.pdf: 952368 bytes, checksum: ce15137e3eda929260a105c871adbf0a (MD5) / Made available in DSpace on 2017-09-19T13:39:00Z (GMT). No. of bitstreams: 1 DISS_2014_Larissa Finger.pdf: 952368 bytes, checksum: ce15137e3eda929260a105c871adbf0a (MD5) Previous issue date: 2014-08-29 / CAPES / A redução na ingestão de proteínas e aumento na ingestão de carboidratos perfaz um padrão alimentar muito presente no estilo de vida atual da população mundial e está associado à incidência de patologias tais como diabetes mellitus, obesidade, hipertensão, entre outras. A ingestão de quantidades insuficientes de proteínas também está associada ao desenvolvimento de estresse oxidativo, o que contribui para o estabelecimento de lesões teciduais que podem culminar em prejuízo funcional de diversos órgãos. Diante destes fatos, o nosso objetivo foi investigar os possíveis danos renais decorrentes da administração da dieta hipoproteica-hiperglicídica a ratos no início da fase de crescimento. Ratos Wistar machos (~30 dias e 100g) foram divididos nos grupos: 1) Controle, alimentado com uma dieta com 17% de proteína e 63% de carboidrato por 15 (C15) ou 45 dias (C45); 2) LPHC, ratos alimentados com uma dieta contendo 6% de proteína e 74% de carboidrato por 15 (LPHC15) ou 45 dias (LPHC45) e 3) reversão, alimentados por 15 dias com a dieta LPHC e por mais 30 dias com a dieta controle (R45). A tolerância à glicose (GTT) foi avaliada pelas áreas sob as curvas (AUC) glicêmicas obtidas pelo método trapezoidal e a tolerância à insulina (ITT) pela constante de decaimento da glicose sérica (Kitt). O estresse oxidativo foi avaliado pela da quantificação do nível de lipoperoxidação através da dosagem do MDA (malondialdeído) nos rins, níveis de GSH (glutationa reduzida) e determinação da atividade das enzimas GPx (glutationa peroxidase), GR (glutationa redutase), catalase e SOD (superóxido dismutase) nos rins, além da quantificação da capacidade antioxidante total (CAT) no plasma. Analisou-se também. A função renal foi avaliada pela da quantificação da creatinina plasmática e análise histológica. Os resultados foram expressos como a média ± E.P.M. e as comparações estatísticas realizadas através do Teste t de Student ou ANOVA uma via, seguida de pós-teste de Tukey (p < 0,05). Os animais LPHC15, apresentaram valores similares ao grupo C15 para GSH, GPx, GR, SOD e catalase. No entanto, o peso dos rins (C15: 5,59 ± 0,21; LPHC15: 4,60 ± 0,08 mg / g de peso corporal) e a CAT (C15: 0,486 ± 0,059; LPHC5: 0,252 ± 0,059 mmol/L) foram menores e a creatinina plasmática (C45: 0,672 ± 0,028; LPHC45: 1,003 ± 0,039 mg/dL) e o nível de MDA (C15: 0,0195 ± 0,001; LPHC15: 0,033 ± 0,001 mmol/g de tecido) foram maiores no grupo LPHC15 em relação ao C15. Após a administração da dieta LPHC por 45 dias, os valores da glicemia de jejum dos animais C45 e LPHC45 foram similares. No entanto, a glicemia dos animais do grupo R45 foi 11% (p0,05) maior que nos demais grupos. No GTT não houve diferença na AUC entre os grupos analisados. O mesmo ocorreu na análise do decaimento da glicose plasmática após administração de insulina entre os diferentes grupos. A atividade das enzimas SOD e catalase também foi similar nos três grupos avaliados, já a atividade das enzimas GPx (C45: 2,730 ± 0,732; LPHC45: 0,928 ± 0,176; R45: 3,290 ± 0,304 U/mg de proteína) e GR (C45: 4,701 ± 0,320; LPHC45: 2,840 ± 0,151; R45: 6,308 ± 1,087 U/mg de proteína) foram menores no grupo LPHC45. A concentração de GSH foi menor no grupo R45 (C45: 0,785 ± 0,034; LPHC45: 0,760 ± 0,047; R45: 0,510 ± 0,024 mmol/g de tecido). O nível de MDA foi maior nos grupos LPHC45 e R45 (C45: 11,170 ± 2,020; LPHC45: 31,030 ± 3,060; R45: 31,540 ± 4,460 mmol/g de tecido). O peso dos rins (C45: 3,72 ± 0,03; LPHC45: 3,17 ± 0,05; R45: 3,66 ± 0,09) e a CAT (C45: 0,583 ± 0,059; LPHC45: 0,135 ± 0,050; R45= 0,407 ± 0,108 mmol/L) foram menores no grupo LPHC45. O nível plasmático de creatinina foi maior nos grupos LPHC45 e R45 (C45: 0,556 ± 0,020; LPHC45: 0,640 ± 0,021; R45: 0,678 ± 0,023 mg/dL). Análise histológica mostrou deposição de lipídeos no interstício dos rins nos grupos LPHC45 e R45, classificada como leve a acentuada. Estes dados permitem concluir que a dieta LPHC introduzida logo após o desmame e administrada por 45 dias não altera a tolerância à glicose nem a sensibilidade à insulina, diferente do que já foi demonstrado em estudo prévio, quando a mesma é administrada por 15 dias, resultando em maior sensibilidade à insulina. No entanto, restrição protéica introduzida logo após o desmame levou a um prejuízo no desenvolvimento dos rins, com possível prejuízo na função renal, associada a acúmulo de lipídeos e ao stresse oxidativo. Embora a reversão da dieta recupere o peso dos rins, os níveis elevados de creatinina sérica e o maior conteúdo de MDA no órgão sugerem que os danos funcionais decorrentes do stress oxidativo são irreversíveis. / The reduction in protein intake and the increase in carbohydrate intake feature a dietary pattern present in the current lifestyle of the population worldwide, and it is associated with the incidence of pathologies such as diabetes mellitus, obesity and high blood pressure among others. Low protein intake is also associated with the development of oxidative stress, which contributes to the establishment of tissue lesions that may result in functional impairment of various organs. In view of these facts, this study aimed to investigate the possible renal damages caused by the administration of a hypoproteic-hyperglycemic diet to rats in the early growth stages. Male Wistar rats (~30 days and 100g) were divided into the following groups: 1) Control, fed on a diet containing 17% protein and 63% carbohydrates for 15 (C15) or 45 (C45) days; 2) LPHC, fed on a diet containing 6% protein and 74% carbohydrates for 15 (LPHC15) or 45 (LPHC45) days, and 3) reversal group, fed on a LPHC diet during 15 days and then fed on a control diet for the following 30 days (R45). Glucose tolerance (GTT) was assessed by the areas under glycemic curves (AUC) obtained by the Trapezoidal Rule and insulin tolerance (ITT) was calculated according to the serum glucose decline rate constant (Kitt). Oxidative stress was evaluated by quantifying the lipid peroxidation level through the dosage of MDA (malondialdehyde) in kidneys, levels of GSH (reduced glutathione) and by determining the activity of the enzymes GPx (glutathione peroxidase), GR (glutathione reductase), catalase and SOD (superoxide dismutase) in the kidneys as well as quantifying the total antioxidant capacity (TAC) in plasma. The renal function was evaluated by the quantification of plasma creatinine and histological analysis. Results were expressed as the mean ± SEM, and statistical comparisons were carried out by means of the Student t Test or one-way ANOVA, followed by Tukey’s post-test (p < 0,05). LPHC15 animals presented similar values to those of the C15 group in reference to GSH, GPx, GR, SOD and catalase. However, the weight of kidneys (C15: 5,59 ± 0,21; LPHC15: 4,60 ± 0,08 mg / g body weight) and TAC values (C15: 0,486 ± 0,059; LPHC5: 0,252 ± 0,059 mmol/L) were lower, while plasma creatinine (C45: 0,672 ± 0,028; LPHC45: 1,003 ± 0,039 mg/dL) and MDA level (C15: 0,0195 ± 0,001; LPHC15: 0,033 ± 0,001 mmol/g tissue) were higher for the LPHC15 group compared with C15. After administering the LPHC diet for 45 days, the values for fasting glycemia in C45 and LPHC45 animals were similar. However, the glycemia level of R45 animals was 11% (p < 0,05) higher than in the other groups. There were no differences in the AUC between groups analyzed for GTT. The same happened when plasma glucose decline was analyzed following insulin administration. The activity of SOD and catalase enzymes was similar in the three groups under evaluation, whereas the activity of GPx (C45: 2,730 ± 0,732; LPHC45: 0,928 ± 0,176; R45: 3,290 ± 0,304 U/mg protein) and GR (C45: 2,730 ± 0,732; LPHC45: 0,928 ± 0,176; R45: 3,290 ± 0,304 U/mg protein) was lower in the LPHC45 group. GSH concentration was lower in the R45 group (C45: 0,785 ± 0,034; LPHC45: 0,760 ± 0,047; R45: 0,510 ± 0,024 mmol/g of tissue). The level of MDA was higher in the LPHC45 and R45 groups (C45: 11,170 ± 2,020; LPHC45: 31,030 ± 3,060; R45: 31,540 ± 4,460 mmol/g of tissue). The weight of kidneys (C45: 3,72 ± 0,03; LPHC45: 3,17 ± 0,05; R45: 3,66 ± 0,09) and TAC (C45: 0,583 ± 0,059; LPHC45: 0,135 ± 0,050; R45= 0,407 ± 0,108 mmol/L) showed lower values in the LPHC45 group. LPHC45 and R45 groups presented higher levels of plasma creatinine (C45: 0,556 ± 0,020; LPHC45: 0,640 ± 0,021; R45: 0,678 ± 0,023 mg/dL). Histological analysis showed interstitial lipid deposition in kidneys for LPHC45 and R45 groups, graded from mild to marked. These data lead to the conclusion that the LPHC diet, when introduced immediately after weaning and administered along 45 days, does not alter either glucose tolerance or insulin sensitivity. This conclusion is different from what was concluded in a previous study where LPHC diet administered during 15 days resulted in greater insulin sensitivity. Yet protein restriction, introduced soon after weaning, has led to damage in kidney development, which may result in impaired renal function associated to increased fat deposition and oxidative stress. Even though the diet reversal may recover kidney weight, the increased levels of serum creatinine and higher content of MDA in the organ suggest that functional damages resulting from oxidative stress are irreversible.