Ο Σακχαρώδης Διαβήτης (ΣΔ) είναι μια μεταβολική διαταραχή, που χαρακτηρίζεται από χρόνια υπεργλυκαιμία ως αποτέλεσμα διαταραχής στην έκκριση της ινσουλίνης ή τη δράση της ή και στα δύο αυτά χαρακτηριστικά. Οι μακροπρόθεσμες επιπτώσεις της χρόνιας υπεργλυκαιμίας στον ΣΔ διακρίνονται σε μικροαγγειακές και μακροαγγειακές επιπλοκές. Η μικροαγγειακή νόσος οδηγεί σε αμφιβληστροειδοπάθεια, νεφρική ανεπάρκεια και νευροπάθεια, ενώ η σχετιζόμενη με τον ΣΔ μακροαγγειακή νόσος προκαλεί αυξημένο κίνδυνο για έμφραγμα μυοκαρδίου, αγγειακά εγκεφαλικά επεισόδια και ακρωτηριασμούς των άκρων. Έχει βρεθεί ότι η υπεργλυκαιμία είναι η κύρια αιτία της μικροαγγειακής νόσου, ενώ στην παθογένεια της μακροαγγειακής νόσου συμμετέχει η υπεργλυκαιμία, αλλά και η αντίσταση στην ινσουλίνη. Ο σύνδεσμος ανάμεσα στην χρόνια υπεργλυκαιμία και την αγγειακή βλάβη έχει αποδοθεί σε τέσσερα ανεξάρτητα βιοχημικά μονοπάτια: 1. Αυξημένη δραστηριότητα του μονοπατιού της πολυόλης 2. Συσσώρευση τελικών προϊόντων προχωρημένης γλυκοζυλίωσης (Advanced Glycation Endproducts: AGEs) 3. Ενεργοποίηση της πρωτεϊνικής κινάσης C (PKC) και 4. Αυξημένη δραστηριότητα του μονοπατιού της εξοζαμίνης. Αυτά τα φαινομενικά μη σχετιζόμενα μεταξύ τους μοριακά μονοπάτια έχουν έναν υποκείμενο κοινό μηχανισμό : την υπερπαραγωγή ριζών υπεροξειδίου από τη μιτοχονδριακή αλυσίδα μεταφοράς ηλεκτρονίων. Οι μιτοχονδριακές ελεύθερες ρίζες οξυγόνου, μέσω ενεργοποίησης της πολυμεράσης της πολυ-ADP-ριβόζης, μερικώς αναστέλλουν το γλυκολυτικό ένζυμο αφυδρογονάση της 3-φωσφορικής γλυκεραλδεΰδης, με αποτέλεσμα τη συσσώρευση των γλυκολυτικών ενδιάμεσων προϊόντων, όπως της 3-φωσφορικής γλυκεραλδεΰδης και της 6-φωσφορικής φρουκτόζης, που αποτελούν υποστρώματα για τα τέσσερα παραπάνω βιοχημικά μονοπάτια. Το αποτέλεσμα της αντίστασης στην ινσουλίνη, όσον αφορά τις μακροαγγειακές επιπλοκές, είναι η αυξημένη ροή των ελεύθερων λιπαρών οξέων από τα λιποκύτταρα προς τα αρτηριακά ενδοθηλιακά κύτταρα. Η αυξημένη οξείδωση των ελεύθερων λιπαρών οξέων στα μιτοχόνδρια και η μιτοχονδριακή υπερπαραγωγή ελευθέρων ριζών οξυγόνου οδηγούν στην ενίσχυση των τεσσάρων μοριακών μονοπατιών με τον ίδιο ακριβώς μηχανισμό που έχει περιγραφεί παραπάνω για την υπεργλυκαιμία. Στην αντίσταση στην ινσουλίνη έχει παρατηρηθεί, επίσης, η μερική αναστολή του μονοπατιού της κινάσης της 3-φωσφατιδυλινοσιτόλης, που τελικά προάγει την ενίσχυση των αθηρογόνων και την καταστολή των αντι-αθηρογόνων ιδιοτήτων της ινσουλίνης. Ο κύριος στόχος αυτής της βιβλιογραφικής εργασίας είναι η περιγραφή των μονοπατιών που οδηγούν στη δημιουργία των, επαγόμενων από την υπεργλυκαιμία αλλά και την αντίσταση στην ινσουλίνη, διαβητικών αγγειακών επιπλοκών, καθώς και του κοινού μηχανισμού (παραγωγής ελευθέρων ριζών οξυγόνου) που βρίσκεται πίσω από αυτά τα μονοπάτια, παρέχοντας πλέον μια καινούρια βάση για μελλοντική έρευνα και ανακάλυψη φαρμάκων, προληπτικών και θεραπευτικών της διαβητικής αγγειοπάθειας. / Diabetes Mellitus is a metabolic disorder characterized by chronic hyperglycemia, due to decreased secretion of insulin and/ or decreased tissue sensitivity to insulin. The sequelae of chronic hyperglycemia in diabetes of all phenotypes are divided into microvascular and macrovascular complications. Microvascular disease causes blindness, renal failure, and neuropathy, and diabetes-accelerated macrovascular disease causes excessive risk for myocardial infarction, stroke, and lower limb amputation. Strict glycemic control has been shown to reduce both microvascular and macrovascular complications of diabetes. However, in contrast to diabetic microvascular disease, it is believed that hyperglycemia is not the major determinant of diabetic macrovascular disease : a large part of cardiovascular disease risk is due to insulin resistance. The link between chronic hyperglycemia and vascular damage has been established by four independent biochemical abnormalities : increased polyol pathway flux, increased formation of Advanced Glycation End-products (AGEs), activation of Protein Kinase C (PKC), and increased hexosamine pathway flux. These seemingly unrelated pathways have an underlying common denominator : overproduction of superoxide by the mitochondrial electron transport chain. Mitochondrial reactive oxygen species (ROS) partially inhibit the glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenase, which diverts increased substrate flux from glycolysis to pathways of glucose overutilization. As for insulin resistance, it causes increased free fatty acid flux from adipocytes into endothelial cells and increased free fatty acid oxidation in macrovascular endothelial cells, resulting in mitochondrial overproduction of ROS by exactly the same mechanism described above about hyperglycemia. Furthermore, metabolic insulin resistance is characterized by pathway-specific impairment in phosphatidylinositol 3-kinase-dependent signaling, which also causes endothelial dysfunction. Preliminary experimental evidence in vivo suggests that these mechanisms leading to diabetic microvascular and macrovascular complications offer a novel basis for research and drug development, targeting to prevention and treatment of angiopathy in Diabetes Mellitus.

Many diets of different compositions are available for use in animal experiments and have been used as standard, but they may induce adverse metabolic effects, compromising the comparison between the results of several studies. The literature records many reports of changes related to the use of these diets, however it lacks studies that compare metabolic effects of consumption of the different standard diets in animal experiments. Accordingly, the objective of this dissertation was to evaluate the metabolic effects of the consumption of diets considered standard widely used in animal research, being presented in the form of two articles: a review of the literature that gathers evidence yet little discussed by the scientific community on the feeding of laboratory animals; the second article refers to an experimental study with rats newly weaned, who received two types of diets: a commercial cereal-based, Nuvilab®, and another purified proposal by the American Institute of Nutrition, the AIN-93. Under the experimental conditions established, coefficients of protein and feeding efficiency presented significantly higher in group AIN-93 than in group Nuvilab®. The AIN-93 showed significantly higher lipid and protein digestibility than Nuvilab®. The different diets did not cause weight difference evolution of animals and histological analysis to the optical microscope of the kidneys, heart, spleen, stomach and small intestine showed no changes in the structures of these bodies, despite the different treatments. Animals fed the AIN-93 diet, regardless of age, had hepatic steatosis in frequency significantly higher than the animals that received the commercial Nuvilab®. The different diets did not cause influence on the absolute and relative weights of organs of animals, except for the absolute weight of the liver among younger animals and relative weight of the intestine among older animals. There was no influence of different diets on biochemical parameters evaluated, and the differences detected possibly resulting from the interaction between age and length of exposure of animals to diets. The markers of damage of kidney and liver function were similar and serum creatinine varied according to age. It was shown that both diets, AIN-93 and Nuvilab ®, are able to promote the growth of rats for a period of study considered subchronic. However, the occurrence of hepatic steatosis in animals fed the AIN-93 diet in pellets, reinforces the importance of tracking the standard protocols of experimentation and is indicative of nutritional inadequacies by imposing the need for further investigations to clarify which components or characteristics of this diet, widely used in animal experiments, may have contributed to this result. For instance, it is suggested that diets in the form of flour are used preferably those pellets, particularly on protocols to investigate metabolic effects. / Fundação de Amparo a Pesquisa do Estado de Alagoas / Várias dietas de diferentes composições estão disponíveis para o uso em experimentação animal e têm sido utilizadas como padrão, mas podem induzir efeitos metabólicos distintos, comprometendo a comparação entre os resultados dos diversos estudos. A literatura registra inúmeros relatos de alterações relacionadas ao uso dessas dietas, porém, há carência de estudos que comparem os efeitos metabólicos do consumo das diferentes dietas padrão utilizadas em experimentos animais. Assim sendo, o objetivo da presente dissertação foi avaliar as repercussões metabólicas do consumo de dietas consideradas padrão, amplamente utilizadas na pesquisa animal, sendo apresentada na forma de dois artigos: uma revisão da literatura, que reúne evidências ainda pouco debatidas pela comunidade científica relativas à alimentação de animais de laboratório, e um segundo artigo, que se refere a um estudo experimental com ratos Wistar recémdesmamados, que receberam dois tipos de dieta: uma comercial à base de cereais, Nuvilab®, e outra purificada proposta pelo American Institute of Nutrition, a AIN-93. Nas condições experimentais estabelecidas, coeficientes de eficiências protéica e alimentar apresentaram-se significativamente maiores no grupo AIN-93 que no grupo Nuvilab®. A AIN-93 apresentou digestibilidades lipídica e protéica significativamente maiores que a Nuvilab®. As diferentes dietas não causaram diferença na evolução ponderal dos animais e a análise histológica ao microscópio óptico dos rins, coração, baço, estômago e intestinos não evidenciou alterações nas estruturas desses órgãos, apesar dos diferentes tratamentos. Os animais alimentados com a dieta AIN-93, independente da idade, apresentaram esteatose hepática em uma frequência significativamente maior que os animais que receberam a comercial Nuvilab®. As diferentes dietas não exerceram influência sobre os pesos absoluto e relativo dos órgãos dos animais, com exceção do peso absoluto do fígado, entre os animais mais jovens, e do peso relativo do intestino, entre os animais mais velhos. Não se observou influência das diferentes dietas sobre os parâmetros bioquímicos avaliados, sendo as diferenças detectadas possivelmente resultantes da interação entre a idade e o tempo de exposição dos animais às dietas. Os marcadores de lesão e função hepática e renal foram similares e a creatinina sérica variou em função da idade. Demonstrou-se que ambas as dietas, AIN-93 e Nuvilab®, são capazes de promover o crescimento de ratos Wistar por um período de estudo considerado subcrônico. Porém, a ocorrência de esteatose hepática nos animais alimentados com a dieta AIN-93 peletizada, reforça a importância de monitoramento de protocolos padrão de experimentação e é indicativa de inadequações nutricionais, impondo a necessidade de investigações adicionais para esclarecer que componentes ou características dessa dieta, amplamente utilizada em experimentação animal, podem ter contribuído para tal resultado. Por hora, sugere-se que a dieta AIN-93 seja oferecida preferencialmente na forma de farinha, particularmente em protocolos que investiguem efeitos metabólicos.

No diabete melito, produtos de glicação avançada (AGE) reduzem o efluxo de colesterol celular o que agrava o desenvolvimento da aterosclerose. Neste estudo, investigou-se o papel da albumina modificada por glicação avançada (albumina-AGE) sobre a sensibilização de macrófagos à resposta inflamatória e o impacto da secreção de citocinas, quimocinas e moléculas de adesão sobre o efluxo de colesterol mediado por apolipoproteína A-I e subfrações de HDL. Além disso, determinou-se a capacidade da HDL em modular a resposta inflamatória em macrófagos tratados com albumina-AGE. Macrófagos de peritônio de camundongo foram tratados com ou sem sobrecarga de colesterol, na presença de 2 mg/mL de albumina-controle (albumina-C) ou albumina-AGE, por 72 h, seguindo-se de incubação, por 24 h, com calgranulina S100B (20 g/mL) ou lipopolissacarídeo (LPS; 1 g/mL). Em comparação com albumina-C, a albumina-AGE, isenta em endotoxinas, isoladamente não alterou a secreção de citocinas em macrófagos. No entanto, a albumina-AGE sensibilizou macrófagos não enriquecidos em colesterol a uma maior secreção de interleucina 6 (IL-6), fator de necrose tumoral alfa (TNF-), proteína quimoatraente de monócitos 1 (MCP-1), interlucina 1 beta (IL-1) e molécula de adesão celular vascular 1 (VCAM-1) após estimulação com S100B ou LPS, o que foi potencializado pela sobrecarga de colesterol celular. Em macrófagos não estimulados, o meio condicionado, advindo das incubações de células com albumina-AGE e S100B (meio enriquecido em citocinas), reduziu o efluxo de 14C-colesterol mediado por apoA-I, HDL2 e HDL3 em, respectivamente, 23%, 43% e 20%, em comparação com células incubadas com meio isolado do tratamento com albumina-C e S100B. De forma similar, o efluxo de 14C-colesterol mediado por apoA-I, HDL2 e HDL3 foi reduzido em macrófagos tratados com meio advindo de incubações com albumina-AGE e LPS, respectivamente, 37%, 47% e 8,5% em comparação ao tratamento com albumina-C e LPS. Em macrófagos tratados com albumina-C e S100B, a incubação prévia com HDL reduziu a secreção de IL-6, TNF-, MCP-1 e VCAM-1 em, respectivamente, 72%, 57%, 50% e 41% quando comparada à incubação na ausência de HDL. Em incubações com albumina-C, a secreção de IL-6, TNF-, MCP-1, IL-1 e VCAM-1 induzida por LPS foi respectivamente, 58%, 54%, 42%, 74% e 45% menor mediante incubação com HDL, em comparação a incubações similares, porém na ausência desta lipoproteína. Por outro lado, em macrófagos tratados com albumina-AGE e S100B, a HDL não foi capaz de reduzir a secreção de TNF-, IL-1 e VCAM-1 e aumentou a secreção de IL-6 (54%) e MCP-1 (20%). Nas células tratadas com albumina-AGE e LPS, a HDL também não reduziu a secreção de TNF-, MCP-1 e IL-1 e aumentou a secreção de IL-6 (16%) e VCAM-1 (20%). Redução na secreção de mediadores inflamatórios foi observada em macrófagos tratados com albumina-AGE apenas quando a HDL foi incubada juntamente com S100B ou LPS. Em conclusão, a albumina-AGE sensibiliza macrófagos à resposta inflamatória induzida por calgranulina S100B e LPS, prejudicando o transporte reverso de colesterol de macrófagos. Além disso, a albumina-AGE reduz as propriedades anti-inflamatórias da HDL, o que pode agravar a aterosclerose no diabete melito / In diabetes mellitus, advanced glycation end products (AGE) reduces the cholesterol efflux from cells, which aggravates the development of atherosclerosis. In this study, we investigated the role of advanced glycated albumin (AGE-albumin) on macrophage inflammatory response and the impact of cytokines, chemokines and adhesion molecules secretion on cholesterol efflux mediated by apolipoprotein A-I (apoA-I) and HDL subfractions. Furthermore, the HDL ability in modulating inflammatory response in macrophages treated with AGE-albumin was also determined. Mouse peritoneal macrophages previously enriched or not with cholesterol were treated in the presence of 2 mg/mL of control-albumin (C-albumin) or AGE-albumin, for 72 h, followed by incubation, for 24 h, with S100B calgranulin (20 g/mL) or lipopolysaccharide (LPS; 1 g/mL). In comparison to free endotoxin-C-albumin, AGE-albumin, by itself did not alter cytokine secretion by macrophages. However, AGE-albumin primed non-cholesterol enriched macrophages to a higher secretion of interleukin -6 (IL-6), tumor necrosis factor alpha (TNF-), monocyte chemotactic protein 1 (MCP-1), interleukin 1 beta (IL-1) and vascular cell adhesion molecule 1 (VCAM-1) after stimulation with S100B or LPS, which was potentiated by cell cholesterol overload. In non-stimulated macrophages, conditioned medium, derived from incubation with AGE-albumin and S100B (cytokine enriched-medium), reduced the 14C-cholesterol efflux mediated by apoA-I, HDL2 and HDL3 in, respectively, 23%, 43% and 20%, in comparison to cells incubated with conditioned medium isolated from treatment with C-albumin and S100B. Similarly, 14C-cholesterol efflux mediated by apoA-I, HDL2 and HDL3 was reduced in macrophages treated with medium derived from incubation with AGE-albumin and LPS, respectively, 37%, 47% and 8,5% in comparison to treatment with C-albumin and LPS. In macrophages treated with C-albumin and S100B, previous incubation with HDL reduced the secretion of IL-6, TNF-, MCP-1 and VCAM-1 in, respectively 72%, 57%, 50% and 41% in comparison to incubation in the absence of HDL. In incubations with C-albumin, the secretion of IL-6, TNF-, MCP-1, IL-1 and VCAM-1 induced by LPS was respectively, 58%, 54%, 42%, 74% and 45% lower in cells treated with HDL in comparison to similar incubations in the absence of this lipoprotein. On the other hand, in macrophages treated with AGE-albumin and S100B, HDL was unable to reduce the TNF-, IL-1 and VCAM-1 secretion and increased the secretion of IL-6 (54%) and MCP-1 (20%). In cells treated with AGE-albumin and LPS, HDL was unable to reduce the secretion of TNF-, MCP-1 and IL-1 and increased IL-6 (16%) and VCAM-1 (20%). Reduction in inflammatory mediators was observed in macrophages treated with AGE-albumin only when HDL was incubated simultaneously with S100B or LPS. In conclusion, AGE-albumin primes macrophages to an inflammatory response elicited by S100B calgranulin and LPS, impairing macrophage reverse cholesterol transport. Moreover, AGE-albumin impairs the HDL anti-inflammatory properties, which can aggravate the atherosclerosis in diabetes mellitus

The advanced glycation endproducts (AGEs) constitute a large variety of compounds formed from nonenzymatic amino-carbonyl interactions between reducing sugars or oxidized lipids and proteins, nucleic acids or aminophospholipids. The AGEs formation in foods and biological systems is a topic of increasing interest for the science, since they are involved in proinflammatory and pro-oxidative effects related to the pathogenesis of chronic degenerative diseases such as diabetes, Alzheimer's disease and renal failure. The Nε-carboxymethyllisine (CML) was the first AGE identified in foods and, since then, has been the compound of choice in studies on which a single product is used as an AGE marker. Immunochemical or instrumental methods are available for the AGEs determination, but they present limitations and there is not an ideal method yet. Thus, in order to compare and optimize different analytical methods, this study aimed to determine the CML content on enteral and parenteral nutrition formulas by ELISA and LC-MS/MS. So, in order to compare and optimize different analytical methods, this study aimed to determine the content of CML in nutritional enteral and parenteral formulas by ELISA and by liquid chromatography coupled to tandem mass spectrometry (LC-MS / MS). Thus, 5 parenteral and 17 enteral commercially available formulations were investigated. All samples studied showed detectable levels of CML, regardless of the method of analysis used. The parenteral formulations presented CML contents measured by ELISA ranging from 529.9 ± 33.47 to 1948.88 ± 3.68 ng CML/mL of sample and showed positive linear correlations to their content in lipids (0.9259) and carbohydrates (0.9426), but they were not submitted to the LC-MS/MS analysis due to the impossibility of applying the established purification protocol for this group of samples. Enteral formulations analyzed by ELISA presented CML contents ranging from 1076.91 ± 76.87 to 55950.71 ± 1891.29 ng CML/mL of sample and showed positive correlations to its contents in carbohydrate (0.6057), lipid (0.5264) and protein (0.6157). They showed a variation from 0.09 to 0.503 μg CML/mg of protein of the diets when analyzed by LC-MS/MS and there was no correlation between CML and the variables "lipid", "carbohydrate" or "protein" calculated for this group. The investigation conducted during the samples preparation prior to injection into the LC-MS/MS showed a significant loss of CML during the different stages of the protocol, compromising the reliability of the results obtained through this method of analysis, while the comparison between the results obtained through different methods applied to similar samples showed the reliability of the anti-CML ELISA test used in this experiments. The results of this study indicate the need for improvement of AGEs analysis protocols in food and should guide future research on this area. The determination of reliable methods of detection, which enable the measurement of AGEs structures in body fluids, tissues and also in food, in a sensitive, specific, fast and not too expensive way is a challenge that remains present on this field. / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Os produtos da glicação avançada (AGEs, do inglês Advanced Glycation Endproducts) constituem grande variedade de substâncias formadas a partir de interações amino-carbonilo, de natureza não-enzimática, entre açúcares redutores ou lipídeos oxidados e proteínas, aminofosfolipídeos ou ácidos nucléicos. A formação de AGEs nos alimentos e em sistemas biológicos constitui tema de crescente interesse, desde que estão associados a efeitos pró-oxidativos e pró-inflamatórios envolvidos na patogênese de diversas doenças crônico-degenerativas como o diabetes, o mal de Alzheimer, a insuficiência renal. A Nε-carboximetilisina (CML) foi o primeiro AGE a ser identificado em alimentos e tem sido o composto de escolha em estudos em que um único produto é usado como marcador de AGEs de um sistema. Métodos imunoquímicos ou instrumentais estão disponíveis para a determinação da CML, mas ambos apresentam limitações, não havendo ainda um método considerado ideal. Assim, a fim de comparar e otimizar diferentes métodos analíticos, o presente estudo teve como objetivo determinar o conteúdo em CML de fórmulas nutricionais enterais e parenterais através das técnicas de ELISA e de cromatografia líquida acoplada à espectrometria de massa tandem (LC-MS/MS). Para tanto, foram investigadas 5 formulações parenterais e 17 enterais comercialmente disponíveis. Todas as amostras investigadas apresentaram níveis detectáveis de CML neste estudo, independentemente do método de análise utilizado. As fórmulas parenterais apresentaram conteúdos mensurados através de ELISA que variaram de 529,9 ± 33,47 a 1948,88 ± 3,68 ng de CML/mL de amostra e apresentaram correlações lineares positivas quanto aos seus conteúdos em lipídeos (0,9259) e em carboidratos (0,9426), mas não foram submetidas às análises através do LC-MS/MS devido à inviabilidade da aplicação para esse grupo de amostras do protocolo de purificação estabelecido nesta investigação. As formulações enterais apresentaram conteúdos em CML que variaram entre 1076,91 ± 76,87 e 55950,71 ± 1891,29 ng de CML/ mL de amostra e evidenciaram correlações positivas quanto aos seus conteúdos em carboidratos (0,6057), lipídeos (0,5264) e proteínas (0,6157), quando analisadas através de ELISA, e apresentaram uma variação de 0,09 e 0,503 μg CML/ mg de proteína das dietas quando analisadas através do LC-MS/MS, não havendo correlações entre a CML e as variáveis “lipídeos”, “carboidratos” ou “proteínas” para esse grupo. A investigação conduzida durante o processo de preparo das amostras, anterior à injeção no LC-MS/MS, evidenciou uma expressiva perda da CML durante as diferentes etapas do protocolo, comprometendo a confiabilidade dos resultados obtidos através desse método de análise, enquanto a comparação entre os resultados encontrados através dos diferentes métodos aplicados a amostras semelhantes em composição e preparo demonstrou a confiabilidade do teste de ELISA anti-CML utilizado nas condições deste experimento Os resultados do presente estudo apontam a necessidade de aperfeiçoamento dos protocolos de análise de AGEs em alimentos e deverão guiar futuras investigações nesta área. Dentre os desafios que permanecem presentes no campo de estudo dos AGEs está a definição de métodos de detecção confiáveis, que possibilitem a mensuração de estruturas nos fluidos ou tecidos corporais e nos alimentos, de maneira sensível, específica, rápida e não muito dispendiosa.

Produtos de glicação avançada (AGE) alteram o metabolismo de lípides e, em especial, o efluxo de colesterol de macrófagos, por meio da redução dos receptores ABCA-1 e ABCG-1. Isto prejudica o transporte reverso de colesterol, sistema que favorece o fluxo de colesterol de macrófagos arteriais ao fígado, permitindo sua excreção na bile e eliminação fecal. Óxidos de colesterol modulam favoravelmente a homeostase lipídica em macrófagos e favorecem o transporte reverso de colesterol, embora o acúmulo de 7-cetocolesterol, 7-hidroxicolesterol e 7-hidroxicolesterol associe-se à aterogênese e morte celular. Neste estudo, avaliou-se o efeito do tratamento com glicolaldeído (GAD; oxoaldeído que induz rápida geração intracelular de AGE), em macrófagos sobrecarregados com LDL oxidada e incubados com HDL ou HDL e indutor de LXR (T0901317) sobre: 1) a distribuição seletiva de óxidos de colesterol e o conteúdo total de esteróis intracelulares e 2) o conteúdo de ABCA-1 e ABCG-1. Colesterol total e os diversos subtipos de óxidos de colesterol foram determinados por cromatografia a gás acoplada à espectrômetro de massa. O conteúdo dos receptores de HDL (ABCA-1 e ABCG-1) foi avaliado por imunoblot. Em macrófagos controles (C), a adição de HDL ou HDL + T0901317 promoveu redução no conteúdo de esteróis totais (colesterol + óxidos de colesterol), colesterol e 7-cetocolesterol. No entanto, isto não foi observado em macrófagos GAD. Nas diversas condições experimentais, não houve diferença no conteúdo intracelular dos outros subtipos de óxidos de colesterol, em células C e GAD. Macrófagos GAD apresentaram menor conteúdo de ABCA-1 (45%), quando comparados aos macrófagos C, mesmo após adição de HDL ou HDL + T0901317. O conteúdo de ABCG-1 foi 36,6% menor em macrófagos GAD, na presença de HDL, em comparação às células C. Em conclusão, em macrófagos sobrecarregados com LDL oxidada, o tratamento com glicolaldeído diminui a exportação celular de colesterol e 7-cetocolesterol, mediada pela HDL. Isto é decorrente do menor conteúdo dos receptores ABCA-1 e ABCG-1 em macrófagos tratados com glicolaldeído, e pode contribuir para o desenvolvimento de aterosclerose no diabete melito / Advanced glycation end products (AGE) alter lipid metabolism and reduce the macrophage expression of ABCA-1 and ABCG-1 which impairs the reverse cholesterol transport, a system that drives cholesterol from arterial wall macrophages to the liver, allowing its excretion into the bile and feces. Oxysterols favors lipid homeostasis in macrophages and drive the reverse cholesterol transport, although the accumulation of 7-ketocholesterol, 7- hydroxycholesterol and 7- hydroxycholesterol is related to atherogenesis and cell death. We evaluated the effect of glycolaldehyde treatment (GAD; oxoaldehyde that induces a fast formation of intracellular AGE) in macrophages overloaded with oxidized LDL and incubated with HDL alone or HDL plus LXR agonist (T0901317) in: 1) the intracellular content of oxysterols and total sterols and 2) the contents of ABCA-1 and ABCG-1. Total cholesterol and oxysterol subspecies were determined by gas chromatography/mass spectrometry and HDL receptors content by immunoblot. In control macrophages (C), incubation with HDL or HDL + T0901317 reduced the intracellular content of total sterols (total cholesterol + oxysterols), cholesterol and 7-ketocholesterol, which was not observed in GAD macrophages. In all experimental conditions no changes were found in the intracellular content of other oxysterol subspecies comparing C and GAD macrophages. GAD macrophages presented a 45% reduction in ABCA-1 protein level as compared to C cells, even after the addition of HDL or HDL + T0901317. The content of ABCG-1 was 36.6% reduced in GAD macrophages in the presence of HDL as compared to C macrophages. In conclusion, in macrophages overloaded with oxidized LDL, glycolaldehyde treatment reduces the HDL-mediated cholesterol and 7-ketocholesterol efflux which is ascribed to the reduction in ABCA-1 and ABCG-1 protein level. This may contribute to atherosclerosis in diabetes mellitus

Le glaucome est une maladie neurodégénérative qui se définit par une perte progressive en fibres nerveuses rétiniennes et un rétrécissement du champ visuel. Il s’agit de la première cause de cécité irréversible dans le monde et le principal facteur de risque est la pression intraoculaire (PIO). L’étude ALIENOR (Antioxydants, Lipides Essentiels, Nutrition et maladies OculaiRes) est une étude épidémiologique qui a pour but de déterminer l’incidence des différentes pathologies oculaires liées à l’âge avec les facteurs nutritionnels, démographiques ou environnementaux dans une population représentative de la région de Bordeaux. En 2009-2010, 624 sujets âgés de plus de 74 ans ont bénéficié d’un examen ophtalmologique complet incluant un examen du nerf optique en rétinophotographie et en tomographie à cohérence optique spectral-domain (SD-OCT), d’une mesure la PIO au tonomètre à air et d’une évaluation des propriétés biomécaniques de la cornée. Une mesure de l’accumulation cutanée de produits de glycation avancée a été réalisée par autofluorescence. Le diagnostic de glaucome a été réalisé en utilisant les critères de la classification ISGEO (International Society for Epidemiologic and Geographical Ophthalmology). Les paramètres biomécaniques de la cornée étaient modifiés avec l’âge et chez les sujets ayant une histoire résidentielle à des latitudes plus exposées aux ultraviolets ambiants. L’épaisseur de cornée était plus élevée chez les sujets anciennement fumeurs. L’autofluorescence cutanée ≥ 2.7 UA (Unité Arbitraire) était indépendamment associée au glaucome. Les paramètres d’épaisseur en fibres nerveuses rétiniennes du SD-OCT présentaient de bonnes performances diagnostiques pour discriminer les sujets glaucomateux des témoins et la base normative présentait de bonnes performances discriminatives lorsqu’au moins un des paramètres était considéré comme anormal. Notre étude apporte des résultats originaux en termes de facteurs de risque de glaucome ou de déterminants des facteurs de risque de glaucome. De plus les performances diagnostiques du SD-OCT pourraient fournir des informations utiles pour optimiser les stratégies de dépistage du glaucome dans une population générale âgée. / Glaucoma is a neurodegenerative disease defined by a progressive loss of optic nerve axons and retinal ganglion cells resulting in a characteristic enlargement of the optic nerve head cup and associated visual field defects. It remains the first cause of irreversible blindness worldwide and intraocular pressure (IOP) is the main risk factor. The ALIENOR (Antioxydants, Lipides Essentiels, Nutrition et maladies OculaiRes) study is a population-based study. It aims to assess the associations of age-related eye diseases with nutritional, demographic and environmental factors in a representative population of the Bordeaux area. In 2009-2010, 624 subjects, aged 74 years or more, underwent a complete eye examination, including an optic nerve head evaluation using retinophotography and a spectral-domain optical coherence tomography (SD-OCT), an IOP measurement using air-puff tonometry and an evaluation of biomechanical properties of the cornea. A measurement of skin accumulation of advanced glycation end-products was performed using an autofluorescence reader. Glaucoma diagnosis was made using ISGEO (International Society for Epidemiologic and Geographical Ophthalmology) criteria. Biomechanical properties of the cornea were modified by increasing age and in subjects having a higher lifetime ambient ultraviolet exposure. Central corneal thickness was thicker in former smokers. Skin autofluorescence values ≥ 2.7 AU (Arbitrary Unit) were independently associated with glaucoma. SD-OCT retinal nerve fiber layer thickness parameters had good diagnostic performances for discriminating glaucoma and control subjects and the normative database had good diagnostic performances if at least one parameter was considered abnormal by the machine. Our study provides new insights on glaucoma risk factors and determinants of glaucoma risk factors. Furthermore diagnostic performances of SD-OCT may provide valuable information in a screening strategy to optimize glaucoma detection in a general population of elderly people.

Dans un contexte de vieillissement de la population et d’accroissement des maladies chroniques liées à l’âge comme le diabète, de nouveaux biomarqueurs de l’état de santé à long terme doivent être étudiés. Les produits de glycation avancée (AGE) sont des molécules témoins de la charge métabolique accumulée au cours du temps, dénommée "mémoire métabolique". Les AGE jouent un rôle important dans les lésions à long terme dans le diabète et dans le déclin du métabolisme global lié au vieillissement. L’accumulation cutanée des AGE peut être mesurée par autofluorescence (AF) de manière instantanée et non invasive grâce à l’AGE-READER. Les objectifs de cette thèse étaient d’évaluer la valeur de l’AF cutanée des AGE en tant que marqueur de mémoire métabolique chez des personnes âgées de la cohorte des 3-Cités et parallèlement d’évaluer la valeur pronostique de l’AF pour les complications du diabète chez des patients porteurs de diabète de type 1. Chez les personnes âgées, nous avons montré que l’AF reflétait les statuts glycémique et rénal 10 ans avant la mesure. Chez les patients atteints de diabète de type 1, l’AF était associée à la présence d’une neuropathie 4 ans plus tard. De plus, dans cette même population, nous avons décrit l’évolution de l’AF sur 4 ans de suivi. Nous avons montré que les principaux déterminants de son évolution étaient la fonction rénale et le traitement par pompe à insuline. Enfin nous avons trouvé que l’augmentation de l’AF sur 4 ans de suivi était associée à la survenue de la maladie rénale. Ces travaux soulèvent de nouvelles perspectives de recherche quant à l’intérêt de l’AF à différents âges clés de la vie en tant que biomarqueur de pathologies qui évoluent sur des dizaines d’années. / In the context of the ageing of the population and the increase of age related diseases such as diabetes, new biomarquers of the long-term health status should be considered. Advanced glycation end products (AGE) are molecules indicators of the metabolic burden over time, called “metabolic memory”. AGE play an important role in long term diabetes injuries and in the global decline of the metabolism related to ageing. Skin accumulation of AGE can be measured by autofluorescence instantly and non-invasivly with a tool called AGE-READER. The objectives of my dissertation were to evaluate the value of the skin autofluorescence (sAF) of AGE as marker of metabolic memory in elderly people from the 3-City cohort and in parallel, in patients with type 1 diabetes, evaluate the prognostic value of sAF for diabetes complications. In the elderly population, we showed that sAF reflected glycemic and renal status of 10 years before. In patients with type 1 diabetes, sAF was associated to the presence of neuropathy 4 years later. Moreover, in this same population, we described the evolution of sAF in 4 years of follow-up and we showed that the principal determinants of the evolution of sAF were kidney function and insulin pump therapy. Finally, we also found that increase of sAF in 4 years was associated with the occurrence of kidney disease. This work rises new research opportunities about the interest of sAF at differents key ages as biomarker of pathologies which evolve in several decades.

Advanced glycation end products the (AGE) comprise a series of compounds, quite different, whose activity is related to their ability to modify the chemical and functional properties of diverse biological structures. Several studies, including that used animals, indicate that AGE restriction in the diet can have many protective effects. Furthermore, the use of certain medicinal plants appears to offer additional benefits, especially to individuals suffering from disorders associated with excessive accumulation of AGE. It is believed that plants combiam antiglicantes and antioxidant properties, have positive effects on the inhibition of glycation. This fact has attracted the interest of the scientific community, as it can provide important therapeutic potential. Although any further component is particularly suitable as reducing AGEs in the context of the human body, some substances in vitro studies demonstrated the anti-AGE effects important and worth further investigation such as pyridoxamine, alilcisteína (garlic extract component), phenolics, vitamins C and E, thiamin, taurine and carnosine, recognized for its antioxidant properties. Therefore the aim of this proposal was to determine the antiglicante activity of extracts of plants native to the brasileleira flora of recognized antioxidant activity. All the studied extracts are part of the bank of plant extracts belonging to the Laboratory for Research on Natural Resources (LPqRN), the Institute of Chemistry and Biotechnology - UFAL. Therefore, this thesis was divided into three parts, which generated three articles. The first titled "Antioxidant activity of native plant extracts of flora by ABTS methods, DPPH, FRAP and ORAC", aimed to investigate the antioxidant capacity of different plant extracts, by different in vitro assays (ABTS, DPPH, FRAP , ORAC), and determine whether there is correlation between the tested methods. In this proposal, 49 plant samples were studied, the tests of antioxidant capacity, DPPH; ABTS; FRAP and ORAC. Further tests were conducted to see if there was a correlation between the methods. The results were screened for to follow up the research, specifically, the antiglicante activity, believed to be related to the antioxidant activity. So the second article entitled "antiglicante in vitro activity of native plant extracts of flora", aimed to determine the antiglicante activity of native plant extracts of brazilian flora, with recognized antioxidant activity and the possibility of correlation between tests, antiglicante and antioxidants. For this 18 extracts, which had been classified as important sources of antioxidant compounds were tested for activity antiglicante. From the most promising extract, Auxemma oncocalyx Taub. (Bark) for activity, an isolated quinone thereof was tested in Oncocalixona A. This may be an important antiglicante agent, which resulted in the third section, "Oncocalyxone The functions of an anti-glycation agent in vitro", that this was submitted to the journal FOOD CHEMISTRY, ISSN: 0308-8146, Impact factor 3.259, on 02/12/14, in the form of Short communication. The particular this thesis is of importance, since investigations of this nature favors the development of research investigating potential therapeutic products, and that in turn may be useful in treating diseases associated with excessive accumulation of AGE / Os produtos de glicação avançada (AGE) englobam uma série de compostos, bastante diferentes entre si, cuja atividade está relacionada à capacidade destes de modificar as propriedades químicas e funcionais das mais diversas estruturas biológicas. Diversos estudos, inclusive que utilizaram animais, apontam que a restrição de AGE na dieta pode exercer diversos efeitos protetores. Além disso, o uso de determinadas plantas medicinais parece oferecer benefícios adicionais, especialmente, para indivíduos que sofrem com transtornos associados ao acúmulo exagerado de AGE. Acredita-se que plantas que combinam propriedades antiglicantes e antioxidantes, exercem efeitos positivos sobre a inibição da glicação. Este fato tem atraído o interesse da comunidade científica, visto que pode oferecer importante potencial terapêutico. Embora nenhum componente seja ainda especificamente indicado como redutor de AGEs no contexto do corpo humano, em estudos in vitro algumas substâncias demonstraram efeitos anti-AGE importantes e merecem investigações adicionais, tais como piridoxamina, alilcisteína (componente do extrato de alho), compostos fenólicos, vitaminas C e E, tiamina, taurina e carnosina, reconhecidos por suas propriedades antioxidantes. Por esta razão o objetivo da presente proposta foi determinar a atividade antiglicante de extratos de plantas nativas da flora brasileira, que tenham reconhecida atividade antioxidante. Todos os extratos estudados fazem parte do banco de extratos vegetais pertencentes ao Laboratório de Pesquisas em Recursos Naturais (LPqRN), do Instituto de Química e Biotecnologia – UFAL. Para tanto, a presente tese foi dividida em três partes, que geraram três artigos. O primeiro deles entitulado “Atividade antioxidante de extratos de plantas nativas da flora brasileira pelos métodos ABTS, DPPH, FRAP e ORAC”, teve como objetivo investigar a capacidade antioxidante de diferentes extratos de plantas, por diferentes ensaios in vitro (ABTS, DPPH, FRAP, ORAC), e determinar se existe correlação entre os métodos testados. Nesta proposta, foram estudadas 49 amostras vegetais, pelos ensaios de capacidade antioxidante, DPPH; ABTS; FRAP e ORAC. Adicionalmente foram realizados testes para verificar se existia correlação entre os métodos. Os resultados foram uma triagem para dar sequência às pesquisas, especificamente, da atividade antiglicante, que se acredita haver relação com a atividade antioxidante. Assim o segundo artigo entitulado “Atividade antiglicante in vitro de extratos de plantas nativas da flora brasileira”, teve como objetivo determinar a atividade antiglicante de extratos de plantas nativas da flora barasileira, com reconhecida atividade antioxidante e a possibilidade de correlação entre os testes, antiglicante e antioxidantes. Para isto 18 extratos, que haviam sido classificados como importantes fontes de componentes antioxidantes, foram testados para atividade antiglicante. A partir do extrato mais promissor, Auxemma oncocalyx Taub. (entrecasca) para a atividade, foi testada uma quinona isolada do mesmo, a Oncocalixona A. Esta demonstrou ser um importante agente antiglicante, o que resultou no terceiro artigo, “Oncocalyxone A functions as an anti-glycation agent in vitro”, este que foi submetido à revista FOOD CHEMISTRY, ISSN: 0308-8146, Fator de impacto 3.259, no dia 02.12.14, na forma de Short communication. A presente tese se reveste de particular importância, uma vez que investigações desta natureza favorecem o desenvolvimento de pesquisas que investiguem potenciais produtos terapêuticos, e que por sua vez, possam ser úteis no tratamento de doenças associadas ao acúmulo exagerado de AGE

博士(理学) / Doctor of Philosophy in Science / 同志社大学 / Doshisha University

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