Dissertations / Theses on the topic 'Diabetes Iran'

Background and Methods: The involvement of iron in the risk for, and complications of, type 2 diabetes has generated substantial interest over the past 15 years, initially sparked by an association with raised serum ferritin, and the observation that people with iron overload diseases frequently develop diabetes. Considerable advances have since been made in understanding the effect glucose has on molecules, cells, and tissues; and the role that oxidative stress plays in the development of the pathologies of long-term diabetes. Poorly liganded iron is potentially both a contributor to, and consequence of, these complications. In vitro experiments with glucose-incubated transferrin by earlier workers have demonstrated loss of function with increasing glycation, leading to the suggestion that the failure of this key iron-binding protein may contribute to diabetic pathology, via the presence of redox active non-transferrin-bound iron (NTBI). In vitro glycated transferrin is examined here by ultrafiltration, to assess loss of function and possible oxidative fragmentation. Mass spectrometry is used to identify a range of amino acid glycation sites on in vitro glycated transferrin for the first time. Finally, several groups have previously measured NTBI in people with diabetes, finding little agreement in results. NTBI is measured here in a cohort of people with type 2 diabetes, using a new adaptation of earlier NTBI assays. NTBI is also assessed in pre-dialysis chronic kidney disease (CKD) stages I to III for the first time. Results and Conclusions: Experiments with glycated transferrin in vitro demonstrate oxidative fragmentation, explaining the loss of function reported by earlier groups. In vitro glycated transferrin examined by mass spectrometry reveals a substantial number and range of amino acids subject to glycation. Comparison with in vivo glycated transferrin suggests that many of the in vitro glycation sites are not glycated in vivo, and that there are many oxidized methionine residues which are potential artefacts, or likely to be repaired by methionine sulphoxide reductases in vivo. A study of people with type 2 diabetes finds no direct association between NTBI and protein glycation. Unexpected correlations between NTBI and LDL, and LDL and haemoglobin with increasing protein glycation, are reported for the first time. NTBI is suggested to be iron sourced from haemoglobin or haem, from erythrocyte haemolysis prior to sample collection. In people with pre-dialysis CKD stages I to III no significant difference in NTBI level compared to controls is seen, or correlations with markers of renal function. No link between NTBI and kidney function at this stage of disease is indicated.

Hyperglycaemia stimulates a variety of biochemical abnormalities. The area of particular interest in this study is the influence of non-enzymatic glycation of proteins on iron homeostasis, and particularly on non-transferrin-bound iron (NTBI) and its possible relation to atherogenesis in both Type 2 diabetic and obese non diabetic subjects. The link between non-enzymatic glycation of proteins and iron homeostasis, and development of macrovascular disease may be mechanistically different in Type 2 diabetic and obese non diabetic subjects due to a difference in the protein glycation pattern. Because the following in vivo study required storage of samples for up to two years to complete the processing of all the samples, a storage study was carried out using different anticoagulants and addition of reduced glutathione (GSH) to samples to study the effects of storage, thawing and freezing of the samples on the level of malondialdehyde (MDA), a biomarker of lipid peroxidation. This storage study showed that EDTA attenuated the action of lipid oxidation compared with lithium heparin (LiH). A combination of GSH with either EDTA or LiH added more protection from lipid peroxidation in the first week of storage, but due to the thawing and freezing of the sample the action of GSH diminished through its autooxidation, meaning that addition of GSH to samples in the following in vivo study would be useless. An in vivo study was carried out on iron-related parameters in three subject groups: control (non-diabetic, non-obese), Type 2 diabetic and obese non diabetic. Glycated haemoglobin (HbA1C) was strongly correlated with NTBI in the diabetic group. Also the level of NTBI was significantly increased in Type 2 diabetic subjects compared with other groups while the level of total iron was significantly decreased. The study showed a strong positive correlation between NTBI and a biomarker of endothelium dysfunction (E-selectin) in all groups studied. Although it is not possible from the current data to know if there is a causal relationship between these two parameters, it remains a possibility that iron released from its binding sites could initiate oxidative damage to the endothelial cells and begin the process of atherogenesis. Positive correlation at the 90% confidence level between NTBI and a biomarker of inflammation, high sensitivity C-reactive protein, is another indicator in this study of a link between increases in NTBI, inflammation, endothelium dysfunction and atherosclerosis. This study also showed for first time that NTBI is present in higher levels in the plasma of obese subjects compared to controls despite the obese subjects having significantly lower total iron. An in vitro study found that glycation of transferrin half saturated with iron increased with increasing glucose concentration, leading to decreased capacity of transferrin to hold iron and increased release of free iron. Also co-incubation of transferrin half saturated with iron with low density lipoprotein (LDL) and glucose showed oxidation of LDL (measured as MDA). This may be explained by the effect of glycation, leading to release of free iron, which catalyses oxidation of LDL. In addition, glycation of LDL may enhance the oxidation of LDL catalysed by iron. Both studies indicate that the glycation of proteins has a major impact on iron homeostasis leading to release of non-enzymatic glycation and contributing to one of the most common complications of Type 2 diabetes, atherosclerosis.

Antecedents: El consum de ferro hemo i els dipòsits de ferro s'han associat amb el risc de diabetis mellitus tipus 2 (DMT2) en cohorts nord-americans, xineses, i del nord d'Europa, però aquestes relacions no han estat estudiades en poblacions del sud d'Europa. Objectius: Avaluar, prospectivament, l'efecte de la ingesta elevada de ferro i l'estat elevat de ferro en l'aparició de la DMT2 en població mediterrània adulta, i estudiar la relació entre l'estat del ferro i l'osteocalcina. Mètodes: Els participants van ser homes i dones de 55 a 80 anys de l'estudi PREDIMED. La DMT2 va ser diagnosticada utilitzant els criteris de l'ADA. Es van recollir dades sociodemogràfiques, antropomètriques, d'estil de vida, estat de ferro, perfil lipídic, metabolisme de la glucosa i inflamació. En un estudi observacional en 1.073 persones, 131 diabètics incidents, es va avaluar l'associació entre la ingesta de ferro i la DMT2. Es va analitzar transversalment en 423 subjectes la relació entre l'estat de ferro i l'osteocalcina. Finalment, un estudi casos-control niat va ser dissenyat per avaluar l'associació entre l'estat de ferro, mesurat mitjançant la ferritina sèrica (FS) i el receptor soluble de transferrina (sTfR), i el risc de DMT2 en 459 participants, 153 diabètics incidents. Resultats: Es va trobar que una ingesta elevada de ferro hemo augmenta el risc de DMT2 en un 30%. A més, els valors de FS>257μg/L en homes i >139μg/L en dones es van associar amb DMT2. També es va trobar associació entre la ràtio sTfR: ferritina i la DMT2, però no amb el sTfR. Tant la FS com el sTfR es relacionen amb l'osteocalcina. Conclusions: En una població mediterrània adulta d'alt risc cardiovascular, una ingesta elevada de ferro hemo i uns dipòsits elevats de ferro augmenten el risc de DMT2. La relació entre l'estat de ferro i l'osteocalcina és controvertida.
Antecedentes:El consumo de hierro hemo y los depósitos de hierro se han asociado con el riesgo de diabetes mellitus tipo 2 (DMT2) en cohortes estadounidenses, chinas, y del norte de Europa, sin embargo estas relaciones no han sido estudiadas en poblaciones del sur de Europa. Objetivos:Evaluar, prospectivamente, el efecto de la ingesta elevada de hierro y el estado elevado de hierro en la aparición de la DMT2 en población mediterránea adulta, y estudiar la relación entre el estado del hierro y la osteocalcina. Métodos:Los participantes fueron hombres y mujeres de 55 a 80 años del estudio PREDIMED. La DMT2 fue diagnosticada utilizando los criterios de la ADA. Se recogieron datos sociodemográficos, antropométricos, de estilo de vida, estado de hierro, perfil lipídico, metabolismo de la glucosa e inflamación. En un estudio observacional en 1.073 personas, 131 diabéticos incidentes, se evaluó la asociación entre la ingesta de hierro y la DMT2. Se analizó transversalmente en 423 sujetos la relación entre el estado de hierro y la osteocalcina. Finalmente, un estudio casos-control anidado fue diseñado para evaluar la asociación entre el estado de hierro, medido mediante la ferritina sérica (FS) y el receptor soluble de transferrina (sTfR),y el riesgo de DMT2 en 459 participantes, 153 diabéticos incidentes. Resultados:Se encontró que una ingesta elevada de hierro hemo aumenta el riesgo de DMT2 en un 30%. Además, los valores de FS>257μg/L en varones y >139μg/L en mujeres se asociaron con DMT2. También se encontró asociación entre el ratio sTfR:ferritina y la DMT2, pero no con el sTfR. Tanto SF como el sTfR se relacionan con la osteocalcina. Conclusiones:En una población mediterránea adulta de alto riesgo cardiovascular, una ingesta elevada de hierro hemo y unos depósitos elevados de hierro aumentan el riesgo de DMT2. La relación entre el estado de hierro y la osteocalcina es controvertida.
Background: The intake of haem iron intake as well as body iron stores have been associated with the risk of developing type 2 diabetes mellitus (T2DM) in U.S., Chinese, and northern European cohorts, however these relationships have not been studied in southern European populations. Objectives: To assess, prospectively, the effect of elevated dietary iron intake and body iron status on the onset of T2DM in an adult Mediterranean population, and to evaluate the relationship between iron status and osteocalcin as a potential mechanism for explaining iron-induced T2DM. Methods: Participants were men and women aged 55 to 80 years from the PREDIMED study. New-onset T2DM during follow-up was diagnosed using the criteria of the American Diabetes Association. Socio-demographic, anthropometric, lifestyle, dietary intake, iron status, lipid profile, glucose metabolism, inflammation variables were collected at baseline. An observational cohort study was conducted in 1,073 individuals, 131 incident diabetics, to assess the association between iron intake and T2DM. A cross-sectional study was carried out in 423 subjects to evaluate the relationship between iron status and osteocalcin. Finally, a prospective nested case-control study was design to assess the association between body iron status, measured as serum ferritin (SF) and soluble transferrin receptor (sTfR) and its ratio, with T2DM in 459 participants, 153 incident diabetics. Results: We found that a high haem iron intake increases the risk of T2DM by 30%. In addition, SF values >257µg/L in males and >139µg/L in females were associated with T2DM. We also found association between sTfR:ferritin ratio and T2DM, but not with sTfR. Both SF and sTfR were related to osteocalcin. Conclusions: In an adult Mediterranean population at high cardiovascular risk, high dietary haem iron and body iron stores increase the risk of T2DM after adjustment for potential confounding variables. The relationship between iron status and serum osteocalcin levels is controversial.

Les maladies auto-immunes chroniques sont la conséquence de la reconnaissance par le système immunitaire d'auto-antigènes comme élément étranger, entraînant une destruction des tissus et organes cibles. Le diabète de type 1 (DT1), la maladie auto-immunes chronique la plus courante, est caractérisé par un insuffisance en insuline due à la destruction sélective des cellules bêta productrices d'insuline. Lors de l'apparition des signes cliniques, plus de 70% de la masse des cellules bêta peut être détruite. Par conséquent, le diagnostic précoce est un objectif majeur afin de limiter l'agression auto-immune, et de créer une fenêtre thérapeutique pour améliorer la survie ou la régénération des cellules bêta. Les approches spécifiques d'antigène (Ag) sont attrayantes du fait de la spécificité de leur mécanisme limité à l'organe cible. Cependant, bien que la prévention du développement du diabète via l'utilisation d'autoantigènes chez la souris non obèse diabétique (NOD) ait été étudiée, les essais cliniques chez l'homme ont produit des résultats décevants. Par conséquent, des approches combinant deux stratégies thérapeutiques pourraient être envisagées. Une stratégie potentielle consiste à co-administrer des auto-antigènes à un traitement antiinflammatoire afin que les deux traitements soient présentés au même moment dans l'environnement des cellules immunitaires auto-réactives. La première partie de ce travail consiste à caractériser physico-chimiquement le vecteur transportant les deux traitements afin d'optimiser la charge médicamenteuse tout en maintenant la biocompatibilité et la stabilité du véhicule de délivrance. Ainsi, des nanoparticules (NPs) d'oxydes de fer superparamagnétiques (USPIO) ont été fonctionnalisées en surface avec des polymères de phosphonate polyéthylène glycol (USPIO-PEG). Les fonctions acide carboxylique ont été utilisées pour lier par covalence un autoantigène DT1. Une molécule antiinflammatoire est piégée par interactions hydrophobes dans les chaines de polymères. Après avoir mis en évidence l'internalisation cellulaire et la non toxicité sur des cellules dendritiques dérivées de la moelle osseuse murine (BMDCs), nous avons entrepris des études de biodistribution et de pharmacocinétique en utilisant le modèle NOD qui partage de nombreuses caractéristiques avec la pathologie humaine. Différentes techniques ont été utilisées à savoir l'IRM, l'histologie et de la magnétométrie sur organe isolé. Les NPs s'accumulent préférentiellement dans le pancréas des souris NOD via un effet de perméabilité et de rétention accrue (EPR effect). Cette bioaccumulation pourrait être exploitée pour la délivrance ciblée du traitement. La deuxième partie du travail consiste à évaluer l'effet thérapeutique de telles nanoparticules tolérogènes sur des souris diabétiques NOD. Des souris diabétiques ont été injectées par voie intraveineuse. Les souris contrôles traitées avec des nanoparticules « nues » ont atteint un niveau de glucose sanguin de 600 mg/dL, considéré comme point limite de l'expérience, en 4-6 jours. Les nanoparticules portant soit la molécule tolérogène soit l'autoantigène ont retardé la progression du diabète jusqu'à 40 jours. Les NP complètes quant à elles, ont montré des effets synergiques. En effet, 50% des souris traitées étaient encore en vie 65 jours après l'apparition de la maladie, et deux souris montrèrent une normoglycémie stable plus de 300 jours après l'apparition de la pathologie. Les nanoparticules tolérogènes induisent une splénomégalie principalement due à la prolifération des lymphocytes B. Les cellules B stimulées par des nanoparticules sécrètent des cytokines anti-inflammatoires, à savoir IL-10 et TGF-bêta. Des cellules B similaires sont également produites in vitro lors de l'incubation avec des nanoparticules. Notre stratégie au potentiel thérapeutique prometteur pourrait être appliquée, en utilisant des antigènes appropriés, à un plus large éventail de maladies auto-immunes
Chronic autoimmune diseases are the consequence of self-antigens recognition as foreign by the adaptive immune system, resulting in inflammation and potential destruction of targeted tissues and organs. Type 1 diabetes (T1D) is one of the most common chronic autoimmune diseases. It is characterized by insulin deficiency due to selective destruction of insulin-producing beta-cells. At clinical onset, more than 70% of beta-cell mass can be destroyed. Consequently, early diagnosis is a major objective in order to avoid, limit or reverse autoimmune aggression, and to create opportunities for strategies enhancing beta-cell survival or regeneration. Antigen (Ag)-specific approaches are appealing because their effects are expected to be limited to cells expressing the chosen antigen, ideally the target organ. However, while treatment with beta -cell Ags can prevent disease in the model of the Non-Obese Diabetic (NOD) mouse, clinical trials in humans have produced disappointing results. Consequently, combinatorial approaches may be required for reversal and prevention of T1D. A potential strategy is to associate self-antigens with signals inducing a tolerogenic phenotype. Co-delivery ensures that both compounds get delivered at the same time and presented in the same cellular environment to auto-reactive immune cells. The first part of this work consisted in undertaking a thorough physicochemical characterization of a new drug vector, aiming to establish quantitative methods to optimize drug loading while maintaining biocompatibility and stability of the delivery vehicle. In this work, 9nm Ultra-small superparamagnetic iron-oxide (USPIO) nanoparticles were surface functionalized with phosphonate polyethylene glycol molecules (USPIO-PEG). Carboxylic acid functions were used to covalently bind a T1D autoantigen. PEG brush allows for the co-packaging of hydrophobic tolerogenic drug molecules, trapped between PEG chains through hydrophobic interactions. We carefully characterized protein and tolerogenic drug loading, and studied cell labeling, toxicity, integrity of loaded protein and tolerogenic drug, and activity of our nanoplatform on murine Bone Marrow Derived Dendritic Cells (BMDCs). We undertook biodistribution and pharmacokinetics studies using the NOD model that shares numerous features with human T1D. Biokinetic studies were performed both qualitatively using MRI 7T and histological Perls staining analyses and quantitatively using magnetometry for NP quantification. USPIO accumulate preferentially in NOD mice pancreas via Enhanced Permeability retention (EPR) effect thus, allowing us to distinguish pre-diabetic mice from non-diabetic controls. This result suggests that vascular leakage could be exploited for NP bioaccumulation, for therapeutic agent delivery and for imaging using MRI agents to monitor treatment. The second part of the work consisted in evaluating the therapeutic effect of such tolerogenic nanoparticles (NPS) on NOD diabetic mice. Diabetic mice were injected intravenously at diabetes onset. USPIO-PEG and vehicle treated mice reached 600mg/dL blood glucose level, considered limit for sacrificing mice, within a couple of days. NPs carrying either the tolerogenic drug or the autoantigen delayed diabetes progression up to 40 days. Complete NPs showed synergistic effects. In fact, 50% of treated mice were still alive 65 days after disease onset, and two mice reverted to stable normoglycemia for more than 300 days. To identify the underlying mechanism, the immune response to NPs in lymphoid organs was investigated. It was found that tolerogenic NPs induce splenomegaly mainly due to B cell proliferation. NP-stimulated B cells secrete anti-inflammatory cytokines namely IL-10 and TGF-beta. Similar B cells could be produced in vitro upon incubation of with NPs. Our strategy has promising therapeutic potential and could be applied, using relevant antigens, to a wider range of autoimmune diseases

A maior expectativa de vida dos pacientes com talassemia maior, as repetidas transfusões de concentrado de hemácias como parte do tratamento, podem ocasionar maior deposição de ferro nos órgãos, e consequentemente, as comorbidades. Dessa forma, dentre as comorbidades endócrinas, temos o diabetes mellitus como uma das principais, sendo necessário conhecer o panorama em nossa realidade. O estudo tem como objetivos determinar a prevalência do diagnóstico de diabetes em pacientes com talassemia maior, caracterizar e comparar os pacientes com talassemia maior, e diabetes mellitus, segundo as variáveis sociodemográficas e clínicas.Trata-se de um estudo descritivo e transversal, realizado em um centro de referência no tratamento de talassemia do interior paulista. A amostra foi constituída por 31 pacientes com talassemia maior. Para a coleta de dados utilizou-se um instrumento subdividido em duas partes. Os dados sociodemográficos e clínicos foram obtidos por meio de entrevista dirigida e os resultados de exames laboratoriais pelo prontuário eletrônico do paciente, no período de junho a agosto de 2015. Os dados foram digitados e importados para o programa SPSS for Windows, versão 17.0 e submetidos à análise estatística descritiva. O projeto foi aprovado pelo Comitê de Ética em Pesquisa da Escola de Enfermagem de Ribeirão Preto da Universidade de São Paulo (CEP/EERP-USP), sob Protocolo nº 41912415.3.0000.5393. Dos 31 pacientes com talassemia maior, 5 (16,1%) tinham diabetes mellitus. Em relação as variáveis sociodemográficas, não houve diferenças na distribuição dos pacientes entre os sexos, a maioria eram solteiros e cursaram até o ensino médio completo. A idade variou de cinco a 48 anos, com média de 24,9 anos de idade, a maioria recebia até 10 salários mínimos, eram estudantes e possuíam carteira de trabalho assinada. No que tange às variáveis clínicas, temos que o tratamento utilizado predominante foi o quelante oral deferasirox, e naqueles pacientes com diabetes, além do deferasirox, a maioria utilizava a insulina. O esquema transfusional predominante foi o de 15 a 22 dias. O índice de massa corpórea foi classificado como eutrófico e a pressão arterial, considerada ótima para a maioria dos pacientes. Quanto aos achados dos exames laboratoriais, os pacientes com diabetes e talassemia apresentaram valores alterados de glicemia de jejum e transaminase, já os pacientes sem diabetes apresentaram valores alterados de ferritina sérica. No que se refere aos achados dos exames de imagem, nenhum paciente com diabetes e Talassemia maior apresentou massa óssea adequada, o que reforça a importância de seu monitoramento. Destaca-se o aumento de sobrecarga cardíaca para os pacientes com diabetes e talassemia. Para os pacientes com talassemia e sem diabetes, a maioria apresentou grave sobrecarga de ferro hepático na ressonância magnética do fígado, enquanto para a maioria dos pacientes com diabetes foi considerado normal. Dessa forma, conhecer as características clínicas dos pacientes com talassemia maior e diabetes permite subsidiar a assistência de enfermagem qualificada e contribuir com a saúde dos pacientes com condições crônicas. Contudo, os nossos achados corroboram com as taxas de prevalência de diabetes em pacientes com talassemia maior encontradas na literatura nacional e internacional
The longer life expectancy of patients with thalassemia major, repeated red blood cell transfusions as part of the treatment, may lead to increased iron deposition in the organs, and consequently, comorbidities. Thus, among the endocrine comorbidities, we have diabetes mellitus as one of the main ones, being necessary to know the panorama in our reality. The aim of the study was to determine the prevalence of diabetes in patients with thalassemia major and to characterize and compare patients with thalassemia major, and diabetes mellitus, according to sociodemographic and clinical variables.This is a descriptive and cross-sectional study, carried out in a reference center in thalassemia treatment of São Paulo countryside. The sample consisted of 31 patients with thalassemia major. For data collection, an instrument was divided into two parts. Sociodemographic and clinical data were obtained by means of a directed interview and the laboratory tests results by the patient\'s electronic record, from June to August 2015. Data were loaded into SPSS for Windows software, version 17.0 and submitted to descriptive statistical analysis. The project was approved by the Research Ethics Committee of the University of São Paulo at Ribeirão Preto College of Nursing (CEP / EERP-USP) under Protocol No. 41912415.3.0000.5393. Of the 31 patients with thalassemia major, 5 (16,1%) had diabetes mellitus. Regarding the sociodemographic variables, there were no differences in the distribution of the patients between the sexes, most of them were single and enrolled in high school. The age ranged from five to 48 years, with a mean of 24,9 years of age, the majority received up to 10 minimum wages, were students and had a work contract. Regarding the clinical variables, we have that the predominant treatment was the oral chelator deferasirox, and in those patients with diabetes, in addition to deferasirox, the majority used insulin. The predominant transfusion regimen was 15 to 22 days. Body mass index was classified as eutrophic and blood pressure was considered optimal for most patients. Regarding the laboratory findings, patients with diabetes and thalassemia had altered values of fasting glycemia and transaminase, whereas patients without diabetes had altered values of serum ferritin. Regarding imaging exams findings, no patient with diabetes and major thalassemia presented adequate bone mass, which reinforces the importance of their monitoring. The increase in cardiac overload for patients with diabetes and thalassemia stands out. For patients with thalassemia and without diabetes, the majority had severe hepatic iron overload in the magnetic resonance imaging of the liver, while for most patients with diabetes it was considered normal. Thus, knowing the clinical characteristics of patients with thalassemia major and diabetes allows subsidizing qualified nursing care and contributing to the health of patients with chronic conditions. However, our findings corroborate the prevalence rates of diabetes in patients with thalassemia major found in the national and international literature

Alzheimer's disease (AD) is the most common form of dementia, afflicting almost 5 million patients in the US, and impacting millions more, financially, physically and emotionally. Coming in as the 6th leading cause of death in the US, and showing no signs of slowing its annually increasing rates, the world is in desperate need of improved understanding of the disease's multifaceted pathogenesis and progression, more accurate forms of detection and diagnosis, and more effective prevention and treatment. While many are focused on the noble pursuit of understanding the genetic contributions to the appearance of the pathological amyloid beta (Aβ)) plaques and tau tangles seen in AD, the majority of cases are not explained by genes or allele risk. Instead environmental, dietary and lifestyle contributors may be the key to understanding, diagnosing and treating this awful disease. Diet especially may impact the body's ability to regulate oxidative stress, which will cause damage within the cell and lead to further dysregulation of iron storage and metabolism. Iron storage is heavily monitored through cellular mechanisms, and the way in which the body reacts involves creation of the Aβ plaques and tau tangles as receptacles for the molecule it has deemed as the cause of the problem, iron. We have aptly named our theory, the Iron Hypothesis, and in the following document will outline the evidence for this hypothesis, and the experiments designed and performed to prove it.First, we aimed to examine the impacts that various treatments would have on a transgenic in-vivo model, examining the cohorts' behavior over several time points. We report a significant difference in the behavioral measures of time, distance, errors and failed trials in the radial arm maze existed between genotype, treatment and sex of the mice. Tissue of the experimental mice was collected, but will be processed and analyzed at a later date.Secondly, we aimed to examine the same cohorts of the in-vivo mouse model for minute anatomical changes that took place over the course of the aforementioned behavioral trials using novel MRI scanning sequences sensitive to the low levels of iron build up. We report significant differences in the UTE scan measures for our western diet treatment at TE's of 1.2ms. Additionally, further investigation and optimization of the protocol may be required to further expand the findings.

The aim of this thesis is to contribute to the understanding of iron metabolism, as a factor associated with cardiometabolic risk, by undertaking secondary data analyses. The objectives were to identify gaps in existing knowledge in terms of populations studied and alternative iron markers, and to attempt to fill the gaps with additional analyses and interpretation. Serum ferritin was the most widely available measure of iron status but the role of serum transferrin and soluble transferrin receptor (sTfR) levels was considered where available. I have taken a life-course approach with analyses in childhood and adulthood, and have included both intermediate factors such as the metabolic syndrome (MetS), and disease diagnoses of diabetes and cardiovascular disease as outcomes. Chapter one presents a review of empirical research literature on the relationship between iron metabolism and cardiometabolic risk, concepts surrounding iron markers and the study outcomes. This chapter also describes the gaps in understanding the iron-cardiometabolic risk relationship, which are subsequently explored in chapters two to six. Chapter two explores the link between serum ferritin and transferrin and MetS in cross-sectional and prospective studies of 725 Spanish children and 567 Chilean adolescents. I found associations between both ends of the ferritin distribution and MetS or glucose metabolism markers in different paediatric populations. For instance, whereas in the Spanish children there was a decrease of 0.02 SD units in the change of MetS score over time for every SD unit increase in ferritin, in the Chilean male adolescents being in the highest tertile of ferritin (v. the lowest) was associated with an increase of 0.25 SD units of MetS score. Furthermore, sustained high ferritin levels at various time points and gradual increase of ferritin during childhood were associated with higher MetS score in adolescence. The third chapter describes the association between serum ferritin status and MetS in adults in two cross-sectional studies of Scottish populations (2,047 individuals from Shetland Islands and 8,563 subjects from the Scottish Health Surveys (SHeS) 1995- 1998). I also examined the overall association between ferritin, MetS and each MetS component in adults, by conducting a meta-analysis and investigating potential relevant sources of heterogeneity for the association. Interestingly, ferritin levels were positively associated with MetS in the Scottish populations, but the association was not independent of the effect of covariates, mainly body mass index (BMI) and transaminase levels [Men Odds ratio (OR) 95% confidence interval (CI) 1.43(0.83- 2.46); Postmenopausal women OR (95%CI) 1.09(0.62-1.90); Premenopausal women OR (95%CI) 1.02(0.42-2.46), P > 0.05]. The meta-analysis supported this finding by describing hepatic injury markers and BMI as the major attenuating factors of the ferritin-MetS association. Chapter four investigates the association between sTfR or ferritin, and MetS in 725 Croatian adults in a cross-sectional study. There was no evidence of an association between sTfR and MetS [Men OR (95%CI) 1.35(0.90-2.02); Postmenopausal women OR (95%CI) 0.73(0.47-1.15); Premenopausal women OR (95%CI) 0.87(0.66-1.17), P > 0.05]. In contrast serum ferritin, was positively and independently associated with MetS in men and postmenopausal women (P < 0.05) [Men OR (95%CI) 1.78(1.31- 2.42); Postmenopausal women OR (95%CI) 1.71(1.12-2.62); Premenopausal women OR (95%CI) 1.24(0.85-1.80)]. These contrasting results suggest that different iron markers reflect different physiological processes other than iron metabolism. Chapter five evaluates the longitudinal association between serum ferritin and several cardiometabolic disease outcomes (CMDs) in the nationally representative SHeS 1995 and 1998 (n = 6,497). I found an independent positive longitudinal association between ferritin and cerebrovascular disease (CEVD), which was strengthened by using higher cut-points for increased ferritin [higher v. lowest sextile fully adjusted Hazard ratio(HR) 95%CI 2.08 (1.09-3.94), P=0.024], and a not significant association with coronary heart disease (CHD) after adjustment for covariates. My analyses confirmed the widely established association with type 2 diabetes (T2D) [whole sample fully adjusted HR 95% CI 1.59(1.10-2.34), P=0.006], even with serum ferritin within the normal range. The above set of observations confirm ferritin as biomarker mainly related to the development of T2D and identifies the need to investigate the association between ferritin and CEVD in other populations. Chapter six investigates whether ferritin is associated with risk for cardiovascular complications among people with T2D using cross-sectional study designs in two populations with differing baseline cardiovascular risk (Spanish study SIDIAP n=38,617) and (Edinburgh Type 2 Diabetes Study (ET2DS) n= 821) with additional analysis of follow-up data for ET2DS. Interestingly, ferritin levels were negatively associated with prevalence of cardiovascular disease, mainly CHD, in people with T2D in both studies [ET2DS OR (95%CI): 0.80(0.67-0.96), P=0.020; SIDIAP study: 0.85(0.83-0.88), P < 0.001). Ferritin was also negatively associated with incident cardiovascular disease in ET2DS: HR 95% CI: 0.39(0.16-0.93), P=0.035. Therefore, the association between iron status and CMD risk in people with T2D appears to differ from that in general populations in which a positive association has been more commonly described. In conclusion, serum ferritin is associated with cardiometabolic risk in different ways in a variety of populations. Inconsistent associations for other iron markers suggest that iron biomarkers reflect factors other than iron homeostasis that influence cardiometabolic risk. The association between iron markers and MetS appears to differ between populations. This thesis illustrates the complex relationship between iron metabolism markers, MetS and CMD, and identifies the need for further research on the topic in order to extend knowledge about pathophysiology and the potential for measures of iron status as biomarkers for CMD.

Arachidonic acid (AA), docosahexaenoic acid (DHA) and iron are important nutrients for infants and are hypothesized to be compromised in infants of diabetic mothers (IDM). The objective of this study was to compare AA, DHA and iron status in IDM with those measured in infants of non-diabetic mothers born both appropriate for gestational age (AGA) and large for gestational age (LGA); and to determine if infants that have compromised AA and DHA status also have a compromised iron status. Analyses of covariance between AA, DHA and iron revealed that AA in infants was positively correlated with iron status overall, but this was not the case for DHA. In IDM, the relationship between iron and AA status was more pronounced. These data suggest iron is linked to AA status in all infants and that IDM are at higher risk for low AA status and consequently impaired development.
L'acide arachidonique (AA), l'acide docosahexaénoïque (DHA) et le fer sont d'importants nutriments pour le nouveau-né. Il semblerait que leurs niveaux sont compromis chez les enfants de mères diabétiques (EMD). L'objectif de cette étude est de comparer les niveaux d'AA, de DHA et de fer chez les EMD avec ceux mesurés chez les enfants de mères non diabétiques qui sont nés avec un poids moyen ou gros pour l'âge gestationnelle pour déterminer si les niveaux de gras insaturés à longues chaînes et de fer sont tous les deux amoindris chez l'enfant. L'analyse de covariance entre le fer et les gras insaturés a montré que le niveau de fer est positivement corrélé avec le niveau d'AA mais pas avec celui de DHA. Chez les EDM, le lien entre le fer et l'AA est encore plus marquant. Ces résultats suggèrent que le niveau de fer est relié avec le niveau d'AA chez tous les nouveau-nés et qu'il y a un plus grand risque chez les EDM d'avoir un niveau d'AA amoindri et par conséquent de souffrir de problèmes de croissance.

[Truncated abstract. Please see the pdf format for the complete text.] The discovery of the C282Y mutation and its role in the development of hereditary haemochromatosis has allowed a greater understanding into the effects of iron overload and its involvement in other conditions such as diabetes and heart disease. It has also allowed the better classification of heterozygotes, who were previously only diagnosed through the use of family studies. There are however, areas of conflict between phenotyping and genotyping methods. My research involved examining the relationship between Haemochromatosis and certain diseases such as diabetes and heart disease; genotyping versus phenotyping discrepancies and the possible interaction of secondary mutations. In Chapter 3 a population study was undertaken with the aim of comparing genotyping versus phenotyping methods as well as increasing general practitioner awareness regarding hereditary haemochromatosis and its diagnosis. It was determined that a minimum of 5000 subjects would be required to give the study sufficient power. Individuals were to be between the ages of 20—40 years, and thus presumably presymptomatic. Participation was entirely voluntary and a consent form was to be signed. Recruitment of subjects proved to be difficult and there was a selective bias towards individuals already displaying symptoms of haemochromatosis. In total less than a 100 subjects were recruited for the study. There were several issues encountered in the implementation of this study. Firstly the number of GPs participating was probably insufficient to recruit the subjects required. A more extensive campaign was probably required to enroll more GPs. Secondly it is very difficult for a busy GP to find the time necessary to explain the study to each of his patients and to get them to sign the consent form. Finally a bias developed in some of the requests. The subjects participating in this study were supposed to be random but in many cases the GPs had enrolled them in the study because they had symptoms of iron overload. In effect the biggest obstacle this study faced was the recruitment of subjects. Due to the small number of subjects little statistical data could be obtained from this study. It was noted, however, that genotyping methods detected two individuals who were homozygous for the C282Y mutation. Both also had increased transferrin saturation levels. Phenotyping detected 5 individuals with increased transferrin saturation. The three others detected via phenotyping were C282Y heterozygotes. Haemochromatosis has long been though to be related to the development of diabetes due to the effect of iron overload on the pancreas. If this is so it would be logical to assume that the prevalence of haemochromatosis would be higher in a diabetic population. Chapter 4 examined the possibility that diabetics have a higher frequency of the C282Y mutation. A population group consisting of 1355 diabetics was genotyped for the C282Y mutation and iron studies were performed on all heterozygotes and C282Y homozygotes. Initial findings indicated that there was a significant difference between the diabetic and control population. However, this finding was the opposite of what was expected, there seemed to be a decreased frequency of the Y allele in the diabetic population rather than an increased one. The control and diabetic populations were not matched in terms of ethnicity. The removal of the ethnic bias in the diabetic population altered the statistics so there was no longer a significant difference between the two groups. This study highlighted the importance of using appropriate control populations as comparison groups. The final results of the study indicated that there was no significant difference between the diabetic population and the control population. This would seem to indicate that there is not an increased occurrence of the C282Y mutation in the diabetic population when compared to the control group. Chapter 5 considered the possible association between C282Y heterozygosity and cardiovascular disease as well as the potential for early mortality. Several recent studies have indicated that C282Y heterozygosity may be a risk factor for the development of atherosclerosis, possibly on the basis of increased iron loading. Using a control population and a population of individuals with known coronary events the incidence of the C282Y mutation was compared against other risk factors. C282Y heterozygosity did not appear to be a risk factor for atherosclerosis. There was however, a statistically significant link between increased ferritin in women and carotid plaques. A population of elderly women was genotyped in order to examine the effects of C282Y heterozygosity on longevity. The first hypothesis addressed in chapter 5 was that C282Y heterozygosity was a risk factor for the development of coronary heart disease.

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Dissertação (Mestrado em Tecnologia Nuclear)
IPEN/D
Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

Thesis (Ph.D. in Epidemiology) -- University of Colorado at Denver and Health Sciences Center, 2006.
Typescript. Includes bibliographical references (leaves 168-182). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;

Des études ont récemment mis en évidence le rôle des lipopolysaccharides (LPS) des bactéries à Gram négatif de la flore intestinale dans le processus de l’inflammation conduisant à l’obésité et au diabète de type 2.Le présent travail est réalisé dans le cadre d’une collaboration entre les équipes du Dr. Caroff (U. Paris-Sud, Orsay) et du Pr. Zhao (U. Jiao Tong, Shanghai). Les expériences présentées ont été réalisées lors de séjours dans les deux laboratoires.Il a été démontré en Chine que des bactéries Sulfato-réductrices (SRB) à Gram négatif étaient présentes en plus forte proportion dans la flore intestinale chez les souris suivant un régime gras. Les mêmes résultats ont été observés chez l'homme. L’hypothèse selon laquelle des SRB seraient à l’origine de grandes quantités d’endotoxines chez les obèses et les patients diabétiques a été émise. Plusieurs souches de SRB isolées de la flore intestinale humaine d’un sujet sain et d’un sujet diabétique ont été cultivées en Chine. Des études de relation structure/activité des LPS isolés de ces bactéries ont été réalisées dans le laboratoire Français pour déterminer leur rôle dans le développement des maladies métaboliques. Les souches isolées des deux sujets ont pu être classées dans le genre Desulfovibrio. Les LPS correspondants ont été extraits et purifiés par des méthodes mises au point dans l’équipe d’Orsay. La structure chimique a été élucidée par les méthodes suivantes : Electrophorèse, Chromatographie sur couche mince, Chromatographie en phase gazeuse et Spectrométrie de masse MALDI. C’est ainsi que des spectres de masse ont été obtenus et que la structure des lipides A, principes actifs des LPS, isolés de SRB a été décrite pour la première fois. Les activités biologiques testées (TNFα, IL-6) varient en fonction du nombre d’acides gras présents. Les LPS de SRB du patient sain ont une structure variable (Smooth versus Rough) en fonction de la quantité de fer présent dans le milieu, et ceux isolés du patient diabétique présentent des structures atypiques qui ne sont pas toutes inflamogènes. Une molécule membranaire inconnue, que nous avons nommée « Glycosyl’X » était co-extraite avec les LPS. Elle joue apparemment un rôle important dans la croissance des SRB et a été étudiée après des étapes de purification complexes. Les structures et le pouvoir inflammatoire de ces molécules dont la structure varie avec les souches, et qui chélatent le fer, ont été étudiées. Elles sont de nature principalement osidique et fixées à la membrane. La proportion de ces molécules par rapport aux LPS varie avec la quantité de fer disponible dans le milieu. Un milieu riche en fer favorise la croissance des Desulfovibrio portant les Glycosyl’X qui n’ont pas de pouvoir inflammatoire eux-mêmes, mais entrent en compétition avec les LPS, modulant ainsi indirectement l’activité de ces derniers. L’augmentation du nombre de Desulfovibrio conduisant à l’augmentation des molécules Glycosyl’X pourrait aussi moduler positivement (par présentation) ou négativement (par élimination des bactéries) l’adsorption du fer dans les intestins dont l’équilibre est essentiel pour l’homéostasie métabolique.Par ailleurs, la croissance des Desulfovibrio augmente la production d’Hydrogène Sulfuré connu pour son action délétère sur les cellules. Nous favorisons l’hypothèse selon laquelle son action sur la disjonction des cellules épithéliales permettrait le passage des différents LPS relargués par la flore Gram-négative intestinale, et même des bactéries entières, vers la circulation sanguine
Recent studies have highlighted the role of lipopolysaccharide (LPS) in the intestinal flora (gut microbiota) which could contribute to the inflammation process leading to obesity and type 2 diabetes. This thesis is part of a collaborative project between the laboratories of Dr. Caroff (U. Paris -Sud, Orsay, France) and Prof. Zhao (U. Jiao Tong , Shanghai, China). It has been shown by Pr.Zhao’s team in 2010 that the Sulfate -Reducing Bacteria (SRB) were presented in greater proportion in the intestinal mice flora following a fat diet compared to mice following a normal diet. The same results were observed in humans. The starting hypothesis was that SRB could produce a large amount of endotoxin in obese and diabetic patients and play a role in the development of metabolic diseases. Several SRB strains isolated from the human intestinal flora of a healthy subject and of a diabetic subject were grown in the Chinese laboratory. Studies of their LPS structure / activity relationships were carried out in the French laboratory. The aim of this study was to determine their roles in the development of metabolic diseases.Strains isolated from the two subjects could be classified in the Desulfovibrio genus. The corresponding LPS were extracted and purified by the methods developed in the French laboratory. The chemical structure was elucidated by the following methods: Electrophoresis, Thin layer chromatography, Gas chromatography and MALDI mass spectrometry. The mass spectra were obtained and the structure of lipid A, the active part of LPS isolated from SRB was described here for the first time. The biological activities test (TNFα, IL-6) vary depending on the number of fatty acids present in their lipid A structure. The LPS of SRB isolated from the healthy patient had a variable structure (Smooth versus Rough) depending on the amount of iron present in the medium, and those isolated from diabetic patients had atypical structures are not all inflamogenic .An unknown membrane molecule, which we named "Glycosyl'X" was co-extracted with the LPS. It apparently plays an important role in the growth of SRB was investigated after complex purification steps. The structures and the inflammatory power of these molecules variying with strains chelating iron were studied. They are mainly of glycosidic nature and linked to the bacterial membrane.The proportion of these molecules relatively to LPS varies with the amount of iron in the medium. An environment rich in iron promotes the growth of Desulfovibrio Glycosyl'X, molecules but competes with LPS and indirectly modulates the activity of the latter. The increase number of Desulfovibrio leading to increased Glycosyl'X molecules may also modulate positively (by presentation) or negatively (by killing bacteria) the absorption of iron in the intestines which balance is essential for metabolic homeostasis.Furthermore, the growth of Desulfovibrio increasing the production of Hydrogen Sulfide is known for its deleterious effects on the cells. We favor the hypothesis that its action on the separation of epithelial cells favors the passage of different LPS released by the Gram- negative of intestinal flora and even whole cell bacteria into the bloodstream

abstract: The effects of iron and chromium blood concentrations have been linked to blood glucose control in diabetics. It is suggested that iron causes oxidative stress in the beta cells of the pancreas and adipocytes creating insulin insufficiency and resistance. Chromium is believed to increase the action of insulin through its biologically active molecule chromodulin. Both of these mechanisms are not clear. This 20 week case study tests the feasibility of combining iron depletion therapy followed by chromium supplementation to improve insulin sensitivity. This single case study followed a protocol of two blood donations separated by eight weeks followed by chromium supplementation of 250 µg of chromium picolinate once a day four weeks after the second blood donation. Fasting blood draws were taken at baseline, post blood draws and pre and post chromium supplementation. Results were not promising for the first hypothesis of lowering HbA1c, but the results were promising for the second hypothesis of improving insulin sensitivity by lowering the HOMA score.
Dissertation/Thesis
Masters Thesis Nutrition 2015

The goal of this study was to compare different anthropometric measures in terms of their ability to predict T2DM and to determine whether predictive ability was modified by ethnicity. Anthropometrics were measured at baseline on 1073 non-Hispanic Whites (nHW), African Americans (AA) and Hispanics (HA), of which 146 developed T2DM after 5.2 years. Logistic regression models were used with areas under the receiver operator characteristic curve (AROC) comparing the prediction of models. Overall, there was no clear distinction between measures of overall and central obesity in terms of T2DM prediction. Waist-height ratio (AROC=0.678) was the most predictive measure, followed by BMI (AROC=0.674). Results were similar in nHW and HA, although, in AA, central adiposity measures best predicted T2DM. Measures of central and overall adiposity predicted T2DM to a similar degree, except in AA where central measures were most predictive.

Dissertação de mestrado em Farmacologia Aplicada, apresentada à Faculdade de Farmácia da Universidade de Coimbra
O glucagon-like peptide I (GLP-1) corresponde a um dos principais, e mais recentes, alvos terapêuticos para o tratamento da Diabetes Mellitus tipo II, pois desempenha várias funções metabólicas benéficas para o tratamento desta patologia, tais como, a potenciação da secreção de insulina induzida pela entrada de nutrientes no organismo (“efeito incretina”), a estimulação da biossíntese de insulina, a inibição da secreção de glucagina e a melhoria da função das células beta-pancreáticas. O GLP-1 é também caraterizado por promover cardioproteção, induzir a saciedade, inibir o esvaziamento gástrico e a secreção ácida gástrica, e ainda, inibir a motilidade gastrintestinal através da ativação de recetores (GLP-1R) presentes em neurónios entéricos e por via da libertação de óxido nítrico. Têm sido desenvolvidos novos fármacos agonistas do recetor do GLP-1 (GLP-1RA) que mimetizam os efeitos do agonista endógeno e que são resistentes à degradação enzimática pela DPP-IV. Os GLP-1RA também têm sido aplicados na terapêutica da obesidade devido ao efeito significativo desta classe farmacológica na inibição do apetite e consequente perda de peso. O objetivo deste projeto consistiu no estudo do efeito dos GLP-1RA, GLP-1 e Liraglutido, sobre a motilidade gástrica e de que forma esta está alterada na presença da diabetes tipo II. Para esse fim, foram usadas tiras de fundo de estômago isolado de ratos Wistar (grupo controlo) e Goto-Kakizaki (rato GK, modelo animal diabético do tipo II não obeso), com dezasseis semanas de idade, para a realização de estudos funcionais de registo da contração isométrica de curvas cumulativas concentração-resposta (CR) dos GLP-1RA (0,05 nM - 111,1 nM) após pré-contração com carbacol 5 μM. Ambos os GLP-1RA induziram contração tónica, não colinérgica e dependente da concentração, sendo que o GLP-1 foi mais eficaz na indução da contração de fundo gástrico isolado de rato GK comparativamente ao rato Wistar, enquanto os dois GLP-1RA foram equivalentes no rato Wistar. Por outro lado, o Liraglutido foi o mais potente nos animais controlo, sendo que ambos os GLP-1RA foram equipotentes nos animais diabéticos. Comparando ambos os modelos animais, o GLP-1 foi mais eficaz em rato diabético (comparado com rato Wistar). Este aumento de eficácia da resposta contrátil ao agonista endógeno pode relacionar-se com a tendência para o aumento da expressão do GLP-1R observada por Western Blotting em lisados de fundo de estômago isolado de rato GK. O aumento da expressão do recetor pode ser promovido pela patologia como mecanismo compensatório, uma vez que foi comprovada anteriormente por outro grupo de investigadores a diminuição dos níveis plasmáticos e pós-prandiais de GLP-1 em rato GK (comparado com o rato Wistar). Para a caracterização farmacológica da resposta contrátil aos GLP-1RA, realizaram-se segundas curvas CR ao GLP-1 em tiras de estômago de ratos Wistar, na presença e na ausência de 300 nM de Exendin-3 (antagonista seletivo do recetor do GLP-1) e curvas cumulativas CR ao Liraglutido na presença e na ausência de 250 M de NG-nitro-L-arginina (L-NNA, inibidor não seletivo da sintase do óxido nítrico), ambos adicionados ao banho de órgãos trinta minutos antes da execução das curvas. Na presença do antagonista Exendin-3, foi observada uma redução estatisticamente significativa (p <0,05) de 55% do efeito máximo de contração induzida pelo GLP-1, sem alteração da potência, concluindo-se que a resposta contrátil aos GLP-1RA é mediada parcialmente pelo seu recetor específico. Na presença do inibidor L-NNA, não foram observadas alterações estatisticamente significativas na resposta contrátil ao Liraglutido, e portanto, esta não parece ser dependente da sintase do óxido nítrico. Procurando compreender melhor o observável efeito contráctil dos GLP-1RA na nossa preparação, procedeu-se à avaliação do efeito do Liraglutido no relaxamento induzido por um dador de óxido nítrico, o nitroprussiato de sódio e pela noradrenalina, em tiras de fundo de estômago isoladas a partir de ratos Wistar e ratos GK tratados com o fármaco. Para tal, animais com catorze semanas de idade foram divididos em quatro grupos: ratos Wistar e ratos GK administrados com Liraglutido (200 μg/kg s.c.), duas vezes por dia e durante catorze dias; paralelamente ratos Wistar e ratos GK foram administrados com soro fisiológico NaCl 0,9% (grupo controlo). Foram realizadas medições diárias do peso corporal e a ingestão calórica foi avaliada semanalmente. Os parâmetros bioquímicos, glicemia em jejum (de seis horas), triglicerídeos e colesterol total, e a sensibilidade periférica à insulina foram avaliados no primeiro e no último dia de tratamento com Liraglutido. Após o tratamento e sacrifício dos animais, foram realizadas curvas cumulativas CR de relaxamento isométrico ao SNP e à NA (0,01 μM - 631 μM) em tiras de fundo de estômago isolado de ratos Wistar e GK tratados e não tratados com Liraglutido e pré-contraídas com carbacol 5 μM. Comprovámos que este GLP-1RA induziu melhorias no perfil glicémico e lipídico e na sensibilidade periférica à insulina nos animais diabéticos sujeitos ao fármaco, e também, redução da ingestão calórica e perda de peso nos ratos tratados. Portanto, estes resultados são bons indicadores dos efeitos benéficos dos GLP-1RA para o tratamento da diabetes tipo II e da obesidade. Quanto à resposta contrátil do órgão isolado, uma diminuição no relaxamento induzido pelo SNP foi observada em ratos Wistar e GK tratados com Liraglutido (este efeito foi mais acentuado nos animais controlo). A perda de eficácia do efeito do SNP com o tratamento pode resultar de um possível efeito do Liraglutido na diminuição da libertação ou na degradação do óxido nítrico, sendo que um efeito direto do GLP-1RA sobre a síntese deste neurotransmissor é improvável, uma vez que não foram observadas alterações significativas na expressão da nNOS e da p-nNOS em lisados de fundo gástrico isolado de ratos controlo e diabéticos, tratados e não tratados com Liraglutido. Além disso, é possível que os GLP-1RA atuem por via da ativação da eNOS, em detrimento da nNOS. Deste modo, a redução da expressão do GLP-1R, como foi demonstrada em ratos Wistar e GK tratados com Liraglutido, pode resultar numa redução da ativação da eNOS, o que por sua vez, resultaria numa diminuição da síntese e da libertação de óxido nítrico e na consequente inibição do relaxamento do músculo liso gástrico. De fato observou-se imunomarcação do GLP-1R em células endoteliais de vasos sanguíneos co-localizados no plexo nervoso deste órgão. Contudo, há que salientar que não foram observadas alterações significativas na curva CR ao Liraglutido na presença de L-NNA, um inibidor não seletivo da NOS. Sobre a redução da expressão do GLP-1R em fundo gástrico isolado de ratos tratados com Liraglutido, esta pode ser consequência de uma possível internalização do GLP1-1R induzida por uma estimulação prolongada por parte do GLP-1RA. Além disso, também é possível que a perda de peso e a inibição do apetite observada nos ratos tratados com Liraglutido resulte num aumento da secreção de GLP-1 e consequente diminuição da expressão de GLP-1R (mecanismo de feedback negativo). O efeito do SNP é equipotente em ratos Wistar tratados e não tratados com Liraglutido, no entanto, foi observada perda de potência do efeito relaxante do SNP em animais diabéticos tratados com o GLP-1RA, sendo que esta pode ser explicada pela perda de neurónios nitrérgicos, como consequência da diabetes tipo II, e deste modo, diminui o relaxamento por diminuição da síntese e libertação de óxido nítrico endógeno. De facto no nosso estudo observou-se uma tendência para diminuição da expressão da nNOS e/ou p-nNOS no rato GK, relativamente ao rato Wistar. No caso da noradrenalina, foi observada uma perda significativa de potência relativamente à situação controlo, determinada quer pela patologia, quer pelo tratamento. Sendo que a perda de potência da noradrenalina com o tratamento pode relacionar-se com a sub-regulação da expressão do GLP-1R observada nos ratos tratados com Liraglutido, o que por sua vez, pode resultar numa redução da ativação de vias nervosas adrenérgicas. Enquanto a perda de potência da noradrenalina com a patologia pode ser explicada por uma possível sub-regulação da expressão de recetores beta-adrenérgicos em fundo gástrico isolado de rato diabético. Em ambos os casos, a ação da NA estaria diminuída, assim como, o relaxamento induzido por esta. Por fim, a imunomarcação do GLP-1R foi detetada nas células parietais das glândulas gástricas, nas células musculares lisas, nas células ganglionares do plexo nervoso e nas células endoteliais dos vasos sanguíneos, em fundo gástrico isolado de rato. Finalmente, a realização de novos estudos é necessária para compreender melhor a real contribuição do óxido nítrico no mecanismo de ação subjacente ao efeito dos GLP1-RA sobre a motilidade gástrica. E além disso, o uso de animais diabéticos com uma idade mais avançada, ou até mesmo o uso de um outro modelo animal de diabetes tipo II, poderia ajudar a entender melhor as alterações promovidas pela patologia na motilidade gástrica.
Glucagon-like peptide I (GLP-1) has been studied in the development of new therapeutic strategies for the treatment of type II diabetes because of its metabolic effects, like stimulation of insulin secretion induced by nutrients (incretin effect), stimulation of insulin biosynthesis, inhibition of glucagon secretion and improvement of beta-cell function. GLP-1 also promotes cardioprotection, induces satiety, delays gastric emptying, inhibits acid gastric secretion and inhibits gut motility through activation of GLP-1R on enteric neurons and via nitric oxide release. GLP-1R agonists (GLP-1RA) are drugs that mimic the effects of GLP-1 and they are resistant to enzymatic degradation by DPP-IV. GLP-1RA have also been used in the treatment of obesity because of its significant effects in inhibition of appetite and weight loss. The main aim of this project was to study the effects of GLP-1RA, GLP-1 and Liraglutide, in gastric motility and its alterations in the presence of type II diabetes. To this end, gastric fundus strips from Wistar rats (control group) and Goto-Kakizaki rats (GK rat, animal model of spontaneous type II diabetes and non-obese), with sixteen weeks of age, were used in functional studies. Isometric contractile cumulative concentration-response (CR) curves for GLP-1RA were performed (0,05 nM - 111,1 nM) and all gastric fundus strips were pre-contracted with carbachol 5 μM. Both GLP1-RA induced concentration-dependent, non-cholinergic and tonic contraction of isolated rat gastric fundus. GLP-1 was the most effective in GK rat (compared to Wistar rat) and both GLP-1RA were equivalent in Wistar rat. Inversely, Liraglutide was the most potent in the control group and both agonists were equipotent in diabetic rats. Comparing both animal models, GLP-1 was more effective in diabetic rats than in control rats. This increased efficacy of GLP-1 in diabetic rats can be explained, in part, by the tendency of up-regulation of the GLP-1R expression determined, by Western Blotting, in lysates from isolated gastric fundus from GK rat (compared to Wistar rat). This increased GLP-1R expression can be promoted by type II diabetes and can result from a compensation mechanism since a reduction in plasma and post-prandial levels of GLP-1 was reported in GK rat (compared to Wistar rat). xvi For pharmacological characterization of the contractile response to GLP-1RA, second cumulative CR curves for GLP-1 in gastric fundus strips from Wistar rats were performed in the absence and in the presence of Exendin-3, 300 nM (selective GLP-1R antagonist) and cumulative CR curves for Liraglutide were performed in the absence and in the presence of NG-nitro-L-Arginine, 250 μM (L-NNA, non-selective inhibitor of nitric oxide synthase). Both (Exendin-3 and L-NNA) were added to the organ bath thirty minutes before the CR curves. The GLP-1R antagonist, Exendin-3, caused a statistically significant (p <0,05) reduction of the contractile response induced by GLP-1, without changes in potency, so we can conclude that contraction of gastric fundus induced by GLP-1RA is partially mediated by its specific receptor. No significant alterations in contractile response to Liraglutide were observed in the absence and in the presence of inhibitor L-NNA, therefore gastric fundus contraction induced by GLP-1RA is independent of nitric oxide synthase. To better understand the contractile response to GLP-1RA, the effect of the treatment with Liraglutide in isometric relaxation induced by a nitric oxide donor, sodium nitroprusside (SNP) and by noradrenaline, in isolated gastric fundus from Wistar and GK rats was evaluated. To this end, Wistar and GK rats with fourteen weeks of age were divided into four groups: Wistar and GK rats treated with Liraglutide (200 μg/kg s.c.) twice daily and for fourteen days; and Wistar and GK rats treated with saline (NaCl 0,9% s.c.) during the same period. Body weight and caloric intake were evaluated daily and weekly, respectively. Biochemical analyses (fasting glycaemia, triglycerides and total cholesterol) and insulin tolerance evaluation were performed on the first and on the last day of the treatment. After treatment with Liraglutide, cumulative CR curves for SNP and NA were performed (0,01 μM - 631 μM) and all gastric fundus strips from Wistar and GK, treated and non-treated, rats were pre-contracted with carbachol 5 μM. We proved that Liraglutide induced an improvement of biochemical profile and peripheral insulin tolerance in diabetic rats, and also, a significant decrease in caloric intake and body weight in treated-rats. Therefore, we can conclude that GLP-1RA are beneficial for the treatment of type II diabetes and obesity. xvii A decrease in relaxation induced by SNP was observed in Wistar and GK rats treated with Liraglutide and this effect was more significant in Wistar rats. The loss of efficacy of SNP effect determined by the treatment with Liraglutide can be explained by a possible effect of this GLP-1RA in reduction of release or in degradation of nitric oxide. A direct effect in nitric oxide synthesis is unlikely to occur because no significant alterations were observed in nNOS and p-nNOS expression in isolated gastric fundus from treated and non-treated rats. Besides that, if GLP1-RA could act through eNOS activation, instead of nNOS, a reduction in GLP-1R expression (demonstrated, in this study, in Wistar and GK rats treated with Liraglutide) could result in a reduction of eNOS activation and consequently, in a reduction of nitric oxide release and relaxation of gastric smooth muscle. In fact GLP-1R was detected (by immunohistochemical studies) in endothelial cells of blood vessels co-localized in nervous plexus of gastric fundus. But no significant alterations were observed in CR curves to Liraglutide in the presence of L-NNA (a non-selective inhibitor of NOS). About the significant reduction of GLP-1R expression in isolated gastric fundus from rats treated with Liraglutide (compared to non-treated rats), it can be explained by a compensation mechanism between a probable increase of plasmatic levels of GLP-1 induced by weight loss and inhibition of appetite and the reduction of GLP-1R expression in treated-rats (negative feedback mechanism). Besides that, it’s possible that Liraglutide could promote GLP-1R internalization induced by a prolonged stimulation to this GLP1-RA. SNP was equipotent in Wistar treated and non-treated rats, but a loss of potency of SNP effect was observed in diabetic and treated-rats and it can be explained by the fact that diabetes promotes loss of nitrergic neurons and subsequent reduction of nitric oxide synthesis and release, so relaxation of gastric smooth muscle is inhibited. In fact, in our study, a tendency to decreased nNOS and p-nNOS expression in lysates from gastric fundus was observed in GK rat (compared to Wistar rat). Noradrenaline was less potent in Wistar and GK rats treated with Liraglutide, indicating loss of potency with the treatment. Comparing control and diabetic rats, NA was significantly less potent in GK rat, suggesting loss of potency determined by type II diabetes. Loss of potency of noradrenaline with the treatment can be related to the down-regulation of GLP-1R expression in gastric fundus from treated-rats, since GLP1-1RA can act xviii through GLP-1R and subsequent sympathetic adrenergic pathways activation. In this case, a decrease in GLP-1R expression could result in a decrease in activation of sympathetic adrenergic pathways and consequently, in inhibition of noradrenaline action. Loss of potency of NA with the pathology can be explained by a possible down-regulation of beta-adrenergic receptors in gastric fundus induced by type II diabetes and therefore, noradrenaline action would be reduced and also relaxation induced by NA. Finally, GLP-1R immunoreactivity was observed in parietal cells of gastric glands, in smooth muscle cells, in ganglion cells of nervous plexus and in endothelial cells of blood vessels, in isolated rat gastric fundus. To conclude, new studies are necessary to better understand the real contribution of nitric oxide in the action mechanism of GLP-1RA effect in gastric motility. And the use of older diabetic animals or even using another animal model of type II diabetes could help understanding gut motility alterations caused by diabetes.