Précédemment, nous avons montré que la surexpression de l'échangeur Na/Ca NCX1), une protéine responsable de la sortie de calcium (Ca2+) des cellules, augmentait la mort cellulaire programmée ou « apoptose » et réduisait la prolifération des cellules β. Afin d’étudier plus en profondeur le rôle de l’échangeur dans les cellules β in vivo, nous avons développé et caractérisé des souris présentant une inactivation de NCX1.<p>Des méthodes biologiques et morphologiques (imagerie du Ca2+, capture de Ca2+, métabolisme du glucose, sécrétion d'insuline et morphométrie par comptage de points) ont été utilisées pour évaluer la fonction de la cellule β in vitro. Les taux de glucose et d'insuline dans le sang ont été mesurés afin de déterminer le métabolisme du glucose et la sensibilité à l’insuline in vivo. Des îlots ont été transplantés sous la capsule rénale pour évaluer leur capacité à corriger le diabète chez les souris rendues diabétiques par l’alloxane.<p>L'inactivation hétérozygote de Ncx1 chez les souris provoque une augmentation de la sécrétion d’insuline induite par le glucose avec un renforcement important à la fois de la première et de la deuxième phase. Ces résultats s’accompagnent d’une augmentation de la masse et de la prolifération des cellules β. La mutation augmente également le contenu en insuline, l’immunomarquage de la proinsuline, la capture de Ca2+ induite par le glucose et la résistance à l'hypoxie des cellules β. En outre, les îlots de souris Ncx1+/- montrent une capacité à compenser le diabète 2 à 4 fois plus élevé que les îlots de souris Ncx1+/+ lorsque transplantés chez des souris diabétiques.<p>En conclusion, l’inactivation de l'échangeur Na/Ca conduit à une augmentation de la fonction de la cellule β, de sa prolifération, de sa masse et de sa résistance au stress physiologique, à savoir à divers changements de fonction des cellules β opposés aux principales anomalies rencontrées dans le diabète de type 2 (Type 2 Diabetes Mellitus,T2DM). Ceci nous procure un modèle unique pour la prévention et le traitement du dysfonctionnement des cellules β dans le T2DM et pour la transplantation d'îlots.<p> / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

Diabetes Mellitus (DM) is a metabolic disorder in which the body fails to achieve glucose homeostasis, due to either insulin resistance or reduced insulin secretion or both. This inadequate glucose control leads to hyperglycemia which, if left unchecked, leads to secondary complications like nephropathy, neuropathy, retinal degeneration and other serious conditions. In non-disease state, normal glucose level in the blood is maintained by pancreatic β-cells, which secrete insulin. However, during diabetes development, there is loss of β-cell mass and function; resulting in decreased insulin secretion which is the ultimate cause of hyperglycemia. The ability to non-invasively monitor changes in the β-cell mass during the development or treatment of diabetes would be a significant advance in diabetes management. However, a primary limitation for analysis of β-cell mass and developing dysfunction is the lack of specificity of β-cell targeting agents. Our novel approach for achieving the required specificity for a usable β-cell targeted contrast agent is to target a set of receptors on the cell surface that, as a combination, are unique to that cell. Through genetic screening, Glucagon Like Peptide-1 Receptor (GLP-1R) and α2Adrenergic Receptor (α2AR) were chosen as a potential molecular barcode for β-cells since their combination expression is relatively unique to the β-cells. GLP-1R and α2AR are both G-protein couple receptors (GPCRs) that, apart from being a β-cell specific combination, play an important role in regulating fundamental downstream signaling pathways in β-cells. To target these receptors effectively, we synthesized a multivalent ligand composed of Yohimbine (Yhb), an α2 adrenergic receptor (α2AR) antagonist, linked to an active Glucagon-like Peptide 1 analog (GLP-1₇₋₃₆). In this manuscript, I describe the synthesis and characterization of binding selectivity and signaling ability of GLP-1/Yhb at the cellular level. Using high throughput binding assays, we observed high affinity binding of GLP-1/Yhb to βTC3 cells, a β-cell mimetic line expressing both receptors, at a Kd of ~3 nM. Using microscopy, we observed significant Cy5-tagged GLP-1/Yhb binding and rapid internalization in cells expressing the complementary receptor pair at low concentrations, as low as 1 nM and 5 nM. When one of the receptors was made inaccessible due to presence of saturating quantities of a single unlabeled monomer, GLP-1/Yhb-Cy5 failed to bind to the cells at low concentrations (<10 nM). Similarly, in cells where either GLP-1R or α2AR were knocked down (using shRNA), binding of GLP-1/Yhb was significantly reduced (≤half of cells with both receptors), indicating strong selectivity of the ligand to cells expressing the combination of receptors. We also observed that GLP-1/Yhb construct modulates downstream signaling inβ TC3 cells resulting in enhanced Glucose Stimulated Insulin Secretion (GSIS). In presence of stimulatory glucose, GLP-1/Yhb significantly potentiated GSIS with a half-maximal effective dose of 2.6 nM. Compared to GLP-1₇₋₃₆ alone or GLP-1₇₋₃₆ and Yhb monomers added together, only GLP-1/Yhb could significantly potentiate GSIS at 1 nM, demonstrating that GLP-1/Yhb could translate high affinity binding to increased efficacy for GSIS potentiation. Unlike for insulin secretion, high affinity divalent binding did not translate to increased cAMP production at low concentrations, with significant increases above baseline seen only at 10 nM and higher. Nevertheless, these data show that GLP-1/Yhb binds selectively to β-cells and affects signaling, demonstrating its potential for targeted β-cell imaging and therapy. Overall, our work indicates that synthetic heterobivalent ligands, such as GLP-1/Yhb can be developed to increase cellular specificity and sensitivity making them a strong candidate for both noninvasive imaging and targeted therapy.

Os genes relógio são responsáveis pelo ritmo circadiano e homeostase de diversos sistemas biológicos, incluindo o pâncreas endócrino. Nas células &beta; são de grande importância para a regulação do metabolismo e da secreção de insulina (SI), e sua ausência pode levar ao desenvolvimento do diabetes. A NADPH oxidase (NOX) é um complexo enzimático responsável pela produção do ânion superóxido através da redução do oxigênio molecular. Em ilhotas pancreáticas, a NOX participa da regulação do metabolismo da glicose e da SI, através da modulação do estado redox intracelular. O objetivo do nosso estudo foi verificar se a desregulação dos genes relógio mediada pela ausência de Bmal1 seria capaz de modular a NOX e o estado redox nas células &beta; pancreáticas, influenciando assim a SI. Observamos que a ausência de Bmal1 alterou a atividade e expressão da NOX, desregulando o estado redox intracelular. Essas alterações levaram à redução da viabilidade celular e mudanças na resposta à estimulação com glicose, resultando em uma deficiência na principal função da célula &beta; a SI. / Clock genes are responsible for homeostasis and circadian rhythm in various biological systems, including endocrine pancreas. In &beta; -cells, they are important for the regulation of metabolism and insulin secretion (IS), and its absence can lead to development of diabetes. NADPH oxidase (NOX) is an enzymatic complex responsible for production of superoxide anion by reducing molecular oxygen. In pancreatic islets, NOX regulates glucose metabolism and IS through modulation of the intracellular redox state. The aim of our study was to investigate whether dysregulation of clock genes mediated by Bmal1 suppression would be able to modulate NOX activity and redox state in pancreatic &beta; cells, thus influencing the SI. In this work, the lack of Bmal1 altered the activity and expression of NOX, deregulating the intracellular redox state. These changes led to reduced cell viability and changes in cell response after stimulation with glucose, resulting in a deficiency in &beta; cell main function, GSIS.

The present thesis will be largely focused on identifying and understanding the scope and mechanistic details associated with the tetrabutylammonium fluoride (TBAF) mediated cyclization of alkynyl hydrazines and (O)-hydroxylamines. Also, the synthesis of 2-(2-carboxyethyl)-4-methyl-5-propylfuran-3-carboxylic acid (CMPF) and its analogs will be discussed along with an analysis of their effects on insulin secretion. Chapter 1 will present the importance of developing isoxazoline and pyrazoline type heterocycles given that they are continually demonstrated to possess a variety of biological activities. Further, the scope of the reaction in terms of functional group tolerability, scalability and mild conditions will be shown. To expand the importance of this work, a route to access non-racemic heterocycles is also noted. With the heterocycles in hand, new methods were developed to generate more complex frameworks in the form of a novel one pot deprotection/functionalization reaction. Chapter 2 will focus on mechanistic investigations of the cyclization. From the initial discovery of the reaction, its actual mechanism was unknown and a main point of interest. What appeared unusual is that a nucleophilic attack occurs on an unactivated triple bond. Given the identity of the products, a reasonable proposal was a 5-endo-dig type cyclization. However, such a pathway would result in the generation of a vinyl anion intermediate which is well known to be of very high energy and it would seem unlikely to occur under mild conditions. Various trapping experiments were used to demonstrate that the vinyl anion forms and a 5-endo-dig-cyclization is the operative mechanism. Chapter 3 analyzes the importance of the tetrabutylammonium fluoride reagent. During optimization studies, it became clear that this base is the ideal reagent to facilitate the cyclization although other bases can also enable the transformation at much slower rates. Addition of non-basic ammonium salt additives to bases such as KF and CsF had a dramatic effect on the rate of the reaction. To determine whether the observed rate differences were merely a phase transfer effect or something more, both empirical and Raman spectroscopy data were collected. Based on this, the first evidence for an ammonium-alkyne cation-pi type interaction was shown. Chapter 4 will summarize the work on the synthesis of 2-(2-carboxyethyl)-4-methyl-5-propylfuran-3-carboxylic acid (CMPF) and its analogs in order to be used in various biological assays. The main goals were to determine a possible structure activity relationship between the substrates and insulin secretion in beta cells and also determine the fate of CMPF in vivo. Several 13C labeled analogs of CMPF were synthesized and successfully used to show for the first time that CMPF in metabolized in vivo in mice. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2017. / FAU Electronic Theses and Dissertations Collection

A via de amplificação da secreção de insulina estimulada por glicose (GSIS) é um fenômeno discutido na literatura, cujos componentes são amplamente debatidos. Evidências sugerem que a condutância a Cl- compõe parte desta via. Porém, o mecanismo pelo qual essa condutância desempenharia papel na via de amplificação ainda é debatido, e, além disso, as ferramentas farmacológicas para estudo dessas afeta o transporte de outros ânions, como bicarbonato (HCO3-). Buscamos neste trabalho compreender a contribuição do transporte desses ânions para a via de amplificação da GSIS levando em consideração a distribuição de Cl- e HCO3- extracelular em células INS-1E. Concluímos que o transporte de ânions nas células INS-1E não contribui para a via de amplificação da GSIS, porém essas células não expressaram os canais CFTR e Anoctamina 1 que foram relacionados com esse fenômeno. Acreditamos que em células secretoras de insulina que expressem esses canais, o transporte de ânions possua alguma relevância funcional. / The amplification pathway of glucose stimulated insulin secretion (GSIS) is a phenomenon discussed in the literature, which components are broadly debated. Evidence suggests that Cl- conductance composes part of this pathway. However, the mechanism that this conductance would play role on the amplification pathway still is debated, and, besides that, the pharmacological tools to study these affects transport of other anions, such as bicarbonate (HCO3-). We aimed in this study to understand the contribuition of anion transport for the amplification of GSIS considering the Cl- and HCO3- extracellular distribuition in INS-1E cells. We concluded that anion transport in INS-1E cell line do not contribute for the amplification pathway of GSIS, however those cells do not express CFTR and Anoctamin 1 channels which were related with this phenomenon. We believe that in insulin secretin cells that express those channels, the anion transport may have a functional relevance.

Avaliamos importância da NADPH oxidase 2 (NOX2) na produção de espécies reativas de oxigênio (EROs) em ilhotas de ratas alimentadas ou em jejum, incubadas na presença de 2,8 mM ou 16,7 mM de glicose, associada ou não a leucina, com ou sem inibição da NOX2. As ilhotas dos animais alimentados ou em jejum apresentaram reduzida secreção de insulina e altas concentrações de EROs na presença de 2,8 mM de glicose. Esses parâmetros foram invertidos pela adição de inibidores da NOX2. A leucina, que é metabolizada no Ciclo dos Ácidos Tricarboxílicos, também aumentou a secreção de insulina por aumento de ATP, e diminuiu as EROs, devido ao aumento de NADPH, um substrato do sistema antioxidante. Desse modo, quando as ilhotas são submetidas ao jejum, a diminuição da atividade secretória é fundamental para impedir que quantidades maiores de hormônio sejam secretadas, podendo levar a uma hipoglicemia. Porém, na presença de alta concentração de glicose, a ativação das defesas antioxidantes da célula b atenua o excesso de EROS, liberando a secreção de insulina. / We sought to evaluate the importance of NADPH oxidase 2 (NOX2) in the production of reactive oxygen species (ROS) in islets from rats fed or fasted, incubated in the presence of glucose 2.8 mM or 16.7 mM, with or without leucine or inhibition of NOX2. Islets of fed or fasted animals showed reduced insulin secretion and high concentration of ROS in the presence of 2.8 mM glucose. These parameters were reversed by addition of inhibitors NOX2. Leucine that is metabolized in the cycle of Tricarboxylic Acids (TCA) also increased insulin secretion, by increasing ATP, and ROS decreased due to the increase of NADPH, a substrate of the antioxidant system. Thus, when the islets are subjected to fasting, decreased secretory activity is essential to prevent amounts of the hormone be secreted and may lead to hypoglycaemia. However, in the presence of high glucose levels, activation of antioxidant defenses of b cell attenuates the excess of ROS, releasing insulin secretion.

Apart from CFRD, low cognitive skill index (CSI) was also found in CF patients and was attributed the lacking of vitamin E. Since it is known that insulin plays a role in the learning and memory, decreased plasma insulin level in CF patients is an alternative mechanism for impaired cognitive function. Although numerous studies have found that insulin can improve learning and memory, the mechanism of it is not well understood. In this study, we investigated the effect of insulin on the expression of hippocampal early-phase long-term potentiation (E-LTP) in the immature rats. Hippocampal brain slices were acutely prepared from 10-12 days and 2 months old rats and field excitatory postsynaptic potentials (tEPSCs) were recorded from CA1 region by a multi-electrode in vitro recording system. In the control group, the hippocampal slices of neonatal rats showed no increase in the magnitude of fEPSC after conventional high frequency stimulation (HFS). After pretreatment of the slices with 0.08ng/ml insulin for over one hour, there was no significant change in the magnitude of E-LTP. However, when the insulin concentration increased to 0.8ng/ml, a significant increase in the magnitude of E-LTP was observed. On the contrary, any doses of insulin failed to affect the magnitude of E-LTP of mature rats. These results suggested that insulin could dose-dependently facilitate the production of E-LTP in the hippocampus of infant rats. Application of AG-1024, an inhibitor of insulin receptor, largely abolished the insulin-dependent E-LTP in immature rats rather than adult rats, indicating the involvement of insulin signaling pathway in the insulin effect. On the other hand, increasing the concentration of glucose from 11mM to 22 or 33 mM did not facilitate the E-LTP and application of indinavir, a blocker of insulin-sensitive glucose transporter-4, did not inhibit the effect of insulin. Therefore, it is unlikely that the facilitory action of insulin on E-LTP is via an indirect effect on glucose homeostasis or utilization. Pretreatment with the MAPK pathway inhibitor PD98059 blocked insulin-mediated E-LTP facilitation. Furthermore, the tetanic stimulation induced a significant increase in the level of phosphorylated p42MAPK in the insulin-treated hippocampus than that in the control group. In conclusion, our results suggested that insulin could facilitate the production of hippocampal E-LTP in infant rats, which may play an important role in modulating the expression of LTP in the developing brain and perhaps is an underlying mechanism for the improving effect of insulin on learning and memory. Since insulin plays an important role in the developing brain, perhaps the deficiency of insulin effect resulted from CF patients induces the impairment of cognitive function. / Cystic fibrosis (CF), which is caused by the deficiency of cystic fibrosis transmembrne conductance regulator (CFTR), is the most common autosomal recessive systemic disease with an incidence of 1: 2500 in Caucasians. Cystic fibrosis-related diabetes (CFRD), as one of the complications of CF patients, is regarded as one of the leading co-morbidity in CF patients. The mechanism ofCFRD is attributed to the reduced number of islets due to pancreatic fibrosis caused by the loss of CFTR in pancreatic duct. However, the above mechanism failed to explain the dynamics of insulin secretion induced by glucose tolerance test (GTT) in some CF patients and therefore, we were forced to re-consider the mechanism for the pathogenesis of CFRD. Interestingly, the following facts imply that perhaps there is another mechanism for the onset of CFRD: decreased insulin secretion and decreased plasma HCO3 - concentration was observed in the metabolic acidosis disease, plasma HCO3- level increased accompanied by the elevation of plasma insulin after food intake and CFTR accounted for HCO3 - transport in many epithelial cells. These facts promoted us to hypothesize that the loss of HCO3--induced insulin secretion resulting from the deficiency of CFTR is an alternative mechanism for the onset of CFRD. Our results showed that HCO3- could induce insulin secretion of isolated islets from rats. Ca2+ imaging revealed that HCO3- dose-dependently induced an increase in intracellular Ca2+ ([Ca2+] i) in RIN-5F cells, an insulin-secreting cell line. Removal of extracellular Ca2+ or addition of nifedipine, the blocker of L-type Ca 2+ channel, decreased the effect of HCO3- significantly, indicating the activation of L-type Ca2+ channel during HCO3- stimulation. The inhibitory effect of BaCl2 implied the involvement of K+ channel. The results that HCO3--induced increase in [Ca 2+]i was reduced by PKA inhibitor and sAC blocker demonstrated that the pathway of sAC-cAMP-PKA-ATP-sentitive K+ channel (K ATP channel) was responsible for the effect of HCO3 -. The reduction of extracellular Cl- or the inhibitor of anion exchanger (AE) inhibited the [Ca2+]i increase induced by HCO3- significantly but the omission of external Na+ failed. The facts that CFTR blocker decreased the effect of HCO3- markedly and the expression of CFTR in RIN-5F cells revealed by western blotting suggested the CFTR-mediated HCO3- transport. These results suggested that HCO 3- could induce insulin secretion in a CFTR-dependent manner, which provided a new insight into the understanding of pathogenesis of CFRD and paved the way for the therapy of CFRD. / Zhao, Wenchao. / "November 2010." / Advisers: Chang Chan; Wing Ho Yung. / Source: Dissertation Abstracts International, Volume: 73-04, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 115-138). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

A crescente incidência de disfunções metabólicas, como resistência à insulina e diabetes mellitus tipo 2 (T2DM), está correlacionada com a elevação da ocorrência de obesidade e sobrepeso. Em busca da melhora da saúde e de um corpo ideal segundo os padrões estéticos propagados atualmente, um número cada vez maior de indivíduos adere às dietas da moda que prometem rápida redução do peso corporal, ao invés de adotar uma alimentação balanceada e a prática regular de exercícios físicos. Uma dieta bastante divulgada e até recomendada por profissionais da saúde é o jejum intermitente (JI), que consiste em alternar períodos de jejum de até 24 horas com períodos de ingestão alimentar. Diversos estudos experimentais têm relatado alterações metabólicas em consequência do JI, como modificações da glicemia e da tolerância à glicose, porém, os resultados encontrados na literatura são conflitantes e, além disso, o impacto do jejum intermitente, em longo prazo, sobre as ilhotas pancreáticas ainda não foi devidamente elucidado. Desta forma, o presente estudo teve como objetivo caracterizar os impactos de doze semanas de JI em ratas Wistar. Para tal, ratas Wistar com 30 dias de idade foram distribuídas aleatoriamente em dois grupos: controle, com livre acesso à ração balanceada; e jejum intermitente, submetido a 24 horas de jejum intercalado com 24 horas de livre acesso à ração balanceada. Foi observado que os animais submetidos ao JI, apresentaram menor ganho de peso corporal, redução do comprimento da tíbia e da distância naso-anal, e alteração da composição corporal, incluindo diminuição da massa muscular e aumento do tecido adiposo. Em média, o consumo de ração do grupo JI foi menor, porém, no dia que era disponibilizado alimento, os animais apresentaram hiperfagia o que resultou em grande aumento das dimensões do estômago. O jejum intermitente reduziu os valores plasmáticos do colesterol total, triglicérides, LDL, HDL e glicemia, e aumentou a concentração basal da insulina plasmática, bem como a secreção da insulina após o estímulo com glicose. Foi observada redução significativa da massa de ilhotas pancreáticas e aumento da porcentagem de células dispersas de ilhotas em apoptose. Ainda nas células dispersas de ilhotas pancreáticas, houve aumento do conteúdo de espécies reativas de oxigênio mitocondrial e total, e do peróxido de hidrogênio (H2O2), além de aumento da expressão do sistema antioxidante. Assim, nossos dados sugerem que esse protocolo estudado de 24 horas de jejum intercalados com 24 horas de alimentação à vontade não seja saudável em longo prazo. Mais estudos em longo prazo são necessários para investigar qual seria o melhor protocolo de jejum intermitente de forma a reduzir os efeitos colaterais e melhorar a saúde, para então o JI ser considerado uma boa alternativa para perda e manutenção do peso. / The increasing incidence of metabolic dysfunctions, such as insulin resistance and type 2 diabetes mellitus (T2DM), is correlated with increased occurrence of obesity and overweight. In pursuit of improved health and an ideal body according to today\'s aesthetic standards, an increasing number of individuals adhere to fad diets that promise a rapid reduction of body weight, instead of adopting a balanced diet and regular practice of physical exercises. A well-publicized diet and even recommended by health professionals is intermittent fasting (IF), which consists of alternating fasting periods of up to 24 hours with periods of food intake. Several experimental studies have reported metabolic changes as a consequence of IF, such as changes in glucose and glucose tolerance, but the results found in the literature are conflicting and, in addition, the impact of intermittent fasting in the long term on pancreatic islets has not yet been properly elucidated. Thus, the present study aimed to characterize the effects of twelve-week IF on Wistar rats. For this, 30-day-old Wistar rats were randomly assigned to two groups: control, with free access to balanced chow; and intermittent fasting, subjected to 24-hour fast intercalated with 24 hours of free access to the balanced chow. It was observed that the animals submitted to IF presented lower body weight gain, reduced tibia length and naso-anal distance, and altered body composition, including decreased muscle mass and increased adipose tissue. On average, the dietary intake of the IF group was lower, but on the day that food was available, the animals presented hyperphagia which resulted in a large increase in the stomach size. Intermittent fasting reduced plasma levels of total cholesterol, triglycerides, LDL, HDL, and glycaemia, and increased basal plasmatic insulin level, as well as insulin secretion after stimulation with glucose. We observed significant reduction in pancreatic islet mass and increase in percentage of islet-dispersed cells in apoptosis. Still in islet-dispersed cells, there was an increase in mitochondrial and total reactive oxygen species content, and of hydrogen peroxide (H2O2), in addition to an increase in expression of antioxidant system. Thus, our data suggest that this protocol of 24-hour fasting intercalated with 24-hour feed at will is not healthy in the long run. More long-term studies are needed to investigate the best intermittent fasting protocol in order to reduce side effects and improve health, so IF be considered a good alternative for weight loss and maintenance.

The development of microarray technology has provided an unprecedented wealth of data on gene expression in various tissue and cell types. Few studies have, however, taken full advantage of these data by selecting and then extensively characterising the functions of particular genes chosen from these microarray datasets. In this study, after analysing differentially-regulated genes revealed by microarray analysis of human mast cells activated via Fc??RI cross-linking, we chose two promising gene candidates for further research, A20 and Gem. Our group??s extensive gene expression database of major leukocytes showed that both A20 and Gem were up-regulated in other leukocyte types, and yet neither of these genes has been extensively explored in mast cells or in the immune system prior to our study. In order to investigate the first of these genes selected for further study, A20, we utilised both A20-deficient mast cells and mast cells in which A20 was over-expressed. Our findings establish for the first time that A20 is an important regulator of mast cell inflammatory responses to both LPS and Fc??RI cross-linking, and that it plays a novel role in mast cell proliferation. Our study of the second gene chosen for investigation, Gem, was conducted in a Gemdeficient mouse model developed by our group. In this study, we investigated the effect of Gem deficiency in two key immune cell types, macrophages and T-cells, complementing the work of a previous group member who investigated Gem deficiency in mast cells. Our results clearly exclude a role for Gem in macrophage and T-cell effector responses, and further establish that Gem is dispensable for in vivo inflammatory responses in models of delayed-type hypersensitivity and allergic airway inflammation. In addition to these findings, and given that the physiological role of Gem was not yet understood prior to our study, we extended our investigation to explore a potential function for Gem in the metabolic system. Using Gem-deficient mice, we found that Gem is necessary for insulin secretion from pancreatic islets. These findings confirm the potential for microarray expression data to reveal excellent gene candidates for further research and functional characterisation.

Identification of modifiable risk factors for Alzheimer’s disease (AD) is crucial in order to diminish suffering from this devastating disease. The aim of this thesis was to investigate if different aspects of glucose metabolism, insulin, fatty-acid composition or other vascular risk factors predict the future development of AD and dementia. This thesis is based on the Uppsala Longitudinal Study of Adult Men (ULSAM) cohort, which started in 1970. A total of 2322 men at age 50 were examined with focus on vascular risk factors. The cohort was re-examined at ages 60, 71, 77, 82 and 88. Incident diagnoses of AD, vascular dementia, other dementias and cognitive impairment were assessed in 2005–2010. The risk of AD was increased in subjects with lower early insulin response measured with both an intravenous glucose tolerance test at 50 years and an oral glucose tolerance test at 71 years of age. The presence of vascular risk factors such as hypertension, obesity, hypercholesterolemia and smoking increased the risk of future vascular dementia but not of AD. Furthermore, saturated fatty acids at midlife were inversely associated with risk of AD. No evidence of a protective effect of omega-3 fatty acids against dementia was found. The susceptibility allele, APOE ε4, was the strongest individual risk factor. APOE ε4 carriers with vascular risk factors had the greatest risk of developing dementia. Low insulin response was a risk factor for AD mainly in APOE ε4 non-carriers. Disturbances in insulin and glucose metabolism, vascular risk factors and fatty acids are linked differentially to the pathogenesis of AD and vascular dementia. These observations should be considered when future clinical approaches are planned to prevent and postpone the onset of dementia. / ULSAM