A hallmark of type 2 diabetes (T2D) is β-cell dysfunction and the eventual loss of functional β-cell mass. Therefore, mechanisms that improve or preserve β-cell function could be used to improve the quality of life of individuals with T2D. Studies have shown that monomeric, oligomeric and polymeric cocoa flavanols have different effects on obesity, insulin resistance and glucose tolerance. We hypothesized that these cocoa flavanols may have beneficial effects on β-cell function. INS-1 832/13 derived β-cells and primary rat islets cultured with a monomeric catechin-rich cocoa flavanol fraction demonstrated enhanced glucose-stimulated insulin secretion, while cells cultured with total cocoa extract, oligomeric, or polymeric procyanidin-rich fractions demonstrated no improvement. The increased glucose-stimulated insulin secretion in the presence of the monomeric catechin-rich fraction corresponded with enhanced mitochondrial respiration, suggesting improvements in β-cell fuel utilization. Mitochondrial complex III, IV and V components were upregulated after culture with the monomer-rich fraction, corresponding with increased cellular ATP production. The monomer-rich fraction improved cellular redox state and increased glutathione concentration, which corresponds with Nrf2 nuclear localization and expression of Nrf2 target genes, including NRF-1 and GABPA, essential genes for increasing mitochondrial function. We propose a model by which monomeric cocoa catechins improve the cellular redox state, resulting in Nrf2 nuclear migration and upregulation of genes critical for mitochondrial respiration, and, ultimately, enhanced glucose-stimulated insulin secretion and β-cell function. These results suggest a mechanism by which monomeric cocoa catechins exert their effects as an effective complementary strategy to benefit T2D patients.

Genetically engineered cells have the potential to solve the cell availability problem in developing a pancreatic tissue substitute for the treatment of insulin-dependent diabetes (IDD). These cells can be beta-cells genetically engineered so that they can be grown in culture, such as the betaTC3 and betaTC tet mouse insulinomas developed by Efrat et al; or, they can be non-beta cells genetically engineered to secrete insulin constitutively or under transcriptional regulation. The aim of this work was to thoroughly characterize and improve the secretion dynamics of pancreatic substitutes based on genetically engineered cells. One issue involved with the continuous beta-cell lines is the remodeling of the cells inside an encapsulated cell system, which may affect the insulin secretion dynamics exhibited by the construct. To evaluate the effect of remodeling on the secretion properties of the construct, we used a single-pass perfusion system to characterize the insulin secretion dynamics of different alginate beads in response to step-ups and downs in glucose concentration. Results indicated that the secretion dynamics of beads indeed changed after long-term culture. On the other hand, data with a growth-regulated cell line, betaTC tet cells, showed that the secretion profile of beads can be retained if the cell growth is suppressed. A major concern associated with genetically engineered cells of non-beta origin is that they generally exhibit sub-optimal insulin secretion characteristics relative to normal pancreatic islets. Instead of relying on molecular tools such as manipulating gene elements, our approach was to introduce a glucose-responsive material acting as a control barrier for insulin release from a device containing constitutively secreting cells. Proof-of-concept experiments were performed with a disk-shaped prototype based on recombinant HepG2 hepatomas or C2C12 myoblasts, which constitutively secreted insulin, and concanavalin A (con A)-based glucose-responsive material as the control barrier. Results demonstrated that the a hybrid pancreatic substitute consisting of constitutively secreting cells and glucose-responsive material has the potential to provide a more physiologic regulation of insulin release than the cells by themselves or in an inert material.

Loss of glucose-stimulated insulin secretion (GSIS) from the pancreatic beta-cells is one of the earliest detectable defects in the pathogenesis of type 2 diabetes. However, despite its relevance, the mechanisms that govern GSIS are still not completely understood. ARNT/HIF-1β is a member of the bHLH-PAS family of transcription factors, with a prominent role in the transcriptional regulation of enzymes required for the metabolism of xenobiotics as well as regulation of genes that are critical for cellular responses to hypoxia. Recent research has uncovered a previously unknown function for ARNT/HIF-1β in the pancreatic beta-cells, where the gene was found to be 90% down-regulated in human type 2 diabetic islets and loss of ARNT/HIF-1β protein leads to defective GSIS in pancreatic beta-cells of mice. The main focus of this thesis was to understand the mechanisms by which ARNT/HIF-1β maintains normal GSIS from pancreatic beta-cells and understand how loss of ARNT/HIF-1β leads to beta-cell dysfunction and type 2 diabetes in mice. ARNT/HIF-1β was found to positively regulate GSIS in both INS-1 derived 832/13 cell line and mice islets. In the 832/13 cells, loss of ARNT/HIF-1β leads to a reduction in glycolysis without affecting the glucose oxidation and the ATP/ADP ratio suggesting that the regulation of GSIS takes place in a manner that is independent of the KATP channels. In order to further assess the mechanism of lowered GSIS in the absence of ARNT/HIF-1β in the 832/13 cells, a metabolite profiling was performed which revealed a significant reduction in the metabolite levels of glycolysis and the TCA cycle intermediates and glucose-induced fatty acid production, suggesting the involvement of ARNT/HIF-1β in regulating glucose-stimulated anaplerosis, which is believed to play a key role in the regulation of GSIS from the pancreatic beta-cells. The changes in metabolite levels in the absence of ARNT/HIF-1β were associated with corresponding changes in the gene expression pattern of key enzymes regulating glycolysis, the TCA cycle and fatty acid synthesis in beta-cells. In an attempt to understand how loss of ARNT/HIF-1β leads to beta-cell dysfunction and type 2 diabetes in mice, a pancreatic beta-cell specific ARNT/HIF-1β knock out mouse (β-ARNT KO) was generated using the Cre-loxP technology. Functional characterization of islets from both male and female β-ARNT KO mice revealed a significant impairment in GSIS, which was attributed due to a small, but significant reduction in rise in intracellular calcium upon glucose stimulation. Further analysis revealed reduced secretory response to glucose in the presence of KCl and diazoxide indicating a defect in the amplifying pathway of GSIS in β-ARNT KO islets. Expression of pyruvate carboxylase (PC) was significantly reduced in β-ARNT KO islets suggesting possible impairments in anaplerosis and consistent with this, defect in GSIS in β-ARNT KO islets could be almost completely rescued by treatment with membrane permeable TCA intermediates. Surprisingly, both male and female β-ARNT KO mice have normal glucose homeostasis. In an attempt to assess how β-ARNT KO mice maintained normal blood glucose levels, indirect calorimetry was used to understand changes in whole-body energy expenditure. This investigation revealed that β-ARNT KO mice exhibited a small but significant increase in respiratory exchange ratio (RER), suggesting a preference in utilizing carbohydrates as a fuel source, possibly leading to improved glucose uptake from the blood stream. Response to exogenous insulin was completely normal in β-ARNT KO mice suggesting intact functioning of the skeletal muscles. To conclude, based on our in vitro data, we believe that ARNT/HIF-1β plays an indispensable role in maintaining normal beta-cell secretory function, however, results from β-ARNT KO mice indicates that these mice are protected from the adverse effects of hyperglycemia. Although loss of ARNT/HIF-1β alone is not sufficient for the genesis of type 2 diabetes, it creates a perfect storm in the pancreatic beta-cells that may eventually lead to an imbalance in the whole body glucose homeostasis. Our study provides significant information to the scientific community that engages in assessing the pharmacological potential of gene targets for the treatment of type 2 diabetes.

A thorough understanding of beta-cell proliferation, function, death and regeneration under normal condition as well as in the progression of diabetes is crucial to the conquest of this disease. The work presented in this thesis aimed to investigate the expression and role of a novel transcription factor, Zinc finger BED domain-containing protein 6 (ZBED6), in beta-cells. ZBED6 was present in mouse βTC-6 cells and human islets as a double nuclear band at 115/120 kDa and as a single cytoplasmic band at 95-100 kDa, which lacked N-terminal nuclear localization signals. Lentiviral shRNA-mediated stable silencing of ZBED6 in βTC-6 cells resulted in altered morphology, decreased proliferation, a partial S/G2 cell cycle arrest, increased expression of beta-cell specific genes, and higher rates of apoptosis. ChIP sequencing of human islets showed that ZBED6 binding was preferentially to genes that control transcription, macromolecule biosynthesis and apoptosis. We proposed that ZBED6 supported proliferation and survival of beta-cells, possibly at the expense of specialized beta-cell function, i.e. insulin production. To further investigate the role of ZBED6 in beta-cells, ChIP sequencing and whole transcriptome analysis were performed using MIN6 cells. More than 4000 putative target genes of ZBED6 were identified, including Pdx1, MafA and Nkx6.1. ZBED6-silencing resulted in differential expression of more than 700 genes, which was paralleled by an increase in the content and release of insulin in response to a high glucose concentration. Altered morphology/growth patterns as indicated by increased cell clustering were observed in ZBED6 silenced cells. We found also that ZBED6 decreased the ratio between N- and E-cadherin. A lower N- to E-cadherin ratio may hamper the formation of three-dimensional beta-cell clusters and cell-to-cell junctions with neural crest stem cells, and instead promote efficient attachment to a laminin support and monolayer growth. Thus, by controlling beta-cell adhesion and cell-to-cell junctions, ZBED6 might play an important role in beta-cell differentiation, proliferation and survival.

Diss. (sammanfattning) Stockholm : Karol. inst. / Titel på diss.-titelbl.: Islet glucose metabolism and insulin secretion in two animal models of glucose intolerance. Härtill 5 uppsatser.

ABSTRACT Insulin Dynamic Measures and Weight Change By Noreen Kloc B.S. Computer Information Technology, Purdue University December 7, 2015 INTRODUCTION: Weight gain and obesity are risk factors for insulin resistance that can lead to type 2 diabetes and cardiovascular disease; however, there is a complicated interplay between insulin sensitivity (SI), fasting insulin, acute insulin response (AIR), and disposition index (DI) and the relationship of these dynamic measures with weight change is not well understood. AIM: The aim of this study was to investigate the relationships between insulin dynamic measures, SI, fasting insulin, AIR, and DI, with weight change during a 5-years follow-up period in the multi-ethnic cohort of the Insulin Resistance Atherosclerosis Study (IRAS). METHODS: Data on 879 men and women of Hispanic, non-Hispanic White, and African-American race/ethnicity aged 40-69 years were obtained at baseline (1992-1994) and at 5 year follow-up. Crude associations between the insulin dynamic measures and weight change were evaluated using Kruskal-Wallis test and the relationships between log-transformed insulin-related variables were examined using Spearman rank-order analysis. Multivariate regression models evaluated associations of interest adjusted for age, sex, ethnicity, and diabetes status in a time-dependent manner using mixed models. RESULTS: Insulin sensitivity SI inversely coevolves with weight, i.e. greater weight is predicted by lower SI at any time point. To answer the question whether SI is the cause or a consequence of weight change, we examined the associations with the baseline values and a change in SI. In this model, both the baseline SI and change in SI were inversely correlated with weight gain. A similar approach showed that baseline values and change in fasting insulin were directly associated with weight gain. Weight change over time was associated with AIR, i.e. increases in AIR and greater AIR at baseline predicted weight gain. We did not find strong relationships between DI and weight change. DISCUSSION: These results suggest that insulin sensitivity and insulin secretion can modulate weight in a non-diabetic population.

Orientadores: Helena Coutinho Franco de Oliveira, Antonio Carlos Boschero / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-08T19:29:15Z (GMT). No. of bitstreams: 1 Bonfleur_MariaLucia_D.pdf: 1633140 bytes, checksum: 765548b2268a05188490bc90cf1939b8 (MD5) Previous issue date: 2007 / Resumo: Neste trabalho, investigamos se a hipercolesterolemia primária per se, independente de dieta rica em gordura, afeta a homeostase glicêmica e a secreção de insulina estimulada por vários secretagogos em animais knockout para o receptor de LDL (LDLR-/-). Além disso, investigamos os possíveis mecanismos envolvidos na liberação deste hormônio neste modelo animal. Podemos resumir nossos achados da seguinte maneira: camundongos LDLR-/- apresentam hiperglicemia pós-prandial, hipoinsulinemia, intolerância à glicose e sensibilidade periférica à insulina normal. Nós demonstramos que, as alterações na homeostase glicêmica ocorrem em parte, por uma diminuição da sensibilidade das ilhotas à glicose. A secreção de insulina é normal na presença de baixa concentração de glicose, entretanto na presença de 11,1 mmol/l, as ilhotas de animais LDLR-/- liberam menos insulina que as ilhotas controles. A secreção de insulina estimulada por outros secretagogos metabolizáveis (leucina e KIC) também está reduzida nas ilhotas dos animais knockout. O conteúdo total de insulina e DNA são similares entre os grupos, sugerindo que as alterações na secreção de insulina não ocorrem devido a diferenças no tamanho e/ou número de células b. Observamos uma redução na primeira e segunda fase de secreção de insulina estimulada por 11,1 mmol/l de glicose. A oxidação da glicose está reduzida, enquanto a metabolização da leucina está aumentada. Quando adicionamos agentes despolarizantes (KCl, Arginina e Tolbutamida), observamos uma redução da secreção de insulina tanto em concentrações basais quanto estimulatórias de glicose. Na presença de 11,1 mmol/l de glicose e carbacol (agonista colinérgico) ou PMA (ativador da proteína-quinase C), a secreção de insulina foi semelhante entre os grupos LDLR- /- e controles. Entretanto, quando estimulamos a secreção com forskolin ou IBMX, que aumentam os níveis de AMPc, observamos redução na liberação de insulina pelas ilhotas dos animais LDLR-/- em comparação com os controles. A expressão protéica da fosfolipase C (PLCb2) está aumentada enquanto que a expressão da proteína-quinase A (PKA) está reduzida nas ilhotas dos animais LDLR-/-. Assim, observamos que camundongos LDLR-/- apresentam alterações na homeostase glicêmica independente de dieta rica em gordura, provocada por redução na secreção de insulina devido, em parte à redução do metabolismo da glicose, bem como, redução na expressão da PKA / Abstract: In this work, we investigated whether primary hyperlipidemia per se, independently of a high-fat diet, affects glycemia and insulin secretion stimulated by several secretagogues in hypercholesterolemic low-density lipoprotein receptor knockout mice (LDLR-/-). In addition, we investigated the possible mechanisms involved in the release of this hormone. We found that, besides higher total cholesterol and triglyceride plasma concentrations, glucose plasma levels were increased and insulin decreased in LDLR-/- compared to the wild type (WT) mice. LDLR-/- mice presented impaired glucose tolerance, but normal whole body insulin sensitivity. In addition, we also demonstrate that the main cause of the impaired glucose homeostasis is a reduced pancreatic islet insulin secretion ability following fuel secretagogue stimuli. LDLR-/- mice have impaired insulin secretion in response to glucose without alterations in the pancreatic total insulin and DNA contents. These findings support the idea that the decreased response to glucose cannot be explained by differences islet size or number of beta cells, but it is probably caused by a defect in the secretory process. Glucose oxidation was 30% lower and L-leucine oxidation 60% higher in LDLR-/- islets than in WT islets. At basal (2.8 mmol/l) and stimulatory (11.1 mmol/l) glucose concentrations, the insulin secretion rates induced by depolarizing agents such as KCl, L-arginine and tolbutamide were significantly reduced in LDLR-/- when compared with WT islets. Insulin secretion induced by the PKA activators, forskolin and IBMX, in the presence of 11.1 mmol/l glucose, was lower in LDLR-/- islets, and it was normalized in the presence of the PKC pathway activators, carbachol and PMA. Western blotting analysis showed that phospholipase C-b2 expression was increased and PKA-a decreased in LDLR-/- compared with WT islets. In conclusion, we demonstrate that genetic hypercholesterolemia, due to complete deficiency of LDLR, impairs the beta cell insulin secretion, leading to hyperglycemia without affecting body insulin sensitivity. The lower insulin secretion in LDLR-/- mice islets may be explained by reduced glucose metabolism and expression of PKA / Doutorado / Fisiologia / Doutor em Biologia Funcional e Molecular

Orientador: Everardo Magalhães Carneiro / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-24T14:27:08Z (GMT). No. of bitstreams: 1 Borck_PatriciaCristine_M.pdf: 1384245 bytes, checksum: 89fa526347ae455e0cfcf6e3bd414018 (MD5) Previous issue date: 2014 / Resumo: Os processos fisiológicos como ciclo sono-vigília e o metabolismo estão sujeitos a oscilações circadianas e são regulados por um conjunto de genes conhecidos como genes do relógio, ou clock genes. Mutação nesses genes em camundongos reduz a secreção de insulina e a proliferação das células ? pancreáticas promovendo intolerância a glicose e hiperglicemia. Distúrbios nutricionais em fases iniciais da vida estão associados com o aparecimento do Diabetes Mellitus tipo 2 na vida adulta. Camundongos submetidos a restrição proteica intrauterina apresentam expressão alterada dos clock genes e maior suscetibilidade ao ganho de peso e intolerância a glicose. Neste trabalho tivemos como objetivo determinar a expressão diária dos clock genes em tecidos periféricos, hipotálamo e ilhotas pancreáticas de camundongos submetidos a restrição proteica. Avaliamos também o perfil oscilatório da secreção de insulina estimulada por glicose e pelo agonista colinérgico carbacol nesse modelo animal. Camundongos submetidos a restrição proteica (R) apresentaram características típicas de desnutrição como menor peso corpóreo, hipoinsulinemia, hipoproteinemia e maior tolerância a glicose e a insulina. Camundongos R apresentaram maior consumo alimentar, acompanhado de alterações no perfil oscilatório de genes hipotalâmicos Pomc (Pro-opiomelanocortina) e Npy (Neuropeptídeo Y). Nesse tecido, somente o gene do relógio Rev-erb? teve sua expressão influenciada pela restrição proteica. Camundongos R apresentaram, no fígado e músculo perda do perfil oscilatório para os genes Bmal1 e Clock. Ainda, no fígado e ilhotas pancreáticas a expressão de Rev-erb? foi alterada, com redução no conteúdo de mRNA. Em relação ao gene Per1, camundongos R exibiram adiantamento na expressão desse gene no tecido adiposo. No músculo e ilhotas houve perda da oscilação diária para esse gene. Camundongos R exibiram, no músculo e tecido adiposo, adiantamento na expressão do gene Per2. Ilhotas isoladas de camundongos controle (C) apresentaram padrão oscilatório de secreção de insulina sendo os maiores níveis atingido nos ZT 2 e 14 e redução no ZT 8. Contudo, camundongos R apresentaram redução na secreção de insulina estimulada por glicose, e perda do seu perfil oscilatório. O grupo R não apresentou alteração na liberação de insulina na presença do agonista e antagonista de Rev-erb?. Além disso, a expressão dos genes Sintaxina, Sinaptotagmina, e Insulina, estão reduzidos em camundongos R. O grupo R também apresentou perda oscilatória da secreção de insulina na presença de glicose associada ao Carbacol e redução na expressão do Receptor Muscarínico de Acetilcolina. Com os presentes resultados podemos concluir que camundongos submetidos a restrição proteica apresentaram características típicas de desnutrição com alteração na homeostase glicêmica e secreção de insulina. Ademais, camundongos R exibiram perda do perfil de secreção desse hormônio ao longo de 24 horas, o qual está relacionado com as alterações na expressão de Rev-erb?. Além disso, houve alteração no perfil de expressão dos genes clock, em especial Rev-erb?, Per1 e Per2 nos tecidos periféricos, fato que pode estar relacionado com as alterações na tolerância a glicose e insulina em camundongos R / Abstract: The physiological processes such as sleep-wake cycle and metabolism are subject to circadian fluctuations and are regulated by a group of genes known as clock genes or genes clock. Mutations in these genes in mice reduces insulin secretion and ?-pancreatic cell proliferation promoting impaired glucose tolerance and hyperglycemia. Nutritional disorders in the early stages of life are associated with the onset of type 2 diabetes in adulthood. Mice subjected to intrauterine protein restriction have altered expression of clock genes and increased susceptibility to weight gain and glucose intolerance. In this study we aimed to determine the daily expression of clock genes in peripheral tissues, hypothalamus and pancreatic islets of mice subjected to protein restriction. We also evaluated the oscillatory profile of the glucose stimulated insulin secretion and the cholinergic agonist carbachol in this animal model. Mice subjected to protein restriction (R) showed typical features of malnutrition as lower body weight , hypoinsulinemia , hypoproteinemia and increased glucose tolerance and insulin. R mice had higher food consumption, accompanied by changes in the oscillatory profile to Pomc and Npy gene in the hypothalamus. In this tissue, only the expression Rev- erb? gene was influenced by protein restriction. Mice R showed in the liver and muscle loss of the oscillatory profile to Clock and Bmal1 gene. Still, in liver and pancreatic islets the expression of Rev- erb? was changed, with reduction in mRNA content. Regarding the Per1 gene, R mice exhibited advance in the expression of this gene in adipose tissue. In muscle and islets there was loss of daily fluctuation for this gene. R mice exhibited, muscle and adipose tissue, in advance of Per2 gene expression. Islets isolated from control mice (C) showed oscillatory pattern of insulin secretion with the highest levels attained in the ZT 2 e 14 and reduction in the ZT 8. However, R mice had reduced glucose stimulated insulin secretion and loss of its oscillatory profile. R group showed no change in insulin release in the presence of Rev- erb? agonist and antagonist. Furthermore, the expression of Syntaxin, Synaptotagmin, and Insulin genes are reduced in R mice. R group also exhibited oscillatory loss of insulin secretion in the presence of glucose linked Carbachol and the reduction in the expression of Muscarinic Acetylcholine Receptor. With these results we can conclude that mice subjected to protein restriction showed typical features of malnutrition with alterations in glucose homeostasis and insulin secretion. Moreover, R mice exhibited loss of secretion of this hormone profile over 24 hours, which is associated with changes in the expression of Rev- erb?. In addition, there were changes in expression profile of clock genes, especially Rev-erb?, Per1 and Per2 in peripheral tissues, which may be related to changes in glucose tolerance and insulin in R mice / Mestrado / Fisiologia / Mestra em Biologia Funcional e Molecular

Orientadores: Everardo Magalhães Carneiro, Rosane Aparecida Ribeiro / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-22T08:39:55Z (GMT). No. of bitstreams: 1 Vettorazzi_JeanFranciesco_M.pdf: 1578894 bytes, checksum: 8f7aa3029a184083fe57d566c9524755 (MD5) Previous issue date: 2013 / Resumo: No acoplamento estímulo/secreção das células ?, os canais de K+ sensíveis ao ATP (KATP) e os canais de Ca2+ sensíveis à voltagem (Cav) contribuem para a geração e sustentação do potencial de ação. Alterações na sua expressão/atividade resultam em prejuízos na secreção de insulina. A restrição protéica e dieta hiperlipídica alteram o fluxo iônico e secreção de insulina nas células ?, e o aminoácido taurina (Tau) regula o fluxo iônico. O objetivo desse estudo foi caracterizar a expressão e o funcionamento dos KATP e Cav, assim como proteínas de extrusão dos grânulos de insulina em ilhotas pancreáticas de camundongos desnutridos alimentados com dieta hiperlipídica e suplementados com Tau. Camundongos machos C57Bl/6J receberam dieta controle (C) ou dieta lipoproteica (R) por 6 semanas. Em sequência metade desses grupos foi alimentados por 8 semanas com dieta hiperlipídica sem (CH e RH) ou com suplementação de 5% de Tau (CHT e RHT). Foi verificado que camundongos R tiveram menor crescimento e desenvolvimento dos órgãos. Camundongos RH e CH apresentaram aumento de peso corporal e estoques de gordura, hipercolesterolemia, intolerância à glicose e resistência à insulina. A suplementação com Tau preveniu o acúmulo de gordura e prejuízos na homeostase da glicose apenas no grupo CHT. Ilhotas isoladas de camundongos R secretaram menos insulina em resposta a 2,8 mM ou 11,1mM de glicose em associação com 30 mM de K+ ou 100 ?M de tolbutamida (Tolb). O influxo de Ca2+ em resposta à Tolb não diferiu entre ilhotas R e C. Ainda, ilhotas R tiveram redução do conteúdo total de insulina, da expressão protéica da SNAP-25, e aumento da subunidade Kir6.2 dos KATP e da sua atividade em resposta à glicose. Ilhotas CH secretaram mais insulina frente a 11,1 mM de glicose e agentes despolarizantes, associada à maior inibição dos KATP e ativação dos Cav em resposta à glicose. O grupo RH apresentou menor secreção de insulina frente a 2,8 mM de glicose e 30 mM de K+, e secreção similar ao grupo C frente a 11,1 mM de glicose e agentes despolarizantes, com aumento na expressão da SNAP-25 e inibição dos KATP em resposta à glicose. No grupo CHT houve normalização da liberação de insulina e aumento na expressão das subunidades ?1.2 e ?2 do Cav, SNAP-25 e sintaxina. Nas ilhotas RHT houve aumento na secreção de insulina frente à glicose e agentes despolarizantes, maior expressão da sintaxina e da subunidade Kir6.2 do KATP, bem como de sua inibição em resposta à glicose. Prejuízos na secreção de insulina no grupo R estão associados à menor expressão de proteínas envolvidas no processo exocitótico e aumento da expressão e atividade dos KATP. O grupo RH, com resistência à insulina similar ao grupo CH, não apresenta a mesma adaptação funcional das células ?, sem hipersecreção nesse grupo. A melhora na capacidade secretória do grupo RHT pode decorrer da ação da Tau sobre os KATP contribuindo para o adequado acoplamento estímulo/secreção e a extrusão dos grânulos de insulina via sintaxina / Abstract: In ? cells stimulus/secretion coupling, ATP-sensitive K+ channels (KATP) and voltage-sensitive Ca2+ channel (Cav) contribute to generating and sustaining the potential action. Changes in the expression or activity of these channels lead to insulin secretion impairment. Protein restriction as well as high-fat diet alters ?-cells ionic handling modifying insulin secretory profile. Taurine (Tau) regulates ion flux and improves ?-cell function. The aim of this study was to characterize the expression and function of KATP and Cav channels, as well as exocitotic proteins in isolated islets from malnourished mice submitted to high-fat diet and supplemented with Tau. Male C57Bl/6J mice received control (14% protein-C) or low-protein diet (6% protein-R) for 6 weeks. After, half of these groups were submitted to high-fat diet for 8 weeks without (CH and RH) or combined with 5% of Tau (CHT and RHT). Protein restricted mice showed lower growth and organs development. Both, CH and RH mice presented higher body weight, fat stores, hypercholesterolemia, glucose intolerance and insulin resistance. Tau supplementation prevented fat accumulation and glucose intolerance only in CHT group. Isolated islets from malnourished mice secreted less insulin in response to 2.8 mM glucose in combination with 30 mM K + or 100 ?M tolbutamide (Tolb). However, Ca2+ influx in these conditions did not differ. R group also released less insulin in response to 11.1 mM glucose in combination or not with depolarizing agents. In addition, R islets showed lower insulin and SNAP-25 protein content, whereas an increased protein expression of the Kir6.2 subunit of the KATP channel, as well as its activation during glucose stimulus. However, CH islets presented increased hormone release in the presence of 11.1 mM glucose in combination or not with depolaring agents, effect associated with enhanced KATP inhibition and Cav activation in response to glucose. RH group showed a lower insulin secretion in response to 2.8 mM glucose plus K+, and a similar secretion to C group upon 11.1 mM glucose together or not with depolarizing agents. Also, RH islets showed increased SNAP-25 protein levels. In CHT islets, Tau supplementation normalized insulin release and increased protein amount of ?1.2 and ?2 subunits of the Cav without modify its activity, and enhanced islet SNAP-25 and syntaxin protein levels. But, in RHT islets, enhanced insulin secretion in response to glucose and depolarizing agents, and increased syntaxin and Kir6.2 protein expression. In addition, RHT islets showed improved KATP inhibition and Cav activation in the presence of the sugar. In conclusion, insulin secretory dysfunction in R islets was associated with the lower expression of the exocytotic proteins and altered protein expression and activity of the KATP . Whereas, despite RH mice showed insulin resistance in a similar extent of that observed in CH mice, they did not present ?-cells functional adaptations to this condition, since RH islets did not hypersecret insulin. In addition, RHT islets presented a better secretory capacity probably due to the higher content of KATP and it inhibition induced by Tau which contributes to enhance the insulin exocytosis via syntaxin protein / Mestrado / Fisiologia / Mestre em Biologia Funcional e Molecular

Orientadores: Everardo Magalhães Carneiro, Rosane Aparecida Ribeiro / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-26T15:27:40Z (GMT). No. of bitstreams: 1 Santos-Silva_JuniaCarolinaRebelo_D.pdf: 1918048 bytes, checksum: 70dc34ec9c47cef2caa60225a914173e (MD5) Previous issue date: 2014 / Resumo: A obesidade é um importante fator que predispõe ao desenvolvimento do diabetes mellitus tipo 2 (T2DM). A transição da obesidade para o T2DM é precedida pela resistência à insulina que pode se manifestar em resposta a influências ambientais, como a ingestão de dietas ricas em gorduras, ou por fatores genéticos, como a deficiência na produção do hormônio leptina. Camundongos obesos ob/ob são caracterizados pela ausência da produção do hormônio leptina e apresentam obesidade por hiperfagia. A taurina (Tau) é um aminoácido sulfatado não essencial e dentre suas várias ações biológicas destaca-se seu envolvimento na regulação da homeostase glicêmica e metabolismo energético. Nosso objetivo foi avaliar o efeito da suplementação com Tau sobre o desenvolvimento da obesidade, homeostase da glicose, estrutura e função do pâncreas endócrino em camundongos ob/ob. Foram utilizados camundongos C57 e ob/ob que beberam água (C e ob) ou 5% de Tau (CT e obT) dos 30 aos 90 dias de vida. Camundongos ob apresentaram massivo acúmulo de gordura corporal, intolerância à glicose e resistência à insulina. Essas alterações na homeostase à glicose foram acompanhadas por alterações morfológicas e funcionais adaptativas na ilhota pancreática dos camundongos ob, sendo evidenciada hipersecreção de insulina em resposta à glicose associada ao maior influxo de Ca 2+ e redução da ação da somatostina sobre a célula ?. Essa maior resposta secretória também pode estar associada à maior ação do sistema nervoso parassimpático, visto que ilhotas ob secretaram mais insulina em resposta ao agonista colinérgico, carbacol (Cch), e quando as proteínas kinases (PK)-A e C foram ativadas. Contudo, ilhotas de fêmeas ob não apresentaram redução da secreção de insulina em resposta à fenilefrina, o que sugere menor sensibilidade simpática nesse grupo. A suplementação com Tau não preveniu o desenvolvimento da obesidade, mas melhorou a tolerância à glicose, reduziu a produção hepática de glicose e aumentou a sensibilidade à insulina. Ilhotas de camundongos obT apresentaram atenuação da hipersecreção de insulina em resposta à glicose. Tal efeito pode estar associado à melhora na sensibilidade da ilhota obT à somatostatina e ao menor influxo de Ca 2+ em resposta a glicose, bem como redução da amplificação da secreção em resposta à ação da via colinérgica/PKC e a maior sensibilidade simpática na ilhota de fêmeas obT. Ainda, foi observada maior secreção de glucagon em ilhotas de camundongos ob. Essa alteração pode ser devida a alterações na excitação da célula ?, pois o grupo ob apresentou aumento da frequência de oscilações de Ca 2+ em resposta a concentrações basais e estimulatórias de glicose. Todas as alterações fisiológicas da ilhota pancreática foram acompanhadas por aumento da massa de ilhotas, células ? e ? no pâncreas de camundongos ob. A Tau reduziu a massa de ilhotas e células ?, contudo no pâncreas do grupo obT houve maior massa de células ? e ?. As modificações morfofuncionais da ilhota ob foram acompanhadas por redução da expressão gênica do glucagon, somatostatina, do GLUT-2, TRPM5, do SSTR2, e MafB, porém aumento do conteúdo de mRNA de insulina, PAX6, PDX-1 e Ngn3. A Tau normalizou a expressão dos genes do glucagon, Glut-2 e TRPM5 e aumentou a expressão de MafA, Ngn3 e NeuroD. Nossos resultados, pela primeira vez, demonstraram que a suplementação com Tau melhorou a homeostase glicêmica regulando a morfo-fisiologia das células ?, ? e ? em camundongos geneticamente obesos, indicando um possível papel do aminoácido na preservação da função pancreática endócrina e também da interação parácrina de seus hormônios na obesidade e T2DM / Abstract: Obesity is an important factor that predisposes to the development of type 2 diabetes mellitus (T2DM). The transition from obesity to T2DM is preceded by insulin resistance which can manifest in response to environmental influences such as the intake of high-fat diets or genetic factors, such as deficiency in the production of leptin. Obese (ob/ob) mice are characterized by the absence of production of leptin and become obese due to severe hyperfagia. Taurine (Tau) is a non-essential amino acid and among its various biological actions stands out its involvement in the regulation of glucose homeostasis and energy metabolism. Our aim was to evaluate the effect of Tau supplementation upon the development of obesity, glucose homeostasis, structure and function of the endocrine pancreas in ob/ob mice. We used C57 and ob/ob who drank water (C and ob) or 5% of Tau (CT and obT) from 30 to 90 days of age. Ob mice showed massive accumulation of body fat, glucose intolerance and insulin resistance. These changes in glucose homeostasis were followed by adaptive morphological and functional changes in the pancreatic islet from ob mice with hypersecretion of insulin in response to glucose associated with increased Ca 2+ influx and a reduction in somatostatin action on the ? cell. This increased secretory response was also associated with increased activity of the parasympathetic nervous system, as ob islets secrete more insulin in response to the cholinergic agonist carbachol (Cch), and when protein kinases (PK) -A and C pahtways were activated. However, female ob islets showed no reduction in insulin secretion in response to phenylephrine, suggesting diminished sympathetic sensitivity in this group. Tau supplementation did not prevent the development of obesity, but improved glucose tolerance, reduced hepatic glucose production and increased insulin sensitivity. Islets from obT mice showed attenuation of hypersecretion of insulin in response to glucose. This effect may be associated with improved sensitivity of obT islet to somatostatin and lower Ca 2+ influx in response to glucose and reduced the amplification of insulin secretion in response to cholinergic/PKC pathway and increased sympathetic sensitivity in islets from female obT mice. Moreover, we observed higher glucagon secretion in islets from ob mouse and this change may be due to changes in the excitement of ? cell because the ob group increased frequency of Ca 2+ oscillations in response to basal and stimulatory concentrations of glucose. All physiological changes were accompanied by an increase in islet, ? and ? cells masses in the pancreas from ob mice. Tau reduced the mass of islets and ? cells, however there was a higher content of ? and ? cells masses in obT. Morphological and functional modifications in islets from ob mice were accompanied by reduced gene expression of glucagon, somatostatin, GLUT-2, TRPM5, SSTR2, and MafB, but increased mRNA content of insulin, PAX6, PDX-1 and Ngn3. Tau normalized gene expression of glucagon, TRPM5 and GLUT-2 and increased the expression of MafA, Ngn3 and NeuroD. Our results indicate, for the first time, that supplementation with Tau improves glucose homeostasis by regulating the morphology and physiology of ?, ? and ? cells in ob mice, indicating a possible therapeutic role for preservation of pancreatic endocrine function in obesity and T2DM / Doutorado / Fisiologia / Doutora em Biologia Funcional e Molecular