Xenotransplantation, i.e. transplantation between different species, could be a possible solution to the present shortage of organ donors. The immunological response to a xenograft is strong and difficult to suppress. It is driven both by the humoral and cellular part of the immune system. The aim of this thesis was to characterise and modulate this response in a concordant mouse-to-rat model, using both vascularised and non-vascularised grafts. Exposure of mouse cells or tissue to the circulation of a rat, either through transplantation or transfusions, easily evoked an immune response, consisting of IgM antibodies. A response that was aimed both at antigens present on mouse mononuclear cells and on erythrocytes. A non-immunosuppressed rat rejected a mouse heart graft within three days. The combined use of cyclosporine A (CyA) and deoxyspergualin (DSG) as immunosuppression prevented the rejection of vascularised heart transplants as well as of non-vascularised pancreatic islet grafts. This acceptance was sustained for the heart transplant also after the termination of DSG treatment, but not for the pancreatic islet graft. Furthermore, a second heart graft was accepted when transplanted under monotherapy with CyA 56-154 days after the first transplantation. This finding was interpreted as a humoral unresponsiveness, which could not be reproduced when the primary heart was substituted with a cellular graft, consisting of pancreatic islets or heart cells, or by blood transfusions. However, the rejection of a mouse heart after blood transfusions occurred in the absence of antibodies directed against mouse erythrocytes, in contrast to the observations in non-transfused animals. This indicates that a partial humoral tolerance restricted to the response against erythrocytes can be induced. This mechanism may offer a possibility to induce total humoral tolerance against a xenograft if the appropriate antigens are administered in conjunction with CyA and DSG.

Enterovirus (EV) infections have been associated with the pathogenesis of Type 1 Diabetes (T1D). However, the pathway(s) by which EV may induce or accelerate diabetes is not well understood. The purpose of this thesis was to obtain new information on the mechanism by which EV infections, with different strains of EV, could cause damage to the insulin-producing β-cells in isolated human islets and in a rat insulin-producing cell line (RINm5F). Infection with EV strains isolated from T1D patients revealed replication/cell destruction in human islets and EV-like particles in the cytoplasm of the β-cell and infection with the isolates affected the release of insulin in response to glucose stimulation as early as three days post infection, before any decrease in cell viability was observed. A decrease in the induction/secretion of the chemokine RANTES in human islets during EV infection was also detected. When islets were cultured with nicotinamide (NA) the secretion of RANTES was increased irrespectively if the islets were infected or not. In addition, the degree of virus-induced cytolysis of human islets was reduced by NA, suggesting an antiviral effect of NA. Infection with EV strains revealed permissiveness to islet-derived cells. All EV strains used for infection were able to replicate in the RIN cell clusters (RCC) but not in the RIN cells that were cultured as a monolayer. This might be due to the differences in expression of the Coxsackie-adenovirus receptor (CAR), which only could be detected on the RCC. Infection of RCC with a CBV-4 strain did not affect cell viability and did not induce nitric oxide (NO) production alone or with the addition of IFN-γ. This was in contrast to the results obtained with synthetic dsRNA, poly(IC), which induced NO, suggesting that synthetic dsRNA does not mimic enteroviral intermediate dsRNA. During analyses performed with the samples from a family where the mother and one son where diagnosed with T1D on the same day, the results showed that the whole family had a proven EV infection at the time diagnosis. To conclude, the ability of EV strains to replicate in RIN cells is dependent on the growth pattern of the cells and this may be due to the upregulation and/or changed expression pattern of CAR in these cells. In the RIN cells, contrary to artificial dsRNA, viral dsRNA does not induce NO. The isolated EV virus strains used were able to infect and affect human pancreatic islets in vitro. The chemokine RANTES is reduced during an EV infection of human pancreatic islets and NA causes upregulation of RANTES in infected and uninfected islets.

The bank vole (Microtus arvalis) develops glucose intolerance both when kept in captivity and in the wild state. Glucose intolerant bank voles kept in captivity exhibited polydipsia, polyuria, hyperglycemia, hyperinsulinemia, islet autoantibodies and a markedly changed islet structure resembling so–called hydropic degeneration. Islets showing hydropic degeneration have reduced β–cell mass. However, the relative islet size to total pancreas area was not changed. Pancreatic islet isolated from glucose intolerant bank voles had an altered islet function showing signs of being exposed to an increased functional demand on their β–cells. Also, islets from male bank voles seem more affected than the islets from females. Islets isolated from glucose tolerant male bank voles cultured for 5 days at 28 mM glucose did not reveal any change in insulin gene expression or insulin biosynthesis rate. However, islets from female bank voles displayed a glucose concentration dependent response. This suggests that there is gender difference in that, islets of female more easily than islets of males adapt to elevated glucose concentration. Furthermore, islets isolated from glucose tolerant males had reduced insulin gene expression after exposure to proinflammatory cytokines for 48 hrs. This effect seemed to be NO-independent since only a minor elevation of nitrite accumulation in the medium was seen, and the use of iNOS inhibitor could not counteract the cytokine effect. The observed response seen in bank vole islets upon exposure to various glucose concentrations or proinflammatory cytokines is similar to those seen in studies of human islets. The bank vole may therefore represent a novel animal model for the study of diabetes. An unresolved issue is the role of the Ljungan virus which is found in the bank vole colony. Bank voles developing glucose intolerance display features of both human type 1 and type 2 diabetes, where environmental factors seems to play an important role as determinant. Our findings suggest that bank voles bred in the laboratory may develop more of a type 2 diabetes. However, bank voles caught in nature instead may rather develop a type 1 form of the disease.

Patients with type-1 diabetes lose their β-cells after autoimmune attack. Islet transplantation is a co-option for curing this disease, but survival of transplanted islets is poor. Thus, methods to enhance β-cell viability and function as well as methods to expand β-cell mass are required. The work presented in this thesis aimed to study the roles of neural crest stem cells or their derivatives in supporting β-cell proliferation, function, and survival. In co-culture when mouse boundary cap neural crest stem cells (bNCSCs) and pancreatic islets were in direct contact, differentiating bNCSCs strongly induced β-cell proliferation, and these proliferating β-cells were glucose responsive in terms of insulin secretion. Moreover, co-culture of murine bNCSCs with β-cell lines RIN5AH and β-TC6 showed partial protection of β-cells against cytokine-induced β-cell death. Direct contacts between bNCSCs and β-cells increased β-cell viability, and led to cadherin and β-catenin accumulations at the bNCSC/β-cell junctions. We proposed that cadherin junctions supported signals which promoted β-cell survival. We further revealed that murine neural crest stem cells harvested from hair follicles were unable to induce β-cell proliferation, and did not form cadherin junctions when cultured with pancreatic islets. Finally, we discovered that the presence of bNCSCs in co-culture counteracted cytokine-mediated insulin-producing human EndoC-βH1 cell death. Furthermore, these two cell types formed N-cadherin, but not E-cadherin, junctions when they were in direct contact. In conclusion, the results of these studies illustrate how neural crest stem cells influence β-cell proliferation, function, and survival which may improve islet transplantation outcome.

O diabetes mellitus tipo 1 é uma doença autoimune causada pela destruição das células β produtoras de insulina, presentes nas ilhotas pancreáticas, por células autorreativas do sistema imune. A opção de tratamento mais utilizada são injeções diárias de insulina exógena, o que configura um tratamento não curativo. Para alcançar a independência de insulina, alternativas como o transplante de ilhotas vêm sendo estudadas. Entretanto, a disponibilidade de pâncreas de doadores cadavéricos para o isolamento destas ilhotas é pequena e os métodos de isolamento, pouco eficazes, sendo necessários de 2 a 4 doadores para atingir o número adequado de ilhotas. Além disso, o transplante apresenta problemas relacionados à enxertia, devidos principalmente à baixa vascularização, o que leva à morte de células β nos primeiros dias pós-transplante. Desta forma, estudos explorando alternativas que aumentem a sobrevivência e a funcionalidade dos transplantes e diminuam o número de ilhotas exigido por receptor fazem-se muito necessários. As células-tronco mesenquimais apresentam propriedades interessantes para aplicação em terapia celular. Entre elas, destaca-se o efeito parácrino, que exerce diversas funções benéficas, como o aumento da vascularização, nos locais onde estas células estão presentes. Sendo assim, este trabalho explorou o co-transplante de ilhotas pancreáticas com células-tronco mesenquimais derivadas de tecido adiposo, para o tratamento do diabetes mellitus em modelo murino. Os resultados mostraram que a presença destas células no grupo que recebeu o co-transplante não aumentou a taxa de cura, em relação ao grupo que recebeu somente ilhotas. No entanto, o fenômeno de reversão do diabetes foi antecipado no grupo co-transplantado, o que sugere um possível efeito angiogênico das células-tronco adiposo-derivadas presentes neste grupo. Desta forma, conclui-se que estas células podem exercer atividades benéficas, quando co-transplantadas com ilhotas pancreáticas, para o tratamento do diabetes. / Type 1 diabetes mellitus is an autoimmune disease caused by destruction of insulin-producing β cells, present in pancreatic islets, by auto-reactive cells of the immune system. The most widely used treatment option are daily injections of insulin, which configures a non-curative treatment. To achieve insulin independence, alternatives such as islet transplantation have been studied. However, the availability of pancreas from cadaveric donors for the isolation of these islets is poor and the methods for isolation, ineffective, requiring 2 to 4 donors to achieve the appropriate number of islets. In addition, transplantation presents problems related to engraftment, mainly due to poor vascularization, which leads to β cell death in the first days after transplantation. Thus, studies exploring alternatives that increase the survival and function of transplants and reduce the number of islets required by the recipient are very necessary. Mesenchymal stem cells have interesting properties for application in cell therapy. Among them is the paracrine effect, which has several beneficial functions, such as promoting vascularization in the tissues where these cells are present. Thus, the present study explored the co-transplantation of pancreatic islets with mesenchymal stem cells derived from adipose tissue for the treatment of diabetes mellitus in mice. The results showed that the presence of these cells in the group that received co-transplantation did not increase the cure rate, compared to the group that received islets alone. However, the phenomenon of diabetes reversion was anticipated in co-transplanted animals, which suggests a possible angiogenic effect of adipose-derived stem cells present in this group. Thus, we conclude that these cells may exert beneficial functions when co-transplanted with pancreatic islets for the treatment of diabetes.

O diabetes mellitus tipo 1 é uma doença autoimune causada pela destruição das células β produtoras de insulina, presentes nas ilhotas pancreáticas, por células autorreativas do sistema imune. A opção de tratamento mais utilizada são injeções diárias de insulina exógena, o que configura um tratamento não curativo. Para alcançar a independência de insulina, alternativas como o transplante de ilhotas vêm sendo estudadas. Entretanto, a disponibilidade de pâncreas de doadores cadavéricos para o isolamento destas ilhotas é pequena e os métodos de isolamento, pouco eficazes, sendo necessários de 2 a 4 doadores para atingir o número adequado de ilhotas. Além disso, o transplante apresenta problemas relacionados à enxertia, devidos principalmente à baixa vascularização, o que leva à morte de células β nos primeiros dias pós-transplante. Desta forma, estudos explorando alternativas que aumentem a sobrevivência e a funcionalidade dos transplantes e diminuam o número de ilhotas exigido por receptor fazem-se muito necessários. As células-tronco mesenquimais apresentam propriedades interessantes para aplicação em terapia celular. Entre elas, destaca-se o efeito parácrino, que exerce diversas funções benéficas, como o aumento da vascularização, nos locais onde estas células estão presentes. Sendo assim, este trabalho explorou o co-transplante de ilhotas pancreáticas com células-tronco mesenquimais derivadas de tecido adiposo, para o tratamento do diabetes mellitus em modelo murino. Os resultados mostraram que a presença destas células no grupo que recebeu o co-transplante não aumentou a taxa de cura, em relação ao grupo que recebeu somente ilhotas. No entanto, o fenômeno de reversão do diabetes foi antecipado no grupo co-transplantado, o que sugere um possível efeito angiogênico das células-tronco adiposo-derivadas presentes neste grupo. Desta forma, conclui-se que estas células podem exercer atividades benéficas, quando co-transplantadas com ilhotas pancreáticas, para o tratamento do diabetes. / Type 1 diabetes mellitus is an autoimmune disease caused by destruction of insulin-producing β cells, present in pancreatic islets, by auto-reactive cells of the immune system. The most widely used treatment option are daily injections of insulin, which configures a non-curative treatment. To achieve insulin independence, alternatives such as islet transplantation have been studied. However, the availability of pancreas from cadaveric donors for the isolation of these islets is poor and the methods for isolation, ineffective, requiring 2 to 4 donors to achieve the appropriate number of islets. In addition, transplantation presents problems related to engraftment, mainly due to poor vascularization, which leads to β cell death in the first days after transplantation. Thus, studies exploring alternatives that increase the survival and function of transplants and reduce the number of islets required by the recipient are very necessary. Mesenchymal stem cells have interesting properties for application in cell therapy. Among them is the paracrine effect, which has several beneficial functions, such as promoting vascularization in the tissues where these cells are present. Thus, the present study explored the co-transplantation of pancreatic islets with mesenchymal stem cells derived from adipose tissue for the treatment of diabetes mellitus in mice. The results showed that the presence of these cells in the group that received co-transplantation did not increase the cure rate, compared to the group that received islets alone. However, the phenomenon of diabetes reversion was anticipated in co-transplanted animals, which suggests a possible angiogenic effect of adipose-derived stem cells present in this group. Thus, we conclude that these cells may exert beneficial functions when co-transplanted with pancreatic islets for the treatment of diabetes.

Acredita-se que a diminuição progressiva da massa de células observada durante a evolução do diabetes mellitus tipo 2 (DM 2) ocorra por apoptose deste tipo celular. As tiazolidinedionas (TZDs), uma classe de medicamentos utilizada no tratamento do DM 2, atuam como ligantes dos receptores ativados por proliferadores de peroxissomos (PPAR) e e promovem diminuição da resistência periférica à insulina. Embora existam estudos controversos, tem se especulado que as TZDs possam exercer efeitos diretos sobre as células pancreáticas, prevenindo a perda por apoptose e melhorando a sua viabilidade. O objetivo deste estudo foi avaliar os efeitos diretos da Pioglitazona (PIO) na concentração de 10 M sobre a viabilidade funcional e o índice de apoptose de ilhotas pancreáticas isoladas de ratos Wistar expostas a concentrações fisiológica (5,6 mM) e suprafisiológica (23 mM) de glicose durante 24, 48 e 72 horas. A viabilidade funcional foi avaliada pela análise da secreção de insulina estimulada por glicose e do conteúdo total de insulina nas ilhotas. O índice de apoptose foi avaliado pela medida da fragmentação do DNA, da expressão do RNAm dos genes Bcl2 (anti-apoptótico) e Bax (pró-apoptótico) e da atividade proteolítica da caspase-3 em ilhotas tratadas e não tratadas com a PIO. Em 5,6 mM de glicose, não se observou efeito significativo sobre a secreção de insulina, mas a avaliação do conteúdo total de insulina evidenciou uma diminuição transitória nas ilhotas tratadas com PIO por 24 horas, seguida por um aumento no conteúdo de insulina quando as ilhotas foram cultivadas por 48 e 72 horas em presença da droga. Em relação à avaliação da apoptose, observou-se uma diminuição na expressão do RNAm do gene Bax nas ilhotas tratadas com PIO por 24 horas, entretanto, após 48 e 72 horas, houve um aumento da expressão do RNAm deste gene nas ilhotas tratadas com a droga. Não foram observadas diferenças estatisticamente significativas na expressão do RNAm do gene Bcl2 em nenhum dos tempos estudados e a avaliação da apoptose determinada pela medida da fragmentação do DNA somente demonstrou uma diminuição do índice de apoptose após 48 horas de tratamento com a PIO. Em 23 mM de glicose, a PIO promoveu um aumento transitório na secreção de insulina estimulada por glicose e no conteúdo total de insulina (após 48 horas), no entanto, após 72 horas, observou-se diminuição significativa no conteúdo total de insulina. Em relação à apoptose, o tratamento com PIO determinou um aumento do índice de apoptose medido pela fragmentação do DNA e da atividade proteolítica da caspase-3 após 48 e 72 horas e uma diminuição da expressão do RNAm do gene Bcl2 nos tempos 24 e 48 horas. Os resultados do presente estudo sugerem que os efeitos diretos da PIO sobre as ilhotas pancreáticas murídeas em cultura variam de acordo com a concentração de glicose a qual as ilhotas estão expostas: em concentração fisiológica de glicose, a PIO parece exercer efeitos diretos benéficos, enquanto em concentração suprafisiológica de glicose, ela exerce efeitos diretos deletérios sobre a viabilidade funcional e o índice de apoptose de ilhotas pancreáticas murídeas em cultura. / The progressive decrease in -cell mass observed during the evolution of type 2 diabetes (T2DM) is believed to occur due to cell apoptosis. Thiazolidinediones (TZDs), a class of agents used for the treatment T2DM, act as ligands of the peroxisome proliferator-activated receptor (PPAR) and and decrease peripheral insulin resistance. Although still controversial, some studies have shown a direct effect of TZDs on pancreatic -cell, preventing cell loss due to apoptosis and improving their viability. The objective of this study was to evaluate the direct effects of 10 M Pioglitazone (PIO) on functional viability and apoptosis rate of islets isolated from Wistar rats exposed to physiological (5.6 mM) and supraphysiological (23 mM) glucose concentrations during 24, 48 and 72 hours. The functional viability was evaluated by the analysis of insulin secretion after glucose challenge and of islet total insulin content. Apoptosis rate was evaluated by measurement of DNA fragmentation, of Bcl2 (antiapoptotic) and Bax (proapoptotic) mRNA expression and of proteolytic activity of caspase-3 in pancreatic islets treated or not with PIO. At 5.6 mM glucose concentration, no significant effects in insulin secretion were observed, while a transitory decrease (after 24 hours) followed by an increase in total insulin content was observed in islets treated with PIO for 48 and 72 hours. Regarding apoptosis, a lower expression of Bax mRNA was detected in islets treated with PIO for 24 hours, followed, however, by an increase in the expression of this gene after 48 and 72 hours of drug exposition. PIO treatment did not promote significant changes in Bcl2 mRNA expression, while decreased the apoptosis rate measured by DNA fragmentation only after 48 hours of exposition. At 23 mM glucose concentration, PIO treatment elicited a transitory increase in insulin secretion after glucose challenge and in islet total insulin content after 48 hours followed by a decrease in the islet total insulin content after 72 hours. Concerning apoptosis, PIO treatment determined an increase in the apoptose rate measured by DNA fragmentation and by proteolytic activity of caspase-3 after 48 and 72 hours and a decrease in Bcl2 mRNA expression after 24 and 48 hours. These findings suggest that the direct effects of PIO on pancreatic islets depend on glucose concentration to which they are exposed: while under physiological glucose concentration the direct effects seem to be beneficial, under supraphysiological glucose concentration, PIO exerts direct deleterious effects on the functional viability and on the apoptosis rate of murine pancreatic islets.

Orientador: Antonio Carlos Boschero / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-13T00:36:09Z (GMT). No. of bitstreams: 1 Rezende_LuizFernandode_D.pdf: 9509933 bytes, checksum: fc8e235ac3d4242d8e48c28911285e25 (MD5) Previous issue date: 2009 / Resumo: Introdução: O CNTF pertence à família da IL-6, e sendo assim, sinaliza pelo complexo receptor gp130, ativando diversas vias de sinalização dependendo do tipo celular, principalmente as vias STAT3, MAPK e PI3K. Seus efeitos incluem diferenciação e/ou sobrevivência neuronal, e é diferencialmente expresso ao longo da vida do animal. As ilhotas pancreáticas, por sua vez, são ricamente enervadas e expressam receptores para NGFs, podem apresentar respostas neurotípicas e expressam o CNTF. O objetivo desse estudo foi investigar os possíveis efeitos do CNTF sobre a diferenciação e/ou sobrevivência de ilhotas pancreáticas de ratos neonatos, qual(is) via(s) de sinalização ele ativa nessas ilhotas e como é expresso nelas ao longo da vida dos animais. Material e Métodos: Ilhotas pancreáticas de ratos neonatos (1-2 dias) foram isoladas pelo método de colagenase e cultivadas por 3 dias em meio RPMI com (CNTF) ou sem (CTL) 1nM de CNTF. Após isso, foram analisados a secreção de insulina estimulada por glicose (RIE), metabolismo (MTS, produção de NADPH), metabolismo de glicose (produção de 14CO2), expressão gênica (RTPCR), protéica (Western-Blot), atividade de caspase-3 (DEVD) e apoptose (fragmentação de DNA). Resultados: O CNTF reduziu a secreção de insulina estimulada por glicose e o metabolismo de ilhotas pancreáticas, não alterando o metabolismo de glicose e expressão de proteínas cruciais para a função das ilhotas. Por outro lado, o CNTF aumentou a expressão de proteínas relacionadas à sobrevivência das ilhotas pancreáticas, como Cx36, PAX4, e BCL-2, reduziu a atividade da caspase-3 e a apoptose das ilhotas. O CNTF também aumentou a fosforilação de STAT3, sua translocação ao núcleo e expressão de genes-alvo, como a SOCS-3, levando à redução da GSIS e sobrevivências observadas, apesar de não ativar as vias da MAPK e PI3K. Mais ainda, a expressão do CNTF é aumentada em ilhotas pancreáticas de ratos de 2 meses de idade, e assim permanecendo até os 20 meses de idade. Conclusão: O CNTF não promove a maturação de ilhotas pancreáticas, mas sim sua sobrevivência, e esses efeitos são mediados através da via JAK/STAT3, sem ativar as vias MAPK ou PI3K. Finalmente, o CNTF possui expressão diferenciada ao longo da vida do animal / Abstract: Introduction: CNTF belongs to the IL-6 cytokine family and as such, it signals through gp130 receptor complex, activating many pathways depending on the cell-type, mainly STAT3, MAPK and PI3K. Its effects include increased neuron differentiation and/or survival, and are differentially expressed throughout the animal life. Meanwhile, pancreatic islets are richly innervated and express receptors for NGFs, may undergo neurotypic responses, and express CNTF. The aim of this study was to investigate possible effects of CNTF on neonatal rat pancreatic islet differentiation and/or survival, which signalling pathway (s) it activates on pancreatic islets and how it is expressed in the pancreatic islets throughout the animal life. Methods: Pancreatic islets from neonatal rats (1-2 d old) were isolated by the collagenase method and cultured for 3 days in RPMI medium with (CNTF) or without (CTL) 1nM of CNTF. Thereafter, glucose-stimulated insulin secretion (RIA), general metabolism by (NAD(P)H production) (MTS), glucose metabolism (14CO2 production), gene (RT-PCR), protein expression (Western-Blot), caspase-3 activity (DEVD), and apoptosis (DNA fragmentation) were analysed. Results: CNTF reduced pancreatic islets GSIS and metabolism, whereas not affecting glucose metabolism and the expression of proteins crucial for the islets function. Conversely, CNTF significantly expression of proteins related pancreatic islets survival, such as Cx36, PAX4, and BCL-2, reduced caspase-3 activity and islet-cells apoptosis. CNTF also increases STAT3 fosforilation, translocation to the nuclei and expression of target genes, resulting in the reduced GSIS and survival observed, although not affecting MAPK and PI3K pathways. Moreover, CNTF expression is increased in rats pancreatic islets after 2 months of age, and it remains so until 20 months of age. Conclusion: CNTF has no effect over maturation of pancreatic islets function, whereas it improves pancreatic islets survival, and also that these effects are mediated through JAK/STAT3 but not through MAPK or PI3K pathways. Finally, CNTF is differentially expressed in rat pancreatic islets throughout the animal life / Doutorado / Fisiologia / Doutor em Biologia Funcional e Molecular

Não é claro como o receptor de glicocorticóide (GR) contrarregula a atividade do STAT5 na transição da gestação para a lactação. Dexras1 é uma pequena proteína G ativada por dexametasona (DEX), que regula morfologia celular, crescimento, etc. Neste estudo detectamos a expressão de Dexras1 em células beta de ilhotas pancreáticas. DEX induz a expressão de Dexras1 em células RINm5F, que está aumentada na gestação e diminuída na lactação. A expressão protéica de 11<font face=\"Symbol\">&#946HSD1, enzima ativadora de glicocorticóides (GCs), segue esse perfil. A ligação tanto do GR como do STAT5, analisada por ChIP assay, ao promotor do gene da Dexras1 aumentada por DEX é revertida por prolactina (PRL), e está diminuída e aumentada na gestação e lactação, respectivamente. DEX induz a associação ao GR, fosforilação e translocação nuclear do STAT5b. O silenciamento gênico de Dexras1 promoveu aumento da secreção de insulina, e aumentou os níveis de pERK1/2, pCREB, pPKC<font face=\"Symbol\">&#948 e PKA. Sendo assim, a regulação de Dexras1 por PRL e GCs contribui para a secreção de insulina característica do periparto. / It is not clear how glucocorticoid receptor (GR) counteracts STAT5 activity during the transition of pregnancy to lactation. Dexras1 is a small G protein activated by dexamethasone (DEX) that controls cell morphology, growth, etc. In the present study we detected Dexras1 expression in pancreatic beta cell. DEX induces Dexras1 expression in RINm5F cells, which is increased in pregnancy and decreased in lactation. The expression of 11<font face=\"Symbol\">&#946HSD1, the glucocorticoids (GCs) activating enzyme, followed Dexras1 profile in pancreatic islet. Both GR and STAT5b bindings to Dexras1 gene promoter, analyzed by ChIP assay, are increased by DEX and PRL counteracts this effect. Both bindings are decreased in pregnancy and increased in lactation. DEX induces STAT5b association to GR, phosphorylation and nuclear translocation. Dexras1 knockdown using small interference RNA (si-RNA) promoted an increase in insulin secretion, as well as increased levels of pERK1/2, pCREB, pPKC<font face=\"Symbol\">&#948 and PKA. Thus, Dexras1 regulation by PRL and GCs contributes to insulin secretion during peripartum.

Orientador: Mario José Abdalla Saad / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-21T17:03:51Z (GMT). No. of bitstreams: 1 Araujo_TiagoGomes_D.pdf: 9136785 bytes, checksum: 25c68d3dd56714d164986b415f98070d (MD5) Previous issue date: 2012 / Resumo: A resistência à insulina está presente na obesidade e na diabetes tipo 2, e está associada à hiperplasia das ilhotas pancreáticas e hiperinsulinemia como resposta compensatória, entretanto, as forças motrizes por trás desse mecanismo compensatório não são totalmente compreendidos. Dados anteriores sugeriram o envolvimento de um fator circulante desconhecido que na resistência à insulina atua como um fator de crescimento das células ?. Neste contexto, procurando por candidatos a serem este fator circulante, percebemos que o fator de crescimento de hepatócitos (HGF) é um forte candidato a ser este elo entre a resistência à insulina e o aumento da massa de ilhotas / hiperinsulinemia. Nossa abordagem teve como objetivo mostrar uma possível relação de causa-efeito entre o aumento dos níveis circulantes de HGF e a hiperplasia da ilhota / hiperinsulinemia compensatória, assim mostrando a força da associação. Ainda, se esta associação, apresenta ou não uma resposta dose-dependente, temporalidade, consistência, plausibilidade e reversibilidade. Nesse sentido, os nossos dados mostraram: a) uma correlação forte e consistente entre o HGF e o mecanismo de compensação em três modelos animais de resistência à insulina; b) o HGF aumenta a massa de célula ? de uma forma dose-dependente; c) o bloqueio do HGF interrompe os mecanismos de compensação; d) o aumento nos níveis de HGF precede a resposta compensatória associada com a resistência à insulina, indicando que estes eventos ocorrem em um modo sequencial. Além disso, o bloqueio do receptor de HGF (Met) piorou a já prejudicada sinalização da insulina no fígado de ratos obesos induzidos por dieta. Em geral, os nossos dados indicam que o HGF é um fator de crescimento que desempenha um papel fundamental no aumento da massa de ilhotas e hiperinsulinemia em ratos obesos induzidos por dieta, e sugerem um efeito protetor da interação HGF-Met na sinalização de insulina no fígado / Abstract: Insulin resistance is present in obesity and in type 2 diabetes, and is associated with islet cell hyperplasia and hyperinsulinemia but the driving forces behind this compensatory mechanism are incompletely understood. Previous data have suggested the involvement of an unknown circulating insulin resistance-related ?-cell growth-factor. In this context, looking for candidates to be a circulating factor, we realized that hepatocyte growth factor (HGF) is a strong candidate as a link between insulin resistance and increased mass of islets/hyperinsulinemia. Our approach aimed to show a possible cause-effect relationship between increase in circulating HGF levels and compensatory islet hyperplasia/hyperinsulinemia by showing the strength of the association, whether or not is a dose-dependent response, the temporality, consistency, plausibility and reversibility of the association. In this regard, our data showed: a) a strong and consistent correlation between HGF and the compensatory mechanism in three animal models of insulin resistance; b) HGF increases ?-cell mass in a dose-dependent manner; c) blocking HGF shuts down the compensatory mechanisms; d) an increase in HGF levels seems to precede the compensatory response associated with insulin resistance, indicating that these events occur in a sequential mode. Additionally, blockages of HGF receptor (Met) worsen the impaired insulin-induced insulin signaling in liver of diet-induced obesity rats. Overall, our data indicate that HGF is a growth factor playing a key role in islet mass increase and hyperinsulinemia in diet-induced obesity rats, and suggest a protective effect of the HGF-Met axis on insulin signaling in the liver / Doutorado / Medicina Experimental / Doutor em Ciências