Fetal and neonatal pancreatic islets present a lower insulin secretory response as compared with adult islets. Prolonged culturing leads to an improvement of the glucose-induced insulin secretion response in neonatal pancreatic islets that may involve regulation of gap junction mediated cell communication. In this study, we investigated the effect of culturing neonatal islet cells for varying periods of time and with different glucose medium concentrations on the cellular expression of the endocrine pancreatic gap junction associated connexin (Cx) 36 and Cx43. We report here that the 7-d culture induced upregulation of the expression of these junctional proteins in neonatal islets in a time-dependent manner. A correlation was observed between the increased mRNA and protein expression of Cx36 and Cx43 and the increased insulin secretion following islet culturing. In addition, increasing glucose concentration within the culture medium induced a concentration-dependent enhancement of Cx36 islet expression, but not of Cx43 expression in cultured neonatal islets. In conclusion, we suggest that the regulation of gap junctional proteins by culture medium containing factors and glucose may be an important event for the maturation process of beta cells observed at in vitro conditions.

Cell-cell contacts mediated by intercellular junctions are crucial for proper insulin secretion in the endocrine pancreas. The biochemical composition of the intercellular junctions in this organ and the role of junctional proteins in endocrine pancreatic dysfunctions are still unclear. In this study, we investigated the expression and cellular location of junctional and cytoskeletal proteins in cultured neonatal rat pancreatic islets. Neonatal B-cells had an impaired insulin secretion compared to adult cells. Cultured neonatal islets showed a time-dependent increase in the glucose-induced secretory response. The maturation of B-cells in vitro was accompanied by upregulation of the expression of some junctional proteins in islet cells. Neonatal islets cultured for only 24 h showed a low expression and a diffuse cytoplasmic location of the tight junctional proteins occludin and ZO-1 and of the adherens junctional proteins alpha- and beta-catenins, as demonstrated by immunoblotting and immunocytochemistry. Culturing islets for up to 8 days significantly increased the cell expression of these junctional proteins but not of the cytoskeletal proteins vinculin and alpha-actinin. A translocation of ZO-1 and catenins to the cell-cell contact region, as well as a higher association of F-actin with the intercellular junction, were also observed in neonatal islets following prolonged culturing. ZO-1 and beta-catenin were immunolocated in the endocrine pancreas of adult rats indicating that these junctional proteins are also expressed in this organ in situ. In conclusion, endocrine pancreatic cells express several junctional proteins that are upregulated following differentiation of the endocrine pancreas in vitro.

parte dos requisitos necessários para a obtenção do título de Mestre em Biologia Vegetal. Campinas 2005 iv Dedico aos meus pais, Francisco e Francisca. v AGRADECIMENTOS Sou muito grato a todos que contribuíram, direta ou indiretamente, para realização deste trabalho, em especial: À CAPES e ao CNPq pela bolsa de estudo; À FAEP/UNICAMP pelo auxílio financeiro durante as fases iniciais do projeto; Ao Fernando pela orientação, confiança e por estar sempre presente quando precisei; Aos membros da pré-banca e banca pelas valiosas sugestões que enriqueceram este trabalho: Flavio Santos, José Eugênio, Dalva Matos, Luciana Alves e Kikyo Yamamoto; Ao professor Flavio (Flavião), que agradeço especialmente, pela ajuda com materiais e sugestões em várias etapas do trabalho; Aos administradores e funcionários do Condomínio Colinas do Atibaia, especialmente ao Jorge e ao Marcelo (portaria) por serem sempre tão prestativos; Às pessoas que me ajudaram no trabalho de campo pelo empenho e dedicação: Fabiano Berlini, Karina Degaki, Mirela e Thiago; À Karina pelo amor e companheirismo; À Professora Cláudia por ter me cedido espaço para cultivar as plântulas na casa de vegetação; Ao Fabiano Michelletto pela amizade e por ter sido meu anfitrião no departamento de Fisiologia Vegetal;Ao professor Jorge Tamashiro pelas dicas de reconhecimento das plântulas; Ao Professor Ivany pela boa vontade e pela cessão do sensor de luz; Aos colegas do departamento de Botânica pelos momentos mais variados possíveis que compartilhamos: Aneliza (que foi quase uma co-orientadora!),

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