Búsqueda de dianas terapéuticas en la célula beta-pancreática para el tratamiento de la diabetes

  1. López, José Francisco
Dirixida por:
  1. Germán M. Perdomo Hernández Co-director
  2. Irene Cozar Castellano Co-director

Universidade de defensa: Universidad de Valladolid

Fecha de defensa: 13 de decembro de 2013

Tribunal:
  1. Ana Obeso Caceres Presidenta
  2. Ana Isabel Rojas González Secretario/a
  3. Adolfo García Ocaña Vogal
  4. David Antonio Cano González Vogal
  5. Eduard Montanya Mías Vogal
Departamento:
  1. Bioquímica y Biología Molecular y Fisiología

Tipo: Tese

Resumo

There is an urgency to find new therapeutic targets and effective drugs for diabetes mellitus treatment, to improve glycemic control and diabetes complications. Pancreatic beta-cells viability and function are impaired in the two most common forms of diabetes, type 1 and type 2. Regeneration of functional pancreatic beta-cell mass has been proposed as a potential therapy for diabetes. Therefore, we have proposed two aims, first, the regeneration and protection of pancreatic beta-cell mass; and second, to study proteins that could be crucial for beta-cell function, hence, potential therapeutic targets for diabetes. In the first part of this thesis project, we screened a collection of marine products looking for beta-cell proliferation induction. One unique compound (epoxypukalide) showed capability to induce beta-cell replication, both in the INS1 832/13 cell line and primary cultures of pancreatic islets. Epoxypukalide was used to study beta-cell proliferation by [3H]thymidine incorporation and BrdU incorporation followed by BrdU/insulin staining in primary cultures of rat islets. AKT and ERK1/2 signalling pathways were analyzed. Cell cycle activators were detected by western-blot. Apoptosis was studied by TUNEL and cleaved caspase 3. Pancreatic beta-cell function was measured by glucose-stimulated insulin secretion (GSIS). Epoxypukalide induced 2.5-fold increase in beta-cell proliferation; this effect was mediated by activation of ERK1/2 signalling pathway and upregulation of the cell cycle activators, cyclin D2 and cyclin E. Interestingly, epoxypukalide showed protection from basal (40% lower versus control) and cytokine-induced apoptosis (80% lower versus control). Finally, epoxypukalide did not impair beta-cell function when measured by GSIS in vitro. In conclusion, our study showed that epoxypukalide has regenerative and protective properties on the beta-cell. These findings may be translated into new treatments for diabetes. In the second part, we studied the role of Insulin-degrading enzyme (IDE) in the pancreatic beta-cell under physiologic and pathophysiologic conditions. IDE is an ubiquitous metalloprotease that degrades insulin. It is localized in a susceptibility locus for type 2 diabetes in humans. At the beginning of this study there were not publications on the role of IDE in the pancreatic beta-cell, and its function was completely unknown. To this end, we studied IDE expression levels in islets of hyperinsulinemic db/db mice. We measured IDE expression in INS-1E cells and primary cultures of rat islets, under physiological and pathophysiological levels of glucose and insulin. To understand the physiological role of IDE in the pancreatic beta-cell, we used siRNA-IDE in INS-1E cells, and 1,10¿Phenantroline (an inhibitor of IDE activity) in rat islets, performing glucose-stimulated insulin secretion (GSIS) to study beta-cell function. We also studied the expression of proteins involved in the secretory machinery of the beta-cell. We showed that IDE expression was increased in islets of hyperinsulinemic db/db mice, correlating to plasma insulin levels. IDE expression was markedly downregulated by hyperglycemia in INS-1E cells and rat islets. In contrast, IDE was upregulated by hyperinsulinemia. Surprisingly, IDE loss-of-function reduced GSIS by 50%, whereas beta-cell insulin content remained unchanged. In parallel, secreted C-peptide levels were decreased by 50%. To study the molecular mechanisms underlying these effects, we measured glut2, glucokinase, sur1 and kir6.2 levels by RT-PCR, showing no changes in expression after IDE loss-of-function. In conclusion, our results are consistent with recently published work where IDE-null mice showed a decreased GSIS due to impaired replenishment of the releasable pool of insulin granules. Together, we have unraveled an unexpected role of IDE on beta-cell GSIS. These data suggest that IDE could be a therapeutic target for diabetes treatment.