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

Resumen

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.