Identification and characterization of pancreatic beta-cell survival factors

Programmed β-cell death plays an important role in both type 1 and type 2 diabetes, but analysis of candidate survival factors has yielded a few hormones and growth factors exhibiting modest β-cell protection against various stresses. Most of what is known about the mechanisms of β-cell death comes from single time-point, single parameter measurements of bulk populations of mixed cells, which are inadequate for studying the heterogeneity in death mechanisms. We simultaneously measured the kinetics of six distinct cell death mechanisms by using a caspase-3 sensor and three vital dyes, together with bright-field imaging. This allowed the characterization of the timing and order of molecular events associated with cell death in single β-cells under multiple diabetic stress conditions. Using this approach, we identified several cell death modes where the order of events that typically define apoptosis were not observed. It is becoming increasingly apparent that islets release and respond to more secreted factors than previously thought and systematic analyses of their pro-survival effects can assist in therapeutic developments. Novel putative autocrine/paracrine signalling loops in islets were identified by compiling results from gene expression datasets. Factors best known for their roles in axon guidance, Netrin and Slit families, were further characterized for their pro-survival roles in adult β-cells. With the development of the live-cell imaging-based, high-throughput screening methods capable of identifying factors that modulate β-cell death, we screened the Prestwick library of small molecules and a custom library of endogenous factors. Carbamazepine, a Na+ channel inhibitor, down-regulated the pro-apoptotic and ER-stress signalling induced by cytotoxic cytokines pointing to Na+ channels as a novel therapeutic target in diabetes. Whether specific cellular stresses associated with type 1 or type 2 diabetes require specific β-cell survival factors remains unknown. Our comparison of 206 endogenous soluble factors, predicted to act on islet cells, under 5 diabetes-relevant stress conditions revealed unique sets of protective survival factors for each stress and identified a cluster of survival factors that exhibited generalized protective effects. Since diabetes results from a deficiency in functional β-cell mass, these studies are important steps towards developing novel therapies to improve β-cell survival and function.Medicine, Faculty ofGraduat