The overall objective of this proposal is to provide better insight into mechanisms that cause beta-cell apoptosis in Type 1 and Type 2 diabetes mellitus. Human beta-cell death signaling mechanisms are incompletely understood and very little is known about early events in human beta-cell apoptosis. The endoplasmic reticulum (ER) and mitochondria play essential roles in apoptosis in many types of cells. Apoptosis is associated with activation of caspases, a family of cysteine proteases, many of which function as cell executioners. ER stress increases expression of the transcription factor CHOP (C/EBP homologous protein also known as GADD153) gene and activates specific members of the caspase family. Mitochondria membrane permeability is increased during apoptosis and release of proteins from mitochondria is an early and necessary step in cell death. The temporal and causal interrelationships between ER stress, mitochondria function, and human beta-cell death are not understood. Our preliminary experiments in MIN6 cells and in mouse islets of Langerhans indicate that ER stress induced by disruption of sarcoendoplasmic reticulum Ca2+-ATPase (SERCA) activity and ER Ca2+ homeostasis causes apoptosis. The proposed experiments will: [1] identify molecular signals associated with apoptosis induced by ER stress and mitochondria dysfunction in human beta-cells; [2] determine whether proinflammatory cytokines induce human beta-cell ER stress and changes in mitochondrial membrane permeability; and [3] define the temporal and causal interrelationships between ER stress and mitochondria-induced cell death signaling in human islets of Langerhans. The following hypotheses will be tested: [1] ER stress activates human beta-cell apoptosis via caspase- and CHOP-dependent pathways; [2] ER stress induces mitochondrially-derived cell death signals; and [3] cytotoxic cytokines perturb beta-cell ER and mitochondrial Ca2+ homeostasis, induce release of pro-apototic proteins (cytochrome c and Smac/DIABLO) from mitochondria, activate caspases and induce CHOP expression. Human islets and beta-cells will be studied. Experimental procedures will include visualization of subcellular Ca2+ concentration gradients with genetically targeted Ca2+ biosensors, real-time measurements of caspase activity with fluorescent biosensors, cytochrome c and Smac/DIABLO release, and application of real-time quantitative PCR to monitor SERCA and CHOP expression in islets. The proposed studies will provide new insights into mechanisms regulating human beta-cell viability and information essential in advancing our understanding of the pathogenesis of both Type 1 and Type 2 diabetes mellitus.