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OBJECTIVE-Endoplasmic reticulum (ER) stress has been implicated in the pathogenesis of diabetes, but the roles of specific ER Ca^sup 2+^ release channels in the ER stress-associated apoptosis pathway remain unknown. Here, we examined the effects of stimulating or inhibiting the ER-resident inositol trisphosphate receptors (IP^sub 3^Rs) and the ryanodine receptors (RyRs) on the induction of β-cell ER stress and apoptosis.
RESEARCH DESIGN AND METHODS-Kinetics of β-cell death were tracked by imaging propidium iodide incorporation and caspase-3 activity in real time. ER stress and apoptosis were assessed by Western blot. Mitochondrial membrane potential was monitored by flow cytometry. Cytosolic Ca^sup 2+^ was imaged using fura-2, and genetically encoded fluorescence resonance energy transfer (FRET)-based probes were used to measure Ca^sup 2+^ in ER and mitochondria.
RESULTS-Neither RyR nor IP^sub 3^R inhibition, alone or in combination, caused robust death within 24 h. In contrast, blocking sarco/endoplasmic reticulum ATPase (SERCA) pumps depleted ER Ca^sup 2+^ and induced marked phosphorylation of PKR-like ER kinase (PERK) and eukaryotic initiation factor-2α (eIF2α), C/EBP homologous protein (CHOP)-associated ER stress, caspase-3 activation, and death. Notably, ER stress following SERCA inhibition was attenuated by blocking IP^sub 3^Rs and RyRs. Conversely, stimulation of ER Ca^sup 2+^ release channels accelerated thapsigargin-induced ER depletion and apoptosis. SERCA block also activated caspase-9 and induced perturbations of the mitochondrial membrane potential, resulting eventually in the loss of mitochondrial polarization.
CONCLUSIONS-This study demonstrates that the activity of ER Ca^sup 2+^ channels regulates the susceptibility of β-cells to ER stress resulting from impaired SERCA function. Our results also suggest the involvement of mitochondria in β-cell apoptosis associated with dysfunctional β-cell ER Ca^sup 2+^ homeostasis and ER stress. Diabetes 58:422-432, 2009
Inappropriate activation of cell death pathways in the pancreatic β-cell is involved in the pathogenesis of type 1 diabetes, type 2 diabetes, and rare diabetic disorders such as maturity-onset diabetes of the young, Wolcott-Rallison syndrome, and Wolfram syndrome (1-5). β-Cell apoptosis also hampers clinical islet transplantation (6). The endoplasmic reticulum (ER) plays a key role in multiple programmed cell death pathways (7-9). Apoptosis caused by ER stress has been associated with diabetes (1,2,5,10) and can be induced by the accumulation of unfolded proteins resulting from disrupted Ca^sup 2+^-dependent chaperone function in the ER (1,11). Both thapsigargin, a potent and specific inhibitor...