Abstract

Objective: Endoplasmic reticulum (ER)-stress has been implicated in the pathogenesis of diabetes, but the roles of specific ER Ca²⁺ 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 (IP₃R) and the ryanodine receptors (RyR) 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 Ca²⁺ was imaged using Fura-2 and genetically encoded FRET-based probes were used to measure Ca²⁺ in ER and mitochondria.

Results: Neither RyR nor IP₃R inhibition, alone or in combination, caused robust death within 24 hours. In contrast, blocking SERCA pumps depleted ER Ca²⁺, induced marked phosphorylation of Perk and eIF2α, Chop-associated ER stress, Caspase-3 activation and death. Notably, ER stress following SERCA inhibition was attenuated by blocking IP₃Rs and RyRs. Conversely, stimulation of ER Ca²⁺ 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 Ca²⁺ channels regulates the susceptibility of β-cells to ER stress resulting from impaired SERCA function. Our results also suggests the involvement of mitochondria in β-cell apoptosis associated with dysfunctional β-cell ER Ca²⁺ homeostasis and ER stress.