Luminal H2O2 promotes ER Ca2+ dysregulation and toxicity of palmitate in insulin‐secreting INS‐1E cells

The endoplasmic reticulum (ER) lumen is not only the major site for the assembly and folding of newly synthesized proteins but also the main intracellular Ca2+ store. Ca2+ ions are involved in versatile biochemical processes, including posttranslational processing and folding of nascent proteins. Disruption of ER Ca2+ homeostasis is usually accompanied by an ER stress response that can ultimately lead to apoptosis if unresolved. Abnormal ER Ca2+ depletion has been linked to pancreatic β‐cell dysfunction and death under lipotoxic conditions. However, the underlying mechanisms how the β‐cell toxic saturated free fatty acid palmitate perturbs ER Ca2+ homeostasis and its interplay with other organelles are not fully understood. In the present study, we demonstrate that treatment of insulin‐secreting INS‐1E cells with palmitate diminished ER Ca2+ levels, elevated cytosolic/mitochondrial Ca2+ content, lowered the mitochondrial membrane potential, and ATP content. In addition, palmitate‐pretreated β‐cells contained significantly less luminal Ca2+, revealed a severely impaired ER Ca2+ reuptake rate, and substantially lower insulin content. Importantly, detoxification of luminal H2O2 by expression of the ER‐resident glutathione peroxidase 8 (GPx8) abrogated the lipotoxic effects of palmitate. Moreover, GPx8 supported oxidative protein folding and preserved insulin content under lipotoxic conditions. A direct involvement of luminal H2O2 in palmitate‐mediated ER Ca2+ depletion could be corroborated by the ectopic expression of an ER‐luminal active catalase. Our data point to the critical role of luminal H2O2 in palmitate‐mediated depletion of ER Ca2+ through redox‐dependent impairment of Ca2+ ATPase pump activity upstream of mitochondrial dysfunction in insulin‐secreting INS‐1E cells.