Luminal H2 O2 promotes ER Ca2+ dysregulation and toxicity of palmitate in insulin-secreting INS-1E cells

Abstract

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 H₂ O₂ 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 H₂ O₂ 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 H₂ O₂ 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.

Keywords: beta cell death; calcium homeostasis; glutathione peroxidase; lipotoxicity; mitochondrial dysfunction; type 2 diabetes.