Mitochondrial oxidative stress mediates high-phosphate-induced secretory defects and apoptosis in insulin-secreting cells

Inorganic phosphate (P_i) plays an important role in cell signaling and energy metabolism. In insulin-releasing cells, P_i transport into mitochondria is essential for the generation of ATP, a signaling factor in metabolism-secretion coupling. Elevated P_i concentrations, however, can have toxic effects in various cell types. The underlying molecular mechanisms are poorly understood. Here, we have investigated the effect of P_i on secretory function and apoptosis in INS-1E clonal β-cells and rat pancreatic islets. Elevated extracellular P_i (1~5 mM) increased the mitochondrial membrane potential (Δ ψ_m), superoxide generation, caspase activation, and cell death. Depolarization of the Δ ψ_m abolished P_i-induced superoxide generation. Butylmalonate, a nonselective blocker of mitochondrial phosphate transporters, prevented Δ ψ_m hyperpolarization, superoxide generation, and cytotoxicity caused by P_i. High P_i also promoted the opening of the mitochondrial permeability transition (PT) pore, leading to apoptosis, which was also prevented by butylmalonate. The mitochondrial antioxidants mitoTEMPO or MnTBAP prevented P_i-triggered PT pore opening and cytotoxicity. Elevated extracellular P_i diminished ATP synthesis, cytosolic Ca²⁺ oscillations, and insulin content and secretion in INS-1E cells as well as in dispersed islet cells. These parameters were restored following preincubation with mitochondrial antioxidants. This treatment also prevented high- P_i-induced phosphorylation of ER stress proteins. We propose that elevated extracellular P_i causes mitochondrial oxidative stress linked to mitochondrial hyperpolarization. Such stress results in reduced insulin content and defective insulin secretion and cytotoxicity. Our data explain the decreased insulin content and secretion observed under hyperphosphatemic states.