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

Inorganic phosphate (Pi) 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 beta-cells and rat pancreatic islets. Elevated extracellular P-i (1 similar to 5 mM) increased the mitochondrial membrane potential (Delta Psi(m)), superoxide generation, caspase activation, and cell death. Depolarization of the Delta Psi(m) abolished Pi-induced superoxide generation. Butylmalonate, a nonselective blocker of mitochondrial phosphate transporters, prevented Delta Psi(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.