Mitochondrial phosphate transport during nutrient stimulation of INS-1E insulinoma cells

Here, we have investigated the role of inorganic phosphate (P_i) transport in mitochondria of rat clonal β-cells. In α-toxin-permeabilized INS-1E cells, succinate and glycerol-3-phosphate increased mitochondrial ATP release which depends on exogenous ADP and P_i. In the presence of substrates, addition of P_i caused mitochondrial matrix acidification and hyperpolarisation which promoted ATP export. Dissipation of the mitochondrial pH gradient or pharmacological inhibition of P_i transport blocked the effects of P_i on electrochemical gradient and ATP export. Knock-down of the phosphate transporter P_iC, however, neither prevented P_i-induced mitochondrial activation nor glucose-induced insulin secretion. Using ³¹P NMR we observed reduction of P_i pools during nutrient stimulation of INS-1E cells. Interestingly, P_i loss was less pronounced in mitochondria than in the cytosol. We conclude that matrix alkalinisation is necessary to maintain a mitochondrial P_i pool, at levels sufficient to stimulate energy metabolism in insulin-secreting cells beyond its role as a substrate for ATP synthesis.