| Abstract: | In the pancreatic beta-cell, insulin secretion is stimulated by glucose metabolism resulting in membrane potential-dependent elevation of cytosolic Ca2+ (Ca2+]c). This cascade involves the mitochondrial membrane potential (delta psim]) hyperpolarization and elevation of mitochondrial Ca2+ (Ca2+]m) which activates the Ca(2+)-sensitive NADH-generating dehydrogenases. Metabolism-secretion coupling requires unidentified signals, other than Ca2+]c, possibly generated by the mitochondria through the rise in Ca2+]m. To test this paradigm, we have established an alpha-toxin permeabilized cell preparation permitting the simultaneous monitoring of Ca2+] with mitochondrially targeted aequorin and insulin secretion under conditions of saturating ATP] (10 mM) and of clamped Ca2+]c at substimulatory levels (500 nM). The tricarboxylic acid (TCA) cycle intermediate succinate hyperpolarized delta psi(m), raised Ca2+]m up to 1.5 microM and stimulated insulin secretion 20-fold, without changing Ca2+]c. Blockade of the uniporter-mediated Ca2+ influx into the mitochondria abolished the secretory response. Moreover, glycerophosphate, which raises Ca2+]m by hyperpolarizing delta psi(m) without supplying carbons to the TCA cycle, failed to stimulate exocytosis. Activation of the TCA cycle with citrate evoked secretion only when combined with glycerophosphate. Thus, mitochondrially driven insulin secretion at permissive Ca2+]c requires both a substrate for the TCA cycle and a rise in Ca2+]m. Therefore, mitochondrial metabolism generates factors distinct from Ca2+ and ATP capable of inducing insulin exocytosis. |
