MITOCHONDRIAL ACTIVATION DIRECTLY TRIGGERS THE EXOCYTOSIS OF INSULIN IN PERMEABILIZED PANCREATIC BETA-CELLS

In the pancreatic beta-cell, insulin secretion is stimulated by glucos e metabolism resulting in membrane potential-dependent elevation of cy tosolic Ca2+ ([Ca2+](c)), This cascade involves the mitochondrial memb rane potential (Delta psi(m)) hyperpolarization and elevation of mitoc hondrial Ca2+ ([Ca2+](m)) which activates the Ca2+-sensitive NADH-gene rating dehydrogenases, Metabolism-secretion coupling requires unidenti fied signals, other than [Ca2+](c), possibly generated by the mitochon dria through the rise in [Ca2+](m). To test this paradigm, we have est ablished an alpha-toxin permeabilized cell preparation permitting the simultaneous monitoring of [Ca2+] with mitochondrially targeted aequor in and insulin secretion under conditions of saturating [ATP] (10 mM) and of clamped [Ca2+](c) at substimulatory levels (500 nM). The tricar boxylic acid (TCA) cycle intermediate succinate hyperpolarized Delta p si(m), raised [Ca2+](m) up to 1.5 mu M and stimulated insulin secretio n 20-fold, without changing [Ca2+](c). Blockade of the uniporter-media ted Ca2+ influx into the mitochondria abolished the secretory response , Moreover, glycerophosphate, which raises [Ca2+](m) by hyperpolarizin g Delta psi(m) without supplying carbons to the TCA cycle, failed to s timulate exocytosis. Activation of the TCA cycle with citrate evoked s ecretion only when combined with glycerophosphate, Thus, mitochondrial ly driven insulin secretion at permissive [Ca2+](c) requires both a su bstrate for the TCA cycle and a rise in [Ca2+](m), Therefore, mitochon drial metabolism generates factors distinct from Ca2+ and ATP capable of inducing insulin exocytosis.