Mitochondrial H(2)O(2) as an enable signal for triggering autophosphorylation of insulin receptor in neurons

BACKGROUND: Insulin receptors are widely distributed in the brain, where they play roles in synaptic function, memory formation, and neuroprotection. Autophosphorylation of the receptor in response to insulin stimulation is a critical step in receptor activation. In neurons, insulin stimulation leads to a rise in mitochondrial H(2)O(2) production, which plays a role in receptor autophosphorylation. However, the kinetic characteristics of the H(2)O(2) signal and its functional relationships with the insulin receptor during the autophosphorylation process in neurons remain unexplored to date. RESULTS: Experiments were carried out in culture of rat cerebellar granule neurons. Kinetic study showed that the insulin-induced H(2)O(2) signal precedes receptor autophosphorylation and represents a single spike with a peak at 5–10 s and duration of less than 30 s. Mitochondrial complexes II and, to a lesser extent, I are involved in generation of the H(2)O(2) signal. The mechanism by which insulin triggers the H(2)O(2) signal involves modulation of succinate dehydrogenase activity. Insulin dose–response for receptor autophosphorylation is well described by hyperbolic function (Hill coefficient, n(H), of 1.1±0.1; R(2)=0.99). N-acetylcysteine (NAC), a scavenger of H(2)O(2), dose-dependently inhibited receptor autophosphorylation. The observed dose response is highly sigmoidal (Hill coefficient, n(H), of 8.0±2.3; R(2)=0.97), signifying that insulin receptor autophosphorylation is highly ultrasensitive to the H(2)O(2) signal. These results suggest that autophosphorylation occurred as a gradual response to increasing insulin concentrations, only if the H(2)O(2) signal exceeded a certain threshold. Both insulin-stimulated receptor autophosphorylation and H(2)O(2) generation were inhibited by pertussis toxin, suggesting that a pertussis toxin-sensitive G protein may link the insulin receptor to the H(2)O(2)-generating system in neurons during the autophosphorylation process. CONCLUSIONS: In this study, we demonstrated for the first time that the receptor autophosphorylation occurs only if mitochondrial H(2)O(2) signal exceeds a certain threshold. This finding provides novel insights into the mechanisms underlying neuronal response to insulin. The neuronal insulin receptor is activated if two conditions are met: 1) insulin binds to the receptor, and 2) the H(2)O(2) signal surpasses a certain threshold, thus, enabling receptor autophosphorylation in all-or-nothing manner. Although the physiological rationale for this control remains to be determined, we propose that malfunction of mitochondrial H(2)O(2) signaling may lead to the development of cerebral insulin resistance.