The Protective Effect of Insulin on Rat Cortical Neurons in Oxidative Stress and Its Dependence on the Modulation of Akt, GSK-3beta, ERK1/2, and AMPK Activities.

ABSTRACT

Insulin is a promising drug for the treatment of diseases associated with brain damage. However, the mechanism of its neuroprotective action is far from being understood. Our aim was to study the insulin-induced protection of cortical neurons in oxidative stress and its mechanism. Immunoblotting, flow cytometry, colorimetric, and fluorometric techniques were used. The insulin neuroprotection was shown to depend on insulin concentration in the nanomolar range. Insulin decreased the reactive oxygen species formation in neurons. The insulin-induced modulation of various protein kinase activities was studied at eight time-points after neuronal exposure to prooxidant (hydrogen peroxide). In prooxidant-exposed neurons, insulin increased the phosphorylation of GSK-3beta at Ser9 (thus inactivating it), which resulted from Akt activation. Insulin activated ERK1/2 in neurons 5-30 min after cell exposure to prooxidant. Hydrogen peroxide markedly activated AMPK, while it was for the first time shown that insulin inhibited it in neurons at periods of the most pronounced activation by prooxidant. Insulin normalized Bax/Bcl-2 ratio and mitochondrial membrane potential in neurons in oxidative stress. The inhibitors of the PI3K/Akt and MEK1/2/ERK1/2 signaling pathways and the AMPK activator reduced the neuroprotective effect of insulin. Thus, the protective action of insulin on cortical neurons in oxidative stress appear to be realized to a large extent through activation of Akt and ERK1/2, GSK-3beta inactivation, and inhibition of AMPK activity increased by neuronal exposure to prooxidant.