Neuroprotective Effect of Insulin on Rat Cortical Neurons in Oxidative Stress Is Mediated by Autophagy and Apoptosis Inhibition in vitro

Although insulin is one of the most promising neuroprotectors,it is still unknown whether it is able to prevent autophagic neuronalcell death. In this work, we aimed to evaluate the contribution ofautophagy and apoptosis to oxidative stress-induced death of ratcortical neurons in culture, as well as to explore the ability ofinsulin to prevent this death by inhibiting autophagy and apoptosisin neurons. To do this, we explored the effect of hydrogen peroxideand insulin on the levels of two main autophagy markers (LC3B-IIand SQSTM1/p62) and apoptosis marker (cleaved caspase-3, CC3). Neuronalviability was assessed via the MTT assay, marker protein levelswere measured by Western blotting. It was found that oxidative stresscauses the activation of autophagy and apoptosis in neurons, asmanifested in a significant increase in the respective markers LC3B-IIand CC3 and a decrease in the SQSTM1/p62 level. The content of SQSTM1/p62,which is involved in autophagosome formation, declined with autophagyactivation because this protein degrades in lysosomes. Hydrogenperoxide induced autophagic and apoptotic death of neurons, as the inhibitorsof autophagy (3-methyl adenine) and apoptosis (z-DEVD-FMK) increasedneuronal viability under conditions of oxidative stress. Insulin,in its turn, inhibited autophagy, causing a decrease in the levelof the lipidated LC3B-II form and an increase in SQSTM1/p62 level;insulin inhibited apoptosis as well, reducing CC3 level and thuspreventing oxidative stress-induced neuronal death. The protectiveeffect of insulin was mediated by the activation of specific signaling pathwaysassociated with insulin receptors and IGF-1, as the inhibitor ofthese receptors BMS-754807 completely blocked the neuroprotectiveeffect of insulin. Thus, a pronounced activation of autophagy underoxidative stress is one of the main causes of neuronal death, while insulin-mediated neuronal protection is due to suppression of notonly apoptotic but also autophagic neuronal cell death.