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 neuronal cell death. In this work, we aimed to evaluate the contribution of autophagy and apoptosis to oxidative stress-induced death of rat cortical neurons in culture, as well as to explore the ability of insulin to prevent this death by inhibiting autophagy and apoptosis in neurons. To do this, we explored the effect of hydrogen peroxide and insulin on the levels of two main autophagy markers (LC3B-II and SQSTM1/p62) and apoptosis marker (cleaved сaspase-3, CC3). Neuronal viability was assessed via the MTT assay, marker protein levels were measured by Western blotting. It was found that oxidative stress causes the activation of autophagy and apoptosis in neurons, as manifested in a significant increase in the respective markers LC3B-II and CC3 and a decrease in the SQSTM1/p62 level. The content of SQSTM1/p62, which is involved in autophagosome formation, declined with autophagy activation because this protein degrades in lysosomes. Hydrogen peroxide induced autophagic and apoptotic death of neurons, as the inhibitors of autophagy (3-methyl adenine) and apoptosis (z-DEVD-FMK) increased neuronal viability under conditions of oxidative stress. Insulin, in its turn, inhibited autophagy, causing a decrease in the level of the lipidated LC3B-II form and an increase in SQSTM1/p62 level; insulin inhibited apoptosis as well, reducing CC3 level and thus preventing oxidative stress-induced neuronal death. The protective effect of insulin was mediated by the activation of specific signaling pathways associated with insulin receptors and IGF-1, as the inhibitor of these receptors BMS-754807 completely blocked the neuroprotective effect of insulin. Thus, a pronounced activation of autophagy under oxidative stress is one of the main causes of neuronal death, while insulin-mediated neuronal protection is due to suppression of not only apoptotic but also autophagic neuronal cell death. [ABSTRACT FROM AUTHOR]