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.
Highlights
The excessive activation of free radical reactions in brain nerve cells is one of the main causes of their injury and death in various diseases concerned with brain damage [1,2,3]
The aim of the present work is to study the protective and antioxidant effects of insulin, to find out how the protective effect of insulin depends on its concentration, and to study the modulatory effect of insulin on the activity of protein kinase B (Akt), ERK1/2, GSK-3beta, and AMPK, on the ratio of proapoptotic to antiapoptotic proteins (Bax/Bcl-2) in the course of oxidative stress development in brain cortical neurons exposed to hydrogen peroxide
Insulin was shown to increase the viability of cultured rat brain cortical neurons exposed to hydrogen peroxide in a large range of concentrations, We used 100 μM hydrogen peroxide as a prooxidant because it is a natural prooxidant; its content in the brain may be increased in ischemic or other unfavorable conditions
Summary
The excessive activation of free radical reactions in brain nerve cells is one of the main causes of their injury and death in various diseases concerned with brain damage [1,2,3] Such natural antioxidants as carnosine, flavonoids, components of vitamin E, gangliosides, insulin, and others exert their protective and antioxidant action on neurons and other cells mainly by modulating the activity of signaling pathways. Effective use in clinical practice of insulin and other natural bioactive substances with antioxidant activity still requires a more in-depth understanding of the way they increase the viability of neurons and other cells modulating the activity of various signaling pathways. Such studies may identify the compounds which markedly enhance the protective effect of each other
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