Espinosa A, Garcia A, Härtel S, Hidalgo C & E Jaimovich.
JBC. 284, pp:2568-2575. [Epub 2008 Nov 20]
Skeletal muscle cells are a main physiological target of insulin. This hormone triggers production of reactive species of oxygen (ROS) in different cell types. ROS are considered a second messenger and can produce redox modifications in proteins such as ion channels inducing changes in their probability of opening. Insulin can produce intracellular calcium release from intracellular stores in myotubes. In this work we studied the relationshipt between ROS generation and calcium increase induced by insulin and the mechanism involved in both events. Using the probe 2′, 7′-dichlorofluorescein diacetate (DCFH-DA) we show ROS generation by insulin in myotubes wich could be abolished by the NADPH oxidase inhibitor apocynin and by siRNA against p47phox; a regulatory subunit of NADPH oxidase. We studied the translocation of p47phox from cytoplasm to plasma membrane as a marker of the activation of NADPH oxidase. In order to study the main pathway involved in the activation of this enzyme, we tested the effects of bisindolylmaleimide (BIM); a non specific inhibitor of PKC and Ly290042; an inhibitor of PI3K. Both drugs modified the generation of ROS by insulin. We measured intracellular calcium increase in the presence of BIM, LY290042, apocynin, siRNA against p47phox and two drugs that interfere in IP3 pathway; xestospongin B and U73122. Calcium signals were inhibited by all of these treatments. Results indicate that insulin induces ROS generation trough NADPH activation. ROS participate in calcium increase dependent on IP3 receptors. Both PI3K and PKC are also involved in ROS and calcium increases induced by insulin.