Effect of Insulin on Hydrogen Peroxide Production by Human Polymorphonuclear Leukocytes

Abstract

This study evaluated the effect of insulin on the respiratory burst of human polymorphonuclear leukocytes (PMNLs) and the signalling pathways involved in this process, especially the involvement of protein kinase C (PKC). Isolated human PMNLs from healthy volunteers were incubated with different concentrations of insulin (10^-10-10^-7 mol/l) and for different durations of incubation (5-90 min). The intracellular production of hydrogen peroxide (H₂O₂) was detected employing a previously validated flow cytometric assay using 2’,7’-dichlorofluorescein-diacetate (DCFH-DA) as a marker for H₂O₂production. Specificity of insulin action was verified using an insulin antagonist (the monoclonal antibody MA-10). To identify the signalling pathway involved, we used: (a) monoclonal antibody MA-5, directed against the α-subunit of the insulin receptor, that partially mimics insulin without activating tyrosine kinase; (b) H7, an inhibitor of PKC involved in O₂^- production in PMNLs, and (c) phorbol myristate acetate (PMA) that binds and stimulates PKC. Insulin caused a dose- and time-dependent stimulation of H₂O₂ release by human PMNLs. The effect of insulin was blocked by MA-10. The actions of insulin and PMA on H₂O₂ release were additive, whereas the actions of MA-5 and PMA were not. H7 partially inhibited the H₂O₂ production stimulated by insulin and completely inhibited MA-5 action. We conclude that insulin stimulates, in a dose- and time-related manner, the respiratory burst of human PMNLs. PKC activation can only partially account for the intracellular mechanisms involved in this process.