Effect of insulin on hydrogen peroxide production by human polymorphonuclear leukocytes: Studies with monoclonal anti-insulin receptor antibodies, and an agonist and an inhibitor of protein kinase C

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.