EFFECT OF INSULIN ON HYDROGEN-PEROXIDE PRODUCTION BY HUMAN POLYMORPHONUCLEAR LEUKOCYTES - STUDIES WITH MONOCLONAL ANTIINSULIN 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 pathway s involved in this process, especially the involvement of protein kina se C (PKC). Isolated human PMNLs from healthy volunteers were incubate d with different concentrations of insulin (10(-10)-10(-7) mol/l) and for different durations of incubation (5-90 min). The intracellular pr oduction of hydrogen peroxide (H2O2) was detected employing a previous ly validated flow cytometric assay using 2',7'-dichlorofluorescein-dia cetate (DCFH-DA) as a marker for H2O2 production. Specificity of insul in action was verified using an insulin antagonist (the monoclonal ant ibody MA-10). To identify the signalling pathway involved, we used: (a ) monoclonal antibody MA-5, directed against the alpha-subunit of the insulin receptor, that partially mimics insulin without activating tyr osine kinase; (b) H7, an inhibitor of PKC involved in O-2- production in PMNLs, and (c) phorbol myristate acetate (PMA) that binds and stimu lates PKC. Insulin caused a dose- and time-dependent stimulation of H2 O2 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 man ner, the respiratory burst of human PMNLs. PKC activation can only par tially account for the intracellular mechanisms involved in this proce ss.