title: 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.
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 H202 production. 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 alpha-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 H202 release by human PMNLs. The effect of insulin was blocked 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.
authors.affiliation: Department of Pediatrics Tor Vergata University, Rome, Italy.
authors.forename: A
authors.initials: A
authors.lastname: Spagnoli
authors.forename: G L
authors.initials: GL
authors.lastname: Spadoni
authors.forename: G
authors.initials: G
authors.lastname: Sesti
authors.forename: D
authors.initials: D
authors.lastname: Del Principe
authors.forename: D
authors.initials: D
authors.lastname: Germani
authors.forename: B
authors.initials: B
authors.lastname: Boscherini
mesh_terms: D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D000328:Adult; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D005260:Female; D005434:Flow Cytometry; D005452:Fluoresceins; D006801:Humans; D006861:Hydrogen Peroxide / Q000097:blood*; D007328:Insulin / Q000494:pharmacology*; D007546:Isoquinolines / Q000494:pharmacology; D008297:Male; D009504:Neutrophils / Q000187:drug effects* / Q000378:metabolism*; D010879:Piperazines / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000097:blood; D011972:Receptor, Insulin / Q000276:immunology / Q000502:physiology; D016897:Respiratory Burst / Q000187:drug effects*; D015398:Signal Transduction; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
chemical_list: D000911:Antibodies, Monoclonal; D005452:Fluoresceins; D007328:Insulin; D007546:Isoquinolines; D010879:Piperazines; C029569:diacetyldichlorofluorescein; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D006861:Hydrogen Peroxide; D011972:Receptor, Insulin; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate