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

Anna Spagnoli, Gian Luigi Spadoni, Giorgio Sesti, Domenico Del Principe, Daniela Germani, Brunetto Boscherini

Research output: Contribution to journalArticle

29 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)286-293
Number of pages8
JournalHormone Research in Paediatrics
Volume43
Issue number6
DOIs
StatePublished - 1995

Keywords

  • Hydrogen peroxide
  • Insulin
  • Insulin receptor
  • Polymorphonuclear leukocytes
  • Protein kinase C

ASJC Scopus subject areas

  • Pediatrics, Perinatology, and Child Health
  • Endocrinology, Diabetes and Metabolism
  • Endocrinology

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