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Objective: This study investigated the effects of insulin on the phagocytosis of C3bi - and IgG-opsonized yeast particles in normal human neutrophils., Methods: Neutrophils were incubated in different insulin concentrations for 30 minutes and stimulated by C3bi - or IgG-opsonized yeast particles. Phagocytosis was quantified by both light microscopy and FACscan flow cytometry. Laser confocal microscopy was used for quantification of F-actin levels., Results: Elevated insulin concentrations decreased neutrophil phagocytosis of both types of targets. This defect was shown to be in part due to a delayed phagocytosis in the presence of insulin. Following a 30 minute incubation, insulin was found to increase the accumulation of cortical F-actin, without affecting the total cellular F-actin content. The specific PKCalpha/beta inhibitor, Go6976, abolished the insulin-mediated increase in cortical F-actin content and both Go6976 and the PKCalpha/beta/delta/epsilon-specific inhibitor GF109203X reversed the inhibitory effects of insulin on phagocytosis., Conclusion: Hyperinsulinemia in vitro can inhibit phagocytosis of opsonized targets in normal human neutrophils. This effect of insulin is dependent on activation of PKCalpha and/or PKCbeta, and these insulin signals may interfere with the dynamic assembly/disassembly and/or distribution of F-actin, which is required for the phagocytosis process.

Objective and Design: To further understand the mechanisms behind defective neutrophil function in diabetes mellitus, the ability of insulin to affect the production and metabolism of reactive oxygen metabolites in normal human neutrophils was studied., Materials and Methods: Neutrophil granulocytes from healthy adults were studied for their N-formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe)-stimulated production of reactive oxygen metabolites and release of myeloperoxidase or elastase following treatment with insulin., Results: Preincubation of the neutrophils for 30 min with insulin (40-320 microU/ml), before activation with 1 microM fMet-Leu-Phe, revealed that the hormone at 80 and 160 microU/ml reduced the luminol-enhanced chemiluminescence without affecting the superoxide secretion. The insulin-induced reduction of the chemiluminescence response was reversed by the addition of exogenous peroxidase and was also paralleled by a reduced myeloperoxidase activity, with no effect on the elastase activity, in cell-free supernatants from fMet-Leu-Phe-stimulated neutrophils. Superoxide dismutase removed the inhibitory effect of insulin on myeloperoxidase activity., Conclusions: These results suggest that elevated levels of insulin do not affect the NADPH-oxidase activity but, together with superoxide anions, interfere with myeloperoxidase availability and a subsequent myeloperoxidase-dependent generation of reactive oxygen metabolites in fMet-Leu-Phe-stimulated normal human neutrophils.