Per-Arne Oldenborg
Studies on the effects of glucose and insulin on the function of neutrophil granulocytesABSTRACT
Per-Arne Oldenborg, Department of Histology and Cell Biology, Umeå University, S-901 87 Umeå, Sweden.Neutrophil granulocytes play an important role in the host defence against invading microorganisms and constitute the front line of defence in the cellular limb of the immune system. Circulating neutrophils are resting cells but when activated they should respond quickly, adhere to the vascular wall, leave the blood vessel and migrate to the infected area. Defective regulation of the recruitment of neutrophil granulocytes to the site of infection is coupled to decreased defence against infection, as seen in patients with diabetes mellitus. In neutrophils from diabetic patients, impairment of most cellular functions, including an increased resting activity of circulating neutrophils, have been described, although the underlying mechanisms are not fully understood. The aim of the present study was, therefore, to further investigate the effects of glucose and insulin on cellular mechanisms behind neutrophil locomotion and production of reactive oxygen metabolites.
D-glucose dose-dependently, but not dependent on insulin and not through an osmotic effect, inhibited the expression of a polarized locomotory morphology in response to the chemotactic peptide N-formyl-Methionyl-Leucyl-Phenylalanine (fMet-Leu-Phe) in non-adherent normal human neutrophils. This effect of glucose may impair neutrophil chemotaxis on a subtratum, as the chemotactic response is intimately coupled to the expression of this cell shape. D-glucose, up to 15 mM, was also found to stimulate random locomotion of human and mouse neutrophils, whereas the rate of locomotion was reduced by glucose above 15 mM. Neutrophils from Umeå ob/ob mice (chronically obese, hyperglycemic and hyperinsulinemic) showed an elevated locomotor activity at 4-21 mM glucose, as compared with lean control mice, although the resting production of reactive oxygen meatbolites was similar in the neutrophils from ob/ob or control mice at 0-15 mM D-glucose.
The chemokinesis of normal human neutrophils was dose-dependently stimulated by insulin (0-160 µU/ml) at 5 mM D-glucose, an effect found to be abolished by 15 mM D-glucose but also sensitive to inhibitors of tyrosine kinase (genistein) and phosphatidylinositol 3-kinase (wortmannin) but insensitive to protein kinase C (PKC) inhibition by bisindolylmaleimide (BIM). That specific inhibition of PKC by BIM restored the chemokinetic effect of insulin at 15 mM glucose, suggests that the inhibitory effect of 15 mM glucose on insulin-signaling is mediated by a PKC-dependent mechanism. fMet-Leu-Phe-stimulated luminol-enhanced chemiluminescence but not superoxide secretion from normal human neutrophils was inhibited by insulin at 80-160 µU/ml, an effect associated with a reduced myeloperoxidase (MPO) activity in the extracellular medium during fMet-Leu-Phe-stimulation. As the MPO activity was not changed by preincubation with insulin per se, the inhibitory effect of insulin may be on the fMet-Leu-Phe-stimulated release of MPO from the neutrophils, indicating an inhibitory effect of insulin at 80-160 µU/ml on the important MPO-H2O2-halide system.
Key words: neutrophils, locomotion, cell shape, glucose, insulin, respiratory burst, fMet-Leu-Phe, PMA, bisindolylmaleimide, myeloperoxidase This thesis is based on the following papers, which will be referred to in the text by using their Roman numerals:
I. Oldenborg, P.-A. and Sehlin, J. (1997) D-glucose but not insulin reduces N-formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe)-induced shape changes in suspended human neutrophils. Biosci. Rep. 17, 475-486.
II. Oldenborg, P.-A. and Sehlin, J. (1997) Effects of D-glucose on chemokinesis and resting production of reactive oxygen species in neutrophil granulocytes of lean or obese-hyperglycemic mouse. Biosci. Rep. 17, 487-498.
III. Oldenborg, P.-A. and Sehlin, J. (1998) Insulin-stimulated chemokinesis in normal human neutrophils is dependent on the glucose concentration and sensitive to inhibitors of tyrosine kinase and phosphatidylinositol 3-kinase.
J. Leukoc. Biol. 63, 203-208.
IV. Oldenborg, P.-A. and Sehlin, J. (1998) Hyperglycemia in vitro attenuates insulin-stimulated chemokinesis in normal human neutrophils. Role of protein kinase C activation. Manuscript V. Oldenborg, P.-A. (1998) Effects of insulin on N-formyl-methionyl-leucyl- phenylalanine (fMet-Leu-Phe)-stimulated production of reactive oxygen metabolites from normal human neutrophils. Submitted to Biochim. Biophys. Acta
