Kinetics of respiratory burst and its regulation in bone marrow granulocytes of diabetic db/db mice

Diabetic injury and complications with NADPH oxidase involvement are being studied using the homozygous db/db mouse model associated with obesity. Altered regulation of phagocyte NADPH oxidase (NOX2) and other isoforms (NOX1/4/) is partially known in db/db mice. In the current work the kinetics of NADPH oxidase-related reactive species (RS) generation in response to microbial agents in peripheral blood and bone marrow granulocytes (BMGs) of db/db mice were studied. The kinetics were recorded with chemiluminescent probe and analyzed by statistical methods. The expression of p-p67phox was assessed by Western blot. Kinetic parameters (response rate, amplitude, RS production) were higher while Tmax was lower in response to opsonized zymosan in blood samples and BMGs from db/db mice than in controls. In blood, RS production was the most significant predictor to discriminate the groups. Spearman correlations between baseline and other parameters were absent in db/db mice, and correlations between RS production and amplitude or response rate were very high in this group and low or moderate in controls, suggesting different mechanisms involved in phagocytosis-related regulation of NADPH oxidase. The IC50 for tyrphostin 51, tyrosine kinase inhibitor, was lower in db/db mice, and the IC50 for wortmannin, PI3K inhibitor, was not different between groups. Despite the overall insulin resistance of the organism, as determined by a high triglycerides/glucose index value, the cells responded to insulin, which altered significantly the kinetics of response to fMLF and increased p-p67phox level. Thus, a more reactive phagocyte phenotype and altered NADPH oxidase regulation were revealed in db/db mice.