Changed regulation of granulocyte NADPH oxidase activity in the mouse model of obesity-induced type 2 diabetes mellitus.

NADPH oxidase is a target of hyperglycemia in type 2 diabetes mellitus (T2DM), which causes dysregulation of enzyme. Alterations in regulation of NADPH oxidase activity mediated receptor and non-receptor signaling in bone marrow granulocytes of mice with obesity-induced T2DM were studied. The animals fed high fat diet (516 kcal/100 g) for 16 weeks. NADPH oxidase-related generation of reactive species (RS) at normo- and hyperthermia was estimated using chemiluminescent analysis. The redox status of the cells was assessed by Redox Sensor Red CC-1. Baseline biochemical indicators in blood (glucose, cholesterol, HDL and LDL levels) were significant higher in T2DM mice versus controls. Using specific inhibitors, signaling mediated by formyl peptide receptors (FPRs) to NADPH oxidase was shown to involve PLC, PKC, cytochrome p450 in both control and T2DM groups and PLA2 in controls. In T2DM regulation of NADPH oxidase activity via mFpr1, a high-affinity receptors, occurred with a significant increase of the role of PKC isoforms and suppression of PLA2 participation. Significant differences between this regulation via mFpr2, low-affinity receptors, were not found. Non-receptor activation of NADPH oxidase with ionomycin (Ca2+ ionophore) or phorbol ester (direct activator of PKC isoforms) did not revealed differences in the kinetic parameters between groups at 37 °C and 40 °C. When these agents were used together (synergistic effect), lower sensitivity of cells to ionophore was observed in T2DM at both temperatures. Redox status in responses to opsonized zymosan was higher in T2DM mice at 37 °C and similar to control levels at 40 °C. ROC-analysis identified Tmax, RS production and effect of opsonized zymosan as the most significant predictors for discriminating between groups. It was concluded that Ca2+-dependent/PKC-mediated regulation of NADPH oxidase activity was altered in BM granulocytes from diabetic mice.

The effect of high temperature on kinetics of reactive species generation in patients with type 2 diabetes.

The excessive amount of reactive species under chronic inflammation, which are accompanied by an increase body temperature, lead to diabetic complications. Phagocyte NADPH oxidase is the key enzyme in these processes. The role of high temperature in its regulation in diabetes is not clear. The aim was to investigate the effect of high temperature on NADPH-oxidase-dependent generation of reactive species in diabetic patients. Chemiluminescent method was applied to assess respiratory burst kinetics initiated by opsonized zymosan in blood or phorbol ester in isolated granulocytes. Analyzing ROC curves, the main predictors and changes in stages of activation of NADPH oxidase were determined. Phosphoisoforms of p47phox and p67phox were quantified by immunoblotting. Response to opsonized zymosan was lower in all subjects at 40 °C vs 37 °C, its kinetic parameters (except Tmax) were higher in blood of patients vs controls. Response rate was the main significant predictor to distinguish groups of subjects at 40 °C indicating NADPH oxidase upregulation in diabetes. Ca2+-dependent generation of reactive species by cells increased in both groups at 40 °C vs 37 °C, kinetic parameters were higher in patients. Initial phospho-p47phox level was higher in patient cells vs ones in controls. It was increased by ionomycin, phorbol ester, or 40 °C in control cells and unchanged in patient ones. Phospho-p67phox level was unchangeable in intact cells of healthy donors and patients at both temperatures. Excessive amounts of reactive species in patient cells were the consequence of granulocyte priming due to p47phox phosphorylation. Thus, high temperature decreased phagocytosis- and enhanced Ca2+-dependent generation of reactive species making the differences between controls and patients less pronounced. The effect of temperature on the generation of reactive species in blood granulocytes is associated with activity of NADPH oxidase that can be a prospective therapeutic target for pathologies accompanied by inflammation including type 2 diabetes.

Modified kinetics of generation of reactive species in peripheral blood of patients with type 2 diabetes.

Level of reactive species in blood is an important pathogenic factor in diabetes mellitus leading to dysfunctions of vascular endothelial and smooth muscle cells and coagulation system abnormality. A massive release of reactive species (respiratory burst), catalyzed by NADPH oxidase in blood phagocytes, is not well understood in diabetes. The work aimed to study kinetics of response to microbial particles in blood to specify changes in regulatory mechanisms of generation of reactive species in patients with type 2 diabetes. Production of reactive species in blood and isolated granulocytes was measured by luminol-dependent chemiluminescence. Respiratory burst was initiated by serum opsonized zymosan in blood samples and phorbol ester in cell samples. Kinetic parameters were calculated from experimental kinetic curves of chemiluminescence intensity. ROC curve analysis and mathematical modeling were used to reveal the most significant predictors and clarify specific mechanisms of NADPH oxidase activation. It was shown that kinetic parameters of response to opsonized zymosan (lag-time, response rate, amplitude, production of reactive species) were higher in blood of patients than controls. Amplitude and response rate were the most statistically significant predictors for distinguishing patients and controls at high glucose. It indicated NADPH oxidase activation was the target of hyperglycemia. Mathematical modeling showed hyperglycemia increased stability of NADPH oxidase complex, decreased synchronization of its assembling and elevated neutrophil capacity to phagocytosis in patients. Weak or no dependence of response kinetics on ionomycin concentration was shown in patients indicating changed Ca2+-dependent mechanism of NADPH oxidase activation. Hyperglycemia in type 2 diabetes causes disturbances in mechanisms of NADPH oxidase activation associated with both phagocytosis and the state of intracellular signaling systems, including Ca2+-dependent. We suggest that NADPH oxidase in blood granulocytes can be a promising target for clinical intervention improving management of diabetic complications associated with inflammation.