Translocation of p47phox and Activation of NADPH Oxidase in Mononuclear Cells

Diabetes mellitus is associated with increased reactive oxygen species (ROS) generation by mononuclear cells (MNC) and an increased oxidative load, which causes oxidative damage to proteins, lipids and DNA (1 –3 ). Recently, we have demonstrated that glucose challenge causes an increase in ROS generation by MNC and polymorphonuclear cells (PMNL). We have also demonstrated that the suppression of ROS generation by MNC by diphenylene iodonium (DPI), a specific inhibitor of NADPH oxidase and the membrane enzyme that generates the superoxide (undefined₂ ^- ) radical, is significantly diminished in the obese and diabetics when compared with normal subjects (4 ). The assay system used in our ROS generation studies largely detects the superoxide radical, which is produced by the conversion of molecular O₂ by NADPH oxidase (5 –6 ). Thus, it would appear that NADPH oxidase activity increases following glucose challenge and that it is relatively non-suppresible in the obese. NADPH oxidase consists of multiple subunits. Equimolar amounts of p47^phox and p67^phox in the cytosol combine to a form a highly basic 250 kDa protein. During activation, this complex migrates from the cytosol to the membrane where this complex combines with cytochrome b₅₅₈ , which in turn, consists of gp91^phox and p22phox .Rac 2, th_e fifth component of the enzyme migrates to the membrane in association with p47^phox , which without it, is unable to reach the membrane (7 –10 ).