Diabetes-Induced Oxidative Stress Is Mediated by Ca2+-Independent Phospholipase A2 in Neutrophils

Abstract

Neutrophils from people with poorly controlled diabetes present a primed phenotype and secrete excessive superoxide. Phospholipase A2 (PLA2)-derived arachidonic acid (AA) activates the assembly of NADPH oxidase to generate superoxide anion. There is a gap in the current literature regarding which PLA2 isoform regulates NADPH oxidase activation. The aim of this study was to identify the PLA2 isoform involved in the regulation of superoxide generation in neutrophils and investigate if PLA2 mediates priming in response to pathologic hyperglycemia. Neutrophils were isolated from people with diabetes mellitus and healthy controls, and HL60 neutrophil-like cells were grown in hyperglycemic conditions. Incubating neutrophils with the Ca2+-independent PLA2 (iPLA2) inhibitor bromoenol lactone (BEL) completely suppressed fMLP-induced generation of superoxide. The nonspecific actions of BEL on phosphatidic acid phosphohydrolase-1, p47phox phosphorylation, and apoptosis were ruled out by specific assays. Small interfering RNA knockdown of iPLA2 inhibited superoxide generation by neutrophils. Neutrophils from people with poorly controlled diabetes and in vitro incubation of neutrophils with high glucose and the receptor for advanced glycation end products ligand S100B greatly enhanced superoxide generation compared with controls, and this was significantly inhibited by BEL. A modified iPLA2 assay, Western blotting, and PCR confirmed that there was increased iPLA2 activity and expression in neutrophils from people with diabetes. AA (10 μM) partly rescued the inhibition of superoxide generation mediated by BEL, confirming that NADPH oxidase activity is, in part, regulated by AA. This study provides evidence for the role of iPLA2 in enhanced superoxide generation in neutrophils from people with diabetes mellitus and presents an alternate pathway independent of protein kinase C and phosphatidic acid phosphohydrolase-1 hydrolase signaling.

Polarization of Macrophages toward M2 Phenotype Is Favored by Reduction in iPLA2β (Group VIA Phospholipase A2)

“…Expression of iPLA 2 ␤ is elevated in pancreatic islets from diabetic subjects and rodent models of diabetes (28,29). Stresses (endoplasmic reticulum, proinflammatory cytokines, oxidative) associated with T1D development that cause ␤-cell death also increase iPLA 2 ␤ expression, and genetic or pharmacologic reduction of iPLA 2 ␤ activity ameliorates ␤-cell apoptosis (30 -35).…”

Insulin Treatment Directly Restores Neutrophil Phagocytosis and Bactericidal Activity in Diabetic Mice and Thereby Improves Surgical Site Staphylococcus aureus Infection

“…Therefore, neutrophil dysfunction may be involved in the high susceptibility of diabetic patients to staphylococcal infection. However, results remain conflicting and equivocal with regard to neutrophil function in diabetic hosts (3,15,40). Although analyses of neutrophil function among diabetic patients may provide information to enable diabetic patients to more effectively overcome infectious complications, it has yet to be fully elucidated how diabetic hyperglycemia affects the neutrophil-mediated host defense and also how insulin treatment affects diabetes-related infections.…”

Glucose initially inhibits and later stimulates blood ROS generation

“…There has already been evidence that glucose can alter blood ROS generation [12][13][14][15][16][17][18][19]: 11 mM glucose reduced the neutrophils' respiratory burst by 28% ± 5% and 56 mM glucose by 74% ± 7% [12]. 50% decreased ROS generation was detected at 25 mM glucose [13].…”