Elevated Mitochondrial Respiration and Superoxide Production in Diabetic PMN

Oxidative stress is a major pathogenic mechanism leading to the complications of diabetes. Zymosan A-elicited PMN isolated from diabetic Akita mice release more superoxide compared to wild type (WT) PMN. Objectives: As mitochondrial superoxide production is increased in endothelial cells in diabetes, we hypothesized that mitochondria contribute to the elevated free radical production in PMN as well. Methods: Oxygen consumption by zymosan A-elicited murine peritoneal PMN was measured using a biological oxygen monitor model 5300 equipped with a 5331 standard oxygen probe (YSI). Mitochondria were labeled with Rhodamine 123 and mitochondrial superoxide production was determined with MitoSox Red staining followed by flow cytometry (FACScan). Results: Oxygen consumption is increased in Akita PMN (0.23 ± 0.16 nmol O₂/min*10⁶ cells) compared to WT (0.11 ± 0.09 nmol O₂/min*10⁶ cells, P < 0.05, n = 7). The number of mitochondria per PMN as determined with Rhodamine 123 staining and flow cytometry is similar in Akita and WT PMN (WT: 27.46 ± 6 Akita: 23.07 ± 3.85 arbitrary fluorescence units, P = 0.96, n = 6). Mitochondrial superoxide production is elevated in Akita PMN by 23%, approaching statistical significance as determined with MitoSox Red staining followed by flow cytometry (WT: 39.28 ± 11.58, Akita 48.44 ± 10.98 arbitrary fluorescence units, n = 6, P = 0.055). Conclusion: Mitochondrial respiration is accelerated in diabetic Akita PMN. The relative number of mitochondria in PMN is not altered by chronic hyperglycemia. However, the accelerated respiratory rate results in increased superoxide production. Thus, leukocytes may contribute to micro vascular free radical damage not only with superoxide from NADPH oxidase, but also with superoxide of mitochondrial origin. Supported by NIH NIDCR R01 DE016933 and DE15566.