Mitochondrial superoxide production in neutrophils of diabetic Akita mice
Kim, Seok Yong
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Diabetes Mellitus, characterized by chronic hyperglycemia, is a widespread chronic disease. Hyperglycemia induces microvascular and macrovascular complications in diabetics through different pathways, including free radical damage. In neutrophils, NADPH oxidase is a main source of superoxide production.In the diabetic endothelium, studies have focused on mitochondrial superoxide release. This study is to identify the source of superoxide release in neutrophils of wild type and diabetic Akita mice. METHODS: Neutrophils were isolated from bone marrow from Akita and age-matched wild type mice with discontinuous Histopaque separation followed by stimulation with phorbol 12-myristate 13-acetate (PMA). Cells were fractionated into cytoplasmic and membrane fractions, electrophoresed onto gel, and transferred to PVDF membrane. Western blotting was performed using p47 subscript phox antibody and visualized by chemiluminiscence reagents. For superoxide experiments, neutrophils were isolated from intraperitoneal cavity from Akita and wild type mice 2 hr after Zymosan A injection in vivo followed by centrifugation. Superoxide was measured with cytochrome c reduction assay in the presence or absence of CCCP and PMA. RESULTS: The expression of p47 superscript phox in Akita neutrophils is higher than wild type neutrophils at baseline (wild type: 0.13[plus or minus]0.03 arbitrary units, AU Akita mice: 0.63[plus or minus]0.34 AU). However, there is no difference in superoxide production in neutrophils from bone marrow in the baseline between wild type and Akita mice. In vivo Zymosan A stimulation promotes significantly more superoxide production in neutrophils from Akita mice than from wild type (wild type: 0.337[plus or minus]0.119 [mega]mole/10 superscipt 6 cells, Akita mice: 0.410[plus or minus]0.129 [mega]mole/10 superscipt 6 cells at 15 min). Excess superoxide production in Akita neutrophils can be inhibited by CCCP, an uncoupler of mitochondrial respiration. CONCLUSION: In addition to the NADPH oxidase, there are other sources of superoxide production in neutrophils. Our data that excess superoxide production in neutrophils from Akita mice is inhibited by CCCP indicate that mitochondria are a significant source of superoxide in diabetes.
PLEASE NOTE: This work is protected by copyright. Downloading is restricted to the BU community: please click Download and log in with a valid BU account to access. If you are the author of this work and would like to make it publicly available, please contact firstname.lastname@example.org.Thesis (MSD) --Boston University, Henry M. Goldman School of Dental Medicine, 2009 (Department of Oral Biology and Periodontology).Includes bibliographic references: leaves 47-58.
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