Figure 6.

Nampt alleviates oxidative stress in an NADK-dependent manner. (A) Nampt prevents H₂O₂-induced Prdx1 oxidation in an NADK-dependent manner. (B) Nampt prevents H₂O₂-induced intermolecular disulfide bond formation in AMPKα and mTOR in an NADK-dependent manner (Ox: oxidized, Red: reduced, LE: long exposure, SE: short exposure). (C) Nampt prevents H₂O₂-induced mTOR inhibition in an NADK-dependent manner. After 1 day of adenovirus vector (Nampt) transduction, shNADK was transfected into cardiomyocytes. After 1 or 2 days of transfection, cells were treated with 100 μM H₂O₂ for 30 minutes. Western blot analyses were performed following SDS-PAGE under reducing (A and C) and non-reducing (B) conditions using indicated antibodies. (D) Nampt prevents H₂O₂-induced myocyte cell death, an effect that is partly abolished by inhibition of NADK, GSH synthesis or Trx1. (Left) After adenovirus transduction and shRNA transfection as described above, cells were treated with 100 μmol/L H₂O₂ for 6 hours. Cell viability was assessed by trypan blue dye exclusion. (Middle) Cells were treated with buthionine sulfoximine (BSO, 100 μmol/L), a GSH synthesis inhibitor, for 16 hours prior to H₂O₂ treatment. (Right) Adenovirus vector harboring shTrx1 was transduced together with that for Nampt. (E) Sirt1, but not Sirt3, mediates the protective effects of Nampt against H₂O₂-induced myocyte cell death. After adenovirus transduction and shRNA transfection, cells were treated with 100 μmol/L H₂O₂ for 6 hours. Cell viability was assessed by trypan blue dye exclusion. (F) Western blot showing knockdown of Trx1, Sirt1 and Sirt3. Statistical significance was determined with ANOVA (A-E).