Improvement of the Mitochondrial Antioxidant Defense Status Prevents Cytokine-Induced Nuclear Factor-κB Activation in Insulin-Producing Cells

  1. Anna Karenina Azevedo-Martins1,
  2. Stephan Lortz1,
  3. Sigurd Lenzen1,
  4. Rui Curi2,
  5. Decio L. Eizirik3 and
  6. Markus Tiedge1
  1. 1Institute of Clinical Biochemistry, Hannover Medical School, Hannover, Germany
  2. 2Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil
  3. 3Laboratory of Experimental Medicine, Free University Brussels, Brussels, Belgium


    Proinflammatory cytokines (interleukin-1β [IL-1β], tumor necrosis factor-α [TNF-α], and γ-interferon [IFN-γ]) initiate a variety of signal cascades in pancreatic β-cells that affect the expression level of genes involved in both the destruction and the protection of the β-cell. The generation of nitric oxide (NO) via the inducible NO synthase (iNOS) and oxygen free radicals play a key role in cytokine-mediated β-cell destruction. Within these signal cascades, the activation of the transcription factor nuclear factor-κB (NF-κB) is crucial, and many cytokine-sensitive genes contain binding sites for this transcription factor in their promoter regions. The aim of this study was to characterize the cytokine-mediated activation of NF-κB and the subsequent expression of iNOS protein in insulin-producing RINm5F cells with an improved antioxidant defense status by overexpression of the cytoprotective enzymes catalase (Cat), glutathione peroxidase (Gpx), and the cytoplasmic Cu/Zn superoxide dismutase (Cu/ZnSOD). RINm5F cells with diverse mitochondrial antioxidative defense status were generated by stable overexpression of MnSOD constructs in sense (MnSOD sense) and antisense orientation (MnSOD antisense). Cytokine-induced (IL-1β or cytokine mix consisting of IL-1β + TNF-α + IFN-γ) activation of NF-κB in RINm5F cells was reduced by >80% through overexpression of MnSOD. The activity of the iNOS promoter remained at basal levels in cytokine-stimulated MnSOD sense cells. In contrast, the suppression of MnSOD gene expression in cytokine-stimulated MnSOD antisense cells resulted in a threefold higher activation of NF-κB and a twofold higher activation of the iNOS promoter as compared with control cells. The iNOS protein expression was significantly reduced after a 6- and 8-h cytokine incubation of MnSOD sense cells. The low activity level of MnSOD in RINm5F MnSOD antisense cells increased the iNOS protein expression in particular during the early phase of cytokine-mediated toxicity. Cat, Gpx, and the cytoplasmic Cu/ZnSOD did not affect the activation of NF-κB and the iNOS promoter. In conclusion, the overexpression of MnSOD, which inactivates specifically mitochondrially derived oxygen free radicals, significantly reduced the activation of NF-κB in insulin-producing cells. As a consequence of this protective effect in the early cytokine signaling pathways, the induction of iNOS, an important event in the β-cell destruction process, was also significantly reduced. The results provide evidence that mitochondrially derived reactive oxygen species (ROS) play a critical role in the activation of the cytokine-sensitive transcription factor NF-κB. Overexpression of MnSOD may thus be beneficial for β-cell survival through suppression of oxygen free radical formation, prevention of NF-κB activation, and iNOS expression.


    • Address correspondence and reprint requests to Dr. Markus Tiedge, Institute of Clinical Biochemistry, Hannover Medical School, D-30623 Hannover, Germany. E-mail: tiedge.markus{at}

      Received for publication 12 June 2002 and accepted in revised form 30 September 2002.

      Cat, catalase; EMSA, electrophoretic mobility shift assay; Gpx, glutathione peroxidase; IFN-γ, γ-interferon; IL-1β, interleukin-1β; iNOS, inducible nitric oxide synthase; NF-κB, nuclear factor-κB; ROS, reactive oxygen species; SEAP, secreted alkaline phosphatase; SOD, superoxide dismutase; TNF-α, tumor necrosis factor-α.

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