Importance of mitochondrial superoxide dismutase expression in insulin-producing cells for the toxicity of reactive oxygen species and proinflammatory cytokines.

ABSTRACT

AIMS/HYPOTHESIS: Free radicals generated in mitochondria play a crucial role in the toxic effects of cytokines upon insulin-producing cells. This study therefore investigated the role of manganese superoxide dismutase (MnSOD) in cytokine-mediated toxicity in insulin-producing cells. METHODS: MnSOD was either stably overexpressed (MnSODsense) or stably suppressed (MnSODantisense) in insulin-producing RINm5F cells. Cell viability was quantified after incubation with different chemical reactive oxygen species (ROS) generators and with cytokines (IL-1beta alone or a mixture of IL-1beta, TNF-alpha and IFN-gamma). Additionally, cell proliferation and endogenous MnSOD protein expression were determined after exposure to cytokines. RESULTS: After incubation with hydrogen peroxide (H(2)O(2)) or hypoxanthine/xanthine oxidase no significant differences were observed in viability between control and MnSODsense or MnSODantisense clones. MnSOD overexpression reduced the viability of MnSODsense cells after exposure to the intracellular ROS generator menadione compared with control and MnSODantisense cells. MnSODsense cells also showed the highest susceptibility to cytokine toxicity with more than 75% loss of viability and a significant reduction of the proliferation rate after 72 h of incubation with a cytokine mixture. In comparison with control cells (67% viability loss), the reduction of viability in MnSODantisense cells was lower (50%), indicating a sensitising role of MnSOD in the progression of cytokine toxicity. The cell proliferation rate decreased in parallel to the reduction of cell viability. The MnSOD expression level after exposure to cytokines was also significantly lower in MnSODantisense cells than in control or MnSODsense cells. CONCLUSIONS/INTERPRETATION: The increase of the mitochondrial imbalance between the superoxide- and the H(2)O(2)-inactivating enzyme activities corresponds with a greater susceptibility to cytokines. Thus optimal antioxidative strategies to protect insulin-producing cells against cytokine toxicity may comprise a combined overexpression of H(2)O(2)-inactivating enzymes or suppression of MnSOD activity.