Mitochondrial Catalase Overexpression Protects Insulin-Producing Cells Against Toxicity of Reactive Oxygen Species and Proinflammatory Cytokines

Abstract

Insulin-producing cells are known for their extremely low antioxidant equipment with hydrogen peroxide (H 2 O 2 )-inactivating enzymes. Therefore, catalase was stably overexpressed in mitochondria and for comparison in the cytoplasmic compartment of insulin-producing RINm5F cells and analyzed for its protective effect against toxicity of reactive oxygen species (ROS) and proinflammatory cytokines. Only mitochondrial overexpression of catalase provided protection against menadione toxicity, a chemical agent that preferentially generates superoxide radicals intramitochondrially. On the other hand, the cytoplasmic catalase overexpression provided better protection against H 2 O 2 toxicity. Mitochondrial catalase overexpression also preferentially protected against the toxicity of interleukin-1␤ (IL-1␤) and a proinflammatory cytokine mixture (IL-1␤, tumor necrosis factor-␣ [TNF-␣], and ␥-interferon [IFN-␥])that is more toxic than IL-1␤ alone. Thus, it can be concluded that targeted overexpression of catalase in the mitochondria provides particularly effective protection against cell death in all situations in which ROS are generated intramitochondrially. The observed higher rate of cell death after exposure to a cytokine mixture in comparison with the weaker effect of IL-1␤ alone may be due to an additive toxicity of TNF-␣ through ROS formation in mitochondria. The results emphasize the central role of mitochondrially generated ROS in the cytokine-mediated cell destruction of insulin-producing cells.

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

“…These experiments allow the conclusion that an increased superoxide-radical-inactivation capacity in the MnSOD sense cells results in an enhanced H 2 O 2 production, the inactivation of which is limited, however, by the low H 2 O 2 detoxification capacity in insulin-producing cells [10,25]. On the other hand, mitochondrial superoxide radical dismutation is apparently not rate-limiting in insulin-producing cells, since the viability was not reduced through MnSOD suppression in the MnSODantisense cells.…”

“…Reduced MnSOD gene expression in MnSODantisense cells left the toxicity of IL-1β unaffected and in the case of the more toxic cytokine mixture it even provided protection. The greater toxicity of the cytokine mixture is apparently a result of TNF-α-induced ROS formation in the mitochondria [10,[26][27][28][29]. The observed protective effect of a reduced MnSOD gene expression was evident not only in the MTT viability assay but also in the measurements of cell proliferation.…”

“…The very low expression level of H 2 O 2 -inactivating enzymes in these cells [8,9] prevents a fast and efficient removal of toxic H 2 O 2 generated intramitochondrially through the action of MnSOD. Through overexpression of catalase within the mitochondrial compartment, we were able to protect insulin-producing cells against cytokine-induced toxicity and thus demonstrate the deleterious effects of mitochondrially accumulated H 2 O 2 [10]. Enhanced toxicity due to increased MnSOD protein expression was also observed in MnSOD-overexpressing fibroblasts [7,30,31], in skeletal muscle cells [32], in fibroblasts and in brain of patients suffering from neuronal ceroid lipofuscinosis type 6 [33], in the ovarian cancer cell line SK-OV-3 [34] and in transformed human bronchial epithelial cells [35].…”

“…This fact must also be kept in mind when devising therapeutic measures in order to strengthen resistance against the toxic actions of ROS and proinflammatory cytokines. A possible strategy for correction of this imbalance might be an increase of catalase activity in the mitochondrial compartment, thereby preventing the accumulation of H 2 O 2 by elimination of this bottle-neck in the ROSinactivation chain [10]. This strategy may be combined with the overexpression of MnSOD to ensure a balanced elimination of mitochondrially generated radicals.…”

“…In particular the mitochondrial targeting of catalase proved to be an efficient strategy to protect the cells against cytokine-mediated cell death, acting against the imbalance between superoxide dismutation and the inactivation of H 2 O 2 [10]. Also manganese SOD (MnSOD) overexpression proved to be an efficient principle to ameliorate the activation of nuclear factor kappa B (NF-κB) and inducible nitric oxide (NO) synthase after cytokine exposure in insulin-producing RINm5F cells in the early phase of cytokine signalling [11].…”