No abstract

Antioxidant enzyme expression was determined in rat pancreatic islets and RINm5F insulin-producing cells on the level of mRNA, protein, and enzyme activity in comparison with 11 other rat tissues. Although superoxide dismutase expression was in the range of 30% of the liver values, the expression of the hydrogen peroxide-inactivating enzymes catalase and glutathione peroxidase was extremely low, in the range of 5% of the liver. Pancreatic islets but not RINm5F cells expressed an additional phospholipid hydroperoxide glutathione peroxidase that exerted protective effects against lipid peroxidation of the plasma membrane. Regression analysis for mRNA and protein expression and enzyme activities from 12 rat tissues revealed that the mRNA levels determine the enzyme activities of the tissues. The induction of cellular stress by high glucose, high oxygen, and heat shock treatment did not affect antioxidant enzyme expression in rat pancreatic islets or in RINm5F cells. Thus insulin-producing cells cannot adapt the low antioxidant enzyme activity levels to typical situations of cellular stress by an upregulation of gene expression. Through stable transfection, however, we were able to increase catalase and glutathione peroxidase gene expression in RINm5F cells, resulting in enzyme activities more than 100-fold higher than in nontransfected controls. Catalase-transfected RINm5F cells showed a 10-fold greater resistance toward hydrogen peroxide toxicity, whereas glutathione peroxidase overexpression was much less effective. Thus inactivation of hydrogen peroxide through catalase seems to be a step of critical importance for the removal of reactive oxygen species in insulin-producing cells. Overexpression of catalase may therefore be an effective means of preventing the toxic action of reactive oxygen species.

To determine the importance of different antioxidative enzymes for the defense status of insulin-producing cells, the effects of stable overexpression of glutathione peroxidase (Gpx), catalase (Cat), or Cu/Zn superoxide dismutase (SOD) in insulin-producing RINm5F cells on the cytotoxicity of hydrogen peroxide (H2O2), hypoxanthine/xanthine oxidase (H/XO), and menadione have been investigated. Single overexpression of Cat or Gpx provided less protection than the combined expression of Cat plus SOD or Cat plus Gpx, while single overexpression of SOD either had no effect on the toxicity of the test compounds or increased it. RINm5F cells were also susceptible to butylalloxan, a lipophilic alloxan derivative that is selectively toxic to pancreatic beta-cells. Overexpression of enzymes, both alone and in combination, did not protect against butylalloxan-induced toxicity while SOD overexpression increased it, as evident from a half maximally effective concentration (EC50) value. The addition of Cat to the culture medium completely prevented the toxic effects of H2O2 and H/XO but had no significant effect on the toxicity of menadione or butylalloxan. Extracellular SOD had no effect on the toxicity of any of the test compounds. The results of this study show the importance of a combination of antioxidant enzymes in protecting against the toxicity of reactive oxygen species. Thus, overexpression of Cat and Gpx, alone or in combination with SOD, by use of molecular biology techniques can protect insulin-producing cells against oxidative damage. This may represent a strategy to protect pancreatic beta-cells against destruction during the development of autoimmune diabetes and emphasizes the importance of optimal antioxidative enzyme equipment for protection against free radical-mediated diseases.

Nitric oxide (NO) and reactive oxygen species (ROS) are crucial elements in cytokine-mediated ␤-cell destruction. In insulin-producing RINm5F cells, overexpression of cytoprotective enzymes provides significant protection against the synergistic toxicity of NO and ROS. We therefore examined whether overexpression of catalase (Cat), glutathione peroxidase (Gpx), and Cu/Zn superoxide dismutase (SOD) can provide protection for bioengineered RINm5F cells against cytokine-mediated toxicity. A 72-h exposure of RINm5F control cells to interleukin-1␤ (IL-1␤) alone or a combination of IL-1␤, tumor necrosis factor-␣, and ␥-interferon resulted in a time-and concentration-dependent decrease of cell viability in the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) cytotoxicity assay. Although IL-1␤ alone caused only a moderate reduction of viability in the range of 25%, the cytokine mixture induced a significant loss of viability of >75%. This increased toxicity of the cytokine mixture compared with that of IL-1␤ alone could be explained by a higher rate of NO generation within the early 24-48 h incubation period that would favor the toxic synergism of NO and oxygen free radicals. Overexpression of Cat, Gpx, and Cu/Zn SOD protected against toxicity of the cytokine mixture but not against that of IL-1␤ alone. The reduction of cytokine-mediated toxicity was evident also because of an increased proliferation rate and a drastic decrease in the cell death rate. The improved antioxidant defense status did not prevent the activation of iNOS after cytokine exposure. However, RINm5F cells overexpressing cytoprotective enzymes showed a significantly lower level of ROS-damaged protein residues. Thus, protection through Cat, Gpx, and Cu/Zn SOD overexpression was apparently because of an inactivation of ROS generated in the signal cascades of the cytokines. Overexpression of cytoprotective enzymes thus represents a feasible strategy to protect insulin-producing cells against cytokine-mediated cytotoxicity.

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.