Complementary action of antioxidant enzymes in the protection of bioengineered insulin-producing RINm5F cells against the toxicity of reactive oxygen species.

Abstract

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.

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

“…Effects of cytokines on the proliferation rate of MnSOD sense and MnSODantisense insulin-producing RINm5F cells From previous studies it was evident that the cell growth rate of RINm5F cells was significantly affected by cytokines and thus represents another valid parameter of the toxic effects of cytokines [14,19,21]. Therefore, in addition to the viability loss observed in the MTT assay, the proliferation rate of the different cell clones after incubation with IL-1β and with the cytokine mixture was quantified.…”

“…Insulinproducing RINm5F cells have an antioxidative defence status with an antioxidant enzyme equipment which does not differ from primary islet cells [9,14].…”

“…The cells were exposed to 50 μmol/l H 2 O 2 for 2 h in HEPES (20 mmol/l)-supplemented Krebs-Ringer bicarbonate medium with 5 mmol/l glucose and, after removal of H 2 O 2 , for another 18 h in RPMI 1640 medium. In another experimental protocol, cells were incubated with a mixture of 125 μmol/l HX and 2.5 mU/ml XO in RPMI 1640 medium for 18 h as described before [14]. This system continuously generates superoxide radicals and H 2 O 2 through electron transversion from the substrate HX [16].…”

“…H 2 O 2 accumulated in the culture medium depending on the HX concentration, reaching a saturation level 3 h after initiation of the HX/XO reaction. Menadione was freshly dissolved and cells were exposed at a final concentration of 15 μmol/l in RPMI 1640 medium for 18 h as described before [14].…”

Oxidative and nitrosative stress induces peroxiredoxins in pancreatic beta cells

“…Since there are only low concentrations of SOD, CAT and GPX in pancreatic beta cells [6,7,8,9], peroxiredoxins could play an important role as an antioxidant system particularly because, in contrast to these well characterized antioxidant enzymes [8,9,64], gene expression of peroxiredoxins is adjustable by oxidative and nitrosative stress agents. The induction of cellular stress by high glucose, high oxygen, and heat shock treatment has been shown not to affect CAT, SOD or GPX expression in rat pancreatic islets or in RINm5F cells [8].…”