Preconditioning with millimolar concentrations of Vitamin C or N-acetylcysteine protects L6 muscle cells insulin-stimulated viability and DNA synthesis under oxidative stress

Abstract

The effect of reactive oxygen/nitrogen species (ROS/RNS)(hydrogen peroxide — H 2O 2, superoxide anion radical O 2 − and hydroxyl radical OH — the reaction products of hypoxanthine/xanthine oxidase system), nitric oxide (NO from sodium nitroprusside — SNP), and peroxynitrite (ONOO − from 3-morpholinosydnonimine — SIN-1) on insulin mitogenic effect was studied in L6 muscle cells after one day pretreatment with/or without antioxidants. ROS/RNS inhibited insulin-induced mitogenicity (DNA synthesis). Insulin (0.1 μM), however, markedly improved mitogenicity in the muscle cells treated with increased concentrations (0.1, 0.5, 1 mM) of donors of H 2O 2, O 2 − , OH, ONOO − and NO . Cell viability assessed by morphological criteria was also monitored. Massive apoptosis was induced by 1 mM of donors of H 2O 2 and ONOO −, while NO additionally induced necrotic cell death. Taken together, these results have shown that ROS/RNS provide a good explanation for the developing resistance to the growth promoting activity of insulin in myoblasts under conditions of oxidative or nitrosative stress. Cell viability showed that neither donor induced cell death when given below 0.5 mM. In order to confirm the deleterious effects of ROS/RNS prior to the subsequent treatment with ROS/RNS plus insulin one day pretreatment with selected antioxidants (sodium ascorbate — ASC (0.01, 0.1, 1 mM), or N-acetylcysteine — NAC (0.1, 1, 10 mM) was carried out. Surprisingly, at a low dose (micromolar) antioxidants did not abrogate and even worsened the concentration-dependent effects of ROS/RNS. In contrast, pretreatment with millimolar dose of ASC or NAC maintained an elevated mitogenicity in response to insulin irrespective of the ROS/RNS donor type used.