Preincubation with sodium ascorbate potentiates insulin-dependent PKB/Akt and c-Jun phosphorylation in L6 rat myoblasts challenged with reactive oxygenitrogen species

摘要

Previously, we reported that mitogenicity in L6 muscle cells was Stimulated by insulin but inhibited by reactive oxygenitrogen species (ROS/RNS; [Orzechowski, A., Lokociejewska, M., Muras, P., Hocquette, J.-F., 2002a. Preconditioning with millimolar concentrations of vitamin C or N-acetylcysteine protects L6 muscle cells insulin-stimulated viability and DNA synthesis under oxidative stress. Life Sciences 71. 1793-.-1808]) and that preincubation with sodium ascorbate (ASC) protected from either the impaired DNA synthesis and/or loss of cell viability. Now, we addressed the question how ascorbate (AA) rescued DNA synthesis in L6 muscle cells being challenged with ROS/RNS. We assumed that AA might be able to influence insulin signaling, We found that insulin elevated the protein levels of both PKB/Akt kinase phosphorylated at Serine 473 (pS473-Akt), and c-Jun phosphorylated at Serine(63), Serine(73) (pS63, pS73-c-Jun) residues, respectively. A short-term treatment experiment (0 - 45 min) revealed that either insulin (0.1 mu M) or hydrogen peroxide (0.1, 0.5 mM, H2O2) increased the pS473-Akt and pS63, pS73-c-Jun protein levels, although the effect of ROS/RNS peaked earlier (5 min) than that of insulin (45 min). Astonishingly, the elevated levels of both pS473-Akt and pS63, pS73-c-Jun in response to insulin were reduced by the concomitant treatment with H2O2 in a dose-dependent fashion. In contrast, a 4-hour preincubation with ASC (I mM) augmented the signal from pS473-Akt and pS63, pS73-c-Jun, when both insulin and H2O2 were added. Moreover, a 24 It preincubation with ASC also elevated the pS473-Akt and pS63, pS73-c-Jun levels in response to insulin irrespective to ROS/RNS co-treatment. During chronic treatment studies, ROS/RNS stimulated neither phosphorylation of Akt nor c-Jun, indicating that ROS/RNS-dependent activation of the above-mentioned proteins was short-term and transient. Furthermore, higher levels of pS473 Akt and pS63, pS73-c-Jun after preincubation with ASC suggest that by this route AA could protect insulin-induced mitogenicity. Basal levels of Akt and its target p70(S6K) remained constant regardless of treatment. These results suggest that AA defends the insulin-stimulated mitogenicity hampered by ROS/RNS most likely by the amplification of insulin signal at the level of pS473-Akt and pS63, pS73-c-Jun, respectively. (c) 2005 Elsevier Inc. All rights reserved.