P055 Evaluation of cytokines in C2C12 myotubes treated with leucine and exposed to walker factor proteolysis-inducing factor-like

Abstract

Introduction The proteolysis-inducing factor (PIF), produced by tumours, has been widely studied for cellular signalling processes, especially, for understanding the molecular mechanism of skeletal muscle protein catabolism induced by cachexia. Several studies also focus on muscle protein synthesis inhibition and its process during cachexia. In our studies, we have demonstrated that muscular protein breakdown increased, and the protein synthesis decreased under the effect of proteolysis-inducing factor (PIF) and also under treatment with Walker Factor, a molecule similar to PIF, in myotubes and myoblasts. Leucine plays an important role on muscle metabolism, increasing protein synthesis and decreasing muscle proteolysis. Methods The aim of this work was to investigate the cytokine profile in C2C12 myotubes culture under WF exposure and leucine modulation. C2C12 myoblast cells were maintained in DMEM supplemented with 10% foetal calf serum at 37 °C, and differentiated in myotubes after replaced medium (DMEM supplemented with 2% horse serum) for 4 days prior to the experimental procedures. C2C12 myotubes previously treated with 25 μM or 50 μM leucine for 2 h were exposed to different concentrations of WF (5 and 25 μg WF/well) for 24, 48 and 72 h. After 24 h, the culture medium and cells were collected to measure the amount of cytokines interferon-γ, interleukin-4, interleukin-6, interleukin-10 and TNF-α. Results The cytokine analysis of the DMEM medium, and also media supplemented with Factor Walker and/or leucine were measured and stabilised as control standard for each cytokine (pg/mL culture medium). Results showed that the culture of C2C12 myotube changed the cytokine concentration presented in standard medium; the control cells medium had an increase in INFγ and IL-6, reduction in IL-10 and IL-4, and similar TNF-α content. The results of experimental cells showed that the C2C12 myotubes exposed to WF had different cytokine pattern in medium culture compared to control cells. The C2C12 myotubes under high WF concentration (25 μg/mL) had a decrease in INFγ, IL-10, IL-4 and IL-6 content in the culture medium when compared to medium from control cells. These cytokine patterns for IL-10, IL-4 and IL-6 content had been reverted in these myotubes after previous 25 and 50 μM leucine exposition. Evaluating the C2C12 myotube cells, we suggest the higher IL-6 levels in the culture medium from these cells could be related probably to the normal cell activity. However, the WF exposition added to decrease IL-6 content changed cell activity and also leaded to protein wasting in these cells (increased proteasome enzymes activity and low myosin content). In C2C12 myotubes exposed to WF, the leucine treatment minimized those effects restoring the total protein net (inhibited the protein degradation and enhanced the protein synthesis). Conclusion In tumour-bearing animals, the IL-6 was higher and could be related among other pro-inflammatory cytokines with host wasting. This in vitro experiment suggested that this IL-6 cytokine can be related to hypertrophy and myogenesis during control state, but its lower release from C2C12 cells could be also leading to pro-cachectic process as an effect of Walker Factor, which could be modulated by leucine treatment. Statistical support Dr. J Marcondes; Financial support Fapesp #2006/6007-6, #2010/11328-9.