Effects of Leucine Starvation or Supplementation on Oxidative Stress and Markers of Apoptosis in C2C12 Myotubes

Abstract

L-leucine (LEU) deprivation decreases protein synthesis and increases the rates of protein degradation. Whey protein hydrolysate (WPH) is a commonly used supplement that has been shown to prevent LEU-deficient myofiber atrophy. However, the effects of LEU deprivation or LEU/WPH supplementation on oxidative stress and apoptosis in myotubes have not been elucidated. Therefore, the aim of the current experiment was to investigate the response of C2C12 myotubes to either LEU starvation or supplementation for oxidative stress and apoptosis. C2C12 myoblasts were grown in growth media (GM) to 70% confluency and GM was replaced with differentiation media (DM) for 5 days. Then, myotubes were treated with one of the following media for two days: 1) DM (0.10g/L LEU); 2) LEU-free (LF) DM; 3) LF DM containing 0.90 g/L of added LEU (LF+LEU); or 4) LF DM containing 2.01 g/L WPH (LF+WPH) (0.22g/L LEU); n=8 plates per group. Cells were subsequently harvested and analyzed for primary antioxidant protein levels, oxidative damage, and apoptosis signaling markers. LEU starvation resulted in an increase in the protein levels of SOD1 and SOD2 (p<0.05). LF+LEU prevented the aforementioned increases (p<0.05). Interestingly, WPH induced higher levels of SOD2 compared to DM, LF, and LF+LEU (p<0.05). Catalase, glutathione peroxidase, and 4 hydroxynonenal (4HNE) protein levels were not significantly different among groups (p>0.05). Protein levels of select apoptotic markers (pBAD, p53, and PARP) were also not different among groups (p>0.05). In conclusion, 2 days of LEU starvation (i.e., cell stress) increases antioxidant enzymes potentially due to increased oxidant production; however there were no changes in the three markers of apoptosis investigated.