Figure 5

Effects of tumor necrosis factor (TNF)-α on protein metabolism in L6 myotubes are prevented by ceramide-synthesis inhibition. (A) Myotubes were treated for 12 hours with TNF-α, in the presence or absence of 100 nmol/l myriocin, or 1 μmol/l OMS. The rate of protein synthesis was measured by adding [³H]-tyrosine to the culture medium, and counting the radioactivity present in trichloroacetic acid (TCA) precipitates of the cells, in relation to the total protein content. The results are the mean ± SE of three determinations. ++ Different from control: P = 0.001; *different from TNF-α alone: P≤ 0.05. (B) Proteolysis was measured in L6 myotubes labeled with [³H]-tyrosine for 48 h, and treated for 12 hours with TNF-α in the presence or absence of 100 nmol/l myriocin, 1 μmol/l OMS, or both. TCA-soluble radioactivity released in the medium was measured and related to the total incorporated radioactivity. The results are the mean ± SE of three determinations. *Different from TNF-α alone: P < 0.05. (C) The mRNA levels of Atrogin-1 and LC3b were measured by reverse transcription quantitative PCR in myotubes treated or not with TNF-α in the presence of 100 nmol/l myriocin, 10 μmol/l GW4869, or 1 μmol/l OMS, and normalized to the TATA box binding protein mRNA. Results are the mean ± SE of 3 to 5 measurements in duplicate. **Different from control without drug: P = 0.01; +++ different from TNF-α alone: P≤ 0.001, + P < 0.05. (D) Myotubes were treated for 3 days with or without TNF-α, in the presence of 100 nmol/l myriocin. Phospho- Ser253 Foxo3 in cell extracts was analyzed by western blotting. Results were normalized by total Foxo3 protein amounts. *Different from all other values: P < 0.02.