Abstract
The purpose of the present study was to test the hypothesis that endogenous NO negatively affects translation in skeletal muscle cells after exposure to a combination of endotoxin (LPS) and interferon-gamma (IFN-gamma). Individually, LPS and IFN-gamma did not alter protein synthesis, but in combination, they inhibited protein synthesis by 80% in C2C12 myotubes. The combination of LPS and IFN-gamma dramatically downregulated the autophosphorylation of the mammalian target of rapamycin and its substrates S6K1 and 4EBP-1. The phosphorylation of ribosomal protein S6 was decreased, whereas phosphorylation of elongation factor 2 and raptor was enhanced, consistent with defects in both translation initiation and elongation. Reduced S6 phosphorylation occurred 8 to 18 h after LPS/IFN-gamma and coincided with a prolonged upregulation of NOS2 messenger RNA and protein. NOS2 protein expression and the LPS/IFN-gamma-induced fall in phosphorylated S6 were prevented by the proteasome inhibitor MG-132. The general NOS inhibitor, L-NAME, and the specific NOS2 inhibitor, 1400W, also prevented the LPS/IFN-gamma-induced decrease in protein synthesis and restored translational signaling. LPS/IFN-gamma downregulated the phosphorylation of multiple Akt substrates, including the proline-rich Akt substrate 40, while enhancing the phosphorylation of raptor on a 5'-AMP-activated kinase (AMPK)-regulated site. The negative effects of LPS/IFN-gamma were blunted by the AMPK inhibitor compound C. The data suggest that, in combination, LPS and IFN-gamma induce a prolonged expression of NOS2 and excessive production of NO that reciprocally alter Akt and AMPK activity and consequently downregulate translation via reduced mammalian target of rapamycin signaling.