ENDOTOXIN AND INTERFERON-γ INHIBIT TRANSLATION IN SKELETAL MUSCLE CELLS BY STIMULATING NITRIC OXIDE SYNTHASE ACTIVITY

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.