title: Endotoxin and interferon-gamma 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.
authors.affiliation: Department of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033, USA. rfrost@psu.edu
authors.forename: Robert A
authors.initials: RA
authors.lastname: Frost
authors.forename: Gerald J
authors.initials: GJ
authors.lastname: Nystrom
authors.forename: Charles H
authors.initials: CH
authors.lastname: Lang
mesh_terms: D055372:AMP-Activated Protein Kinases / Q000378:metabolism; D000578:Amidines / Q000494:pharmacology; D000818:Animals; D001596:Benzylamines / Q000494:pharmacology; D015153:Blotting, Western; D002352:Carrier Proteins / Q000378:metabolism; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D004731:Endotoxins / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D007371:Interferon-gamma / Q000494:pharmacology*; D051379:Mice; D032342:Muscle Cells / Q000187:drug effects* / Q000201:enzymology* / Q000378:metabolism; D018485:Muscle Fibers, Skeletal / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D018482:Muscle, Skeletal / Q000166:cytology*; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010078:Oxazines / Q000494:pharmacology; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism; D014176:Protein Biosynthesis / Q000187:drug effects; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D058570:TOR Serine-Threonine Kinases
chemical_list: D000578:Amidines; D001596:Benzylamines; D002352:Carrier Proteins; D004731:Endotoxins; C104820:N-(3-(aminomethyl)benzyl)acetamidine; D010078:Oxazines; D007371:Interferon-gamma; C085142:oxadin; D019001:Nitric Oxide Synthase; D017853:Phosphotransferases (Alcohol Group Acceptor); C546843:mTOR protein, mouse; D051057:Proto-Oncogene Proteins c-akt; D058570:TOR Serine-Threonine Kinases; D055372:AMP-Activated Protein Kinases; D019331:NG-Nitroarginine Methyl Ester