Leucine ameliorates endotoxin‐induced alterations in protein‐protein interactions within mTORC1 complex in neonatal piglets

Abstract

Sepsis decreases protein synthesis in skeletalmuscle in part by limiting the phosphorylation of translation initiationregulatory factors, eukaryotic initiation factor (eIF) 4E‐binding protein (4EBP1)and S6 kinase (S6K1), that are downstream of the mammalian target of rapamycincomplex 1 (mTORC1). Previous work inmature rats suggests that decreased raptor binding activity to eIF3b mayunderline the sepsis‐induced reduction in protein synthesis in skeletal muscle. Raptor‐eIF3b interaction is crucial for thestimulation of protein translation and its alteration during endotoxemia inmature animals appears to block the anabolic response to the branched‐chainamino acid, leucine (Leu). We have shown in healthy neonatal pigs that Leu supplementation increases muscle protein synthesis by enhancing the activation of mTORC1 and its downstream targets, 4EBP1 and S6K1. To determine if supplemental Leu can stimulate raptor‐eIF3b binding and muscle protein synthesis in theneonate during endotoxemia, overnight fasted neonatal pigs (n=6–8/group) were infused for 8 h with LPS or saline while plasma amino acids, glucose, and insulin were maintained at fasting levels during pancreatic‐substrate clamps. Leu (400 μmol·kg−1·hr−1) or saline was infused during the last h. Phosphorylation of 4EBP1 and S6K1, eIF4E‐eIF4G complex formation, raptor‐eIF3b binding, protein synthesis rates, ribosomal translational capacity (Cs) and translational efficiency (KRNA) were determined in longissimus dorsi (LD), gastrocnemius (Gast) and soleus muscles. LPS decreased protein synthesis and KRNA in LD, Gast, and soleus muscles (P<0.05) while Cs did not change compared to controls. Leu infusion in the LPS group increased 4EBP1 and S6K1 phosphorylation, eIF4E‐eIF4G complex formation, KRNA, and the binding of raptor to eIF3b (by 2 to 3 fold) and protein synthesis (by over 80%) in LD, Gast, and soleus muscles compared to LPS infusion alone (P<0.05). Compared to saline, Leu infusion increased protein synthesis, KRNA, raptor‐eIF3b interaction, 4EBP1 and S6K1 phosphorylation and eIF4E·eIF4G complex formation in all muscles (P<0.05). Raptor‐eIF3b binding showed a linear correlation with 4EBP1 and S6K1 phosphorylation, eIF4E‐eIF4G formation, and protein synthesis in LD, Gast, and soleus muscles that was independent of treatment(P<0.01). These findings suggest that parenteral Leu supplementation attenuates the reduction in muscle protein synthesis induced by endotoxemia by stimulating the interaction of raptor with eIF3 within the mTORC1 complex in the neonate.Support or Funding InformationSupported by NIH AR444474, USDA/ARS 6250‐51000‐055, NIH HD072891, and USDA NIFA 2013‐67015‐20438