The objectives of the studies described herein are to identify the loci responsible for the inhibition of protein synthesis in skeletal muscle during sepsis and to establish the mechanism(s) by which the inhibition can be reversed in order to develop treatment strategies to combat the severe muscle wasting associated with the septic process. Sustained muscle wasting contributes to the morbidity and mortality associated with sepsis. The defect in protein synthesis is localized to an impaired translation of mRNA at the level of peptide-chain initiation. Translation initiation is regulated at two steps: formation of the 43S pre-initiation complex (controlled by eukaryotic initiation factor 2 (eIF2) and eIF2B); and the binding of mRNA to the 40S ribosome (controlled by elF4E). We have identified a decreased activity of eIF2B as one defect in peptide-chain initiation and have shown that the muscle content of eIF2B protein is diminished 40 percent by sepsis. Therefore, reduced expression of eIF2B appeared a likely cause of the sepsis-induced inhibition of peptide-chain initiation in muscles of septic rats. However, protein synthesis can be stimulated 2-fold by perfusion of muscles from septic rats with buffer containing either IGF-I or elevated concentrations of amino acids by accelerating peptide-chain initiation without increasing the muscle content of eIF2B. Thus, effects of a reduced eIF2B expression on protein synthesis can be overridden, but the mechanisms responsible remain unknown. The hypothesis to be tested is that altered regulation of eIF2B and/or eIF4E mediates the changes in protein synthesis in sepsis.
The specific aims of the studies proposed for the next project period are: (1) to evaluate the role of altered phosphorylation of eIF2B activity in controlling translation initiation during sepsis; (2) to investigate the effect of sepsis on eIF4E by measuring the amount of eIF4E found in the inactive 4E-BPI eIF4E complex and the active eIF4G eIF4E complex in muscle; (3) to investigate the mechanisms by which IGF-I stimulates translation initiation and contrast the response of skeletal muscle protein synthesis to IGF-I with that of insulin during sepsis; (4) to investigate the mechanisms by which amino acids stimulate translation initiation, and hence protein synthesis, during sepsis; and (5) to investigate the mechanisms by which chronic infusion of TNF or IL-1 cause an inhibition of protein synthesis in skeletal muscle. The research design will be to correlate changes in eukaryotic factor activity with rates of protein synthesis to establish which control mechanisms are important for regulating protein synthesis in skeletal muscle during sepsis.
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