Muscle catabolism in sepsis is caused by inhibition of amino acid uptake and protein synthesis and increased protein degradation. Recent studies showed that sepsis-induced muscle protein breakdown mainly reflects degradation of myofibrillar proteins. In contrast, the influence of sepsis on myofibrillar protein synthesis is not known. Mediators and mechanisms of muscle catabolism in sepsis also remain to be determined. We propose to: 1) determine the effect of sepsis on myofibrillar protein synthesis; 2) define mediators and mechanisms of muscle catabolism in sepsis, in particular the role of glucocorticoids and cytokines; 3) characterize and isolate the plasma factor which inhibits amino acid transport in sepsis. Sepsis is induced in rats by cecal ligation and puncture. Protein synthesis in vivo is measured following a flooding dose of l4C-leucine. Myofibrillar and sarcoplasmic protein synthesis is determined after separation by differential salt solubility. The influence of sepsis on transcription of myofibrillar proteins is determined by measuring mRNA levels for actin and myosin. For protein synthesis in vitro, extensor digitorum longus and soleus muscles of rats or biopsies from the rectus abdominis muscle of patients are incubated for 2 h in physiologic buffer and incorporation of 14C-phenylalanine into total proteins or into myofibrillar and sarcoplasmic protein pools is determined. For protein degradation, tyrosine and 3-methylhistidine release by incubated muscles is measured by HPLC. The role of glucocorticoids in muscle catabolism is tested by treating septic rats with the glucocorticoid receptor antagonist RU 38486. The role of cytokines is tested by measuring plasma and muscle levels of interleukin-1 (IL-1) and tumor necrosis factor (TNF), by administering IL-1 or TNF to normal rats and by treating septic rats with IL-1 or TNF antiserum or IL-1 receptor antagonist. The contribution of hypoperfusion and reduced energy levels to sepsis-induced changes in protein turnover is determined. To identify the amino acid transport inhibiting factor in septic plasma, muscles are incubated with 3H-alpha-aminoisobutyric acid (AIB) and with fractions of septic or endotoxic plasma from rats or patients. Following gel filtration on Sephadex columns, peptides are separated by ion-exchange or reverse-phase chromatography, using HPLC. Structure and amino acid sequence of the isolated amino acid transport inhibiting factor in plasma will be determined, the goals being to synthesize the peptide, produce antibodies, and test the effect on sepsis metabolism.
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