Protein breakdown in skeletal muscle is accelerated during sepsis, resulting in increased peripheral release of amino acids. Concomitantly, hepatic amino acid uptake and protein synthesis are stimulated. Since metabolic events in skeletal muscle during sepsis may provide the liver with substrates for increased protein synthesis, determination of these effects is of great importance. Therefore, we propose to: 1) Determine sequential changes in muscle protein metabolism during sepsis and to define mechanism(s) of altered protein turnover; 2) Test the hypothesis that protein turnover is similarly regulated in septic and normal skeletal muscle; and 3) Determine the effect of sepsis on amino acid transport in skeletal muscle and to define mechanism(s) and agents of sepsis-induced changes. Sepsis is induced in rats by cecal ligation and puncture (CLP). Control animals are fasted and sham-operated. Intact extensor digitorum longus (EDL) and soleus (SOL) muscles are incubated in a medium consisting of physiologic buffer and glucose and the medium is gassed with O2:CO2. For the study of protein synthesis, 14C-phenylalanine is added to the medium and amount of phenylalanine incorporated into protein is calculated. For the study of protein degradation, cycloheximide is added to the incubation medium and amount of tyrosine released into the medium is measured. When muscle amino acid uptake is studied, 3H-Alpha-aminoisobutyric acid ([3H]-AIB) and 14C-inulin are added to the medium and intracellular and extracellular volume are measured, determined as 14C-inulin space. AIB uptake is expressed as distribution ratio of [3H]-AIB in intracellular and extracellular fluid after a 2-hr. incubation. In other experiments, attempts will be made to determine the chemical signal(s) for increased protein breakdown and reduced AIB uptake. To test the hypothesis that the alterations are caused by a circulating factor(s), muscles will be incubated in the presence of whole septic plasma, or various fractions of septic plasma containing solutes with different molecular weights. In another set of experiments, we will compare the effects of leucine, Alpha-ketoisocaproic acid, insulin, and leupeptin (substances which are all known to regulate protein turnover in normal skeletal muscle) on protein synthesis and degradation in normal and septic muscle.
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