We propose to continue work on three separate but related projects. 1) We have partially purified from skeletal muscle a heat and acid stable non-dialysable factor, presumed to be a peptide, which inhibits protein synthesis in rabbit reticulocyte lysates, apparently at the level of translation initiation. Moreover, rat hemidiaphragms incubated with glucose alone yield significantly more inhibitor activity than those incubated in media supplemented with insulin and amino acids. Proposed studies include: a) confirm and extend these findings, confirm peptide nature of the muscle derived translational inhibitor and site of action on initiation and delineate respective role of insulin and amino acids in its regulation; b) purify and characterize peptide(s) in the muscle extract which inhibit translation initiation and are regulated by insulin and/or amino acids; c) study the mode of action of this inhibitor(s) and mechanism of suppression by insulin and/or amino acids. Working hypotheses are that the inhibitor promotes the phosphorylation of eIF-2Alpha and that insulin treatment of muscle reduces inhibitor activity; d) study physiological and pathological conditions which may modulate the activity and/or suppressibility of the inhibitor in vivo e.g. the effects of diabetes, hormones, muscle work and denervation; 2) exercise enhances insulin sensitivity of muscles, while immobilization or short-term denervation causes insulin resistance. We have studied isolated soleus muscles 6-24 hours after cutting the sciatic nerve and observed decreased insulin binding, decreased sensitivity to insulin of glucose transport and marked resistance to insulin of glycogen synthesis, indicating receptor and post-receptor defects. The molecular mechanisms by which muscle activity modulates the insulin receptor and receptor-function coupling will be investigated in different types of muscle, and with respect to several parameters of insulin action: e.g. glucose transport glycogen synthesis, glycolysis, amino acid transport and protein synthesis. 3) Studies concerning the regulation of branched chain amino acid (BCAA) catabolism will be continued in 2 areas: a) feedback regulation of branched chain keto acid dehydrogenase, b) mechanisms mediating accelerated oxidation of BCAA by muscles in diabetes, fasting and during exercise. These studies are relevant to understanding of altered metabolism in diabetes and other diseases associated with insulin resistance and protein wasting.
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