Cleveland BM, Weber GM. Effects of insulin-like growth factor-I, insulin, and leucine on protein turnover and ubiquitin ligase expression in rainbow trout primary myocytes. The effects of insulin-like growth factor-I (IGF-I), insulin, and leucine on protein turnover and pathways that regulate proteolytic gene expression and protein polyubiquitination were investigated in primary cultures of 4-day-old rainbow trout myocytes. Supplementing media with 100 nM IGF-I increased protein synthesis by

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... (P Ͻ 0.05) and decreased protein degradation by 14% (P Ͻ 0.05). Treatment with 1 M insulin increased protein synthesis by 13% (P Ͻ 0.05) and decreased protein degradation by 17% (P Ͻ 0.05). Supplementing media containing 0.6 mM leucine with an additional 2.5 mM leucine did not increase protein synthesis rates but reduced rates of protein degradation by 8% (P Ͻ 0.05). IGF-I (1 nM-100 nM) and insulin (1 nM-1 M) independently reduced the abundance of ubiquitin ligase mRNA in a dose-dependent manner, with maximal reductions of ϳ70% for muscle atrophy F-box (Fbx) 32, 40% for Fbx25, and 25% for muscle RING finger-1 (MuRF1, P Ͻ 0.05). IGF-I and insulin stimulated phosphorylation of FOXO1 and FOXO4 (P Ͻ 0.05), which was inhibited by the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor wortmannin, and decreased the abundance of polyubiquitinated proteins by 10 -20% (P Ͻ 0.05). Supplementing media with leucine reduced Fbx32 expression by 25% (P Ͻ 0.05) but did not affect Fbx25 nor MuRF1 transcript abundance. Serum deprivation decreased rates of protein synthesis by 60% (P Ͻ 0.05), increased protein degradation by 40% (P Ͻ 0.05), and increased expression of all ubiquitin ligases. These data suggest that, similar to mammals, the inhibitory effects of IGF-I and insulin on proteolysis occur via P I3-kinase/protein kinase B signaling and are partially responsible for the ability of these compounds to promote protein accretion. atrogin-1; forkhead box O; insulin-like growth factor-I; protein synthesis Address for reprint requests and other correspondence: B. M. Cleveland, 11861 Leetown Rd.,