Effects of insulin-like growth factor-I, insulin, and leucine on protein turnover and pathways that regulate ubiquitin ligase expression in rainbow trout primary myocytes

Authors: Cleveland, Beth; Weber, Gregory - Greg

Submitted to: American Journal of Physiology - Regulatory Integrative & Comparative Physiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/2/2009
Publication Date: 2/1/2010
Citation: Cleveland, B.M., Weber, G.M. 2010. Effects of insulin-like growth factor-I, insulin, and leucine on protein turnover and pathways that regulate ubiquitin ligase expression in rainbow trout primary myocytes. American Journal of Physiology - Regulatory Integrative & Comparative Physiology. 298:R341-350.

Interpretive Summary: Protein represents the main component of the biomass of the rainbow trout, therefore rates of protein turnover can have tremendous impacts on the growth, weight loss, and feed efficiency of the fish. Protein turnover is determined by the rates of protein synthesis as well as rates of protein degradation. Our objective was to better understand the mechanisms by which anabolic compounds promote protein accretion through the reduction of protein degradation. Primary muscle cells from rainbow trout were incubated with IGF-I, insulin, and leucine and the effects on protein degradation and gene expression were determined. These compounds reduced protein degradation and decreased expression of genes that promote protein degradation by acting through known signaling pathways. These findings provide a better understanding of how fish regulate protein turnover, which is critical for the development of feeding strategies or husbandry practices that optimize growth and feed efficiency. Additionally, these data suggest that the correlation between plasma IGF-I and growth performance in fish is partially a result of the ability of IGF-I to slow protein degradation, thereby promoting protein accretion and growth.

Technical Abstract: 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 four day old rainbow trout myocytes. Supplementing media with 100 nM IGF-I increased protein synthesis by 13% (P<0.05) and decreased protein degradation by 14% (P<0.05). Treatment with 1 uM insulin increased protein synthesis by 13% (P<0.05) and decreased protein degradation by 17% (P<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<0.05). IGF-I (1 nM - 100 nM) and insulin (1 nM - 1uM) independently reduced the abundance of ubiquitin ligase mRNA in a dose-dependent manner, with maximal reductions of approximately 70% for Fbx32, 40% for Fbx25, and 25% for MuRF1 (P<0.05). IGF-I and insulin stimulated phosphorylation of FOXO1 and FOXO4 (P<0.05), which was inhibited by the PI3K inhibitor wortmannin, and decreased the abundance of polyubiquitinated proteins by 10%-20% (P<0.05). Supplementing media with leucine reduced Fbx32 expression by 25% (P<0.05) but did not affect Fbx25 nor MuRF1 transcript abundance. Serum starvation decreased rates of protein synthesis by 60% (P<0.05), increased protein degradation by 40% (P<0.05), and increased expression of all ubiquitin ligases. These data indicate that, in rainbow trout, the inhibitory effects of IGF-I, insulin, and leucine on signaling pathways that regulate proteolysis are partially responsible for their anabolic effects, which is similar to what has been observed in mammals.