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 13% (P < 0.05) and decreased protein degradation by 14% (P < 0.05). Treatment with 1 microM 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-1 microM) independently reduced the abundance of ubiquitin ligase mRNA in a dose-dependent manner, with maximal reductions of approximately 70% for muscle atrophy F-box (Fbx) 32, 40% for Fbx25, and 25% for muscle RING finger-1 (MuRF1, P < 0.05). IGF-I and insulin stimulated phosphorylation of FOXO1 and FOXO4 (P < 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 < 0.05). Supplementing media with leucine reduced Fbx32 expression by 25% (P < 0.05) but did not affect Fbx25 nor MuRF1 transcript abundance. Serum deprivation 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 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.
Cleveland
Beth M
BM
United States Department of Agriculture, Agricultural Research Service, National Center for Cool and Cold Water Aquaculture, Kearneysville, West Virginia, USA. beth.cleveland@ars.usda.gov
Weber
Gregory M
GM
D045930:Anabolic Agents / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D002402:Cathepsin D / Q000096:biosynthesis / Q000235:genetics; D056668:Cathepsin L / Q000096:biosynthesis / Q000235:genetics; D002469:Cell Separation; D003470:Culture Media; D016895:Culture Media, Serum-Free; D004305:Dose-Response Relationship, Drug; D044783:F-Box Proteins / Q000378:metabolism; D007004:Hypoglycemic Agents / Q000494:pharmacology*; D007328:Insulin / Q000494:pharmacology*; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology*; D007930:Leucine / Q000494:pharmacology*; D009000:Monocytes / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D017686:Oncorhynchus mykiss / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D011506:Proteins / Q000378:metabolism*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D044767:Ubiquitin-Protein Ligases / Q000096:biosynthesis*
D045930:Anabolic Agents; D003470:Culture Media; D016895:Culture Media, Serum-Free; D044783:F-Box Proteins; D007004:Hypoglycemic Agents; D007328:Insulin; D011506:Proteins; D012333:RNA, Messenger; D007334:Insulin-Like Growth Factor I; D044767:Ubiquitin-Protein Ligases; D019869:Phosphatidylinositol 3-Kinases; D056668:Cathepsin L; D002402:Cathepsin D; D007930:Leucine