Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 5/22/2015
Publication Date: 8/27/2015
Citation: Ramsay, T.G., Stoll, M.J., Caperna, T.J., Blomberg, L. 2015. The immune system modulator a1-acid glycoprotein inhibits insulin and IGF1 induced protein synthesis in C2C12 myotubes. Meeting Abstract. 064:40.
Technical Abstract: Alpha-1 acid glycoprotein (AGP) has previously been demonstrated by our laboratory to be negatively correlated with growth rate in newborn piglets. However, a mechanism of action for AGP in growth has not been identified. Previous research has demonstrated that AGP can modify adipose tissue metabolism in swine by altering insulin action. The present study was undertaken to determine if AGP can modify muscle metabolism by examining protein turnover in the C2C12 murine muscle cell line. Cells were thawed from frozen stocks and propagated to confluency in 6 well tissue culture plates with DMEM containing 10% fetal bovine serum and antibiotics/antimycotics. Medium was changed to 5% horse serum in DMEM at confluency and cells were permitted to fuse into myotubes. Cells were used for experiments 4 days post fusion. Medium containing serum was removed from culture wells which were rinsed with serum free DMEM prior to addition of treatment media. Treatment media were comprised of serum free DMEM, 0.5% BSA, and 0, 0.1, 1.0 or 10.0 µg/mL AGP for the initial experiments. Treatment media were added to cultures for 1 hour, then supplemented with 3H-thymidine/mL for an additional 2 hours. Wells were washed three times with ice cold 10% trichloroacetic acid, with the last wash permitted to incubate with the cells overnight at 4°C. Cells were then scraped from wells and transferred to centrifuge tubes. Cells were centrifuged and supernatant removed. One mL of 0.5 M NaOH + 0.1% Triton X-100 was then added and tubes were incubated at 50°C for 1 hour prior to counting. Treatment of C2C12 myotubes with AGP had no detectable effect on protein synthesis (p > 0.05; n = 4 trials). Insulin (10 nM) increased 3H-tyrosine incorporation by 27% (P < 0.05; n = 3 trials). However, addition of 1 µg murine AGP/mL medium reduced the insulin induced 3H-tyrosine incorporation by 56% (P< 0.01, n = 3 trials). Incubation with a higher concentration of insulin (1 µm) stimulated a 50% increase in 3H-tyrosine incorporation (P < 0.01, n = 3 trials). Addition of 1 µg AGP/mL medium did not affect the incorporation induced by 1 µM insulin (P > 0.05, n = 3 trials). Treatment with IGF1 (20 ng /mL) increased 3H-tyrosine incorporation by 53% (P < 0.01, n = 3 trials); while addition of AGP was associated with a 40% reduction in 3H-tyrosine incorporation that was induced by IGF1 (P < 0.01, n = 3 trials). These data indicate that AGP can indirectly reduce protein synthesis in skeletal muscle cells by altering the efficacy of physiological concentrations of insulin or IGF1. Key Words: Alpha-1 acid glycoprotein; IGF1, insulin; myotube, protein synthesis