IGF-I binding in primary culture of muscle cells of rainbow trout: changes during in vitro development

Abstract

To characterize and study the variations of IGF-I binding during the development of trout muscle cells, in vitro experiments were conducted using myocyte cultures, and IGF-I binding assays were performed in three stages of cell development: mononuclear cells ( day 1), small myotubes ( day 4), and large myotubes ( day 10). Binding experiments were done by incubating cells with IGF-I for 12 h at 4°C. Specific IGF-I binding increased with the concentration of labeled IGF-I and reached a plateau at 32 pM. The displacement of cold human and trout IGF-I showed a very similar curve (EC50 = 1.19 ± 0.05 and 0.95 ± 0.05 nM, respectively). IGF binding proteins did not interfere significantly because displacement of labeled IGF-I by either cold trout recombinant IGF-I or Des (1–3) IGF-I resulted in similar curves. Insulin did not displace labeled IGF-I even at very high concentrations (>1 μM), which indicates the specificity of IGF-I binding. The amount of receptor (R0) increased from 253 ± 51 fmol/mg DNA on day 1 to 766 ± 107 fmol/mg DNA on day 10. However, the affinity ( K d) of IGF-I receptors did not change significantly during this development (from 1.29 ± 0.19 to 0.79 ± 0.13 nM). On the basis of our results, we conclude that rainbow trout muscle cells in culture express specific IGF-I receptors, which increase their number with development from mononuclear cells to large myotubes.