Interaction of insulin-like growth factor I with turkey satellite cells and satellite cell-derived myotubes

Abstract

Satellite cells, isolated from the superficial pectoralis muscle of growing Nicholas tom turkeys, were cloned to obtain a pure population of myogenic cells. These cells proliferated rapidly and differentiated (fused) into myotubes typically containing 92–98% fused nuclei. Competitive binding assays were performed on near-confluent satellite cell or myotube cultures in 35 mm diameter wells by adding [ 125I]IGF-I along with increasing concentrations of unlabeled IGF-I, IGF-II, or insulin. Following incubation, the cultures were washed to remove the unbound hormones, solubilized with 0.5 N NaOH, and the radioactivity specifically bound was determined. Total and fused nuclei number as well as total protein were determined in parallel cultures. Our results indicate that turkey satellite cell and myotube cultures possess specific binding sites for IGF-I. Displacement of [ 125I]IGF-I was in the order of IGF-I > IGF-II ⩾ insulin. Although the [ 125I]IGF-I association constants were similar for turkey satellite cells and myotubes, a 2.8-fold decrease in the number of receptors per nuclei was observed as satellite cells differentiated into myotubes. The 50% inhibition constants for IGF-I, IGF-II, and insulin were 3.7 × 10 −9 M, 7.5 × 10 −8 M, and 8.7 × 10 −8 M for satellite cells and 3.1 × 10 −9 M, 7.5 × 10 −8 MM, and 9.6 × 10 −8 M for myotubes, respectively. Receptor cross-linking analysis using disucinimidyl suberate was performed on near-confluent satellite cell cultures incubated with [ 125I]IGF-I in the presence or absence of 1 × 10 −7 M IGF-I, IGF-II, or insulin. Receptor subunit species of M r 130 kDa and 98 kDa were observed under reducing conditions (100 mM dithiothreitol) and at a M r > 300 kDa (native receptor tetramer) under non-reduced conditions. Autoradiographic bands were displaced with IGF-I but not with equimolar levels of IGF-II or insulin. The results suggest that turkey satellite cells possess a type I IGF receptor.