Insulin and IGF-I Binding in Isolated Trout Cardiomyocytes

Abstract

Insulin and IGF-I receptor binding were characterized in cardiac muscle cells isolated from the brown trout,Salmo trutta fario.Cardiomyocyte suspensions obtained by perfusion of ventricles with collagenase showed a high degree of viability as judged by trypan blue exclusion, LDH leakage, and morphology. Specific insulin binding was 2.88 ± 0.28%/10 mg cells after overnight incubation at 4°. Scatchard analysis indicated the presence of high affinity insulin binding sites with an apparent dissociation constant (Kd) of 0.285 ± 0.043 nMand a binding site density of 1.61 ± 0.19 × 108/mg cells. Specificity of insulin binding was determined by displacing labeled insulin with increasing concentrations of IGF-I, and theKdvalue obtained was 4.77 ± 2.82 nM, 17-fold higher thanKdvalues for displacement of insulin tracer by nonlabeled insulin. The percentage of IGF-I specific binding (6.70±1.42%/10 mg cells), affinity (Kd= 0.163 ± 0.023 nM), and binding site density (4.00 ± 1.13 × 108/mg cells) were higher than those of insulin. Displacement curves of labeled IGF-I with nonlabeled insulin (Kd= 33.6 ± 9.9 nM), indicated a high specificity of the IGF-I binding site. High concentrations of cold insulin and IGF-I were able to decrease markedly the specific binding to their own receptor. Incubation with cold IGF-I also induced a diminution in insulin binding in agreement with the lower specificity of the insulin receptor. These data suggest that insulin and IGF-I are able to down-regulate their own receptor number in cardiac muscle cells. The present results demonstrate that the isolated cardiac myocyte preparation from brown trout is a useful model for studying insulin and IGF-I binding in fish heart tissue.