Insulin and IGF-I binding in isolated trout cardiomyocytes

Gen Comp Endocrinol. 1996 Sep;103(3):264-72. doi: 10.1006/gcen.1996.0121.

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 degrees. Scatchard analysis indicated the presence of high affinity insulin binding sites with an apparent dissociation constant (Kd) of 0.285 +/- 0.043 nM and a binding site density of 1. 61 +/- 0.19 x 10(8)/mg cells. Specificity of insulin binding was determined by displacing labeled insulin with increasing concentrations of IGF-I, and the Kd value obtained was 4.77 +/- 2.82 nM, 17-fold higher than Kd values 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 x 10(8)/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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Binding, Competitive
  • Catheterization
  • Cell Survival
  • Cells, Cultured
  • Humans
  • Insulin / analysis
  • Insulin / metabolism*
  • Insulin-Like Growth Factor I / analysis
  • Insulin-Like Growth Factor I / metabolism*
  • Iodine Radioisotopes
  • Microscopy, Interference
  • Myocardium / cytology
  • Myocardium / metabolism*
  • Radioligand Assay / methods
  • Receptor, IGF Type 1 / metabolism
  • Receptor, Insulin / metabolism
  • Recombinant Proteins / analysis
  • Recombinant Proteins / metabolism
  • Swine
  • Trout

Substances

  • Insulin
  • Iodine Radioisotopes
  • Recombinant Proteins
  • Insulin-Like Growth Factor I
  • Receptor, IGF Type 1
  • Receptor, Insulin