Insulin and IGF-I action on insulin receptors, IGF-I receptors, and hybrid insulin/IGF-I receptors in vascular smooth muscle cells.
Insulin and insulin-like growth factor I (IGF-I) are known to affect cardiovascular disease. We have investigated ligand binding and the dose-response relationship for insulin and IGF-I on vascular smooth muscle cells (VSMCs) at the receptor level. VSMCs from rat thoracic aorta were serum starved, stimulated with IGF-I or insulin, lysed, immunoprecipitated, and analyzed by Western blot. d-[U-(14)C]Glucose accumulation and [6-(3)H]thymidine incorporation into DNA were also measured. Specific binding of both insulin and IGF-I was demonstrated, being higher for IGF-I. Both IGF-I receptor (IGF-IR) and insulin receptor (IR) beta-subunits were detected and coprecipitated after immunoprecipitation (IP) against either of the two. No coprecipitation was found after reduction of disulphide bonds with dithiotreitol before IP. After stimulation with 10(-10)-10(-9) M IGF-I, IP of the IGF-IR, or IR beta-subunit and immunoblot with anti-phosphotyrosine antibody, we found two distinct bands indicating phosphorylation of both the IGF-IR and the IR beta-subunit. Stimulation with 10(-10)-10(-9) M insulin and IP against the IGF-IR did not show phosphorylation of either beta-subunit, whereas after IP of the IR we found phosphorylation of the IR beta-subunit. [(14)C]Glucose accumulation and [(3)H]thymidine incorporation were elevated in cells stimulated with IGF-I at 10(-10)-10(-7) M, reaching maximum by 10(-9) M. Insulin stimulation showed measurable effects only at supraphysiological concentrations, 10(-8)-10(-7) M. In conclusion, coprecipitation of both the IGF-IR and the IR beta-subunit indicates the presence of hybrid insulin/IGF-I receptors in VSMC. At a physiological concentration, insulin activates the IR but does not affect either glucose metabolism or DNA synthesis, whereas IGF-I both activates the receptor and elicits biological effect.
Johansson
Git S
GS
Department of Biomedicine and Surgery, Division of Cell Biology, Linköping University, Linköping, Sweden. git.johansson@ibk.liu.se
Arnqvist
Hans J
HJ
D000818:Animals; D001013:Aorta, Thoracic / Q000166:cytology; D002250:Carbon Radioisotopes; D002478:Cells, Cultured; D019281:Dimerization; D005947:Glucose / Q000493:pharmacokinetics; D007004:Hypoglycemic Agents / Q000378:metabolism* / Q000494:pharmacology; D007328:Insulin / Q000378:metabolism* / Q000494:pharmacology; D007334:Insulin-Like Growth Factor I / Q000378:metabolism* / Q000494:pharmacology; D007457:Iodine Radioisotopes; D008024:Ligands; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism*; D010766:Phosphorylation; D051381:Rats; D017207:Rats, Sprague-Dawley; D017526:Receptor, IGF Type 1 / Q000737:chemistry / Q000378:metabolism*; D011972:Receptor, Insulin / Q000737:chemistry / Q000378:metabolism*; D013936:Thymidine / Q000493:pharmacokinetics; D014316:Tritium
D002250:Carbon Radioisotopes; D007004:Hypoglycemic Agents; D007328:Insulin; D007457:Iodine Radioisotopes; D008024:Ligands; D014316:Tritium; D007334:Insulin-Like Growth Factor I; D017526:Receptor, IGF Type 1; D011972:Receptor, Insulin; D005947:Glucose; D013936:Thymidine