Insulin and IGF-I action on insulin receptors, IGF-I receptors, and hybrid insulin/IGF-I receptors in vascular smooth muscle cells

Abstract

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-14C]Glucose accumulation and [6-3H]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) β-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 10−10–10−9M IGF-I, IP of the IGF-IR, or IR β-subunit and immunoblot with anti-phosphotyrosine antibody, we found two distinct bands indicating phosphorylation of both the IGF-IR and the IR β-subunit. Stimulation with 10−10–10−9M insulin and IP against the IGF-IR did not show phosphorylation of either β-subunit, whereas after IP of the IR we found phosphorylation of the IR β-subunit. [14C]Glucose accumulation and [3H]thymidine incorporation were elevated in cells stimulated with IGF-I at 10−10–10−7M, reaching maximum by 10−9M. Insulin stimulation showed measurable effects only at supraphysiological concentrations, 10−8–10−7M. In conclusion, coprecipitation of both the IGF-IR and the IR β-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.