Johansson Git S, Arnqvist Hans J
Department of Biomedicine and Surgery, Division of Cell Biology, Linköping University, Linköping, Sweden.
Am J Physiol Endocrinol Metab. 2006 Nov;291(5):E1124-30. doi: 10.1152/ajpendo.00565.2005. Epub 2006 Jun 27.
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.
已知胰岛素和胰岛素样生长因子I（IGF-I）会影响心血管疾病。我们已经在受体水平研究了胰岛素和IGF-I对血管平滑肌细胞（VSMC）的配体结合及剂量反应关系。取自大鼠胸主动脉的VSMC血清饥饿后，用IGF-I或胰岛素刺激，然后裂解、免疫沉淀，并通过蛋白质印迹法进行分析。还测量了d-[U-(14)C]葡萄糖摄取和[6-(3)H]胸苷掺入DNA的情况。证实了胰岛素和IGF-I的特异性结合，IGF-I的结合更高。在针对两者之一进行免疫沉淀（IP）后，检测到并共沉淀了IGF-I受体（IGF-IR）和胰岛素受体（IR）的β亚基。在IP前用二硫苏糖醇还原二硫键后未发现共沉淀。在用10(-10)-10(-9) M IGF-I刺激、对IGF-IR或IRβ亚基进行IP并用抗磷酸酪氨酸抗体进行免疫印迹后，我们发现两条不同的条带，表明IGF-IR和IRβ亚基均发生了磷酸化。用10(-10)-10(-9) M胰岛素刺激并针对IGF-IR进行IP，未显示任何一个β亚基的磷酸化，而在对IR进行IP后，我们发现IRβ亚基发生了磷酸化。在用10(-10)-10(-7) M IGF-I刺激的细胞中，[(14)C]葡萄糖摄取和[(3)H]胸苷掺入增加，在10(-9) M时达到最大值。胰岛素刺激仅在超生理浓度10(-8)-10(-7) M时显示出可测量的效应。总之，IGF-IR和IRβ亚基的共沉淀表明VSMC中存在胰岛素/IGF-I杂合受体。在生理浓度下，胰岛素激活IR，但不影响葡萄糖代谢或DNA合成，而IGF-I既能激活受体又能引发生物学效应。
