Characterisation of receptors for IGF-I and insulin; evidence for hybrid insulin/IGF-I receptor in human coronary artery endothelial cells

Abstract

Objective Coronary artery disease is a prevalent cause of morbidity and mortality in diabetes. Little is known about insulin-like growth factor-I receptors (IGF-IR) and insulin receptors (IR) in human coronary endothelium. Our aim was to characterize IGF-IR and IR in human coronary artery endothelial cells (HCAEC). Design Cultured human coronary artery endothelial cells were used. Gene expression was measured by quantitative real-time RT-PCR analysis and receptor affinity by ligand binding. Receptor protein, phosphorylation of IGF-IR and IR β-subunit as well as the presence of hybrid insulin receptor/Insulin-like growth factor-I receptor (Hybrid IR/IGF-IR) was analyzed by immunoprecipitation and Western blot. Postreceptor effects of insulin and IGF-I were assed by 3H-thymidine incorporation. Results The gene expression of IGF-IR was several folds higher than that of IR. and insulin receptor isoform A (IR-A) was 20-fold more expressed than insulin receptor isoform B (IR-B) in HCAEC. The specific binding of 125I-IGF-I was higher than that of 125I-insulin. Insulin and the new long acting insulin analog, glargine, interacted with the IGF-IR with over thousand and 100-fold less potency than IGF-I itself, whereas IGF-II had 6 times lower potency than IGF-I. Phosphorylation of the IGF-IR β-subunit was obtained by concentrations of 10 −10–10 −8 M IGF-I, 10 −6 M of insulin, inconsistently by 10 −8 M insulin and not at all by 10 −10–10 −9 M insulin. The IR β-subunit was phosphorylated by insulin and IGF-I at concentrations of 10 −9–10 −8 M. When immunoprecipitating with specific monoclonal anti-IR or anti-IGF-IR α-subunit antibodies we found bands situated in slightly different positions suggesting the presence of Hybrid IR/IGF-IR. IGF-I, IGF-II and insulin (10 −9–10 −7 M) had no significant effect on 3H-thymidine incorporation into DNA. Conclusions Human coronary endothelial cells express more IGF-IR than IR, mainly IR-A, and also Hybrid IR/IGF-IR. Both IGF-I and insulin phosphorylate their receptors, but only IGF-I seems to phosphorylate Hybrid IR/IGF-IR. Our study provides experimental evidence for a possible role of IGF-IR, IR and Hybrid IR/IGF-IR in human coronary artery endothelial cells.