IGF-I receptors, insulin receptors and insulin/IGF-I hybrid receptors in human endothelial cells : with special reference to diabetes

Sammanfattning: Patients with diabetes mellitus are known to develop vascular complications, which occur as macroangiopathy, atherosclerosis and mediasclerosis, as well as microangiopathy, e.g. retinopathy and nephropathy. The precise mechanisms causing these complications have not yet been elucidated. The microvascular complications are closely associated with the glycaemic control, which also is a risk factor for the diabetic macroangiopathy. The possible roles of insulin and the related peptide IGF-I, whose levels are affected by diabetes mellitus, are not clear. This study aims to characterise the presence and function of insulin and IGF-I receptors in human endothelial cells.Two types of human endothelial cells were studied; human umbilical vein endothelial cells (HUVEC) and human coronary artery endothelial cells (HCAEC). The presence of insulin receptors and IGF-I receptors was studied at mRNA level by real-time PCR and at protein level by ligand binding and by Western blot analysis after immunoprecipitation. Receptor activation was determined as tyrosine phosphorylation.Both HUVEC and HCAEC were found to express IGF-I receptors and insulin receptors at mRNA and protein levels. The amount of IGF-I receptor mRNA exceeded insulin receptor mRNA by 3.5 and 14-fold in HUVEC and HCAEC, respectively. In HUVEC, the higher expression of IGF-I receptor mRNA compared to insulin receptor mRNA was present in both freshly isolated and cultured cells. Ligand binding studies showed a higher specific binding of 125I-IGF-I than of 125I-insulin which also suggest the presence of more IGF-I receptors than insulin receptors. In HUVEC, the specific binding was 0.64 ± 0.25% (mean ± SEM) for 125I-IGF-I and 0.25 ± 0.092% for 125I-insulin. The EC50 for 125I-IGF-I displacement was 3.6 x 10-10 M for IGF-I vs. 8.25 x 10-8 M for insulin. The EC50 for 125I-insulin displacement was 2.6 x 10-10 M for insulin and 7.39 x 10-9 M for IGF-I. In HCAEC, the specific binding was 1.37 ± 0.09% and for insulin 0.17 ± 0.03%. The EC50 value for IGF-I displacement were 6.9 X 10-10 M for IGF-I, 8.7 X 10-6 M for insulin and 7.5 X 10-8 M for the insulin analogue glargine. Due to the very low specific binding of 125I-insulin, it was not possible to calculate the concentration needed to give half-maximal displacement, EC50, of 125I-labelled insulin. Both cell types expressed insulin/IGF-I hybrid receptors. Receptor phosphorylation studies showed that IGF-I receptor could be activated by 10-10 to 10-8 M IGF-I in both cell types. Insulin receptors were activated by 10-9 to 10-8 M insulin in HUVEC and HCAEC. IGF-I was able to activate insulin receptor phosphorylation at low concentrations, 10-9 to 10-8 M, which also is an indication of the presence of hybrid receptors.In conclusion, two types of human endothelial cells, HUVEC and HCAEC, express more IGF-I receptors than insulin receptors, and they also express insulin/IGF-I hybrid receptors. IGF-I and insulin were found to phosphorylate their own receptor while IGF-I also seemed to be able to phosphorylate hybrid receptors. The results suggest an important role of the IGF-I receptor in human endothelial cells.