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


METHODS
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 beta-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 (3)H-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 (125)I-IGF-I was higher than that of (125)I-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 beta-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 beta-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 alpha-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 (3)H-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.
Chisalita
Simona I
SI
Diabetes Research Centre and Division of Cell Biology, Department of Biomedicine and Surgery, Faculty of Health Sciences, Linköping University, S-581 85 Linköping, Sweden.
Nitert
Marloes Dekker
MD
Arnqvist
Hans J
HJ
D015153:Blotting, Western; D002478:Cells, Cultured; D003331:Coronary Vessels / Q000166:cytology / Q000378:metabolism*; D004247:DNA / Q000096:biosynthesis; D042783:Endothelial Cells / Q000378:metabolism*; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods; D007328:Insulin / Q000378:metabolism; D007334:Insulin-Like Growth Factor I / Q000378:metabolism*; D050506:Mutant Chimeric Proteins / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D011869:Radioligand Assay / Q000379:methods; D017526:Receptor, IGF Type 1 / Q000378:metabolism*; D011972:Receptor, Insulin / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D013936:Thymidine / Q000032:analysis
D007328:Insulin; D050506:Mutant Chimeric Proteins; D007334:Insulin-Like Growth Factor I; D004247:DNA; D017526:Receptor, IGF Type 1; D011972:Receptor, Insulin; D013936:Thymidine