ABSTRACT
Whether insulin, at physiological concentrations, has direct effects on vascular smooth muscle cells (VSMCs) remains controversial. Our aim was to characterize the mechanism for insulin resistance in VSMCs. For comparison, the effects of IGF1 and IGF2 were also studied. Cultured human aortic smooth muscle cells (HASMC) were used. Receptor mRNA was analyzed by quantitative reverse transcription PCR and receptor protein by ELISA and western blot. Biological effects were studied by thymidine incorporation and glucose accumulation. In HASMC, both mRNA and protein expression of IGF1 receptors (IGF1R) were fivefold higher compared to insulin receptor (IR). IR isoform A mRNA was 13-fold more expressed than IR isoform B. IR and IGF1R co-precipitated, indicating the presence of hybrid IR/IGF1R. Phosphorylation of the IGF1R beta-subunit was obtained by IGF1 10(-9)-10(-8) mol/l and IGF2 10(-8) mol/l. IR beta-subunit was phosphorylated by IGF1 10(-8) mol/l but not by insulin. IGF1 stimulated IR substrate-1 and AKT at 10(-8) mol/l and extracellular signal-regulated kinases 1 and 2 at 10(-9)-10(-8) mol/l respectively. IGF1 and 2 at a concentration of 10(-8)-10(-7) mol/l significantly stimulated (3)H-thymidine incorporation, whereas insulin did not. (14)C-Glucose accumulation was stimulated by IGF1 or IGF2 10(-8)-10(-7) mol/l, and also by insulin 10(-7) mol/l. Our results suggest that IGF1R and hybrid IR/IGF1R are activated by physiological concentrations of IGF1 and 2 in HASMC and this propagates downstream signaling and biological effects, while insulin has no effect on its receptor or downstream signaling probably due to a preponderance of IGF1R and incorporation of IR into hybrid IR/IGF1R.
Subject(s)
Aorta/cytology , Insulin-Like Growth Factor II/pharmacology , Insulin-Like Growth Factor I/pharmacology , Insulin/pharmacology , Muscle, Smooth, Vascular/cytology , Myocytes, Smooth Muscle/drug effects , Myocytes, Smooth Muscle/metabolism , Blotting, Western , Cells, Cultured , Electrophoresis, Polyacrylamide Gel , Enzyme-Linked Immunosorbent Assay , Humans , Models, Genetic , Phosphorylation/drug effects , Protein Isoforms/genetics , Protein Isoforms/metabolism , Receptor, IGF Type 1/genetics , Receptor, IGF Type 1/metabolism , Receptor, Insulin/genetics , Receptor, Insulin/metabolism , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
The aim of the study was to investigate if the insulin analogue glargine, with an increased affinity for the IGF-I receptor (IGF-IR), affects the cell growth to a larger extent than human insulin in malignant cells expressing IGF-IRs. The breast cancer cell lines MCF-7 and SKBR-3, and the osteosarcoma cell line SaOS-2 were used. Gene expression was determined by real-time RT-PCR and receptor protein quantified by ELISAs. Receptor phosphorylation was assessed by immunoprecipitation and Western blot. Mitogenic effect was determined as (3)H-thymidine incorporation into DNA. The gene expression of insulin receptor (IR) varied between 4.3-7.5 x 10(-3) and the expression of IGF-IR between 7.7-147.7 x 10(-3) in relation to GAPDH (glyceraldehyde-3-phosphate dehydrogenase). Insulin receptor and IGF-IR protein varied between 2.0-4.1 ng/mg protein and 2.0-40.4 ng/mg protein, respectively. The IGF-IR was phosphorylated by IGF-I at a concentration of 10(-10)-10(-9) M. All three polypeptides stimulated DNA synthesis in MCF-7, SKBR-3, and SaOS-2 cells. SaOS-2 cells were more sensitive to IGF-I than to insulin and glargine. MCF-7 cells were more sensitive to des(1-3)IGF-I than to IGF-I. In SKBR-3 and SaOS-2 cells, glargine tended to be more potent than human insulin to stimulate DNA synthesis. Our results suggest that glargine, compared to human insulin, has little or no increased mitogenic effect in malignant cells expressing IGF-IRs.
