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IGF-I receptors, insulin receptors and insulin/IGF-I hybrid receptors in human endothelial cells: with special reference to diabetes
Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
2005 (English)Licentiate thesis, comprehensive summary (Other academic)
Abstract [en]

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.

Place, publisher, year, edition, pages
Linköping: Linköpings universitet , 2005. , 39 p.
Series
Linköping Studies in Health Sciences. Thesis, ISSN 1100-6013 ; 70
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-31179Local ID: 16919ISBN: 91-7393-857-3 OAI: oai:DiVA.org:liu-31179DiVA: diva2:252002
Available from: 2009-10-09 Created: 2009-10-09 Last updated: 2013-09-10
List of papers
1. IGF-I/insulin hybrid receptors in human endothelial cells
Open this publication in new window or tab >>IGF-I/insulin hybrid receptors in human endothelial cells
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2005 (English)In: Molecular and Cellular Endocrinology, ISSN 0303-7207, E-ISSN 1872-8057, Vol. 229, no 1-2, 31-37 p.Article in journal (Refereed) Published
Abstract [en]

Vascular complications are common in diabetes. IGF-I receptors (IGF-IR) and insulin receptors (IR) in endothelial cells might respond to altered levels of IGF-I and insulin, resulting in altered endothelial function in diabetes. We therefore studied IGF-IR and IR gene expression, ligand binding, receptor protein, and phosphorylation in human umbilical vein endothelial cells (HUVEC). IGF-IR mRNA was more abundant than IRmRNAin freshly isolatedHUVEC(IGF-IR/IR ratio 7.1±1.5) and in culturedHUVEC(ratio 3.5±0.51). Accordingly, specific binding of 125I-IGF-I (0.64±0.25%) was higher than that of 125I-insulin (0.25±0.09%). Protein was detected for both  eceptors and IGF-I/insulin hybrid receptors. IGF-IR phosphorylation was stimulated by 10−10 to 10−8M IGF-I. IR were activated by 10−9 to 10−8M insulin and IGF-I. We conclude that HUVEC express more IGF-IR than IR, and also express hybrid receptors. Both IGF-I and insulin phosphorylate their own receptors but only IGF-I seems to phosphorylate hybrid receptors.

Keyword
IGF-I receptor; Insulin receptor; Insulin/IGF-I hybrid receptor; Endothelial cells; Human
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-12553 (URN)10.1016/j.mce.2004.10.003 (DOI)000226596400005 ()
Available from: 2008-09-15 Created: 2008-09-15 Last updated: 2013-09-10
2. Characterisation of receptors for IGF-I and insulin: evidence for hybrid insulin/IGF-I receptor in human coronary artery endothelial cells
Open this publication in new window or tab >>Characterisation of receptors for IGF-I and insulin: evidence for hybrid insulin/IGF-I receptor in human coronary artery endothelial cells
2006 (English)In: Growth Hormone & IGF Research, ISSN 1096-6374, Vol. 16, no 4, 258-266 p.Article in journal (Refereed) Published
Abstract [en]

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 RTPCR analysis and receptor affinity by ligand binding. Receptor protein, phosphorylation of IGF-IR and IR b-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 125Iinsulin. 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 b-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 b-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 a-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.

Keyword
Human endothelial cells; Insulin-like growth factor-I; Insulin
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-12555 (URN)10.1016/j.ghir.2006.06.003 (DOI)000240947800006 ()
Available from: 2008-09-15 Created: 2008-09-15 Last updated: 2013-09-10

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