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Expression and function of IGF-I and insulin receptors in human micro- and macrovascular cells
Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Local Health Care Services in Central Östergötland, Department of Acute Internal Medicine.
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Insulin-like growth factor and insulin are phylogenetically closely related polypeptides and have large structural and biological similarities. Low circulating insulin-like growth factor-I (IGF-I), diabetes as well as insulin resistance have been implicated in the pathogenesis of cardiovascular disease, but the mechanisms involved are still not clear. Furthermore, little is known about direct effects of insulin-like growth factor-I (IGF-I) and insulin on human micro- and macrovascular cells.

In these studies we investigated the expression and function of insulin-like growth factor-I receptors (IGF-IR) and insulin receptors (IR) in human micro- and macrovascular endothelial cells and in human coronary artery smooth muscle cells.

Our results showed expression of both IGF-IR and IR in human dermal microvascular (HMVEC), aortic (HAEC) umbilical vein (HUVEC) and coronary artery (HCAEC) endothelial cells as well as in human coronary artery smooth muscle cells (HASMC). The gene expression of IGF-IR was several times more abundant than that of IR. Ligand binding studies confirmed that the IGF-IR was severalfold more abundant than the IR. It also demonstrated that insulin and glargine interacted with the IGF-IR with thousand- and hundredfold, respectively, less potency than IGF-I itself. The presence of IGF-IR and IR proteins and activation of their β-subunits was revealed by immunoprecipitation and Western blot analysis in human macrovascular endothelial cells and in coronary artery smooth muscle cells. At physiological concentrations (≤10-9 M) IGF-I and insulin activated their cognate receptors. The presence of hybrid IR/IGF-IR was shown through detection of the β-subunit for IGF-IR and IR on the same membrane by Western blot after immunoprecipitation with specific antibodies against either IGF-IR or IR, implying coprecipitation of the IGF-IR β-subunit and the IR β-subunit. The inability of physiological concentrations of insulin to phosphorylate IR β-subunit immunoprecipitated with IGF-IR antibodies and that IGF-I at physiological concentration activates the IR β-subunit is another evidence for the presence of the hybrid IR/IGF-IR. At physiological concentrations (≤10-9 M) IGF-I stimulated DNA synthesis and glucose incorporation into human coronary artery smooth muscle cells (HCASMC) and DNA synthesis in microvascular endothelial cells (HMVEC), but not in human macrovascular endothelial cells (HCAEC or HUVEC). No effect of insulin was found. Although physiological concentrations of insulin (≤10-9 M) were able to activate IR, insulin had no biological effects on the vascular cells studied. A possible explanation is that the insulin receptor signalling is too attenuated due to the presence of hybrid IR/IGF-IR and low number of IR expressed in the cells studied. Regarding the safety in the use of glargine, we show that glargine has 10-fold higher affinity for IGF-IR than human insulin. However, the glargine concentrations obtained in vivo during diabetes treatment is too low to affect the IGF-IR.

In conclusions our studies provide experimental evidence that human micro- and macrovascular endothelial and vascular smooth muscle cells express both IGF-IR and IR. Our in vitro data suggest that the cells studied are sensitive to IGF-I, but insensitive to insulin and this is due to the preponderance of IGF-IR and presence of hybrid IR/IGF-IR.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press , 2008. , 89 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1045
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-12558ISBN: 978-91-7393-972-0 (print)OAI: oai:DiVA.org:liu-12558DiVA: diva2:1701
Public defence
2008-03-14, Berzeliussalen (hus 463, ingång 65), Hälsouniversitet, Campus US, Linköpings universitet, Linköping, 09:00 (Swedish)
Opponent
Supervisors
Available from: 2008-09-15 Created: 2008-09-15 Last updated: 2009-08-21Bibliographically approved
List of papers
1. Insulin-like growth factor I receptors are more abundant than insulin receptors in human micro- and macrovascular endothelial cells
Open this publication in new window or tab >>Insulin-like growth factor I receptors are more abundant than insulin receptors in human micro- and macrovascular endothelial cells
2004 (English)In: American Journal of Physiology. Endocrinology and Metabolism, ISSN 0193-1849, E-ISSN 1522-1555, Vol. 286, E896-E901 p.Article in journal (Refereed) Published
Abstract [en]

Micro- and macroangiopathy are major causes of morbidity and mortality in patients with diabetes. Our aim was to characterize IGF-I receptor (IGF-IR) and insulin receptor (IR) in human micro- and macrovascular endothelial cells. Cultured human dermal microvascular endothelial cells (HMVEC) and human aortic endothelial cells (HAEC) were used. Gene expression was measured by quantitative real-time RT-PCR and receptor protein by ligand-binding assay. Phosphorylation of IGF-IR ß-subunit was analyzed by immunoprecipitation and Western blot. Glucose metabolism and DNA synthesis was assessed using [3H]glucose and [3H]thymidine incorporation, respectively. We detected gene expression of IGF-IR and IR in HAEC and HMVEC. IGF-IR gene expression was severalfold higher than that of IR. The specific binding of 125I-IGF-I was higher than that of 125I-insulin in HAEC and HMVEC. Insulin and the new, long-acting insulin analog glargine interacted with the IGF-IR with thousand- and hundred-fold less potency than IGF-I itself. Phosphorylation of the IGF-IR ß-subunit was shown in HAEC for IGF-I (10-8 M) and insulin (10-6 M) and in HMVEC for IGF-I and glargine (10-8 M, 10-6 M). IGF-I 10-7 M stimulated incorporation of [3H]thymidine into DNA, and 10-9–10-7 M also the incorporation of [3H]glucose in HMVEC, whereas glargine and insulin had no significant effects at 10-9–10-7 M. Human micro- and macrovascular endothelial cells express more IGF-IR than IR. IGF-I and high concentrations of glargine and insulin  ctivates the IGF-IR. Glargine has a higher affinity than insulin for the IGF-IR but probably has no effect on DNA synthesis at concentrations reached in vivo.

Keyword
Human endothelial cells, receptor, insulin, glargine
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-12552 (URN)10.1152/ajpendo.00327.2003 (DOI)
Available from: 2008-09-15 Created: 2008-09-15 Last updated: 2017-12-14
2. 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
Show others...
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: 2017-12-14
3. 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, E-ISSN 1532-2238, 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: 2017-12-14
4. Expression and function of receptors for insulin-like growth factor-I and insulin in human coronary artery smooth muscle cells
Open this publication in new window or tab >>Expression and function of receptors for insulin-like growth factor-I and insulin in human coronary artery smooth muscle cells
2005 (English)In: Diabetologia, ISSN 0012-186X, E-ISSN 1432-0428, Vol. 48, no 10, 2155-2161 p.Article in journal (Refereed) Published
Abstract [en]

Aims/hypothesis: Hyperinsulinaemia and insulin resistance, as well as low IGF-I, have been implicated in the pathogenesis of cardiovascular disease. Little is known about direct effects of IGF-I and insulin on human coronary artery smooth muscle cells (HCASMCs). Our aim was to characterise the expression and function of IGF-I receptor (IGF-IR) and insulin receptor (IR) in HCASMCs. Materials and methods: Cultured HCASMCs were used. mRNA expression was measured by quantitative real-time RT-PCR analysis. Receptor proteins, phosphorylation of β-subunits and the presence of hybrid IR/IGF-IR were analysed by immunoprecipitation and western blotting. DNA synthesis and glucose metabolism were assessed using [3H]thymidine incorporation and D-[U-14C]glucose accumulation respectively. Results: The mRNA expression of IGF-IR was approximately eight-fold higher than that of IR in HCASMCs. The presence of IGF-IR and IR could be demonstrated by immunoprecipitation and western blot analysis. Phosphorylation of the IGF-IR β-subunit was obtained by IGF-I at 10−10–10−8 mol/l and insulin at 10−8 mol/l. Insulin and IGF-I at 10−10–10−9 mol/l phosphorylated the IR β-subunit. When immunoprecipitated with monoclonal anti-IR α-subunit or IGF-IR α-subunit antibodies, we found bands in slightly different positions, suggesting the presence of hybrid IR/IGF-IR. IGF-I at 10−9–10−8 mol/l significantly stimulated [3H]thymidine incorporation and at a concentration of 10−9–10−7 mol/l also D-[U-14C]glucose accumulation in HCASMCs. Insulin at 10−9–10−7 mol/l had no effect on DNA synthesis, but increased glucose accumulation at 10−7 mol/l. Conclusions/interpretation: Our study provides experimental evidence that IGF-IR and possibly hybrid IR/IGF-IR play a role in HCASMCs.

Keyword
Human coronary artery smooth muscle cells, Hybrid insulin receptor/IGF-I receptor, IGF-I receptor, Insulin receptor
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-12556 (URN)10.1007/s00125-005-1890-4 (DOI)
Available from: 2008-09-15 Created: 2008-09-15 Last updated: 2017-12-14

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Chisalita, Simona I.

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