Diabetes is associated with microcirculatory dysfunction and heart failure and changes in insulin and IGF1 levels. Whether human cardiac microvascular endothelial cells (HMVEC-Cs) are sensitive to insulin and/or IGF1 is not known. We studied the role of insulin receptors (IRs) and IGF1 receptors (IGF1Rs) in metabolic, mitogenic and anti-inflammatory responses to insulin and IGF1 in HMVEC-Cs and human umbilical vein endothelial cells (HUVECs). IR and IGF1R gene expression was studied using real-time RT-PCR. Receptor protein expression and phosphorylation were determined by western blot and ELISA. Metabolic and mitogenic effects were measured as glucose accumulation and thymidine incorporation. An E-selectin ELISA was used to investigate inflammatory responses. According to gene expression and protein in HMVEC-Cs and HUVECs, IGF1R is more abundant than IR. Immunoprecipitation with anti-IGF1R antibody and immunoblotting with anti-IR antibody and vice versa, showed insulin/IGF1 hybrid receptors in HMVEC-Cs. IGF1 at a concentration of 10(-8) mol/l significantly stimulated phosphorylation of both IGF1R and IR in HMVEC-Cs. In HUVECs IGF1 10(-8) mol/l phosphorylated IGF1R. IGF1 stimulated DNA synthesis at 10(-8) mol/l and glucose accumulation at 10(-7) mol/l in HMVEC-Cs. TNF-alpha dramatically increased E-selectin expression, but no inflammatory or anti-inflammatory effects of insulin, IGF1 or high glucose were seen. We conclude that HMVEC-Cs express more IGF1Rs than IRs, and mainly react to IGF1 due to the predominance of IGF1Rs and insulin/IGF1 hybrid receptors. TNF-alpha has a pronounced pro-inflammatory effect in HMVEC-Cs, which is not counteracted by insulin or IGF1.

The immune system of the neonate is influenced by maternal immunity during pregnancy and lactation. An altered microbial exposure, possibly underlying the increase of allergic diseases in affluent societies, may affect maternal breast milk immune composition. Secretory IgA (SIgA), IL-4, IL-10, IL-13, IFN-γ, TGF-β1, and TGF-β2 were analyzed with ELISA in colostrum and 1-mo mature milk from mothers from Estonia (n = 39) and Sweden (n = 60), the two geographically adjacent countries with different living conditions and allergy incidence. The IL-10 and IFN-γ levels were higher in colostrum from Estonian than Swedish mothers, whereas the opposite was true for TGF-β2. In mature milk, higher SIgA and IFN-γ levels but lower TGF-β1 and TGF-β2 levels were observed in Estonian than Swedish mothers. Interestingly, in Sweden but not Estonia, the TGF-β1 and TGF-β2 levels correlated inversely with environmental endotoxin concentrations, whereas positive correlations to microbial load were observed for IL-4, IL-10, and IFN-γ. High colostral IL-13 levels were associated with allergic sensitization during infancy in Sweden. In conclusion, Estonian mothers have lower breast milk levels of TGF-β, particularly TGF-β2, but higher levels of SIgA, IL-10, and IFN-γ than Swedish mothers, possibly because of differences in microbial load.

Whether insulin, in physiological concentrations, has direct effects on vascular smooth muscle cells (VSMC) remains controversial. Our aim was to characterize the mechanism for insulin resistance in VSMCs. For comparison, effects of insulin-like growth factor (IGF)-I and IGF-II were also studied. Cultured human aortic smooth muscle cells (HASMC) were used. Receptor mRNA was analysed by quantitative RT-PCR and receptor protein by ELISA and Western Blot. The biological effects were studied by thymidine incorporation and glucose accumulation.

In HASMC both mRNA and protein expression of IGF-I receptors (IGF-IR) were 5 fold higher compared to insulin receptor (IR). IR isoform A mRNA was 13 times more expressed than IR isoform B. Immunoprecipitation and Western blot showed co precipitation of IR and IGF-IR indicating the presence of hybrid IR/IGF-IR.

Phosphorylation of the IGF-IR β-subunit was obtained by IGF-I 10^-9-10^-8mol l^-1 and IGF-II 10^-8mol l^-1. IR β-subunit was phosphorylated by IGF-I 10^-8mol l^-1 but not by insulin. IGF-I stimulated IRS-I at 10^-8mol l^-1, Akt and Erk 1/2 at 10^-9-10^-8mol l^-1, respectively. IGF-II stimulated Akt at 10^-8mol l^-1 whereas insulin had no effect. IGF-I and IGF-II at a concentration of 10^-8-10^-7mol l^-1 significantly stimulated ³H-thymidine incorporation, whereas insulin did not. 14C-Glucose accumulation was stimulated by IGF-I or IGF-II 10^-8-10^-7mol l^-1, and also by insulin 10^-7mol l^-1.

Our results suggest that IGF-IR and hybrid IR/IGF-IR are activated by physiological concentrations of IGF-I and IGF-II in HASMC and this causes downstream signaling and biological effects, while insulin has no effect on its receptor or downstream signaling probably due to a preponderance of IGF-IR and incorporation of IR into hybrid IR/IGF-IR.

Diabetic complications largely affect the circulation and are associated with resistance to insulin and altered levels of insulin-like growth factor-I (IGF-I). Insulin resistance and altered IGF-I levels are also associated with vascular disease. Insulin and IGF-I are highly homologous peptides and can cross react with each others respective receptors, insulin receptors (IR) and IGF-I receptors (IGFIR), which also share homology to a large extent and can form hybrid IR/IGF-IR. Cultured endothelial and vascular smooth muscle cells from different vascular beds express considerably more IGF-IR than IR. Since the direct action of insulin and IGFs on the vasculature remains poorly understood, our aim was to study mechanisms behind insulin resistance and IGF-I sensitivity and the possible impact of hybrid IR/IGF-IR in vascular cells.

This thesis is based on four papers investigating the presence of IR and IGF-IR in cultured endothelial and vascular smooth muscle cells, and in tissue specimens from human left internal mammary artery (LIMA). We examined, in cultured vascular smooth muscle cells and endothelial cells, the phosphorylation of IR and IGF-IR, and IR and IGF-IR mediated actions, i e subsequent downstream signalling and biological effects, in response to physiologic and supraphysiologic concentrations of insulin, IGF-I and IGF-II. We also examined the presence of insulin/IGF-I hybrid receptors in these cell types. To compare our results in vitro with the in vivo situation we investigated the relative gene expression of IGF-IR to IR in LIMA.

We conclude that: 1) the relative abundance of IGF-IR is considerably higher than IR in vascular cells in vitro and in vivo; 2) in addition to IR and IGF-IR, hybrid IR/IGF-IR are present in vascular cells; 3) IR activation at physiological concentrations (≤10-9M) does not propagate downstream signalling and biological effects in endothelial and vascular smooth muscle cells; 4) low concentrations of IGF-I activate IGF-IR, as well as IR due to the presence of hybrid IR/IGF-IR, and propagate downstream signalling and biological effects in endothelial and vascular smooth muscle cells; and 5) the biological effects mediated by IGF-II suggests a role for IGF-II in vascular smooth muscle cells.

The papers included in this thesis provide new insight on how IGFs and insulin act in the vasculature. The preponderance of IGF-IR relative to IR in addition to sequestration of IR into hybrid IR/IGF-IR contributes to an insulin resistance located at the receptor level in endothelial and vascular smooth muscle cells.

Hence, our results suggest that IGFs rather than insulin have an impact on vascular function.

Diabeteskomplikationer drabbar till stor del cirkulationen och är associerade med resistans för insulin samt ändrade nivåer av insulin-liknande tillväxtfaktor typ I (IGF-I). Insulinresistans och ändrade IGF-I nivåer är också associerade med hjärt-kärlsjukdom. Insulin och IGF-I är mycket lika och kan vid höga koncentrationer korsreagera med varandras respektive receptorer, insulinreceptorer(IR) och IGF-I-receptorer(IGF-IR), som också är mycket lika och kan bilda hybridreceptorer (hybrid IR/IGF-IR). Odlade endotelceller och glatta muskelceller från olika kärlbäddar uttrycker betydligt fler IGF-IR jämfört med IR. Eftersom direkt verkan av insulin och IGF-I i kärlvägg är dåligt utredd, var vårt mål att studera mekanismer bakom insulinresistans och IGF-I känslighet samt möjlig betydelse av hybrid IR/IGF-IR i kärlväggens celler.

Den här avhandlingen baseras på fyra arbeten som undersökt förekomst av IR och IGF-IR i odlade endotelceller och glatta muskelceller från kärl, samt i vävnad från human vänster inre bröstartär (LIMA). I odlade endotelceller och glatta muskelceller från kärl har vi undersökt fosforylering av IR och IGF-IR, samt IR -och IGF-IR medierade svar, dvs vidare signalering nedströms och biologiska effekter, till följd av stimulering med insulin, IGF-I och IGF-II vid fysiologiska och suprafysiologiska koncentrationer. Vi har också undersökt förekomst av hybrid IR/IGF-IR i dessa celltyper. För att relatera våra in vitro försök till situationen in vivo, undersökte vi även det relativa genuttrycket av IGF-IR jämfört med IR i LIMA.

Vi drar följande slutsatser: 1) den relativa förekomsten av IGF-IR är betydligt högre än IR i kärlceller in vitro och in vivo; 2) förutom IR och IGF-IR förekommer i celler från kärlvägg även hybrid IR/IGF-IR; 3) IR aktiverad vid fysiologiska koncentrationer (≤10-9 M) propagerar inte vidare intracellulär signalering eller biologiska effekter, i endotelceller och glatta muskelceller från kärl; 4) låga koncentrationer av IGF-I aktiverar IGF-IR, och även IR tack vare förekomsten av hybrid IR/IGF-IR, samt propagerar vidare intracellulär signalering och biologiska effekter i endotelceller och glatta muskelceller från kärl; och 5) de biologiska effekterna till följd av IGF-II-stimulering föreslår en roll för IGF-II i glatta muskelceller från kärl.

Arbetena inkluderade i den här avhandlingen bidrar med nya insikter i hur IGFs och insulin verkar i kärlvägg. Det övervägande antalet IGF-IR relativt IR samt inkorporeringen av IR in i hybrid IR/IGFIR orsakar en insulinresistens lokaliserad på receptornivå i endotelceller och glatta muskelceller från kärl. Sammantaget talar våra resultat för att IGFs snarare än insulin är av vikt för kärlväggens funktion.

Human microvascular endothelial cells (HMVEC) are sensitive to IGF-I but insulin resistant and express several times more IGF-I receptors (IGF-IR) than insulin receptors (IR). Our aim was to investigate the mechanism of this insulin resistance in cultured HMVEC by studying receptor activation and signal propagation downstream.

propagation downstream. The IGF-IR β-subunit and the IR β-subunitwere detected and found to co-precipitate. IRAwas themajor IR isoformexpressed in HMVEC. IGF-I 10⁻⁹ to 10⁻⁸M phosphorylated its cognate receptor β-subunit. IGF- I also phosphorylated the IR β-subunit at 10⁻⁹ M. Phosphorylation of insulin receptor substrate 1 was obtained by IGF-I 10−9 to 10⁻⁸ M. Akt was phosphorylated by IGF-I at 10⁻⁸ to 10⁻⁷M and by insulin 10−7M. IGF-I at 10⁻⁸ to 10⁻⁶M significantly increased DNA-synthesis. We conclude that microvascular endothelial cells are sensitive to IGF-I but resistant to insulin due to a preponderance of IGF-I receptors and sequestration of insulin receptors into insulin/IGF-I hybrid receptors.

Insulin and insulin-like growth factor I (IGF-I) are known to affect cardiovascular disease. We have investigated ligand binding and the dose-response relationship for insulin and IGF-I on vascular smooth muscle cells (VSMCs) at the receptor level. VSMCs from rat thoracic aorta were serum starved, stimulated with IGF-I or insulin, lysed, immunoprecipitated, and analyzed by Western blot. D-[U-¹⁴C]Glucose accumulation and [6-³H]thymidine incorporation into DNA were also measured. Specific binding of both insulin and IGF-I was demonstrated, being higher for IGF-I. Both IGF-I receptor (IGF-IR) and insulin receptor (IR) β-subunits were detected and coprecipitated after immunoprecipitation (IP) against either of the two. No coprecipitation was found after reduction of disulphide bonds with dithiotreitol before IP. After stimulation with 10^–10–10^–9 M IGF-I, IP of the IGF-IR, or IR β-subunit and immunoblot with anti-phosphotyrosine antibody, we found two distinct bands indicating phosphorylation of both the IGF-IR and the IR β-subunit. Stimulation with 10^–10–10^–9 M insulin and IP against the IGF-IR did not show phosphorylation of either β-subunit, whereas after IP of the IR we found phosphorylation of the IR β-subunit. [¹⁴C]Glucose accumulation and [³H]thymidine incorporation were elevated in cells stimulated with IGF-I at 10^–10–10^–7 M, reaching maximum by 10^–9 M. Insulin stimulation showed measurable effects only at supraphysiological concentrations, 10^–8–10^–7 M. In conclusion, coprecipitation of both the IGF-IR and the IR β-subunit indicates the presence of hybrid insulin/IGF-I receptors in VSMC. At a physiological concentration, insulin activates the IR but does not affect either glucose metabolism or DNA synthesis, whereas IGF-I both activates the receptor and elicits biological effect.

Aims/hypothesis: Cultured endothelial and vascular smooth muscle cells from different vascular beds express several fold more IGF-I receptors (IGF-IR) than insulin receptors (IR). The aim of this study was to investigate the gene expression of IR and IGF-IR in tissue samples from the left internal mammary artery (LIMA) to see if there is a higher gene expression of IGF-IR compared to IR in arterial tissue in vivo.

Methods: Samples from LIMA (n=16) were obtained at coronary bypass surgery, immediately frozen in liquid nitrogen and stored at -70°C until analysis. The samples were thawed, placed on ice and dissected free from surrounding connective tissue and fat, homogenized and total RNA was extracted. Receptor mRNA was analyzed by quantitative real time RT-PCR and comparison of C_T-values.

Results: LIMA samples were obtained from 16 patients, 10 non-diabetic and 6 diabetic patients. Gene expression of IGF-IR was detected in all LIMA samples, however, the insulin receptor mRNA was under the detection limit in 4 samples. Compared to IR the gene expression of IGF-IR was 36 fold higher (n=12. p <0.0001). The relative gene expression of IGF-IR to IR in LIMA from non-diabetic patients (n=9) and diabetic patients (n=3) was similar, 39 and 32 fold, respectively.

Conclusions/interpretation: Our results in mammary artery samples indicate that IGF-IR are more expressed than IR in arterial tissue in vivo.

Diabetes is associated with coronary microcirculatory dysfunction and heart failure as well as changes in insulin and IGF-I levels. Our aim was to study the role of insulin receptors and IGF-I receptors in metabolic, mitogenic and anti-inflammatory responses to insulin and IGF-I in human cardiac microvascular endothelial cells (HMVEC-C) and, for comparison, also human umbilical vein endothelial cells (HUVEC). Insulin receptor (IR) and IGF-I receptor (IGF-IR) gene expression was studied with real-time RT-PCR. Receptor protein expression and phosphorylation was determined with Western blot and ELISA. The metabolic and mitogenic effects were measured as glucose accumulation and thymidine incorporation. An E-selectin ELISA was used to investigate the anti-inflammatory responses. IGF-IR was more abundant than IR both regarding gene expression and protein in HMVEC-C and HUVEC. Immunoprecipitation with anti-IGF-IR antibody and immunoblotting with anti-IR antibody and vice versa, showed insulin/IGF-I hybrid receptors in these cells. IGF-I 10^-8 M significantly stimulated phosphorylation of both IGF-IR and IR in HMVEC-C. In HUVEC IGF-I 10^-8 M phosphorylated IGF-IR. IGF-I also stimulated DNA synthesis at 10^-8 M and glucose accumulation at 10^-7 M. TNF-α significantly increased E-selectin expression whereas no effects were found by insulin, IGF-I or high glucose.

We conclude that HMVEC-C express more IGF-I receptors than insulin receptors and at physiological concentrations of insulin and IGF-I mainly reacts to IGF-I probably due to the predominance of IGF-I receptors and insulin/IGF-I hybrid receptors. TNF-α has a pronounced pro-inflammatory effect in HMVEC-C which is not counteracted by insulin or IGF-I.