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Am J Physiol Endocrinol Metab 291: E1124-E1130, 2006. First published June 27, 2006; doi:10.1152/ajpendo.00565.2005
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Insulin and IGF-I action on insulin receptors, IGF-I receptors, and hybrid insulin/IGF-I receptors in vascular smooth muscle cells

Git S. Johansson1,2 and Hans J. Arnqvist1,2

1Department of Biomedicine and Surgery, Division of Cell Biology; and 2Diabetes Research Centre, Faculty of Health Sciences, Linköping University, Linköping, Sweden

Submitted 18 November 2005 ; accepted in final form 20 June 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
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) beta-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 beta-subunit and immunoblot with anti-phosphotyrosine antibody, we found two distinct bands indicating phosphorylation of both the IGF-IR and the IR beta-subunit. Stimulation with 10–10–10–9 M insulin and IP against the IGF-IR did not show phosphorylation of either beta-subunit, whereas after IP of the IR we found phosphorylation of the IR beta-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 beta-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.

insulin-like growth factor I; ligand binding; receptor phosphorylation; immunopreciptation; dexoyribonucleic acid synthesis; glucose metabolism

INDIVIDUALS WITH DIABETES have a two- to fourfold increased risk of developing cardiovascular disease (23). Insulin resistance and hyperinsulinemia without overt diabetes are also risk factors for atherosclerosis (18, 30). The development of atherosclerotic lesions includes migration and proliferation of smooth muscle cells (29), where the proliferation has been shown to be further accelerated in diabetes (35). Vascular smooth muscle cells (VSMCs) are also involved in restenosis after balloon angioplasty (24). IGF-I is a chemoattractant for VSMC and also stimulates proliferation (5, 7). Low IGF-I concentrations have been associated with the pathogenesis of atherosclerotic disease, such as ischemic heart diseases (22), myocardial infarction (10), ischemic stroke (21), and angina pectoris (11). Low-tissue IGF-I levels and reduced IGF-I receptor (IGF-IR) expression have been found in atherosclerotic plaques (5). Hence, there is evidence (36) that both high and low levels of IGF-I can be harmful to the vasculature, high levels by acting directly on the smooth muscle cells and low levels possibly by a decreased stimulation of nitric oxide production in the endothelial cells.

Insulin and IGF-I are structurally alike (32) and initiate their biological effects by binding to their respective cell surface receptors, i.e., insulin receptors (IRs) and IGF-IRs (7). The receptors share structural and functional homology to a large extent and are activated by their own cognate ligands, but at high concentrations the ligands can also cross-react with each other's receptors (20). The receptors are tyrosine kinases composed of two {alpha}beta-heterodimers, where each {alpha}beta-heterodimer is made up of an {alpha}-subunit and a beta-subunit. Hybrid receptors, consisting of an insulin receptor {alpha}beta-dimer and an IGF-IR {alpha}beta-dimer, are found in tissues where cells coexpress IRs and IGF-IRs (3). Earlier studies have demonstrated both IRs and IGF-IRs in VSMCs, with IGF-IRs being more abundant than IRs (1). We have recently published evidence (9) for the existence of hybrid IGF-I/insulin receptors in human VSMCs. Effects of IGF-I on VSMCs has been well documented (1, 12). Whether insulin in physiological concentrations has effects on vascular smooth muscle is controversial. Due to the cross-reactivity of insulin with the IGF-IRs at high concentrations, effects found when stimulating with high insulin concentrations may actually be due to activation of the IGF-IRs (4, 6, 31).

Because hyperinsulinemia, insulin resistance, and IGF-I are known risk factors for vascular disease and insulin and IGF-I levels are altered in diabetes mellitus (13), it is of great interest to study their direct action on the vascular wall. In the present study, the presence of IGF-I receptors, insulin receptors, and hybrid insulin/IGF-I receptors (HRs) and the ability of insulin and IGF-I to activate their receptors was studied in VSMCs cultured from rat thoracic aorta.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Isolation and culture of VSMCs from rat thoracic aorta. Rat VSMCs were isolated from thoracic aorta of 5- to 6-wk-old male Sprague-Dawley rats according to a modified method of Nilsson and Thyberg (27). Small pieces of the aorta were digested in 0.1% (wt/vol) collagenase (C0130; Sigma-Aldrich, Stockholm, Sweden) diluted in GIBCO's F-12 (Ham) medium (Invitrogen, Göteborg, Sweden) for 1 h at 37°C and then for another 18–20 h in new F-12 with collagenase at 37°C. The cell suspension was filtered through a 22-µm filter and washed in F-12 medium and then transferred to new F-12 medium supplemented with 50 µg/ml ascorbic acid (Sigma-Aldrich), 10 µg/ml gentamicin, 2 µg/ml fungizone, and 10% newborn calf serum (Invitrogen). The cells were cultured in 75-cm2 culturing flasks (Invitrogen) and kept at 37°C in a humidified atmosphere of 5% CO2 in air. The medium was changed 1–2 times/wk, and the cells were passaged using trypsin-EDTA (0.05% trypsin and 0.53 mM EDTA·4 Na) (Invitrogen). The cells used in the experiments were taken from passages 4–15. All experiments were performed on near-confluent cultures that had been made quiescent by incubation for 18–24 h in serum-free F-12 medium otherwise supplemented as described above. The cells were morphologically identified as smooth muscle cells.

Binding of IGF-I and insulin. Confluent rVSMCs grown in six-well plates were incubated for 2 h at room temperature (RT) in HEPES binding buffer (pH 7.8) containing 100 mM HEPES, 120 mM NaCl, 5 mM KCl, 1.2 mM MgSO4, 8 mM glucose, and 1% (wt/vol) BSA with 125I-IGF-I or 125I-insulin and unlabeled peptides at indicated concentrations. The cells were then washed four times with ice-cold phosphate buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, and 1.5 mM KH2PO4, pH 7.4) and lysed with 1.5 ml 0.1% (wt/vol) SDS for 10 min at RT. One milliliter of the cell solution was measured in a {gamma}-counter.

Activation of insulin and IGF-I receptors. After the cells were starved for 18–20 h in serum-free F-12 medium, the insulin and IGF-I receptor tyrosine kinases were activated by stimulation with insulin (Sigma-Aldrich) or IGF-I (Gropep, Adelaide, Australia). First, the cells were washed in warm serum-free F-12 medium with 0.1% (wt/vol) BSA (Sigma-Aldrich) and then preincubated with 50 µM sodium vanadate in F-12-BSA medium on ice for 30 min. Next, the cells were stimulated with IGF-I or insulin in warm F-12-BSA medium at indicated concentrations ranging from 10–10 to 10–8 M for 10 min at 37°C. After stimulation the medium was discarded and the cells were solubilized with ice-cold cell lysis buffer [20 mM Tris, 150 mM NaCl, 5 mM EDTA, 0.5% (wt/vol) sodium deoxycholate, and 0.5% (vol/vol) Triton X-100, pH 7.5] supplemented with protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 1.5 µg/ml leupeptin, 1.5 µg/ml aprotinin) and a phosphotyrosine phosphatase inhibitor (1 mM sodium vanadate), all chemicals from Sigma-Aldrich, for 30 min on ice. The lysed cells were scraped and further disrupted by passage through a syringe needle. The lysates were then centrifuged at 15,000 g for 15 min at 4°C, and the supernatants were stored at –70°C until further analysis. The protein content of the lysates was determined using the bicinchoninic acid protein method (Pierce, Rockford, IL).

Immunoprecipitation of insulin and IGF-I receptor proteins. Prior to SDS-PAGE and Western blot studies of the insulin and IGF-I receptor proteins, the cell lysates were immunoprecipitated using antibodies directed against either receptor beta-subunit together with protein A-Sepharose (Pharmacia-Upjohn, Uppsala, Sweden). Primary antibodies used for immunoprecipitation (IP) included polyclonal rabbit anti-IR beta-subunit (C-19) and polyclonal rabbit anti-IGF-IR beta-subunit (C-20) from Santa Cruz Biotechnology (Santa Cruz, CA). The lysate was incubated with ~0.4–0.6 µg antibody/mg of total protein content for 2 h on a rocker at 4°C. A 50% slurry of protein A-Sepharose beads in ice-cold lysis buffer with 0.1% (wt/vol) BSA was prepared, of which 50 µl/mg of total protein content in the lysate was added and incubated with the samples on a rocker overnight at 4°C to capture the immunocomplexes. The next day, the immunoprecipitates were collected by a 5-min centrifugation at 4°C, whereupon the supernatant fractions were removed and stored at –70°C. The remaining pellets of Sepharose-bound proteins were washed five times by repeated centrifugation and aspiration with ice-cold lysis buffer without phosphatase inhibitors. They were then respuspended in 50 µl of 2x Laemmli sample buffer [0.0125 M Trizma-Base (pH 6.8), 2% (wt/vol) SDS, 20% (vol/vol) glycerol, 0.002% (wt/vol) bromophenol blue, and 2% (vol/vol) beta-mercaptoethanol]. The samples were boiled for 3 min to dissociate the immunocomplexes from the beads and enable the beta-mercapoethanol to reduce the disulphide bridges holding the receptor subunits together. The immunoprecipitates were then centrifuged and stored at –20°C until further analysis with SDS-PAGE and Western blot. Because earlier studies (28) have shown that receptors are in such high excess that it is possible to reuse the supernatants from immunoprecipitated lysates and perform the same IP assay several times without noticing any decrease in receptor protein content through SDS-PAGE and Western blot analysis, we saved the supernatants of the immunoprecipitated lysates in –70°C and reused them for IP of the other receptor.

IP of insulin and IGF-IR proteins after reduction of receptors into {alpha}beta-dimers. To control for unspecific binding of the antibodies used for IP, part of the cell lysate was treated with dithiotreitol (DTT) to separate the receptor beta-subunits from each other before IP. This was done according to a modified method of Moxham et al. (26). The cell lysate was reduced with 1 mM DTT in lysis buffer at pH 8.5 for 30 min, breaking up the class 1 disulfide bridges holding the two receptor {alpha}beta-dimers together. The reduction was then terminated with 3 mM N-ethylmaleimide in lysis buffer at pH 7.5. After reduction, the cell lysate was immunoprecipitated with antibodies directed against either receptor beta-subunit as described above, although the supernatants were not reused. The immunoprecipitates were further analyzed with SDS-PAGE and Western blot.

SDS-PAGE and Western blot analysis of insulin and IGF-I receptor beta-subunit proteins. Levels of total and activated insulin and IGF-I receptor beta-subunit proteins were analyzed with SDS-PAGE and Western blot analysis using standard methods. The samples were thawed, vortexed, and centrifuged and subsequently loaded onto a precast Tris·HCl gel with 7.5% (vol/vol) polyacrylamide (Bio-Rad, Sundbyberg, Sweden), where the proteins were separated by SDS-PAGE. After electrophoresis the proteins were transferred onto a polyvinylidene difluoride membrane (Bio-Rad) and incubated in block solution on a shaker for 1 h at room temperature. When immunoblotting with anti-phosphotyrosine (PY20) antibody, 3% (wt/vol) BSA in Tris-buffered saline (TBS; 0.05 M TrizmaBase, 0.15M NaCl at pH 7.5) with 0.1% (vol/vol) Tween-20 (TBS-T) was used as blocking agent, and for the anti-receptor beta-subunit antibodies 5% (wt/vol) nonfat milk in TBS-T was used. The membrane was then incubated in primary antibody solution on a rocker overnight at 4°C. Primary antibodies used for immunoblotting studies included monoclonal mouse PY20 antibody, polyclonal rabbit anti-IR beta-subunit antibody (C-19), and polyclonal rabbit anti-IGF-IR beta-subunit antibody (C-20), all purchased from Santa Cruz Biotechnology. The next day, the membranes were washed four times in TBS-T and incubated with a horseradish peroxidase (HRP)-conjugated antibody for 1 h on a shaker at room temperature. The activated receptor beta-subunits were detected with 0.2 µg/ml PY20 antibody and 0.17 µg/ml HRP-conjugated sheep anti-mouse antibody (Amersham Biosciences, Uppsala, Sweden). The total amount of receptor beta-subunit proteins was detected using 0.2 µg/ml anti-IGF-IR (C-20) or anti-IR (C-19) together with 0.018 µg/l HRP-conjugated goat anti rabbit antibody bought from Zymed (San Francisco, CA). The proteins were visualized with enhanced chemiluminescence (ECL) detection reagents and exposure to Hyperfilm ECL, both from Amersham Biosciences. After detection of activated receptor beta-subunits with PY20 the membranes were stripped through incubation in stripping buffer [2% (wt/vol) SDS, 62.5 mM Tris·HCl, and 0.1 M beta-mercaptoethanol] for 30 min at 60°C and then reprobed with anti-receptor beta-subunit antibodies to detect the total amounts of receptor beta-subunit proteins.

D-[U-14C]glucose accumulation. The effect of hormones on glucose metabolism was analyzed as accumulation of D-[U-14C]glucose (Amersham Biosciences) into the cells. Near-confluent cultures of VSMCs were grown on six-well plates and starved for 16–24 h in serum-free DMEM. Next, they were incubated with new serum-free DMEM with D-[U-14C]glucose (0.2 µCi/ml) and hormones at indicated concentrations for 3 h. The cells were then washed three times with PBS and lysed with 0.5 ml 0.1% (wt/vol) SDS for 10 min at room temperature. Of this cell solution, 0.4 ml were added to 4 ml UltimaGold scintillation fluid (CiAB, Lidingö, Sweden), and the radioactivity was measured in a liquid scintillation counter (Rackbeta 1217; LKB Wallac). The data were expressed as percentage above unstimulated control cell radioactivity.

[6-3H]thymidine incorporation. DNA synthesis was quantified as [6-3H]thymidine (Amersham Biosciences) incorporation into DNA according to a modified method of Nilsson and Thyberg (27). The cells were grown on 12-well plates and, when near confluent, serum starved for 18–20 h in serum-free F-12 medium. They were then stimulated for 17 h with new serum-free F-12 medium supplemented with [6-3H]thymidine (2 µCi/ml) and hormones at indicated concentrations. The cells were washed free of medium with PBS, and the DNA was precipitated with ice-cold 5% (wt/vol) trichloroacetic acid for 20 min at 4°C. The cells were then solubilized in 0.5 ml of 0.1 M potassium hydroxide for 1–2 h at room temperature. To 4 ml of UltimaGold scintillation fluid, 0.4 ml of cell solution were added, and the radioactivity was measured in a liquid scintillation counter (Rackbeta 1217). The data were expressed as percentage above unstimulated control cell radioactivity.

Statistical analysis. Statistical comparisons of the means ± SE were performed with SPSS 12.0.1 for Windows (Chicago, IL) using one-way ANOVA and post hoc testing with Bonferroni. A P value of <0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Binding of IGF-I and insulin. Specific binding of both 125I-IGF-I and 125I-insulin could be demonstrated (Fig. 1). The specific binding of 125I-IGF-I (0.79%) was several-fold higher than that of 125I-insulin (0.20%). Due to low binding of insulin, the confidence interval of half-maximal displacement of 125I-insulin by insulin (3.9·10–12 M) was very large (7.4·10–14–2.0·10–10 M). For IGF-I, the concentration needed for half-maximal displacement of 125I-IGF-I was 2.6·10–9 M with a confidence interval of (1.6·10–9–4.2·10–9 M).


Figure 1
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  		Fig. 1. Binding studies were performed with 125I-IGF-I or 125I-insulin together with unlabeled IGF-I and insulin at indicated concentrations. Results are means ± SE from 3 independent experiments with IGF-I and 2 experiments with insulin.

 
Presence of insulin and IGF-I receptor proteins. After IP with anti-IGF-IR antibodies and immunoblotting with anti-IGF-IR beta-subunit antibodies, a 97-kDa protein corresponding to the IGF-IR beta-subunits could be detected in the rat VSMCs (Fig. 2, top left). When anti-IR antibodies were used for IP and the IR immunoprecipitates were immunoblotted with anti IR antibodies, a 95-kDa protein corresponding to the IR beta-subunit was found (Fig. 2, bottom right). After the membrane containing the IGF-IR immunoprecipitates was stripped and reprobed with anti-IR beta-subunit antibodies, we could detect a band corresponding to the IR beta-subunit (Fig. 2, top right), implying coprecipitation of the IGF-IR beta-subunit and the IR beta-subunit. Also, when the membrane containing the immunoprecipitated IR with anti-IGF-IR beta-subunit antibodies was reprobed, we showed a band with the molecular size of the IGF-IR beta-subunit (Fig. 2, bottom left).


Figure 2
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  		Fig. 2. Presence of the IGF-I and insulin receptor beta-subunits in xxxxx vascular smooth muscle cells (rVSMCs), implying coprecipitation. After immunoprecipitation (IP) of rVSMC lysate with polyclonal antibodies against either the IGF-I receptor (IGF-IR) beta-subunit (C-20) or the insulin receptor (IR) beta-subunit (C-19), we detected both the IGF-IR beta-subunit and the IR beta-subunit on the same membrane by Western blot, using the same antibodies. This implies coprecipitation of IGF-IR beta-subunits and IR beta-subunits. Top left: IP with anti IGF-IR beta-subunit antibodies (ab) and subesquent immunoblot (IB) of the IGF-IR beta-subunit showed distinct bands corresponding to the IGF-IR beta-subunit. Bottom left: IP with anti-IGF-IR beta-subunit ab and subesquent IB of the IR beta-subunit showed distinct bands corresponding to the IR beta-subunit. Top right: IP with anti-IR beta-subunit ab and subesquent IB of the IGF-IR beta-subunit showed distinct bands corresponding to the IGF-IR beta-subunit. Bottom right: IP with anti-IR beta-subunit ab and subesquent IB of the IR beta-subunit showed distinct bands corresponding to the IR beta-subunit. Experiment was repeated 3 times with similar results.

 
Presence of insulin and IGF-I receptor proteins after reduction of receptors into {alpha}beta-dimers. Before IP, part of the lysate was treated with DTT to reduce the disulfide bridges linking the two {alpha}beta-heterodimers of the receptors. When immunoprecipitating and immunoblotting the reduced lysates with antibodies against the IGF-IR beta-subunit, bands corresponding to the IGF-IR beta-subunit were found (Fig. 3, top left). After reprobing the same membrane with IR beta-subunit antibodies, no or faint IR beta-subunit bands could be detected (Fig. 3, bottom left). Vice versa was true for reduced samples immunoprecipitated with anti-IR beta-subunit antibodies (Fig. 3, top and bottom, right). This indicates that the coprecipitation mentioned above was not due to cross-reactivity of the antibodies used for IP or that the receptors were kept together by membrane fragments.


Figure 3
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  		Fig. 3. Presence of IGF-I and IR proteins after reduction of receptors into {alpha}beta-dimers. rVSMC lysates were treated with dithiotreitol (DTT) to reduce the receptors into {alpha}beta-dimers, thereby separating the receptor beta-subunits from each other. Reduced lysates were then immunoprecipitated with polyclonal ab against either the IGF-IR beta-subunit (C-20) or the IR beta-subunit (C-19). The presence of each receptor beta-subunit in the reduced immunoprecipitates was analyzed by anti-receptor IB using the same antibodies. Top left: in reduced lysate immunoprecipitated against IGF-IR beta-subunit, IGF-IR beta-subunit proteins could be detected. Bottom left: IB of the IGF-IR immunoprecipitates made on reduced lysate with anti-IR beta-subunit antibodies showed no or faint IR beta-subunit bands. Bottom right: in IR immunoprecipitates made on reduced lysate, IB with anti-IR beta-subunit ab showed presence of IR beta-subunit proteins. Top right: IB of the IR immunoprecipitates made on reduced lysate with anti-IGF-IR beta-subunit ab showed no or faint bands corresponding to the IGF-IR beta-subunit. IBs are representative of 3 different experiments.

 
Activation of insulin and IGF-I receptors. To detect activated receptor beta-subunits, receptor immunoprecipitates were immunoblotted with PY20. After the lysates were immunoprecipitated with anti IGF-IR antibodies and immunoblotted with PY20, cells stimulated with 10–10–10–9 M IGF-I showed phosphorylation of two distinct bands with molecular sizes corresponding to the IGF-IR beta-subunit and the IR beta-subunit (Fig. 4A). The phosphorylation was dose dependent and the band corresponding to the IGF-IR beta-subunit was more intense than that of the IR beta-subunit. Following a similar procedure, i.e., immunoblotting IR immunoprecipitates with PY20, cells stimulated with 10–10–10–9 M IGF-I again showed phosphorylation of two distinct bands with increasing intensity in a dose-dependent manner (Fig. 4B). Although the phosphorylation of the IGF-IR beta-subunit was still stronger than that of the IR beta-subunit, the two bands showed a more equal intensity compared with the bands detected with PY20 in IGF-IR immunoprecipitates. Stimulation with insulin at 10–10–10–9 M on IGF-IR immunoprecipitates did not induce any detectable phosphorylation above basal of either receptor beta-subunit when immunoblotted with PY20. Looking at the lysates immunoprecipitated against IR stimulation with insulin at 10–10–10–9 M induced phosphorylation of the IR beta-subunit only.


Figure 4
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  		Fig. 4. IGF-I and insulin activates IGF-I and IRs in rVSMCs. Serum-starved rVSMCs were stimulated with IGF-I or insulin at indicated concentrations for 10 min at 37°C and then lysed. A: IP with anti IGF-IR beta-subunit ab and subsequent IB with anti-phosphotyrosine (PY20) showed phosphorylation of 2 distinct bands with molecular sizes corresponding to the IGF-IR beta-subunit and the IR beta-subunit. Phosphorylation was dose dependent and the band corresponding the IGF-IR beta-subunit was more intense than that of the IR beta-subunit. Stimulation with insulin at 10–10–10–9 M did not induce any detectable phosphorylation above basal of either receptor beta-subunit. B: on IP with anti-IR beta-subunit ab and subsequent IB with PY20, we could again observe that stimulation with 10–10–10–9 M IGF-I had induced phosphorylation of 2 distinct bands, and this time the 2 bands showed a similar level of phosphorylation. The doublet also had an increasing intensity in a dose-dependent manner. Stimulation with insulin at 10–10–10–9 M induced phosphorylation of the IR beta-subunit only. The 2 control lanes with IBs of IGF-I and insulin receptor beta-subunit proteins show that the increased tyrosine phosphorylation in a dose-dependent manner is not due to altered receptor protein levels in the different wells. Similar results were obtained in 3 different experiments.

 
Activated insulin and IGF-I receptor proteins after reduction of receptors into {alpha}beta-dimers. When immunoblotting the reduced lysates with PY20, we detected single bands showing dose-dependent phosphorylation of only the receptor immunoprecipitated, which is in agreement with the above-mentioned findings of receptor proteins in reduced samples (Fig. 5).


Figure 5
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  		Fig. 5. Activation of insulin and IGF-IRs in rVSMCs after reduction of receptors into {alpha}beta-dimers. Cell lysate from rVSMCs was reduced with DTT to separate the receptor {alpha}beta-dimers from each other before IP. Top: in the reduced cell lysate immunoprecipitated with anti-IGF-IR beta-subunit ab, IB with PY20 showed clear phosphorylation of the IGF-IR beta-subunit in cells stimulated with IGF-I 10–9 M, whereas no visible phosphorylation of the IGF-IR beta-subunit could be observed in cells stimulated with insulin 10–9 M. No visible bands corresponding to phosphorylation of the IR beta-subunit could be detected after stimulation with either IGF-I 10–9 M or insulin 10–9 M. Bottom: in the reduced cell lysate immunoprecipitated with anti-IR beta-subunit ab, IB with PY20 showed clear phosphorylation of the IR beta-subunit in cells stimulated with IGF-I 10–9 M, and a modest phosphorylation of the IR beta-subunit could also be seen in cells stimulated with insulin 10–9 M. Stimulation with either IGF-I 10–9 M or insulin10–9 M did not give rise to any visible phosphorylation of the IGF-IR beta-subunit. Results shown in the blots are representative of 3 different experiments.

 
D-[U-14C]glucose accumulation. The effect of ligands on glucose metabolism was analyzed as accumulation of D-[U-14C]glucose. The glucose accumulation was markedly elevated in cells stimulated with IGF-I 10–10–10–7 M, and the maximum level reached at 10–9 M, whereas insulin stimulation did not reach maximum effect until at 10–7 M (Fig. 6).


Figure 6
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  		Fig. 6. Insulin and IGF-I stimulated glucose metabolism in rVSMCs measured by [14C]glucose accumulation. Effect of ligands on glucose metabolism was analyzed as accumulation of D-[U-14C]glucose into the cells. Graph shows that glucose accumulation was markedly elevated in cells stimulated with IGF-I at 10–10–10–7 M, with IGF-I reaching its maximum effect at stimulation with 10–9 M, whereas insulin did not have a maximum effect until stimulation with 10–7 M. [14C]glucose accumulation graph data are expressed as %radioactivity above basal, i.e., unstimulated control cell radioactivity. Values are means of replicates from 4 independent experiments expressed as means ± SE of %incorporation above basal. *P < 0.05 vs. basal; **P < 0.01 vs. basal.

 
[6-3H]thymidine incorporation. DNA synthesis was measured as [6-3H]thymidine incorporation into DNA. A pronounced effect on [6-3H]thymidine incorporation could be seen already at a low concentration, 10–10 M, in cells stimulated with IGF-I, reaching a maximum level at 10–9 M. Stimulation with insulin showed a measurable effect only at high concentrations, 10–8–10–7 M (Fig. 7).


Figure 7
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  		Fig. 7. Insulin and IGF-I stimulated DNA synthesis in rVSMCs assessed by [3H]thymidine incorporation. Graph shows a pronounced effect of IGF-I stimulation on [6-3H]thymidine incorporation already at a low concentration, 10–10 M, reaching a maximum effect at 10–9 M. Stimulation with insulin showed a measurable effect only at high concentrations, 10–8–10–7 M. [3H]Thymidine incorporation graph data are expressed as percentage of radioactivity above basal, i.e., unstimulated control cell radioactivity. Results are means ± SE of replicates from 4 independent experiments expressed as %incorporation above basal. *P < 0.05 vs. basal; **P < 0.01 vs. basal.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
We show here the presence of IR, IGF-IR, and HRs in rVSMCs using IP and Western blot. The receptors were activated using insulin and IGF-I at physiological concentrations, but only IGF-I elicited biological effects at these concentrations.

The existence of HRs was shown through detection of both the IR and the IGF-IR beta-subunit on the same membrane by Western blot after IP with specific antibodies against either of the two, implying coprecipitation of the IGF-IR beta-subunit and the IR beta-subunit. To make sure the coprecipitation was not due to cross reactivity of the antibodies used for IP or that the receptors were kept together by membrane fragments, reduction of receptors, i.e., separation of the beta-subunits from each other with DTT (26), was performed before IP on part of the lysate. The results from these experiments, showing detection of only the receptor beta-subunit immunoprecipitated in contrast to the finding of both receptor beta-subunits in the nonreduced lysate, proves that the coprecipitation was not due to cross-reactivity of the antibodies.

Stimulation with IGF-I at a low concentration, 10–10 M, caused phosphorylation not only of IGF-IR beta-subunits but also of IR beta-subunits after IP against either receptor beta-subunit and immunoblotting with PY20. This further confirms the existence of hybrid receptors, which in other cells have been shown to have a high affinity for IGF-I but not for insulin (33). It is also in agreement with recent results from our group in studies made on human coronary artery smooth muscle cells (HCASMCs) (9), where IGF-I at 10–10 M phosphorylated both IGF-IR beta-subunits and IR beta-subunits, whereas insulin at 10–10 M could only phosphorylate IR beta-subunits. In rVSMCS we could clearly detect the IR beta-subunit after IP IGF-IR, but we could not see any activation by insulin stimulation of the IR beta-subunit after IP of IGF-IR. This supports the concept that the IR beta-subunits immunoprecipitated with the anti-IGF-IR antibody were assembled into HRs, since the HRs as mentioned above are known to have a low affinity for insulin similar to IGF-IRs. This is in agreement with an earlier study in preadipocytes and a recent study on bovine aortic endothelial cells where insulin was able to initiate IGF-IR phosphorylation in cells immunoprecipitated against IGF-IR only at high concentrations >10–9 M (14, 25). This present study is to our knowledge the first one to show clear evidence of the existence of hybrids in VSMCs. The significance of hybrid receptors still remains unclear, but studies in human skeletal muscle have shown the formation of HRs to be linked to insulin resistance (15) and diabetes (16).

Stimulation with IGF-I at low concentrations, 10–10 M and 10–9 M, which phosphorylated both IGF-IR and IR, also had biological effects on glucose metabolism and DNA synthesis, in agreement with previous reports (1, 12). The physiological concentration of free IGF-I in humans is about 10–10 M (17) and probably somewhat higher in rats (8). Although we could activate IR with insulin at a physiological concentrations, 10–10 M, we could not detect any biological effects on rVSMCs. Activation of IR at physiological concentrations and even cell-signaling substrates downstream of the IR have been reported in VSMCs (38) and endothelial cells (25). However, in recent studies on HCASMCs and human coronary artery endothelial cells, no effects of insulin at 10–10–10–9 M on either apoptosis or DNA synthesis was found (34), in agreement with our results on DNA synthesis. In this study, insulin stimulation of accumulation of [14C]glucose and [3H]thymidine incorporation could be seen only at high concentrations, i.e., 10–8–10–7 M. In reviewing studies where insulin effects on growth and metabolism in VSMCs have been reported, most of them have been performed using insulin at high supraphysiological concentrations (1, 19, 37), implying that the resulting insulin effects are probably due to activation of IGF-IRs (2, 4) and possibly also HRs.

In agreement with earlier results on rVSMCs, we found specific binding for both 125I-IGF-I and 125I-insulin, with a higher specific binding for 125I-IGF-I (6). For IGF-I, half-maximal displacement of 125I-IGF-I was 2.6·10–9 M, somewhat higher than we previously reported (6). In plasma membranes isolated from bovine mesenteric arteries we previously found binding characteristics for IGF-I and insulin (5a), similar to our results on cultured VSMCs. This indicates a similar ratio of IGF-IR and IRs in smooth muscle cells in vivo. In cultured human smooth muscle cells, we recently demonstrated an eight times higher abundance of IGF-IRs compared with IRs in HCASMCs using RT-PCR measurements of receptor mRNA expression (9). Due to the higher abundance of IGF-IRs compared with IRs, and because some of the IR {alpha}beta-heterodimers are sequestered into hybrid receptors, the signal generated by insulin through IR homoreceptors may be too weak to elicit biological effects.

In conclusion, we show the presence of insulin receptors, IGF-I receptors, and hybrid insulin/IGF-I receptors in rVSMCS. Stimulation with IGF-I at physiological concentrations activates both IGF-IRs and HRs and also results in biological effects. Insulin activates its own cognate receptor at a low physiological concentration, but this stimulation does not induce any biological effects. Many of the insulin effects reported using high concentrations in in vitro studies are probably propagated through the IGF-IR and could only occur in vivo due to IGF-I stimulation. Our results suggest that IGF-I rather than insulin has an impact on vascular smooth muscle function in vivo.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
Financial support was obtained from Landstinget i Östergötland, the Swedish Research Council (04952), the Swedish Diabetes Association, and Barndiabetesfonden.


   ACKNOWLEDGMENTS
 
We are grateful to Anna-Kristina Granath for excellent technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: G. Johansson, Dept. of Biomedicine and Surgery, Division of Cell Biology, Linköping University, S-581 85 Linköping, Sweden (e-mail: git.johansson{at}ibk.liu.se)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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