Insulin at Physiological Concentrations Selectively Activates Insulin But Not Insulin-Like Growth Factor I (IGF-I) or Insulin/IGF-I Hybrid R
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《内分泌学杂志》
Division of Endocrinology and Metabolism, Department of Internal Medicine, University of Virginia Health System, Charlottesville, Virginia 22908-1410
Abstract
In muscle, physiologic hyperinsulinemia, presumably acting on endothelial cells (ECs), dilates arterioles and regulates both total blood flow and capillary recruitment, which in turn influences glucose disposal. In cultured ECs, however, supraphysiological (e.g. 10 nM) insulin concentrations are typically used to study insulin receptor (IR) signaling pathways and nitric oxide generation. IGF-I receptors (IGF-IRs) are more abundant than IR in ECs, and they also respond to high concentrations of insulin. To address whether IR mediates responses to physiologic insulin stimuli, we examined the insulin concentration dependence of IR and IGF-IR-mediated insulin signaling in bovine aortic ECs (bAECs). We also assessed whether insulin/IGF-I hybrid receptors were present in bAECs. Insulin, at 100–500 pM, significantly stimulated the phosphorylation of IR, Akt1, endothelial isoform of nitric oxide synthase, and ERK 1/2 but not the IGF-IR subunit. At concentrations 1–5 nM or greater, insulin dose-dependently enhanced the tyrosine phosphorylation of IGF-IR, and this was inhibited by IGF-IR neutralizing antibody. In addition, immunoprecipitation of IR pulled down the IGF-IR, and the IR immunocytochemically colocalized with IGF-IR, suggesting that ECs have insulin/IGF-I hybrid receptors. We conclude that: 1) insulin at physiological concentrations selectively activates IR signaling in bAECs; 2) bAECs express IGF-IR and insulin/IGF-I hybrid receptors in addition to IR; 3) high concentrations of insulin (1–5 nM) activate IGF-IR and hybrid receptors as well as IR; and 4) this crossover activation can confound interpretation of studies of insulin action in ECs when high insulin concentrations are used.
Introduction
AMONG THEIR MULTIPLE functions, endothelial cells (ECs) are thought to mediate many of the vascular actions of insulin. These actions include increasing the compliance of conduit blood vessels (1), dilation of resistance arterioles (2, 3), and relaxation of small precapillary arterioles (4, 5). The latter two actions can affect glucose disposal (6, 7, 8). Furthermore, insulin’s vascular actions are impaired in both naturally occurring (e.g. obesity, type 2 diabetes) (9, 10, 11) and experimentally induced (free fatty acid infusion, TNF treatment) (12, 13, 14, 15) states of insulin resistance. Clinically, endothelial dysfunction is an early marker of vascular disease, which is itself more prevalent in the diabetic population (16, 17). However, our mechanistic understanding of insulin’s direct actions on the endothelium remains poorly characterized. This arises in part because the measures used to characterize insulin action in vivo only indirectly report on the role of the endothelium. Thus, insulin-induced smooth muscle relaxation may be due to endothelial action of insulin; insulin acting directly on smooth muscle; or, in the case of skeletal muscle, insulin acting on skeletal muscle that in turn affects vasodilation. Each of these cells possesses insulin receptors (IRs) (18) and one or another isoform of nitric oxide synthase (19). We recently reported that insulin-mediated capillary recruitment preceded activation of insulin signaling pathways within rat muscle, thereby arguing against myocyte involvement in insulin-mediated capillary recruitment (7). The relaxation of resistance arterioles and increases of total muscle blood flow occur slowly and require up to several hours of insulin exposure (18, 20); hence, nonendothelial effects of insulin are not only possibly but also very likely involved.
This underscores the importance of direct examination of insulin’s action on ECs. Evidence for a direct action of insulin on the ECs comes first from the demonstration that these cells possess IR, insulin can stimulate receptor autophosphorylation, and insulin also affects downstream signaling pathways including the phosphatidylinositol 3-kinase (PI3-kinase) pathway (21, 22, 23, 24, 25). Activation of this pathway leads to the activation of protein kinase B (or Akt), which can phosphorylate and activate the endothelial isoform of nitric oxide synthase (eNOS). These findings support a direct role of the EC in the vascular actions of insulin described above. However, it is also true that the majority of studies of insulin action on ECs have used unphysiologically high concentrations of insulin (typically 10 nM) (21, 22, 23, 24, 25, 26). This alone would raise questions regarding the physiological relevance of observations made in these cells. This concern is rendered all the more relevant by the observation that ECs express greater numbers of receptors for IGF-I than insulin (27), and insulin at supraphysiological concentrations can cross-react with IGF-I receptors (IGF-IR). In addition, a recent report suggests that human umbilical vein ECs (HUVEC), like many other insulin-sensitive tissues, express hybrid receptors (28) that are composed of one - and one -subunit of the IR and a corresponding - and -subunit of the IGF-IR. When studied in other tissues, these hybrid receptors appear to have binding characteristics toward insulin more akin to that of the IGF-IR (29, 30, 31).
The major purpose of the present study was to address whether ECs could respond to insulin at physiologically relevant concentrations at which action through the IGF-IR could be excluded. We report that insulin selectively stimulates the tyrosine (Tyr) phosphorylation of its own receptor -subunit and activates downstream signaling pathways in cultured bovine aortic ECs (bAECs). This occurs at insulin concentrations that do not Tyr-phosphorylate the IGF-IR -subunit. We also confirm that bAECs, like several other insulin sensitive tissues, may possess hybrid insulin/IGF-I receptors. These findings underscore that dissecting the specific actions of insulin within the ECs requires careful attention to the insulin concentration to avoid confounding contributions from the IGF-IR or insulin/IGF-I hybrid receptors.
Materials and Methods
Culture of bAECs
Cells in primary culture were purchased from Cambrex BioSciences Walkersville, Inc. (Walkersville, MD) and cultured in endothelial basic media, which was supplemented with 5% fetal bovine serum, bovine brain extract, human EGF (10 ng/ml), gentamicin sulfate (50 μg/ml), amphotericin-B (50 ng/ml), and hydrocortisone (1 μg/ml). Cells between passages 3 and 8 were used for experiments after growing to 75–80% confluence, with or without serum starvation for 16–18 h. Cells were washed with ice-cold 1x PBS solution twice and then lysed and sonicated using an XL2020 sonicator (Fisher Scientific, Pittsburgh, PA) in ice-cold lysis buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Nonidet P-40, 0.25% sodium deoxycholate, 1 mM EGTA, 1 mM sodium orthovanadate, 1 mM NaF, 1 μg/ml aprotinin, 1 μg/ml leupeptin, 1 μg/ml pepstatin, and 1 mM phenylmethylsulfonyl fluoride]. Cell lysates were centrifuged for 10 min at 4 C (20,000 x g), and the supernatants were used for either immunoprecipitation or Western blotting.
Immunoprecipitation of IR and IGF-IR
Aliquots of supernatant containing 500-1000 μg protein in 1000 μl lysis buffer were incubated with 25 μl primary antibody against either IR or IGF-IR (2.5 μg/ml) overnight at 4 C. Protein A/G plus IgG-agarose was then added and the mixture was kept at 4 C for 1 h with gentle rocking. After washing six times with lysis buffer, the beads were spun down (1000 x g for 30 sec), resuspended in 50 μl 2x sample buffer [375 mM Tris-HCl (pH 6.8), 12% sodium dodecyl sulfate, 60% glycerol, 300 mM dithiothreitol, and 0.06% bromophenol blue], and boiled for 5 min.
Immunoblotting
Equal amount of IR or IGF-IR immunoprecipitate or aliquots of cell lysate supernatant containing approximately 100 μg protein were diluted with an equal volume of sodium dodecyl sulfate sample buffer and electrophoresed on a 10% polyacrylamide gel, transferred to nitrocellulose, and blocked with 5% low-fat milk in Tris-buffered saline plus Tween 20. Subsequently membranes were probed with antibodies against IR, IGF-IR, phospho-Tyr (p-Tyr) (Santa Cruz Biotechnology, Santa Cruz, CA), phospho-Akt1 (Ser 473) (Upstate Cell Signaling, Lake Placid, NY), phospho-ERK1/2, or phospho-eNOS (Ser1177) (New England BioLabs, Beverly, MA) for 1 h at 4 C. This was followed by a donkey antirabbit IgG coupled to horseradish peroxidase, and the blots were developed using enhanced chemiluminescence detection (Amersham Life Sciences, Piscataway, NJ). To assure equal loading of proteins, membranes probed with antibodies against above-mentioned phosphoproteins were stripped with restore Western blot stripping buffer (Pierce Chemical Co., Rockford, IL) and reprobed with antibodies against IR, IGF-IR, Akt1, ERK1/2, or eNOS as appropriate.
Immunocytochemical staining
The double-staining protocols were the same as we described previously (32, 33). Briefly, bAECs were grown in slide chambers as described above. Cells were fixed with cold methanol for 10 min at –20 C, washed three times in PBS, permeabilized in PBS containing 0.05% Triton X-100 and 1% horse serum for 30 min at room temperature, and incubated with mouse monoclonal anti-IR antibody (Chemicon International, Temecula, CA; 1:50) and rabbit polyclonal antibody against IGF-IR (Santa Cruz Biotechnology; 1:50) (double labeling) overnight at 4 C. After washing three times with PBS, cells were then incubated with species-specific secondary antibodies conjugated with a fluorochrome (either Cy2 or Cy3) (Jackson ImmunoResearch, West Grove, PA) at 1:200 dilutions for 45 min at room temperature, washed three more times with PBS, and coverslipped with the antifade mounting medium.
Confocal imaging
The double-immunocytochemical labeling was examined simultaneously using a two-color BX50 WI confocal microscope (Olympus, Tokyo, Japan) equipped with krypton and argon laser as described previously (32, 33). An x-y-z-axis scanning method was employed. The images were scanned through up to x100 objectives, acquired at a resolution of 1024 x 1024 pixels, and stored in 24-bit TIFF format. To address whether the immunoreactivity was located within the cells, a series of optical sections at a thickness of less than 0.1 μm was acquired from the top to the bottom of the cells along the z-axis.
Results
Time course of insulin action in cultured bAECs
To examine the time course of IR activation in cultured bAECs, cells were incubated with 100 nM insulin for 0, 10, 20, 30, 40, and 50 min. We used 100 nM insulin because insulin at this concentration has repeatedly been shown to strongly stimulate phosphorylation of Akt, eNOS, and other insulin-activated pathways. Cells were then processed as described above, immunoprecipitated with anti-IR, and immunoblotted with anti-p-Tyr. As shown in Fig. 1, insulin stimulated IR Tyr phosphorylation within 10 min, and this reached a maximum at 30 min and declined thereafter. Based on this finding, we used 30 min incubation time for all subsequent experiments.
Insulin at physiological concentrations stimulates IR Tyr phosphorylation
Inasmuch as nearly all previous studies have used pharmacologic insulin concentrations to study insulin’s actions on ECs, we next examined whether insulin at physiological concentrations stimulates IR Tyr phosphorylation in ECs and, if so, whether this is associated with its downstream signaling. Cells were incubated with insulin at 0, 0.1, 0.2, 0.5, 1, 2, 5, 10, and 100 nM for 30 min and then lysed and immunoprecipitated with anti-IR antibody. The immunoprecipitate was electrophoresed and then immunoblotted with anti-p-Tyr antibody. As shown in Fig. 2A, insulin at concentrations between 0.1 and 0.5 nM strongly stimulated IR Tyr phosphorylation, and higher physiological or pharmacological concentrations did not increase signal intensity further. No differences were seen in IR protein with these short duration insulin exposures.
We then examined whether this IR Tyr phosphorylation was associated with downstream signaling of insulin action. As shown in Fig. 2 and similar to IR Tyr phosphorylation, insulin at concentrations between 0.1 and 0.5 nM potently stimulated the phosphorylation of Akt1 (Fig. 2B), ERK1/2 (Fig. 2C), and eNOS (Fig. 2D) without significant changes in the quantity of each of these proteins present.
bAECs express both IGF-IR and insulin/IGF-I hybrid receptors
ECs possess abundant IGF-IR (27), and studies done in cell lines other than ECs have demonstrated a cross-activation of IGF-IR when insulin is present at high concentrations. Also, a recent report suggests that HUVEC, like muscle and adipose tissue, expresses insulin/IGF-I hybrid receptors (28). To ascertain whether bAECs also express insulin/IGF-I hybrid receptors, we immunoprecipitated cell lysates with either anti-IR or anti-IGF-IR and then probed with antibodies against either IR or IGF-IR. As shown in Fig. 3, IGF-IR clearly coprecipitated with IR, and a significant amount of IR coprecipitated with IGF-IR. Because the IR and IGF-IR antibodies used do not cross-react with IGF-IR and IR, respectively, our data suggest that in addition to IR and IGF-IR, insulin/IGF-I hybrid receptors are present in cultured bAECs.
Immunohistochemical double staining of the bAECs prepared for confocal microscopy as described in Materials and Methods demonstrated a significant spatial colocalization of IR and IGF-IR that is explicable by either distinct IR and IGF-IR localized to specific cellular domains or insulin/IGF-I hybrid receptors in bAECs (Fig. 4).
Insulin at high concentrations activates IGF-IR and insulin/IGF-I hybrid receptors in bAECs
To examine the dose response of IGF-IR and insulin/IGF-I hybrid receptors to insulin, bAECs were serum starved for 16 h and incubated with various concentrations of insulin (0, 0.1, 0.2, 0.5, 1, 2, 5, 10, and 100 nM) for 30 min. Cells were then lysed and immunoprecipitated with anti-IGF-IR. The immunoprecipitation products were electrophoresed and immunoblotted first with anti-p-Tyr and then with anti-IGF-IR after membrane stripping. Unlike IR response, insulin did not significantly stimulate the Tyr phosphorylation of IGF-IR at concentrations less than 1 nM. However, between 1 and 5 nM insulin effectively stimulated IGF-IR phosphorylation, and this increased progressively as the insulin concentration was further raised (Fig. 5A).
To further ascertain that the insulin-induced Tyr phosphorylation was indeed from Tyr phosphorylation of IGF-IR (in either the IGF-IR or hybrid receptors), but not from that of IR, we neutralized the IGF-IR with its specific neutralizing antibody IGF-I receptor (Ab-3) (EMD Biosciences, San Diego, CA) before insulin treatment. Cells were incubated with insulin at 0, 0.2, 2, 10, and 100 nM for 30 min with or without pretreatment of cells with IGF-I receptor (Ab-3) antibody (1 μg/ml) for 30 min, lysed, immunoprecipitated with anti-IR or anti-IGF-IR, and then immunoblotted with anti-p-Tyr. As shown in Fig. 5B, insulin potently stimulated the Tyr phosphorylation of IR at all concentrations, and this was not affected by the pretreatment of cells with IGF-I receptor (Ab-3) antibody. However, insulin-stimulated Tyr phosphorylation of IGF-IR at 2, 10, and 100 nM was completely abolished by preincubating the cells with IGF-I receptor (Ab-3) antibody. Despite this, insulin-stimulated Akt1 phosphorylation was retained in cells pretreated with IGF-I receptor (Ab-3) antibody (Fig 5B), suggesting insulin indeed acted via IR.
Discussion
The current study provides the first clear demonstration that insulin, at concentrations well within the physiologic range, can stimulate the tyrosine phosphorylation of its own receptors on ECs and in addition phosphorylates relevant regulatory proteins within the PI3-kinase and ERK signaling pathways. Both pathways are downstream of IR and are pivotal in mediating insulin’s multiple cellular actions. Whereas we also confirmed that ECs possess IGF-IR and hybrid insulin/IGF-I receptors in addition to IR, these receptors are likewise activated by insulin but at higher insulin concentrations.
Considering first what was seen when the ECs were exposed to physiologic insulin concentrations, it is clear that this was sufficient to substantially activate downstream insulin signaling including at least Akt1 and eNOS as well as ERK1/2. This was seen with insulin concentrations that had no effect to enhance the tyrosine phosphorylation of the IGF-IR subunit. This certainly suggests that these actions result from insulin stimulation of its own receptor tyrosine kinase. That preincubation of ECs with IGF-IR neutralizing antibody completely abolished insulin-stimulated IGF-IR Tyr phosphorylation but not IR Tyr phosphorylation or Akt1 serine phosphorylation confirms that insulin indeed can activate its receptor kinase activity and downstream signaling independent of IGF-IR.
These results support the hypothesis that insulin’s physiologic actions on the vasculature, which contribute to insulin-mediated glucose disposal, may indeed result, at least in part, from direct actions on the ECs. For example, recent results from in vivo studies in our laboratory indicate that very modest increases in plasma insulin concentration from 60 to 300 pM exert a nitric oxide (NO)-dependent action to recruit capillaries within skeletal muscle (6, 7, 34). These effects occur very promptly, being significant by 10 min and fully established by 30 min, a time course similar to that observed here for Tyr phosphorylation of IR. Furthermore, these in vivo responses precede any discernible metabolic effect of insulin on skeletal muscle, implying a more direct effect on the vasculature. Previous reports that insulin augments endothelial NO production or phosphorylation of Akt or eNOS have involved using insulin at concentrations of 10 nM or greater (21, 22, 24, 25). Thus, the current results in cultured bAECs are quite consistent with what has been seen in vivo and would be consonant with the hypothesis that insulin can act directly on the endothelium at physiologic concentrations to exert NO-mediated biologic effects. These observations, of course, in no way exclude the possibilities that insulin might also affect smooth muscle cells and that direct actions on these cells may be importantly involved in insulin’s in vivo vascular action.
The concept that insulin has an important physiologic action on the ECs is perhaps challenged by the observation that mice deficient in IR selectively in the ECs (VENIRKO mice) are insulin resistant only when studied on a low-salt diet (35). However, because targeted deletion of IR in skeletal muscle also minimally affects glucose metabolism, it is clear that either other developmental compensation can occur (expression of both eNOS and endothelin-1 are altered in VENIRKO mice) or our ability to finely assess significant but not gross dysfunction is not in hand. It is obviously easier to dismiss the action of insulin on ECs as of no physiologic significance if it is seen only at supraphysiologic insulin concentrations. Insulin’s action on ECs has previously been most thoroughly studied by Quon and colleagues using HUVECs (21, 22, 24, 25). That laboratory demonstrated that insulin induces phosphorylation of IR, Akt, and eNOS. The latter two actions are blunted by inhibition of PI-3-kinase. However, in their studies no significant effect was seen at insulin concentrations less than 10 nM, and its effect was half maximal at approximately 500 nM. Whether this much reduced insulin sensitivity, compared with our current work, relates to differences between bovine aortic and human venous cells or other specific aspects of the experimental protocol is uncertain. In a number of other studies using either pulmonary arterial (26) or aortic ECs (36, 37), clear demonstration of effects of insulin again were seen only with unphysiologically high concentrations. We would emphasize that findings from our current work in bAECs fully support the conclusions reached by Quon and colleagues from studies using higher insulin concentrations in HUVECs.
Our observation that higher concentrations of insulin (1–5 nM) significantly stimulate IGF-IR tyrosine phosphorylation confirmed the crossover effect of insulin described in other tissues. Generally, in vitro studies of the IGF-IR suggest that it has a more than 100 times lesser relative affinity for insulin than does IR, and the insulin/IGF-I hybrid receptors behave similarly to IGF-IR in insulin or IGF-I binding affinity (38, 39). In the current study, we saw stimulation of the tyrosine phosphorylation of IGF-IR in ECs when insulin concentrations were between 1 and 5 nM or 10- to 50-fold above the lowest insulin dose that stimulated IR Tyr phosphorylation. It is possible that this reflects, at least in part, a greater abundance of IGF-IR and insulin/IGF-I hybrid receptors, compared with IR in the ECs. IGF-I, like insulin, can stimulate NO production in cultured ECs (40) and increase skeletal muscle blood flow in humans (41).
Others have reported that the IGF-IR is as much as 10-fold more abundant than IR on ECs (at least in HUVECs) (42). The contribution of hybrid receptors to this estimate is not known. Our results from IGF-IR and IR reciprocal immunoprecipitation and immunoblotting study (Fig. 3) and immunocytochemical staining/confocal microscopy (Fig. 4) suggested that indeed there are ample insulin/IGF-I hybrid receptors in ECs as well. However, this technique cannot exclude the possibility that the IGF-I and insulin receptors are part of a macromolecular complex that is not disrupted by the lysis procedure and they are being jointly immunoprecipitated as part of a larger complex. We did not attempt to quantitate the relative proportions of insulin, IGF-I, and insulin/IGF-I hybrid receptors present on ECs. We did, however, attempt to define the contribution of IR and IGF-IR or hybrid receptors to insulin signaling by varying insulin concentrations throughout the physiologic and pharmacologic range. Interestingly, we did not observe a further increase in IR Tyr phosphorylation when insulin was present at pharmacological concentrations. One might assume that Tyr phosphorylation of the IR subunits from the insulin/IGF-I hybrid receptors should result in higher signal intensity. Whether this is due to the semiquantitative nature of the Western blotting technique, some more complex interaction between the subunits of the hybrid receptor, or a decline in native IR phosphorylation at very high insulin concentrations was not investigated.
We note that whereas insulin-stimulated tyrosine phosphorylation of its own receptor appeared to reach a maximum between 0.2 and 0.5 nM, phosphorylation of Akt and eNOS continued to increase up to insulin concentrations of 100 nM. This also appeared to occur in the presence of IGF-IR blocking antibody (Fig. 5B). Several factors may account for this. First, because the insulin concentration is further raised, it is possible that insulin is competing with the IGF-I blocking antibody because both compounds associate reversibly with the IGF-IR; second, the hybrid receptors we report (see also Ref.28) may be less sensitive to the IGF-IR blocking antibody than is the native IGF-IR, allowing some further stimulation of the IGF-I/insulin signal transduction pathways at high insulin concentrations. In addition, because there is both amplification of signal within the PI3-kinase/Akt pathway and cross talk between the various arms of IR signaling cascades (e.g. the ERK pathway and the Akt pathway), there may not be a one-to-one quantitative correspondence between IR phosphorylation and phosphorylation of downstream signaling molecules. Finally, Western blotting is by its nature a semiquantitative method that also might prevent a one-to-one correspondence between IR and Akt or eNOS phosphorylation.
In conclusion, bAECs express IGF-IR and insulin/IGF-I hybrid receptors in addition to IR, but the phosphorylation/activation of IR alone is very sensitive to physiological doses of insulin. High doses of insulin cross-activate IGF-IR and insulin/IGF-I hybrid receptors, and this may confound interpretation of previous studies of high-dose insulin action on ECs. The ability of insulin to both phosphorylate its receptor and activate downstream signaling pathways at insulin concentrations well within the physiological range supports the consideration of these cells as a potentially important insulin target tissue. Nevertheless, our data do not clarify whether the vascular action of insulin is impaired in pathological conditions associated with insulin resistance, (e.g. obesity and type 2 diabetes mellitus). Much further study is needed to define this relationship, if any.
Footnotes
This work was supported by a research grant from the American Diabetes Association (to Z.L.) and National Institutes of Health Grants RR-15540 (to Z.L.), DK-38578 and DK-54058 (to E.J.B.), and P30-DK063609 (to the University of Virginia Diabetes Endocrinology Research Center).
Abbreviations: bAEC, Bovine aortic EC; EC, endothelial cell; eNOS, endothelial isoform of nitric oxide synthase; HUVEC, human umbilical vein EC; IGF-IR, IGF-I receptor; IR, insulin receptor; NO, nitric oxide; PI3-kinase, phosphatidylinositol 3-kinase; p-Tyr, phospho-Tyr.
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Bar RS, Boes M, Dake BL, Booth BA, Henley SA, Sandra A 1988 Insulin, insulin-like growth factors, and vascular endothelium. Am J Med 85:59–70(Guolian Li, Eugene J. Bar)
Abstract
In muscle, physiologic hyperinsulinemia, presumably acting on endothelial cells (ECs), dilates arterioles and regulates both total blood flow and capillary recruitment, which in turn influences glucose disposal. In cultured ECs, however, supraphysiological (e.g. 10 nM) insulin concentrations are typically used to study insulin receptor (IR) signaling pathways and nitric oxide generation. IGF-I receptors (IGF-IRs) are more abundant than IR in ECs, and they also respond to high concentrations of insulin. To address whether IR mediates responses to physiologic insulin stimuli, we examined the insulin concentration dependence of IR and IGF-IR-mediated insulin signaling in bovine aortic ECs (bAECs). We also assessed whether insulin/IGF-I hybrid receptors were present in bAECs. Insulin, at 100–500 pM, significantly stimulated the phosphorylation of IR, Akt1, endothelial isoform of nitric oxide synthase, and ERK 1/2 but not the IGF-IR subunit. At concentrations 1–5 nM or greater, insulin dose-dependently enhanced the tyrosine phosphorylation of IGF-IR, and this was inhibited by IGF-IR neutralizing antibody. In addition, immunoprecipitation of IR pulled down the IGF-IR, and the IR immunocytochemically colocalized with IGF-IR, suggesting that ECs have insulin/IGF-I hybrid receptors. We conclude that: 1) insulin at physiological concentrations selectively activates IR signaling in bAECs; 2) bAECs express IGF-IR and insulin/IGF-I hybrid receptors in addition to IR; 3) high concentrations of insulin (1–5 nM) activate IGF-IR and hybrid receptors as well as IR; and 4) this crossover activation can confound interpretation of studies of insulin action in ECs when high insulin concentrations are used.
Introduction
AMONG THEIR MULTIPLE functions, endothelial cells (ECs) are thought to mediate many of the vascular actions of insulin. These actions include increasing the compliance of conduit blood vessels (1), dilation of resistance arterioles (2, 3), and relaxation of small precapillary arterioles (4, 5). The latter two actions can affect glucose disposal (6, 7, 8). Furthermore, insulin’s vascular actions are impaired in both naturally occurring (e.g. obesity, type 2 diabetes) (9, 10, 11) and experimentally induced (free fatty acid infusion, TNF treatment) (12, 13, 14, 15) states of insulin resistance. Clinically, endothelial dysfunction is an early marker of vascular disease, which is itself more prevalent in the diabetic population (16, 17). However, our mechanistic understanding of insulin’s direct actions on the endothelium remains poorly characterized. This arises in part because the measures used to characterize insulin action in vivo only indirectly report on the role of the endothelium. Thus, insulin-induced smooth muscle relaxation may be due to endothelial action of insulin; insulin acting directly on smooth muscle; or, in the case of skeletal muscle, insulin acting on skeletal muscle that in turn affects vasodilation. Each of these cells possesses insulin receptors (IRs) (18) and one or another isoform of nitric oxide synthase (19). We recently reported that insulin-mediated capillary recruitment preceded activation of insulin signaling pathways within rat muscle, thereby arguing against myocyte involvement in insulin-mediated capillary recruitment (7). The relaxation of resistance arterioles and increases of total muscle blood flow occur slowly and require up to several hours of insulin exposure (18, 20); hence, nonendothelial effects of insulin are not only possibly but also very likely involved.
This underscores the importance of direct examination of insulin’s action on ECs. Evidence for a direct action of insulin on the ECs comes first from the demonstration that these cells possess IR, insulin can stimulate receptor autophosphorylation, and insulin also affects downstream signaling pathways including the phosphatidylinositol 3-kinase (PI3-kinase) pathway (21, 22, 23, 24, 25). Activation of this pathway leads to the activation of protein kinase B (or Akt), which can phosphorylate and activate the endothelial isoform of nitric oxide synthase (eNOS). These findings support a direct role of the EC in the vascular actions of insulin described above. However, it is also true that the majority of studies of insulin action on ECs have used unphysiologically high concentrations of insulin (typically 10 nM) (21, 22, 23, 24, 25, 26). This alone would raise questions regarding the physiological relevance of observations made in these cells. This concern is rendered all the more relevant by the observation that ECs express greater numbers of receptors for IGF-I than insulin (27), and insulin at supraphysiological concentrations can cross-react with IGF-I receptors (IGF-IR). In addition, a recent report suggests that human umbilical vein ECs (HUVEC), like many other insulin-sensitive tissues, express hybrid receptors (28) that are composed of one - and one -subunit of the IR and a corresponding - and -subunit of the IGF-IR. When studied in other tissues, these hybrid receptors appear to have binding characteristics toward insulin more akin to that of the IGF-IR (29, 30, 31).
The major purpose of the present study was to address whether ECs could respond to insulin at physiologically relevant concentrations at which action through the IGF-IR could be excluded. We report that insulin selectively stimulates the tyrosine (Tyr) phosphorylation of its own receptor -subunit and activates downstream signaling pathways in cultured bovine aortic ECs (bAECs). This occurs at insulin concentrations that do not Tyr-phosphorylate the IGF-IR -subunit. We also confirm that bAECs, like several other insulin sensitive tissues, may possess hybrid insulin/IGF-I receptors. These findings underscore that dissecting the specific actions of insulin within the ECs requires careful attention to the insulin concentration to avoid confounding contributions from the IGF-IR or insulin/IGF-I hybrid receptors.
Materials and Methods
Culture of bAECs
Cells in primary culture were purchased from Cambrex BioSciences Walkersville, Inc. (Walkersville, MD) and cultured in endothelial basic media, which was supplemented with 5% fetal bovine serum, bovine brain extract, human EGF (10 ng/ml), gentamicin sulfate (50 μg/ml), amphotericin-B (50 ng/ml), and hydrocortisone (1 μg/ml). Cells between passages 3 and 8 were used for experiments after growing to 75–80% confluence, with or without serum starvation for 16–18 h. Cells were washed with ice-cold 1x PBS solution twice and then lysed and sonicated using an XL2020 sonicator (Fisher Scientific, Pittsburgh, PA) in ice-cold lysis buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Nonidet P-40, 0.25% sodium deoxycholate, 1 mM EGTA, 1 mM sodium orthovanadate, 1 mM NaF, 1 μg/ml aprotinin, 1 μg/ml leupeptin, 1 μg/ml pepstatin, and 1 mM phenylmethylsulfonyl fluoride]. Cell lysates were centrifuged for 10 min at 4 C (20,000 x g), and the supernatants were used for either immunoprecipitation or Western blotting.
Immunoprecipitation of IR and IGF-IR
Aliquots of supernatant containing 500-1000 μg protein in 1000 μl lysis buffer were incubated with 25 μl primary antibody against either IR or IGF-IR (2.5 μg/ml) overnight at 4 C. Protein A/G plus IgG-agarose was then added and the mixture was kept at 4 C for 1 h with gentle rocking. After washing six times with lysis buffer, the beads were spun down (1000 x g for 30 sec), resuspended in 50 μl 2x sample buffer [375 mM Tris-HCl (pH 6.8), 12% sodium dodecyl sulfate, 60% glycerol, 300 mM dithiothreitol, and 0.06% bromophenol blue], and boiled for 5 min.
Immunoblotting
Equal amount of IR or IGF-IR immunoprecipitate or aliquots of cell lysate supernatant containing approximately 100 μg protein were diluted with an equal volume of sodium dodecyl sulfate sample buffer and electrophoresed on a 10% polyacrylamide gel, transferred to nitrocellulose, and blocked with 5% low-fat milk in Tris-buffered saline plus Tween 20. Subsequently membranes were probed with antibodies against IR, IGF-IR, phospho-Tyr (p-Tyr) (Santa Cruz Biotechnology, Santa Cruz, CA), phospho-Akt1 (Ser 473) (Upstate Cell Signaling, Lake Placid, NY), phospho-ERK1/2, or phospho-eNOS (Ser1177) (New England BioLabs, Beverly, MA) for 1 h at 4 C. This was followed by a donkey antirabbit IgG coupled to horseradish peroxidase, and the blots were developed using enhanced chemiluminescence detection (Amersham Life Sciences, Piscataway, NJ). To assure equal loading of proteins, membranes probed with antibodies against above-mentioned phosphoproteins were stripped with restore Western blot stripping buffer (Pierce Chemical Co., Rockford, IL) and reprobed with antibodies against IR, IGF-IR, Akt1, ERK1/2, or eNOS as appropriate.
Immunocytochemical staining
The double-staining protocols were the same as we described previously (32, 33). Briefly, bAECs were grown in slide chambers as described above. Cells were fixed with cold methanol for 10 min at –20 C, washed three times in PBS, permeabilized in PBS containing 0.05% Triton X-100 and 1% horse serum for 30 min at room temperature, and incubated with mouse monoclonal anti-IR antibody (Chemicon International, Temecula, CA; 1:50) and rabbit polyclonal antibody against IGF-IR (Santa Cruz Biotechnology; 1:50) (double labeling) overnight at 4 C. After washing three times with PBS, cells were then incubated with species-specific secondary antibodies conjugated with a fluorochrome (either Cy2 or Cy3) (Jackson ImmunoResearch, West Grove, PA) at 1:200 dilutions for 45 min at room temperature, washed three more times with PBS, and coverslipped with the antifade mounting medium.
Confocal imaging
The double-immunocytochemical labeling was examined simultaneously using a two-color BX50 WI confocal microscope (Olympus, Tokyo, Japan) equipped with krypton and argon laser as described previously (32, 33). An x-y-z-axis scanning method was employed. The images were scanned through up to x100 objectives, acquired at a resolution of 1024 x 1024 pixels, and stored in 24-bit TIFF format. To address whether the immunoreactivity was located within the cells, a series of optical sections at a thickness of less than 0.1 μm was acquired from the top to the bottom of the cells along the z-axis.
Results
Time course of insulin action in cultured bAECs
To examine the time course of IR activation in cultured bAECs, cells were incubated with 100 nM insulin for 0, 10, 20, 30, 40, and 50 min. We used 100 nM insulin because insulin at this concentration has repeatedly been shown to strongly stimulate phosphorylation of Akt, eNOS, and other insulin-activated pathways. Cells were then processed as described above, immunoprecipitated with anti-IR, and immunoblotted with anti-p-Tyr. As shown in Fig. 1, insulin stimulated IR Tyr phosphorylation within 10 min, and this reached a maximum at 30 min and declined thereafter. Based on this finding, we used 30 min incubation time for all subsequent experiments.
Insulin at physiological concentrations stimulates IR Tyr phosphorylation
Inasmuch as nearly all previous studies have used pharmacologic insulin concentrations to study insulin’s actions on ECs, we next examined whether insulin at physiological concentrations stimulates IR Tyr phosphorylation in ECs and, if so, whether this is associated with its downstream signaling. Cells were incubated with insulin at 0, 0.1, 0.2, 0.5, 1, 2, 5, 10, and 100 nM for 30 min and then lysed and immunoprecipitated with anti-IR antibody. The immunoprecipitate was electrophoresed and then immunoblotted with anti-p-Tyr antibody. As shown in Fig. 2A, insulin at concentrations between 0.1 and 0.5 nM strongly stimulated IR Tyr phosphorylation, and higher physiological or pharmacological concentrations did not increase signal intensity further. No differences were seen in IR protein with these short duration insulin exposures.
We then examined whether this IR Tyr phosphorylation was associated with downstream signaling of insulin action. As shown in Fig. 2 and similar to IR Tyr phosphorylation, insulin at concentrations between 0.1 and 0.5 nM potently stimulated the phosphorylation of Akt1 (Fig. 2B), ERK1/2 (Fig. 2C), and eNOS (Fig. 2D) without significant changes in the quantity of each of these proteins present.
bAECs express both IGF-IR and insulin/IGF-I hybrid receptors
ECs possess abundant IGF-IR (27), and studies done in cell lines other than ECs have demonstrated a cross-activation of IGF-IR when insulin is present at high concentrations. Also, a recent report suggests that HUVEC, like muscle and adipose tissue, expresses insulin/IGF-I hybrid receptors (28). To ascertain whether bAECs also express insulin/IGF-I hybrid receptors, we immunoprecipitated cell lysates with either anti-IR or anti-IGF-IR and then probed with antibodies against either IR or IGF-IR. As shown in Fig. 3, IGF-IR clearly coprecipitated with IR, and a significant amount of IR coprecipitated with IGF-IR. Because the IR and IGF-IR antibodies used do not cross-react with IGF-IR and IR, respectively, our data suggest that in addition to IR and IGF-IR, insulin/IGF-I hybrid receptors are present in cultured bAECs.
Immunohistochemical double staining of the bAECs prepared for confocal microscopy as described in Materials and Methods demonstrated a significant spatial colocalization of IR and IGF-IR that is explicable by either distinct IR and IGF-IR localized to specific cellular domains or insulin/IGF-I hybrid receptors in bAECs (Fig. 4).
Insulin at high concentrations activates IGF-IR and insulin/IGF-I hybrid receptors in bAECs
To examine the dose response of IGF-IR and insulin/IGF-I hybrid receptors to insulin, bAECs were serum starved for 16 h and incubated with various concentrations of insulin (0, 0.1, 0.2, 0.5, 1, 2, 5, 10, and 100 nM) for 30 min. Cells were then lysed and immunoprecipitated with anti-IGF-IR. The immunoprecipitation products were electrophoresed and immunoblotted first with anti-p-Tyr and then with anti-IGF-IR after membrane stripping. Unlike IR response, insulin did not significantly stimulate the Tyr phosphorylation of IGF-IR at concentrations less than 1 nM. However, between 1 and 5 nM insulin effectively stimulated IGF-IR phosphorylation, and this increased progressively as the insulin concentration was further raised (Fig. 5A).
To further ascertain that the insulin-induced Tyr phosphorylation was indeed from Tyr phosphorylation of IGF-IR (in either the IGF-IR or hybrid receptors), but not from that of IR, we neutralized the IGF-IR with its specific neutralizing antibody IGF-I receptor (Ab-3) (EMD Biosciences, San Diego, CA) before insulin treatment. Cells were incubated with insulin at 0, 0.2, 2, 10, and 100 nM for 30 min with or without pretreatment of cells with IGF-I receptor (Ab-3) antibody (1 μg/ml) for 30 min, lysed, immunoprecipitated with anti-IR or anti-IGF-IR, and then immunoblotted with anti-p-Tyr. As shown in Fig. 5B, insulin potently stimulated the Tyr phosphorylation of IR at all concentrations, and this was not affected by the pretreatment of cells with IGF-I receptor (Ab-3) antibody. However, insulin-stimulated Tyr phosphorylation of IGF-IR at 2, 10, and 100 nM was completely abolished by preincubating the cells with IGF-I receptor (Ab-3) antibody. Despite this, insulin-stimulated Akt1 phosphorylation was retained in cells pretreated with IGF-I receptor (Ab-3) antibody (Fig 5B), suggesting insulin indeed acted via IR.
Discussion
The current study provides the first clear demonstration that insulin, at concentrations well within the physiologic range, can stimulate the tyrosine phosphorylation of its own receptors on ECs and in addition phosphorylates relevant regulatory proteins within the PI3-kinase and ERK signaling pathways. Both pathways are downstream of IR and are pivotal in mediating insulin’s multiple cellular actions. Whereas we also confirmed that ECs possess IGF-IR and hybrid insulin/IGF-I receptors in addition to IR, these receptors are likewise activated by insulin but at higher insulin concentrations.
Considering first what was seen when the ECs were exposed to physiologic insulin concentrations, it is clear that this was sufficient to substantially activate downstream insulin signaling including at least Akt1 and eNOS as well as ERK1/2. This was seen with insulin concentrations that had no effect to enhance the tyrosine phosphorylation of the IGF-IR subunit. This certainly suggests that these actions result from insulin stimulation of its own receptor tyrosine kinase. That preincubation of ECs with IGF-IR neutralizing antibody completely abolished insulin-stimulated IGF-IR Tyr phosphorylation but not IR Tyr phosphorylation or Akt1 serine phosphorylation confirms that insulin indeed can activate its receptor kinase activity and downstream signaling independent of IGF-IR.
These results support the hypothesis that insulin’s physiologic actions on the vasculature, which contribute to insulin-mediated glucose disposal, may indeed result, at least in part, from direct actions on the ECs. For example, recent results from in vivo studies in our laboratory indicate that very modest increases in plasma insulin concentration from 60 to 300 pM exert a nitric oxide (NO)-dependent action to recruit capillaries within skeletal muscle (6, 7, 34). These effects occur very promptly, being significant by 10 min and fully established by 30 min, a time course similar to that observed here for Tyr phosphorylation of IR. Furthermore, these in vivo responses precede any discernible metabolic effect of insulin on skeletal muscle, implying a more direct effect on the vasculature. Previous reports that insulin augments endothelial NO production or phosphorylation of Akt or eNOS have involved using insulin at concentrations of 10 nM or greater (21, 22, 24, 25). Thus, the current results in cultured bAECs are quite consistent with what has been seen in vivo and would be consonant with the hypothesis that insulin can act directly on the endothelium at physiologic concentrations to exert NO-mediated biologic effects. These observations, of course, in no way exclude the possibilities that insulin might also affect smooth muscle cells and that direct actions on these cells may be importantly involved in insulin’s in vivo vascular action.
The concept that insulin has an important physiologic action on the ECs is perhaps challenged by the observation that mice deficient in IR selectively in the ECs (VENIRKO mice) are insulin resistant only when studied on a low-salt diet (35). However, because targeted deletion of IR in skeletal muscle also minimally affects glucose metabolism, it is clear that either other developmental compensation can occur (expression of both eNOS and endothelin-1 are altered in VENIRKO mice) or our ability to finely assess significant but not gross dysfunction is not in hand. It is obviously easier to dismiss the action of insulin on ECs as of no physiologic significance if it is seen only at supraphysiologic insulin concentrations. Insulin’s action on ECs has previously been most thoroughly studied by Quon and colleagues using HUVECs (21, 22, 24, 25). That laboratory demonstrated that insulin induces phosphorylation of IR, Akt, and eNOS. The latter two actions are blunted by inhibition of PI-3-kinase. However, in their studies no significant effect was seen at insulin concentrations less than 10 nM, and its effect was half maximal at approximately 500 nM. Whether this much reduced insulin sensitivity, compared with our current work, relates to differences between bovine aortic and human venous cells or other specific aspects of the experimental protocol is uncertain. In a number of other studies using either pulmonary arterial (26) or aortic ECs (36, 37), clear demonstration of effects of insulin again were seen only with unphysiologically high concentrations. We would emphasize that findings from our current work in bAECs fully support the conclusions reached by Quon and colleagues from studies using higher insulin concentrations in HUVECs.
Our observation that higher concentrations of insulin (1–5 nM) significantly stimulate IGF-IR tyrosine phosphorylation confirmed the crossover effect of insulin described in other tissues. Generally, in vitro studies of the IGF-IR suggest that it has a more than 100 times lesser relative affinity for insulin than does IR, and the insulin/IGF-I hybrid receptors behave similarly to IGF-IR in insulin or IGF-I binding affinity (38, 39). In the current study, we saw stimulation of the tyrosine phosphorylation of IGF-IR in ECs when insulin concentrations were between 1 and 5 nM or 10- to 50-fold above the lowest insulin dose that stimulated IR Tyr phosphorylation. It is possible that this reflects, at least in part, a greater abundance of IGF-IR and insulin/IGF-I hybrid receptors, compared with IR in the ECs. IGF-I, like insulin, can stimulate NO production in cultured ECs (40) and increase skeletal muscle blood flow in humans (41).
Others have reported that the IGF-IR is as much as 10-fold more abundant than IR on ECs (at least in HUVECs) (42). The contribution of hybrid receptors to this estimate is not known. Our results from IGF-IR and IR reciprocal immunoprecipitation and immunoblotting study (Fig. 3) and immunocytochemical staining/confocal microscopy (Fig. 4) suggested that indeed there are ample insulin/IGF-I hybrid receptors in ECs as well. However, this technique cannot exclude the possibility that the IGF-I and insulin receptors are part of a macromolecular complex that is not disrupted by the lysis procedure and they are being jointly immunoprecipitated as part of a larger complex. We did not attempt to quantitate the relative proportions of insulin, IGF-I, and insulin/IGF-I hybrid receptors present on ECs. We did, however, attempt to define the contribution of IR and IGF-IR or hybrid receptors to insulin signaling by varying insulin concentrations throughout the physiologic and pharmacologic range. Interestingly, we did not observe a further increase in IR Tyr phosphorylation when insulin was present at pharmacological concentrations. One might assume that Tyr phosphorylation of the IR subunits from the insulin/IGF-I hybrid receptors should result in higher signal intensity. Whether this is due to the semiquantitative nature of the Western blotting technique, some more complex interaction between the subunits of the hybrid receptor, or a decline in native IR phosphorylation at very high insulin concentrations was not investigated.
We note that whereas insulin-stimulated tyrosine phosphorylation of its own receptor appeared to reach a maximum between 0.2 and 0.5 nM, phosphorylation of Akt and eNOS continued to increase up to insulin concentrations of 100 nM. This also appeared to occur in the presence of IGF-IR blocking antibody (Fig. 5B). Several factors may account for this. First, because the insulin concentration is further raised, it is possible that insulin is competing with the IGF-I blocking antibody because both compounds associate reversibly with the IGF-IR; second, the hybrid receptors we report (see also Ref.28) may be less sensitive to the IGF-IR blocking antibody than is the native IGF-IR, allowing some further stimulation of the IGF-I/insulin signal transduction pathways at high insulin concentrations. In addition, because there is both amplification of signal within the PI3-kinase/Akt pathway and cross talk between the various arms of IR signaling cascades (e.g. the ERK pathway and the Akt pathway), there may not be a one-to-one quantitative correspondence between IR phosphorylation and phosphorylation of downstream signaling molecules. Finally, Western blotting is by its nature a semiquantitative method that also might prevent a one-to-one correspondence between IR and Akt or eNOS phosphorylation.
In conclusion, bAECs express IGF-IR and insulin/IGF-I hybrid receptors in addition to IR, but the phosphorylation/activation of IR alone is very sensitive to physiological doses of insulin. High doses of insulin cross-activate IGF-IR and insulin/IGF-I hybrid receptors, and this may confound interpretation of previous studies of high-dose insulin action on ECs. The ability of insulin to both phosphorylate its receptor and activate downstream signaling pathways at insulin concentrations well within the physiological range supports the consideration of these cells as a potentially important insulin target tissue. Nevertheless, our data do not clarify whether the vascular action of insulin is impaired in pathological conditions associated with insulin resistance, (e.g. obesity and type 2 diabetes mellitus). Much further study is needed to define this relationship, if any.
Footnotes
This work was supported by a research grant from the American Diabetes Association (to Z.L.) and National Institutes of Health Grants RR-15540 (to Z.L.), DK-38578 and DK-54058 (to E.J.B.), and P30-DK063609 (to the University of Virginia Diabetes Endocrinology Research Center).
Abbreviations: bAEC, Bovine aortic EC; EC, endothelial cell; eNOS, endothelial isoform of nitric oxide synthase; HUVEC, human umbilical vein EC; IGF-IR, IGF-I receptor; IR, insulin receptor; NO, nitric oxide; PI3-kinase, phosphatidylinositol 3-kinase; p-Tyr, phospho-Tyr.
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