Skip to main content
NCBI home page
Endocrinology logoLink to Endocrinology
. 2009 May 7;150(8):3475–3482. doi: 10.1210/en.2009-0172

Insulin and Insulin-Like Growth Factor-I Receptors Differentially Mediate Insulin-Stimulated Adhesion Molecule Production by Endothelial Cells

Guolian Li 1, Eugene J Barrett 1, Seung-Hyun Ko 1, Wenhong Cao 1, Zhenqi Liu 1
PMCID: PMC2717867  PMID: 19423756

Abstract

Patients with type 2 diabetes are hyperinsulinemic and insulin resistant and develop premature atherosclerosis. High concentrations of insulin stimulate the production of adhesion molecules by endothelial cells (ECs). ECs express abundant IGF-I receptors as well as insulin receptors. Whether IGF-I receptors contribute to insulin-induced endothelial production of adhesion molecules is unknown. Bovine aortic ECs (BAECs) were incubated with insulin (100 nm) for 24 h. The cellular content of vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) was measured, and monocyte adhesion to ECs was quantified. Insulin increased both VCAM-1 (P < 0.001) and ICAM-1 (P < 0.0002) content, which was accompanied by an increased number of monocytes adherent to BAECs (P = 0.0001). Inhibition of either MAPK kinase-1 or p38 MAPK but not phosphatidylinositol 3-kinase abolished insulin-mediated production of adhesion molecules. Insulin receptor small interfering RNA knockdown abolished insulin-stimulated increases of ICAM-1 but not VCAM-1. Conversely, IGF-I receptor blockade with either a neutralizing antibody or specific small interfering RNA eliminated insulin-induced VCAM-1 but not ICAM-1 production. Blockade of signaling via either the insulin or IGF-I receptors decreased monocyte adherence to BAECs (P < 0.01 for each). We conclude that insulin and IGF-I receptors differentially mediate the production of adhesion molecules by ECs and monocyte adhesion onto the vascular endothelium in response to the hyperinsulinemic state. Dual-receptor activation may most effectively contribute to the pathogenesis of atherosclerotic disease in diabetes.

Insulin at pharmacological concentrations directly stimulates the production of adhesion molecules from the endothelial cells (EC) and the adhesion of monocytes to the EC, and both insulin and IGF-I receptors are involved in mediating these processes.

Patients with type 2 diabetes are insulin resistant and hyperinsulinemic and experience an increased morbidity and mortality from accelerated atherosclerotic disease. Strong evidence indicates that insulin resistance and endothelial dysfunction are key players early in the pathogenesis of atherosclerosis (1,2,3,4). Endothelial expression of cellular adhesion molecules, including intercellular adhesion molecule-1 (ICAM)-1, vascular cell adhesion molecule-1 (VCAM)-1, and E-selectin, is critical in modulating cell-cell interactions between circulating leukocytes and vascular endothelium and subsequent migration of leukocytes across the endothelium (5,6,7,8). In otherwise healthy volunteers, circulating concentrations of E-selectin, ICAM-1, and VCAM-1 significantly correlate with the degree of insulin sensitivity (9), and plasma concentrations of these adhesion molecules are elevated in patients with insulin resistance (10,11,12,13,14). Recent evidence has implicated an important role of insulin in this process.

In response to insulin, endothelial cells (ECs) produce nitric oxide (NO) via the phosphatidylinositol 3-kinase (PI3-kinase)/protein kinase B (Akt)/endothelial nitric oxide synthase pathway as well as various adhesion molecules via the MAPK pathway (3,8,15,16,17,18,19,20). In insulin-resistant states, insulin action through the PI3-kinase/Akt pathway is blunted (3), leading to a compensatory increase in plasma insulin concentrations. Because signaling through the MAPK pathway remains intact or is enhanced in insulin-resistant states (3,21,22), elevated plasma insulin concentrations may enhance the production of various adhesion molecules and thereby predispose insulin-resistant patients to atherosclerosis. Indeed, in cultured human umbilical vein ECs (HUVECs), high concentrations of insulin induce a dose-dependent increase of VCAM-1 on the EC surface and increase monocyte adhesion to the ECs (23). This insulin-stimulated endothelial expression of adhesion molecules uses a MAPK-dependent but PI3K-independent signaling pathway (23,24). Moreover, blockade of PI3K-dependent pathways further enhances the effects of insulin or vascular endothelial growth factor to increase the expression of the adhesion molecules (24).

We and others (20,25) have previously reported that ECs express abundant IGF-I receptors as well as insulin/IGF-I hybrid receptors in addition to insulin receptors. Insulin, at high concentrations, activates not only insulin receptors but also IGF-I receptors (20). However, the physiological roles of insulin and IGF-I receptors in the regulation of EC function remains to be fully defined. In particular, whether insulin regulates the production of adhesion molecules by ECs via insulin and/or IGF-I receptors is not known.

In the current study, we examined the contributions of insulin and IGF-I receptors to insulin-stimulated endothelial content of adhesion molecules and monocyte adhesion to the ECs. VCAM-1 and ICAM-1 are examined because they are both expressed by the endothelium and play crucial roles in mediating monocyte-endothelium interactions and participate in inflammation and atherosclerosis (6,26). We here report for the first time that insulin and IGF-I receptors differentially regulate endothelial production of adhesion molecules and monocyte adhesion to the ECs in the presence of high concentrations of insulin and this may contribute to the pathogenesis of accelerated atherosclerosis in patients with diabetes/insulin resistance.

Materials and Methods

Culture of ECs

Bovine aortic ECs (BAECs) were purchased from Lonza Walkersville, Inc. (Walkersville, MD). Cells in primary culture were cultured in endothelial basic media supplemented with 5% fetal bovine serum, bovine brain extract, human epithelial growth factor (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 and serum starvation for 16–18 h. Cells were then treated with insulin at a final concentration of 100 nm for 24 h. This concentration was selected to maximally stimulate both insulin receptors and IGF-I receptors (20). For some experiments, IGF-I receptor neutralizing antibody (AB-3, 100 ng/ml) wortmannin (PI3-kinase inhibitor, 100 nm), PD98059 [MAPK kinase (MEK)-1 inhibitor, 25 μm], or SB203580 (p38 MAPK inhibitor, 10 μm) was added 30 min before the addition of insulin. Wortmannin and PD98059 were purchased from Sigma-Aldrich (St. Louis, MO). SB203580 was purchased from EMD Bioscience (San Diego, CA). Cells were then used for immunoprecipitation and/or Western blotting or monocyte adhesion analysis.

Insulin receptor and IGF-I receptor knockdown using small interfering RNA (siRNA)

The cognate siRNA against insulin receptors (sc-29370), scrambled siRNA and siRNA transfection reagent (sc-29528) were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Insulin receptor siRNA or scrambled siRNA was introduced into cultured BAECs according to the manufacturer’s instructions. The IGF-I receptor SMARTpool siRNA (M-003012), scrambled siRNA and siIMPORTER (64-101) were obtained from Upstate Cell Signaling (Lake Placid, NY). IGF-I receptor siRNA or scrambled siRNA was introduced into cultured BAECs according to the manufacturer’s instructions. Cells in passages 3–6 were seeded in six wells (1 × 104 cells/cm2) 1 d before transfection. Protein knockdown efficiencies were assessed at 24, 48, 72, and 96 h after transfection (see Fig. 3).

Figure 3.

Figure 3

Open in a new tab

Insulin and IGF-I receptor siRNA knockdown. BAECs were transfected with specific siRNAs against either insulin receptors or IGF-I receptors or scrambled siRNA as described in Materials and Methods. A, Time course of insulin receptor siRNA effect. B, Time course of IGF-I receptor siRNA effect. P < 0.001 for both IR and IGF-IR (ANOVA). IR, Insulin receptors. IGF-IR, IGF-I receptors.

Immunoprecipitation and Western blotting

Cells were washed twice with ice-cold 1× PBS solution and lysed by sonication using a Fisher 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 5 min at 4 C (13,000 × g), and the supernatants were used for immunoprecipitation and/or Western blotting.

For immunoprecipitation of insulin receptor β-subunit (IRβ) and IGF-I receptor β-subunit (IGF-Iβ), aliquots of supernatant containing 500-1000 μg protein in 1000 μl lysis buffer were incubated with 25 μl primary antibody against either IRβ or IGF-IRβ (Santa Cruz Biotechnology) (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 × g for 30 sec), resuspended in 50 μl 2× 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.

For immunoblotting, equal amounts of IRβ or IGF-IRβ immunoprecipitate or aliquots of cell lysate supernatant containing approximately 100 μg protein were diluted with an equal volume of sodium dodecyl sulfate sample buffer. The samples were electrophoresed on a 10% polyacrylamide gel, transferred to nitrocellulose, and blocked with 5% low-fat milk in Tris-buffered saline plus 0.001% Tween 20. Subsequently membranes were probed with antibodies against IRβ, IGF-IRβ, β-actin (Santa Cruz Biotechnology), phospho-tyrosine (p-Tyr) (Upstate), VCAM-1, ICAM-1(R&D Systems, Inc., Minneapolis, MN) 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). Autoradiographic films were scanned densitometrically and quantitated using Imagequant 3.3 (Molecular Dynamics, Piscataway, NJ). Densities were quantitated and the ratios of the protein of interest to β-actin calculated.

Monocyte adhesion assay

U937 monocytes (American Tissue Culture Collection, Manassas, VA) were grown in RPMI 1640 medium with 1 mm sodium pyruvate, 4.5 g/liter glucose, 1.5 g/liter sodium bicarbonate, 2 mm glutamine, and 10% fetal bovine serum. Monocytes were labeled with calcein AM for 15 min at 37 C. For adhesion assays, BAECs were grown to confluence in eight-well slide chambers, and an aliquot of U937 cell suspension containing approximately 50,000 cells was added to each well. After the mixture being incubated for 30 min at 37 C, each well was rinsed six times with endothelial basic media containing 2% fetal calf serum to remove unbound U937 monocytes. Cells were then fixed in 1% glutaraldehyde. Labeled U937 monocytes bound to ECs were counted using epifluorescence microscopy (Olympus 13 × 51, Hauppauge, NY). For each experiment, a total of 10 views (×40 magnifications) were counted and the numbers averaged.

Statistical analysis

Results are expressed as mean ± sem. Statistical analysis was performed using one-way repeated-measures ANOVA with Bonferroni post hoc testing or Student t test as appropriate. P < 0.05 was considered statistically significant.

Results

We first examined the time course of insulin-stimulated endothelial production of VCAM-1 and ICAM-1. After incubating BAECs with 100 nm insulin for 0, 12, 20, 24, and 48 h, protein contents of VCAM-1 and ICAM-1 were quantitated in cell homogenates. We opted to use an insulin concentration of 100 nm because we intended to examine the contributory role of insulin and IGF-I receptors in insulin-mediated endothelial production of adhesion molecules, and we have previously shown that insulin at this concentration maximally activates both the insulin and IGF-I receptors (20) (Fig. 1A). Insulin potently increased the protein contents of both VCAM-1 and ICAM-1 after 12 h of incubation, and the levels remained elevated for all time points examined. For consistency and convenience, all subsequent studies were performed using the incubation time of 24 h.

Figure 1.

Figure 1

Open in a new tab

Insulin stimulates endothelial production of VCAM-1 and ICAM-1 and increases monocyte adhesion to the ECs. Cells were serum starved for 16 h and then incubated with insulin (100 nm, to maximally stimulate both insulin and IGF-I receptors) for 24 h. Cells were then harvested as detailed in the text. A, Representative Western blots illustrating insulin activation of both IRβ and IGF-Iβ (incubation time: 30 min). IP, Immunoprecipitation; p-Tyr, phospho-Tyr. B, Representative Western blots of VCAM-1 and ICAM-1. C, Quantitative analysis of VCAM-1 production. D, Quantitative analysis of ICAM-1 production. E, Representative immunofluorescent images of monocyte adhesion to endothelial cells (×40 magnifications). F, Dose response of insulin effect on ICAM-1 and VCAM-1 production. Results are representative of three separate experiments. *, P < 0.001, **, P < 0.0002 compared with control.

As shown in Fig. 1, insulin incubation for 24 h markedly increased the EC content of both VCAM-1 (0.36 ± 0.02 vs. 0.81 ± 0.04, P < 0.001; Fig. 1, B and C) and ICAM-1 (0.26 ± 0.02 vs. 0.76 ± 0.03, P < 0.0002. Fig. 1 B and D). This was accompanied by a significant increase in monocyte adhesion to the ECs (Fig. 1E). The insulin-stimulated expressions of VCAM-1 and ICAM-1 were insulin concentration dependent (Fig. 1F). At physiological concentrations (0.2 and 0.5 nm), insulin increased ICAM-1 but not VCAM-1 expression. At high concentrations (10 and 100 nm), both ICAM-1 and VCAM-1 expressions were increased. We have previously shown that insulin at physiological concentrations activates only insulin receptors but not IGF-I receptors, and at pharmacological concentrations both receptors are activated by insulin (20).

To examine the potential signaling pathways involved in the insulin-mediated production of adhesion molecules, we incubated BAECs with insulin in the absence or presence of 100 nm wortmannin (PI3-kinase inhibitor), 25 μm PD98059 (MEK1 inhibitor), or 10 μm SB203580 (a specific p38 MAPK inhibitor). Wortmannin was without effect on insulin-stimulated production of VCAM-1 (P = 0.0001) and ICAM-1 (P < 0.0001) (Fig. 2), suggesting that insulin’s effect was PI3-kinase independent as reported by other laboratories (23,24). However, inhibition of either MEK1 or p38 MAPK completely abrogated insulin-stimulated production of both VCAM-1 and ICAM-1 (Fig. 2). Thus, insulin increases endothelial production of adhesion molecules via a PI3-kinase-independent, but MEK1- and p38 MAPK-dependent pathway.

Figure 2.

Figure 2

Open in a new tab

Insulin stimulates endothelial production of VCAM-1 and ICAM-1 via MEK1- and p38 MAPK-dependent pathways. Cells were serum starved for 16 h and then incubated with insulin (100 nm) for 24 h. Cells were then harvested as detailed in Materials and Methods. Top panel, Representative Western blots of VCAM-1 and ICAM-1. Middle panel, Quantitative analysis of VCAM-1 production. Lower panel, Quantitative analysis of ICAM-1 production. Results are representative of three to four separate experiments. *, P < 0.001, **, P < 0.0002, #, P = 0.0001, ##, P < 0.0001 compared with respective control (noninsulin treated cells).

Because insulin exerts its biological actions via the activation of insulin receptors and/or IGF-I receptors, we next examined the contributory roles of these receptors in insulin-mediated production of adhesion molecules and monocyte adhesion. Using a siRNA technique to knock down insulin and IGF-I receptors, respectively, the knockdown efficiency results are shown in Fig. 3. Insulin receptor siRNA decreased the protein content of insulin receptors between 48 and 96 h (P < 0.001, ANOVA) without affecting IGF-I receptor expression. At 72 h, insulin receptor protein content was decreased by 81% vs. control (P = 0.0002). Similarly, IGF-I receptor siRNA markedly decreased the protein content of IGF-I receptors between 24 and 96 h (P < 0.001, ANOVA) without affecting the protein expression of insulin receptors. Similar to the insulin receptor siRNA effect, the knockdown was most dramatic at 72 h, with IGF-I receptor protein content being only 12% of the control cell values (P < 0.0001). As such, all subsequent siRNA studies were executed using a transfection time of 72 h.

To examine the role of insulin receptors in the insulin-mediated adhesion molecule production by the ECs, we incubated BAECs transfected with insulin receptor siRNA with insulin for 24 h. Like in control cells, insulin significantly stimulated the production of both VCAM-1 (P < 0.01) and ICAM-1 (P < 0.02) in cells transfected with scrambled siRNA (Fig. 4A). However, insulin receptor knockdown markedly inhibited insulin-stimulated increase in ICAM-1 production without affecting the production of VCAM-1 (P < 0.01). This was accompanied by a blunted response in insulin-stimulated monocyte adhesion to the cultured ECs (398 ± 27 vs. 245 ± 12, scrambled siRNA vs. insulin receptor siRNA, P < 0.002, Fig. 4B).

Figure 4.

Figure 4

Open in a new tab

Effect of insulin receptor knockdown on insulin-stimulated endothelial production of adhesion molecules and monocyte adhesion. Cells were transfected with the IR siRNA or scrambled siRNA as described in the text. A, Insulin-stimulated endothelial production of VCAM-1 and ICAM-1. *, P = 0.0001, **, P < 0.01, #, P < 0.005, ##, P < 0.02 compared with respective control (noninsulin-treated cells). B, Insulin-mediated monocyte adhesion to ECs (×40 magnifications). *, P < 0.001 compared with respective control (noninsulin-treated cells); #, P < 0.002 compared with insulin-treated scrambled control. IR, Insulin receptors.

We next examined whether IGF-I receptors are involved in insulin-mediated endothelial production of adhesion molecules. Cells were transfected with IGF-I receptor or scrambled siRNA before incubation with insulin. As shown in Fig. 5A, IGF-I receptor knockdown essentially abolished the insulin-stimulated increase in VCAM-1 production. However, insulin-mediated increases of ICAM-1 production were unaffected (P < 0.002). Similar to cells after insulin receptor knockdown, the insulin-stimulated increase in monocyte adhesion to the IGF-I receptor knockdown cells was also significantly blunted (336 ± 7 vs. 197 ± 4, P < 0.0001, Fig. 5B).

Figure 5.

Figure 5

Open in a new tab

Effect of IGF-IR knockdown on insulin-stimulated endothelial production of VCAM-1 and ICAM-1 and monocyte adhesion. Cells were transfected with the IGF-IR SMARTpool siRNA (Upstate Cell Signaling) or scrambled siRNA as described in the text. A, Effect on insulin-stimulated endothelial production of VCAM-1 and ICAM-1. *, P < 0.001, #, P < 0.04, **, P < 0.002 compared with respective control (noninsulin-treated cells). B, Effect on insulin-mediated monocyte adhesion to ECs (×40 magnifications). *, P < 0.0001 compared with respective control (noninsulin-treated cells); #, P < 0.0001 compared with insulin-treated scrambled control. Results are representative of three to five separate experiments. IGF-IR, IGF-I receptor.

To further confirm that IGF-I receptors are indeed solely responsible for mediating insulin-stimulated production of VCAM-1, we also examined the effect of incubating cells with an antibody (AB-3) specific for the exofacial domain of the IGF-I receptors to block insulin binding to the IGF-I receptors before and during insulin incubation. We previously demonstrated that AB-3 antibody at the concentration used completely blocks insulin-stimulated IGF-IRβ subunit tyrosine phosphorylation (20). As expected, AB-3 per se did not change the basal levels of either VCAM-1 or ICAM-1. However, it completely blocked insulin-mediated production of VCAM-1 without affecting ICAM-1 (P < 0.001, ANOVA, Fig. 6A). This again was associated with a marked decrease in insulin-mediated monocyte adhesion to the ECs (#, P < 0.01, Fig. 6B).

Figure 6.

Figure 6

Open in a new tab

Effect of IGF-IR neutralization on insulin-stimulated endothelial production of VCAM-1 and ICAM-1 and monocyte adhesion. Cells were serum starved for 16 h and then incubated with insulin (100 nm) for 24 h in the presence or absence of AB-3 antibody. Cells were then harvested as detailed in Materials and Methods. A, IGF-IR neutralization abrogated insulin-stimulated endothelial production of VCAM-1 but not ICAM-1. *, P < 0.001. B, IGF-IR neutralization decreased insulin-mediated monocyte adhesion to ECs. * P < 0.001, ** P < 0.03 compared with respective control (noninsulin-treated cells); #, P < 0.01 compared with insulin-treated control. C, IGF-IR neutralization did not affect the effect of IR siRNA knockdown on insulin-stimulated endothelial production of VCAM-1 and ICAM-1. *, P = 0.015. Results are representative of three independent experiments. IR, Insulin receptor; IGF-IR, IGF-I receptor.

Because we demonstrated that insulin and IGF-I receptors differentially modulate insulin-mediated endothelial production of VCAM-1 and ICAM-1, we finally examined whether blockade of one receptor signaling pathway would affect insulin effect on adhesion molecule production via the other receptor signaling pathway. We neutralized the IGF-I receptors using the antibody AB-3 in cells transfected with insulin receptor siRNA before incubating these cells with insulin. Similar to control cells without siRNA treatment, we observed a complete abrogation of insulin-mediated increase in VCAM-1 production (Fig. 6C). However, like cells with insulin receptor knockdown but intact and functioning IGF-I receptors (Fig. 4), insulin-stimulated ICAM-1 production in these cells was also significantly blunted (Fig. 6C). These results suggest that insulin actions through the two receptor systems are mutually exclusive.

Discussion

Many studies have shown that insulin-resistant states are associated with elevated plasma concentrations of adhesion molecules and accelerated formation of atherosclerotic plaques. We have previously shown that insulin (at concentrations seen in the insulin resistant states) potently activate both the insulin and IGF-I receptors in ECs (20). In the current study, we demonstrated that insulin at high concentrations activated both insulin and IGF-I receptors, increased endothelial VCAM-1 and ICAM-1 content, and promoted the adhesion of monocytes to the ECs. Our results indicate that this insulin action is mediated via the ERK1/2 and p38 MAPK pathways, and both insulin and IGF-I receptors contributed to these processes.

Consistent with previous reports, we show here that insulin-stimulated endothelial production of VCAM-1 and ICAM-1 is PI3-kinase independent and ERK1/2 and p38 MAPK dependent. This is true for both insulin receptor- and IGF-I receptor-mediated effects. Because both receptor actions are inhibited by blockade of MEK1 or p38 MAPK, the locus for differential expression of ICAM-1 or VCAM-1 is not resolved by our study. However, it is likely that this may involve the gene transcription process because both ERK1/2 and p38 MAPK play important roles in regulating gene expressions. Because independent inhibition of either p38 MAPK or MEK1 signaling is equally effective in blocking insulin-stimulated cell adhesion molecule expression in control cells, further studies are needed to explore the responsible upstream signaling events and to characterize the signaling pathways downstream of the receptors related to insulin-stimulated cell adhesion molecule expression. Our findings do suggest that hyperinsulinemia, acting via both receptor types and MAPKs, can enhance the production of adhesion molecules and contribute to initiating the atherosclerotic process. This is clinically significant due to the high prevalence of insulin-resistant diseases and the marked morbidity and mortality associated with these diseases.

Although in the current study we used chemical inhibitors only to examine the roles of PI3-kinase, ERK1/2, and p38 MAPK and these compounds may have additional effects beyond inhibition of their respective kinase, we have previously shown that these chemical compounds are kinase specific and at the concentrations used do not interfere with insulin action through other pathways in our experimental setting (22). Our observation that inhibition of p38 MAPK completely abrogated insulin-mediated adhesion molecule production suggests that, other than ERK1/2, p38 MAPK also plays a pivotal role in this process. Indeed, Madonna et al. (23) have shown that insulin (1–100 nm) dose-dependently induced the surface expression of VCAM-1 (by nearly 2-fold) in cultured HUVECs, which was paralleled by increased U937 cell adhesion, and these effects were completely abolished with p38 MAPK inhibition. The p38 MAPK belongs to the MAPK superfamily; is a stress-activated serine/threonine protein kinase with major functions in apoptosis, cytokine production, transcriptional regulation, and cytoskeletal reorganization; and plays a pivotal role in vascular inflammation and endothelial dysfunction/repair (27,28). We have previously shown that insulin potently stimulates the phosphorylation of p38 MAPK, which correlates with enhanced kinase activity (29). Furthermore, selective inhibition of p38 MAPK dose-dependently reduces TNF-α or lipopolysaccharide-induced ICAM-1 expression in cultured HUVECs and restores nitric oxide-mediated endothelium-dependent relaxation in spontaneously hypertensive-stroke prone rats (30). Thus, it is not surprising that p38 MAPK mediates insulin-stimulated production of adhesion molecules. Together with our recent findings of p38 MAPK mediating TNF-α-induced insulin resistance and fatty acid-induced apoptosis in the cultured ECs (22,31), our results strongly suggest that p38 MAPK plays an important role in accelerated development of atherosclerosis in patients with insulin resistance.

Our findings that insulin stimulated ICAM-1 production via its action on the insulin receptors and VCAM-1 production through the IGF-I receptors suggest that both receptors are involved in the production of adhesion molecules and monocyte adhesion to the ECs in the presence of high concentrations of insulin. Insulin receptor knockdown using its specific siRNA significantly decreased insulin-induced production of ICAM-1 but not VCAM-1. On the other hand, blockade of IGF-I receptor signaling by using either specific siRNA or neutralizing antibody AB-3 each abolished insulin-mediated production of VCAM-1 without affecting that of ICAM-1. The result is a significantly decreased monocyte adhesion to ECs. The pathophysiological outcomes of insulin and IGF-I receptors differentially mediating the production of ICAM-1 and VCAM-1 by ECs remain to be further defined. Both VCAM-1 and ICAM-1 are expressed in ECs and play important but different roles in leukocyte recruitment in that VCAM-1 is more important in leukocyte arrest and rolling and ICAM-1 in arrest and migration (5,6,32). Inasmuch as blockade of either insulin receptors or IGF-I receptors each independently decreased insulin-stimulated monocyte adhesion to the ECs, our data strongly implicate the dual involvement of both insulin and IGF-I receptors in the pathogenesis of atherosclerosis in the insulin-resistant states.

That insulin receptor knockdown with siRNA only partially abrogated the increased production of ICAM-1 suggests that the production of ICAM-1 is either partially regulated through the insulin receptors or due to residual insulin receptors present on the endothelial membranes. As shown in Fig. 3A, insulin receptor siRNA maximally knocked down only 81% of insulin receptors at 72 h. Thus, it is very likely that these residual insulin receptors might be able to mediate part of the insulin action. Another possibility is that this portion of the ICAM-1 production occurs via the IGF-I receptors because insulin receptor siRNA had no effect on IGF-I receptor expression (Fig. 3B). However, this is highly unlikely because we demonstrated (Fig. 6C) that in the presence of both insulin receptor siRNA and IGF-I receptor neutralizing antibody AB-3, the blunted production of ICAM-1 persisted. At the concentrations used, AB-3 completely abolishes insulin signaling via the IGF-I receptors (20).

A limitation of the current study is whether the findings that insulin and IGF-I receptors differentially mediate the expression of ICAM-1 and VCAM-1 can be generalized to humans due to the species-dependent nature of the relative distribution of insulin and IGF-I receptors in endothelium. Indeed, our results differ from a previous report by Aljada et al. (33), who found that insulin incubation (6 nm) for 2 d reduced the expression of ICAM-1 through an increase in the expression of endothelial NO synthase and NO generation in cultured human aortic ECs. In the current study, we observed a marked increase in the expression of both VCAM-1 and ICAM-1 after incubating cells with insulin for 6–48 h. There are several important differences between the two studies that might have contributed to this difference, including the use of different ECs (BAECs vs. human aortic ECs), different insulin concentrations (100 vs. 6 nm), and the use of fetal bovine serum (serum starvation vs. 2%). We used higher insulin concentrations to ensure maximal activation of both the insulin and IGF-I receptors because our intention was to dissect the contributory roles of these receptors, not dose response. Consistent with our findings, several reports from different laboratories previously reported that insulin directly stimulates endothelial expression of various adhesion molecules. Madonna et al. (23) demonstrated that high concentrations of insulin selectively promote the expression of VCAM-1 in cultured human ECs, whereas Montagnani et al. (24) showed that insulin increased monocyte rolling on ECs by about 30% and potently enhanced the mRNA expression of both VCAM-1 and E-selectin in the presence of PI3-kinase blockade. Additionally, Balaram et al. (34,35) demonstrated that incubating HUVECs with IGF-I significantly elevates VCAM-1 and ICAM-1 production in as little as 2 h. This is not surprising because IGF-I cross-activates the insulin receptors (36,37,38).

In conclusion, insulin at pharmacological concentrations directly stimulates the production of adhesion molecules from the ECs and the adhesion of monocytes to the ECs, and both insulin and IGF-I receptors are involved in mediating these processes. As insulin at concentrations seen in the insulin-resistant states is able to activate both the insulin and IGF-I receptors, and VCAM-1, but not ICAM-1, has been shown to play a major role in early atherosclerosis (39), activation of the IGF-I receptors may be important in initiating the atherosclerotic process and dual receptor activation may most effectively contribute to the atherosclerotic diseases in diabetes. These processes may be attenuated by decreasing insulin resistance and its associated hyperinsulinemia clinically.

Footnotes

This work was supported by American Diabetes Association Clinical Research Award 7-07-CR-34 (to Z.L.), and National Institutes of Health Grants DK-DK057878 and DK-073759 (to E.J.B.) and P30-DK063609 (to the University of Virginia Diabetes Endocrinology Research Center).

Disclosure Summary: The authors have nothing to disclose.

First Published Online May 7, 2009

Abbreviations: AB-3, IGF-I receptor neutralizing antibody; BAEC, bovine aortic EC; EC, endothelial cell; HUVEC, human umbilical vein EC; ICAM, intercellular adhesion molecule; IGF-Iβ, IGF-I receptor β-subunit; IRβ, insulin receptor β-subunit; MEK, MAPK kinase; NO, nitric oxide; PI3-kinase, phosphatidylinositol 3-kinase; siRNA, small interfering RNA; VCAM, vascular cell adhesion molecule.

References

  1. Nigro J, Osman N, Dart AM, Little PJ 2006 Insulin resistance and atherosclerosis. Endocr Rev 27:242–259 [DOI] [PubMed] [Google Scholar]
  2. Semenkovich CF 2006 Insulin resistance and atherosclerosis. J Clin Invest 116:1813–1822 [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Kim JA, Montagnani M, Koh KK, Quon MJ 2006 Reciprocal relationships between insulin resistance and endothelial dysfunction: molecular and pathophysiological mechanisms. Circulation 113:1888–1904 [DOI] [PubMed] [Google Scholar]
  4. Kim JA, Koh KK, Quon MJ 2005 The union of vascular and metabolic actions of insulin in sickness and in health. Arterioscler Thromb Vasc Biol 25:889–891 [DOI] [PubMed] [Google Scholar]
  5. Rao RM, Yang L, Garcia-Cardena G, Luscinskas FW 2007 Endothelial-dependent mechanisms of leukocyte recruitment to the vascular wall. Circ Res 101:234–247 [DOI] [PubMed] [Google Scholar]
  6. Galkina E, Ley K 2007 Vascular adhesion molecules in atherosclerosis. Arterioscler Thromb Vasc Biol 27:2292–2301 [DOI] [PubMed] [Google Scholar]
  7. Cybulsky MI, Gimbrone Jr MA 1991 Endothelial expression of a mononuclear leukocyte adhesion molecule during atherogenesis. Science 251:788–791 [DOI] [PubMed] [Google Scholar]
  8. Muniyappa R, Montagnani M, Koh KK, Quon MJ 2007 Cardiovascular actions of insulin. Endocr Rev 28:463–491 [DOI] [PubMed] [Google Scholar]
  9. Chen NG, Holmes M, Reaven GM 1999 Relationship between insulin resistance, soluble adhesion molecules, and mononuclear cell binding in healthy volunteers. J Clin Endocrinol Metab 84:3485–3489 [DOI] [PubMed] [Google Scholar]
  10. Song Y, Manson JE, Tinker L, Rifai N, Cook NR, Hu FB, Hotamisligil GS, Ridker PM, Rodriguez BL, Margolis KL, Oberman A, Liu S 2007 Circulating levels of endothelial adhesion molecules and risk of diabetes in an ethnically diverse cohort of women. Diabetes 56:1898–1904 [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Meigs JB, Hu FB, Rifai N, Manson JE 2004 Biomarkers of endothelial dysfunction and risk of type 2 diabetes mellitus. JAMA 291:1978–1986 [DOI] [PubMed] [Google Scholar]
  12. Bannan S, Mansfield MW, Grant PJ 1998 Soluble vascular cell adhesion molecule-1 and E-selectin levels in relation to vascular risk factors and to E-selectin genotype in the first degree relatives of NIDDM patients and in NIDDM patients. Diabetologia 41:460–466 [DOI] [PubMed] [Google Scholar]
  13. Desideri G, De Simone M, Iughetti L, Rosato T, Iezzi ML, Marinucci MC, Cofini V, Croce G, Passacquale G, Necozione S, Ferri C 2005 Early activation of vascular endothelial cells and platelets in obese children. J Clin Endocrinol Metab 90:3145–3152 [DOI] [PubMed] [Google Scholar]
  14. Hak AE, Pols HA, Stehouwer CD, Meijer J, Kiliaan AJ, Hofman A, Breteler MM, Witteman JC 2001 Markers of inflammation and cellular adhesion molecules in relation to insulin resistance in nondiabetic elderly: the Rotterdam study. J Clin Endocrinol Metab 86:4398–4405 [DOI] [PubMed] [Google Scholar]
  15. Taniguchi CM, Emanuelli B, Kahn CR 2006 Critical nodes in signalling pathways: insights into insulin action. Nat Rev Mol Cell Biol 7:85–96 [DOI] [PubMed] [Google Scholar]
  16. Zeng G, Quon MJ 1996 Insulin-stimulated production of nitric oxide is inhibited by wortmannin. Direct measurement in vascular endothelial cells. J Clin Invest 98:894–898 [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Montagnani M, Ravichandran LV, Chen H, Esposito DL, Quon MJ 2002 Insulin receptor substrate-1 and phosphoinositide-dependent kinase-1 are required for insulin-stimulated production of nitric oxide in endothelial cells. Mol Endocrinol 16:1931–1942 [DOI] [PubMed] [Google Scholar]
  18. Montagnani M, Chen H, Barr VA, Quon MJ 2001 Insulin-stimulated activation of eNOS is independent of Ca2+ but requires phosphorylation by Akt at Ser1179. J Biol Chem 276:30392–30398 [DOI] [PubMed] [Google Scholar]
  19. Zeng G, Nystrom FH, Ravichandran LV, Cong LN, Kirby M, Mostowski H, Quon MJ 2000 Roles for insulin receptor, PI3-kinase, and Akt in insulin-signaling pathways related to production of nitric oxide in human vascular endothelial cells. Circulation 101:1539–1545 [DOI] [PubMed] [Google Scholar]
  20. Li G, Barrett EJ, Wang H, Chai W, Liu Z 2005 Insulin at physiological concentrations selectively activates insulin but not insulin-like growth factor I (IGF-I) or insulin/IGF-I hybrid receptors in endothelial cells. Endocrinology 146:4690–4696 [DOI] [PubMed] [Google Scholar]
  21. Cusi K, Maezono K, Osman A, Pendergrass M, Patti ME, Pratipanawatr T, DeFronzo RA, Kahn CR, Mandarino LJ 2000 Insulin resistance differentially affects the PI 3-kinase- and MAP kinase-mediated signaling in human muscle. J Clin Invest 105:311–320 [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Li G, Barrett EJ, Barrett MO, Cao W, Liu Z 2007 Tumor necrosis factor-α induces insulin resistance in endothelial cells via a p38 mitogen-activated protein kinase-dependent pathway. Endocrinology 148:3356–3363 [DOI] [PubMed] [Google Scholar]
  23. Madonna R, Pandolfi A, Massaro M, Consoli A, De Caterina R 2004 Insulin enhances vascular cell adhesion molecule-1 expression in human cultured endothelial cells through a pro-atherogenic pathway mediated by p38 mitogen-activated protein-kinase. Diabetologia 47:532–536 [DOI] [PubMed] [Google Scholar]
  24. Montagnani M, Golovchenko I, Kim I, Koh GY, Goalstone ML, Mundhekar AN, Johansen M, Kucik DF, Quon MJ, Draznin B 2002 Inhibition of phosphatidylinositol 3-kinase enhances mitogenic actions of insulin in endothelial cells. J Biol Chem 277:1794–1799 [DOI] [PubMed] [Google Scholar]
  25. Chisalita SI, Arnqvist HJ 2004 Insulin-like growth factor I receptors are more abundant than insulin receptors in human micro- and macrovascular endothelial cells. Am J Physiol Endocrinol Metab 286:E896–E901 [DOI] [PubMed] [Google Scholar]
  26. Kevil CG, Patel RP, Bullard DC 2001 Essential role of ICAM-1 in mediating monocyte adhesion to aortic endothelial cells. Am J Physiol Cell Physiol 281:C1442–C1447 [DOI] [PubMed] [Google Scholar]
  27. Zarubin T, Han J 2005 Activation and signaling of the p38 MAP kinase pathway. Cell Res 15:11–18 [DOI] [PubMed] [Google Scholar]
  28. Liu Z, Cao W 2009 p38 mitogen-activated protein kinase: a critical node linking insulin resistance and cardiovascular diseases in type 2 diabetes mellitus. Endocr Metab Immune Disord Drug Targets 9:38–46 [DOI] [PubMed] [Google Scholar]
  29. Li J, Hu X, Selvakumar P, Russell 3rd RR, Cushman SW, Holman GD, Young LH 2004 Role of the nitric oxide pathway in AMPK-mediated glucose uptake and GLUT4 translocation in heart muscle. Am J Physiol Endocrinol Metab 287:E834–E841 [DOI] [PubMed] [Google Scholar]
  30. Ju H, Behm DJ, Nerurkar S, Eybye ME, Haimbach RE, Olzinski AR, Douglas SA, Willette RN 2003 p38 MAPK inhibitors ameliorate target organ damage in hypertension: Part 1. p38 MAPK-dependent endothelial dysfunction and hypertension. J Pharmacol Exp Ther 307:932–938 [DOI] [PubMed] [Google Scholar]
  31. Chai W, Liu Z 2007 p38 mitogen-activated protein kinase mediates palmitate-induced apoptosis but not inhibitor of nuclear factor-κB degradation in human coronary artery endothelial cells. Endocrinology 148:1622–1628 [DOI] [PubMed] [Google Scholar]
  32. Blankenberg S, Barbaux S, Tiret L 2003 Adhesion molecules and atherosclerosis. Atherosclerosis 170:191–203 [DOI] [PubMed] [Google Scholar]
  33. Aljada A, Saadeh R, Assian E, Ghanim H, Dandona P 2000 Insulin inhibits the expression of intercellular adhesion molecule-1 by human aortic endothelial cells through stimulation of nitric oxide. J Clin Endocrinol Metab 85:2572–2575 [DOI] [PubMed] [Google Scholar]
  34. Balaram SK, Agrawal DK, Allen RT, Kuszynski CA, Edwards JD 1997 Cell adhesion molecules and insulin-like growth factor-1 in vascular disease. J Vasc Surg 25:866–876 [DOI] [PubMed] [Google Scholar]
  35. Balaram SK, Agrawal DK, Edwards JD 1999 Insulin like growth factor-1 activates nuclear factor-κB and increases transcription of the intercellular adhesion molecule-1 gene in endothelial cells. Cardiovasc Surg 7:91–97 [DOI] [PubMed] [Google Scholar]
  36. Johansson GS, Arnqvist HJ 2006 Insulin and IGF-I action on insulin receptors, IGF-I receptors, and hybrid insulin/IGF-I receptors in vascular smooth muscle cells. Am J Physiol Endocrinol Metab 291:E1124–E1130 [DOI] [PubMed] [Google Scholar]
  37. Entingh-Pearsall A, Kahn CR 2004 Differential roles of the insulin and insulin-like growth factor-I (IGF-I) receptors in response to insulin and IGF-I. J Biol Chem 279:38016–38024 [DOI] [PubMed] [Google Scholar]
  38. Denley A, Carroll JM, Brierley GV, Cosgrove L, Wallace J, Forbes B, Roberts Jr CT 2007 Differential activation of insulin receptor substrates 1 and 2 by insulin-like growth factor-activated insulin receptors. Mol Cell Biol 27:3569–3577 [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Cybulsky MI, Iiyama K, Li H, Zhu S, Chen M, Iiyama M, Davis V, Gutierrez-Ramos JC, Connelly PW, Milstone DS 2001 A major role for VCAM-1, but not ICAM-1, in early atherosclerosis. J Clin Invest 107:1255–1262 [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Endocrinology are provided here courtesy of The Endocrine Society

RESOURCES