Low concentrations of insulin-like growth factor (IGF) binding protein-1 (IGFBP1) are associated with insulin resistance, diabetes, and cardiovascular disease. We investigated whether increasing IGFBP1 levels can prevent the development of these disorders. Metabolic and vascular phenotype were examined in response to human IGFBP1 overexpression in mice with diet-induced obesity, mice heterozygous for deletion of insulin receptors (IR+/−), and ApoE−/− mice. Direct effects of human (h)IGFBP1 on nitric oxide (NO) generation and cellular signaling were studied in isolated vessels and in human endothelial cells. IGFBP1 circulating levels were markedly suppressed in dietary-induced obese mice. Overexpression of hIGFBP1 in obese mice reduced blood pressure, improved insulin sensitivity, and increased insulin-stimulated NO generation. In nonobese IR+/− mice, overexpression of hIGFBP1 reduced blood pressure and improved insulin-stimulated NO generation. hIGFBP1 induced vasodilatation independently of IGF and increased endothelial NO synthase (eNOS) activity in arterial segments ex vivo, while in endothelial cells, hIGFBP1 increased eNOS Ser1177 phosphorylation via phosphatidylinositol 3-kinase signaling. Finally, in ApoE−/− mice, overexpression of hIGFBP1 reduced atherosclerosis. These favorable effects of hIGFBP1 on insulin sensitivity, blood pressure, NO production, and atherosclerosis suggest that increasing IGFBP1 concentration may be a novel approach to prevent cardiovascular disease in the setting of insulin resistance and diabetes.
Insulin resistance, obesity, and type 2 diabetes are associated with reduced bioavailability of endothelial nitric oxide (NO) and predisposition to atherosclerosis. Cardiovascular disease develops ∼15 years earlier in the presence of diabetes and accounts for the majority of deaths in these individuals (1). The current epidemic of diabetes (2) and the predicted increase in diabetes prevalence by the year 2030 (3) represent major challenges to global healthcare resources. Disappointing results from recent clinical trials investigating a strategy of intensive blood glucose control to reduce cardiovascular events argue for novel approaches to reduce cardiovascular risk in individuals with diabetes (4).
Insulin-like growth factor (IGF) binding proteins (IGFBPs) comprise a family of proteins that modulate IGF bioactivity through high-affinity binding (5). IGFBP1 is a 30 kDa circulating protein expressed predominantly in the liver that has been implicated in reproductive physiology and metabolic homeostasis (5–7). Both inhibitory and stimulatory effects of IGFBP1 on IGF bioactivity are recognized. In addition, in certain cell types, IGFBP1 regulates cellular actions independently of IGFs (8). Specifically, IGFBP1 modulates cell migration independently of IGF via an interaction of the Arg-Gly-Asp sequence in its COOH-terminal domain with cell surface integrin receptors (9–12). Under physiological conditions, insulin-mediated suppression of hepatic IGFBP1 production in response to nutritional cues confers dynamic regulation of IGF bioavailability (13). IGFBP1 has therefore been proposed as a player in glucose counterregulation and metabolic homeostasis (14,15). In cross-sectional studies, circulating IGFBP1 concentrations correlate with insulin sensitivity (16–22), while in longitudinal studies, low circulating IGFBP1 concentrations predict the development of glucose intolerance and diabetes (23,24). In addition to these data supporting a role of IGFBP1 in metabolism, accumulating evidence suggests that IGFBP1 may contribute to cardiovascular pathophysiology. First, IGFBP1 concentrations are inversely associated with cardiovascular risk (16), carotid artery intima thickening (25), and overt macrovascular disease (26) in cross-sectional studies in humans. Second, we previously reported that transgenic (tg) overexpression of human IGFBP1 in mice was associated with increased vascular NO generation and lower blood pressure (27). Therefore, we hypothesized that low IGFBP1 levels may be permissive for the development of both metabolic diseases and related cardiovascular complications and that IGFBP1 could be a target for therapeutic manipulation.
Here, we investigated whether increasing IGFBP1 in mice subjected to obesity, insulin resistance, or atherosclerosis could ameliorate the development of overt metabolic and cardiovascular disease.
RESEARCH DESIGN AND METHODS
Mice and diet.
The tg mice overexpressing human (h)IGFBP1 (GenBank Accession Number NM_000596.2; transgene copy number ∼20) and generated as previously described (28) were backcrossed for a minimum of six generations on to a C57BL/6 background. Heterozygous hIGFBP1tg mice were used for experiments. Sequence similarity between human and murine IGFBP1 is 66%. However, because the NH2- and COOH-terminal domains of IGFBP1 (which participate in interactions with IGFs) remain highly conserved between species, hIGFBP1 is fully compatible with the murine-IGF axis (28). IR+/− mice were bred from a colony originally obtained from the Medical Research Council (Harwell, U.K.). IGF1Rfloxed mice were from Columbia University (New York, NY) and were crossed with Tie2-Cre tg mice (The Jackson Laboratory) for two to six generations to generate mice homozygous for the floxed allele and heterozygous for the Tie-2-Cre transgene. ApoE−/− mice were from a colony at our institution originally purchased from The Jackson Laboratory. Mice were housed in a conventional animal facility (12-h light/dark cycle) at constant temperature (22°C). Genotype was determined by PCR of ear samples. All mice were established on a C57BL/6 background or were backcrossed onto a C57BL/6 background for at least six generations. To avoid confounding by alterations in sex hormones, experiments were performed exclusively in male mice with littermates used as controls.
To induce obesity, mice received a high-fat/high-energy diet (F3282; Bio-Serv, Frenchtown, NJ) with the following composition: protein 20.5%, fat 36%, ash 3.5%, carbohydrate 35.7%, moisture <10%, and energy 5.49 kcal/g (60% of energy from fat). Control mice received standard rodent chow (BK001; Special Diets Services, Witham, Essex, U.K.) composed of protein 19.64%, fat 7.52%, ash 6.21%, moisture 10%, fiber 3.49%, nitrogen-free extract 54.90%, and energy 3.29 kcal/g. In accordance with our previous observation that diet F3282 rapidly induces an adverse cardiometabolic phenotype in mice (29), the diet was administered ad libitum from weaning for 8 weeks. Food intake was estimated by weighing food on a daily basis during the first 2 weeks of the feeding period. To induce atherosclerotic plaques, ApoE−/− mice received Western diet containing 21% pork fat and supplemented with 0.15% cholesterol (Special Diets Services). Animal experiments were approved by the ethical review committee at the University of Leeds and were performed in accordance with the Animals (Scientific Procedures) Act 1986.
Metabolic studies and plasma assays.
In glucose tolerance tests, mice were fasted overnight before injection with 1 mg/kg glucose i.p. For insulin tolerance tests, mice were fasted for 4 h before injection with 0.75 units/kg recombinant human insulin i.p. (Actrapid; Novo Nordisk). Blood glucose was measured with a portable glucometer. Plasma insulin was quantified by an ultrasensitive mouse ELISA kit (Crystal Chem, Downers Grove, IL), IGFBP1 was measured by an ELISA kit (Diagnostic Systems Laboratories, Webster, TX) that is unaffected by phosphorylation status and does not cross-react with other binding proteins, and IGF-I was quantified by a mouse/rat Quantikine ELISA kit (R&D Systems, Minneapolis, MN). Lipoprotein analyses were carried out in terminal blood samples using an automated analyzer in the Department of Clinical Chemistry, Leeds Teaching Hospitals National Health Service Trust, U.K.
Blood pressure measurement.
Blood pressure was measured by tail volume-pressure recording (CODA2; Kent Scientific, Torrington, CT) in conscious animals in a temperature-controlled restrainer as previously described (27,30,31). Animals were acclimatized to the restrainer during six training sessions before blood pressure measurement.
Body mass was measured after 4 and 8 weeks of high-fat diet. Epididymal fat pads were weighed as a conventional measure of obesity in rodents. Samples of epididymal adipose tissue were fixed in 10% formalin and embedded in paraffin. Adipocyte area was calculated by hematoxylin-eosin stained sections as previously described (32)
Segments of thoracic aorta were suspended in physiological salt solution from strain gauge in an eight-chamber organ bath at 37°C as described (27,30,31). Dose-response curves were constructed (Chart V5.6, ADInstruments) for cumulative addition of phenylephrine after preincubation with insulin (100 mU/mL Actrapid; Novo Nordisk), hIGFBP1 (500 ng/mL with 1 mg/mL BSA carrier protein or BSA alone; Novozymes Biopharma AU Limited, Thebarton, Australia), IGF-I (100 nmol/L), NG-monomethyl-l-arginine (l-NMMA; 0.1 mmol/L), or LY294002 (10 μmol/L).
Human umbilical vein endothelial cells (HUVECs) and human coronary artery endothelial cells (HCAECs) (PromoCell) were cultured in M199 medium supplemented with 20% FCS, 15 μg/mL endothelial cell growth factor, 1% l-glutamine, 20 mmol/L HEPES, 1 μmol/L sodium pyruvate, 5 units/mL heparin, 100 units/mL penicillin, 100 μg/mL streptomycin, and 25 μg/mL amphotericin and maintained in a humidified incubator with 5% CO2 at 37°C.
Immunoblotting and immunofluorescence microscopy.
Protein expression and phosphorylation status were analyzed in cell lysates or homogenized aorta by immunoblotting using the following antibodies: endothelial NO synthase (eNOS), Ser1177 p-eNOS (BD Biosciences), Akt, Ser473 p-Akt, GSK-3β, Ser21/9 p-GSK-α/β (Cell Signaling Technology), and β-actin (Santa Cruz Biotechnology). All antibodies were used at 1:1,000 dilution with the exception of β-actin at 1:3,000. Horseradish peroxidase–conjugated secondary antibodies were visualized using an enhanced chemiluminescence kit (Pierce). For immunofluorescence, intact cells were fixed using 4% paraformaldehyde, permeabilized using 0.5% Triton X-100, blocked with fish-skin gelatin, and probed with primary anti-Ser1177 phospho-eNOS antibodies, using anti-mouse IgG (Invitrogen) for immunofluorescence detection. Slides were imaged under ×100 magnification (Olympus, Hamburg, Germany). Fluorescence intensity was estimated using CellB software (Version 3.0, Olympus Soft Imaging Solutions, Hamburg, Germany) by placing regions of interest within the cytoplasm of ∼100 cells per high-power field. Exposure times and number of cells counted were standardized across experiments.
A total of 0.5 μCi/mL 14C-arginine was added for 5 min to quiesced washed cells bathed in 0.25% HEPES-BSA, followed by hIGFBP1 (500 ng/mL) for 30 min. Chilled 5 mmol l-arginine per 4 mmol/L EDTA was used to stop the reaction, and cells were trypsinized, alcohol denatured, and centrifuged. Cell pellets were dissolved in 20 mmol/L HEPES-Na+ (pH 5.5) and soluble component separated by centrifuge. Equilibrated DOWEX resin was used to separate Arg from citrulline, with the latter then quantified by liquid-scintillation counting and normalized to total cell protein content.
ApoE−/− mice were crossed with hIGFBP1tg mice to generate ApoE−/− hIGFBP1tg mice. After attaining 20 g body mass, mice were fed Western diet for 12 weeks, which was shown in preliminary experiments to be optimal to induce atherosclerotic lesions (data not shown). Aortas were carefully excised after perfusion/fixation, opened longitudinally, fixed in paraformaldehyde overnight, and stained with Oil Red O. Washed aortas were opened and mounted en face and images digitally captured to allow quantification of plaque burden using Image-Pro software (version 7.0, Media Cybernetics). Paraffin-embedded sections of aortic sinus were stained with Miller stain, and plaque area was quantified by planimetry using Image-Pro software (version 7.0, Media Cybernetics). Necrotic core was quantified by planimetry of acellular regions of plaques covered by a fibrous cap. Fibrous cap thickness was quantified by measuring the mean thickness of plaques in each section.
IGFBP1 mRNA expression.
RNA was isolated from freshly harvested aorta or liver using Tri Reagent (Sigma-Aldrich). After DNAse treatment, 1 μg RNA was reverse transcribed using High Capacity RNA to cDNA kit. Real-time PCR was performed (Applied Biosystems) using SYBR Green PCR Master Mix and the following primers: human IGFBP: Forward: AGGCTCTCCATGTCACCAACA, Reverse: CTCCTGATGTCTCCTGTGCCTT; mouse IGFBP Forward: CAGAGATGACAGAGGAGCAGCT, Reverse: TGCTGCTATAGGTGCTGATGG. Results were normalized to expression of β-actin.
Data are presented as mean ± SEM. Data were compared using Student t test, with the exception of aortic concentration-response curves, which were compared using two-way ANOVA followed by the application of post hoc Bonferroni test. P < 0.05 was considered significant.
Increasing hIGFBP1 concentrations improves insulin sensitivity in obesity.
To investigate whether increasing IGFBP1 levels can reverse the metabolic and vascular sequelae of obesity, we fed an obesogenic diet to mice overexpressing hIGFBP1. In control mice, obesity was associated with suppression of hepatic IGFBP1 mRNA expression (Fig. 1A) and a marked reduction in total circulating IGFBP1 concentration (Fig. 1B). As previously published, tg overexpression of hIGFBP1 in chow-fed animals results in a fivefold increase in circulating IGFBP1 concentration (28). In obesity, we found that tg overexpression of hIGFBP1 similarly increased plasma IGFBP1 concentration (Fig. 1C) without altering total plasma IGF-I (Fig. 1D). hIGFBP1tg mice developed a similar increase in body mass compared with wild-type (WT) littermates (Fig. 1E). Because constitutive overexpression of IGFBP1 has been reported to inhibit adipocyte expansion (33), we measured adipocyte diameter in epididymal fat depots after induction of obesity. In our mice, in which hIGFBP1 is expressed under the control of its native promoter and regulatory sequences, we found no effect on epididymal fat pad mass (28 ± 9 in WT vs. 34 ± 2 mg in tg mice; P = 0.47) or adipocyte cross-sectional area (Fig. 1F–I). Food intake was similar in hIGFBP1 mice and WT controls receiving the obesogenic diet (2.54 ± 0.07 vs. 2.57 ± 0.12 g/day; P = 0.85). hIGFBP1tg mice were at least partially protected from the metabolic consequences of obesity. Diet-induced obesity increased fasting blood glucose and induced glucose intolerance in WT animals; these effects were ameliorated in hIGFBP1tg mice (Fig. 2A). Insulin sensitivity, as indicated by an insulin tolerance test, was significantly enhanced in hIGFBP1tg animals (Fig. 2B). Plasma insulin concentrations were not significantly altered by IGFBP1 overexpression (Fig. 2C).
Increasing hIGFBP1 concentrations enhances vascular NO production and lowers blood pressure in obesity.
We next tested whether favorable effects on vascular function accompanied the protective effect of hIGFBP1 on glucose regulation in obesity. Impaired insulin-mediated NO production (34) is implicated in insulin resistance–associated vascular dysfunction. In nonobese mice, insulin induces a hypocontractile response to phenylephrine in aorta by enhancing NO generation (30,31). This effect was absent in aorta harvested from obese mice (Fig. 2D), consistent with vascular insulin resistance. Overexpression of hIGFBP1 restored hypocontractility to insulin in vessels from obese mice (Fig. 2E), indicating increased insulin-induced NO production in tg animals. Vasodilation of aorta to acetylcholine (Fig. 2F) or sodium nitroprusside (not shown) was unchanged. Insulin stimulates NO production via phosphorylation of eNOS at Ser residues (35). Aortic Ser1177 eNOS phosphorylation after insulin injection was significantly upregulated in hIGFBP1tg animals (Fig. 2G). hIGFBP1 overexpression ameliorated the increase in systolic blood pressure observed in WT obese animals (Table 1).
Increasing hIGFBP1 concentrations enhances vascular NO production and lowers blood pressure in insulin resistance.
We next examined whether increasing hIGFBP1 improves vascular function in a nonobese model of insulin resistance. We crossed hIGFBP1tg mice with mice heterozygous for deletion of the insulin receptor (IR+/−). IR+/− mice have increased blood pressure and endothelial dysfunction despite maintaining normal glucose regulation through increased insulin secretion (31). Glucose tolerance and insulin sensitivity were unchanged when hIGFBP1 was overexpressed in IR+/− mice (Fig. 3A and B), but systolic blood pressure was increased (120 ± 3 vs. 102 ± 4 mmHg; P = 0.01). Overexpression of hIGFBP1 protected IR+/− mice from increased blood pressure (Table 1). Insulin did not alter the contractile response of aorta from IR+/− mice (Fig. 3C), in keeping with vascular insulin resistance. Overexpression of hIGFBP1 restored insulin-mediated hypocontractility (Fig. 3D), indicating that IGFBP1 reverses vascular insulin resistance. Vasodilatation to acetylcholine or sodium nitroprusside (data not shown) was unchanged by hIGFBP1 overexpression.
hIGFBP1 increases endothelial NO production independently of IGF-I by stimulating eNOS phosphorylation via the phosphatidylinositol 3-kinase pathway.
To determine whether these phenotypes resulted from a direct effect of hIGFBP1, we carried out experiments in isolated segments of WT mouse aorta. Incubation of aorta with hIGFBP1 reduced contractility to phenylephrine (Fig. 4A). This was abolished by endothelial denudation (Fig. 4B) and coincubation with l-NMMA (Fig. 4C), indicating that hIGFBP1 stimulates endothelial NO generation. Coincubation with LY294022, an inhibitor of phosphatidylinositol 3-kinase (PI3K), abolished this response (Fig. 4D), indicating that PI3K activation is required for hIGFBP1-induced NO production. These effects were observed in a serum-free preparation, suggesting that hIGFBP1 stimulates vascular NO production independently of IGFs. To exclude a contribution from IGF contamination or local IGF secretion, we repeated our experiments in mice with endothelial-specific deletion of type 1 IGF receptors generated by crossing IGF1Rfloxed mice (36,37) with Tie2-Cre tg mice (36). As expected, aortas from these animals did not vasodilate in response to IGF-I (Fig. 4E). However, hIGFBP1 did reduce contractility to phenylephrine (Fig. 4F), reaffirming that hIGFBP1-induced NO generation is not mediated by type 1 IGF receptors.
To confirm a direct effect of hIGFBP1 on endothelial NO production, we undertook experiments in primary human endothelial cells. hIGFBP1 induced concentration- and time-dependent eNOS Ser1177 phosphorylation in HCAECs (Fig. 5A–D). hIGFBP1 similarly stimulated dose-dependent Ser473 phosphorylation of Akt and Ser9 phosphorylation of its downstream substrate GSK-3β (Fig. 5E and F) but had no effect on total levels of expression of eNOS, Akt, or GSK-3β. hIGFBP1 induced Ser1177 eNOS phosphorylation in HUVECs (Fig. 5G–I), an effect blocked by coincubation with LY294022 (Fig. 5J). To confirm that the changes in eNOS phosphorylation resulted in changes in NO production, we demonstrated that hIGFBP1 stimulated LY294022-inhibitable eNOS activity in HUVECs (Fig. 5K).
IGFBP1 overexpression reduces atherosclerosis.
Finally, we hypothesized that increasing IGFBP1 levels would result in protection from atherosclerosis. To address this, we crossed hIGFBP1tg mice with ApoE−/− mice. After feeding a Western-type diet for 12 weeks, plaque burden was significantly reduced in ApoE−/−IGFBP1tg mice, both in en face preparations of aorta (Fig. 6A and B) and in aortic sinus sections (Fig. 6C and D). Overexpression of hIGFBP1 did not affect plaque complexity as measured by analysis of necrotic core area (33.8 ± 3.7% in ApoE−/− vs. 29.1 ± 3.2% in ApoE−/−IGFBP1tg; P = 0.32) or fibrous cap thickness (36 ± 4 μm in ApoE−/− vs. 28 ± 4 μm in ApoE−/−IGFBP1tg; P = 0.17). There were no significant differences in plasma lipoprotein concentrations between ApoE−/−IGFBP1tg mice and ApoE−/− mice (Fig. 6E).
To ascertain whether IGFBP1 is expressed in the vascular wall, we carried out real-time PCR to assess expression of IGFBP1 in atherosclerotic aorta. Murine IGFBP1 expression was detectable at low levels in the aorta of ApoE−/− and ApoE−/−IGFBP1tg mice (Fig. 6F). hIGFBP1 was detectable only in aorta from hIGFBP1tg mice (Fig. 6F), as expected.
During the past 2 decades, a series of epidemiological studies has linked low circulating IGFBP1 concentrations with insulin resistance, type 2 diabetes, and cardiovascular disease. We previously demonstrated that hIGFBP1 overexpression increased vascular NO generation in mice (27), suggesting that low IGFBP1 concentrations may play a mechanistic role in vascular pathophysiology. However, whether the effects of IGFBP1 on the vascular endothelium were direct or indirect, and whether increasing IGFBP1 concentrations could be of therapeutic potential in preventing disease, was unknown. In this study, we demonstrate for the first time, using a series of mouse models, that elevating IGFBP1 levels improves whole-body insulin sensitivity and glucose tolerance, lowers blood pressure, enhances vascular NO production, and reduces susceptibility to atherosclerosis.
A critical novel finding of the current study is that hIGFBP1 has a direct effect on vascular endothelium, resulting in increased NO generation. Mechanistically, although the proximal signaling steps are yet to be elucidated, we have shown that hIGFBP1 stimulates dose-dependent NO production in isolated murine vessels and in primary human endothelial cells. hIGFBP1-stimulated NO generation is mediated by activation of the PI3K/Akt signaling pathway, resulting in Ser phosphorylation of eNOS (Fig. 7). This mirrors the pathway by which insulin and growth factors stimulate NO generation in endothelial cells (38). hIGFBP1 overexpression did not alter arterial responses to acetylcholine, suggesting that calcium/calmodulin-mediated activation of eNOS is not affected by IGFBP1. It is important to note that hIGFBP1-induced NO production was demonstrated in the absence of IGF-I and in vessels in which endothelial type 1 IGF receptors were deleted. Although IGFBP1 has been shown to directly modulate cell migration in other cell types (9–12), this is the first time that IGFBP1 has been demonstrated to act independently of IGFs as a vasoactive molecule in its own right in cells of the vascular wall. Our findings parallel the recent observation that IGFBP3, another member of the IGFBP family, can affect vascular pathophysiology by modulation of NO (39). Although hepatic IGFBP1 production accounts for the majority of circulating IGFBP1, we found expression of both the native murine transcript and the human transgene in the vascular wall. Because IGFBP1 is known to be expressed in human atherectomy specimens (40), though, interestingly, not in primary culture of human vascular smooth muscle (41), our findings raise the possibility that increased local expression of hIGFBP1 as well as increased concentrations of hIGFBP1 in the circulation may contribute to the vascular phenotypes we observed. While other potential contributory factors cannot be excluded, it is tempting to speculate that increased NO generation promoted by elevated circulating IGFBP1 concentrations, or enhanced insulin-induced vascular hypocontractility, accounted for the reduction in blood pressure we observed. It is noteworthy that the plasma concentrations of IGFBP1 achieved through tg overexpression in our obesity model, which were similar to those recorded in insulin-resistant humans (18,19), remained lower than the circulating levels in nonobese WT mice. This suggests that a modest increase in IGFBP1 is sufficient to generate a favorable phenotypic effect.
The contribution of IGFBP1 to glucose regulation has previously been attributed to modulation of IGF-I bioavailability (7). Our observations, however, indicate that hIGFBP1 functions as an insulin-sensitizing peptide per se. In contrast to our findings with hIGFBP2 (32), hIGFBP1 overexpression improved whole-body insulin sensitivity and reduced glucose intolerance in the setting of obesity in the absence of change in body weight or fat cell size. hIGFBP1 overexpression did not affect glucose regulation in our model of genetic insulin resistance (IR+/− mice). However, whole-body glucose homeostasis is relatively preserved in this model, in which we have demonstrated that insulin receptor deletion has a disproportionately greater effect on endothelial insulin sensitivity than on whole-body insulin sensitivity (31). In contrast, enhanced endothelial sensitivity to insulin, a critical determinant of vascular homeostasis (42), was demonstrated in both hIGFBP1tg obese mice and genetically insulin-resistant mice, in which overexpression of hIGFBP1 enhanced insulin-induced arterial relaxation and eNOS phosphorylation. Because IGFBP1 was not present in the organ bath salt solution, these enhanced vascular responses to insulin observed in murine vessels are likely to result from chronic exposure to increased IGFBP1 concentrations in vivo (Fig. 7). Defining the mechanistic basis for the insulin sensitization we observed is beyond the focus of this investigation. However, one can speculate that enhanced insulin-mediated NO generation may have contributed to the improvement in systemic insulin sensitivity (43) or that higher circulating IGFBP1 levels may have upregulated insulin signaling in metabolically active tissues in a similar manner to that we observed in the endothelium. Despite the systemic insulin sensitization we demonstrated in hIGFBP1-overexpressing nutritionally obese mice, it is noteworthy that plasma insulin concentrations were not lower in tg mice than in controls. This may be attributable to a direct effect of hIGFBP1 overexpression on pancreatic insulin secretion, considering that IGFBP1 has been shown to amplify glucose-stimulated insulin secretion in intact islets (44).
The favorable metabolic phenotypes of hIGFBP1 overexpression observed here are discordant with other murine models in which IGFBP1 was overexpressed under the control of heterologous promoters (45,46). Differences in tissue restriction of expression or absolute plasma IGFBP1 concentrations achieved may explain this discrepancy. Alternatively, it is possible that maintained responsiveness of IGFBP1 to nutritional cues, allowing dynamic regulation of plasma levels (achieved in our mouse but using the native promoter sequence to drive transgene expression), is required. It is intriguing that mice with knockout of IGFBP1 do not have any short-term major metabolic dysfunction (47), implying other factors can compensate for the lack of IGFBP1. Effects of IGFBP1 deletion on vascular function and long-term metabolism have not been studied.
The robust effect of IGFBP1 overexpression we observed on atherosclerotic plaque development in ApoE−/− mice was striking. It is tempting to speculate that the inhibition of atherosclerosis we observed in ApoE−/− mice overexpressing hIGFBP1 is attributable to enhanced NO bioavailability as a result of upregulation of Akt-mediated eNOS phosphorylation. Certainly, the evidence that Akt signaling and eNOS-derived NO play fundamental roles in atherosclerosis is compelling (48,49). However, we cannot exclude the possibility that other factors may have contributed to the reduced atherosclerosis we observed in hIGFBP1tg mice.
Although our observations of increased NO generation, reduced blood pressure, and reduced atherosclerotic plaque in IGFBP1tg mice are in keeping with the inverse associations between IGFBP1 and cardiovascular disease demonstrated in cross-sectional studies, we acknowledge that not all human data support a favorable association between IGFBP1 concentrations and cardiovascular risk. Specifically, high circulating concentrations of IGFBP1 have been linked with the development of heart failure in the elderly (50) and with an adverse prognosis after myocardial infarction (51,52). Associated changes in COOH-terminal provasopressin (copeptin) may explain the apparent prognostic importance of IGFBP1 concentrations highlighted in these trials (53); however, further studies of the molecular effects of IGFBP1 in these clinical scenarios are clearly warranted.
In conclusion, our findings demonstrate that hIGFBP1 has insulin-sensitizing, blood pressure–lowering, and antiatherosclerotic properties. We have identified a novel function of hIGFBP1 as a vasomodulatory molecule in its own right, here acting as a regulator of endothelial NO production independent of the type 1 IGF receptor by activating the PI3K/Akt/phospho-eNOS pathway. Our observations have significant clinical implications, raising the possibility that enhancing IGFBP1 levels may be a therapeutic option to protect individuals from insulin resistance, hypertension, and atherosclerosis.
This work was funded by the British Heart Foundation and supported in part through a Yorkshire Enterprise Fellowship for S.B.W.; S.B.W. is a British Heart Foundation Intermediate Clinical Research Fellow. A.R., V.E., E.R.D., R.M.C., M.B.K., A.Ab., and A.Az. held British Heart Foundation Clinical Research Training Fellowships. A.M.S. is a British Heart Foundation Professor of Cardiology.
No potential conflicts of interest relevant to this article were reported.
A.R. and M.B.K. prepared the manuscript and performed the in vitro and in vivo experiments. V.E., J.S., N.Y.Y., E.R.D., M.G., H.I., A.Ab., H.V., A.Az., and P.S. performed in vitro and in vivo experiments. A.V.-P., P.J.G., and K.E.P. supervised the project. R.M.C. and J.K.S. critically reviewed the manuscript. S.X. generated the IGF1Rfloxed mouse. A.M.S. and M.T.K. conceived the study, secured funding, designed the experiments, and supervised the project. S.B.W. conceived the study, secured funding, designed the experiments, supervised the project, and prepared the manuscript. S.B.W. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.