ORIGINAL ARTICLE Increasing Circulating IGFBP1 Levels Improves Sensitivity, Promotes Nitric Oxide Production, Lowers Blood Pressure, and Protects Against Atherosclerosis Adil Rajwani,1 Vivienne Ezzat,2 Jessica Smith,1 Nadira Y. Yuldasheva,1 Edward R. Duncan,2 Matthew Gage,1 Richard M. Cubbon,1 Matthew B. Kahn,1 Helen Imrie,1 Afroze Abbas,1 Hema Viswambharan,1 Amir Aziz,1 Piruthivi Sukumar,1 Antonio Vidal-Puig,3 Jaswinder K. Sethi,3 Shouhong Xuan,4 Ajay M. Shah,2 Peter J. Grant,1 Karen E. Porter,1 Mark T. Kearney,1 and Stephen B. Wheatcroft1

Low concentrations of insulin-like (IGF) binding results from recent clinical trials investigating a strategy of -1 (IGFBP1) are associated with insulin resistance, dia- intensive blood glucose control to reduce cardiovascular betes, and cardiovascular disease. We investigated whether in- events argue for novel approaches to reduce cardiovas- creasing IGFBP1 levels can prevent the development of these cular risk in individuals with diabetes (4). disorders. Metabolic and vascular phenotype were examined in Insulin-like growth factor (IGF) binding (IGFBPs) response to human IGFBP1 overexpression in mice with diet- comprise a family of proteins that modulate IGF bioactivity induced obesity, mice heterozygous for deletion of insulin recep- fi +/2 2/2 through high-af nity binding (5). IGFBP1 is a 30 kDa cir- tors (IR ), and ApoE mice. Direct effects of human (h) culating protein expressed predominantly in the liver that IGFBP1 on nitric oxide (NO) generation and cellular signaling has been implicated in reproductive physiology and were studied in isolated vessels and in human endothelial cells. – IGFBP1 circulating levels were markedly suppressed in dietary- metabolic homeostasis (5 7). Both inhibitory and stimu- induced obese mice. Overexpression of hIGFBP1 in obese mice latory effects of IGFBP1 on IGF bioactivity are recog- reduced blood pressure, improved insulin sensitivity, and in- nized. In addition, in certain cell types, IGFBP1 regulates creased insulin-stimulated NO generation. In nonobese IR+/2 cellular actions independently of IGFs (8). Specifically, mice, overexpression of hIGFBP1 reduced blood pressure and IGFBP1 modulates cell migration independently of IGF via improved insulin-stimulated NO generation. hIGFBP1 induced an interaction of the Arg-Gly-Asp sequence in its COOH- vasodilatation independently of IGF and increased endothelial terminal domain with cell surface receptors (9–12). NO synthase (eNOS) activity in arterial segments ex vivo, while 1177 Under physiological conditions, insulin-mediated suppres- in endothelial cells, hIGFBP1 increased eNOS Ser phosphor- sion of hepatic IGFBP1 production in response to nutritional ylation via phosphatidylinositol 3-kinase signaling. Finally, in ApoE2/2 mice, overexpression of hIGFBP1 reduced atheroscle- cues confers dynamic regulation of IGF bioavailability (13). rosis. These favorable effects of hIGFBP1 on insulin sensitivity, IGFBP1 has therefore been proposed as a player in glucose blood pressure, NO production, and atherosclerosis suggest that counterregulation and metabolic homeostasis (14,15). In increasing IGFBP1 concentration may be a novel approach to cross-sectional studies, circulating IGFBP1 concentrations prevent cardiovascular disease in the setting of insulin resis- correlate with insulin sensitivity (16–22), while in longitu- tance and diabetes. dinal studies, low circulating IGFBP1 concentrations pre- dict the development of glucose intolerance and diabetes (23,24). In addition to these data supporting a role of IGFBP1 in metabolism, accumulating evidence suggests nsulin resistance, obesity, and type 2 diabetes are that IGFBP1 may contribute to cardiovascular pathophysi- associated with reduced bioavailability of endothe- ology. First, IGFBP1 concentrations are inversely associated lial nitric oxide (NO) and predisposition to athero- with cardiovascular risk (16), carotid artery intima thickening ; Isclerosis. Cardiovascular disease develops 15 years (25), and overt macrovascular disease (26) in cross- earlier in the presence of diabetes and accounts for the sectional studies in humans. Second, we previously re- majority of deaths in these individuals (1). The current ported that transgenic (tg) overexpression of human epidemic of diabetes (2) and the predicted increase in di- IGFBP1 in mice was associated with increased vascular abetes prevalence by the year 2030 (3) represent major NO generation and lower blood pressure (27). Therefore, challenges to global healthcare resources. Disappointing we hypothesized that low IGFBP1 levels may be permis- sive for the development of both metabolic diseases and related cardiovascular complications and that IGFBP1 From the 1Division of Cardiovascular and Diabetes Research, Multidisciplin- could be a target for therapeutic manipulation. ary Cardiovascular Research Centre, University of Leeds, Leeds, U.K.; the 2Department of Cardiology, Cardiovascular Division, Kings College London Here, we investigated whether increasing IGFBP1 in British Heart Foundation Centre of Excellence, London, U.K.; the 3Depart- mice subjected to obesity, insulin resistance, or athero- ment of Clinical Biochemistry, University of Cambridge, Cambridge, U.K.; sclerosis could ameliorate the development of overt met- and 4Department of Genetics and Development, Columbia University Med- ical Center, New York, New York. abolic and cardiovascular disease. Corresponding author: Stephen B. Wheatcroft, [email protected]. Received 11 July 2011 and accepted 29 December 2011. RESEARCH DESIGN AND METHODS DOI: 10.2337/db11-0963 Ó 2012 by the American Diabetes Association. Readers may use this article as Mice and diet. The tg mice overexpressing human (h)IGFBP1 (GenBank long as the work is properly cited, the use is educational and not for profit, Accession Number NM_000596.2; transgene copy number ;20) and generated and the work is not altered. See http://creativecommons.org/licenses/by as previously described (28) were backcrossed for a minimum of six gen- -nc-nd/3.0/ for details. erations on to a C57BL/6 background. Heterozygous hIGFBP1tg mice were diabetes.diabetesjournals.org DIABETES 1 Diabetes Publish Ahead of Print, published online February 22, 2012 INCREASING IGFBP1 CONCENTRATION used for experiments. Sequence similarity between human and murine IGFBP1 Metabolic studies and plasma assays. In glucose tolerance tests, mice were is 66%. However, because the NH2- and COOH-terminal domains of IGFBP1 fasted overnight before injection with 1 mg/kg glucose i.p. For insulin tolerance (which participate in interactions with IGFs) remain highly conserved between tests, mice were fasted for 4 h before injection with 0.75 units/kg recombinant species, hIGFBP1 is fully compatible with the murine-IGF axis (28). IR+/2 mice human insulin i.p. (Actrapid; Novo Nordisk). Blood glucose was measured with were bred from a colony originally obtained from the Medical Research Council a portable glucometer. Plasma insulin was quantified by an ultrasensitive mouse (Harwell, U.K.). IGF1Rfloxed mice were from Columbia University (New York, ELISA (Crystal Chem, Downers Grove, IL), IGFBP1 was measured by an NY) and were crossed with Tie2-Cre tg mice (The Jackson Laboratory) for ELISA kit (Diagnostic Systems Laboratories, Webster, TX) that is unaffected by two to six generations to generate mice homozygous for the floxed allele and phosphorylation status and does not cross-react with other binding proteins, and heterozygous for the Tie-2-Cre transgene. ApoE2/2 mice were from a colony at IGF-I was quantified by a mouse/rat Quantikine ELISA kit (R&D Systems, Min- our institution originally purchased from The Jackson Laboratory. Mice were neapolis, MN). Lipoprotein analyses were carried out in terminal blood samples housed in a conventional animal facility (12-h light/dark cycle) at constant using an automated analyzer in the Department of Clinical Chemistry, Leeds temperature (22°C). Genotype was determined by PCR of ear samples. All Teaching Hospitals National Health Service Trust, U.K. mice were established on a C57BL/6 background or were backcrossed onto Blood pressure measurement. Blood pressure was measured by tail volume- a C57BL/6 background for at least six generations. To avoid confounding by pressure recording (CODA2; Kent Scientific, Torrington, CT) in conscious animals alterations in sex hormones, experiments were performed exclusively in male in a temperature-controlled restrainer as previously described (27,30,31). Animals mice with littermates used as controls. were acclimatized to the restrainer during six training sessions before blood To induce obesity, mice received a high-fat/high-energy diet (F3282; Bio- pressure measurement. Serv, Frenchtown, NJ) with the following composition: protein 20.5%, fat 36%, Obesity analysis. Body mass was measured after 4 and 8 weeks of feeding ash 3.5%, carbohydrate 35.7%, moisture ,10%, and energy 5.49 kcal/g (60% of high-fat diet. Epididymal fat pads were weighed as a conventional measure of energy from fat). Control mice received standard rodent chow (BK001; Special obesity in rodents. Samples of epididymal adipose tissue were fixed in 10% Diets Services, Witham, Essex, U.K.) composed of protein 19.64%, fat 7.52%, ash formalinandembeddedinparaffin. Adipocyte area was calculated by hematoxylin- 6.21%, moisture 10%, fiber 3.49%, nitrogen-free extract 54.90%, and energy 3.29 kcal/g. eosin stained sections as previously described (32) In accordance with our previous observation that diet F3282 rapidly induces an Vasomotor studies. Segments of thoracic aorta were suspended in physio- adverse cardiometabolic phenotype in mice (29), the diet was administered ad logical salt solution from strain gauge in an eight-chamber organ bath at 37°C as libitum from weaning for 8 weeks. Food intake was estimated by weighing food on described (27,30,31). Dose-response curves were constructed (Chart V5.6, a daily basis during the first 2 weeks of the feeding period. To induce athero- ADInstruments) for cumulative addition of phenylephrine after preincubation sclerotic plaques, ApoE2/2 mice received Western diet containing 21% pork fat and with insulin (100 mU/mL Actrapid; Novo Nordisk), hIGFBP1 (500 ng/mL with supplemented with 0.15% cholesterol (Special Diets Services). Animal experiments 1 mg/mL BSA carrier protein or BSA alone; Novozymes Biopharma AU Limited, G were approved by the ethical review committee at the University of Leeds and Thebarton, Australia), IGF-I (100 nmol/L), N -monomethyl-L-arginine (L-NMMA; were performed in accordance with the Animals (Scientific Procedures) Act 1986. 0.1 mmol/L), or LY294002 (10 mmol/L).

FIG. 1. Diet-induced obesity and IGFBP1 expression and circulating plasma concentrations in C57BL/6 mice fed high-fat diet or chow (n =8per group). Hepatic murine IGFBP1 expression was quantified by real-time RT-PCR (A), and plasma concentrations were measured by enzyme-linked immunosorbent assay (ELISA) (B). *P < 0.02 vs. chow. C and D: Plasma IGFBP1 and IGF-I concentrations, measured using ELISA, in WT and hIGFBP1tg (TG) diet-induced obese mice (n = 8 per group). **P = 0.01 vs. WT. E: Body mass of WT and TG mice during induction of dietary obesity. F–I: Representative sections of epididymal fat pads (magnification 310) and distribution of adipocyte cross-sectional area in obese WT (F and H) and TG (G and I) mice (n = 8 per group). (A high-quality digital representation of this figure is available in the online issue.)

2 DIABETES diabetes.diabetesjournals.org A. RAJWANI AND ASSOCIATES

Cell culture. Human umbilical vein endothelial cells (HUVECs) and human optimal to induce atherosclerotic lesions (data not shown). Aortas were coronary artery endothelial cells (HCAECs) (PromoCell) were cultured in M199 carefully excised after perfusion/fixation, opened longitudinally, fixed in medium supplemented with 20% FCS, 15 mg/mL endothelial cell growth factor, paraformaldehyde overnight, and stained with Oil Red O. Washed aortas were 1% L-glutamine, 20 mmol/L HEPES, 1 mmol/L sodium pyruvate, 5 units/mL opened and mounted en face and images digitally captured to allow quantifi- heparin, 100 units/mL penicillin, 100 mg/mL streptomycin, and 25 mg/mL cation of plaque burden using Image-Pro software (version 7.0, Media Cy- amphotericin and maintained in a humidified incubator with 5% CO2 at 37°C. bernetics). Paraffin-embedded sections of aortic sinus were stained with Miller Immunoblotting and immunofluorescence microscopy. Protein expression stain, and plaque area was quantified by planimetry using Image-Pro software and phosphorylation status were analyzed in cell lysates or homogenized aorta (version 7.0, Media Cybernetics). Necrotic core was quantified by planimetry by immunoblotting using the following antibodies: endothelial NO synthase of acellular regions of plaques covered by a fibrous cap. Fibrous cap thickness (eNOS), Ser1177 p-eNOS (BD Biosciences), Akt, Ser473 p-Akt, GSK-3b,Ser21/9 was quantified by measuring the mean thickness of plaques in each section. p-GSK-a/b (Cell Signaling Technology), and b-actin (Santa Cruz Biotech- IGFBP1 mRNA expression. RNA was isolated from freshly harvested aorta or nology). All antibodies were used at 1:1,000 dilution with the exception of liver using Tri Reagent (Sigma-Aldrich). After DNAse treatment, 1 mg RNA was b-actin at 1:3,000. Horseradish peroxidase–conjugated secondary antibodies reverse transcribed using High Capacity RNA to cDNA kit. Real-time PCR was were visualized using an enhanced chemiluminescence kit (Pierce). For im- performed (Applied Biosystems) using SYBR Green PCR Master Mix and the munofluorescence, intact cells were fixed using 4% paraformaldehyde, per- following primers: human IGFBP: Forward: AGGCTCTCCATGTCACCAACA, meabilized using 0.5% Triton X-100, blocked with fish-skin gelatin, and probed Reverse: CTCCTGATGTCTCCTGTGCCTT; mouse IGFBP Forward: CAGAG- with primary anti-Ser1177 phospho-eNOS antibodies, using anti-mouse IgG ATGACAGAGGAGCAGCT, Reverse: TGCTGCTATAGGTGCTGATGG. Results (Invitrogen) for immunofluorescence detection. Slides were imaged under were normalized to expression of b-actin. 3100 magnification (Olympus, Hamburg, Germany). Fluorescence intensity Statistical analysis. Data are presented as mean 6 SEM. Data were com- was estimated using CellB software (Version 3.0, Olympus Soft Imaging Sol- pared using Student t test, with the exception of aortic concentration-response utions, Hamburg, Germany) by placing regions of interest within the cyto- curves, which were compared using two-way ANOVA followed by the appli- plasm of ;100 cells per high-power field. Exposure times and number of cells cation of post hoc Bonferroni test. P , 0.05 was considered significant. counted were standardized across experiments. eNOS activity. A total of 0.5 mCi/mL 14C-arginine was added for 5 min to quiesced washed cells bathed in 0.25% HEPES-BSA, followed by hIGFBP1 RESULTS (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 Increasing hIGFBP1 concentrations improves insulin centrifuged. Cell pellets were dissolved in 20 mmol/L HEPES-Na+ (pH 5.5) and sensitivity in obesity. To investigate whether increasing soluble component separated by centrifuge. Equilibrated DOWEX resin was IGFBP1 levels can reverse the metabolic and vascular se- fi used to separate Arg from citrulline, with the latter then quanti ed by liquid- quelae of obesity, we fed an obesogenic diet to mice over- scintillation counting and normalized to total cell protein content. Atherosclerosis. ApoE2/2 mice were crossed with hIGFBP1tg mice to gen- expressing hIGFBP1. In control mice, obesity was associated erate ApoE2/2 hIGFBP1tg mice. After attaining 20 g body mass, mice were fed with suppression of hepatic IGFBP1 mRNA expression Western diet for 12 weeks, which was shown in preliminary experiments to be (Fig. 1A) and a marked reduction in total circulating

FIG. 2. hIGFBP overexpression improves whole-body and vascular insulin sensitivity in diet-induced obesity. A and B: Glucose and insulin tol- erance test in fasted animals fed chow (lean) or high-fat diet (obese). A: Glucose tolerance test: obese hIGFBP1tg (TG) mice had lower fasting glucose concentrations than obese WT mice (6.3 6 0.3 vs. 7.6 6 0.6 mmol/L, P = 0.02) and were more glucose tolerant. Area under the curve: 1287 6 53 mmol/L per 120 min in obese TG vs. 1645 6 42 mmol/L per 120 min in obese WT mice (n = 8 per group). *P = 0.02. B: Insulin tolerance test: obese TG animals were more insulin sensitive than obese WT animals. Area under the curve: 753 6 28 mmol/L per 120 min in obese TG vs. 926 6 31 mmol/L per 120 min in obese WT mice (n = 8 per group). *P =0.01.○, obese WT; ●, obese TG; □,leanWT;■,leanTG.C: Plasma insulin concentrations in fasting animals and repeated 30 min after injection of glucose (1 mg/g i.p.) (n =8pergroup).D–F: Isometric tension studies in thoracic aorta from obese WT and TG mice (n = 8 per group). Phenylephrine (PE)-induced constriction was unaltered by insulin preincubation in WT mice (D)butwas attenuated by insulin preincubation in TG mice (E). *P = 0.01 vs. WT two-way ANOVA. F: Relaxation of preconstricted aorta to acetylcholine (Ach) was unaltered. G: Ser phosphorylation of eNOS was quantified by immunoblotting of aorta harvested from diet-induced obese mice 15 min after injection of insulin (0.75 IU/kg i.p.) or vehicle. Ser1177 phospho-eNOS expression was normalized to expression of total eNOS and b-actin. *P < 0.05 for insulin-induced eNOS phosphorylation in TG mice vs. WT. diabetes.diabetesjournals.org DIABETES 3 INCREASING IGFBP1 CONCENTRATION

TABLE 1 of obesity. In our mice, in which hIGFBP1 is expressed hIGFBP1 overexpression lowers blood pressure in obese and under the control of its native promoter and regulatory insulin-resistant mice sequences, we found no effect on epididymal fat pad mass (28 6 9 in WT vs. 34 6 2 mg in tg mice; P = 0.47) or adi- Systolic blood pressure (mmHg) pocyte cross-sectional area (Fig. 1F–I). Food intake was Control hIGFBP overexpression P value similar in hIGFBP1 mice and WT controls receiving the 6 6 P Chow diet 103 6 3 102 6 3 0.58 obesogenic diet (2.54 0.07 vs. 2.57 0.12 g/day; = Nutritional obesity 122 6 2 108 6 5 0.03 0.85). hIGFBP1tg mice were at least partially protected IR+/2 120 6 3 105 6 4 0.01 from the metabolic consequences of obesity. Diet-induced obesity increased fasting blood glucose and induced glu- Data are mean 6 SEM unless noted otherwise. Systolic blood pres- cose intolerance in WT animals; these effects were ame- sure was measured by tail-cuff plethysmography in control and liorated in hIGFBP1tg mice (Fig. 2A). Insulin sensitivity, as hIGFBP1tg mice (n =6–7 per group). indicated by an insulin tolerance test, was significantly enhanced in hIGFBP1tg animals (Fig. 2B). Plasma insulin IGFBP1 concentration (Fig. 1B). As previously published, concentrations were not significantly altered by IGFBP1 tg overexpression of hIGFBP1 in chow-fed animals results overexpression (Fig. 2C). in a fivefold increase in circulating IGFBP1 concentration Increasing hIGFBP1 concentrations enhances vascular (28). In obesity, we found that tg overexpression of NO production and lowers blood pressure in obesity. hIGFBP1 similarly increased plasma IGFBP1 concentra- We next tested whether favorable effects on vascular func- tion (Fig. 1C) without altering total plasma IGF-I (Fig. 1D). tion accompanied the protective effect of hIGFBP1 on glu- hIGFBP1tg mice developed a similar increase in body cose regulation in obesity. Impaired insulin-mediated NO mass compared with wild-type (WT) littermates (Fig. 1E). production (34) is implicated in insulin resistance–associated Because constitutive overexpression of IGFBP1 has been vascular dysfunction. In nonobese mice, insulin induces reported to inhibit adipocyte expansion (33), we measured a hypocontractile response to phenylephrine in aorta by adipocyte diameter in epididymal fat depots after induction enhancing NO generation (30,31). This effect was absent in

FIG. 3. hIGFBP1 overexpression lowers blood pressure and enhances vascular insulin sensitivity in hemizygous insulin receptor null mice. A and B: Glucose and insulin tolerance tests. Blood glucose was measured at intervals after injection of glucose (1 mg/g i.p.) or insulin (0.75 IU/kg i.p.) in mice hemizygous for deletion of insulin receptors (IR+/2) and littermate IR+/2 mice with tg overexpression of hIGFBP1 (IR+/2hIGFBP1tg) (n =6–8 per group). There was no difference in the response to glucose tolerance test (area under the curve: 993 6 42 vs. 840 6 39 mmol/min/L) or insulin tolerance test (area under the curve: 513 6 21 vs. 503 6 18 mmol/min/L) between IR+/2 and IR+/2hIGFBP1tg mice. C and D: Ex vivo isometric tension studies in thoracic aorta from IR+/2 and IR+/2hIGFBP1tg mice (n =6–8 per group). Phenylephrine (PE)-induced constriction was unaltered by insulin preincubation in IR+/2 mice (C) but was attenuated by insulin preincubation in IR+/2IGFBP1tg mice (D). *P = 0.01 vs. IR+/2 by two-way ANOVA.

4 DIABETES diabetes.diabetesjournals.org A. RAJWANI AND ASSOCIATES aorta harvested from obese mice (Fig. 2D), consistent with hypocontractility (Fig. 3D), indicating that IGFBP1 re- vascular insulin resistance. Overexpression of hIGFBP1 verses vascular insulin resistance. Vasodilatation to ace- restored hypocontractility to insulin in vessels from obese tylcholine or sodium nitroprusside (data not shown) was mice (Fig. 2E), indicating increased insulin-induced NO unchanged by hIGFBP1 overexpression. production in tg animals. Vasodilation of aorta to acetyl- hIGFBP1 increases endothelial NO production in- choline (Fig. 2F) or sodium nitroprusside (not shown) was dependently of IGF-I by stimulating eNOS phosphor- unchanged. Insulin stimulates NO production via phos- ylation via the phosphatidylinositol 3-kinase pathway. phorylation of eNOS at Ser residues (35). Aortic Ser1177 To determine whether these phenotypes resulted from a eNOS phosphorylation after insulin injection was signifi- direct effect of hIGFBP1, we carried out experiments in cantly upregulated in hIGFBP1tg animals (Fig. 2G). hIGFBP1 isolated segments of WT mouse aorta. Incubation of aorta overexpression ameliorated the increase in systolic blood with hIGFBP1 reduced contractility to phenylephrine pressure observed in WT obese animals (Table 1). (Fig. 4A). This was abolished by endothelial denudation Increasing hIGFBP1 concentrations enhances vascular (Fig. 4B) and coincubation with L-NMMA (Fig. 4C), indi- NO production and lowers blood pressure in insulin cating that hIGFBP1 stimulates endothelial NO generation. resistance. We next examined whether increasing hIGFBP1 Coincubation with LY294022, an inhibitor of phosphatidyl- improves vascular function in a nonobese model of insulin inositol 3-kinase (PI3K), abolished this response (Fig. 4D), resistance. We crossed hIGFBP1tg mice with mice het- indicating that PI3K activation is required for hIGFBP1- erozygous for deletion of the insulin receptor (IR+/2). IR+/2 induced NO production. These effects were observed in a mice have increased blood pressure and endothelial dys- serum-free preparation, suggesting that hIGFBP1 stimulates function despite maintaining normal glucose regulation vascular NO production independently of IGFs. To exclude through increased insulin secretion (31). Glucose tolerance a contribution from IGF contamination or local IGF secre- and insulin sensitivity were unchanged when hIGFBP1 tion, we repeated our experiments in mice with endothelial- was overexpressed in IR+/2 mice (Fig. 3A and B), but sys- specific deletion of type 1 IGF receptors generated by tolic blood pressure was increased (120 6 3 vs. 102 6 4 crossing IGF1Rfloxed mice (36,37) with Tie2-Cre tg mice mmHg; P = 0.01). Overexpression of hIGFBP1 protected (36). As expected, aortas from these animals did not vaso- IR+/2 mice from increased blood pressure (Table 1). Insulin dilate in response to IGF-I (Fig. 4E). However, hIGFBP1 did did not alter the contractile response of aorta from IR+/2 reduce contractility to phenylephrine (Fig. 4F), reaffirming mice (Fig. 3C), in keeping with vascular insulin resistance. that hIGFBP1-induced NO generation is not mediated by Overexpression of hIGFBP1 restored insulin-mediated type 1 IGF receptors.

FIG. 4. hIGFBP1 induces vasodilatation by increasing NO bioavailability in isolated vessels. A–D: Ex vivo isometric tension studies in thoracic aorta from WT mice (n =6–8 per group). In A, phenylephrine (PE)-induced constriction was attenuated by preincubation with hIGFBP1 (500 ng/mL 2 h). *P = 0.02 vs. vehicle control containing 1 mg/mL BSA. This vasodilatory effect was blocked by mechanical removal of the endothelium (B) and by coincubation with the NO synthase inhibitor L-NMMA (0.1 mmol/L) (C) or the PI3K inhibitor LY294002 (10 mmol/L) (D). E and F: Isometric tension studies were repeated in aorta from mice with deletion of type 1 IGF receptors in endothelial cells (generated by crossing IGF1Rfloxed mice with Tie2-Cre tg mice) (n = 6 per group). PE-induced constriction was unaltered by incubation with IGF-I (100 nmol/L) (E) but was attenuated by incubation with hIGFBP1 (500 ng/mL) (F). *P = 0.01. diabetes.diabetesjournals.org DIABETES 5 INCREASING IGFBP1 CONCENTRATION

To confirm a direct effect of hIGFBP1 on endothelial NO vs. 29.1 6 3.2% in ApoE2/2IGFBP1tg; P = 0.32) or fibrous production, we undertook experiments in primary hu- cap thickness (36 6 4 mminApoE2/2 vs. 28 6 4 mmin man endothelial cells. hIGFBP1 induced concentration- ApoE2/2IGFBP1tg; P = 0.17). There were no significant and time-dependent eNOS Ser1177 phosphorylation in differences in plasma lipoprotein concentrations between HCAECs (Fig. 5A–D). hIGFBP1 similarly stimulated dose- ApoE2/2IGFBP1tg mice and ApoE2/2 mice (Fig. 6E). dependent Ser473 phosphorylation of Akt and Ser9 phos- To ascertain whether IGFBP1 is expressed in the vascular phorylation of its downstream substrate GSK-3b (Fig. 5E wall, we carried out real-time PCR to assess expression of and F) but had no effect on total levels of expression of IGFBP1 in atherosclerotic aorta. Murine IGFBP1 expression eNOS, Akt, or GSK-3b. hIGFBP1 induced Ser1177 eNOS was detectable at low levels in the aorta of ApoE2/2 and phosphorylation in HUVECs (Fig. 5G–I), an effect blocked ApoE2/2IGFBP1tg mice (Fig. 6F). hIGFBP1 was detectable by coincubation with LY294022 (Fig. 5J). To confirm that only in aorta from hIGFBP1tg mice (Fig. 6F), as expected. the changes in eNOS phosphorylation resulted in changes in NO production, we demonstrated that hIGFBP1 stimulated LY294022-inhibitable eNOS activity in HUVECs (Fig. 5K). DISCUSSION IGFBP1 overexpression reduces atherosclerosis. Fi- During the past 2 decades, a series of epidemiological nally, we hypothesized that increasing IGFBP1 levels would studies has linked low circulating IGFBP1 concentrations with result in protection from atherosclerosis. To address this, insulin resistance, type 2 diabetes, and cardiovascular disease. we crossed hIGFBP1tg mice with ApoE2/2 mice. After We previously demonstrated that hIGFBP1 overexpression feeding a Western-type diet for 12 weeks, plaque burden increased vascular NO generation in mice (27), suggesting was significantly reduced in ApoE2/2IGFBP1tg mice, both that low IGFBP1 concentrations may play a mechanistic in en face preparations of aorta (Fig. 6A and B)andin role in vascular pathophysiology. However, whether the aortic sinus sections (Fig. 6C and D). Overexpression effects of IGFBP1 on the vascular endothelium were direct of hIGFBP1 did not affect plaque complexity as measured or indirect, and whether increasing IGFBP1 concentrations by analysis of necrotic core area (33.8 6 3.7% in ApoE2/2 could be of therapeutic potential in preventing disease,

FIG. 5. hIGFBP1 increases eNOS activity in human endothelial cells by stimulating eNOS phosphorylation via a PI3K/Akt-mediated signaling pathway. A–C: eNOS phosphorylation in HCAECs was assessed by immunoblotting (b-actin is shown as a loading control). hIGFBP1 induced dose- dependent (A) and time-dependent (B) Ser1177 eNOS phosphorylation, which was partially blocked by the PI3K inhibitor wortmannin (C). D–F: Mean data for hIGFBP1-induced dose-dependent phosphorylation of eNOS, Akt, and GSK-3b, expressed as a ratio of the phosphorylated to nonphosphorylated form, each normalized for b-actin. *P < 0.03; **P < 0.05 vs. control. G–J: hIGFBP1-induced eNOS phosphorylation was con- firmed in HUVECs with fluorescence microscopy using anti–phospho-Ser1177-eNOS antibodies. Representative untreated cells are shown in G, with hIGFBP1-stimulated cells shown in H. Mean fluorescence was increased by incubation with hIGFBP1 (500 ng/mL) (20.3 6 1.9 fluorescence units vs. 16.4 6 1.1 units in untreated [Con] cells, P = 0.03) (I). This was blocked by coincubation with LY294002 (10 mmol/L) (J). K: hIGFBP1 (500 ng/mL) increased eNOS activity in HUVECs by 46%. *P = 0.001 vs. control cells. This effect was blocked by coincubation with LY294002 (10 mmol/L). (A high-quality digital representation of this figure is available in the online issue.)

6 DIABETES diabetes.diabetesjournals.org A. RAJWANI AND ASSOCIATES

FIG. 6. hIGFBP1 overexpression reduces atherosclerosis in ApoE-null mice. A: Representative images of Oil Red O–stained aorta demonstrating atherosclerotic lesions in ApoE2/2 mice and ApoE2/2hIGFBP1tg mice (magnification 3200) (n =7–15 per group). B: Mean aortic plaque area (expressed as percent of total vessel area) was significantly reduced in ApoE2/2hIGFBP1tg mice compared with ApoE2/2 mice. *P < 0.001. C: Representative images of aortic sinus sections stained with Miller stain (magnification 3400) (n =9–13 per group). D: Mean aortic sinus plaque area (expressed as percent of total sinus area) was significantly reduced in ApoE2/2hIGFBP1tg mice compared with ApoE2/2 mice. **P < 0.05. E: Plasma total cholesterol (TC), LDL, HDL, and triglyceride concentrations were similar in ApoE2/2 and ApoE2/2hIGFBP1tg mice. F: hIGFBP1 mRNA was readily detectable in aorta from ApoE2/2hIGFBP1tg mice but was undetectable in aorta from ApoE2/2 mice, as expected. Murine (m)IGFBP1 mRNA was expressed at low levels in aorta from both ApoE2/2 and ApoE2/2hIGFBP1tg mice. (A high-quality digital representation of this figure is available in the online issue.) was unknown. In this study, we demonstrate for the first of IGFs as a vasoactive molecule in its own right in cells of time, using a series of mouse models, that elevating the vascular wall. Our findings parallel the recent obser- IGFBP1 levels improves whole-body insulin sensitivity vation that IGFBP3, another member of the IGFBP family, and glucose tolerance, lowers blood pressure, enhances can affect vascular pathophysiology by modulation of NO vascular NO production, and reduces susceptibility to (39). Although hepatic IGFBP1 production accounts for atherosclerosis. the majority of circulating IGFBP1, we found expression A critical novel finding of the current study is that of both the native murine transcript and the human trans- hIGFBP1 has a direct effect on vascular endothelium, re- in the vascular wall. Because IGFBP1 is known to be sulting in increased NO generation. Mechanistically, although expressed in human atherectomy specimens (40), though, the proximal signaling steps are yet to be elucidated, we interestingly, not in primary culture of human vascular have shown that hIGFBP1 stimulates dose-dependent NO smooth muscle (41), our findings raise the possibility that production in isolated murine vessels and in primary hu- increased local expression of hIGFBP1 as well as increased man endothelial cells. hIGFBP1-stimulated NO generation concentrations of hIGFBP1 in the circulation may contrib- is mediated by activation of the PI3K/Akt signaling path- ute to the vascular phenotypes we observed. While other way, resulting in Ser phosphorylation of eNOS (Fig. 7). potential contributory factors cannot be excluded, it is This mirrors the pathway by which insulin and growth tempting to speculate that increased NO generation pro- factors stimulate NO generation in endothelial cells (38). moted by elevated circulating IGFBP1 concentrations, or en- hIGFBP1 overexpression did not alter arterial responses to hanced insulin-induced vascular hypocontractility, accounted acetylcholine, suggesting that calcium/calmodulin-mediated for the reduction in blood pressure we observed. It is activation of eNOS is not affected by IGFBP1. It is impor- noteworthy that the plasma concentrations of IGFBP1 tant to note that hIGFBP1-induced NO production was achieved through tg overexpression in our obesity model, demonstrated in the absence of IGF-I and in vessels in which were similar to those recorded in insulin-resistant which endothelial type 1 IGF receptors were deleted. Al- humans (18,19), remained lower than the circulating levels though IGFBP1 has been shown to directly modulate cell in nonobese WT mice. This suggests that a modest increase migration in other cell types (9–12), this is the first time in IGFBP1 is sufficient to generate a favorable phenotypic that IGFBP1 has been demonstrated to act independently effect. diabetes.diabetesjournals.org DIABETES 7 INCREASING IGFBP1 CONCENTRATION

murine vessels are likely to result from chronic exposure to increased IGFBP1 concentrations in vivo (Fig. 7). De- fining the mechanistic basis for the insulin sensitization we observed is beyond the focus of this investigation. How- ever, one can speculate that enhanced insulin-mediated NO generation may have contributed to the improvement in systemic insulin sensitivity (43) or that higher circulat- ing 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 over- expression 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 FIG. 7. Schematic of proposed effects of IGFBP1 on vascular endothe- lial cells. IGFBP1 forms high-affinity complexes with circulating IGFs in of plasma levels (achieved in our mouse but using the the circulation (1) and confers dynamic regulation of IGF-I activity at native promoter sequence to drive transgene expression), the type 1 IGF receptor (2). IGF-I acts in a similar manner to insulin in is required. It is intriguing that mice with knockout of endothelial cells, stimulating NO generation by activating the PI3K/Akt IGFBP1 do not have any short-term major metabolic dys- signaling pathway (4). In the current study, we have demonstrated that IGFBP1, which is expressed in the vascular wall as well as present in function (47), implying other factors can compensate for the circulation, stimulates NO production independently of IGF and the the lack of IGFBP1. Effects of IGFBP1 deletion on vascular type 1 IGF receptor. Although the receptor for IGFBP1 (6) and the function and long-term metabolism have not been studied. proximal signaling intermediaries are yet to be identified, IGFBP1- stimulated NO production is also mediated through the PI3K/Akt The robust effect of IGFBP1 overexpression we observed 2/2 pathway, culminating in phosphorylation of eNOS (7) and an increase on atherosclerotic plaque development in ApoE mice in NOS activity. In addition to direct effects on endothelial cells, was striking. It is tempting to speculate that the inhibition IGFBP1 also enhances insulin sensitivity in endothelial cells potentially 2/2 by upregulating signaling via the insulin receptor (8). It is likely that this of atherosclerosis we observed in ApoE mice over- is a chronic effect, considering that we observed enhanced insulin sig- expressing hIGFBP1 is attributable to enhanced NO bio- naling in isolated vessels ex vivo in the absence of hIGFBP1. Enhanced availability as a result of upregulation of Akt-mediated eNOS NO bioavailability may contribute to the lower blood pressure, reduced atherosclerosis, and improved insulin sensitivity we observed in phosphorylation. Certainly, the evidence that Akt signaling hIGFBP1-overexpressing mice. It is also possible that IGFBP1 improves and eNOS-derived NO play fundamental roles in athero- whole-body insulin sensitivity by stimulating the PI3K/Akt pathway in sclerosis is compelling (48,49). However, we cannot exclude canonical insulin-sensitive tissues. IRS, insulin receptor substrate. the possibility that other factors may have contributed to the reduced atherosclerosis we observed in hIGFBP1tg mice. Although our observations of increased NO generation, The contribution of IGFBP1 to glucose regulation has pre- reduced blood pressure, and reduced atherosclerotic plaque viously been attributed to modulation of IGF-I bioavail- in IGFBP1tg mice are in keeping with the inverse associ- ability (7). Our observations, however, indicate that hIGFBP1 ations between IGFBP1 and cardiovascular disease dem- functions as an insulin-sensitizing peptide per se. In con- onstrated in cross-sectional studies, we acknowledge that trast to our findings with hIGFBP2 (32), hIGFBP1 over- not all human data support a favorable association between expression improved whole-body insulin sensitivity and IGFBP1 concentrations and cardiovascular risk. Specif- reduced glucose intolerance in the setting of obesity in the ically, high circulating concentrations of IGFBP1 have absence of change in body weight or fat cell size. hIGFBP1 been linked with the development of heart failure in the overexpression did not affect glucose regulation in our elderly (50) and with an adverse prognosis after myocardial model of genetic insulin resistance (IR+/2 mice). However, infarction (51,52). Associated changes in COOH-terminal whole-body glucose homeostasis is relatively preserved in provasopressin (copeptin) may explain the apparent prog- this model, in which we have demonstrated that insulin nostic importance of IGFBP1 concentrations highlighted in receptor deletion has a disproportionately greater effect these trials (53); however, further studies of the molecular on endothelial insulin sensitivity than on whole-body in- effects of IGFBP1 in these clinical scenarios are clearly sulin sensitivity (31). In contrast, enhanced endothelial warranted. sensitivity to insulin, a critical determinant of vascular In conclusion, our findings demonstrate that hIGFBP1 homeostasis (42), was demonstrated in both hIGFBP1tg has insulin-sensitizing, blood pressure–lowering, and anti- obese mice and genetically insulin-resistant mice, in which atherosclerotic properties. We have identified a novel overexpression of hIGFBP1 enhanced insulin-induced function of hIGFBP1 as a vasomodulatory molecule in its arterial relaxation and eNOS phosphorylation. Because own right, here acting as a regulator of endothelial NO pro- IGFBP1 was not present in the organ bath salt solution, duction independent of the type 1 IGF receptor by activating these enhanced vascular responses to insulin observed in the PI3K/Akt/phospho-eNOS pathway. Our observations have

8 DIABETES diabetes.diabetesjournals.org A. RAJWANI AND ASSOCIATES significant clinical implications, raising the possibility that 13. Brismar K, Fernqvist-Forbes E, Wahren J, Hall K. Effect of insulin on the enhancing IGFBP1 levels may be a therapeutic option to hepatic production of insulin-like growth factor-binding protein-1 (IGFBP-1), protect individuals from insulin resistance, hypertension, IGFBP-3, and IGF-I in insulin-dependent diabetes. J Clin Endocrinol Metab 1994;79:872–878 and atherosclerosis. 14. Lewitt MS, Denyer GS, Cooney GJ, Baxter RC. Insulin-like growth factor- binding protein-1 modulates blood glucose levels. Endocrinology 1991;129: – ACKNOWLEDGMENTS 2254 2256 15. Yki-Järvinen H, Mäkimattila S, Utriainen T, Rutanen EM. Portal insulin This work was funded by the British Heart Foundation and concentrations rather than insulin sensitivity regulate serum sex hormone- supported in part through a Yorkshire Enterprise Fellowship binding globulin and insulin-like growth factor binding protein 1 in vivo. for S.B.W.; S.B.W. is a British Heart Foundation Intermediate J Clin Endocrinol Metab 1995;80:3227–3232 Clinical Research Fellow. A.R., V.E., E.R.D., R.M.C., M.B.K., 16. Heald AH, Cruickshank JK, Riste LK, et al. Close relation of fasting insulin-like growth factor binding protein-1 (IGFBP-1) with glucose tol- A.Ab., and A.Az. held British Heart Foundation Clinical erance and cardiovascular risk in two populations. Diabetologia 2001;44: Research Training Fellowships. A.M.S. is a British Heart 333–339 Foundation Professor of Cardiology. 17. Liew CF, Wise SD, Yeo KP, Lee KO. Insulin-like growth factor binding No potential conflicts of interest relevant to this article protein-1 is independently affected by ethnicity, insulin sensitivity, and were reported. leptin in healthy, glucose-tolerant young men. J Clin Endocrinol Metab A.R. and M.B.K. prepared the manuscript and performed 2005;90:1483–1488 18. Maddux BA, Chan A, De Filippis EA, Mandarino LJ, Goldfine ID. IGF- the in vitro and in vivo experiments. V.E., J.S., N.Y.Y., E.R.D., binding protein-1 levels are related to insulin-mediated glucose disposal M.G., H.I., A.Ab., H.V., A.Az., and P.S. performed in vitro and and are a potential serum marker of insulin resistance [corrected in: Di- in vivo experiments. A.V.-P., P.J.G., and K.E.P. supervised the abetes Care 2006;29:2571]. Diabetes Care 2006;29:1535–1537 project. R.M.C. and J.K.S. critically reviewed the manuscript. 19. Mohamed-Ali V, Pinkney JH, Panahloo A, Cwyfan-Hughes S, Holly JM, Yudkin S.X. generated the IGF1Rfloxed mouse. A.M.S. and M.T.K. JS. Insulin-like growth factor binding protein-1 in NIDDM: relationship with conceived the study, secured funding, designed the experi- the insulin resistance syndrome. Clin Endocrinol (Oxf) 1999;50:221–228 20. Rajpathak SN, McGinn AP, Strickler HD, et al. Insulin-like growth factor- ments, and supervised the project. S.B.W. conceived the (IGF)-axis, inflammation, and glucose intolerance among older adults. study, secured funding, designed the experiments, super- Growth Horm IGF Res 2008;18:166–173 vised the project, and prepared the manuscript. S.B.W. is 21. Travers SH, Labarta JI, Gargosky SE, Rosenfeld RG, Jeffers BW, Eckel RH. the guarantor of this work and, as such, had full access to Insulin-like growth factor binding protein-I levels are strongly associated all the data in the study and takes responsibility for the with insulin sensitivity and obesity in early pubertal children. J Clin En- integrity of the data and the accuracy of the data analysis. docrinol Metab 1998;83:1935–1939 22. Yeap BB, Chubb SA, Ho KK, et al. IGF1 and its binding proteins 3 and 1 are differentially associated with metabolic syndrome in older men. Eur REFERENCES J Endocrinol 2010;162:249–257 23. Lewitt MS, Hilding A, Ostenson CG, Efendic S, Brismar K, Hall K. Insulin- 1. Booth GL, Kapral MK, Fung K, Tu JV. Relation between age and cardio- like growth factor-binding protein-1 in the prediction and development of vascular disease in men and women with diabetes compared with non- type 2 diabetes in middle-aged Swedish men. Diabetologia 2008;51:1135– diabetic people: a population-based retrospective cohort study. Lancet 1145 – 2006;368:29 36 24. Sandhu MS, Heald AH, Gibson JM, Cruickshank JK, Dunger DB, Wareham 2. Danaei G, Finucane MM, Lu Y, et al.; Global Burden of Metabolic Risk NJ. Circulating concentrations of insulin-like growth factor-I and develop- Factors of Chronic Diseases Collaborating Group (Blood Glucose). Na- ment of glucose intolerance: a prospective observational study. Lancet 2002; tional, regional, and global trends in fasting plasma glucose and diabetes 359:1740–1745 prevalence since 1980: systematic analysis of health examination surveys 25. Leinonen ES, Salonen JT, Salonen RM, et al. Reduced IGFBP-1 is associ- and epidemiological studies with 370 country-years and 2.7 million par- ated with thickening of the carotid wall in type 2 diabetes. Diabetes Care – ticipants. Lancet 2011;378:31 40 2002;25:1807–1812 3. Shaw JE, Sicree RA, Zimmet PZ. Global estimates of the prevalence of 26. Heald AH, Siddals KW, Fraser W, et al. Low circulating levels of insulin-like – diabetes for 2010 and 2030. Diabetes Res Clin Pract 2010;87:4 14 growth factor binding protein-1 (IGFBP-1) are closely associated with the 4. Tahrani AA, Bailey CJ, Del Prato S, Barnett AH. Management of type 2 presence of macrovascular disease and hypertension in type 2 diabetes. – diabetes: new and future developments in treatment. Lancet 2011;378:182 Diabetes 2002;51:2629–2636 197 27. Wheatcroft SB, Kearney MT, Shah AM, et al. Vascular endothelial function 5. Kelley KM, Oh Y, Gargosky SE, et al. Insulin-like growth factor-binding and blood pressure homeostasis in mice overexpressing IGF binding proteins (IGFBPs) and their regulatory dynamics. Int J Biochem Cell Biol protein-1. Diabetes 2003;52:2075–2082 1996;28:619–637 28. Crossey PA, Jones JS, Miell JP. Dysregulation of the insulin/IGF binding 6. Baxter RC. Insulin-like growth factor binding proteins in the human cir- protein-1 axis in transgenic mice is associated with hyperinsulinemia and culation: a review. Horm Res 1994;42:140–144 glucose intolerance. Diabetes 2000;49:457–465 7. Lee PD, Giudice LC, Conover CA, Powell DR. Insulin-like growth factor 29. Noronha BT, Li JM, Wheatcroft SB, Shah AM, Kearney MT. Inducible nitric binding protein-1: recent findings and new directions. Proc Soc Exp Biol oxide synthase has divergent effects on vascular and metabolic function in Med 1997;216:319–357 obesity. Diabetes 2005;54:1082–1089 8. Wheatcroft SB, Kearney MT. IGF-dependent and IGF-independent actions 30. Duncan ER, Walker SJ, Ezzat VA, et al. Accelerated endothelial dysfunc- of IGF-binding protein-1 and -2: implications for metabolic homeostasis. tion in mild prediabetic insulin resistance: the early role of reactive oxygen Trends Endocrinol Metab 2009;20:153–162 species. Am J Physiol Endocrinol Metab 2007;293:E1311–E1319 9. Chesik D, De Keyser J, Bron R, Fuhler GM. Insulin-like growth factor 31. Wheatcroft SB, Shah AM, Li JM, et al. Preserved glucoregulation but at- binding protein-1 activates integrin-mediated intracellular signaling and tenuation of the vascular actions of insulin in mice heterozygous for migration in oligodendrocytes. J Neurochem 2010;113:1319–1330 knockout of the insulin receptor. Diabetes 2004;53:2645–2652 10. Gleeson LM, Chakraborty C, McKinnon T, Lala PK. Insulin-like growth 32. Wheatcroft SB, Kearney MT, Shah AM, et al. IGF-binding protein-2 protects factor-binding protein 1 stimulates human trophoblast migration by sig- against the development of obesity and insulin resistance. Diabetes 2007; naling through alpha 5 beta 1 integrin via mitogen-activated protein kinase 56:285–294 pathway. J Clin Endocrinol Metab 2001;86:2484–2493 33. Rajkumar K, Modric T, Murphy LJ. Impaired adipogenesis in insulin-like growth 11. Jones JI, Gockerman A, Busby WH Jr, Wright G, Clemmons DR. Insulin- factor binding protein-1 transgenic mice. J Endocrinol 1999;162:457–465 like growth factor binding protein 1 stimulates cell migration and binds to 34. Zeng G, Quon MJ. Insulin-stimulated production of nitric oxide is inhibited the alpha 5 beta 1 integrin by means of its Arg-Gly-Asp sequence. Proc Natl by wortmannin. Direct measurement in vascular endothelial cells. J Clin Acad Sci U S A 1993;90:10553–10557 Invest 1996;98:894–898 12. Perks CM, Newcomb PV, Norman MR, Holly JM. Effect of insulin-like 35. Montagnani M, Chen H, Barr VA, Quon MJ. Insulin-stimulated activation growth factor binding protein-1 on integrin signalling and the induction of of eNOS is independent of Ca2+ but requires phosphorylation by Akt at apoptosis in human breast cancer cells. J Mol Endocrinol 1999;22:141–150 Ser(1179). J Biol Chem 2001;276:30392–30398 diabetes.diabetesjournals.org DIABETES 9 INCREASING IGFBP1 CONCENTRATION

36. Abbas A, Imrie H, Viswambharan H, et al. The insulin-like growth factor-1 characterization and insights into the regulation of IGFBP-1 expression. receptor is a negative regulator of nitric oxide bioavailability and insulin Endocrinology 1994;135:1316–1327 sensitivity in the endothelium. Diabetes 2011;60:2169–2178 46. Rajkumar K, Krsek M, Dheen ST, Murphy LJ. Impaired glucose homeo- 37. Kido Y, Nakae J, Hribal ML, Xuan S, Efstratiadis A, Accili D. Effects of stasis in insulin-like growth factor binding protein-1 transgenic mice. J Clin mutations in the insulin-like growth factor signaling system on embryonic Invest 1996;98:1818–1825 pancreas development and beta-cell compensation to insulin resistance. 47. Leu JI, Crissey MA, Craig LE, Taub R. Impaired hepatocyte DNA synthetic J Biol Chem 2002;277:36740–36747 response posthepatectomy in insulin-like growth factor binding protein 38. Wang Y, Nagase S, Koyama A. Stimulatory effect of IGF-I and VEGF on 1-deficient mice with defects in C/EBP beta and mitogen-activated protein eNOS message, protein expression, eNOS phosphorylation and nitric ox- kinase/extracellular signal-regulated kinase regulation. Mol Cell Biol 2003; ide production in rat glomeruli, and the involvement of PI3-K signaling 23:1251–1259 pathway. Nitric Oxide 2004;10:25–35 48. Fernández-Hernando C, Ackah E, Yu J, et al. Loss of Akt1 leads to severe 39. Kielczewski JL, Jarajapu YP, McFarland EL, et al. Insulin-like growth atherosclerosis and occlusive coronary artery disease. Cell Metab 2007;6: factor binding protein-3 mediates vascular repair by enhancing nitric oxide 446–457 generation. Circ Res 2009;105:897–905 49. Kuhlencordt PJ, Gyurko R, Han F, et al. Accelerated atherosclerosis, 40. Grant MB, Wargovich TJ, Ellis EA, et al. Expression of IGF-I, IGF-I re- aortic aneurysm formation, and ischemic heart disease in apolipoprotein ceptor and IGF binding proteins-1, -2, -3, -4 and -5 in human atherectomy E/endothelial nitric oxide synthase double-knockout mice. Circulation specimens. Regul Pept 1996;67:137–144 2001;104:448–454 41. Patel VA, Zhang QJ, Siddle K, et al. Defect in insulin-like growth factor-1 50. Kaplan RC, McGinn AP, Pollak MN, et al. High insulinlike growth factor survival mechanism in atherosclerotic plaque-derived vascular smooth binding protein 1 level predicts incident congestive heart failure in the muscle cells is mediated by reduced surface binding and signaling. Circ elderly. Am Heart J 2008;155:1006–1012 Res 2001;88:895–902 51. Janszky I, Hallqvist J, Ljung R, Hammar N. Insulin-like growth factor 42. Duncan ER, Crossey PA, Walker S, et al. Effect of endothelium-specific binding protein-1 is a long-term predictor of heart failure in survivors of insulin resistance on endothelial function in vivo [corrected in: Diabetes a first acute myocardial infarction and population controls. Int J Cardiol 2009;58:511]. Diabetes 2008;57:3307–3314 2010;138:50–55 43. Kim JA, Montagnani M, Koh KK, Quon MJ. Reciprocal relationships be- 52. Wallander M, Norhammar A, Malmberg K, Ohrvik J, Rydén L, Brismar K. tween insulin resistance and endothelial dysfunction: molecular and IGF binding protein 1 predicts cardiovascular morbidity and mortality in pathophysiological mechanisms. Circulation 2006;113:1888–1904 patients with acute myocardial infarction and type 2 diabetes. Diabetes 44. Zhang F, Sjöholm K, Zhang Q. Attenuation of insulin secretion by insulin- Care 2007;30:2343–2348 like growth factor binding protein-1 in pancreatic beta-cells. Biochem Bi- 53. Mellbin LG, Rydén L, Brismar K, Morgenthaler NG, Ohrvik J, Catrina SB. ophys Res Commun 2007;362:152–157 Copeptin, IGFBP-1, and cardiovascular prognosis in patients with type 2 45.DaiZ,XingY,BoneyCM,ClemmonsDR,D’Ercole AJ. Human insulin- diabetes and acute myocardial infarction: a report from the DIGAMI 2 trial. like growth factor-binding protein-1 (hIGFBP-1) in transgenic mice: Diabetes Care 2010;33:1604–1606

10 DIABETES diabetes.diabetesjournals.org