The IGF system is increasingly implicated in the development of cardiovascular disease. The effects of circulating IGFs on the vasculature are largely modulated by IGFBPs, which control their access to cell-surface IGF receptors. IGFBP-1 has been proposed as the acute regulator of IGF bioavailability because of its metabolic regulation by glucoregulatory hormones. Posttranslational phosphorylation of IGFBP-1 significantly increases its affinity for IGF-I and therefore represents a further mechanism for controlling IGF bioavailability. We have therefore examined the IGF system and IGFBP-1 phosphorylation status, using specifically developed immunoassays, in a cohort of 160 extensively characterized type 2 diabetic subjects on two occasions 12 months apart. Total IGFBP-1 (tIGFBP-1), which is predominantly highly phosphorylated, was significantly lower in subjects with known macrovascular disease (geometric mean [95% CI], 48.7 μg/l [33.7–63.6]) than in patients with no vascular pathology (80.0 μ g/l [52.2–107]; F = 5.4, P = 0.01). A similar relationship was found for highly phosphorylated IGFBP-1 (hpIGFBP-1) concentration (known macrovascular disease, 45.1 μg/l [35.1–55.2]; no macrovascular disease, 75.8 μg/l [56.2–95.3]; F = 4.8, P = 0.01). Logistic regression showed that for every decrease of 2.73 μg/l in IGFBP-1 concentration, there was a 43% increase in the odds of a subject having macrovascular disease (odds ratio 0.57 [95% CI 0.40–0.83]; P = 0.001). hpIGFBP-1 correlated negatively with systolic blood pressure (ρ = −0.30, P < 0.01), diastolic blood pressure (ρ = −0.45, P < 0.001), and mean arterial pressure (MAP) (ρ = −0.41, P < 0.001). Linear regression modeling showed that 40% of the variance in tIGFBP-1 was accounted for by MAP, triglycerides, and nonesterified fatty acids. In contrast, levels of nonphosphorylated and lesser-phosphorylated IGFBP-1 (lpIGFBP-1) were unrelated to macrovascular disease or hypertension but did correlate positively with fasting glucose concentration (ρ = 0.350, P < 0.01). tIGFBP-1 concentrations were higher in subjects treated with insulin alone (n = 29) than for any other group. This effect persisted after adjustment of tIGFBP-1 levels for BMI, C-peptide, age, and sex (F = 6.5, P < 0.001, ρ = − 0.46). Such an effect was not apparent for lpIGFBP-1. We conclude that low circulating levels of hpIGFBP-1 are closely correlated with macrovascular disease and hypertension in type 2 diabetes, whereas lpIGFBP-1 isoforms are associated with glycemic control, suggesting a dual role for IGFBP-1 in the regulation of IGF actions in type 2 diabetes. Our data suggest that high circulating concentrations of highly phosphorylated IGFBP-1 may protect against the development of hypertension and cardiovascular disease by reducing the mitogenic potential of IGFs on the vasculature.

Type 2 diabetes is associated with a three- to fivefold increased risk of macrovascular disease that cannot be completely explained by known risk factors. IGFs and IGFBPs are increasingly being recognized as contributing to cardiovascular risk (14). In addition, there are well-established links between IGFs and other surrogate markers for cardiovascular risk. IGF-I has been shown to correlate with LDL cholesterol in normal women (5), patients with hypothyroidism (6), and the elderly (6) and with apolipoprotein B and cardiovascular risk in normal subjects (7). There is also evidence that the IGF system is involved in the underlying pathophysiological processes associated with cardiovascular disease. In vivo animal studies suggest a role for IGF-I as a mediator of the hypertrophic responses of vascular smooth muscle cells (VSMCs) in hypertension (8) and implicate IGF-I as a mediator of cardiac hypertrophy in congestive cardiac failure (9), whereas in vitro, IGF-I receptor availability is closely linked with VSMC growth (10).

IGF binding proteins predominantly control the access of IGFs to tissues and cell-surface IGF receptors. Of these, IGFBP-1 is the most likely candidate for acute regulation of IGF actions because of its acute downregulation by insulin and upregulation by other glucoregulatory hormones and cytokines and in catabolic states (1113). Furthermore, transendothelial movement of IGFBP-1 into the tissues is known to occur and to be enhanced by insulin (14). IGFBP-1 thus provides an important link between intermediary metabolism and regulation of IGF actions.

We and others have previously shown that IGFBP-1 is negatively related with insulin resistance, blood pressure, BMI, waist-to-hip ratio, and triglyceride levels, as found previously in a population-based sample and in type 2 diabetes (2,4,15). With the development of type 2 diabetes (15), however, IGFBP-1 concentrations rise again despite persisting hyperinsulinemia, suggesting a complex interaction between pancreatic insulin output and hepatic insulin sensitivity. IGFBP-1 can exist in several phosphorylated isoforms. Phosphorylation of IGFBP-1 significantly alters its affinity for IGF-I (16,17). Under normal circumstances, circulating IGFBP-1 exists primarily in a highly phosphorylated form, which has the highest affinity for IGF-I (17). Changes in the phosphorylation state of IGFBP-1, therefore, could profoundly influence its ability to modulate IGF actions such that lesser-phosphorylated and nonphosphorylated variants would potentially increase, and highly phosphorylated forms reduce, IGF bioavailability. We have previously shown in diabetic pregnancy that increased levels of highly phosphorylated IGFBP-1 are associated with reduced fetal growth, whereas lesser-phosphorylated IGFBP-1 species are more closely associated with maternal weight gain, suggesting that phosphorylation of IGFBP-1 can differentially affect the metabolic and mitogenic actions of IGFs in different tissues (18). It therefore seems likely that in type 2 diabetes, the known dysregulation of the IGF/IGFBP-1 system would result in changes in IGF bioavailability that subsequently could predispose an affected individual to develop premature cardiovascular disease.

In this study, we have examined the relationship between IGFBP-1 phosphorylation status and the IGF system in type 2 diabetic subjects with and without evidence of macrovascular disease.

Subjects and study design.

One hundred sixty subjects were enrolled from two diabetes clinics (Hope Hospital and Manchester Royal Infirmary) in Manchester, U.K. The mean age of the participants was 58.1 ± 9.1 years, and subjects were matched for duration of diabetes (mean 8.2 ± 1.2 years). Previously, individuals had been randomly assigned to type of treatment according to U.K. Prospective Diabetes Study (UKPDS) protocols. Their current treatments were diet alone (n = 20), sulfonylurea (chlorpropamide or glipizide) treatment (n = 71), insulin alone (n = 29), and insulin (once or twice a day) plus sulfonylurea treatment (n = 40). They belonged to three different ethnic groups: European (n = 121), Pakistani (n = 23), and African-Caribbean (n = 16). There was no significant difference between the ethnic groups for age or duration of diabetes. All subjects gave informed consent, and the protocol was approved by local ethics committees.

All blood samples were obtained after an overnight fast, and the patients’ usual medications were withheld until after venesection. Plasma was separated from the remaining blood by centrifugation at 4°C. Plasma samples were stored at −70°C until analysis. Blood samples were taken at baseline and 12 months later.

Blood pressure was the mean of three readings taken with the subject in the supine position. A survey of medical records was carried out to obtain a detailed medical history.

Macrovascular disease was defined as a history of any of the following: ischemic heart disease including previous myocardial infarction and congestive heart failure, intermittent claudication, cerebrovascular accident, transient ischemic attack, peripheral vascular disease (defined by unilateral or bilateral absence of foot pulses), or systolic blood pressure ≥170 mmHg.

Assays.

Levels of IGF-I (19), IGF-II (20), total IGFBP-I (tIGFBP-1) (using monoclonal antibody [mAb] 6303, which detects all IGFBP-1 phosphoforms) (21), and lesser-phosphorylated/nonphosphorylated IGFBP-I (lpIGFBP-1) (using mAb 6305, which detects only the lesser- and nonphosphorylated forms of IGFBP-1) (21) were determined by previously reported antibody-based assays. The assays have respective detection limits of 28 ng/ml, 30 ng/ml, and 3 μg/l and within- and between-assay coefficients of variation (CVs) of <10%. Where necessary, highly phosphorylated IGFBP-1 (hpIGFBP-1) was calculated by subtraction of lpIGFBP-1 from tIGFBP-1 for each subject.

Insulin and insulin C-peptide were measured using the Mercodia (Uppsala, Sweden) enzyme-linked immunosorbent assay (ELISA) for intact insulin and the Dako (Ely, U.K.) ELISA for intact insulin C-peptide, with respective detection limits of 7 and 70 pmol/l and within- and between-assay CVs of <8%. Cross-reactivity of the insulin assay for insulin C-peptide and the C-peptide assay for insulin is <0.1%.

Fasting cholesterol and triglycerides were measured by enzymatic methods using the Cobasintegra autoanalyzer (Roche Diagnostics, Welwyn Garden City, U.K.), with HDL cholesterol measured by a direct method (Cobasintegra autoanalyzer). LDL cholesterol was calculated according to the Friedewald formula (22).

Fasting nonesterified fatty acid (NEFA) concentrations were measured using the Wako (Neuss, Germany) ELISA for total NEFAs. Sensitivity of the assay is 0.05 mEq/l and within- and between-assay CVs <10%. Glucose was assayed using the hexokinase method (Cobasintegra autoanalyzer). HbA1c was measured using the Cobasmira autoanalyzer (Roche Diagnostics).

Nondenaturing PAGE.

To confirm the validity of antibody-based measurements of IGFBP-1 phosphoforms, we also separated and visualized the IGFBP-1 phosphoforms using nondenaturing PAGE (23). Briefly, for a subset of subjects with no manifest macrovascular disease, IGFBP-1 in 250 μl plasma was immunoprecipitated with mAb 6303 (1:1,000; volume 250 μl) (provided by Medix Biochemica, Kauniainen, Finland), which immunoprecipates all phosphoforms of IGFBP-1 (21), and incubated overnight at 4°C. IGFBP-1 in 500 μl plasma from subjects with known macrovascular disease was required for immunoprecipitation to be able to detect lower concentrations. Two controls were used, HepG2 conditioned medium containing all the phosphoforms of IGFBP-1 and amniotic fluid, which contains all the lesser-phosphorylated isoforms but not hpIGFBP-1. After incubation, the antigen-antibody complexes were immunoprecipitated using Saccel rabbit anti-mouse antibody (IDS, Tyne & Wear, U.K.). Immunoprecipitated IGFBP-1 was applied to a 10% native polyacrylamide gel (23). After overnight transfer to nitrocellulose membranes, nonspecific binding was blocked by incubation with 1% BSA in 0.15 mol/l sodium chloride. The proteins were revealed by incubation with 150,000 cpm/ml [125I]IGF-I for 3 h at 25°C in accordance with the method of Hossenlopp et al. (24), followed by phosphor-image analysis (Fuji bio-imaging analyzer) and autoradiography.

Statistics.

Multiple logistic regression was used to assess the risk of macrovascular disease in relation to changes in IGFBP-1 concentration. Spearman correlations, both simple and partial, were calculated to investigate the relation between IGFBP-1 and other metabolic variables, BMI, and blood pressure.

Differences in metabolic variables between treatment groups and ethnic groups were assessed using ANCOVA. Non–normally distributed data were logarithmically transformed before analysis. Post hoc testing used Scheffe’s test. Significant predictors for IGFBP-1 were examined using multiple regression analysis. Comparison of IGFBP-1 at initial assessment and follow-up was carried out using linear regression analysis. All analyses were carried out using the statistical package SPSS, version 9.0.

Relationship of IGFBP-1 with macrovascular disease.

tIGFBP-1 was markedly reduced in subjects (n = 40) with known macrovascular disease (48.7 μg/l [33.7–63.6], geometric mean [95% CI]) compared with patients (n = 120) with no manifest macrovascular pathology (80.0 μg/l [52.2–107]; F = 5.4, P = 0.01) (Table 1). A similar relationship was also found for hpIGFBP-1 concentration (known macrovascular disease, 45.1 μg/l [35.1–55.2]; no macrovascular disease, 75.8 μg/l [56.2–95.3]; F = 4.8, P = 0.01) (Table 1). Logistic regression showed that for every unit decrease in the log tIGFBP-1 (increase of 2.73 μ g/l in the tIGFBP-1 concentration), there was a 43% increase in the odds of the subject having macrovascular disease (odds ratio 0.57 [95% CI 0.40–0.83]; P = 0.001) (Fig. 1).

There was no difference in lpIGFBP-1 by macrovascular disease status. For the whole group, lpIGFBP-1 (12.2 μg/l [10.9–13.5]) was significantly lower than tIGFBP-1 (76.1 μg/l [56.8–95.5]; P < 0.001).

To confirm the assay results, native PAGE was performed on a subset of subjects with high (n = 11) versus low (n = 12) tIGFBP-1. Representative results are shown in Fig. 2. The concentration of hpIGFBP-1 was less in those with macrovascular disease than in those without manifest macrovascular pathology. There was no corresponding difference in lpIGFBP-1 by macrovascular disease status.

tIGFBP-1 correlated negatively with mean arterial pressure (MAP) (Spearman’s ρ = −0.26, P = 0.002). The latter relationship persisted even when corrected for age, sex, BMI, and insulin C-peptide concentration (partial ρ = −0.30, P < 0.001). hpIGFBP-1 correlated negatively with systolic blood pressure (ρ = −0.30, P < 0.01), diastolic blood pressure (ρ = −0.45, P < 0.001), and MAP (ρ = − 0.41, P < 0.001). In contrast to tIGFBP-1 and hpIGFBP-1, no consistent relationships were found with lpIGFBP-1 and macrovascular disease, hypertension, or cardiovascular risk markers.

The relationship between tIGFBP-1 and hypertension was robust. Using multiple regression modeling, 40% of the variance in tIGFBP-1 was accounted for by three variables, MAP (unstandardized coefficient B = −3.4 ± 0.8; P < 0.001), triglycerides (B = −23.1 ± 7.4, P = 0.003), and NEFAs (B = −0.6 ± 0.03, P = 0.04) (Figs. 3A–C) in a model that included IGF-I, IGF-II, insulin C-peptide, cholesterol, triglycerides, NEFAs, fasting glucose, HbA1c, age, sex, BMI, and MAP. When this analysis was repeated by treatment group, the relationship with MAP was greatest in the group treated with insulin alone, where 69% of the variance in IGFBP-1 was accounted for by MAP alone (P < 0.001) (Fig. 3D).

IGFBP-1 and glycemic control.

As anticipated, there was a strong negative correlation of tIGFBP-1 concentration with insulin C-peptide concentration and also with peripheral insulin concentration and triglyceride level (Table 2). However, there was no relationship between tIGFBP-1 and fasting glucose or HbA1c. This result was in complete contrast to lpIGFBP-1, which correlated positively with fasting glucose (ρ = 0.350, P < 0.01).

Effect of different treatments on IGFBP-1 and macrovascular risk.

In agreement with our previously reported findings (25), tIGFBP-1 concentration was higher in subjects treated with insulin alone than in any other group (Table 3; Fig. 4). This difference persisted even when tIGFBP-1 was corrected for BMI and C-peptide in addition to age and sex (F = 6.5, P < 0.001). In contrast, there was no variation in lpIGFBP-1 concentration by treatment group (Table 3). Peripheral insulin levels were higher in those treated with insulin (with or without additional sulfonylureas) than in subjects not receiving insulin, whereas C-peptide levels were lower in subjects treated with insulin (with or without additional sulfonylureas). There was no significant difference in fasting glucose level or HbA1c by treatment group (Table 3).

Relationships of IGF-I and IGF-II with macrovascular disease and type of treatment.

There were no differences in IGF-I or IGF-II levels between subjects with and without evidence of macrovascular disease. IGF-I correlated positively with IGF-II and negatively with NEFAs. Interestingly, IGF-II concentration was strongly positively correlated with elevated total, LDL, and HDL cholesterol levels (Table 2).

IGF-I concentration was not affected by treatment category, but IGF-II levels were lower in subjects treated with insulin alone than in any other group (Table 3). There was a trend for total cholesterol to be lower in subjects treated with insulin alone (F = 2.2, P = 0.06), but otherwise there were no detectable differences in lipid profile by treatment group.

Robustness of IGFBP-1 over a 12-month period.

Because hepatic IGFBP-1 synthesis is acutely regulated by glucoregulatory hormones, we wished to establish whether assessment of fasting IGFBP-1 levels could robustly predict association with macrovascular disease even after a gap of 12 months. Therefore, tIGFBP-1 concentration was assessed at initial visits and 12-month follow-ups as shown in Fig. 5. Linear regression analysis demonstrated that tIGFBP-1 on follow-up was closely related to tIGFBP-1 at initial visit by the equation tIGFBP-1follow-up = 3.2 + (tIGFBP-1initial visit) × 0.92 (r2 = 0.90, P < 0.001). This suggests that fasting tIGFBP-1 concentration is a consistent measure with little variation over a 12-month period.

Effects of ethnicity on the IGF system.

There were no differences in IGFBP-1 levels between the ethnic groups (Table 4). IGF-I concentration was higher in African-Caribbeans than in Europeans or Pakistanis, and IGF-II was higher in Europeans than in African-Caribbeans or Pakistanis. The IGF-I results are in accord with previous findings in nondiabetic subjects (26) and indicate that the onset of type 2 diabetes does not mask underlying differences in circulating IGF concentrations related to ethnicity. Also in keeping with previous studies (2), we found a close correlation between IGF-II and total, LDL, and HDL cholesterol. The significance of the IGF-II/cholesterol relationship is unknown, although similarities in clearance pathways between the β-very low density lipoprotein (β-VLDL) receptor and the mannose-6-phosphate receptor may suggest a possible mechanism (27). Insulin levels were similar in all three ethnic groups, with C-peptide being lower in African-Caribbeans than in Europeans or Pakistanis. There was no significant difference in total cholesterol, LDL cholesterol, or triglyceride levels by ethnic group. There was no difference in age, duration of diabetes, BMI, or fasting glucose between the three ethnic groups.

This is the first study to demonstrate close links between fasting hpIGFBP-1 and the presence of macrovascular disease and hypertension in type 2 diabetes. In addition to the negative correlation between IGFBP-1 and macrovascular disease, we found significant negative relationships between tIGFBP-1 and other known cardiovascular risk factors, namely BMI, triglyceride concentrations, circulating insulin C-peptide, and insulin levels. These relationships were robust and persisted over a 12-month period.

IGFBPs have been clearly shown to modulate the actions of IGFs. IGFBP-1 in particular is accepted as an acute modulator of IGF bioavailability (13). We and others have shown a close relationship between low IGFBP-1 levels and surrogate markers of cardiovascular risk in a nondiabetic population (3,15). We showed that low circulating IGFBP-1 concentrations significantly correlated with impaired glucose tolerance (IGT). Because the subjects in our previous study (15) did not have any evident macrovascular pathology, and because IGT precedes the development of type 2 diabetes, it is probable that a low circulating IGFBP-1 concentration predates the development of macrovascular disease. As proposed previously (15), we believe that the development of peripheral insulin resistance, with maintenance of hepatic insulin sensitivity, explains the decrease in circulating IGFBP-1 (and thus the increase in IGF bioavailability) long before the development of type 2 diabetes. Our current data suggest that this pattern persists in a type 2 diabetic population and further predisposes affected individuals to premature macrovascular disease.

The majority of circulating IGFBP-1 is in the highly phosphorylated state, implying that it is this isoform of IGFBP-1 that is most relevant to the development of hypertension and macrovascular disease (2). hpIGFBP-1 has the highest affinity for IGF-I relative to other isoforms (16,17), and its affinity for ligand is significantly greater than that of the type 1 IGF receptor (28). Thus hpIGFBP-1 has the greatest potential to inhibit IGF-I/IGF receptor interactions.

In nondiabetic hypertensive humans, circulating IGF-I levels independently predict 16% of the variability in left ventricular mass (29). In another study, a reduction in serum IGF-I level in relation to bezafibrate treatment in post–myocardial infarction patients was independently associated with angiographic measures of coronary artery disease regression (30). Furthermore, in acromegaly, a human model of IGF-I excess (where World Health Organization criteria indicate that 37.5% patients have hypertension), blood pressure positively correlates with IGF-I concentration (31). These findings strongly support an endocrine effect of IGF-I in vivo. It is important, however, to recognize that IGFBP-1 is capable of inhibiting endocrine, paracrine, and autocrine IGF effects because of its ability to access the extracellular space and to associate with specific cell-surface integrins (32). Thus a high circulating level of hpIGFBP-1 will inhibit IGF effects whatever the cellular source of the IGF-I.

An elevated concentration of hpIGFBP-1 would therefore serve to protect vascular tissues from the mitogenic potential of circulating IGFs, whereas a consistently reduced level—induced by the diabetic state per se, hyperinsulinemia, or both—would increase IGF availability to susceptible tissues. This situation could be further worsened in type 2 diabetes, where reports of increased IGFBP-3 proteolysis (33) would further contribute to the exposure of the vascular compartment to the mitogenic actions of IGFs. It is interesting in this respect to note that African-Caribbean subjects in this and other studies tend to have higher IGF-I levels and are known to be more susceptible to resistant hypertension than other ethnic groups (34).

There are no studies examining the ability of liver to differentially phosphorylate IGFBP-1 in humans. Peripheral dephosphorylation seems a more likely explanation for the presence of lpIGFBP-1. Evidence for the ability of ectophosphatases (present in many cell types) to dephosphorylate IGFBP-1 has been reported (35). Cell-surface dephosphorylation would significantly affect the bioavailability of IGF-I at the cellular level. Thus IGFBP-1 in the highly phosphorylated state would significantly reduce interaction of IGF-I with its receptors, but with subsequent dephosphorylation could enhance local IGF bioavailability. Thus changes in tissue phosphatase/protease activity would further influence IGFBP-1/IGF-I binding kinetics and so IGF bioavailability at the tissue level (35). Further studies will be required to determine how the regulation of these factors influences cell-specific IGF effects.

In this study, we found that lesser-/nonphosphorylated IGFBP-1 correlated positively with fasting glucose. This finding was unexpected and has not been described previously. It implies that IGFBP-1 phosphorylation status may play an important part in mediating the effects of IGFBP-1 on glucose homeostasis. The finding of a positive correlation between fasting glucose and lpIGFBP-1 may suggest a mechanism to increase insulin-like activity at times of high ambient glucose concentration and, given the lower affinity of lpIGFBP-I for IGF-I, parallels reports of increased IGFBP-3 proteolysis associated with hyperglycemia in type 2 diabetes (35).

This study confirms our earlier findings of higher IGFBP-1 levels in subjects with type 2 diabetes who were treated with insulin alone (25). A simple explanation for this finding would be that a reduced portal insulin concentration in these subjects would remove the tonic inhibition of hepatic IGFBP-1 synthesis, leading to increased circulating levels of IGFBP-1. To address this issue in this study, we measured C-peptide levels, which undergo little hepatic extraction and therefore better represent hepatic insulin exposure via the portal system. There were no significant differences in insulin and insulin C-peptide concentrations between the groups treated with insulin alone or insulin plus sulfonylureas, although IGFBP-1 levels were significantly different, suggesting that it is less likely to be pancreatic β-cell insulin output and more likely to be hepatic insulin resistance that determines the differences in tIGFBP-1 level between treatment groups. This result is in keeping with the multiple linear regression analysis showing that factors other than insulin were important in determining tIGFBP-1 concentration, particularly MAP, NEFAs, and triglycerides.

The close relationship between fasting tIGFBP-1 levels at inception of the study and 1-year follow-up confirms the robustness of fasting tIGFBP-1 measurement over time and points to the utility of tIGFBP-1 as a predictive biomarker for macrovascular disease in type 2 diabetes.

In summary, these studies demonstrate the novel and important finding of close links between low circulating levels of the highly phosphorylated isoform of IGFBP-1 and the presence of macrovascular disease and hypertension in type 2 diabetes. In addition, we have shown that lesser-phosphorylated IGFBP-1 levels are associated with ambient glucose concentration, suggesting that the phosphorylation status of IGFBP-1 may be important in glucose homeostasis. Because IGFBPs largely determine the mitogenic and metabolic potential of IGFs, our findings suggest an etiopathogenic role for IGFBP-1 in modulating the development of large vessel disease in type 2 diabetes.

FIG. 1.

Fasting tIGFBP-1 by macrovascular disease status (age- and sex-adjusted arithmetic means with 95% CIs).

FIG. 1.

Fasting tIGFBP-1 by macrovascular disease status (age- and sex-adjusted arithmetic means with 95% CIs).

FIG. 2.

Native PAGE for IGFBP-1 by macrovascular disease status. Controls used HepG2 conditioned medium containing all the phosphoforms of IGFBP-1 (lane A) and amniotic fluid containing the less-phosphorylated forms but not highly phosphorylated IGFBP-1 (lane B). 1, the position of hpIGFBP-1; 2, the position of lpIGFBP-1.

FIG. 2.

Native PAGE for IGFBP-1 by macrovascular disease status. Controls used HepG2 conditioned medium containing all the phosphoforms of IGFBP-1 (lane A) and amniotic fluid containing the less-phosphorylated forms but not highly phosphorylated IGFBP-1 (lane B). 1, the position of hpIGFBP-1; 2, the position of lpIGFBP-1.

FIG. 3.

Scatter plots of MAP versus IGFBP-1 concentrations (A), triglyceride versus IGFBP-1 concentrations (B), and NEFA versus IGFBP-1 concentrations (C) for all patients in the study and MAP versus IGFBP-1 concentrations for the group treated with insulin alone (D).

FIG. 3.

Scatter plots of MAP versus IGFBP-1 concentrations (A), triglyceride versus IGFBP-1 concentrations (B), and NEFA versus IGFBP-1 concentrations (C) for all patients in the study and MAP versus IGFBP-1 concentrations for the group treated with insulin alone (D).

FIG. 4.

Fasting tIGFBP-1 by treatment group for all patients in the study (age- and sex-adjusted arithmetic means with 95% CIs).

FIG. 4.

Fasting tIGFBP-1 by treatment group for all patients in the study (age- and sex-adjusted arithmetic means with 95% CIs).

FIG. 5.

Scatter plot of fasting tIGFBP-1 concentrations at initial visit and 12-month follow-up.

FIG. 5.

Scatter plot of fasting tIGFBP-1 concentrations at initial visit and 12-month follow-up.

TABLE 1

IGFBP-1 by macrovascular disease status

Macrovascular diseaseNo macrovascular diseaseFP
IGFBP-1 (μg/l) 48.7 (33.7–63.6) 80.0 (52.2–107) 5.4 0.01 
hpIGFBP-1 (μg/l) 45.1 (35.1–55.2) 75.8 (56.2–95.3) 4.8 0.01 
lpIGFBP-1 (μg/l) 12.6 (10.7–14.6) 11.3 (9.9–12.8) 1.2 NS 
Macrovascular diseaseNo macrovascular diseaseFP
IGFBP-1 (μg/l) 48.7 (33.7–63.6) 80.0 (52.2–107) 5.4 0.01 
hpIGFBP-1 (μg/l) 45.1 (35.1–55.2) 75.8 (56.2–95.3) 4.8 0.01 
lpIGFBP-1 (μg/l) 12.6 (10.7–14.6) 11.3 (9.9–12.8) 1.2 NS 

Data are arithmetic means (95% CI).

TABLE 2

Universal correlation matrix for metabolic variables

IGFBP-IlpIGFBP-IIGF-IIGF-IIC-peptide
IGFBP-1 0.48* −0.02 −0.16 −0.46 
lpIGFBP-1 0.48* −0.12 −0.06 −0.17 
IGF-I −0.02 −0.12 0.51 −0.07 
IGF-II −0.16 −0.06 0.51 0.01 
C-peptide −0.46 −0.17 −0.07 0.01 
Insulin −0.23 −0.07 −0.08 −0.12 0.01 
NEFAs 0.03 0.17 −0.19 0.04 0.09 
Total cholesterol −0.13 −0.08 0.05 0.32 −0.03 
LDL cholesterol −0.10 −0.07 0.11 0.23* −0.08 
HDL cholesterol 0.06 −0.01 0.09 0.19 −0.24* 
Triglycerides −0.23 −0.14 −0.13 0.12 0.30 
Glucose 0.08 0.35* 0.01 0.01 0.09 
HbA1c −0.02 0.17 −0.01 −0.01 0.08 
IGFBP-IlpIGFBP-IIGF-IIGF-IIC-peptide
IGFBP-1 0.48* −0.02 −0.16 −0.46 
lpIGFBP-1 0.48* −0.12 −0.06 −0.17 
IGF-I −0.02 −0.12 0.51 −0.07 
IGF-II −0.16 −0.06 0.51 0.01 
C-peptide −0.46 −0.17 −0.07 0.01 
Insulin −0.23 −0.07 −0.08 −0.12 0.01 
NEFAs 0.03 0.17 −0.19 0.04 0.09 
Total cholesterol −0.13 −0.08 0.05 0.32 −0.03 
LDL cholesterol −0.10 −0.07 0.11 0.23* −0.08 
HDL cholesterol 0.06 −0.01 0.09 0.19 −0.24* 
Triglycerides −0.23 −0.14 −0.13 0.12 0.30 
Glucose 0.08 0.35* 0.01 0.01 0.09 
HbA1c −0.02 0.17 −0.01 −0.01 0.08 

Data are Spearman correlations (ρ).

*

P < 0.01;

P < 0.05;

P < 0.001.

TABLE 3

Metabolic variables by treatment group adjusted for age and sex

Diet onlySUInsulin treatmentInsulin + SUFP
n 20 71 29 40   
tIGFBP-1 (μg/l) 55.4 (31.9–78.8) 52 (40.9–63.1) 102.1 (60.3–143.8)* 51.4 (36.6–66.2) 12.5 <0.001 
lpIGFBP-1 (μg/l) 13.6 (8.3–18.9) 11.4 (10.1–12.7) 13.7 (10.3–17.1) 10.3 (8.8–11.8) 1.2 NS 
Insulin (pmol/l) 60.6 (28.3–92.9) 61.7 (48.7–74.7) 148.1 (92.1–204)* 138.4 (95.3–181.4)* 5.0 0.003 
C-peptide (pmol/l) 980 (795–1,165) 959 (871–1047) 721 (480–961)* 706 (608–864)* 2.5 0.03 
IGF-I (ng/ml) 188 (153–224) 199 (172–225) 200 (164–237) 202 (169–237) 1.3 NS 
IGF-II (ng/ml) 667 (553–781) 628 (572–683) 576 (494–659)* 625 (541–709) 3.8 0.003 
Total cholesterol (mmol/l) 5.7 (5.2–6.1) 5.7 (5.4–5.9) 5.5 (5.1–5.8) 5.7 (5.4–6.0) 2.2 0.06 
LDL cholesterol (mmol/l) 3.5 (3.2–3.9) 3.6 (3.4–3.9) 3.4 (3.1–3.8) 3.6 (3.3–4.0) 1.3 NS 
Triglycerides (mmol/l) 2.1 (1.7–2.5) 2.1 (1.8–2.3) 1.9 (1.6–2.2) 2.1 (1.8–2.3) 1.4 NS 
Glucose (mmol/l) 9.2 (8.0–10.5) 10.8 (9.9–11.7) 9.8 (8.1–11.5) 10.2 (9.0–11.4) 1.0 NS 
HbA1c (%) 4.9 (3.9–6.1) 6.3 (5.8–6.8) 5.8 (5.1–6.3) 6.4 (5.7–7.1) 2.4 0.04 
BMI 28.1 (26.3–29.9) 30.4 (29.0–31.9) 29.7 (28.2–31.3) 32.5 (30.8–34.1) 3.8 0.003 
Diet onlySUInsulin treatmentInsulin + SUFP
n 20 71 29 40   
tIGFBP-1 (μg/l) 55.4 (31.9–78.8) 52 (40.9–63.1) 102.1 (60.3–143.8)* 51.4 (36.6–66.2) 12.5 <0.001 
lpIGFBP-1 (μg/l) 13.6 (8.3–18.9) 11.4 (10.1–12.7) 13.7 (10.3–17.1) 10.3 (8.8–11.8) 1.2 NS 
Insulin (pmol/l) 60.6 (28.3–92.9) 61.7 (48.7–74.7) 148.1 (92.1–204)* 138.4 (95.3–181.4)* 5.0 0.003 
C-peptide (pmol/l) 980 (795–1,165) 959 (871–1047) 721 (480–961)* 706 (608–864)* 2.5 0.03 
IGF-I (ng/ml) 188 (153–224) 199 (172–225) 200 (164–237) 202 (169–237) 1.3 NS 
IGF-II (ng/ml) 667 (553–781) 628 (572–683) 576 (494–659)* 625 (541–709) 3.8 0.003 
Total cholesterol (mmol/l) 5.7 (5.2–6.1) 5.7 (5.4–5.9) 5.5 (5.1–5.8) 5.7 (5.4–6.0) 2.2 0.06 
LDL cholesterol (mmol/l) 3.5 (3.2–3.9) 3.6 (3.4–3.9) 3.4 (3.1–3.8) 3.6 (3.3–4.0) 1.3 NS 
Triglycerides (mmol/l) 2.1 (1.7–2.5) 2.1 (1.8–2.3) 1.9 (1.6–2.2) 2.1 (1.8–2.3) 1.4 NS 
Glucose (mmol/l) 9.2 (8.0–10.5) 10.8 (9.9–11.7) 9.8 (8.1–11.5) 10.2 (9.0–11.4) 1.0 NS 
HbA1c (%) 4.9 (3.9–6.1) 6.3 (5.8–6.8) 5.8 (5.1–6.3) 6.4 (5.7–7.1) 2.4 0.04 
BMI 28.1 (26.3–29.9) 30.4 (29.0–31.9) 29.7 (28.2–31.3) 32.5 (30.8–34.1) 3.8 0.003 

Data are arithmetic means (95% CI).

*

Significantly different by post-hoc testing (Scheffe), P < 0.05. SU, salfonylureas.

TABLE 4

Metabolic characteristics by ethnic group

EuropeanAfrican-CaribbeanPakistaniFP
 122 16 23   
IGF-I (ng/ml) 191.3 (171.3–211.3) 255.0 (210.8–299.3)* 198.5 (172.7–224.4) 3.4 0.021 
IGF-II (ng/ml) 625.1 (579.6–670.6)* 545.4 (432.4–618.4) 571.3 (431.6–621.1) 4.3 0.001 
tIGFBP-1 (μg/l) 65.1 (52.3–77.9) 58.6 (32.3–84.8) 45.9 (27.7–64.1) 0.8 NS 
Insulin (pmol/l) 91.7 (73.5–109.9) 70.8 (40.4–101.2) 71.2 (48.7–93.7) 0.3 NS 
C-peptide (pmol/l) 884.2 (796.6–971.7) 546.9 (431.6–662.2) 934 (792.9–1075.2) 2.6 0.03 
EuropeanAfrican-CaribbeanPakistaniFP
 122 16 23   
IGF-I (ng/ml) 191.3 (171.3–211.3) 255.0 (210.8–299.3)* 198.5 (172.7–224.4) 3.4 0.021 
IGF-II (ng/ml) 625.1 (579.6–670.6)* 545.4 (432.4–618.4) 571.3 (431.6–621.1) 4.3 0.001 
tIGFBP-1 (μg/l) 65.1 (52.3–77.9) 58.6 (32.3–84.8) 45.9 (27.7–64.1) 0.8 NS 
Insulin (pmol/l) 91.7 (73.5–109.9) 70.8 (40.4–101.2) 71.2 (48.7–93.7) 0.3 NS 
C-peptide (pmol/l) 884.2 (796.6–971.7) 546.9 (431.6–662.2) 934 (792.9–1075.2) 2.6 0.03 

Data are age = and sex-adjusted arithmetic means (95% CI).

*

Significantly different by post-hoc testing (Scheffe), P < 0.05.

We thank the National Health Service (NHS) North-West Research and Development Directorate for funding the work of A.H.H. in this project and Salford Royal Hospitals NHS Trust for the funding of technical support.

1.
Ferns GA, Morani AS, Anggard EE: The insulin-like growth factors: their putative role in atherogenesis.
Artery
18
:
197
–225,
1991
2.
Gibson JM, Westwood M, Young RJ, White A: Reduced insulin-like growth factor binding protein-1 (IGFBP-1) levels correlate with increased cardiovascular risk in non-insulin dependent diabetes mellitus (NIDDM).
J Clin Endocrinol Metab
81
:
860
–863,
1996
3.
Janssen JA, Stolk RP, Pols HA, Grobbee DE, Lamberts SW: Serum total IGF-I, free IGF-I and IGFBP-1 levels in an elderly population: relation to the cardiovascular risk factors and disease.
Arterioscler Thromb Vasc Biol
18
:
277
–282,
1998
4.
Mohamed-Ali V, Pinkney JH, Panahloo A, Cwyfan-Hughes S, Holly JMP, Yudkin JS: Insulin-like growth factor binding protein in NIDDM.
Clin Endocrinol
50
:
221
–228,
1999
5.
Prewitt TE, Unterman TG, Glick R, Cole TG, Schmeisser D, Bowen PE, Langenberg P: Insulin-like growth factor-I and low density lipoprotein cholesterol in women during high- and low-fat feeding.
Am J Clin Nutr
55
:
381
–384,
1992
6.
Hoogerbrugge van den Linden N, Jansen H, Hülsmann WC, Birkenhäger JC: Relationship between insulin-like growth factor-I and low density lipoprotein cholesterol levels in primary hypothyroidism in women.
J Endocrinol
123
:
341
–345,
1989
7.
Ostlund RE Jr, Staten MA, Kohrt WM, Obert KA, Daughaday WH: Insulin-like growth factor-I and apolipoprotein B.
JAMA
266
:
1937
–1938,
1991
8.
Fath KA, Alexander RW, Delafontaine P: Abdominal coarctation increases insulin-like growth factor I mRNA levels in rat aorta.
Circ Res
72
:
271
–277,
1993
9.
Duerr RL, Huang S, Miraliakbar HR, Clark R, Chien KR, Ross J: Insulin-like growth factor-I enhances ventricular hypertrophy and function during the onset of experimental heart failure.
J Clin Invest
95
:
619
–627,
1995
10.
Ververis JJ, Ku L, Delafontaine P: Regulation of insulin-like growth factor I receptors on vascular smooth muscle cells by growth factors and phorbol esters.
Circ Res
72
:
1285
–1292,
1993
11.
Suikkari AM, Koivisto VA, Rutanen EM, Yki-Järvinen H, Karonen SL, Seppä M: Insulin regulates the serum levels of low molecular weight insulin-like growth factor binding protein.
J Clin Endocrinol Metab
66
:
266
–272,
1988
12.
Conover CA, Lee PDK, Kanaley JA, Clarkson JT, Jensen MD: Insulin regulation of insulin-like growth factor binding protein-1 in obese and nonobese humans.
J Clin Endocrinol Metab
74
:
1355
–1360,
1992
13.
Lee DKL, Giudice LC, Conover CA, Powell DR: Insulin-like growth factor binding protein-1: recent findings and new directions.
Proc Soc Exp Biol Med
216
:
319
–357,
1997
14.
Bar RS, Boes M, Clemmons DR, Busby WH, Sandra A, Dake BL, Booth BA: Insulin differentially alters transcapillary movement of intravascular IGFBP-1, IGFBP-2 and endothelial cell IGF-binding proteins in the rat heart.
Endocrinology
127
:
497
–499,
1990
15.
Heald AH, Cruickshank JK, Riste LK, Cade JE, Anderson S, Greenhalgh A, Sampayo J, Taylor W, Fraser W, White A, Gibson JM: Close relationship of fasting insulin-like growth factor binding protein-1 (IGFBP-1) with glucose tolerance and cardiovascular risk in 2 populations.
Diabetologia
44
:
333
–339,
2001
16.
Westwood M, Gibson JM, Williams AC, Clayton PE, Hamberg O, Flyvbjerg A, White A: Hormonal regulation of circulating insulin-like growth factor phosphorylation status.
J Clin Endocrinol Metab
80
:
3520
–3527,
1995
17.
Westwood M, Gibson JM, White A: Purification and characterization of the insulin-like growth factor-binding protein-1 phosphoform found in normal plasma.
Endocrinology
138
:
1130
–1136,
1997
18.
Gibson JM, Westwood M, Lauszus FF, Klebe JG, Flyvbjerg A, White A: Phosphorylated insulin-like growth factor binding protein 1 is increased in pregnant diabetic subjects.
Diabetes
48
:
321
–326,
1999
19.
Gill MS, Whatmore AJ, Tillman V, White A, Addison GM, Price DA, Clayton PE: Urinary IGF and IGF binding protein-3 in children with disordered growth.
Clin Endocrinol
46
:
483
–492,
1997
20.
Crosby SR, Anderton CD, Westwood M, Holly JMP, Cwyfan Hughes SC, Gibson JM, Morrison CA, Young RJ, White A: Measurement of insulin-like growth factor-II in human plasma using a specific monoclonal antibody-based two-site immunoradiometric assay.
J Endocrinol
137
:
141
–150,
1993
21.
Westwood M, Gibson JM, Davies AJ, Young RJ, White A: The phosphorylation pattern of insulin-like growth factor binding protein-1 in normal plasma is different from that in amniotic fluid and changes during pregnancy.
J Clin Endocrin Metab
79
:
1735
–1741,
1994
22.
Friedewald WT, Levy RI, Frederickson DS: Estimation of the concentration of low-density lipoprotein cholesterol in plasma without use of the preparative ultracentrifuge.
Clin Chem
18
:
499
–502,
1972
23.
Frost RA, Bereket A, Wilson TA, Wojnar MM, Lang CH, Gelato MC: Phosphorylation of insulin-like growth binding protein-1 in patients with insulin-dependent diabetes mellitus and severe trauma.
J Clin Endocrin Metab
78
:
1533
–1535,
1994
24.
Hossenlopp P, Seurin D, Segovia-Quinson B, Hardouin S, Binoux M: Analysis of serum IGFBPs using Western blotting: use of the method for titration of the binding proteins and competitive binding studies.
Anal Chem
154
:
138
–143,
1986
25.
Gibson JM, Westwood M, Crosby SR, Gordon C, Holly JM, Fraser W, Anderson C, White A, Young RJ: Choice of treatment affects plasma levels of insulin-like growth factor-binding protein-1 in noninsulin-dependent diabetes mellitus.
J Clin Endocrinol Metab
80
:
1369
–1375,
1995
26.
Cruickshank JK, Heald AH, Anderson SG, Cade JE, Sampayo J, Riste LK, Greenhalgh A, Taylor W, Fraser W, White A, Gibson JM: The epidemiology of the insulin-like growth factor system in 3 ethnic groups.
Am J Epidemiol
154
:
504
–513,
2001
27.
Gudmensen O, Nenseter MS, Berg T: Endocytosed LDL and beta-VLDL follow different pathways in rat liver.
Biochim Biophys Acta
1210
:
63
–72,
1993
28.
Baxter RC: Insulin-like growth factor (IGF)-binding proteins: interactions with IGFs and intrinsic bioactivities.
Am J Physiol Endocrinol Metab
278
:
E967
–E976,
2000
29.
Verdecchia P, Reboldi G, Schillaci G, Borgioni C, Ciucci A, Telera MP, Santeusanio F, Porcellati C, Brunetti P: Circulating insulin and insulin-like growth factor-I are independent determinants of left ventricular mass and geometry in essential hypertension.
Circulation
100
:
1802
–1807,
1999
30.
Ruotolo G, Bavenholm P, Brismar K, Efendic S, Ericsson CG, de Faire U, Nilsson J, Hamsten A: Serum insulin-like growth factor-I level is independently associated with coronary artery disease progression in young male survivors of myocardial infarction: beneficial effects of bezafibrate treatment.
J Am Coll Cardiol
35
:
647
–654,
2000
31.
Ohtsuka H, Komiya I, Aizawa T, Yamada T: Hypertension in acromegaly: hereditary hypertensive factor produces hypertension by enhancing IGF-I production.
Endocr J
42
:
781
–781,
1995
32.
Jones JI, Gockerman A, Busby WH, Wright G, Clemmons DR: Insulin-like growth factor binding protein-1 stimulates cell migration and binds to the alpha 5 beta 1 integrin by means of its Arg-Gly-Asp sequence.
Proc Natl Acad Sci U S A
90
:
10553
–10557,
1993
33.
Bang P, Brismar K, Rosenfeld RG: Increased proteolysis of insulin-like growth factor binding protein-3 (IGFBP-3) in non-insulin dependent diabetes mellitus serum, with elevation of a 29-kilodalton (kDa) glycosylated IGFBP-1 fragment contained in the approximately 130- to 150-kDa ternary complex.
J Clin Endocrinol Metab
78
:
1119
–1127,
1994
34.
Balajaran R: Ethnicity and variations in the nation’s health.
Health Trends
27
:
114
–119,
1995
35.
Gibson JM, Aplin JD, White A, Westwood M: Regulation of IGF bioavailability in pregnancy.
Mol Hum Reprod
7
:
79
–87,
2001

Address correspondence and reprint requests to Dr. J. Martin Gibson, Department of Diabetes and Endocrinology, University of Manchester, Salford Royal Hospitals University Trust, Hope Hospital, Stott Lane, Salford, Greater Manchester, M6 8HD U.K. E-mail: mgibson@fs1.ho.man.ac.uk.

Received for publication 27 September 2000 and accepted in revised form 1 May 2002.

ELISA, enzyme-linked immunosorbent assay; hpIGFBP-1, highly phosphorylated IGFBP-1; IGFBP, insulin-like growth factor binding protein; IGT, impaired glucose tolerance; lpIGFBP-1, lesser-phosphorylated/nonphosphorylated IGFBP-1; MAP, mean arterial pressure; NEFA, nonesterified fatty acid; tIGFBP-1, total IGFBP-I; VSMC, vascular smooth muscle cell.