The authors state that they have no conflicts of interest.
Version of Record online: 12 MAR 2007
Copyright © 2007 ASBMR
Journal of Bone and Mineral Research
Volume 22, Issue 6, pages 799–807, June 2007
How to Cite
Amin, S., Riggs, B. L., Melton, L. J., Achenbach, S. J., Atkinson, E. J. and Khosla, S. (2007), High Serum IGFBP-2 Is Predictive of Increased Bone Turnover in Aging Men and Women. J Bone Miner Res, 22: 799–807. doi: 10.1359/jbmr.070306
Published online on March 12, 2007;
- Issue online: 4 DEC 2009
- Version of Record online: 12 MAR 2007
- Manuscript Received: 9 OCT 2007
- Manuscript Accepted: 5 MAR 2007
- Manuscript Revised: 18 JAN 2007
- insulin-like growth factors;
- insulin-like growth factor binding protein-2;
- bone resorption;
- bone formation;
- bone turnover markers;
Elevated serum IGFBP-2 is associated with lower BMD in men and women. It is unknown whether IGFBP-2 serves as a negative regulator of bone metabolism by decreasing bone formation or increasing bone resorption. Studying an age-stratified community-based sample of 344 men and 276 women, IGFBP-2 was the strongest predictor of increased bone resorption among the IGF/IGFBPs studied.
Introduction: Serum insulin-like growth factor binding protein-2 (IGFBP-2), which increases with age, is a predictor of low BMD among aging men and women. However, it is unknown whether IGFBP-2 negatively influences bone metabolism by decreasing bone formation or increasing bone resorption. Few have examined the relation between the insulin-like growth factors (IGFs) and their binding proteins (IGFBPs) with bone turnover markers.
Materials and Methods: In an age-stratified, random sample of the community, we examined the association between serum IGF-I, IGF-II, IGFBP-1, −2, and −3, and bone turnover markers before and after adjustment for potential confounders (age, body mass index, bioavailable estradiol and testosterone, and sex hormone binding globulin). Analyses were stratified by sex and menopausal status.
Results: We studied 344 men (age range, 23–90 yr) and 276 women (age range, 21–93 yr; 166 postmenopausal) not on oral contraceptives or hormone replacement. Among the IGF/IGFBPs assessed, IGFBP-2 was the strongest and most consistent predictor of bone turnover in men and women. After adjustment for potential confounders, IGFBP-2 was positively associated with osteocalcin (OC) and urine and serum N-teleopeptide (NTX) in men (r = 0.20, 0.26, and 0.23, respectively; p < 0.001), serum C-telopeptide (CTX) in premenopausal women (r = 0.28; p < 0.01), and OC, urine NTX, and serum CTX in postmenopausal women (r = 0.24, 0.33, and 0.19, respectively; p < 0.05).
Conclusions: Higher serum IGFBP-2, which is predictive of lower BMD, is associated with increased markers of bone resorption, independent of age, body mass, and sex hormones. The association between IGFBP-2 and markers of bone formation may reflect coupling with increased bone resorption, which is not adequate to maintain BMD.
The insulin-like growth factor (IGF) system, a growth promoting regulatory system that includes IGF-I, IGF-II, and six IGF binding proteins (IGFBPs), is known to play an important role in skeletal modeling and growth during pubertal development.(1–3) Serum IGF/IGFBPs levels are known to change with aging, and thus, may also contribute to age-related bone loss,(4,5) although their role is not well understood. Whereas the decline in sex steroid levels are key factors in the pathogenesis of bone loss associated with aging, further understanding on other potential risk factors, including the IGF system, may improve our ability to assess risk and manage this condition.
Studying serum IGF-I, IGF-II, IGFBP-1, IGFBP-2, and IGFBP-3 in a community-based sample of adult men and women, we previously showed that high IGFBP-2 levels were the strongest predictor of low BMD, particularly among men and postmenopausal women, independent of age and bioavailable sex steroids.(6) Although some have found no association between IGFBP-2 and BMD,(7–9) others have shown an inverse association between IGFBP-2 and BMD in both men(10,11) and women.(12,13) Furthermore, elevated serum IGFBP-2 levels were reported to be a predictor of osteoporotic fractures in men.(14)
These findings suggest that elevated serum IGFBP-2 may have a negative effect on bone metabolism in aging men and women. However, it is unknown whether the deleterious effects of IGFBP-2 on bone metabolism reflect a decrease in bone formation or an increase in bone resorption. IGFBP-2 has been shown to have an inhibitory effect on anabolic IGF-I and IGF-II action on bone,(15–17) so elevated levels could lead to a decrease in bone formation by interfering with IGF action. On the other hand, IGFBP-2 is recognized to have IGF-independent actions in some tissues.(18–20) Although such effects have not been well evaluated in bone tissue, it does remain possible that elevated IGFBP-2 could be negatively influencing bone metabolism independent of IGF action, by either decreasing bone formation or increasing bone resorption. Regardless, it remains unknown how IGFBP-2 may be influencing bone turnover in aging men and women.
Relatively few studies have examined the association between the IGF/IGFBPs and markers of bone turnover in both young and old adult men and women. We therefore studied the relationship of serum IGF-I, IGF-II, and the binding proteins IGFBP-1, IGFBP-2, and IGFBP-3 with markers of bone formation and bone resorption among an age-stratified, population-based sample of adult men and women in whom we have previously characterized the associations between these IGF/IGFBPs and BMD.(6) Exploring the role of IGFBP-2, in particular, on markers of bone turnover would help to improve our understanding on the mechanism whereby IGFBP-2 may exert its adverse effects on bone metabolism of aging men and women.
MATERIALS AND METHODS
After approval by Mayo Clinic's Institutional Review Board, subjects were recruited from age-stratified random samples of Rochester, MN, men and women that were selected using the medical records linkage system of the Rochester Epidemiology Project.(21) More than one half of the Rochester population is identified annually in this system, and the majority is seen in any 3-yr period. Thus, the enumerated population (Rochester women seen in 1990 ± 1 yr and men seen in 1991 ± 1 yr) approximates the underlying population of the community, including both free-living and institutionalized individuals. Equal numbers of male and female subjects over the age of 20 yr were recruited to enroll 100 subjects per decade of age stratum. Altogether, 348 men and 351 women were recruited, as described in detail previously.(22) All but 13 men and 2 women were white, reflecting the ethnic composition of the population (96% white in 1990).
Because exogenous sex steroids have been shown to influence serum IGF/IGFBP levels,(23–27) we excluded one man receiving testosterone replacement; another three were excluded because of inexplicably high serum bioavailable estradiol levels (>60 pg/ml). Of the 351 women who participated, 28 premenopausal women on oral contraceptive pills (OCPs) and 47 postmenopausal women on hormone replacement therapy (HRT) were excluded from these analyses.
After providing written informed consent, subjects were interviewed in accordance with a standard protocol to collect clinical, demographic, and lifestyle data. BMD and body composition measurements were performed, and blood samples were drawn for biochemical analyses. Fasting state serum samples were obtained between 8:00 and 9:00 a.m., and for premenopausal women, were collected during the follicular phase of the menstrual cycle. All samples were stored at −70°C until analyzed. In addition, each subject underwent anthropometric assessment at the time of initial interview, which included measurements of height to the nearest 0.1 cm and weight in light clothes without shoes to the nearest 0.1 kg. Body mass index (BMI; kg/m2) was calculated from the height and weight of each subject.
Bone turnover markers:
Bone formation was assessed by measurement of fasting serum levels of bone alkaline phosphatase (BALP) isoenzyme, carboxy-terminal propeptide of type I collagen (PICP), and osteocalcin (OC). Serum BALP was measured by ELISA (Metra Biosystems, Mountain View, CA, USA; interassay CV < 11%).(28) Serum PICP was also measured by ELISA (Prolagen C; Metra Biosystems; interassay CV < 7%). Serum OC was measured by radioimmunoassay (RIA) using antibody G12 (CIS Biointernational, Bedford, MA, USA; interassay CV < 6%).(29) Bone resorption was evaluated through 24-h urine levels of cross-linked N-telopeptides (NTX) of type I collagen, assessed by ELISA (Osteomark; Ostex International, Seattle, WA, USA; interassay CV, 10%). The urine NTX was expressed in units of nanomoles per liter of glomerular filtrate (nM/liter GF), because this represents the more precise correction for alterations in renal function. The glomerular filtration rate was determined from the creatinine clearance and urine creatinine. Results for urine NTX expressed as nM/mM Cr are also provided for comparison; the correlation between these two variables is high (r = 0.91). We also measured fasting serum levels of NTX by an ELISA kit (Osteomark NTX Serum; Ostex International; interassay CV < 17%). For women only, bone resorption was also assessed using fasting serum cross-linked C-telopeptide of type I collagen (CTX; Osteometer BioTech; CV < 14%).
Total IGF-I and IGF-II were each measured by a two-site immunoradiometric assay (IRMA), after separation from their binding proteins with a simple organic solvent extraction (Diagnostic Systems Laboratories, Webster, TX, USA; interassay CVs, 6% for each).(30) IGFBP-1 and IGFBP-3 were also each measured by a two-site IRMA (Diagnostic Systems Laboratories; interassay CV, 7% for IGFBP-1 and 14% for IGFBP-3). IGFBP-2 was measured by a double antibody RIA (Diagnostic Systems Laboratories; interassay CV, 16%).
Fasting serum samples were assayed by RIA for total estradiol and total testosterone (Diagnostic Products Corp., Los Angeles, CA, USA; interassay CVs, 11% for each), and sex hormone binding globulin (SHBG; Wien Laboratories, Succasunna, NJ, USA; interassay CV, 7%). Non–SHBG-bound (bioavailable) fractions of estradiol (BioE2) and testosterone (BioT) were measured using a modification of the techniques of O'Connor et al.(31) and Tremblay and Dube(32) as described previously.(22)
Analyses were stratified by sex. In women, analyses were further stratified by menopausal status. Menopause was defined as either documented bilateral oophorectomy or >6 mo without a menstrual period. The Wilcoxon rank sum test was used to compare differences in medians for all variables by sex and menopausal status. To determine which of the IGF/IGFBPs were more strongly associated with each of the bone turnover markers, we performed age-adjusted stepwise regression analyses, where all IGF/IGFBPs were included as potential predictors. We also used Pearson correlations to examine the relationship between each of the IGF/IGFBPs and bone turnover markers, both unadjusted and adjusted for age. We examined the correlation between each of IGF/IGFBP measures and each of the markers of bone turnover after further adjustment for potential confounders: age, BMI, Bio E2, Bio T, and SHBG. We adjusted for bioavailable sex steroids in our analyses, not total E2 or T, based on work done previously in this cohort.(22) A significance level of p < 0.05 was used in all analyses. IGFBP-1, NTX, and CTX were log-transformed for analyses. Bio E2 was log-transformed for analyses involving postmenopausal women. All analyses were performed using SAS (SAS Institute, Cary, NC, USA).
Our study population for our analyses consisted of 344 men (age range, 23–90 yr; mean, 55.3 ± 19.6 [SD] yr) and 276 women (age range, 21–93 yr; mean, 56.6 ± 19.9 yr; 166 postmenopausal). The characteristics of subjects are outlined in Table 1.
As previously shown in this population, in both men and women, IGF-I and IGFBP-3 levels fell with advancing age, IGFBP-2 levels tended to rise with older age, whereas there was no association between age and either IGF-II or IGFBP-1.(6) In women, but not in men, OC, urine NTX, and serum NTX levels all tended to be higher with older age (Figs. 1 and 2). Serum CTX levels also tended to be higher with increasing age in older women (Fig. 2). As noted in the figures, a few outlier data were noted among the markers of bone turnover in men and women. We have presented our results with outlier data included, but our findings remained similar when outlier data were excluded.
IGF/IGFBPs as predictors of bone turnover markers
Bone formation markers:
In age-adjusted step-wise regression models, where all IGF/IGFBPs were included as potential predictors of each of the bone formation markers, there were differences in the significant (p < 0.05) predictors identified for men and women. In men, high serum IGF-I was a predictor of elevated BALP, high serum IGFBP-2 was a predictor of elevated PICP, whereas high serum levels of both were predictors of elevated levels of OC. In premenopausal women, high serum IGFBP-1 and IGFBP-2 were both predictors of elevated OC, whereas none of the IGF/IGFBPs were predictors of either BALP or PICP. In postmenopausal women, low serum IGFBP-3 was a predictor of both elevated levels of BALP and PICP, whereas both high serum IGFBP-2 and IGFBP-1 were predictors elevated OC.
Bone resoption markers:
In similar analyses examining the bone resorption markers, high serum IGF-I and IGFBP-2 were both predictors of elevated urine and serum NTX in men. In premenopausal women, high serum IGFBP-2 was the only predictor of elevated urine NTX, serum NTX and CTX. In postmenopausal women, high serum IGF-I and IGFBP-2, but low IGFBP-3, were predictors of elevated levels of urine NTX, whereas high serum IGFBP-2 was the only predictor of both increased serum NTX and CTX. Overall, among the IGF/IGFBPs studied, IGFBP-2 emerged as the strongest predictor of all bone resorption markers in men and premenopausal and postmenopausal women.
When we explored for an interaction between IGF-I and IGFBP-2 in these regression models, we found no relevant associations for any of the bone turnover markers, in either men or women.
Multivariate-adjusted correlations for IGF/IGFBPs and bone turnover markers
Unadjusted and adjusted correlations between IGF/IGFBPs and bone turnover markers for all 344 men, 110 premenopausal women, and 166 postmenopausal women are presented in Tables 2, 3, and 4, respectively.
Adjusted for age, high serum IGF-I was associated with increased levels of BALP, OC, urine NTX, and serum NTX in men (r = 0.16, 0.13, 0.14, and 0.12, respectively; all p < 0.05; Table 2), but low levels of BALP in postmenopausal women (r = −0.16, p < 0.05; Table 4). After additional adjustment for BMI, Bio E2, Bio T, and SHBG, high serum IGF-I remained associated with increased BALP, OC, urine NTX, and serum NTX in men only (r = 0.22, 0.15, 0.20, and 0.19, respectively; p < 0.01) (Tables 2–4).
No associations were seen between IGF-II and any of the markers of bone turnover in multivariate adjusted models for either men or women (Tables 2–4).
In multivariate adjusted models, high serum IGFBP-1 was associated with increased levels of OC in men (r = 0.12, p < 0.05; Table 2) and increased levels of both BALP and OC in premenopausal women (r = 0.22 and 0.20, respectively; p < 0.05; Table 3). In multivariate adjusted models, no association was seen between IGFBP-1 and any of the bone turnover markers in postmenopausal women (Table 4).
In age-adjusted models, high serum IGFBP-2 was associated with elevated levels of several markers of bone turnover in men and women (Tables 2–4). In multivariate adjusted models, high serum IGFBP-2 levels remained associated with increased serum OC, urine NTX, and serum NTX in men (r = 0.20, 0.26, and 0.23, respectively; p < 0.001), with increased serum CTX in premenopausal women (r = 0.28; p < 0.01), and with increased serum OC, urine NTX, and serum CTX in postmenopausal women (r = 0.24, 0.33, and 0.19, respectively; p < 0.05) (Tables 2–4).
In this population-based study of adult men and women, we found serum IGFBP-2 to be the strongest and most consistent predictor of bone turnover among the IGF/IGFBPs studied. High serum IGFBP-2 levels were more consistently associated with elevated levels of bone resorption markers, particularly in men and postmenopausal women, independent of age, body mass index, and sex steroid levels.
Although osteocalcin, a bone formation marker, was increased with higher levels of serum IGFBP-2 despite adjustment for confounders, including age, this may be the result from coupling of bone formation with increased bone resorption. However, we did not identify an association between IGFBP-2 and other markers of bone formation after adjustment for potential confounders (age, BMI, and sex steroids). Bone formation and bone resorption markers were correlated with each other and ranged from 0.29 to 0.58 for men, 0.25 to 0.56 for premenopausal women, and 0.39 to 0.64 for postmenopausal women. In men and both groups of women, the weakest correlations observed were between PICP and bone resorption markers, whereas the strongest correlations were seen between OC and bone resorption markers. When we explored whether IGFBP-2 remained an independent predictor of osteocalcin after adjustment for markers of bone resorption, we found that the association was attenuated in men, whereas no further association was identified in women (data not shown). These results would suggest that the association between IGFBP-2 and OC may be confounded by the strong correlation between OC and bone resorption markers. Nevertheless, given the fact that we previously showed that higher serum IGFBP-2 levels are associated with lower BMD in this population,(6) it would seem that any apparent increase in bone formation reflects increased bone turnover, yet is not adequate to maintain BMD.
Whereas IGF-I, IGF-II, and IGFBP-3 are all considered anabolic to bone,(2,19,33) only among men did we find a consistent association between higher serum IGF-I levels and greater markers of bone formation. In our age-adjusted, stepwise regression model where we examined all the IGF/IGFBPs together as potential predictors, both IGF-I and IGFBP-2 were independent predictors of bone turnover in men. The increase in bone resorption markers observed with higher IGF-I levels, again, likely reflects coupling from enhanced bone formation. Unlike in men, we did not identify an association between IGF-I and bone formation markers in women. Men are known to have greater periosteal apposition of bone with aging than women, and we speculate that this may be, in part, under IGF-I control. We found no relevant interactions between IGF-I and IGFBP-2 on markers of bone turnover. Interestingly, we identified a positive association between IGFBP-1 and some bone formation markers among men and premenopausal women. In this same population of men and women, however, we found no association between IGFBP-1 and BMD.(6) Contrary to our expectations, in postmenopausal women, there was a tendency for an inverse association between IGFBP-3 and some bone turnover markers. Although generally considered a potentiator of IGF action, IGFBP-3 has also been shown to have inhibitory effects on osteoblasts.(19) Again, however, we did not find strong associations between the IGF/IGFBPs and BMD in this same study population, with the exception of IGFBP-2,(6) which is consistent with our overall findings on the relation between the IGF/IGFBPs and bone turnover markers.
Our results add further evidence that elevated IGFBP-2 may serve as a negative regulator of bone metabolism.(10–15,34,35) How this occurs is uncertain but open to speculation. Although, in animal and in vitro studies, high IGFBP-2 levels have a negative effect on developing bone by interfering with the anabolic effects of IGF-I and IGF-II,(15–17) this would be an unlikely explanation for our results; we would have anticipated an inverse association with bone formation markers, which we did not find, not a positive association with bone resorption. The mechanism by which elevated IGFBP-2 exerts its negative effect on bone may differ for mature bones. It is possible that IGFBP-2 may be stimulating bone resorption through IGF-independent effects. IGFBP-2 has been shown to independently stimulate the proliferation and activation of peripheral blood mononuclear cells(20); cytokines released from activated peripheral blood mononuclear cells have been implicated in increasing bone resorption.(36–39)
However, in contrast to the evidence for a negative impact of IGFBP-2 on bone, some studies have suggested an anabolic action of IGFBP-2 on bone. Exogenous IGFBP-2 stimulates IGF-II–mediated osteoblast activity in vitro,(40) whereas systemic administration of an IGF-II/IGFBP-2 complex prevented bone loss in rats after sciatic neurectomy.(41) Concomitant elevations in an IGF-II precursor molecule (IGF-IIE) and IGFBP-2 were noted in patients with the rare syndrome of hepatitis C–associated osteosclerosis.(42) These studies would suggest that IGFBP-2 might actually have beneficial effects to bone under certain circumstances. Nonetheless, elevated serum IGFBP-2 may play a different role on bone metabolism in the hormonal milieu of aging men and women.
Our study has limitations. Bone turnover markers were collected at a single point in time; the biologic variation in these different markers could account for some of the lack of associations observed. It is not known whether the observed associations based on serum levels of the IGF/IGFBPs reflect their action at the level of bone tissue. The IGF/IGFBPs are produced by osteoblasts and are known to have autocrine and paracrine effects.(33) Furthermore, we do not have measures of three of the six known IGFBPs (IGFBP-4, IGFBP-5, and IGFBP-6), of which IGFBP-4 and IGFBP-5 have been shown to be important regulators of bone metabolism at the level of bone tissue.(33,43) How IGFBP-2 may interact with IGFBP-4 or IGFBP-5 is unknown. Nevertheless, our study is the first to examine the association between serum IGFBP-2 and bone turnover markers in a relatively large number of both young and older, men and women. We have also been able to evaluate the associations between the IGF/IGFBPs and bone turnover markers, while taking into account other important covariates including sex steroid levels. Although we do not have measures of free estradiol and testosterone, we have previously shown important associations between bioavailable sex steroids and bone metabolism in this same cohort,(22) so adjustment for bioavailable sex steroids were performed in analyses. We can not exclude the fact that residual confounding could still be influencing our associations between the IGF/IGFBPs and bone turnover markers caused by our lack of free estradiol and testosterone measures. Although not all potential confounders could be addressed, we did explore whether results were influenced by season or vitamin D or PTH levels, but they were not (data not shown). Although our study population consisted of a large sample of men and women spanning a wide age range, those excluded from participation were primarily men and women who were cognitively impaired and therefore unable to provide informed consent.(22) Our results, therefore, may not be generalizable to those individuals. Finally, our results are from a cross-sectional, epidemiologic study. Whereas our findings are hypothesis generating, we can not infer causality, and additional studies are needed to clarify the relation between IGF/IGFBPs, including IGFBP-2, on bone metabolism of aging men and women. Our data would suggest the need for further exploring the role of the IGF system on age-related bone loss.
In summary, we identified higher serum IGFBP-2 levels to be associated with increased bone turnover, particularly increased bone resorption, in aging men and women. Our results add further evidence to suggest that elevated serum IGFBP-2 levels may serve as a negative regulator of bone metabolism in older men and women. These findings provide additional support on the likely role of the IGF system in age-related bone loss. Moreover, the association between higher serum IGFBP-2 and enhanced bone resorption would also suggest that IGFBP-2 may have a greater negative influence on trabecular bone than cortical bone. Further evaluation on the influence of IGFBP-2 on aspects on bone microarchitecture in aging men and women would be of interest.
The authors thank Vicki Gathje and Joan Muhs for help in recruiting subjects, Margaret Holets for BMD measurements, Roberta Soderberg for sample handling, and Jim Peterson for assistance with figures. This work was supported by Research Grants AR27065 and RR00585 from the National Institutes of Health, U.S. Public Health Service.
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