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Adiponectin and leptin are adipokines that influence bone metabolism in vitro and in animal models. However, less is known about the longitudinal association of leptin and adiponectin with fracture. We tested the hypothesis that low leptin and high adiponectin levels are each individually associated with fracture risk in a prospective cohort study in Memphis and Pittsburgh, among 3,075 women and men, aged 70–79, from the Health Aging and Body Composition (Health ABC) study. There were 406 incident fractures (334 non-vertebral and 72 vertebral) over a mean of 6.5 ± 1.9 years. Cox regression was used to estimate the hazard ratios for fracture. Sex modified the association between adiponectin and fracture (p for interaction=0.025). Men with the highest adiponectin level (tertile 3) had a 94 % higher risk of fracture (HR=1.94; 95% CI 1.20, 3.16) compared to the lowest tertile (tertile 1), p for trend=0.007 after adjusting age, race, BMI, education, diabetes weight change, and hip BMD. Among women, after adjusting for age and race this association was no longer significant (p for trend=0.369). Leptin did not predict fracture risk in women (p for trend=0.544) or men (p for trend=0.118) in the multivariate models. Our results suggest that adiponectin, but not leptin, may be a novel risk factor for increased fracture risk independent of body composition and BMD and that these relationships may be influenced by sex. More research is needed to understand the physiological basis underlying these sex differences.
Leptin is secreted by adipose cells and has been shown to be highly correlated with body fat mass1, and a regulator of fat metabolism and appetite.2 Leptin may also modulate bone metabolism by enhancing differentiation of bone marrow stroma cells into mature osteoblasts and by inhibiting the differentiation of osteoclasts.3;4 Studies in mice have shown that leptin administration increases BMD.5–7 Some population based cross-sectional studies reported that leptin was positively8–14 associated with BMD, while others have not confirmed this association15–17 or found a negative relationship.18–23
Adiponectin, a hormone exclusively secreted by adipocytes, influences insulin sensitivity and has anti-inflammatory properties.24;25 Adiponectin and its receptors are also expressed in human osteoblasts, suggesting that adiponectin may be a hormone linking bone and fat metabolism.26 However, adiponectin may have negative effects on bone metabolism by stimulating the receptor activator of nuclear factor-κB ligand (RANKL) pathway and inhibiting the production of the decoy receptor for RANKL, osteoprotegerin.27 Several epidemiological studies have found that lower levels of adiponectin are associated with higher BMD9;12;28–30 while others have failed to find an association between adiponectin and BMD.17;19;21;21;24;31–33
There are limited data available on the relationship of leptin14;34 and adiponectin30;35;36 to fracture risk and the results are conflicting. However, several of these studies were cross-sectional, had short follow-up time, low prevalence or incidence of fracture, and failed to adjust for change in weight. The aim of the current analysis was to test the hypothesis that lower serum leptin levels and higher adiponectin levels are associated with a higher risk of a fracture in older adults.
The Health Aging and Body Composition (Health ABC) study consists of 3,075 women and men, aged 70–79, from two field centers, Pittsburgh, PA and Memphis, TN. Among women and men enrolled, 46% and 37% were blacks, respectively. To be eligible to participate in Health ABC, subjects had to report no difficulty walking at least 1/4 mile and or climbing a flight of stairs. Participants were identified from a random sample of white Medicare beneficiaries and all age-eligible black community residents in designated ZIP code areas surrounding Pittsburgh and Memphis. Exclusion criteria included reported difficulty performing basic activities of daily living, obvious cognitive impairment, inability to communicate with the interviewer, intention of moving within 3 years, or participation in a trial involving a lifestyle intervention. The institutional review board (IRB) at each center approved the study protocol, and written informed consent was obtained from all the participants. Baseline data were collected from 1997 to 1998.
Specimens were obtained by venipuncture in the morning after an overnight fast, processed, aliquoted into cryovials, frozen at −70°C, and subsequently shipped to the Health ABC Core Laboratory at the University of Vermont. Baseline leptin (N=3020) concentrations were measured in duplicate using the Sensitive Human Leptin radioimmunoassy (RIA) Kit (product number SHL-81K) from Linco Research, Inc. (St. Charles, MO).37 The assay is a competitive RIA in which the concentration of leptin is determined by competition with 125I-Human Leptin, with a maximum detectable leptin level of 50 ng/ml. The intra-assay CV is 3.7–7.5% and the inter-assay CV is 3.2–8.9%. Adiponectin (N=3044) was assayed using baseline serum specimens that were frozen approximately 6 years earlier.38 Total circulating levels of adiponectin were measured in duplicate by RIA (Linco Research, St. Charles, MO) with an intra-assay coefficient of variation of 1.8–3.6%.
Incident fractures (non-vertebral and vertebral) were assessed every 6 months by self-report and validated by radiology reports. Adjudication of fractures was complete through 2007 for the Pittsburgh clinic, and through 2006 for the Memphis clinic. The mean follow-up time was 6.5 ± 1.9 years. Nontraumatic and traumatic fractures were included since both have been linked to low BMD.39 Pathological fractures and fractures of unknown etiology were excluded.
Demographic variables included self-report of age, race, (black or white), sex, site, and education (<high school (HS), HS graduate, or post secondary). Weight was measured on a standard balance beam scale to the nearest 0.1 kg, and height was measured by a stadiometer to the nearest 0.1 cm. BMI (kg/m2) was calculated by using the formula weight (kg) /height2(m2). Whole body DXA (QDR 4500A; software version 9.03; Hologic, Bedford, MA, USA) was also used to measure total lean body mass (kg), and body fat (kg). A separate scan of the proximal femur was done to measure hip BMD. DXA quality assurance procedures were conducted at both study sites and monitored by the study Coordinating Center, ensuring scanner reliability and identical scan protocols. An anthropometric spine phantom was scanned daily, and a hip phantom was scanned once per week to assess longitudinal performance of the scanners. Annual weight change (through visit 10) was estimated as the weight difference from baseline to the most recent re-assessment divided by the respective time. Lifestyle factors included self-report of smoking (never, current, or former), and alcohol consumption (no consumption in last year, <1 drink per week, 1–7 drinks per week, or >1 drink per day). To assess supplementary intake for vitamin D and calcium, and non-steroidal anti-inflammatory drug (NSAID) use, participants were asked to bring all prescription and over the counter medications, which were coded based on the Iowa Drug Information System.40 To estimate dietary intake of calcium and vitamin D, participants completed a 108-item interviewer-administered FFQ (Block Dietary Data Systems, Berkeley, CA). Physical activity (kcal/week) was determined using the caloric expenditure in the past week for self-reported walking, climbing stairs, and exercise.41 Diabetes was defined using fasting glucose (≥126 mg/dl), self-report, or hypoglycemic medication use. Similarly, subjects were classified as having hypertension through measurement of blood pressure (systolic ≥140 or diastolic ≥90), self-report or antihypertensive medication use. Other medical conditions were determined by asking respondents if they have ever been told by a doctor that they had a specific diagnosis of myocardial infarction (MI) and history of fracture after age 45. Handgrip strength (kg) was measured using a hand-held dynamometer (Jamar; TEC, Clifton, NJ). The dynamometer was adjusted for hand size for each participant, and two trials were performed on each hand with the maximum value recorded as the observation.
Two-sample t-tests (Wilcoxon rank-sum test for non-parametric measures) and chi-square tests were used to evaluate mean and proportion differences by sex, respectively. Leptin and adiponectin means differed in women and men and this difference could not be explained by BMI. Therefore, sex-specific tertiles were used in the analysis. For normally distributed variables, a test of linear trend was performed by treating leptin or adiponectin tertiles as continuous variables. The Cochran-Armitage test for trend was used for dichotomous variables. For non-parametric variables with more than 2 groups the Jonckheere-Terpstra test of trend was performed.
Cox proportional hazards models were used to compare the time to fracture by tertiles of leptin and adiponectin and estimate the hazards ratio while controlling for potential confounders. Secondary analyses were performed separately for non-vertebral and vertebral fractures. Schoenfeld residuals were used to the test the assumption of proportionality.
To further test if there was a linear relationship between adiponectin and fracture risk we performed spline analysis to test for possible inflection points. Restricted cubic spline linear regression was used with knots for adiponectin at the 5th, 25th, 75th and a reference group at the 95th percentile was set to create the spline plot. Threshold effects were evaluated by identifying potential inflection points on the spline and performing a test of equality to determine if the slopes above and below the cut point are equal. We did adjust for any covariates to preserve the shape and smoothness of the spline plot.
A backward elimination procedure was used with age, race, BMI and the exposure of interest forced in all the multivariate models. Diabetes, weight change and hip BMD were added in the final model to determine if these factors further explained our associations. Multi-collinearity was assessed using the variance inflation factor (VIF). Interactions between sex and leptin or adiponectin were also evaluated in the multivariate models. All statistical analyses were performed using the Statistical Analysis System (SAS, version 9.1; SAS Institute, Cary, NC).
Table 1 shows the baseline characteristics by sex. Women had significantly greater serum leptin (21.4 vs. 7.9 ng/ml) and adiponectin levels (13.3 vs. 9.5 µg/ml) than men (p<0.001). Women were also somewhat younger, had higher BMI, and total body fat, lower weight loss and total lean mass, more likely to never smoke or consume alcohol, had lower dietary vitamin D or calcium intake, lower prevalence of diabetes and MI, and lower grip strength than men. Men had significantly higher hip aBMD than women (0.97 ± 0.15 g/cm2 vs. 0.91 ± 0.15 g/cm2; p<0.001) at baseline.
Greater serum leptin levels were significantly associated with higher BMI, no alcohol use in the past year, diabetes, hypertension, and higher aBMD in both women and men (Table 2). Among women only, increasing leptin tertiles were significantly associated with lower age, greater weight loss, <HS education, supplementary calcium intake, lower dietary and supplementary vitamin D intake, lower physical activity, lower history of fracture prevalence, higher NSAID use, and grip strength. The direction of these associations was reversed for adiponectin with the exception of NSAID use in women.
A total of 406 incident fractures (non-vertebral=334 and vertebral=72) occurred over a mean follow-up of 6.5 ± 1.9 years. For women and men the fracture rates per 1000 person-years were: 27.5 and 14.0, respectively. Based on the unadjusted model, greater leptin levels were significantly (p for trend=0.009) associated with lower fracture rates, and higher adiponectin concentration predicted (p for trend=0.003) greater fracture risk in women (Table 3). In men, higher adiponectin was a predictor (p for trend=0.001) of increased fracture risk in the unadjusted models. However, the association of leptin and adiponectin with fracture in women was attenuated after adjusting for age, race, and BMI (p for trend >0.658). Men with the highest adiponectin level (tertile 3) had a 94 % higher risk of fracture (HR=1.94; 95% CI 1.09, 2.77) compared to the lowest tertile (tertile 1), p for trend=0.007 after adjusting for age, race, BMI, education, diabetes, weight change, and hip BMD. Sex modified the association between adiponectin tertiles and fracture risk in the full multivariate model (p for interaction=0.025). However, there was no evidence of a threshold for serum adiponectin and risk of a fracture in men (p for test of threshold>0.05; Figure 1).
Results of the secondary analyses for serum leptin/adiponectin and risk of non-vertebral and vertebral fractures are shown in Table 3. In the full multivariate model, men in tertile 3 for adiponectin had a 133% higher risk of non-vertebral fracture [HR=2.33; 95% CI 1.32, 4.10] compared to tertile 1, p for trend=0.004. However, there was very limited power to detect any significant associations for incident vertebral fractures by serum leptin or adiponectin.
We conducted the largest and most comprehensive analysis of the association of leptin and adiponectin with risk of fracture among older individuals. We found that among older men, higher adiponectin levels were associated with an increased risk of fracture independent of potential confounders including BMI, diabetes, weight change and BMD. This association was not observed among older women and the interaction between adiponectin and sex was statistically significant. On the other hand, the impact of leptin on fracture risk was largely explained by BMI in both groups.
Sex modified the association between adiponectin and fracture. Men in the highest adiponectin tertile had a 94% higher risk of fracture than men in lowest tertile. This association was independent of BMI, weight change and other potentially important covariates. In contrast, adiponectin levels were unrelated to fracture risk in women. Our results are consistent with those from the Rancho Bernardo Study which recently reported an association between adiponectin and fractures [adjusted OR:1.13; 95% CI:1.08–1.23] in men not women.36 Participants of the Rancho Bernardo Study were similar to our subjects in age and smoking prevalence, but had lower BMI, fat mass, adiponectin and prevalence of diabetes. Also, consistent with our findings, a cross-sectional study in Japan among 231 men and 170 post-menopausal Japanese women, reported a one standard deviation difference in adiponectin was associated with higher odds of vertebral fracture in men, but not women.35 In contrast, a recent prospective study (15 year follow-up) in Sweden, did not find an association adiponectin levels and fracture, among 507 men aged 70 and older.30 The results of our spline analysis among men, suggest that this association was linear with no evidence of an adiponectin threshold for fracture risk.
Recombinant adiponectin induces RANKL and inhibits OPG mRNA expression in human osteoblasts in a dose and time dependent manner leading to osteoclast formation.27 Our findings are consistent with these data showing that adiponectin at higher levels may have a direct influence on bone turnover and remodeling. The risk of fracture with greater levels of adiponectin may reflect greater osteoclast activation and bone resorption. However, population based longitudinal studies evaluating the association of adiponectin and markers of bone turnover are needed.
Our results suggest that adiponectin is a unique risk factor for fracture in men. It is unclear why there are gender differences for the association of adiponectin with incident fractures. Adiponectin was 40% higher in women compared to men. However, it is uncertain if sex differences in adiponectin account for these differential associations. BMI and weight change explained fracture risk in women; however in men these variables did not predict fractures. Prior studies suggest that the association between adiponectin and bone may be influenced by sex hormones.36 Future prospective studies that include both adiponectin and sex hormones are needed to further our understanding of adiponectin and fracture.
In contrast to adiponectin, we found no evidence of an association between serum leptin levels and the risk of fracture in this cohort of older adults. A ten year cohort study among 250 Italian women and men with comparatively lower leptin levels reported that subjects in the highest leptin tertile had a significantly reduced risk of a non-traumatic fracture when compared to the lowest tertile group after adjusting for weight and other confounders.34 However, this study included relatively younger participants (mean age 58.5 years), and also included few fracture cases (N=31). Higher leptin levels were also associated with a lower prevalence of vertebral fractures independent of percent body fat among 139 Japanese postmenopausal women aged 48–78.14 These two studies included relatively younger subjects and older age is associated with a greater number of comorbidities. Therefore, age and other potentially confounding variables such as BMI, weight change, and grip strength in our study were able to explain fracture risk better than leptin levels.
Our study had several potential limitations. We measured leptin and adiponectin at baseline only. In addition, survivors may have greater BMD than non-survivors42 and study participants are likely to be healthier than the general population. Also, this assay cannot distinguish high and low molecular weight forms of adiponectin which may have varying biological activities.43 Finally, we adjusted for many covariates, however lacked sufficient data to adjust for insulin (a known correlate of adiponectin), and several bone markers including osteocalcin, crosslinks, PINP and serum CTX. Our study also had notable strengths including its large sample size, reliable measurement of leptin and adiponectin, and validation of fractures, adjustment for many potential confounders, and long follow-up period
In summary, in a large cohort of racially diverse older women and men, higher adiponectin levels were associated with an increased risk of fracture in men, but not women, after controlling for multiple confounders including measures of adiposity and BMD. Serum leptin levels were not associated with fracture risk in either men or women. Our findings suggest that adiponectin may be a novel risk factor for fractures among older men. Future studies are needed to evaluate and understand the physiological basis for the potential sex differences in the relationship between adiponectin and fracture.
Role of Funding Source: The Health Aging and Body Composition Study (Health ABC) includes the contract numbers: N01-AG-6-2101, N01-AG-6-2103, N01-AG-6-2106. This research was supported in part by the Intramural Research Program of the NIH, National Institute on Aging.
Conflict of Interest: All authors have no conflict of interest