0021-972X/01/$03.00/0 The Journal of Clinical Endocrinology & Metabolism Copyright © 2001 by The Endocrine Society
Vol. 86, No. 5 Printed in U.S.A.
Serum Leptin Levels Are Associated with Bone Mass in Nonobese Women* JULIE A. PASCO, MARGARET J. HENRY, MARK A. KOTOWICZ, GREGORY R. COLLIER, MADELEINE J. BALL, ANTONY M. UGONI, GEOFFREY C. NICHOLSON
AND
The University of Melbourne, Department of Clinical and Biomedical Sciences, Barwon Health (J.A.P., M.J.H., M.A.K., G.C.N.), and Department of General Practice and Public Health (A.M.U.), Metabolic Research Unit, School of Health Sciences (G.R.C.), Victoria 3220, Australia; and School of Biomedical Sciences (M. J. B.), University of Tasmania, Tasmania 7250, Australia ABSTRACT Both serum leptin and bone mineral density are positively correlated with body fat, generating the hypothesis that leptin may be a systemic and/or local regulator of bone mass. We investigated 214 healthy, nonobese Australian women aged 20 –91 yr. Bone mineral content, projected bone area, and body fat mass were measured by dual energy x-ray absorptiometry and fasting serum leptin levels by RIA. Associations between bone mineral content (adjusted for age, body weight, body fat mass, and bone area) and the natural logarithm of serum leptin concentrations were analyzed by multiple regression techniques. There was a significant positive association at the lateral spine, two proximal femur sites (Ward’s triangle and trochanter), and
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EPTIN IS A hormone with a diverse range of local and systemic physiological functions. In addition to its role as a satiety factor and involvement in regulating energy balance, leptin modulates the reproductive (1, 2), hematopoietic, and immune systems (3–5); promotes angiogenesis (6, 7); and is involved in brain development (8) and regulation of carbohydrate metabolism (9 –11). In vitro studies have demonstrated a direct effect of leptin on human marrow stromal cells, stimulating osteoblast differentiation and mineralization of bone matrix (12). A role for leptin in fetal bone metabolism has been suggested (13). Leptin has also been implicated in the development of the periosteal envelope in growing bone (1), suggesting an anabolic effect on the skeleton. Both serum leptin (14 –18) and bone mass (19, 20) are positively correlated with body fat. Mechanical loading on the skeleton (21) and/or the actions of a mediator between adipose tissue and bone may contribute to the association between body fat and bone mass (19). We hypothesized that leptin may be such a mediator and, therefore, we have evaluated the relationship between serum leptin concentrations and bone mass in women. Received August 9, 2000. Revision received December 7, 2000. Accepted December 8, 2000. Address all correspondence and requests for reprints to: Dr. J. A. Pasco, The University of Melbourne, Department of Clinical and Biomedical Sciences, Barwon Health, P.O. Box 281, Geelong 3220, Australia. E-mail:
[email protected]. * The project was supported by the Victorian Health Promotion Foundation.
whole body (partial r2 ⫽ 0.019 to 0.036; all P ⬍ 0.05). Similar trends were observed at the femoral neck and posterior-anterior-spine. With bone mineral density the dependent variable (adjusted for age, body weight, and body fat mass), the association with the natural logarithm of leptin remained significant at the lateral spine (partial r2 ⫽ 0.030; P ⫽ 0.011), was of borderline significance at the proximal femur sites (partial r2 ⫽ 0.012 to 0.017; P ⫽ 0.058 to 0.120), and was not significant at the other sites. Our results demonstrate an association between serum leptin levels and bone mass consistent with the hypothesis that circulating leptin may play a role in regulating bone mass. (J Clin Endocrinol Metab 86: 1884 –1887, 2001)
Materials and Methods Subjects Subjects were from a large age-stratified sample of women drawn at random from electoral rolls spanning the Barwon Statistical Division in southeastern Australia for participation in the Geelong Osteoporosis Study (22, 23). Serum leptin levels were determined for a subgroup encompassing a wide range of body mass index (BMI) for involvement in other studies (24, 25). As the exponential relationship between body fat mass and circulating leptin levels in nonobese subjects diminishes among the obese (14, 16, 25), we excluded obese women [BMI ⬎ 30.0 (26)]. Three hundred sixty-five women were eligible to participate in our study. Subjects were also excluded if they were currently exposed to glucocorticoids (n ⫽ 4) or the oral contraceptive pill (n ⫽ 73); were breast-feeding (n ⫽ 18); had a fasting plasma glucose ⬎ 7.0 mmol/liter (n ⫽ 4), incomplete sets of scans (n ⫽ 23); or had prostheses (n ⫽ 7), pacemakers (n ⫽ 1), silicon implants (n ⫽ 2), or nonremovable jewelry (n ⫽ 29) that would affect scan interpretation. None of the subjects was pregnant or using hormone replacement therapy. Of the 214 nonobese subjects included in the study (aged 20 –91 yr), 133 were premenopausal, 67 were postmenopausal, and 14 had indeterminate menopausal status. All were free from drugs and diseases known to affect bone metabolism. The study was approved by the Barwon Health Research and Ethics Advisory Committee, and informed consent was obtained from all participants.
Measurements Body weight and height were measured to the nearest 0.1 kg and 0.1 cm, respectively, and BMI calculated as weight/height2 (kg/m2). Dualenergy x-ray absorptiometry was performed using a Lunar Corp. (Madison, WI). DPX-L densitometer and analyzed with Lunar Corp. DPX-L software version 1.31. Bone mineral content (BMC) (g), areal bone mineral density (BMD) (g/cm2), and projected bone area (cm2) were measured at the spine in the posterior-anterior (PA, L2– 4) and lateral projections (L3), proximal femur (femoral neck, Ward’s triangle, trochanter), whole body, ultradistal (UD), and midforearm sites. In vivo short-term
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SERUM LEPTIN AND BONE MASS IN NONOBESE WOMEN precision for BMC, BMD, and projected area, respectively, was 1.4%, 0.6%, 1.5% for PA-spine; 2.9%, 3.4%, 4.0% for lateral spine; 2.9%, 1.6%, 2.3% for the femoral neck; 3.0%, 2.1%, 2.8% for Ward’s triangle; 4.3%, 1.6%, 3.9% for the trochanter; 0.6%, 0.4%, 0.9% for the whole body; 1.6%, 2.1%, 1.7% for UD-forearm; and 0.8%, 1.1%, 0.9% for the midforearm. Body fat mass (g) was determined from whole-body scans, with a precision of 3.8%. Venous blood samples were collected following an overnight fast, separated by centrifugation and stored at ⫺80 C until analysis. Serum leptin concentrations were determined by a commercial RIA (Linco Research, Inc., St. Louis, MO). The interassay coefficient of variation ranged from 4.1– 8.2%, and the intraassay coefficient of variation was 5%.
Statistics Serum leptin concentrations were transformed to the natural logarithm (ln) to normalize the data before analysis. Regression techniques (27) were used to develop equations for predicting BMC and BMD at each site. Higher than linear adjustments for age, centered about the mean to reduce collinearity (27), were included for the forearm sites. Linear adjustments were made for age at the other sites and for body weight and body fat mass at all sites. BMC was also adjusted for projected bone area to correct for differences in areas scanned. All betweenpredictor correlations were ⬍0.9, as required for valid regression analysis (27). Menopause status was not included in the models for the entire sample because its contribution was negligible. Partial r2 values of the predictors (27) were calculated for each predictor using site-specific models. Significance was set at P ⬍ 0.05 and all statistical analyses were performed using Minitab (release 12) software package.
Results
Table 1 lists subject characteristics. Median serum leptin (range) was 11.3 ng/ml (2.1– 89.3). Univariate analysis indicated that leptin (ln) was correlated with all indices of body fatness: r ⫽ 0.60 for body weight; r ⫽ 0.80 for body fat mass; r ⫽ 0.83 for per cent body fat and r ⫽ 0.74 for BMI (all P ⬍ 0.001). Correlations between leptin (ln) and BMC or BMD in the entire sample and according to menopausal status displayed no consistent pattern (Table 2). At the lateral spine and two proximal femur sites (Ward’s triangle and trochanter), there was an independent positive TABLE 1. Subject characteristics (mean and Mean
Age (yr) Body weight (kg) Height (cm) BMI (kg/m2) Body fat mass (g) Leptin (ln) BMC (g) Femoral neck Ward’s triangle Trochanter PA-spine Lateral spine Whole body UD-forearm Midforearm BMD (g/cm2) Femoral neck Ward’s triangle Trochanter PA-spine Lateral spine Whole body Ultradistal-forearm Midforearm
SD) SD
44.5 62.6 162.5 23.7 21,462 2.40
13.1 8.6 6.4 2.9 6594 0.72
4.34 1.99 9.52 49.99 2.29 2506 1.77 3.06
0.77 0.52 2.54 9.79 0.75 387 0.32 0.40
0.930 0.806 0.786 1.188 0.669 1.132 0.322 0.688
0.141 0.164 0.124 0.169 0.184 0.091 0.054 0.076
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TABLE 2. Correlations between leptin (ln) and BMC or BMD BMC
BMD
r
P
r
P
All Femoral neck Ward’s triangle Trochanter PA-spine Lateral spine Whole body UD-forearm Midforearm
0.040 0.057 0.161 ⫺0.025 ⫺0.074 0.094 ⫺0.092 ⫺0.083
0.557 0.408 0.018 0.713 0.281 0.170 0.179 0.227
⫺0.011 ⫺0.030 0.102 ⫺0.009 ⫺0.103 0.087 ⫺0.072 ⫺0.172
0.877 0.665 0.137 0.898 0.135 0.207 0.291 0.012
Premenopausal Femoral neck Ward’s triangle Trochanter PA-spine Lateral spine Whole body UD-forearm Midforearm
0.074 0.053 0.138 0.079 0.085 0.208 0.059 ⫺0.037
0.397 0.546 0.113 0.364 0.329 0.016 0.497 0.669
0.045 0.005 0.079 0.046 0.081 0.207 0.039 ⫺0.084
0.603 0.951 0.367 0.596 0.355 0.017 0.655 0.336
Postmenopausal Femoral neck Ward’s triangle Trochanter PA-spine Lateral spine Whole body UD-forearm Midforearm
0.142 0.187 0.104 0.020 ⫺0.032 0.167 ⫺0.102 0.028
0.250 0.130 0.400 0.874 0.796 0.178 0.414 0.824
0.106 0.123 0.237 0.141 0.016 0.218 ⫺0.016 ⫺0.047
0.392 0.321 0.053 0.255 0.899 0.076 0.895 0.708
association of leptin (ln) with BMC (Table 3) adjusted for age, body weight, body fat mass and projected bone area (partial r2 ⫽ 0.019 to 0.036; all P ⬍ 0.05). Similar trends were observed at the femoral neck and PA-spine (partial r2 ⫽ 0.013, 0.011; P ⫽ 0.103, 0.137; respectively). At the whole body, body weight and body fat mass were not significant predictors of BMC after adjusting for age and bone area. Whether these variables were forced into the regression equation, leptin (ln) remained a significant predictor of BMC (P ⬍ 0.05). No association was detected between leptin (ln) and BMC at the UD-forearm (P ⫽ 0.825), and a trend toward a negative association was observed at the midforearm (P ⫽ 0.158). When BMD (adjusted for age, body weight, and body fat mass) was analyzed (Table 4), the effect of leptin (ln) remained significant at the lateral spine (partial r2 ⫽ 0.030, P ⫽ 0.011). The association was of borderline significance at the proximal femur sites (partial r2 ⫽ 0.012 to 0.017, P ⫽ 0.058 to 0.120) and was not significant at the other sites (P ⬎ 0.05). Among postmenopausal women, significant positive associations between leptin (ln) and BMC were observed at the PA-spine and whole body (P ⫽ 0.011 and 0.016, respectively); associations at other sites, and using BMD as the dependent variable, were not significant (P ⫽ 0.082– 0.443). No significant associations were observed among premenopausal women. Discussion
To our knowledge, this is the first reported association between bone mass and serum leptin concentrations, independent of body weight, and body fat mass. The regression models we used to test the association between BMC and leptin included adjustments for body weight, to account for
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TABLE 3. Multiple regression analysis with BMC (g) as the dependent variable and age (yr), body weight (kg), body fat mass (g), bone area (cm2), and leptin (ng/mL) (ln) as independent variables in the models for the proximal femur sites (femoral neck, Ward’s triangle, and trochanter), spine (PA and lateral), and whole body for the whole cohort Variable
 coefficient (slope)
P value
Partial r2
Femoral neck Age Body weight Body fat mass Bone area Leptin (ln)
⫺0.019 0.060 ⫺0.000 0.828 0.161
0.000 0.000 0.001 0.000 0.103
0.139 0.114 0.052 0.257 0.013
Ward’s triangle Age Body weight Body fat mass Bone area Leptin (ln)
⫺0.014 0.031 ⫺0.000 0.786 0.118
0.000 0.000 0.002 0.000 0.047
0.193 0.089 0.047 0.398 0.019
Trochanter Age Body weight Body fat mass Bone area Leptin (ln)
⫺0.034 0.113 ⫺0.000 0.931 0.552
0.000 0.000 0.008 0.000 0.025
0.066 0.067 0.033 0.620 0.024
PA-spine Age Body weight Body fat mass Bone area Leptin (ln)
⫺0.193 0.494 ⫺0.001 1.466 1.573
0.000 0.000 0.011 0.000 0.137
0.130 0.063 0.031 0.386 0.011
Lateral spine Age Body weight Body fat mass Bone area Leptin (ln)
⫺0.034 0.028 ⫺0.000 0.622 0.210
0.000 0.001 0.006 0.000 0.006
0.469 0.048 0.036 0.409 0.036
Whole body Age Body weight Body fat mass Bone area Leptin (ln)
⫺4.589 3.314 ⫺0.005 1.498 63.80
0.000 0.429 0.350 0.000 0.018
0.161 0.002 0.004 0.560 0.030
the mechanical loading on the skeleton caused by gravitational forces (21), and bone area, to correct for differences in areas scanned. We also adjusted for body fat mass to allow for the potential influence of other humoral factors associated with adipose tissue (28). Using these models, significant positive associations were demonstrated at two proximal femur sites (Ward’s triangle and trochanter), the lateral spine, and whole body. Leptin may have contributed to the variance attributed to body fat mass; however, leptin alone explained a small proportion (1– 4%) of the variance in adjusted BMC. Significant associations between leptin and adjusted BMC were observed at the PA-spine and whole body among postmenopausal women. Smaller numbers in the separate analyses of premenopausal and postmenopausal women may have limited our ability to detect significant associations at other sites. The high correlations between circulating leptin concentrations and indices of adiposity have been reported previously (14 –18). In a study of 54 postmenopausal women (BMI 15.8 – 42.9 kg/m2), the positive association between plasma leptin
TABLE 4. Multiple regression analysis with areal BMD (g/cm2) as the dependent variable and age (yr), body weight (kg), body fat mass (g), and leptin (ng/ml) (ln) as independent variables in the models for the proximal femur sites (femoral neck, Ward’s triangle, and trochanter), spine (PA and lateral), and whole body for the whole cohort Variable
 coefficient (slope)
P value
Partial r2
Femoral neck Age Body weight Body fat mass Leptin (ln)
⫺0.004 0.012 ⫺0.000 0.034
0.000 0.000 0.001 0.115
0.151 0.112 0.047 0.012
Ward’s triangle Age Body weight Body fat mass Leptin (ln)
⫺0.006 0.012 ⫺0.000 0.046
0.000 0.000 0.002 0.058
0.200 0.088 0.043 0.017
Trochanter Age Body weight Body fat mass Leptin (ln)
⫺0.002 0.012 ⫺0.000 0.031
0.006 0.000 0.002 0.120
0.036 0.126 0.045 0.012
PA-spine Age Body weight Body fat mass Leptin (ln)
⫺0.005 0.015 ⫺0.000 0.029
0.000 0.000 0.002 0.253
0.153 0.131 0.046 0.006
Lateral spine Age Body weight Body fat mass Leptin (ln)
⫺0.010 0.008 ⫺0.000 0.055
0.000 0.001 0.011 0.011
0.493 0.051 0.031 0.030
Whole body Age Body weight Body fat mass Leptin (ln)
⫺0.003 0.007 ⫺0.000 0.015
0.000 0.000 0.031 0.216
0.235 0.126 0.022 0.007
levels and BMC was no longer significant after adjusting for body fat mass (17). The inclusion of obese subjects may have diminished the association. There is a different relationship between body fat mass and circulating leptin levels among the obese, who display a wide range of serum leptin concentrations that overlap those observed in lean subjects (14, 15). The findings in the present study are reported for nonobese women alone for whom there is an exponential relationship between body fat mass and serum leptin. In accordance with results from our study, another study of 94 adult women (18) reported no relationship between leptin and BMD or bone geometry at the distal radius. The reasons for the lack of association at nonweight-bearing sites remain unclear. In cross-sectional studies (17, 18), no correlation was observed between circulating leptin and markers of bone turnover. Because the subjects were likely to be in a steady state with bone formation coupled to resorption (29), it would seem unlikely that any association would be observed. Changes in leptin concentrations and bone turnover at the individual bone remodeling units might not produce measurable systemic changes. However, the hypothesis that there is a relationship between serum leptin and bone turnover could be tested by producing pharmacological alterations in serum leptin and measuring turnover response. Unlike a recent study (30), earlier studies in mice (31) and
SERUM LEPTIN AND BONE MASS IN NONOBESE WOMEN
pubertal girls (1) suggested that the effect of leptin on the skeleton occurs in cortical bone, whereas leptin-treated ob/ob mice were shown to gain both trabecular and cortical bone (32). Our data indicate the associations between bone mass and serum leptin were not as strong when BMD rather than BMC was used as the dependent variable. BMD is influenced by bone size (33). The association between leptin and BMD (i.e. the ratio of BMC/ bone area) is conceptually different from the association with BMC after adjusting for bone area, which partially compensates for the confounding influence of bone size. Furthermore, if leptin promotes periosteal bone apposition, the amount of mineral at the bone-soft tissue interface might increase, resulting in an increase in apparent bone area. Thus, BMD may remain relatively unchanged because any increase in BMC would be offset by increases in bone size. Further studies on the association among leptin concentration, cortical thickness, and medullary width may indicate whether an anabolic effect occurs at the endosteal or periosteal surface of bone. Weight loss or gain in adult women is associated with corresponding changes in circulating leptin levels (14, 34) and bone mass (35). Furthermore, reduced body fat (36), reduced leptin levels (25), and reduced bone mass (37) have been observed among smokers. These patterns may suggest that changes in body fat may, in part, be translated into changes in bone mass through fluctuations in circulating leptin levels and/or other mediators of adipose tissue origin. Raised peripheral leptin levels may favor bone formation while suppressing adipogenesis. At a local level, bone marrow adipocytes produce leptin, which may enhance osteogenic activity and inhibit adipogenic activity (12). Failure to identify leptin receptors or leptin effects on osteoblasts in primary osteoblast cultures from calvaria (30) may reflect changes associated with osteoblast differentiation. Recent data suggest that leptin may also inhibit fetal bone resorption (13). This cross-sectional study of the relationship between bone mass and circulating leptin levels does not address the question as to whether leptin is involved in skeletal growth and the development of peak bone mass. However, the results suggest that serum leptin may play a role in regulating skeletal mass in nonobese adult women, and these findings need to be explored in men. Further studies on the association between circulating leptin levels and bone geometry may provide insight into the mechanism of leptin’s effect on bone. Acknowledgment Our thanks are extended to S. Panahi and B. Skoric for their help scanning subjects, A. de Silva and M. S. Solin for measuring serum leptin concentrations, and B. M. Burgess for assistance in collating data.
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