The skeleton is regarded recently as an endocrine organ that affects energy metabolism. However, there are very limited data available concerning the relationships between the osteoblast-derived hormone osteocalcin, weight status, adiponectin and leptin in obese humans, especially in children.
We analyzed osteocalcin, adiponectin, leptin and insulin resistance (IR) index homeostasis model assessment (HOMA) in 60 obese and 19 age- and gender-matched normal weight children. Furthermore, these parameters were determined in 60 obese children after participating in an outpatient 1-year lifestyle intervention based on exercise, behavior and nutrition therapy.
Sixty obese children had significantly lower osteocalcin levels (26.8±0.8 ng ml−1) than 19 normal weight controls (32.2±2.3 ng ml−1). Boys (29.9±1.1 ng ml−1) showed significantly (P=0.046) higher osteocalcin levels compared with girls (26.4±1.2 ng ml−1). In stepwise multiple linear regression analysis adjusted for age, gender and pubertal stage, osteocalcin was significantly negatively related to leptin and HOMA, but not to adiponectin. Changes of osteocalcin in the course of 1 year correlated significantly negatively with changes of IR index HOMA (r=−0.25), standard deviation score–body mass index (SDS–BMI) (r=−0.33) and leptin (r=−0.50). Substantial weight loss in 29 obese children led to a significant increase in osteocalcin and a significant decrease in leptin and HOMA. In 31 obese children without substantial weight loss, osteocalcin levels did not change significantly in the course of 1 year.
Osteocalcin levels were lower in obese children and were related to IR and leptin both in cross-sectional and longitudinal analyses. Therefore, osteocalcin might be a new promising link between obesity and IR.
The skeleton is regarded as an endocrine organ that affects energy metabolism. A link between bone and energy metabolism has long been suspected on the basis of the observation that obesity is inversely associated with osteoporosis. However, the mediators of such a relationship have not been identified until recently, when leptin, an adipocyte-derived hormone, was shown to be a major regulator of bone turnover.1 Working on the hypothesis that the bone may also exert feedback control on energy homeostasis, Lee et al.2 recently showed that osteocalcin, a marker of bone formation specifically synthesized and secreted by osteoblasts, is the hormone involved.2 Osteocalcin is a cell-specific molecule, synthesized as a pre-promolecule and secreted in the general circulation.3
Recently, a relationship between osteocalcin, obesity and insulin resistance (IR) has been suggested. Mice lacking the gene that encodes osteocalcin (osteocalcin −/−) have an abnormal amount of visceral fat and exhibit glucose intolerance, IR and impaired insulin secretion compared with wild-type mice.2 In humans, lower circulating osteocalcin levels were found in overweight4, 5 and diabetic subjects6, 7 compared with normal weight individuals. Moreover, osteocalcin concentrations increase after weight loss in overweight individuals8, 9 and after improvement in glycemic control in subjects with type 2 diabetes.10 However, another human weight loss study based on diet showed no increase in osteocalcin.11 Serum osteocalcin concentrations were inversely associated with fasting plasma glucose (FPG), insulin and homeostasis model assessment (HOMA) for IR in cross-sectional analyses in humans in some studies,12, 13 whereas another study found a relationship only in lean but not obese subjects.14
The mechanisms linking osteocalcin to obesity and IR remain unclear. Osteocalcin concentrations are suggested to be modulated by the adipocytokine leptin. Leptin is one key regulator of weight status through well-identified pathways in the hypothalamus.15 Moreover, leptin inhibits osteoclast generation16 and enhances osteoblast differentiation in vitro.17 In animals, intracerebroventricular infusion of leptin causes not only weight loss but also bone loss, suggesting that leptin acts through the central nervous system to inhibit bone formation.18 In addition, it has been recently shown that osteocalcin regulates insulin sensitivity through adiponectin in animals.2 However, the findings in humans are controversial.12, 14
Taken together, these data support a regulatory role of the skeleton on glucose and energy homeostasis, which seems to be mediated by osteocalcin. As the data concerning osteocalcin in obesity, in particular in children, are limited and sometimes controversial. We studied osteocalcin levels in obese children participating in a lifestyle intervention as well as the relationship between osteocalcin, IR, adiponectin and leptin in the course of 1 year. Longitudinal studies and studies in obese children seem preferable because (1) cross-sectional studies cannot prove causality and are prone to many confounder, (2) adverse patterns of IR itself begin in childhood, and (3) studies in children have the advantage that there is no potential confusion with other diseases medications or active tobacco smoking. For example, osteocalcin has been reported to vary by age, sex and smoking status.19
We hypothesized that (1) osteocalcin concentrations were lower in obese children compared with normal weight children and that (2) changes in osteocalcin were associated with weight loss and changes in leptin, adiponectin and IR.
We examined anthropometrical markers, fasting serum osteocalcin, adiponectin, leptin and, as parameters of IR, fasting glucose and insulin in 60 obese Caucasian children at baseline and 1 year later after participating in a lifestyle intervention. In addition, these parameters were determined in 19 normal-weight healthy Caucasian children of similar age, gender and pubertal stage. Obese children were studied before and after participating in the 1-year lifestyle intervention ‘Obeldicks,’ which has been described in detail elsewhere.20, 21 Briefly, this outpatient intervention program for obese children is based on physical exercise, nutrition education and behavior therapy including the individual psychological care of the child and his or her family. The nutritional course is based on a fat and sugar-reduced diet compared with every day nutrition of German children.
None of the children in this study suffered from endocrine disorders, premature adrenarche or syndromal obesity.
Height was measured to the nearest centimeter using a rigid stadiometer. Weight was measured unclothed to the nearest 0.1 kg using a calibrated balance scale. Body mass index (BMI) was calculated as weight in kilograms (kg) divided by the square of height in meters (m2). The degree of overweight was quantified using Cole's least mean square method, which normalized the BMI skewed distribution and expressed BMI as a standard deviation score (SDS–BMI).22 Reference data for German children were used.23 All children in the study were obese according to the definition of the International Obesity Task Force.24
Blood sampling was performed in the fasting state at 0800 hours. After clotting, blood samples were centrifuged for 10 min at 8000 rpm. Serum was stored at −81 °C for later determination of osteocalcin (chemiluminescent immunometry, IMMULITE Bühlmann Laboratories AG, Schoenenbuch, Switzerland) and leptin (Leptin (human) ELISA Kit, BioVendor, Alexis Biochemicals, Lausen, Switzerland). Serum adiponectin was determined by enzyme-linked immunosorbent assay (ELISA) (Human Adiponectin ELISA Kit, Linco Research, Inc., St Charles, MO, USA; intra-assay coefficient of variation (CV), 1.0–7.4%; inter-assay coefficient of variation, 2.4–8.4%; sensitivity, 0.5 ng ml−1). Insulin concentrations were measured by microparticle-enhanced immunometric assay (MEIA Abbott, Wiesbaden, Germany). Glucose levels were determined by colorimetric test using a Vitros analyzer (Ortho Clinical Diagnostics, Neckargmuend, Germany). Intra- and inter-assay coefficient of variations were <5% in all these methods. HOMA was used to detect the degree of IR.27 The resistance can be assessed from fasting glucose and insulin concentrations by the following formula: resistance (HOMA)=(insulin (mU l−1) × glucose (mmol l−1))/22.5.
Using the LMS calculation method described above, substantial weight loss in the course of 1 year was defined as a reduction in SDS–BMI of 0.5, because with only reduction of >0.5 in SDS–BMI was an improvement in IR measured in obese children.28
Statistical analyses were performed using the Winstat software package (R. Fitch Software, Bad Krozingen, Germany). All variables were normally distributed as tested by the Kolmogorov–Smirnov test. Osteocalcin was correlated with leptin, adiponectin, anthropometric variables and IR index HOMA by Pearson's correlation. Furthermore, a multiple stepwise linear regression was calculated with osteocalcin as a dependent variable and leptin, adiponectin and HOMA as independent variables adjusted for pubertal stage, age, gender and BMI. Gender and pubertal stage were used as binary variables in this model. To compare variables at baseline or in the course of 1 year, Fisher's exact test and Student's t-test for paired and unpaired observations were used as appropriate. Correlations between changes of osteocalcin, adiponectin, leptin, anthropometric variables and IR index HOMA in the course of 1 year were calculated by Pearson's correlation. Changes were expressed as delta variable calculated by variable at baseline minus variable measured 1 year later. A P-value <0.05 was considered as significant. Data were presented as mean and standard error of the mean (s.e.m.).
Written informed consent was obtained from all children and their parents. The study was approved by the local ethics committee of the University of Witten/Herdecke in Germany.
The osteocalcin and adiponectin concentrations were significantly lower, whereas leptin concentrations and the IR index HOMA were significantly higher in the obese children compared with the normal weight children (see Table 1). Normal weight and obese children did not differ significantly with respect to age, gender or pubertal stage.
Osteocalcin was significantly negatively correlated with BMI (r=−0.36, P<0.001; Figure 1a) and to SDS–BMI (r=−0.32, P=0.007) but not to age (r=−0.04, P=0.352). Boys (29.9±1.1 ng ml−1) showed significantly (P=0.046) higher osteocalcin levels compared with girls (26.4±1.2 ng ml−1). The osteocalcin concentrations did not differ significantly (P=0.888) between prepubertal (28.2±0.7 ng ml−1) and pubertal children (28.0±1.5 ng ml−1).
Osteocalcin was significantly correlated with leptin (r=−0.42, P<0.001; Figure 1b) and to IR index HOMA (r=−0.42, P<0.001; Figure 1d), but not to adiponectin (r=0.04, P=0.364; Figure 1c). In a multiple stepwise linear regression (r2=0.22) adjusted for pubertal stage, age, gender and BMI, osteocalcin was significantly negatively related to leptin (β-coefficient: −0.14±0.06, P=0.037) and HOMA (β-coefficient: −0.78±0.38, P=0.041), but not adiponectin (P=0.102).
In the 1-year follow-up period, the changes in osteocalcin correlated significantly negatively with changes in IR index HOMA (r=−0.25, P=0.028; Figure 2d), changes in SDS–BMI (r=−0.33, P=0.005; Figure 2a) and changes in leptin (r=−0.50, P<0.001; Figure 2b), but not to changes in adiponectin (r=−0.18, P=0.092; Figure 2c).
In 29 obese children with substantial weight loss, osteocalcin increased significantly, and leptin and HOMA decreased significantly, whereas adiponectin increased in tendency (Table 2). In 31 obese children without substantial weight loss, no significant changes could be observed. At baseline, the obese children with and without substantial weight loss did not differ with respect to age (P=0.687), gender (P=0.811), pubertal stage (P=0.380), BMI (P=0.091), SDS–BMI (P=0.428), osteocalcin (P=0.601), adiponectin (P=0.853), leptin (P=0.078) and IR index HOMA (P=0.142).
To the best of our knowledge, this is the first study analyzing both cross-sectional and longitudinal relationships between osteocalcin, obesity, IR, leptin and adiponectin in childhood. Our hypothesis was that circulating osteocalcin levels would be lower in overweight children and inversely correlated with leptin and IR and positively correlated with adiponectin.
In our study, obese children showed significantly lower osteocalcin levels compared with their normal weight peers. Substantial weight loss was associated with an increase in osteocalcin. In concordance, previous cross-sectional studies in animals29 and adult humans4, 8, 13, 30, 31 found inverse correlations between osteocalcin levels and BMI. Furthermore, studies in children showed that normal weight children had significantly higher circulating osteocalcin levels than overweight children.5, 32 Osteocalcin levels have been reported to increase in overweight adults after weight loss.8, 9, 30 The only existing human study with no increase in osteocalcin after weight loss was based on a high protein, high calcium diet, which probably explains this difference.11 In conclusion, our findings together with data from the literature suggest a reversible decrease of osteocalcin in obesity.
The mechanisms associating fat mass with low osteocalcin levels are not fully understood. Leptin, which is produced by the adipose tissue, has important effects on bone metabolism1 and may be the link between obesity and low osteocalcin levels in obesity. Leptin-deficient ob/ob and leptin-resistant db/db mice have elevated osteocalcin levels.33 In concordance with the hypothesis of leptin reducing osteocalcin levels, leptin correlated negatively with osteocalcin levels in our study, both in cross-sectional and longitudinal analyses.
In our study, osteocalcin was associated negatively with IR index HOMA both in cross-sectional and longitudinal analyses. These findings are in line with animal studies showing that osteocalcin differentially regulates β-cells and affects the development of metabolic diseases in wild-type mice.34 Osteocalcin can enhance insulin expression, insulin sensitivity and fat mass.34, 35 Osteocalcin−/− mice are glucose intolerant.2 In cross-sectional analyses of humans, serum osteocalcin concentrations were inversely correlated with fasting plasma glucose, fasting insulin and HOMA for IR,12, 13 whereas another study found a relationship only in lean but not obese subjects.14 However, in the latter study, the number of obese humans was small, probably explaining the negative findings.
Adiponectin has been suggested as the link between low osteocalcin and IR. An in vitro study showed that osteocalcin regulates insulin sensitivity through adiponectin.2 However, we found no significant relationship between adiponectin and osteocalcin both in cross-sectional and longitudinal analyses in our study. These findings are in concordance with most human studies.12, 14 It could be that both adiponectin and osteocalcin are correlated with parameters of IR and obesity involving independent pathways.
In the human life cycle, osteocalcin levels have been described at their highest during adolescence.19, 36 However, we did not find a relationship between age or puberty and osteocalcin concentrations in our study. As most of our patients were obese, and obesity is associated with lower osteocalcin levels, these facts may explain this difference to studies in normal weight children. The lower levels of osteocalcin in obesity reflecting a reduced osteoblast activity during bone formation may have a clinical impact as there is increasing evidence that children who are overweight have less mineralized bones after correction for bone size and are more prone to fracture than non-obese children.37, 38
This study has a few potential limitations. First, BMI percentiles were used to classify overweight. Although BMI is a good measure for overweight, one needs to be aware of its limitation as an indirect measure of fat mass. Second, the HOMA model is only an assessment of IR.39 Clamp studies are actually the gold standard for analyzing IR.39 Third, we are not able to differentiate the effect of diet, increased physical exercise and weight loss on osteocalcin concentrations because of our study protocol. Fourth, the effect of weight loss on HOMA-IR and leptin could have been mediated by an undertermined confounder. In addition, our study protocol does not allow us to determine whether the changes in osteocalcin with weight loss induced changes in HOMA-IR and leptin or vice versa. Therefore, future research is necessary to study the relationships between osteocalcin, leptin and IR. Finally, osteocalcin undergoes post-translational modification whereby three glutamic acid residues are carboxylated.2 Increased levels of uncarboxylated osteocalcin in mice lacking the osteotesticular protein tyrosine phosphatase (OST-PTP) protect them from developing glucose intolerance, IR and obesity when fed with a high-fat diet. This is associated with an increase in β-cell proliferation, insulin secretion and insulin sensitivity.2 Thus, the uncarboxylated form of osteocalcin may exert hormonal activity and affect energy metabolism. In our study, we measured only total osteocalcin and not uncarboxylated osteocalcin.
In summary, osteocalcin concentrations were significantly lower in obese children compared with normal weight children, suggesting an inverse relationship between osteocalcin and fat mass. We hypothesize that leptin may be the link between obesity and osteocalcin, as osteocalcin was significantly negatively related to leptin both in cross-sectional and longitudinal analyses. Osteocalcin levels were independent of age and pubertal stage in our study sample of predominantly obese children. As osteocalcin was significantly negatively related to IR both in cross-sectional and longitudinal analyses, these findings support the hypothesis of a functionally relevant relationship between osteocalcin and IR in obesity. Our data do not support that adiponectin is the link between IR and osteocalcin. Further prospective research is necessary to clarify the role of osteocalcin in the pathogenesis of IR, especially in obese humans as well as related molecular pathways.
We thank Ms K Schark-Zimmer, Children's Hospital University of Bonn, for her kind support and great expertise in the laboratory and Gideon de Sousa for his critical review of the article. TR received grant support (2008–2010) from the Bundesministerium für Bildung und Forschung (Obesity network: LARGE Grant no. 01 GI0839).
CR received grant support (2003–2004) from Bonfor Research Foundation, University of Bonn, Germany.