http://informahealthcare.com/arp ISSN: 1381-3455 (print), 1744-4160 (electronic) Arch Physiol Biochem, 2014; 120(2): 73–79 ! 2014 Informa UK Ltd. DOI: 10.3109/13813455.2013.877488
ORIGINAL ARTICLE
Anil Baran Choudhury1, Purnima Dey Sarkar2, Dilip K. Sakalley3, and Sudhakar B. Petkar4 1
Department of Biochemistry, NSCB Medical College, Jabalpur, Madhya Pradesh, India, 2Department of Biochemistry, MGM Medical College, Agra/ Bombay Road, Indore, Madhya Pradesh, India, 3Department of Forensic Medicine, NSCB Medical College, Jabalpur, Madhya Pradesh, India, and 4 Department of Biochemistry, People’s College of Medical Sciences and Research Centre, Bhanpur Bypass Road, Bhopal, Madhya Pradesh, India Abstract
Keywords
Objective: To analyse the association of osteocalcin with insulin resistance and type 2 diabetes and assess the role of adiponectin in these relationships. Methods: This study comprised 98 newly diagnosed type 2 diabetic women (51 pre-menopausal and 47 post-menopausal) and 102 age and BMI matched controls (53 pre-menopausal and 49 post-menopausal). Insulin resistance was calculated by homeostasis model assessment-insulin resistance (HOMA-IR). Results: Osteocalcin was significantly positively correlated with adiponectin in both premenopausal (p ¼ 0.0026) and post-menopausal diabetic women (p ¼ 0.0357). Significant negative association between osteocalcin and HOMA-IR was observed only in pre-menopausal diabetic women (p ¼ 0.0019), but the association was partially reduced (p ¼ 0.0219) after additional adjustment for adiponectin. Adiponectin slightly attenuated the inverse association between osteocalcin and presence of type 2 diabetes in both pre- and post-menopausal women. Conclusion: The protective action of osteocalcin against the development of insulin resistance and type 2 diabetes in women may be partially mediated through up-regulation of adiponectin secretion.
Adiponectin, glucose homeostasis, HOMA-IR, insulin resistance, osteocalcin, type 2 diabetes
Introduction In addition to bone mineralization and calcium homeostasis, a novel endocrine role of skeleton, affecting energy homeostasis, has recently been reported in animal studies (Ferron et al., 2008; Lee et al., 2007). Osteocalcin, a non-collagenous protein with 49 amino acids (MW 5800 Da), is produced by osteoblast and has been known as a marker of bone turnover (Eastell & Ebeling, 2009; Hauschka et al., 1989). Recently, it has been demonstrated that mice lacking the osteocalcin gene develop a series of phenotypic abnormalities such as decreased beta cell proliferation, decreased insulin secretion, insulin resistance and hyperglycaemia than wild type mice (Lee et al., 2007). Furthermore, administration of recombinant osteocalcin reversed these phenotypic abnormalities and protected them to a large extent against obesity and type 2 diabetes mellitus (Ferron et al., 2008). In humans we and others have reported that serum osteocalcin concentration was negatively correlated with blood glucose and insulin resistance (Hwang et al., 2012; Pittas et al., 2009; Sarkar & Choudhury, 2013; Shea et al., 2009) and inversely associated
Correspondence: Anil Baran Choudhury, Department of Biochemistry, NSCB Medical College, Jabalpur, Madhya Pradesh, India. Tel: +919424443478. E-mail: [email protected]
History Received 17 September 2013 Revised 24 November 2013 Accepted 16 December 2013 Published online 9 January 2014
with type 2 diabetes (Hwang et al., 2012; Sarkar & Choudhury, 2012; Zhou et al., 2009). Prior studies have also shown that type 2 diabetic patients have reduced osteocalcin concentrations compared with non diabetics (Hwang et al., 2012; Zhou et al., 2009). In addition, menopause transition may have an influence on circulating osteocalcin concentration, since osteocalcin concentration is significantly higher among post-menopausal women than premenopausal women (Zhou et al., 2009). However, data concerning the association between osteocalcin and impaired glucose metabolism among pre- or post-menopausal women provided inconsistent results (Im et al., 2008; Kim et al., 2013; Zhou et al., 2009). Adiponectin, an adipocyte derived protein with 244 amino acids (Scherer et al., 1995), plays an important role in the regulation of energy homeostasis and insulin sensitivity (Helio¨vaara et al., 2006; Mantzoros et al., 2005) and predicts the development of type 2 diabetes (Daimon et al., 2003; Spranger et al., 2003). Adiponectin and its receptors are also expressed in osteoblastic cells (Berner et al., 2004) and stimulate osteoblast proliferation and differentiation (Kanazawa et al., 2007). Recent in vitro and in vivo studies have shown that in adipose tissue osteocalcin promotes insulin sensitivity by increasing expression of the gene ADIPOQ encoding adiponectin (Ferron et al., 2008; Lee
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Role of adiponectin in mediating the association of osteocalcin with insulin resistance and type 2 diabetes: a cross sectional study in pre- and post-menopausal women
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et al., 2007). A number of studies have found a positive link between serum osteocalcin and adiponectin (Gravenstein et al., 2011; Sumarac-Dumanovic et al., 2012). However, clinical data that investigated the role of adiponectin in the relationship between osteocalcin and insulin resistance are limited, with conflicting results (Hwang et al., 2012; Shea et al., 2009). Therefore, in this cross sectional study, we evaluated the relationship between serum osteocalcin and insulin resistance among pre- and post-menopausal women with type 2 diabetes. We also determined whether adiponectin could mediate the association of osteocalcin with insulin resistance and type 2 diabetes.
Methods Study subjects This study was conducted in the Department of Biochemistry, Netaji Subhash Chandra Bose (NSCB) Medical College and Hospital, Jabalpur and approved by Institutional Ethical Committee. Ninety eight newly diagnosed type 2 diabetic women (51 pre-menopausal, 47 post-menopausal), aged between 31 to 70 years (mean age ± SD: 50.90 ± 8.84 years), were recruited from the outpatients department of NSCB Medical College and Hospital. One hundred two age and BMI matched healthy asymptomatic women participants (53 pre-menopausal, 49 post-menopausal; mean age ± SD: 50.70 ± 7.82 years), who had come for a routine health checkup in the hospital, were taken as control. Diabetes mellitus was confirmed according to the 1999 World Health Organization (WHO, 1999) criteria. Menopause was defined as the absence of menstruation for 12 consecutive months, which was not due to surgical resection of uterus or ovaries (WHO, 1981). Brief clinical history of present and past illness and medical therapy were recorded from all participants. Written informed consent was obtained from the study group and the controls before entry into the study. Exclusion criteria The exclusion criteria in the patient group were: (1) any systemic disease other than type 2 diabetes, (2) under hypoglycaemic drug or insulin treatment, (3) recent history of fracture, (4) use of any bone active medications such as vitamin D, calcitonin, bisphosphonate or lipid lowering drug, (5) use of hormone replacement therapy, (6) smokers, (7) alcoholics and (8) pregnancy.
Arch Physiol Biochem, 2014; 120(2): 73–79
Laboratory analyses Venous blood samples were collected after an overnight fasting in the morning in an aseptic condition from the antecubital vein. Blood samples were centrifuged at 4 centigrade and serum was stored immediately at 80 centigrade until they were analysed. Fasting blood glucose (FBG), total cholesterol (TC), high density lipoprotein-cholesterol (HDL-C) and triglycerides (TG) were estimated by the standard laboratory kit (Biosystem) method using a fully automated biochemistry analyser (Biosystem A25; Biosystem SA, Barcelona, Spain). Low density lipoprotein-cholesterol (LDL-C) was calculated according to Friedewald’s formula (TC [mg/dl] HDL-C [mg/dl] TG [mg/dl]/5) (Friedewald et al., 1972). Serum osteocalcin level was estimated by ELISA method (Quidel Corporation, San Diego, CA, USA). Serum adiponectin was measured by ELISA method (Ray Biotech Inc, Norcross, GA, USA). Fasting insulin was measured using a commercially available ELISA kit (LDN, Nordhorn). Insulin resistance was calculated by homeostasis model assessment (HOMA) based on the formula: HOMAIR ¼ fasting glucose (mmol/l) fasting insulin (mIU/ml)/22.5 (Matthews et al., 1985). Statistical analyses The Kolmogorov–Smirnov statistical test was used to test the normality of the distribution. Variables with a skewed distribution were log-transformed before performing statistical analyses. Data were expressed as the mean ± standard deviation. Comparisons of baseline anthropometric and biochemical parameters between groups were done by unpaired Student’s t-test. All correlations were analysed with Pearson’s correlation coefficient. To adjust for confounding variables in the correlation analyses, partial correlation coefficients were calculated. Linear regression analyses were done to study the association between HOMA-IR and osteocalcin after multivariate adjustment for potential confounders. Multivariate logistic regression models were used to assess the association between serum osteocalcin and type 2 diabetes with age, BMI, waist circumference, WHR, systolic and diastolic blood pressure, fasting blood glucose, HDLcholesterol and triglycerides, as covariates. To test the role of serum adiponectin as cofactor, the analyses were repeated with the inclusion of serum adiponectin as additional covariates in both linear and logistic regression models. All statistical tests were two-tailed and p value less than 0.05 were considered to be statistically significant. All data were analysed using statistical software SPSS version 16 (SPSS Inc., Chicago, IL, USA).
Anthropometry
Results
Body mass index (BMI) was calculated for all subjects by using the formula weight in kilograms divided by the square of height in metres. Waist (WC) and hip circumference were measured in the standing position using standard techniques and waist to hip ratio (WHR) was calculated as waist circumference divided by hip circumference. Seated systolic (SBP) and diastolic blood pressure (DBP) were measured by manual sphygmomanometer.
The baseline anthropometric and biochemical characteristics of control and type 2 diabetic patients group are presented in Table 1. The diabetic group had significantly higher systolic blood pressure (p50.05), fasting blood glucose, fasting insulin, HOMA-IR, total cholesterol, LDL-cholesterol and triglycerides (p50.0001) than the control group, whereas HDL-cholesterol, adiponectin (7.96 ± 4.26 mg/ml vs. 18.47 ± 7.01 mg/ml; p50.0001) and osteocalcin levels
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DOI: 10.3109/13813455.2013.877488
Role of adiponectin in association of osteocalcin with insulin resistance and type 2 diabetes
with adiponectin (Figure 1), which was true also after adjusting for age and BMI (FBG: r ¼ 0.427; p ¼ 0.0022, HOMA-IR: r ¼ 0.353; p ¼ 0.0130, adiponectin: r ¼ 0.421; p ¼ 0.0026) (data not shown). In post-menopausal diabetic women, serum osteocalcin was significantly negatively correlated with FBG, fasting insulin and HOMA IR and positively with adiponectin levels (Figure 1). However, when adjusted for age and BMI, only correlation with FBG (r ¼ 0.331; p ¼ 0.0265) and adiponectin (r ¼ 0.314; p ¼ 0.0357) remained significant (data not shown). To more precisely define the relationship between osteocalcin and insulin resistance, we further examined this relationship after adjusting for some metabolic factors in addition to age and BMI in linear regression model. As shown in Table 3, the negative relationship between osteocalcin and HOMA-IR remained significant (b ¼ 0.446; p ¼ 0.0019)
(4.24 ± 1.89 ng/ml vs. 9.67 ± 3.24 ng/ml; p50.0001) were significantly lower in the diabetic group (Table 1). When data from diabetic patients were stratified according to menopausal status, the average age ± SD was 44.67 ± 5.91 years and 57.82 ± 4.13 years for the pre- and post-menopausal women, respectively. Post-menopausal women showed significantly higher BMI, WHR, systolic blood pressure, diastolic blood pressure, triglycerides, serum adiponectin (9.20 ± 4.12 mg/ml vs. 6.81 ± 4.10 mg/ml; p ¼ 0.0049) and osteocalcin (4.80 ± 2.06 ng/ml vs. 3.73 ± 1.57 ng/ml; p ¼ 0.0047) levels than in pre-menopausal women. However, there were no significant differences in waist circumference, FBG, fasting insulin, HOMA-IR, total cholesterol, LDL-C and HDL-C levels between the groups (Table 2). In pre-menopausal diabetic women, serum osteocalcin was significantly negatively correlated with FBG and HOMA-IR and positively correlated
Table 1. Anthropometric and biochemical characteristics of control and type 2 diabetic group.
N (Pre-menopausal/post-menopausal women) Age (Years) BMI (kg/m2) Waist circumference (cm) Waist to hip ratio SBP (mmHg) DBP (mmHg) Fasting blood glucose (mmol/l) Fasting insulin (mIU/ml) HOMA-IR Total cholesterol (mg/dl) LDL-C (mg/dl) HDL-C (mg/dl) Triglycerides (mg/dl) Adiponectin (mg/ml) Osteocalcin (ng/ml)
Control
Type 2 diabetes
P
102 (53/49) 50.70 ± 7.82 24.94 ± 3.42 89.06 ± 10.54 0.91 ± 0.08 124.25 ± 9.01 74.19 ± 8.41 4.72 ± 0.90 7.58 ± 3.73 1.59 ± 0.87 171.63 ± 17.34 101.35 ± 18.05 50.52 ± 7.94 98.75 ± 14.12 18.47 ± 7.01 9.67 ± 3.24
98 (51/47) 50.90 ± 8.84 25.50 ± 4.20 88.10 ± 7.58 0.92 ± 0.07 126.84 ± 8.54 75.89 ± 9.77 9.31 ± 2.35 13.72 ± 7.66 5.93 ± 3.99 200.33 ± 49.41 123.84 ± 51.60 42.83 ± 9.82 168.45 ± 20.11 7.96 ± 4.26 4.24 ± 1.89
0.8642 0.3045 0.4608 0.6733 0.0387 0.1920 50.0001 50.0001 50.0001 50.0001 50.0001 50.0001 50.0001 50.0001 50.0001
Data are presented as mean ± SD. BMI, body mass index; SBP, systolic blood pressure; DBP, diastolic blood pressure; HOMA-IR, homeostasis model assessment-insulin resistance; LDL-C, low density lipoprotein-cholesterol; HDL-C, high density lipoprotein-cholesterol.
Table 2. Anthropometric and biochemical characteristics of pre- and post-menopausal women with type 2 diabetes. Type 2 diabetes
N Age (years) BMI (kg/m2) Waist circumference (cm) Waist to hip ratio SBP (mmHg) DBP (mmHg) Fasting blood glucose (mmol/l) Fasting insulin (mIU/ml) HOMA-IR Total cholesterol (mg/dl) LDL-C (mg/dl) HDL-C (mg/dl) Triglycerides (mg/dl) Adiponectin (mg/ml) Osteocalcin (ng/ml)
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Pre-menopausal women
Post-menopausal women
P
51 44.67 ± 5.91 24.40 ± 4.34 87.99 ± 6.49 0.90 ± 0.09 124.86 ± 7.89 73.55 ± 9.12 9.28 ± 2.51 12.29 ± 7.98 5.36 ± 4.14 196.39 ± 46.83 121.02 ± 49.00 44.00 ± 9.55 164.25 ± 17.35 6.81 ± 4.10 3.73 ± 1.57
47 57.82 ± 4.13 26.19 ± 4.22 88.23 ± 6.69 0.94 ± 0.05 128.98 ± 8.76 78.43 ± 9.90 9.34 ± 2.18 15.28 ± 7.06 6.53 ± 3.77 204.60 ± 52.25 126.90 ± 54.64 41.75 ± 10.04 175.36 ± 23.03 9.20 ± 4.12 4.80 ± 2.06
50.0001 0.0411 0.8810 0.0077 0.0167 0.0131 0.9056 0.0536 0.1501 0.4144 0.5758 0.2574 0.0089 0.0049 0.0047
Data are presented as mean ± SD. BMI, body mass index; SBP, systolic blood pressure; DBP, diastolic blood pressure; HOMA-IR, homeostasis model assessment-insulin resistance; LDL-C, low density lipoprotein-cholesterol; HDL-C, high density lipoprotein-cholesterol.
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Arch Physiol Biochem, 2014; 120(2): 73–79
Figure 1. Correlation between serum osteocalcin levels and fasting blood glucose (A), fasting insulin (B), HOMA-IR (C) and adiponectin (D), among pre- () and post-menopausal () women with type 2 diabetes.
also after adjustment for age, BMI, waist circumference, WHR, blood pressure and lipid profile parameters in premenopausal diabetic women. Although osteocalcin tended to be negatively correlated with the HOMA-IR level after adjustment for same confounders, this did not reach statistical significance in post-menopausal diabetic women. Further, the association among pre-menopausal women was weakened but remained statistically significant after additional adjustment for adiponectin (b ¼ 0.350; p ¼ 0.0219). We further investigated whether adiponectin modulated the well-documented inverse association between serum osteocalcin concentrations and type 2 diabetes. When multivariate logistic regression analyses were performed with the presence of diabetes as dependent variable and serum osteocalcin adjusted for age, BMI, waist circumference, WHR, blood pressure, FBG, HDL-C and triglycerides, as independent variable, higher osteocalcin levels were associated with a lower odds of type 2 diabetes in both pre- and postmenopausal women and this association appeared stronger in pre-menopausal than in post-menopausal women (odds ratio (95% CI) was 0.228 (0.122–0.428); p50.0001 in premenopausal and 0.497 (0.376–0.656); p50.0001 in postmenopausal women).The subsequent addition of adiponectin
slightly attenuated the association in both groups (odds ratio (95% CI) was 0.311 (0.167–0.578); p ¼ 0.0002 in premenopausal and 0.584 (0.432–0.790); p ¼ 0.0005 in postmenopausal women) (Table 4).
Discussion In the present study, we have shown a significant inverse association between osteocalcin level and type 2 diabetes in women and the association was stronger in pre-menopausal than in post-menopausal women. Our findings of significant link between osteocalcin and type 2 diabetes are consistent with our previous report in Central Indian men (Sarkar & Choudhury, 2012) and with some other recent studies (Hwang et al., 2012; Zhou et al., 2009) but differed from those of Hwang et al. (2012) that showed no evidence of significant association between osteocalcin and type 2 diabetes. Recently, it has been demonstrated that administration of osteocalcin in mice prevented the development of dietinduced obesity and type 2 diabetes (Ferron et al., 2008). Interestingly, osteocalcin is synthesized by bone gammacarboxyglutamate protein (BGLAP) gene located in the well replicated region of type 2 diabetes linkage on chromosome
DOI: 10.3109/13813455.2013.877488
Role of adiponectin in association of osteocalcin with insulin resistance and type 2 diabetes
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Table 3. Multiple linear regression analyses of the relationship between HOMA-IR and osteocalcin in pre- and post-menopausal women with type 2 diabetes. Type 2 diabetes Pre-menopausal women b
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Independent variables
Osteocalcin (ng/ml) 0.446 Age (years) 0.466 BMI (kg/m2) 0.333 Waist circumference (cm) 0.001 Waist to hip ratio 0.009 SBP (mmHg) 0.008 DBP (mmHg) 0.035 Total cholesterol (mg/dl) 0.403 LDL-C (mg/dl) 0.025 HDL-C (mg/dl) 0.014 Triglycerides (mg/dl) 0.138 After additional adjustment for adiponectin Osteocalcin (ng/ml) 0.350 Age (years) 0.464 BMI (kg/m2) 0.310 Waist circumference (cm) 0.123 Waist to hip ratio 0.028 SBP (mmHg) 0.001 DBP (mmHg) 0.014 Total cholesterol (mg/dl) 0.386 LDL-C (mg/dl) 0.023 HDL-C (mg/dl) 0.076 Triglycerides (mg/dl) 0.155 Adiponectin (mg/ml) 0.252
Post-menopausal women P
b
P
0.0019 0.0003 0.1378 0.9957 0.9441 0.8931 0.4572 0.0039 0.3183 0.9258 0.3934
0.238 0.272 0.129 0.447 0.257 0.042 0.009 0.473 0.378 0.114 0.162
0.0559 0.0252 0.4707 0.0454 0.1926 0.3123 0.1855 0.6960 0.7579 0.6307 0.4992
0.0219 0.0003 0.1610 0.6064 0.8330 0.8101 0.8233 0.0050 0.5731 0.6317 0.3329 0.1355
0.208 0.288 0.116 0.388 0.268 0.002 0.011 0.302 0.215 0.027 0.201 0.131
0.1061 0.0201 0.5180 0.0959 0.1775 0.7322 0.6691 0.8059 0.8626 0.9159 0.4113 0.3822
BMI, body mass index; SBP, systolic blood pressure; DBP, diastolic blood pressure; HOMA-IR, homeostasis model assessmentinsulin resistance; LDL-C, low density lipoprotein-cholesterol; HDL-C, high density lipoprotein-cholesterol. Table 4. Multivariate logistic regression analyses for association of osteocalcin with type 2 diabetes as dependent variable in pre- and post-menopausal women. Pre-menopausal women
Model 1: Model 2: Model 3:
Post-menopausal women
OR
95% confidence interval
P
OR
95% confidence interval
P
0.276 0.228 0.311
0.166–0.459 0.122–0.428 0.167–0.578
50.0001 50.0001 0.0002
0.528 0.497 0.584
0.412–0.676 0.376–0.656 0.432–0.790
50.0001 50.0001 0.0005
Model 1: unadjusted; Model 2: adjusted for age, BMI, waist circumference, waist to hip ratio, blood pressure, FBG, HDL-cholesterol and triglycerides; Model-3: adjusted for variables in Model 2 plus adiponectin; OR: odds ratio.
1q22 (Das & Elbein, 2007). Furthermore, a recent casecontrol study identified coding variants in the fourth exon of human osteocalcin that appeared to correlate with type 2 diabetes in African-American patients (Das et al., 2010). Taken together, our observation suggests that osteocalcin may play a significant role in the pathophysiology of type 2 diabetes. However, further prospective studies are needed to clarify whether low osteocalcin level plays a causal role in the development of type 2 diabetes. In animal experiments, osteocalcin regulates glucose metabolism, modulating both beta cell insulin secretion and peripheral insulin resistance (Lee et al., 2007). In human, lower osteocalcin level has been shown to be associated with altered glucose metabolism. However, studies, which analysed this association in women according to menopausal status, are limited and have been highly variable. More specifically, in a cross-sectional study in type 2 diabetes, osteocalcin showed no association with any of the glucose metabolism related variables in either pre or post-menopausal
women (Zhou et al., 2009). On the other hand, several reports suggested that serum osteocalcin was inversely correlated with fasting insulin and HOMA-IR in healthy non diabetic (Chen et al., 2013; Lee et al., 2012) and diabetic postmenopausal women (Im et al., 2008; Sheng et al., 2013). Kim et al. (2013) also showed similar associations in postmenopausal women but not in pre-menopausal women. Some of these discrepancies may be explained by the underlying differences in the study populations, inclusion criteria and adjustment for potential confounders. In the present study, serum osteocalcin levels were significantly higher in post-menopausal than in pre-menopausal diabetic women, as was reported in a previous study (Zhou et al., 2009). The increased levels of osteocalcin in the postmenopausal group may be attributed to relative estradiol deficiency (Griesmacher et al., 1997). Further, in an age- and BMI-adjusted model, osteocalcin showed significant inverse correlation with HOMA-IR only in pre-menopausal but not in post-menopausal diabetic women. The difference in the
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association between osteocalcin and insulin resistance among pre- and post-menopausal diabetic women remained unexplained in our study. However, it has been reported that the associations of sex steroid hormones with insulin resistance are different depending on the oestrogen status (Matsui et al., 2013). Therefore, it is possible that changes in several sex hormones that occur with the onset of menopause might have affected the association between osteocalcin and insulin resistance in post-menopausal diabetic women, although this is purely speculative as we did not measure sex hormones. Therefore, our results need confirmation in other studies. Nevertheless, these findings point to a functional role of osteocalcin in glucose metabolism, where osteocalcin could provide a signal linking bone and energy homeostasis. Our findings of significant positive correlation between serum osteocalcin and adiponectin level in both pre- and postmenopausal diabetic women are supported by previous studies in post-menopausal women with type 2 diabetes (Kanazawa et al., 2009) and non-diabetes (Chen et al., 2013). Our results also coincide with animal studies demonstrating that adipocytes from wild type mice, co-cultured with osteoblast-conditioned media derived from osteocalcin knockout mice, showed a decrease in adiponectin synthesis and osteocalcin administration regulated adiponectin gene expression in adipocytes (Lee et al., 2007). Recent in vitro studies reported a link between adiponectin and bone homeostasis by demonstrating transcription, translation and secretion of adiponectin as well as expression of its receptors AdipoR1 and AdipoR2 in bone-forming cells (Berner et al., 2004). In adipose tissue, osteocalcin increases expression of the gene ADIPOQ encoding adiponectin, which enhances insulin sensitivity (Lee et al., 2007). In turn, by the presence of adiponectin receptors on osteoblast, adiponectin can induce proliferation and differentiation of the same in a feedback loop (Kanazawa et al., 2007). Adiponectin also promoted the transcriptional activity of Runx2, a key transcription factor in osteogenesis, which binds onto the promoter of target genes for osteocalcin (Lee et al., 2009). However, the molecular mechanisms by which osteocalcin may affect adiponectin gene expression and secretion, particularly in human, are yet to be discovered. Our results provide further in vivo evidence that the effect of osteocalcin on insulin resistance was partially reduced after controlling for adiponectin in pre-menopausal women with type 2 diabetes. Moreover, adjustment for adiponectin slightly attenuated the inverse association between osteocalcin and type 2 diabetes in both pre- and post-menopausal women. This could suggest that plasma adiponectin, to some extent, may be involved in these relationships, supporting the hypothesis of osteocalcin contributing to decreased insulin resistance and thereby lowered type 2 diabetes risks through effects (may be partial) on adiponectin synthesis. We are aware of two prior studies that have evaluated the role of adiponectin in the association between osteocalcin and insulin resistance in human, with contradictory results. Observations made by Shea et al. (2009) in non-diabetic older men and women, revealed that strength of the association between osteocalcin and HOMA-IR was partially attenuated by adiponectin. On the other hand, reports from other authors (Hwang et al., 2012) contradict this result and showed that
Arch Physiol Biochem, 2014; 120(2): 73–79
adiponectin did not mediate the association between the osteocalcin level and insulin secretion and sensitivity in human. In addition, inverse association of osteocalcin with development of type 2 diabetes mellitus was independent of adiponectin level in that study (Hwang et al., 2012). It seems that the role of adiponectin in the pathway linking bone and glucose metabolism in human is complex and may be influenced by other unknown metabolic and genetic risk factors. So, the debate on this issue may continue and some further studies in different populations should be done to achieve a definite conclusion. There are several limitations inherent in our study. First, the relatively small sample size. Second, in recent animal studies, uncarboxylated osteocalcin (ucOC) appears to be the active form (Ferron et al., 2008; Lee et al., 2007). However, Bullo´ et al. (2012) reported that both total osteocalcin and ucOC were related to insulin secretion and sensitivity in humans. As we did not measure ucOC we were unable to determine specific association between ucOC and the investigated variables. Third, we must emphasize the crosssectional nature of our study and therefore, no inferences of causality can be made.
Conclusion In conclusion, the present study suggests that the protective action of osteocalcin against the development of insulin resistance and type 2 diabetes in women may be partially mediated through up-regulation of adiponectin secretion. However, further studies from different populations are warranted to clarify the absence of significant association between osteocalcin and insulin resistance in postmenopausal diabetic women.
Declaration of interest The authors report no declarations of interest.
References Berner HS, Lyngstadaas SP, Spahr A, et al. (2004). Adiponectin and its receptors are expressed in bone-forming cells. Bone, 35:842–9. Bullo´ M, Moreno-Navarrete JM, Ferna´ndez-Real JM, Salas-Salvado´ J. (2012). Total and undercarboxylated osteocalcin predict changes in insulin sensitivity and b cell function in elderly men at high cardiovascular risk. Am J Clin Nutr, 95:249–55. Chen L, Li Q, Yang Z, et al. (2013). Osteocalcin, glucose metabolism, lipid profile and chronic low-grade inflammation in middle-aged and elderly Chinese. Diabet Med, 30:309–17. Daimon M, Oizumi T, Saitoh T, et al. (2003). Decreased serum levels of adiponectin are a risk factor for the progression to type 2 diabetes in the Japanese population: The Funagata study. Diabetes Care, 26: 2015–20. Das SK, Elbein SC. (2007). The search for type 2 diabetes susceptibility loci: The chromosome 1q story. Curr Diab Rep, 7:154–64. Das SK, Sharma NK, Elbein SC. (2010). Analysis of osteocalcin as a candidate gene for type 2 diabetes (T2D) and intermediate traits in Caucasians and African Americans. Dis Markers, 28:281–6. Eastell R, Ebeling PR. (2009). Bone turnover markers: A key tool for understanding osteoporosis. Osteoporos Int, 20:S237–8. Ferron M, Hinoi E, Karsenty G, Ducy P. (2008). Osteocalcin differentially regulates beta cell and adipocyte gene expression and affects the development of metabolic diseases in wild type mice. Proc Natl Acad Sci USA, 105:5266–70. Friedewald WT, Levy RI, Fredrickson DS. (1972). Estimation of the concentration of low-density lipoprotein cholesterol in plasma,
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DOI: 10.3109/13813455.2013.877488
Role of adiponectin in association of osteocalcin with insulin resistance and type 2 diabetes
without use of the preparative ultracentrifuge. Clin Chem, 18: 499–502. Gravenstein KS, Napora JK, Short RG, et al. (2011). Cross-sectional evidence of a signaling pathway from bone homeostasis to glucose metabolism. J Clin Endocrinol Metab, 96:E884–90. Griesmacher A, Peichl P, Pointinger P, et al. (1997). Biochemical markers in menopausal women. Scand J Clin Lab Invest Suppl, 227: 64–72. Hauschka PV, Lian JB, Cole DE, Gundberg CM. (1989). Osteocalcin and matrix Gla protein: Vitamin K-dependent proteins in bone. Physiol Rev, 69:990–1047. Helio¨vaara MK, Strandberg TE, Karonen SL, Ebeling P. (2006). Association of serum adiponectin concentration to lipid and glucose metabolism in healthy humans. Horm Metab Res, 38:336–40. Hwang YC, Jeong IK, Ahn KJ, Chung HY. (2012). Circulating osteocalcin level is associated with improved glucose tolerance, insulin secretion and sensitivity independent of the plasma adiponectin level. Osteoporos Int, 23:1337–42. Hwang YC, Jee JH, Jeong IK, et al. (2012). Circulating osteocalcin level is not associated with incident type 2 diabetes in middle-aged male subjects: Mean 8.4-year retrospective follow-up study. Diabetes Care, 35:1919–24. Im JA, Yu BP, Jeon JY, Kim SH. (2008). Relationship between osteocalcin and glucose metabolism in post-menopausal women. Clin Chim Acta, 396:66–9. Kanazawa I, Yamaguchi T, Yamamoto M, et al. (2009). Serum osteocalcin level is associated with glucose metabolism and atherosclerosis parameters in type 2 diabetes mellitus. J Clin Endocrinol Metab, 94:45–9. Kanazawa I, Yamaguchi T, Yano S, et al. (2007). Adiponectin and AMP kinase activator stimulate proliferation, differentiation, and mineralization of osteoblastic MC3T3-E1 cells. BMC Cell Biol, 8:51. doi: 10.1186/1471-2121-8-51. Kim S, Lee JY, Im JA, et al. (2013). Association between serum osteocalcin and insulin resistance in post-menopausal, but not premenopausal, women in Korea. Menopause, 20:1061–6. Lee HW, Kim SY, Kim AY, et al. (2009). Adiponectin stimulates osteoblast differentiation through induction of COX2 in mesenchymal progenitor cells. Stem Cells, 27:2254–62. Lee NK, Sowa H, Hinoi E, et al. (2007). Endocrine regulation of energy metabolism by the skeleton. Cell, 130:456–69. Lee SW, Jo HH, Kim MR, et al. (2012). Association between obesity, metabolic risks and serum osteocalcin level in post-menopausal women. Gynecol Endocrinol, 28:472–7. Mantzoros CS, Li T, Manson JE, Hu FB. (2005). Circulating adiponectin levels are associated with better glycemic control, more favorable
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lipid profile, and reduced inflammation in women with type 2 diabetes. J Clin Endocrinol Metab, 90:4542–8. Matsui S, Yasui T, Tani A, et al. (2013). Associations of estrogen and testosterone with insulin resistance in pre- and post-menopausal women with and without hormone therapy. Int J Endocrinol Metab, 11:65–70. Matthews DR, Hosker JP, Rudenski AS, et al. (1985). Homeostasis model assessment: Insulin resistance and beta-cell function from fasting plasma glucose and insulin concentrations in man. Diabetologia, 28:412–19. Pittas AG, Harris SS, Eliades M, et al. (2009). Association between serum osteocalcin and markers of metabolic phenotype. J Clin Endocrinol Metab, 94:827–32. Sarkar PD, Choudhury AB. (2012). Relationship of serum osteocalcin levels with blood glucose, insulin resistance and lipid profile in central Indian men with type 2 diabetes. Arch Physiol Biochem, 118: 260–4. Sarkar PD, Choudhury AB. (2013). Relationships between serum osteocalcin levels versus blood glucose, insulin resistance and markers of systemic inflammation in central Indian type 2 diabetic patients. Eur Rev Med Pharmacol Sci, 17:1631–5. Scherer PE, Williams S, Fogliano M, et al. (1995). A novel serum protein similar to C1q, produced exclusively in adipocytes. J Biol Chem, 270:26746–9. Shea MK, Gundberg CM, Meigs JB, et al. (2009). Gamma-carboxylation of osteocalcin and insulin resistance in older men and women. Am J Clin Nutr, 90:1230–35. Sheng L, Cao W, Cha B, et al. (2013). Serum osteocalcin level and its association with carotid atherosclerosis in patients with type 2 diabetes. Cardiovasc Diabetol, 12:22. doi: 10.1186/1475-284012-22. Spranger J, Kroke A, Mo¨hlig M, et al. (2003). Adiponectin and protection against type 2 diabetes mellitus. Lancet, 361:226–8. Sumarac-Dumanovic M, Milin J, Stamenkovic-Pejkovic D, et al. (2012). Association between osteocalcin and insulin resistance in obese women. Endocrine Abstracts, 29:1248. World Health Organization. (1999). Definition, diagnosis and classification of diabetes mellitus and its complications: Report of a WHO consultation. Part 1: Diagnosis and classification of diabetes mellitus. Geneva: World Health Organization. World Health Organization Scientific Group. (1981). Research on the menopause. Technical Report Series 670. Geneva: World Health Organization. Zhou M, Ma X, Li H, et al. (2009). Serum osteocalcin concentrations in relation to glucose and lipid metabolism in Chinese individuals. Eur J Endocrinol, 161:723–9.
ORIGINAL ARTICLE
Anil Baran Choudhury1, Purnima Dey Sarkar2, Dilip K. Sakalley3, and Sudhakar B. Petkar4 1
Department of Biochemistry, NSCB Medical College, Jabalpur, Madhya Pradesh, India, 2Department of Biochemistry, MGM Medical College, Agra/ Bombay Road, Indore, Madhya Pradesh, India, 3Department of Forensic Medicine, NSCB Medical College, Jabalpur, Madhya Pradesh, India, and 4 Department of Biochemistry, People’s College of Medical Sciences and Research Centre, Bhanpur Bypass Road, Bhopal, Madhya Pradesh, India Abstract
Keywords
Objective: To analyse the association of osteocalcin with insulin resistance and type 2 diabetes and assess the role of adiponectin in these relationships. Methods: This study comprised 98 newly diagnosed type 2 diabetic women (51 pre-menopausal and 47 post-menopausal) and 102 age and BMI matched controls (53 pre-menopausal and 49 post-menopausal). Insulin resistance was calculated by homeostasis model assessment-insulin resistance (HOMA-IR). Results: Osteocalcin was significantly positively correlated with adiponectin in both premenopausal (p ¼ 0.0026) and post-menopausal diabetic women (p ¼ 0.0357). Significant negative association between osteocalcin and HOMA-IR was observed only in pre-menopausal diabetic women (p ¼ 0.0019), but the association was partially reduced (p ¼ 0.0219) after additional adjustment for adiponectin. Adiponectin slightly attenuated the inverse association between osteocalcin and presence of type 2 diabetes in both pre- and post-menopausal women. Conclusion: The protective action of osteocalcin against the development of insulin resistance and type 2 diabetes in women may be partially mediated through up-regulation of adiponectin secretion.
Adiponectin, glucose homeostasis, HOMA-IR, insulin resistance, osteocalcin, type 2 diabetes
Introduction In addition to bone mineralization and calcium homeostasis, a novel endocrine role of skeleton, affecting energy homeostasis, has recently been reported in animal studies (Ferron et al., 2008; Lee et al., 2007). Osteocalcin, a non-collagenous protein with 49 amino acids (MW 5800 Da), is produced by osteoblast and has been known as a marker of bone turnover (Eastell & Ebeling, 2009; Hauschka et al., 1989). Recently, it has been demonstrated that mice lacking the osteocalcin gene develop a series of phenotypic abnormalities such as decreased beta cell proliferation, decreased insulin secretion, insulin resistance and hyperglycaemia than wild type mice (Lee et al., 2007). Furthermore, administration of recombinant osteocalcin reversed these phenotypic abnormalities and protected them to a large extent against obesity and type 2 diabetes mellitus (Ferron et al., 2008). In humans we and others have reported that serum osteocalcin concentration was negatively correlated with blood glucose and insulin resistance (Hwang et al., 2012; Pittas et al., 2009; Sarkar & Choudhury, 2013; Shea et al., 2009) and inversely associated
Correspondence: Anil Baran Choudhury, Department of Biochemistry, NSCB Medical College, Jabalpur, Madhya Pradesh, India. Tel: +919424443478. E-mail: [email protected]
History Received 17 September 2013 Revised 24 November 2013 Accepted 16 December 2013 Published online 9 January 2014
with type 2 diabetes (Hwang et al., 2012; Sarkar & Choudhury, 2012; Zhou et al., 2009). Prior studies have also shown that type 2 diabetic patients have reduced osteocalcin concentrations compared with non diabetics (Hwang et al., 2012; Zhou et al., 2009). In addition, menopause transition may have an influence on circulating osteocalcin concentration, since osteocalcin concentration is significantly higher among post-menopausal women than premenopausal women (Zhou et al., 2009). However, data concerning the association between osteocalcin and impaired glucose metabolism among pre- or post-menopausal women provided inconsistent results (Im et al., 2008; Kim et al., 2013; Zhou et al., 2009). Adiponectin, an adipocyte derived protein with 244 amino acids (Scherer et al., 1995), plays an important role in the regulation of energy homeostasis and insulin sensitivity (Helio¨vaara et al., 2006; Mantzoros et al., 2005) and predicts the development of type 2 diabetes (Daimon et al., 2003; Spranger et al., 2003). Adiponectin and its receptors are also expressed in osteoblastic cells (Berner et al., 2004) and stimulate osteoblast proliferation and differentiation (Kanazawa et al., 2007). Recent in vitro and in vivo studies have shown that in adipose tissue osteocalcin promotes insulin sensitivity by increasing expression of the gene ADIPOQ encoding adiponectin (Ferron et al., 2008; Lee
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Role of adiponectin in mediating the association of osteocalcin with insulin resistance and type 2 diabetes: a cross sectional study in pre- and post-menopausal women
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et al., 2007). A number of studies have found a positive link between serum osteocalcin and adiponectin (Gravenstein et al., 2011; Sumarac-Dumanovic et al., 2012). However, clinical data that investigated the role of adiponectin in the relationship between osteocalcin and insulin resistance are limited, with conflicting results (Hwang et al., 2012; Shea et al., 2009). Therefore, in this cross sectional study, we evaluated the relationship between serum osteocalcin and insulin resistance among pre- and post-menopausal women with type 2 diabetes. We also determined whether adiponectin could mediate the association of osteocalcin with insulin resistance and type 2 diabetes.
Methods Study subjects This study was conducted in the Department of Biochemistry, Netaji Subhash Chandra Bose (NSCB) Medical College and Hospital, Jabalpur and approved by Institutional Ethical Committee. Ninety eight newly diagnosed type 2 diabetic women (51 pre-menopausal, 47 post-menopausal), aged between 31 to 70 years (mean age ± SD: 50.90 ± 8.84 years), were recruited from the outpatients department of NSCB Medical College and Hospital. One hundred two age and BMI matched healthy asymptomatic women participants (53 pre-menopausal, 49 post-menopausal; mean age ± SD: 50.70 ± 7.82 years), who had come for a routine health checkup in the hospital, were taken as control. Diabetes mellitus was confirmed according to the 1999 World Health Organization (WHO, 1999) criteria. Menopause was defined as the absence of menstruation for 12 consecutive months, which was not due to surgical resection of uterus or ovaries (WHO, 1981). Brief clinical history of present and past illness and medical therapy were recorded from all participants. Written informed consent was obtained from the study group and the controls before entry into the study. Exclusion criteria The exclusion criteria in the patient group were: (1) any systemic disease other than type 2 diabetes, (2) under hypoglycaemic drug or insulin treatment, (3) recent history of fracture, (4) use of any bone active medications such as vitamin D, calcitonin, bisphosphonate or lipid lowering drug, (5) use of hormone replacement therapy, (6) smokers, (7) alcoholics and (8) pregnancy.
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Laboratory analyses Venous blood samples were collected after an overnight fasting in the morning in an aseptic condition from the antecubital vein. Blood samples were centrifuged at 4 centigrade and serum was stored immediately at 80 centigrade until they were analysed. Fasting blood glucose (FBG), total cholesterol (TC), high density lipoprotein-cholesterol (HDL-C) and triglycerides (TG) were estimated by the standard laboratory kit (Biosystem) method using a fully automated biochemistry analyser (Biosystem A25; Biosystem SA, Barcelona, Spain). Low density lipoprotein-cholesterol (LDL-C) was calculated according to Friedewald’s formula (TC [mg/dl] HDL-C [mg/dl] TG [mg/dl]/5) (Friedewald et al., 1972). Serum osteocalcin level was estimated by ELISA method (Quidel Corporation, San Diego, CA, USA). Serum adiponectin was measured by ELISA method (Ray Biotech Inc, Norcross, GA, USA). Fasting insulin was measured using a commercially available ELISA kit (LDN, Nordhorn). Insulin resistance was calculated by homeostasis model assessment (HOMA) based on the formula: HOMAIR ¼ fasting glucose (mmol/l) fasting insulin (mIU/ml)/22.5 (Matthews et al., 1985). Statistical analyses The Kolmogorov–Smirnov statistical test was used to test the normality of the distribution. Variables with a skewed distribution were log-transformed before performing statistical analyses. Data were expressed as the mean ± standard deviation. Comparisons of baseline anthropometric and biochemical parameters between groups were done by unpaired Student’s t-test. All correlations were analysed with Pearson’s correlation coefficient. To adjust for confounding variables in the correlation analyses, partial correlation coefficients were calculated. Linear regression analyses were done to study the association between HOMA-IR and osteocalcin after multivariate adjustment for potential confounders. Multivariate logistic regression models were used to assess the association between serum osteocalcin and type 2 diabetes with age, BMI, waist circumference, WHR, systolic and diastolic blood pressure, fasting blood glucose, HDLcholesterol and triglycerides, as covariates. To test the role of serum adiponectin as cofactor, the analyses were repeated with the inclusion of serum adiponectin as additional covariates in both linear and logistic regression models. All statistical tests were two-tailed and p value less than 0.05 were considered to be statistically significant. All data were analysed using statistical software SPSS version 16 (SPSS Inc., Chicago, IL, USA).
Anthropometry
Results
Body mass index (BMI) was calculated for all subjects by using the formula weight in kilograms divided by the square of height in metres. Waist (WC) and hip circumference were measured in the standing position using standard techniques and waist to hip ratio (WHR) was calculated as waist circumference divided by hip circumference. Seated systolic (SBP) and diastolic blood pressure (DBP) were measured by manual sphygmomanometer.
The baseline anthropometric and biochemical characteristics of control and type 2 diabetic patients group are presented in Table 1. The diabetic group had significantly higher systolic blood pressure (p50.05), fasting blood glucose, fasting insulin, HOMA-IR, total cholesterol, LDL-cholesterol and triglycerides (p50.0001) than the control group, whereas HDL-cholesterol, adiponectin (7.96 ± 4.26 mg/ml vs. 18.47 ± 7.01 mg/ml; p50.0001) and osteocalcin levels
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DOI: 10.3109/13813455.2013.877488
Role of adiponectin in association of osteocalcin with insulin resistance and type 2 diabetes
with adiponectin (Figure 1), which was true also after adjusting for age and BMI (FBG: r ¼ 0.427; p ¼ 0.0022, HOMA-IR: r ¼ 0.353; p ¼ 0.0130, adiponectin: r ¼ 0.421; p ¼ 0.0026) (data not shown). In post-menopausal diabetic women, serum osteocalcin was significantly negatively correlated with FBG, fasting insulin and HOMA IR and positively with adiponectin levels (Figure 1). However, when adjusted for age and BMI, only correlation with FBG (r ¼ 0.331; p ¼ 0.0265) and adiponectin (r ¼ 0.314; p ¼ 0.0357) remained significant (data not shown). To more precisely define the relationship between osteocalcin and insulin resistance, we further examined this relationship after adjusting for some metabolic factors in addition to age and BMI in linear regression model. As shown in Table 3, the negative relationship between osteocalcin and HOMA-IR remained significant (b ¼ 0.446; p ¼ 0.0019)
(4.24 ± 1.89 ng/ml vs. 9.67 ± 3.24 ng/ml; p50.0001) were significantly lower in the diabetic group (Table 1). When data from diabetic patients were stratified according to menopausal status, the average age ± SD was 44.67 ± 5.91 years and 57.82 ± 4.13 years for the pre- and post-menopausal women, respectively. Post-menopausal women showed significantly higher BMI, WHR, systolic blood pressure, diastolic blood pressure, triglycerides, serum adiponectin (9.20 ± 4.12 mg/ml vs. 6.81 ± 4.10 mg/ml; p ¼ 0.0049) and osteocalcin (4.80 ± 2.06 ng/ml vs. 3.73 ± 1.57 ng/ml; p ¼ 0.0047) levels than in pre-menopausal women. However, there were no significant differences in waist circumference, FBG, fasting insulin, HOMA-IR, total cholesterol, LDL-C and HDL-C levels between the groups (Table 2). In pre-menopausal diabetic women, serum osteocalcin was significantly negatively correlated with FBG and HOMA-IR and positively correlated
Table 1. Anthropometric and biochemical characteristics of control and type 2 diabetic group.
N (Pre-menopausal/post-menopausal women) Age (Years) BMI (kg/m2) Waist circumference (cm) Waist to hip ratio SBP (mmHg) DBP (mmHg) Fasting blood glucose (mmol/l) Fasting insulin (mIU/ml) HOMA-IR Total cholesterol (mg/dl) LDL-C (mg/dl) HDL-C (mg/dl) Triglycerides (mg/dl) Adiponectin (mg/ml) Osteocalcin (ng/ml)
Control
Type 2 diabetes
P
102 (53/49) 50.70 ± 7.82 24.94 ± 3.42 89.06 ± 10.54 0.91 ± 0.08 124.25 ± 9.01 74.19 ± 8.41 4.72 ± 0.90 7.58 ± 3.73 1.59 ± 0.87 171.63 ± 17.34 101.35 ± 18.05 50.52 ± 7.94 98.75 ± 14.12 18.47 ± 7.01 9.67 ± 3.24
98 (51/47) 50.90 ± 8.84 25.50 ± 4.20 88.10 ± 7.58 0.92 ± 0.07 126.84 ± 8.54 75.89 ± 9.77 9.31 ± 2.35 13.72 ± 7.66 5.93 ± 3.99 200.33 ± 49.41 123.84 ± 51.60 42.83 ± 9.82 168.45 ± 20.11 7.96 ± 4.26 4.24 ± 1.89
0.8642 0.3045 0.4608 0.6733 0.0387 0.1920 50.0001 50.0001 50.0001 50.0001 50.0001 50.0001 50.0001 50.0001 50.0001
Data are presented as mean ± SD. BMI, body mass index; SBP, systolic blood pressure; DBP, diastolic blood pressure; HOMA-IR, homeostasis model assessment-insulin resistance; LDL-C, low density lipoprotein-cholesterol; HDL-C, high density lipoprotein-cholesterol.
Table 2. Anthropometric and biochemical characteristics of pre- and post-menopausal women with type 2 diabetes. Type 2 diabetes
N Age (years) BMI (kg/m2) Waist circumference (cm) Waist to hip ratio SBP (mmHg) DBP (mmHg) Fasting blood glucose (mmol/l) Fasting insulin (mIU/ml) HOMA-IR Total cholesterol (mg/dl) LDL-C (mg/dl) HDL-C (mg/dl) Triglycerides (mg/dl) Adiponectin (mg/ml) Osteocalcin (ng/ml)
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Pre-menopausal women
Post-menopausal women
P
51 44.67 ± 5.91 24.40 ± 4.34 87.99 ± 6.49 0.90 ± 0.09 124.86 ± 7.89 73.55 ± 9.12 9.28 ± 2.51 12.29 ± 7.98 5.36 ± 4.14 196.39 ± 46.83 121.02 ± 49.00 44.00 ± 9.55 164.25 ± 17.35 6.81 ± 4.10 3.73 ± 1.57
47 57.82 ± 4.13 26.19 ± 4.22 88.23 ± 6.69 0.94 ± 0.05 128.98 ± 8.76 78.43 ± 9.90 9.34 ± 2.18 15.28 ± 7.06 6.53 ± 3.77 204.60 ± 52.25 126.90 ± 54.64 41.75 ± 10.04 175.36 ± 23.03 9.20 ± 4.12 4.80 ± 2.06
50.0001 0.0411 0.8810 0.0077 0.0167 0.0131 0.9056 0.0536 0.1501 0.4144 0.5758 0.2574 0.0089 0.0049 0.0047
Data are presented as mean ± SD. BMI, body mass index; SBP, systolic blood pressure; DBP, diastolic blood pressure; HOMA-IR, homeostasis model assessment-insulin resistance; LDL-C, low density lipoprotein-cholesterol; HDL-C, high density lipoprotein-cholesterol.
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Figure 1. Correlation between serum osteocalcin levels and fasting blood glucose (A), fasting insulin (B), HOMA-IR (C) and adiponectin (D), among pre- () and post-menopausal () women with type 2 diabetes.
also after adjustment for age, BMI, waist circumference, WHR, blood pressure and lipid profile parameters in premenopausal diabetic women. Although osteocalcin tended to be negatively correlated with the HOMA-IR level after adjustment for same confounders, this did not reach statistical significance in post-menopausal diabetic women. Further, the association among pre-menopausal women was weakened but remained statistically significant after additional adjustment for adiponectin (b ¼ 0.350; p ¼ 0.0219). We further investigated whether adiponectin modulated the well-documented inverse association between serum osteocalcin concentrations and type 2 diabetes. When multivariate logistic regression analyses were performed with the presence of diabetes as dependent variable and serum osteocalcin adjusted for age, BMI, waist circumference, WHR, blood pressure, FBG, HDL-C and triglycerides, as independent variable, higher osteocalcin levels were associated with a lower odds of type 2 diabetes in both pre- and postmenopausal women and this association appeared stronger in pre-menopausal than in post-menopausal women (odds ratio (95% CI) was 0.228 (0.122–0.428); p50.0001 in premenopausal and 0.497 (0.376–0.656); p50.0001 in postmenopausal women).The subsequent addition of adiponectin
slightly attenuated the association in both groups (odds ratio (95% CI) was 0.311 (0.167–0.578); p ¼ 0.0002 in premenopausal and 0.584 (0.432–0.790); p ¼ 0.0005 in postmenopausal women) (Table 4).
Discussion In the present study, we have shown a significant inverse association between osteocalcin level and type 2 diabetes in women and the association was stronger in pre-menopausal than in post-menopausal women. Our findings of significant link between osteocalcin and type 2 diabetes are consistent with our previous report in Central Indian men (Sarkar & Choudhury, 2012) and with some other recent studies (Hwang et al., 2012; Zhou et al., 2009) but differed from those of Hwang et al. (2012) that showed no evidence of significant association between osteocalcin and type 2 diabetes. Recently, it has been demonstrated that administration of osteocalcin in mice prevented the development of dietinduced obesity and type 2 diabetes (Ferron et al., 2008). Interestingly, osteocalcin is synthesized by bone gammacarboxyglutamate protein (BGLAP) gene located in the well replicated region of type 2 diabetes linkage on chromosome
DOI: 10.3109/13813455.2013.877488
Role of adiponectin in association of osteocalcin with insulin resistance and type 2 diabetes
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Table 3. Multiple linear regression analyses of the relationship between HOMA-IR and osteocalcin in pre- and post-menopausal women with type 2 diabetes. Type 2 diabetes Pre-menopausal women b
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Independent variables
Osteocalcin (ng/ml) 0.446 Age (years) 0.466 BMI (kg/m2) 0.333 Waist circumference (cm) 0.001 Waist to hip ratio 0.009 SBP (mmHg) 0.008 DBP (mmHg) 0.035 Total cholesterol (mg/dl) 0.403 LDL-C (mg/dl) 0.025 HDL-C (mg/dl) 0.014 Triglycerides (mg/dl) 0.138 After additional adjustment for adiponectin Osteocalcin (ng/ml) 0.350 Age (years) 0.464 BMI (kg/m2) 0.310 Waist circumference (cm) 0.123 Waist to hip ratio 0.028 SBP (mmHg) 0.001 DBP (mmHg) 0.014 Total cholesterol (mg/dl) 0.386 LDL-C (mg/dl) 0.023 HDL-C (mg/dl) 0.076 Triglycerides (mg/dl) 0.155 Adiponectin (mg/ml) 0.252
Post-menopausal women P
b
P
0.0019 0.0003 0.1378 0.9957 0.9441 0.8931 0.4572 0.0039 0.3183 0.9258 0.3934
0.238 0.272 0.129 0.447 0.257 0.042 0.009 0.473 0.378 0.114 0.162
0.0559 0.0252 0.4707 0.0454 0.1926 0.3123 0.1855 0.6960 0.7579 0.6307 0.4992
0.0219 0.0003 0.1610 0.6064 0.8330 0.8101 0.8233 0.0050 0.5731 0.6317 0.3329 0.1355
0.208 0.288 0.116 0.388 0.268 0.002 0.011 0.302 0.215 0.027 0.201 0.131
0.1061 0.0201 0.5180 0.0959 0.1775 0.7322 0.6691 0.8059 0.8626 0.9159 0.4113 0.3822
BMI, body mass index; SBP, systolic blood pressure; DBP, diastolic blood pressure; HOMA-IR, homeostasis model assessmentinsulin resistance; LDL-C, low density lipoprotein-cholesterol; HDL-C, high density lipoprotein-cholesterol. Table 4. Multivariate logistic regression analyses for association of osteocalcin with type 2 diabetes as dependent variable in pre- and post-menopausal women. Pre-menopausal women
Model 1: Model 2: Model 3:
Post-menopausal women
OR
95% confidence interval
P
OR
95% confidence interval
P
0.276 0.228 0.311
0.166–0.459 0.122–0.428 0.167–0.578
50.0001 50.0001 0.0002
0.528 0.497 0.584
0.412–0.676 0.376–0.656 0.432–0.790
50.0001 50.0001 0.0005
Model 1: unadjusted; Model 2: adjusted for age, BMI, waist circumference, waist to hip ratio, blood pressure, FBG, HDL-cholesterol and triglycerides; Model-3: adjusted for variables in Model 2 plus adiponectin; OR: odds ratio.
1q22 (Das & Elbein, 2007). Furthermore, a recent casecontrol study identified coding variants in the fourth exon of human osteocalcin that appeared to correlate with type 2 diabetes in African-American patients (Das et al., 2010). Taken together, our observation suggests that osteocalcin may play a significant role in the pathophysiology of type 2 diabetes. However, further prospective studies are needed to clarify whether low osteocalcin level plays a causal role in the development of type 2 diabetes. In animal experiments, osteocalcin regulates glucose metabolism, modulating both beta cell insulin secretion and peripheral insulin resistance (Lee et al., 2007). In human, lower osteocalcin level has been shown to be associated with altered glucose metabolism. However, studies, which analysed this association in women according to menopausal status, are limited and have been highly variable. More specifically, in a cross-sectional study in type 2 diabetes, osteocalcin showed no association with any of the glucose metabolism related variables in either pre or post-menopausal
women (Zhou et al., 2009). On the other hand, several reports suggested that serum osteocalcin was inversely correlated with fasting insulin and HOMA-IR in healthy non diabetic (Chen et al., 2013; Lee et al., 2012) and diabetic postmenopausal women (Im et al., 2008; Sheng et al., 2013). Kim et al. (2013) also showed similar associations in postmenopausal women but not in pre-menopausal women. Some of these discrepancies may be explained by the underlying differences in the study populations, inclusion criteria and adjustment for potential confounders. In the present study, serum osteocalcin levels were significantly higher in post-menopausal than in pre-menopausal diabetic women, as was reported in a previous study (Zhou et al., 2009). The increased levels of osteocalcin in the postmenopausal group may be attributed to relative estradiol deficiency (Griesmacher et al., 1997). Further, in an age- and BMI-adjusted model, osteocalcin showed significant inverse correlation with HOMA-IR only in pre-menopausal but not in post-menopausal diabetic women. The difference in the
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association between osteocalcin and insulin resistance among pre- and post-menopausal diabetic women remained unexplained in our study. However, it has been reported that the associations of sex steroid hormones with insulin resistance are different depending on the oestrogen status (Matsui et al., 2013). Therefore, it is possible that changes in several sex hormones that occur with the onset of menopause might have affected the association between osteocalcin and insulin resistance in post-menopausal diabetic women, although this is purely speculative as we did not measure sex hormones. Therefore, our results need confirmation in other studies. Nevertheless, these findings point to a functional role of osteocalcin in glucose metabolism, where osteocalcin could provide a signal linking bone and energy homeostasis. Our findings of significant positive correlation between serum osteocalcin and adiponectin level in both pre- and postmenopausal diabetic women are supported by previous studies in post-menopausal women with type 2 diabetes (Kanazawa et al., 2009) and non-diabetes (Chen et al., 2013). Our results also coincide with animal studies demonstrating that adipocytes from wild type mice, co-cultured with osteoblast-conditioned media derived from osteocalcin knockout mice, showed a decrease in adiponectin synthesis and osteocalcin administration regulated adiponectin gene expression in adipocytes (Lee et al., 2007). Recent in vitro studies reported a link between adiponectin and bone homeostasis by demonstrating transcription, translation and secretion of adiponectin as well as expression of its receptors AdipoR1 and AdipoR2 in bone-forming cells (Berner et al., 2004). In adipose tissue, osteocalcin increases expression of the gene ADIPOQ encoding adiponectin, which enhances insulin sensitivity (Lee et al., 2007). In turn, by the presence of adiponectin receptors on osteoblast, adiponectin can induce proliferation and differentiation of the same in a feedback loop (Kanazawa et al., 2007). Adiponectin also promoted the transcriptional activity of Runx2, a key transcription factor in osteogenesis, which binds onto the promoter of target genes for osteocalcin (Lee et al., 2009). However, the molecular mechanisms by which osteocalcin may affect adiponectin gene expression and secretion, particularly in human, are yet to be discovered. Our results provide further in vivo evidence that the effect of osteocalcin on insulin resistance was partially reduced after controlling for adiponectin in pre-menopausal women with type 2 diabetes. Moreover, adjustment for adiponectin slightly attenuated the inverse association between osteocalcin and type 2 diabetes in both pre- and post-menopausal women. This could suggest that plasma adiponectin, to some extent, may be involved in these relationships, supporting the hypothesis of osteocalcin contributing to decreased insulin resistance and thereby lowered type 2 diabetes risks through effects (may be partial) on adiponectin synthesis. We are aware of two prior studies that have evaluated the role of adiponectin in the association between osteocalcin and insulin resistance in human, with contradictory results. Observations made by Shea et al. (2009) in non-diabetic older men and women, revealed that strength of the association between osteocalcin and HOMA-IR was partially attenuated by adiponectin. On the other hand, reports from other authors (Hwang et al., 2012) contradict this result and showed that
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adiponectin did not mediate the association between the osteocalcin level and insulin secretion and sensitivity in human. In addition, inverse association of osteocalcin with development of type 2 diabetes mellitus was independent of adiponectin level in that study (Hwang et al., 2012). It seems that the role of adiponectin in the pathway linking bone and glucose metabolism in human is complex and may be influenced by other unknown metabolic and genetic risk factors. So, the debate on this issue may continue and some further studies in different populations should be done to achieve a definite conclusion. There are several limitations inherent in our study. First, the relatively small sample size. Second, in recent animal studies, uncarboxylated osteocalcin (ucOC) appears to be the active form (Ferron et al., 2008; Lee et al., 2007). However, Bullo´ et al. (2012) reported that both total osteocalcin and ucOC were related to insulin secretion and sensitivity in humans. As we did not measure ucOC we were unable to determine specific association between ucOC and the investigated variables. Third, we must emphasize the crosssectional nature of our study and therefore, no inferences of causality can be made.
Conclusion In conclusion, the present study suggests that the protective action of osteocalcin against the development of insulin resistance and type 2 diabetes in women may be partially mediated through up-regulation of adiponectin secretion. However, further studies from different populations are warranted to clarify the absence of significant association between osteocalcin and insulin resistance in postmenopausal diabetic women.
Declaration of interest The authors report no declarations of interest.
References Berner HS, Lyngstadaas SP, Spahr A, et al. (2004). Adiponectin and its receptors are expressed in bone-forming cells. Bone, 35:842–9. Bullo´ M, Moreno-Navarrete JM, Ferna´ndez-Real JM, Salas-Salvado´ J. (2012). Total and undercarboxylated osteocalcin predict changes in insulin sensitivity and b cell function in elderly men at high cardiovascular risk. Am J Clin Nutr, 95:249–55. Chen L, Li Q, Yang Z, et al. (2013). Osteocalcin, glucose metabolism, lipid profile and chronic low-grade inflammation in middle-aged and elderly Chinese. Diabet Med, 30:309–17. Daimon M, Oizumi T, Saitoh T, et al. (2003). Decreased serum levels of adiponectin are a risk factor for the progression to type 2 diabetes in the Japanese population: The Funagata study. Diabetes Care, 26: 2015–20. Das SK, Elbein SC. (2007). The search for type 2 diabetes susceptibility loci: The chromosome 1q story. Curr Diab Rep, 7:154–64. Das SK, Sharma NK, Elbein SC. (2010). Analysis of osteocalcin as a candidate gene for type 2 diabetes (T2D) and intermediate traits in Caucasians and African Americans. Dis Markers, 28:281–6. Eastell R, Ebeling PR. (2009). Bone turnover markers: A key tool for understanding osteoporosis. Osteoporos Int, 20:S237–8. Ferron M, Hinoi E, Karsenty G, Ducy P. (2008). Osteocalcin differentially regulates beta cell and adipocyte gene expression and affects the development of metabolic diseases in wild type mice. Proc Natl Acad Sci USA, 105:5266–70. Friedewald WT, Levy RI, Fredrickson DS. (1972). Estimation of the concentration of low-density lipoprotein cholesterol in plasma,
Archives of Physiology and Biochemistry Downloaded from informahealthcare.com by Dalhousie University on 06/27/14 For personal use only.
DOI: 10.3109/13813455.2013.877488
Role of adiponectin in association of osteocalcin with insulin resistance and type 2 diabetes
without use of the preparative ultracentrifuge. Clin Chem, 18: 499–502. Gravenstein KS, Napora JK, Short RG, et al. (2011). Cross-sectional evidence of a signaling pathway from bone homeostasis to glucose metabolism. J Clin Endocrinol Metab, 96:E884–90. Griesmacher A, Peichl P, Pointinger P, et al. (1997). Biochemical markers in menopausal women. Scand J Clin Lab Invest Suppl, 227: 64–72. Hauschka PV, Lian JB, Cole DE, Gundberg CM. (1989). Osteocalcin and matrix Gla protein: Vitamin K-dependent proteins in bone. Physiol Rev, 69:990–1047. Helio¨vaara MK, Strandberg TE, Karonen SL, Ebeling P. (2006). Association of serum adiponectin concentration to lipid and glucose metabolism in healthy humans. Horm Metab Res, 38:336–40. Hwang YC, Jeong IK, Ahn KJ, Chung HY. (2012). Circulating osteocalcin level is associated with improved glucose tolerance, insulin secretion and sensitivity independent of the plasma adiponectin level. Osteoporos Int, 23:1337–42. Hwang YC, Jee JH, Jeong IK, et al. (2012). Circulating osteocalcin level is not associated with incident type 2 diabetes in middle-aged male subjects: Mean 8.4-year retrospective follow-up study. Diabetes Care, 35:1919–24. Im JA, Yu BP, Jeon JY, Kim SH. (2008). Relationship between osteocalcin and glucose metabolism in post-menopausal women. Clin Chim Acta, 396:66–9. Kanazawa I, Yamaguchi T, Yamamoto M, et al. (2009). Serum osteocalcin level is associated with glucose metabolism and atherosclerosis parameters in type 2 diabetes mellitus. J Clin Endocrinol Metab, 94:45–9. Kanazawa I, Yamaguchi T, Yano S, et al. (2007). Adiponectin and AMP kinase activator stimulate proliferation, differentiation, and mineralization of osteoblastic MC3T3-E1 cells. BMC Cell Biol, 8:51. doi: 10.1186/1471-2121-8-51. Kim S, Lee JY, Im JA, et al. (2013). Association between serum osteocalcin and insulin resistance in post-menopausal, but not premenopausal, women in Korea. Menopause, 20:1061–6. Lee HW, Kim SY, Kim AY, et al. (2009). Adiponectin stimulates osteoblast differentiation through induction of COX2 in mesenchymal progenitor cells. Stem Cells, 27:2254–62. Lee NK, Sowa H, Hinoi E, et al. (2007). Endocrine regulation of energy metabolism by the skeleton. Cell, 130:456–69. Lee SW, Jo HH, Kim MR, et al. (2012). Association between obesity, metabolic risks and serum osteocalcin level in post-menopausal women. Gynecol Endocrinol, 28:472–7. Mantzoros CS, Li T, Manson JE, Hu FB. (2005). Circulating adiponectin levels are associated with better glycemic control, more favorable
79
lipid profile, and reduced inflammation in women with type 2 diabetes. J Clin Endocrinol Metab, 90:4542–8. Matsui S, Yasui T, Tani A, et al. (2013). Associations of estrogen and testosterone with insulin resistance in pre- and post-menopausal women with and without hormone therapy. Int J Endocrinol Metab, 11:65–70. Matthews DR, Hosker JP, Rudenski AS, et al. (1985). Homeostasis model assessment: Insulin resistance and beta-cell function from fasting plasma glucose and insulin concentrations in man. Diabetologia, 28:412–19. Pittas AG, Harris SS, Eliades M, et al. (2009). Association between serum osteocalcin and markers of metabolic phenotype. J Clin Endocrinol Metab, 94:827–32. Sarkar PD, Choudhury AB. (2012). Relationship of serum osteocalcin levels with blood glucose, insulin resistance and lipid profile in central Indian men with type 2 diabetes. Arch Physiol Biochem, 118: 260–4. Sarkar PD, Choudhury AB. (2013). Relationships between serum osteocalcin levels versus blood glucose, insulin resistance and markers of systemic inflammation in central Indian type 2 diabetic patients. Eur Rev Med Pharmacol Sci, 17:1631–5. Scherer PE, Williams S, Fogliano M, et al. (1995). A novel serum protein similar to C1q, produced exclusively in adipocytes. J Biol Chem, 270:26746–9. Shea MK, Gundberg CM, Meigs JB, et al. (2009). Gamma-carboxylation of osteocalcin and insulin resistance in older men and women. Am J Clin Nutr, 90:1230–35. Sheng L, Cao W, Cha B, et al. (2013). Serum osteocalcin level and its association with carotid atherosclerosis in patients with type 2 diabetes. Cardiovasc Diabetol, 12:22. doi: 10.1186/1475-284012-22. Spranger J, Kroke A, Mo¨hlig M, et al. (2003). Adiponectin and protection against type 2 diabetes mellitus. Lancet, 361:226–8. Sumarac-Dumanovic M, Milin J, Stamenkovic-Pejkovic D, et al. (2012). Association between osteocalcin and insulin resistance in obese women. Endocrine Abstracts, 29:1248. World Health Organization. (1999). Definition, diagnosis and classification of diabetes mellitus and its complications: Report of a WHO consultation. Part 1: Diagnosis and classification of diabetes mellitus. Geneva: World Health Organization. World Health Organization Scientific Group. (1981). Research on the menopause. Technical Report Series 670. Geneva: World Health Organization. Zhou M, Ma X, Li H, et al. (2009). Serum osteocalcin concentrations in relation to glucose and lipid metabolism in Chinese individuals. Eur J Endocrinol, 161:723–9.
