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Circulating osteocalcin is increased in early-stage diabetes
diabetes research and clinical practice 92 (2011) 181–186
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Diabetes Research and Clinical Practice journ al h omepage: www .elsevier.co m/lo cate/diabres
Circulating osteocalcin is increased in early-stage diabetes Atsushi Aoki a, Takatoshi Muneyuki b,c, Masashi Yoshida a, Hiromi Munakata b, San-e Ishikawa a, Hitoshi Sugawara a, Masanobu Kawakami a, Masafumi Kakei a,* a
First Department of Comprehensive Medicine, Saitama Medical Center, Jichi Medical University School of Medicine, 1-847 Amanuma, Omiya, Saitama 330-8503, Japan b Internal Medicine, Social Insurance General Omiya Hospital, 453 Bonsai, Kita, Saitama 331-0805, Japan c Saitama Citizen Medical Center, 299-1 Shimane, Nishi, Saitama 331-0054, Japan
article info
abstract
Article history:
We aimed to examine whether circulating levels of osteocalcin, bone formation marker
Received 8 November 2010
secreted from osteoblast, are changed in glucose-intolerant subjects without taking glucose
Received in revised form
lowering agent, because bone metabolism is reportedly related to glucose metabolism in
4 January 2011
animal and human studies. According to 75 g oral glucose tolerance test (75 g-OGTT), all
Accepted 10 January 2011
subjects (47.6 10.2 years of age; 45 men and 10 women) were divided into three categories:
Published on line 2 February 2011
normal glucose tolerance (NGT, n = 39), prediabetes (PDM, n = 11) that included impaired
Keywords:
osteocalcin levels were increased in T2DM as compared to NGT. In all the participants,
Osteocalcin
simple regression analysis model revealed positive correlation of osteocalcin with plasma
Leptin
glucose at 120 min, G(120), on 75 g-OGTT, negative with both creatinine and Ln(CRP), but not
Obese
significantly with fasting plasma glucose. Osteocalcin and leptin were independent vari-
Type 2 diabetes mellitus
ables for G(120) (P = 0.026 and 0.035, respectively). In multinomial logistic analysis leptin
fasting glucose (IFG) and impaired glucose tolerance (IGT), and diabetes (T2DM, n = 5). Serum
(PDM vs. NGT: P = 0.02 Odds ratio (OR) of 1.05, 95% confidence intervals, 1.007–1.084) and osteocalcin (T2DM vs. NGT: P = 0.038, OR 10.8, 1.13–102.4) were independently associated. We conclude that circulating osteocalcin and leptin are related to glucose intolerant state. # 2011 Elsevier Ireland Ltd. All rights reserved.
1.
Introduction
Bone remodeling is essential to maintain skeleton structure during adult life. Bone resorption and formation, which are mechanistically repairing processes against both micro crack and macro damage, play a pivotal role as bone remodeling. Resorption mediated by osteoclasts differentiated from progenitor cell of bone marrow initiates osteoblast-mediated bone formation after bone destruction at damaged bone and subsequent repair [1,2]. This biphasic process in bone remodeling by these two types of bone cells is normally balanced in healthy individuals. Aging, postmenopausal state and lean body produce imbalance of bone remodeling cycle
and consequent osteoporosis with future occurrence of fracture. Bone is now identified as an organ regulating energy metabolism via osteocalcin that is secreted by osteoblast and enhances insulin secretion in pancreatic islets and insulin sensitivity. Osteocalcin stimulates insulin expression in bcells, b-cell proliferation and insulin secretion, adiponectin secretion from adipocyte and consequently improvement of insulin sensitivity [3,4]. Osteoblast is regulated via sympathetic nerve that relays energy balance information from adipocyte through hypothalamic leptinergic transmission [1,2,5]. The sympathetic nerve linking to osteoblast uses adrenergic b2 receptor stimulation by two pathways, protein
* Corresponding author. Tel.: +81 486472111; fax: +81 486485188. E-mail address: [email protected] (M. Kakei). 0168-8227/$ – see front matter # 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.diabres.2011.01.009
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diabetes research and clinical practice 92 (2011) 181–186
kinase A and molecular clock. The former mediates bone resorption by osteoclast stimulation via secretion of RANKL (receptor activation of NF-kB ligand that is released by activated osteoblast) and the latter osteoblast proliferation (bone formation). In human studies, it was reported that serum osteocalcin concentration is reciprocally correlated with glucose metabolism: osteocalcin is decreased in patients with type 2 diabetes who had never taken hypoglycemic medication as compared to those with normal glucose tolerance [6]. In cross-sectional study, osteocalcin concentration was inversely associated with fasting plasma glucose (FPG), fasting insulin, homeostasis model assessment for insulin resistance (HOMA-R), C-reactive protein (CRP), adipocytokine, BMI and body fat in elderly [7,8]. However, in gestational diabetes patients (GDM), osteocalcin is reportedly greater than normal glucose tolerant pregnant patients and correlated with insulin secretion parameters [9]. Thus, osteocalcin may produce more insulin secretion to overcome insulin resistance as a consequence of pregnancy and weight increase as has been reported in cellular-based study [3–5]. Diabetic patients, who had been taking insulin or oral antidiabetic dugs, exhibited negative correlation of serum osteocalcin with glucose metabolism and fat mass, and positive with adiponectin levels particularly in women [10].
Uncarboxylated osteocalcin is known to be an active form and stimulates the insulin secretion and proliferation of b-cells in studies with animal [3] and human [11]. In diabetic subjects regardless of whether they were treated by glucose-lowering agents, osteocalcin level is associated with improved glucose tolerance [11,12]. Osteocalcin may further mediate both insulin sensitivity and fasting triglycerides, and changes in visceral fat and leg muscle strength are independent variable for serum osteocalcin [13]. These clinical studies suggest that serum osteocalcin is inversely correlated with type 2 diabetes mellitus (T2DM) except for in GDM. Thus, a plausible idea is that in contrast to overt diabetes serum osteocalcin in earlystage diabetes may adaptively be increased to insulin resistance. We evaluated circulating osteocalcin in these subjects with newly found early-stage T2DM or prediabetes including IFG or IGT in annual health check.
2.
Materials and methods
2.1.
Subjects
We recruited 45 men (mean age of 46.7 7.6 years) and 10 women (55.6 4.6 years) who visited Social Insurance General
Table 1 – Clinical characteristic of subjects.
Age (years) Sex (male) Height (cm) Weight (kg) BMI (kg/m2) Waist (cm) Hip (cm) W/H ratio SBP (mmHg) DBP (mmHg) HbA1c (%) FPG (mg/dl) G(120) (mg/dl) FINS (mU/ml) Ins(120) (mU/ml) HOMA-R HOMA-b TC (mg/dl) TG (mg/dl) HDL-C (mg/dl) LDL-C (mg/dl) CRP (mg/dl) Cre (mg/dl) UN (mg/dl) Ghrelin (fmol/ml) LPL (pg/ml) TNFa (pg/ml) Adiponectin (ng/ml) Leptin (pg/ml) Osteocalcin (ng/ml)
NGT (39)
PDM (11)
T2DM (5)
P
47.9 8.2 33 167.7 7.6 65.9 10.6 23.3 2.6 80.8 7.0 93.3 5.2 0.87 0.04 115.1 14.2 70.7 11.4 5.5 0.2 96.3 6.3 111 20.0 8.3 (3.8–13.0) 46.1 29.7 2.10 (0.9–3.4) 80.3 (42.0–127.4) 194 (176–211) 96.0 (75.8–123.0) 53.0 (47.0–58.0) 123 (107–136) 0.08 0.14 0.80 0.14 14.0 3.2 14.0 9.3 1.7 0.7 0.56 (0.48–0.75) 8.5 (5.0–15.9) 26.0 (13.2–35.0) 4.1 1.3
48.6 8.9 9 169.9 8.3 75.0 18.1 26.0 4.8* 88.5 11.9* 97.8 8.8 0.90 0.04* 124.3 16.1 77.2 11.0 5.7 0.3 105.9 11.6 151.6 23.6** 11.3 (9.6–20.6) 93.6 56.9 3.2 (2.5–4.5) 101.9 (75.0–147.0) 209 (199–244) 111 (83.0–179.0) 55.0 (49.0–56.0) 136 (111.4–136) 0.08 0.09 0.75 0.13 12.1 3.2 9.0 4.6 1.9 0.5 0.63 (0.52–1.10) 7.6 (6.4–17.4) 46.2 (24.0–65.5) 4.4 2.1
50.6 3.4 3 162.9 7.6 69.8 11.0 26.2 2.7 86.4 7.8 96.9 4.1 0.89 0.05 128.8 26.0 79.6 10.9 6.2 0.4**,## 111.4 11.0 232.0 22.4**,## 28.9 (4.2–44.0) 163.1 129.1 7.2 (1.2–12.5) 192.8 (32.2–348.3) 205 (194–233) 129 (74.0–166.5) 61.0 (43.5–73.5) 134 (112–155) 0.14 0.18 0.67 0.19 11.5 1.7 14.8 11.1 1.4 0.3 0.79 (0.56–1.22) 4.4 (2.3–17.1) 53.9 (36.7–106.5) 6.2 1.9*
NS NS NS NS 0.018 0.019 NS 0.036 NS NS < 0.001 0.016 < 0.001 NS 0.039 NS NS NS NS NS NS NS NS NS NS NS NS NS NS 0.02
PDM included either IFG or IGT, or both. FINS: fasting immunoreactive insulin, I(120): IRI at 120 min during 75 g-OGTT. SBP: systolic blood pressure, DBP: diastolic blood pressure. * P < 0.05 vs. NGT (normal glucose tolerance). ** P < 0.01 vs. NGT. ## P < 0.01 vs. PDM (prediabetes).
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diabetes research and clinical practice 92 (2011) 181–186
Omiya Hospital between October, 2008 and December, 2009 to annually have metabolic health checked. Ordinal parameters including anthropometric measures, plasma glucose and serum insulin upon 75 g oral glucose tolerance test (75 gOGTT), serum lipids, liver enzymes and cardiological measures such as chest X-ray and electrocardiogram were checked. The baseline characteristics are shown in Table 1. Exclusion criteria of the subjects were following; taking any of antihypertensive or hypoglycemic agent, diuretics, lipid lowering agent or insulin, being pregnant, and having past history of malignancy, liver or kidney disease. The participants arrived at the hospital at 7:20 am following 10 h overnight fasting. Anthropometric measures of height, weight, waist and hip circumferences were performed. Waist was measured at midlevel between lowest rib and upper edge of superior anterior iliac crest or an umbilicus level. Body mass index (BMI) was calculated as weight (kg) divided by squared height in meters. Blood pressures were measured at sitting position after 15 min rest by sphygmomanometer in mmHg. Insulin sensitivity was calculated by homeostasis model assessment of insulin resistant (HOMA-R) with following equation: HOMA-R ¼
fasting plasma glucose ðFPG; mg=dlÞ fasting insulin ðFINS;mU=mlÞ
405
(140 G(120) < 200). NGT (normal glucose tolerance) indicates none of the above criteria. This work has been approved by the Ethical Committee in Social Insurance General Omiya Hospital where the participants were collected. This study complied with Helsinki Declaration. All subjects were informed about the study and provided written consent before participating the study.
2.2.
:
Data were expressed as mean S.D. unless they were skewed variables that were presented as median (interquartile range 25–75%). Clinical characteristics that followed a normal distribution were compared among the three groups using one-way ANOVA test after F-test or Welch-test. The Pearson correlation coefficients were calculated to assess the strength of the correlations of random pairs of parameters of energy, bone and adipocytokine factors, adiposity, glucose, and lipid metabolism listed above. When parameter revealed skewed distribution, the variable was transformed to logarithmic value (Ln) before performing regression or correlation analyses. Multiple stepwise regression analysis was performed to determine association between G(120) and serum osteocalcin or other metabolic parameters after adjusting for potential confounders. Multivariate logistic regression analysis was conducted using the existence of combination of impaired fasting glucose (IFG), IGT and T2DM as a dependent variable. All reported P values were two tailed, and P values less than
Capability of insulin secretion at basal state was assessed by HOMA-b that was calculated by following equation: HOMA-b ¼
ð360 FINSÞ : ðFPG-63Þ
Blood was sampled at fasting between 8:00 and 9:00 am and then all the participants received 75 g-OGTT with following blood sampling at 60 and 120 min for measurements of plasma glucose and insulin concentrations. Serum concentrations of total cholesterol (TC), triglyceride (TG), HDL-cholesterol, low density lipoprotein cholesterol (LDL-C), CRP, creatinine (Cre), uric acid (UA) and urine nitrogen (UN) were measured by standard biochemical methods. We analyzed following metabolic parameters at fasting using commercially available ELISA Kits: insulin (Yanaihara Institute Inc., Shizuoka, Japan), osteocalcin (Biomedical Technologies Inc., Massachusetts, USA), ghrelin (Mitsubishi Kagaku Iatron, Inc., Tokyo, Japan), lipoprotein lipase (LPL, Sekisui Medical Co. Ltd., Tokyo, Japan), tumor necrosis factor a (TNFa, Invitrogen Corporation, California, USA), adiponectin (Fujirebio Inc., Tokyo, Japan) and leptin (R&D Systems, Minneapolis, USA). The value for HbA1c (%) is estimated as NGSP (National Glycohemoglobin Standardization Program) equivalent value (%) calculated by the formula HbA1c (%) = HbA1c (JDS) (%) + 0.4%, considering the relational expression of HbA1c (JDS) (%) measured by the previous Japanese standard substance and measurement methods and HbA1c (NGSP), according to ‘‘The Committee of Japan Diabetes Society’’ on the diagnostic criteria of diabetes mellitus from Report of the Committee on the Classification and Diagnostic Criteria of Diabetes Mellitus [14]. T2DM was diagnosed when one of FPG 126 mg/dl, G(120) that is the plasma glucose level at 120 min during 75 g-OGTT 200 mg/dl and HbA1c 6.5% was fulfilled. PDM includes IFG (110 FPG < 126) or IGT
Statistical analysis
[()TD$FIG] (ng/ml) 10.0
Osteocalcin A
*
7.5 5.0 2.5 0.0
NGT (pg/ml) 150
IGT/IFG
DM
Leptin B
100
50
0
NGT
IGT/IFG
DM
Fig. 1 – comparisons of serum osteocalcin (A) and serum leptin (B) concentrations in NGT, PDM (IFG and IGT) and T2DM. *P < 0.05 vs. NGT by ANOVA.
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diabetes research and clinical practice 92 (2011) 181–186
[()TD$FIG]
FPG (mg/dl) 150
R=0.25, P=0.063
A
1.5
125
1.0
100
0.5
75 0.0
2.5
5.0
7.5
10.0
Creatinine (mg/dl) C
0.0 0.0
Osteocalcin (ng/ml) G120 (mg/dl)
300
R=0.30, P=0.028
B
R=-0.39, P=0.004
2.5 5.0 7.5 Osteocalcin (ng/ml)
Ln CRP 0 D
10.0
R=-0.30, P=0.024
-1 200
-2 100 0 0.0
-3 2.5 5.0 7.5 Osteocalcin (ng/ml)
10.0
-4 0.0
2.5 5.0 7.5 Osteocalcin (ng/ml)
10.0
Fig. 2 – Simple regression analyses of FPG (A), G(120) (B), creatinine (C) and Ln(CRP) (D) with serum osteocalcin. Pearson correlation coefficients were calculated.
0.05 were considered statistically significant. SPSS1 Statistics 17.0 was used to analyze data.
3.
Results
The clinical characteristics of study participants are shown in Table 1. BMI and waist/hip (W/H) ratio were significantly greater in PDM that was defined when either IFG or IGT or both were present (P = 0.02). HbA1c was greater in T2DM as compared to NGT and PDM. G(120) in PDM and T2DM was greater than those of NGT. FPG and serum insulin at 120 min on 75 g-OGTT, Ins(120) were significantly different among these groups with a tendency of increment in accordance with glucose-metabolism impairment. Serum leptin level had a trend of increase in PDM and T2DM as compared to NGT (P > 0.05) whereas osteocalcin concentration was greater in T2DM than NGT (P = 0.02, Table 1 and Fig. 1). In all the participants, simple regression analysis model revealed positive correlation of osteocalcin with G(120) (r = 0.30, P = 0.028), creatinine (r = 0.39, P = 0.004) and Ln(CRP) (r = 0.30, P = 0.024), but not with FPG (r = 0.25, P = 0.063) in Fig. 2. To further evaluate which variables were independently correlated with serum osteocalcin, multiple stepwise regression analysis was performed and G(120) (b = 0.27, P = 0.038) and Ln(CRP) (b = 0.35, P = 0.008) were associated with osteocalcin. Likewise, we assessed which variables including osteocalcin and other confounding factors are associated with
G(120) that a determinant of IGT and T2DM by using multiple stepwise regression analysis. In Table 2, osteocalcin and leptin were independent parameters for G(120). Furthermore, to determine independent variables for T2DM and PDM, multiple logistic model analysis was performed in Table 3. Age, sex, BMI, CRP, TC, TG, HDL-C, TNFa, adiponectin, systolic blood pressure (SBP) and leptin were included in the model as independent factors. Osteocalcin and leptin were final independent variables being associated with the combined group of PDM and T2DM. Multinomial logistic analysis in the model described above revealed that leptin was associated (PDM vs. NGT: P = 0.02 Odds ratio (OR) of 1.05; 95% confidence intervals, 1.007–1.084) and as to NGT vs. T2DM osteocalcin (P = 0.038, OR 10.8; 1.13–102.4), leptin (P = 0.032, OR 1.1; 1.008– 1.205) and systolic blood pressure (P = 0.046, OR 1.10; 1.002– 1.210) were independently associated with diabetes.
Table 2 – Multiple stepwise model analysis to assess relationship between G(120) and independent variables.
Osteocalcin Leptin
B
sem
Standardized b
P
7.0 0.38
3.1 0.18
0.27 0.30
0.026 0.035
Age, sex, BMI, WHR, SBP, TC, TG, HDL-C, FPG, adiponectin, ghrelin, TNFa, LPL and leptin were included as independent variable but finally excluded in the model. Standard error of mean was revealed with sem.
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diabetes research and clinical practice 92 (2011) 181–186
Table 3 – Multiple logistic model analysis showing independent variables to assess patients with NGT vs., IFG, IGT or T2DM. Independent variables PDM, T2DM vs. NGT
Osteocalcin Leptin
B
sem
P
Exp
0.57 0.058
0.28 0.021
0.041 0.006
1.7 1.1
95% CI 1.02 1.02
3.05 1.10
Comparison between NGT vs. combination of PDM and T2DM was performed. Age, sex, BMI, CRP, TC, TG, HDL-C, TNFa, adiponectin, osteocalcin, leptin and SBP were included in the initial model as independent variables, but all of these except for factors listed above were excluded at the final step.
4.
Discussion
In the present paper, we found that serum osteocalcin concentration is increased in early-stage T2DM subjects, who had never been taking medication. Multinomial logistic analysis revealed positive correlation of osteocalcin with T2DM and positive association of leptin in subjects with both PDM (IFG and IGT) and T2DM as compared to NGT. Osteocalcin was not correlated with leptin. These suggest that osteocalcin is independently related to impairment of glucose tolerance in T2DM at early stage. The several lines of study that examined osteocalcin level in T2DM patients reported that osteocalcin is decreased in these patients regardless of whether treatment with antidiabetic agents is taken [6,10–12]. These human and animal studies [3,4] suggest inversed relation between plasma glucose concentration and osteocalcin in overt diabetes. Presently we could not explain the discrepancy between our findings and these previous reports. In GDM subjects serum osteocalcin is reportedly increased [9]. Taken together, osteocalcin may be related to increase in insulin secretion in early-stage diabetic subjects who have insulin resistance as revealed with increases in Ins(120), W/H ratio and BMI as compared to NGT (Table 1). G(120) was associated with osteocalcin and leptin in multiple stepwise analysis (Table 2). Increase in G(120) rather than FPG may reflect to insulin resistance. In cell-based experiments, picomolar amounts of osteocalcin increase the expression of the insulin genes and b-cell proliferation markers, whereas nanomolar amounts enhance production of adiponectin and expression of Pgc1-a, molecular markers of insulin sensitivity and energy expenditure, in white and brown adipocytes, respectively [15]. In the present study Ins(120) was increase in PDM and T2DM, and conversely adiponectin exhibited a trend of inverse relation with glucosemetabolism impairment. These findings further suggest that in our glucose-metabolism impaired subjects changes in circulating osteocalcin level may be related to G(120) increases in insulin resistant state and to requirement of enhanced bcell function to overcome insulin resistance. In postmenopausal women, osteocalcin was independent reciprocal correlation factor for glucose and HbA1c [16]. Our study included women aging 50 years or more. Osteocalcin level in women was 5.4 2.0 ng/ml (n = 10) compared to that of men (4.1 1.4 ng/ml, n = 45, P = 0.017, unpaired test). Although number of postmenopausal women was hesitant in our data, to a certain extent inclusion of postmenopausal subjects may partly be a cause of osteocalcin difference between both sexes in the present study because postmenopausal women have higher osteocalcin level [6,17]. There was no correlation
between age and osteocalcin levels when only women data were selected for simple regression analysis in the present data. Likewise multivariate analyses in Tables 2 and 3 by adjusting sex and age that are basically included in these two models showed osteocalcin was independent factor for G(120) and for differentiation of combination of PDM and T2DM from NGT. Osteocalcin level was not different between men and women in T2DM subjects. Uncarboxylated osteocalcin (ucOCN) is an active and predominant form of osteocalcin and mediates not only glucose metabolism by enhancing b-cell function but also adipocytokine release from fat cells [1,11]. unOCN is inversely correlated with plasma glucose and fat mass [18,19]. Osteocalcin is expressed in human adipocyte tissue [20] and adiponectin and its receptor also on osteoblast [21,22], suggesting a link between adipose tissue and osteocalcin in metabolic regulation. In the present study, osteocalcin did not correlate with serum adiponectin. We have a few limitations in our study. Firstly, number of subjects collected particularly in diabetic subjects was small. Diabetic and PDM subjects may be in similar pathophysiological state as to glucose-metabolic abnormality in the present subjects. Accordingly, when the subjects of T2DM and PDM were compared with NGT, both osteocalcin and leptin also were predictive markers for glucose intolerance after adjusting confounding factors. However, it may be too early to presently conclude that osteocalcin is a biomarker to reflect glucose intolerance. Secondary, we could not determine how osteocalcin influences glucose intolerance state or whether increases of leptin and osteocalcin are simply consequence of high glucose state. It is unknown why leptin increased in PDM and T2DM but osteocalcin only in T2DM as compared to NTG in multinomial logistic analysis. Osteocalcin showed a trend of increase in circulating level in PDM (Table 1). Thirdly, longitudinal observation of increased osteocalcin is required in subjects intervened by reducing body weight with improvement of dietary habits. We demonstrated that circulating osteocalcin showed positive correlation with glycemia in early-stage T2DM. It is required to await further study to determine when osteocalcin is turned to declining course along with progression of glucose intolerance toward overt diabetes.
Acknowledgments We are grateful to all subjects for participating to this study and cooperation of co-medical staff in Omiya general Hospital. This work was supported by Grants-in-Aid for Scientific
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Research from the Japan Society for the Promotion of Science (JPSP: to M.K.).
[12]
Conflict of interest [13]
The authors declare that they have no conflict of interest.
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Contents lists available at ScienceDirect
Diabetes Research and Clinical Practice journ al h omepage: www .elsevier.co m/lo cate/diabres
Circulating osteocalcin is increased in early-stage diabetes Atsushi Aoki a, Takatoshi Muneyuki b,c, Masashi Yoshida a, Hiromi Munakata b, San-e Ishikawa a, Hitoshi Sugawara a, Masanobu Kawakami a, Masafumi Kakei a,* a
First Department of Comprehensive Medicine, Saitama Medical Center, Jichi Medical University School of Medicine, 1-847 Amanuma, Omiya, Saitama 330-8503, Japan b Internal Medicine, Social Insurance General Omiya Hospital, 453 Bonsai, Kita, Saitama 331-0805, Japan c Saitama Citizen Medical Center, 299-1 Shimane, Nishi, Saitama 331-0054, Japan
article info
abstract
Article history:
We aimed to examine whether circulating levels of osteocalcin, bone formation marker
Received 8 November 2010
secreted from osteoblast, are changed in glucose-intolerant subjects without taking glucose
Received in revised form
lowering agent, because bone metabolism is reportedly related to glucose metabolism in
4 January 2011
animal and human studies. According to 75 g oral glucose tolerance test (75 g-OGTT), all
Accepted 10 January 2011
subjects (47.6 10.2 years of age; 45 men and 10 women) were divided into three categories:
Published on line 2 February 2011
normal glucose tolerance (NGT, n = 39), prediabetes (PDM, n = 11) that included impaired
Keywords:
osteocalcin levels were increased in T2DM as compared to NGT. In all the participants,
Osteocalcin
simple regression analysis model revealed positive correlation of osteocalcin with plasma
Leptin
glucose at 120 min, G(120), on 75 g-OGTT, negative with both creatinine and Ln(CRP), but not
Obese
significantly with fasting plasma glucose. Osteocalcin and leptin were independent vari-
Type 2 diabetes mellitus
ables for G(120) (P = 0.026 and 0.035, respectively). In multinomial logistic analysis leptin
fasting glucose (IFG) and impaired glucose tolerance (IGT), and diabetes (T2DM, n = 5). Serum
(PDM vs. NGT: P = 0.02 Odds ratio (OR) of 1.05, 95% confidence intervals, 1.007–1.084) and osteocalcin (T2DM vs. NGT: P = 0.038, OR 10.8, 1.13–102.4) were independently associated. We conclude that circulating osteocalcin and leptin are related to glucose intolerant state. # 2011 Elsevier Ireland Ltd. All rights reserved.
1.
Introduction
Bone remodeling is essential to maintain skeleton structure during adult life. Bone resorption and formation, which are mechanistically repairing processes against both micro crack and macro damage, play a pivotal role as bone remodeling. Resorption mediated by osteoclasts differentiated from progenitor cell of bone marrow initiates osteoblast-mediated bone formation after bone destruction at damaged bone and subsequent repair [1,2]. This biphasic process in bone remodeling by these two types of bone cells is normally balanced in healthy individuals. Aging, postmenopausal state and lean body produce imbalance of bone remodeling cycle
and consequent osteoporosis with future occurrence of fracture. Bone is now identified as an organ regulating energy metabolism via osteocalcin that is secreted by osteoblast and enhances insulin secretion in pancreatic islets and insulin sensitivity. Osteocalcin stimulates insulin expression in bcells, b-cell proliferation and insulin secretion, adiponectin secretion from adipocyte and consequently improvement of insulin sensitivity [3,4]. Osteoblast is regulated via sympathetic nerve that relays energy balance information from adipocyte through hypothalamic leptinergic transmission [1,2,5]. The sympathetic nerve linking to osteoblast uses adrenergic b2 receptor stimulation by two pathways, protein
* Corresponding author. Tel.: +81 486472111; fax: +81 486485188. E-mail address: [email protected] (M. Kakei). 0168-8227/$ – see front matter # 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.diabres.2011.01.009
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kinase A and molecular clock. The former mediates bone resorption by osteoclast stimulation via secretion of RANKL (receptor activation of NF-kB ligand that is released by activated osteoblast) and the latter osteoblast proliferation (bone formation). In human studies, it was reported that serum osteocalcin concentration is reciprocally correlated with glucose metabolism: osteocalcin is decreased in patients with type 2 diabetes who had never taken hypoglycemic medication as compared to those with normal glucose tolerance [6]. In cross-sectional study, osteocalcin concentration was inversely associated with fasting plasma glucose (FPG), fasting insulin, homeostasis model assessment for insulin resistance (HOMA-R), C-reactive protein (CRP), adipocytokine, BMI and body fat in elderly [7,8]. However, in gestational diabetes patients (GDM), osteocalcin is reportedly greater than normal glucose tolerant pregnant patients and correlated with insulin secretion parameters [9]. Thus, osteocalcin may produce more insulin secretion to overcome insulin resistance as a consequence of pregnancy and weight increase as has been reported in cellular-based study [3–5]. Diabetic patients, who had been taking insulin or oral antidiabetic dugs, exhibited negative correlation of serum osteocalcin with glucose metabolism and fat mass, and positive with adiponectin levels particularly in women [10].
Uncarboxylated osteocalcin is known to be an active form and stimulates the insulin secretion and proliferation of b-cells in studies with animal [3] and human [11]. In diabetic subjects regardless of whether they were treated by glucose-lowering agents, osteocalcin level is associated with improved glucose tolerance [11,12]. Osteocalcin may further mediate both insulin sensitivity and fasting triglycerides, and changes in visceral fat and leg muscle strength are independent variable for serum osteocalcin [13]. These clinical studies suggest that serum osteocalcin is inversely correlated with type 2 diabetes mellitus (T2DM) except for in GDM. Thus, a plausible idea is that in contrast to overt diabetes serum osteocalcin in earlystage diabetes may adaptively be increased to insulin resistance. We evaluated circulating osteocalcin in these subjects with newly found early-stage T2DM or prediabetes including IFG or IGT in annual health check.
2.
Materials and methods
2.1.
Subjects
We recruited 45 men (mean age of 46.7 7.6 years) and 10 women (55.6 4.6 years) who visited Social Insurance General
Table 1 – Clinical characteristic of subjects.
Age (years) Sex (male) Height (cm) Weight (kg) BMI (kg/m2) Waist (cm) Hip (cm) W/H ratio SBP (mmHg) DBP (mmHg) HbA1c (%) FPG (mg/dl) G(120) (mg/dl) FINS (mU/ml) Ins(120) (mU/ml) HOMA-R HOMA-b TC (mg/dl) TG (mg/dl) HDL-C (mg/dl) LDL-C (mg/dl) CRP (mg/dl) Cre (mg/dl) UN (mg/dl) Ghrelin (fmol/ml) LPL (pg/ml) TNFa (pg/ml) Adiponectin (ng/ml) Leptin (pg/ml) Osteocalcin (ng/ml)
NGT (39)
PDM (11)
T2DM (5)
P
47.9 8.2 33 167.7 7.6 65.9 10.6 23.3 2.6 80.8 7.0 93.3 5.2 0.87 0.04 115.1 14.2 70.7 11.4 5.5 0.2 96.3 6.3 111 20.0 8.3 (3.8–13.0) 46.1 29.7 2.10 (0.9–3.4) 80.3 (42.0–127.4) 194 (176–211) 96.0 (75.8–123.0) 53.0 (47.0–58.0) 123 (107–136) 0.08 0.14 0.80 0.14 14.0 3.2 14.0 9.3 1.7 0.7 0.56 (0.48–0.75) 8.5 (5.0–15.9) 26.0 (13.2–35.0) 4.1 1.3
48.6 8.9 9 169.9 8.3 75.0 18.1 26.0 4.8* 88.5 11.9* 97.8 8.8 0.90 0.04* 124.3 16.1 77.2 11.0 5.7 0.3 105.9 11.6 151.6 23.6** 11.3 (9.6–20.6) 93.6 56.9 3.2 (2.5–4.5) 101.9 (75.0–147.0) 209 (199–244) 111 (83.0–179.0) 55.0 (49.0–56.0) 136 (111.4–136) 0.08 0.09 0.75 0.13 12.1 3.2 9.0 4.6 1.9 0.5 0.63 (0.52–1.10) 7.6 (6.4–17.4) 46.2 (24.0–65.5) 4.4 2.1
50.6 3.4 3 162.9 7.6 69.8 11.0 26.2 2.7 86.4 7.8 96.9 4.1 0.89 0.05 128.8 26.0 79.6 10.9 6.2 0.4**,## 111.4 11.0 232.0 22.4**,## 28.9 (4.2–44.0) 163.1 129.1 7.2 (1.2–12.5) 192.8 (32.2–348.3) 205 (194–233) 129 (74.0–166.5) 61.0 (43.5–73.5) 134 (112–155) 0.14 0.18 0.67 0.19 11.5 1.7 14.8 11.1 1.4 0.3 0.79 (0.56–1.22) 4.4 (2.3–17.1) 53.9 (36.7–106.5) 6.2 1.9*
NS NS NS NS 0.018 0.019 NS 0.036 NS NS < 0.001 0.016 < 0.001 NS 0.039 NS NS NS NS NS NS NS NS NS NS NS NS NS NS 0.02
PDM included either IFG or IGT, or both. FINS: fasting immunoreactive insulin, I(120): IRI at 120 min during 75 g-OGTT. SBP: systolic blood pressure, DBP: diastolic blood pressure. * P < 0.05 vs. NGT (normal glucose tolerance). ** P < 0.01 vs. NGT. ## P < 0.01 vs. PDM (prediabetes).
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Omiya Hospital between October, 2008 and December, 2009 to annually have metabolic health checked. Ordinal parameters including anthropometric measures, plasma glucose and serum insulin upon 75 g oral glucose tolerance test (75 gOGTT), serum lipids, liver enzymes and cardiological measures such as chest X-ray and electrocardiogram were checked. The baseline characteristics are shown in Table 1. Exclusion criteria of the subjects were following; taking any of antihypertensive or hypoglycemic agent, diuretics, lipid lowering agent or insulin, being pregnant, and having past history of malignancy, liver or kidney disease. The participants arrived at the hospital at 7:20 am following 10 h overnight fasting. Anthropometric measures of height, weight, waist and hip circumferences were performed. Waist was measured at midlevel between lowest rib and upper edge of superior anterior iliac crest or an umbilicus level. Body mass index (BMI) was calculated as weight (kg) divided by squared height in meters. Blood pressures were measured at sitting position after 15 min rest by sphygmomanometer in mmHg. Insulin sensitivity was calculated by homeostasis model assessment of insulin resistant (HOMA-R) with following equation: HOMA-R ¼
fasting plasma glucose ðFPG; mg=dlÞ fasting insulin ðFINS;mU=mlÞ
405
(140 G(120) < 200). NGT (normal glucose tolerance) indicates none of the above criteria. This work has been approved by the Ethical Committee in Social Insurance General Omiya Hospital where the participants were collected. This study complied with Helsinki Declaration. All subjects were informed about the study and provided written consent before participating the study.
2.2.
:
Data were expressed as mean S.D. unless they were skewed variables that were presented as median (interquartile range 25–75%). Clinical characteristics that followed a normal distribution were compared among the three groups using one-way ANOVA test after F-test or Welch-test. The Pearson correlation coefficients were calculated to assess the strength of the correlations of random pairs of parameters of energy, bone and adipocytokine factors, adiposity, glucose, and lipid metabolism listed above. When parameter revealed skewed distribution, the variable was transformed to logarithmic value (Ln) before performing regression or correlation analyses. Multiple stepwise regression analysis was performed to determine association between G(120) and serum osteocalcin or other metabolic parameters after adjusting for potential confounders. Multivariate logistic regression analysis was conducted using the existence of combination of impaired fasting glucose (IFG), IGT and T2DM as a dependent variable. All reported P values were two tailed, and P values less than
Capability of insulin secretion at basal state was assessed by HOMA-b that was calculated by following equation: HOMA-b ¼
ð360 FINSÞ : ðFPG-63Þ
Blood was sampled at fasting between 8:00 and 9:00 am and then all the participants received 75 g-OGTT with following blood sampling at 60 and 120 min for measurements of plasma glucose and insulin concentrations. Serum concentrations of total cholesterol (TC), triglyceride (TG), HDL-cholesterol, low density lipoprotein cholesterol (LDL-C), CRP, creatinine (Cre), uric acid (UA) and urine nitrogen (UN) were measured by standard biochemical methods. We analyzed following metabolic parameters at fasting using commercially available ELISA Kits: insulin (Yanaihara Institute Inc., Shizuoka, Japan), osteocalcin (Biomedical Technologies Inc., Massachusetts, USA), ghrelin (Mitsubishi Kagaku Iatron, Inc., Tokyo, Japan), lipoprotein lipase (LPL, Sekisui Medical Co. Ltd., Tokyo, Japan), tumor necrosis factor a (TNFa, Invitrogen Corporation, California, USA), adiponectin (Fujirebio Inc., Tokyo, Japan) and leptin (R&D Systems, Minneapolis, USA). The value for HbA1c (%) is estimated as NGSP (National Glycohemoglobin Standardization Program) equivalent value (%) calculated by the formula HbA1c (%) = HbA1c (JDS) (%) + 0.4%, considering the relational expression of HbA1c (JDS) (%) measured by the previous Japanese standard substance and measurement methods and HbA1c (NGSP), according to ‘‘The Committee of Japan Diabetes Society’’ on the diagnostic criteria of diabetes mellitus from Report of the Committee on the Classification and Diagnostic Criteria of Diabetes Mellitus [14]. T2DM was diagnosed when one of FPG 126 mg/dl, G(120) that is the plasma glucose level at 120 min during 75 g-OGTT 200 mg/dl and HbA1c 6.5% was fulfilled. PDM includes IFG (110 FPG < 126) or IGT
Statistical analysis
[()TD$FIG] (ng/ml) 10.0
Osteocalcin A
*
7.5 5.0 2.5 0.0
NGT (pg/ml) 150
IGT/IFG
DM
Leptin B
100
50
0
NGT
IGT/IFG
DM
Fig. 1 – comparisons of serum osteocalcin (A) and serum leptin (B) concentrations in NGT, PDM (IFG and IGT) and T2DM. *P < 0.05 vs. NGT by ANOVA.
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[()TD$FIG]
FPG (mg/dl) 150
R=0.25, P=0.063
A
1.5
125
1.0
100
0.5
75 0.0
2.5
5.0
7.5
10.0
Creatinine (mg/dl) C
0.0 0.0
Osteocalcin (ng/ml) G120 (mg/dl)
300
R=0.30, P=0.028
B
R=-0.39, P=0.004
2.5 5.0 7.5 Osteocalcin (ng/ml)
Ln CRP 0 D
10.0
R=-0.30, P=0.024
-1 200
-2 100 0 0.0
-3 2.5 5.0 7.5 Osteocalcin (ng/ml)
10.0
-4 0.0
2.5 5.0 7.5 Osteocalcin (ng/ml)
10.0
Fig. 2 – Simple regression analyses of FPG (A), G(120) (B), creatinine (C) and Ln(CRP) (D) with serum osteocalcin. Pearson correlation coefficients were calculated.
0.05 were considered statistically significant. SPSS1 Statistics 17.0 was used to analyze data.
3.
Results
The clinical characteristics of study participants are shown in Table 1. BMI and waist/hip (W/H) ratio were significantly greater in PDM that was defined when either IFG or IGT or both were present (P = 0.02). HbA1c was greater in T2DM as compared to NGT and PDM. G(120) in PDM and T2DM was greater than those of NGT. FPG and serum insulin at 120 min on 75 g-OGTT, Ins(120) were significantly different among these groups with a tendency of increment in accordance with glucose-metabolism impairment. Serum leptin level had a trend of increase in PDM and T2DM as compared to NGT (P > 0.05) whereas osteocalcin concentration was greater in T2DM than NGT (P = 0.02, Table 1 and Fig. 1). In all the participants, simple regression analysis model revealed positive correlation of osteocalcin with G(120) (r = 0.30, P = 0.028), creatinine (r = 0.39, P = 0.004) and Ln(CRP) (r = 0.30, P = 0.024), but not with FPG (r = 0.25, P = 0.063) in Fig. 2. To further evaluate which variables were independently correlated with serum osteocalcin, multiple stepwise regression analysis was performed and G(120) (b = 0.27, P = 0.038) and Ln(CRP) (b = 0.35, P = 0.008) were associated with osteocalcin. Likewise, we assessed which variables including osteocalcin and other confounding factors are associated with
G(120) that a determinant of IGT and T2DM by using multiple stepwise regression analysis. In Table 2, osteocalcin and leptin were independent parameters for G(120). Furthermore, to determine independent variables for T2DM and PDM, multiple logistic model analysis was performed in Table 3. Age, sex, BMI, CRP, TC, TG, HDL-C, TNFa, adiponectin, systolic blood pressure (SBP) and leptin were included in the model as independent factors. Osteocalcin and leptin were final independent variables being associated with the combined group of PDM and T2DM. Multinomial logistic analysis in the model described above revealed that leptin was associated (PDM vs. NGT: P = 0.02 Odds ratio (OR) of 1.05; 95% confidence intervals, 1.007–1.084) and as to NGT vs. T2DM osteocalcin (P = 0.038, OR 10.8; 1.13–102.4), leptin (P = 0.032, OR 1.1; 1.008– 1.205) and systolic blood pressure (P = 0.046, OR 1.10; 1.002– 1.210) were independently associated with diabetes.
Table 2 – Multiple stepwise model analysis to assess relationship between G(120) and independent variables.
Osteocalcin Leptin
B
sem
Standardized b
P
7.0 0.38
3.1 0.18
0.27 0.30
0.026 0.035
Age, sex, BMI, WHR, SBP, TC, TG, HDL-C, FPG, adiponectin, ghrelin, TNFa, LPL and leptin were included as independent variable but finally excluded in the model. Standard error of mean was revealed with sem.
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Table 3 – Multiple logistic model analysis showing independent variables to assess patients with NGT vs., IFG, IGT or T2DM. Independent variables PDM, T2DM vs. NGT
Osteocalcin Leptin
B
sem
P
Exp
0.57 0.058
0.28 0.021
0.041 0.006
1.7 1.1
95% CI 1.02 1.02
3.05 1.10
Comparison between NGT vs. combination of PDM and T2DM was performed. Age, sex, BMI, CRP, TC, TG, HDL-C, TNFa, adiponectin, osteocalcin, leptin and SBP were included in the initial model as independent variables, but all of these except for factors listed above were excluded at the final step.
4.
Discussion
In the present paper, we found that serum osteocalcin concentration is increased in early-stage T2DM subjects, who had never been taking medication. Multinomial logistic analysis revealed positive correlation of osteocalcin with T2DM and positive association of leptin in subjects with both PDM (IFG and IGT) and T2DM as compared to NGT. Osteocalcin was not correlated with leptin. These suggest that osteocalcin is independently related to impairment of glucose tolerance in T2DM at early stage. The several lines of study that examined osteocalcin level in T2DM patients reported that osteocalcin is decreased in these patients regardless of whether treatment with antidiabetic agents is taken [6,10–12]. These human and animal studies [3,4] suggest inversed relation between plasma glucose concentration and osteocalcin in overt diabetes. Presently we could not explain the discrepancy between our findings and these previous reports. In GDM subjects serum osteocalcin is reportedly increased [9]. Taken together, osteocalcin may be related to increase in insulin secretion in early-stage diabetic subjects who have insulin resistance as revealed with increases in Ins(120), W/H ratio and BMI as compared to NGT (Table 1). G(120) was associated with osteocalcin and leptin in multiple stepwise analysis (Table 2). Increase in G(120) rather than FPG may reflect to insulin resistance. In cell-based experiments, picomolar amounts of osteocalcin increase the expression of the insulin genes and b-cell proliferation markers, whereas nanomolar amounts enhance production of adiponectin and expression of Pgc1-a, molecular markers of insulin sensitivity and energy expenditure, in white and brown adipocytes, respectively [15]. In the present study Ins(120) was increase in PDM and T2DM, and conversely adiponectin exhibited a trend of inverse relation with glucosemetabolism impairment. These findings further suggest that in our glucose-metabolism impaired subjects changes in circulating osteocalcin level may be related to G(120) increases in insulin resistant state and to requirement of enhanced bcell function to overcome insulin resistance. In postmenopausal women, osteocalcin was independent reciprocal correlation factor for glucose and HbA1c [16]. Our study included women aging 50 years or more. Osteocalcin level in women was 5.4 2.0 ng/ml (n = 10) compared to that of men (4.1 1.4 ng/ml, n = 45, P = 0.017, unpaired test). Although number of postmenopausal women was hesitant in our data, to a certain extent inclusion of postmenopausal subjects may partly be a cause of osteocalcin difference between both sexes in the present study because postmenopausal women have higher osteocalcin level [6,17]. There was no correlation
between age and osteocalcin levels when only women data were selected for simple regression analysis in the present data. Likewise multivariate analyses in Tables 2 and 3 by adjusting sex and age that are basically included in these two models showed osteocalcin was independent factor for G(120) and for differentiation of combination of PDM and T2DM from NGT. Osteocalcin level was not different between men and women in T2DM subjects. Uncarboxylated osteocalcin (ucOCN) is an active and predominant form of osteocalcin and mediates not only glucose metabolism by enhancing b-cell function but also adipocytokine release from fat cells [1,11]. unOCN is inversely correlated with plasma glucose and fat mass [18,19]. Osteocalcin is expressed in human adipocyte tissue [20] and adiponectin and its receptor also on osteoblast [21,22], suggesting a link between adipose tissue and osteocalcin in metabolic regulation. In the present study, osteocalcin did not correlate with serum adiponectin. We have a few limitations in our study. Firstly, number of subjects collected particularly in diabetic subjects was small. Diabetic and PDM subjects may be in similar pathophysiological state as to glucose-metabolic abnormality in the present subjects. Accordingly, when the subjects of T2DM and PDM were compared with NGT, both osteocalcin and leptin also were predictive markers for glucose intolerance after adjusting confounding factors. However, it may be too early to presently conclude that osteocalcin is a biomarker to reflect glucose intolerance. Secondary, we could not determine how osteocalcin influences glucose intolerance state or whether increases of leptin and osteocalcin are simply consequence of high glucose state. It is unknown why leptin increased in PDM and T2DM but osteocalcin only in T2DM as compared to NTG in multinomial logistic analysis. Osteocalcin showed a trend of increase in circulating level in PDM (Table 1). Thirdly, longitudinal observation of increased osteocalcin is required in subjects intervened by reducing body weight with improvement of dietary habits. We demonstrated that circulating osteocalcin showed positive correlation with glycemia in early-stage T2DM. It is required to await further study to determine when osteocalcin is turned to declining course along with progression of glucose intolerance toward overt diabetes.
Acknowledgments We are grateful to all subjects for participating to this study and cooperation of co-medical staff in Omiya general Hospital. This work was supported by Grants-in-Aid for Scientific
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Research from the Japan Society for the Promotion of Science (JPSP: to M.K.).
[12]
Conflict of interest [13]
The authors declare that they have no conflict of interest.
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