Relation between osteocalcin and the energy metabolism in obesity

Background/Aim. Numerous findings have indicated the potential relation between the osteocalcin, the traditional parameter of bone turnover and the regulation of energy metabolism. The aim of this study was to identify the relationship between osteocalcin and calculated indexes, which evaluate insulin sensitivity, insulin resistance and/or secretory capacity of the pancreas, in non-diabetic, obese subjects. Methods. The study included 57 (11 men and 46 women) euglycemic, obese patients (the body mass index – BMI : 41.03 ± 6.61 kg/m2) and 48 healthy individuals, age and sex matched (BMI : 23.15 ± 2.04 kg/m2). Plasma glucose and the insulin levels during the two-hour oral glucose tolerance test (OGTT) were determined in order to calculate the Homeostatic Model Assessment (HOMA) indexes (HOMA-IR, HOMA-B%), EISI (estimated insulin sensitivity index), EFP (estimated first phase) and ESP (estimated second phase). Osteocalcin was measured by using the Electro-chemiluminescence (ECLIA) methodology. Results. Statistically lower osteocalcin was found in the obese subjects (24.72 ± 9.80 vs 33.31 ± 10.89 ng/mL; p < 0.01). Тhere was a statistically significant positive correlation between osteocalcin and EISI (r = 0.340; p < 0.01). The inverse correlations were found between the osteocalcin and HOMA-IR (r = -0.276; p < 0.01), HOMA-B% (r = -0.337; p < 0.01), EFP (r = -0.332; p < 0.01) and ESP (r = -0.266; p < 0.01). Multiple regression showed that the BMI and osteocalcin have a significant inverse prediction with the EISI and HOMA-IR, but the level of prediction of the BMI was substantially higher. Conclusion. The effect of osteocalcin in the glycoregulation is evident, but its contribution is significantly smaller in relation to other obesity associated factors. Therefore, when assessing its position and the role in glycemic control it is always necessary to bear in mind that osteocalcin represents only one of the many contributing factors, some of which exhibit dominant influence than osteocalcin itself.


Introduction
Obesity is defined as a condition of increasing of fat in total body weight and as such represents an important risk factor for the development of many diseases, which are based on insulin resistance (1,2).Dysfunctional adipose tissue has a high degree of metabolic activity, producing a wide range of humoral mediators that are closely related to the level of bone metabolic activity.Based on these findings, the early 21st century has formed the hypothesis that obesity affects the metabolic activity of the bones, and that feedback, bone tissue through humoral factors such as osteocalcin may regulate some aspects of glycemic control (3,4).
Osteocalcin is one of the most important noncollagenous proteins that participates in the process of bone mineralization.Based on studies in animal models, osteocalcin released in circulation, has a prominent role in glycemic control through two main mechanisms: by acting directly on the islet cells of Langerhans, increasing the production and secretion of insulin (5) and by acting through the indirect mechanisms, primarily through adiponectin (6,7) on insulin-sensitive cells of the peripheral tissues (muscle and adipose tissue) (8,9).This was followed by clinical studies that have examined the relationship between osteocalcin and traditional parameters which evaluate glucose metabolism.For the most part, studies were conducted on subjects with already altered glycemic control, with the values of glycemia over the euglycemic range (10).
In accordance with these facts, this study focuses on the analysis and testing of relations between osteocalcin as a traditional marker of bone metabolism and standard laboratory markers of glucose metabolism.

Material and Methods
This cross-sectional study was conducted in the Department of endocrine diagnostics in cooperation with the outpatient department of Clinic for Endocrinology, Diabetes and Metabolic Disorders, Clinical center of Vojvodina, during 2015.The study included 57 obese patients (11 men and 46 women) and 48 healthy, normal weight subjects, which correspond to the study group by age and gender.
Criteria for exclusion from the study were: disorders of glycemic control (elevated fasting glucose, impaired glucose tolerance and/or diabetes mellitus), endocrinologic diseases, liver diseases which exclude steoatohepatitis and include hepatic steatosis), kidney diseases, psychic disorders and the presence of metabolic bone diseases, as well as supplementation with vitamin D and / or calcium preparations.

Anthropometric measurements
To all subjects body height (TV) and body weight (BW), were measured.Body height was measured using Anthropometer according to Martin and is expressed in centimeters (cm); and body weight was determined on decimal scale in kilograms (kg).Body mass index was calculated using the formula: BMI (kg/m²) = BW (kg) / TV² (m²) in order to determine the presence of obesity (BMI > 30 kg/m²) (11).

Oral glucose tolerance test
After an eight-hour, overnight fasting, standard oral glucose tolerance test (OGTT) was perfomed to all subjects in order to measure glucose (Gluc) and insulin (Ins) plasma levels in basal condition as well as in 120.minute after oral administration of 75 g of glucose.
Blood was taken by venipuncture, using Vacuette® system.Hemolytic, lipemic and icteric samples were not taken for analysis.For determination of osteocalcin, obtained serum samples were frozen at -20 ºC and stored until moment of analysis.All other laboratory parameters were determined directly after obtaining serum and plasma samples.

Determination of glucose, insulin and glycated hemoglobin A1c
Glucose was measured by biochemical method (hexokinase) on Abbott Architect c8000 analyzer using commercial kits from the same manufacturer.The reference value for glucose is from 4,1 to 6,1 mmol/L.Insulin was determined by direct Chemiluminescentne technology (CLIA) on the automated system ADVIA Centaur XP.The recommended reference value for insulin in basal conditions is from 3,0 to 25,0 mIU/L.Glycated hemoglobin A1c was measured using immunoturbidimetric method of inhibition of agglutination on microparticles, on the automated system Abbott Architect ci 4100.

Calculating indexes for the evaluation of glycoregulation
In order to evaluate insulin sensitivity, EISI (estimated insulin sensitivity index) was calculated to all subjects.On that occasion the following formula was used (12):  EISI=0.222-0.00333xBMI-0.0000779xIns120-0.000422xage As part of the assessment of the secretory capacity of pancreatic beta cells, EFP (estimated first phase) and ESP (estimated second phase) were calculated using following formulas Also, HOMA (HOMA-IR and HOMA-B%) indexes were determined to all participants using HOMA 2 calculator.The calculator was downloaded from the official website of the Oxford School of Medicine (13), based on measured values of blood glucose and insulin levels in basal conditions.Cut off value for HOMA-IR is defined as a value less than 2.5 (14).

Determination of osteocalcin and crosslaps levels
The total osteocalcin (Osteo) was determined on an automated system, Cobas e 411 Roche Diagnostics.The lower limit of detection is 0.5 ng/mL.Osteocalcin values are expressed in ng/mL, and according to the manufacturer, the following reference values were defined: for women: 11-48 ng/mL and for men: 14-46 ng/mL).Crosslaps was determined on the automated cobas s 411 system (the reference value of women's reproductive period: 299-573 pg/mL for men: 300-704 pg / mL).

Statistical analysis
Data are presented using descriptive statistical methods, continuous variables as mean (x±s.d.).In order to evaluate the differences between groups we used Mann-Whitney U test.The correlations among variables were assessed using the Pearson correlation coefficient.Multiple regression analysis was performed to assess the independent association between BMI, osteocalcin levels and calculated parameters of insulin resistance/sensitivity as well as, insulin secretion.A 2-tailed P < 0.05 was considered statistically significant.Statistical analysis was performed using the Data Analysis Excel (Microsoft Corp., Redmond, WA) and MedCalc 12.1.4.0 statistical software (MedCalc Software, Mariakerke, Belgium).

Results
The examined group consisted of 57 obese subjects (46 female and 11 male) (BMI:41.03±6.61kg/m²).Compared to the control group, obese subjects differed significantly in all monitored parameters except the blood glucose values in basal, as well as in 120.minute of standard OGT test (p=0.689;p =0.714).Based on results, the study group had significantly lower osteocalcin levels (p<0.01)(Table 1).
Multiple regression analysis was used in order to examine an independent predictive potential of osteocalcin in relation to the individual parameters of glycemic control, in all subjects (Table 3).In model A, (R²=0.862,p<0.001) we observed that serum levels of osteocalcin, positively and independently contribute to the EISI values.Also, model B (R²=0.798, p<0.001) showed that serum levels of osteocalcin negatively and independently contribute to the HOMA-IR.In addition to a statistically significant predictive impact of osteocalcin on EISI and HOMA-IR (models A and B), t coefficients indicate that the influence of BMI to specified indexes was more pronounced then of the osteocalcin itself.
Osteocalcin, in the models C, D and E did not show a significant predictive potential in relation to the HOMA-B%, EFP and ESP.

Discussion
The examined group consisted of 57 obese patients (obesity grade II and grade III) .In comparison to healthy controls, the group of obese subjects did not differ significantly in measured values of plasma glucose levels during two-hour OGTT.However, statistically significant elevated HbA1c values may indicate the presence of the modified discrete regime of glycemic control in the group of obese patients.
Our results are in agreement with the known facts that obese subjects compared to normally weighted, have decreased insulin sensitivity and/or increased insulin resistance (15).According to obtained results, examined group had significantly higher levels of insulin in basal conditions and after 120.minutes of OGT-test, which could be responsible for maintaining of plasma glucose levels in the reference range.Also, calculated index that assesses insulin resistance (HOMA-IR) was significantly higher in obese, while the indicator of insulin sensitivity (EISI) were significantly lower in obese compared to control group.Aforementioned index is widely used in everyday clinical practice and represent a measure of quantifying insulin resistance and insulin sensitivity.
Our analysis shows that obesity has a very high degree of correlation with parameters of insulin secretion: HOMA-B%, EFP and ESP (Table 2).Due to the fact, obesity is essentially connected to an increased insulin resistance and elevation of insulin secretion reflects the strong compensatory mechanism in order to maintaine normoglycemia.The obtained results indicate that in obesity, with clearly altered biological effects of insulin on the effector cells, functioning capacity of the islet cells of Langerhans is completely preserved, and operates in the mode of hypersecretion.Since among the examined group, hiperglycemia or decreased glucose tolerance was not registered, this group of obese patients is characterized by an initial, the mildest form of abnormal glucose regulation.
Obese patients showed statistically lower values of osteocalcin, compared to the control group of healthy subjects.Similar results were obtained by Cifuentes M et al. (16).Lucey AJ, et al.Showed a significantly lower value of the total osteocalcin in the patients with a BMI value equal or less than to 34,9 kg/m² in comparison to the women of normal weight (17).
Apart from the already mentioned fact that obese people have lower levels of osteocalcin, we found a significant negative correlationbetween the BMI and osteocalcin levels (Table 2).It is believed that osteocalcin released from the bone tissue in the circulation is becoming a mediator who stimulates the proliferation of pancreatic beta cells and insulin secretion, and on the other hand increases the insulin sensitivity of peripheral tissue (18,19,20).The mechanism which can explain this relation is an abnormal secretion of leptin in obesity which may decrease bone metabolic activity, primarily through the central, sympathetic regulation of bone cells.It is also known that leptin decreases the expression of adiponectin and its concentration in the circulation.In mice, the parenteral administration of osteocalcin increases the expression of adiponectin in white adipose tissue due to the improvement of insulin sensitivity, reduction of triglycerides in the liver and muscles, as well as the inhibition of gluconeogenesis (21,22,23).
Osteocalcin shows a highly significant correlation with EISI and HOMA-IR which makes it the one of indisputable regulators of the biological effects of insulin on the cell level.In respect of osteocalcin, obesity exerts opposite effects in terms of increase insulin resistance and decrease insulin sensitivity.Also, it can be seen that compared to osteocalcin, obesity has a significantly higher level of association with insulin resistance and/or sensitivity.
These results indicate that obesity is predominantly related with increased insulin resistance and reduced insulin sensitivity, while the preferred level of osteocalcin connection to the manifestation of the biological insulin effects is substantially lower (Table 3).The current level of increased insulin resistance and reduced sensitivity in the group of obese, is predominantly due to adverse effects of obesity opposite to protective efects of osteocalcin.
At first glance, a great surprise are correlation results, which showed significant inverse relationship between osteocalcin and the already mentioned indicators of insulin secretion (HOMA-B%, EFP, ESP).These results are in stark contrast to the so far known facts suggesting that osteocalcin stimulates beta cells of Langerhans and insulin secretion (24).This mechanism is mediated by the action of osteocalcin via Gprc6a receptors by stimulating proliferation of beta cells.Also, it is known that through the same receptor at the level of the gastrointestinal tract, osteocalcin stimulates the production of GLP-1 (glucagon like peptide-1), and in that way participate in the preservation of insulin secretion (7,25).The explanation for the obtained relation can be found in the fact that the study was conducted among obese people with minimal disturbances of glycemic control and subsequent insulin hypersecretion.Logical processing of the data shows: greater is obesity, osteocalcin is lower, and the insulin secretion is higher.From this it follows that between osteocalcin and insulin secretion there is an inverse correlation.
In order to comprehensively examine the influence of different factors on the insulin secretion in obese subjects, multiple linear regression analysis was performed.It must be borne in mind that the factor of obesity is complex and comprises a plurality of factors that directly or indirectly influence both insulin secretion, and insulin resistance/sensitivity.This statistical method showed that osteocalcin has no significant prediction of insulin secretion (HOMA-B%, the EFP, ESP), opposite to the complex factor of obesity (BMI) (Table 3).Therefore, it could be concluded that the chosen model of this research, although conducted on the small sample size, is not suitable for studyng the influence of osteocalcin on insulin secretion.

Conclusions
Obtained significantly lower concentrations of osteocalcin in obese subjects compared with those of normal weight,are the consequence of the altered energy metabolism in obesity.
The inverse relationship between osteocalcin and obesity indicate an actual connection between bone metabolism and glycemic control in the complex system of the energy metabolism.
Osteocalcin' s impact on glycemic control is evident, but its share is substancially lower in relation to obesity and other factors associated with obesity.Therefore, when assessing the place and role of osteocalcin in glycemic control, must always bear in mind that osteocalcin as a systemic mediator, is only one of the many other, less influential factors.Legend: Gluc 0 i gluc 120-plasma glucose values in 0. and 120.minute of OGTT; Ins 0 i ins 120-plasma insulin levels in 0. and 120.minute of OGTT; Osteo -osteocalcin; Cslscroslaps; 25OHD -vitamin D; EISIestimated insulin sensitivity index.calculated according to Stumvoll and coworkes; EFPestimated first phase.calculated according to Stumvoll and coworkes; ESPestimated second phase.calculated according to Stumvoll and coworkes; HOMA-IR -homa index for estimation of insulin resistance.using HOMA 2 calculator; HOMA-S% -homa index for estimation of insulin sensitivity.using HOMA 2 calculator; HOMA-B%-homa index for estimation of insulin secretion.using HOMA 2 calculator; p-statistical significance

Table 2 :
Linear correlation analysis

Table 3 :
Multiple regression analysis Adjusted R 2coefficient of determination that is compliant with the number of independent variables included in the model; p-statistical significance Received on March 28, 2017.Revised on April 21, 2017.Accepted on May 22, 2017.Online First May, 2017.