Association of bone turnover markers with glucose metabolism in type 2 diabetes mellitus

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INTRODUCTION
nown as the "silent disease," osteoporosis is a significant public health concern that affects large populations globally.Reduced bone mass and altered microarchitecture are its defining characteristics (1,2).The greatest risk factor for osteoporosis is ageing.Skeletal health is a critical factor for older persons, especially in light of the global trend of population ageing (3).Type 2 Diabetes mellitus type (Type 2 DM) is a significant age-related illness.India is referred to as the global "diabetes capital" (4)(5)(6).Despite having high bone mineral density (BMD), type 2 diabetics are more likely to fracture.Accordingly, skeletal fragility associated with diabetes has been linked to poor bone quality (7).Bone resorption and formation are reflected by bone turnover biomarkers (BTMs) and hence represent the state of bone remodeling, which is the basic mechanism of osteoporosis (8).Due to a potential relationship between BTMs and glucose metabolism, BTM may be a more sensitive surrogate marker than BMD in determining fracture risk among individuals with diabetes (9).Osteocalcin (OC) is an accurate marker of bone turnover in both coupled and uncoupled bone growth and resorption conditions.At three glutamine terminals, OC undergoes gamma carboxylation.It can therefore communicate with hydroxyapatite.It is undercarboxylated (Uc) when there are fewer than three gamma carboxylated terminals.In addition to serving as indicators of bone formation, OC and UcOC have been linked to insulin sensitivity, beta-cell activity, and the possible control of plasma glucose levels (10).
CTx, a marker of bone resorption, has been suggested as a predictor of bone fracture in diabetic patients (11).Despite having normal or higher bone mineral density, people with type 2 diabetes have a friable bony architecture (BMD).Given the rising prevalence of diabetes mellitus, it is essential to understand the fracture risk associated with this population.
Bone is regarded as an endocrine organ that is essential in energy metabolism.Studies on animals have demonstrated that bone plays a vital role in energy metabolism through the production of osteocalcin.It is still debatable if BTMs and human glucose metabolism are related.By examining the relationship between BTM and glucose metabolism markers in type 2 diabetes mellitus, the current study may provide additional light on how bone affects energy metabolism.

MATERIALS AND METHODS
This study was conducted on 100 type 2 DM (Diagnosed as per American Diabetic Association (ADA) criteria and 100 age and sex matched controls in the age group between 30-60 years, visiting Justice KS Hegde Charitable Hospital, Deralakatte, K Mangaluru.Patients with Type 1 DM, treatment with insulin and bisphosphonates, known case of liver and renal disease, malignancy, calcium, and vitamin D supplements were excluded from the study.The institutional ethical committee granted its approval (INST.EC/EC/085/2018-2019).
After obtaining informed consent four mL of venous blood was collected under aseptic conditions from the antecubital vein following an 8-12-hour fast.Samples were centrifuged at 3000 rpm for 10 minutes.Serum obtained was used for the analysis.

Biochemical analysis
Osteocalcin, CTx and insulin was measured by Electrochemiluminescence immunoassay on Cobas e-411 autoanalyzer from Roche Diagnostics.Alkaline phosphatase was measured by colorimetric assay on Cobas c-311 autoanalyzer from Roche diagnostics.

Statistical analysis
The mean + standard deviation was used to express parametric data.Comparison between means of quantitative data was done by using student t test.The median and interquartile range were used to express non-parametric data.The correlation between the obtained metabolic indices and bone turnover markers was calculated using Pearson's correlation, and the significance of the results was assessed using the Student's 't' tests for normally distributed data.
The correlation between the obtained metabolic indices and bone turnover indicators was assessed for significance using Spearman's correlation for nonparametric data.p < 0.05 was used to determine statistical significance.SPSS 16 was used to conduct the statistical analysis.

RESULTS
The mean levels of FBS, ALP and HOMA-IR were significantly increased in Type 2 DM when compared to controls.(p<0.01)The means of osteocalcin, CTx, insulin and HOMA-B were significantly lower in Type 2 DM when compared to controls (p<0.01).The mean age difference between the case and control groups was not statistically significant (Table 1).There was a significant association seen between osteocalcin quartiles and study participants.(p<0.001).Lower quartiles of serum osteocalcin (Q1 & Q2) were predominantly seen in diabetic patients and higher quartiles of serum osteocalcin (Q3) were seen in controls (Table 2).
The various parameters in diabetic patients were compared between first and second quartiles of serum osteocalcin.The third quartile was excluded from the comparison as there was only one individual whose serum osteocalcin was in the range of the third quartile.Serum FBS was increased in the first quartile of serum osteocalcin compared to the second quartile.Serum osteocalcin and CTx were significantly decreased in the first quartile compared to the second quartile of serum osteocalcin (p<0.05;Table 3).and insulin (r=-0.699,p<0.001).HOMA-B showed negative correlation with FBS (r=-0.325,p<0.001),ALP (r=-0.262,p<0.001), whereas a positive correlation was seen with osteocalcin (r=0.320,p<0.001), insulin (r=0.775,p<0.001) and HOMA-IR (r=0.333,p<0.001) (Table 6).

DISCUSSION
The goal of the present study was to investigate the levels of markers associated with bone turnover in individuals with Type 2 DM and controls, as well as the relationship between BTM and glucose metabolism.The present study showed that FBS, ALP and HOMA-IR were higher and osteocalcin, CTx and HOMA-B were lower in Type 2 DM when compared to controls.Levels of osteocalcin revealed a negative relationship with FBS, ALP, HOMA-IR and HOMA-B.CTx levels showed a negative correlation with ALP and FBS.
Lalitha et al., in their study showed that osteocalcin levels are considerably lower in Type 2 DM than in the control group (p= 0.000) which agrees to the findings in our present study (12).A study by Wang, et (17).
Important functions of osteoclasts, including differentiation, fusion, survival, activation, and apoptosis, are regulated by the agonist RANKL (18).Hyperglycemia has been demonstrated to block the RANKL-mediated pathway and may cause a decrease in bone turnover (19).By activating cell surface receptors, insulin functions as an anabolic agent and directly influences the control of osteoblastic activity.Osteoblasts and their progenitor cells are inhibited by decreased insulin in diabetes mellitus (20.)Early identification of decrease in bone mass in diabetic patients may be helpful in preventing bone loss and future fracture risks.

CONCLUSION
Monitoring for diabetes associated with osteopenia is essential.The biochemical markers of bone metabolism can detect even small changes in the bone turnover, thus identifying diabetics who are at risk of developing osteoporosis.Since fracture risk in type 2 diabetics is independent of bone mineral density, study of markers of bone metabolism may identify diabetics who are at high risk for osteoporosis and help in the institution of preventive measures for osteoporosis to preserve a good quality of life in diabetics.

Table 1 :
Comparison of parameters between cases and controls

Table 2 :
Distribution of subjects based on osteocalcin quartiles

Table 3 :
Comparison of various parameters based on osteocalcin quartiles in diabetic patients

Table 4 :
Distribution of study participants based on CTx quartiles

Table 5 :
Comparison of various parameters based on CTx quartiles in diabetic patients

Table 6 :
Correlation of biochemical parameters among the study participants