Bone mineral density and bone metabolism in postmenopausal women with type 2 diabetes in a Chinese community: a cross-sectional study

Background: There is growing evidence of a complex interaction between type 2 diabetes (T2DM) and osteoporosis. The purpose of this study was to further study the relationship between Bone turnover markers (BTMs) and fasting blood glucose (FBG) in postmenopausal patients with type 2 diabetes and to analyze the effect of hyperglycemia on bone metabolism. Methods: Six hundred and twelve (612) postmenopausal women were included, including one hundred and seven (107) subjects with T2DM and �ve hundred and �ve (505) subjects without diabetes. Bone mineral density (BMD) was measured by DXA (dual-energy X-ray absorptiometry). Markers of bone formation Type 1 collagen N-terminal peptide (P1NP) and resorption C-telopeptide of type l collagen (CTX) were quanti�ed. Results: Compared to controls, postmenopausal women with diabetes had a higher prevalence of previous osteoporosis fracture (27.1% vs. 17.4% for diabetic and nondiabetic women, respectively) and a higher BMD. The P1NP level in women with T2DM was 49.451 ng/ml, while in N-DM individuals, it was 58.633 ng/ml, (p = 0.017). The CTX level in women with T2DM was 0.325 ng/ml, while in N-DM individuals, it was 0.412 ng/ml (p=0.039). In addition, P1NP was signi�cantly negatively associated with age (β=-0.590; p= 0.002) and FBG (β=-1.950; p = 0.035). CTX was negatively associated with FBG (β=-0.029; p = 0.015). Conclusions: T2DM was associated with higher BMD and paradoxically, with an increased risk of fracture. Postmenopausal women with T2DM had lower bone turnover than controls. With increased levels


Background
As the population ages, the incidence of osteoporosis and diabetes is increasing.Observational studies in recent years have analyzed several aspects of the association between diabetes and osteoporosis with con icting results.
Bone mineral density (BMD) is the most commonly used measurement in the assessment of osteoporosis status and risk for osteoporosis fracture in the elderly population.The diagnostic criteria for osteoporosis are based on BMD, and thus far, there is less information on the BMD of patients with type 2 diabetes.BMD signi cantly decreases in type 1 diabetes mellitus (T1DM) [1][2][3][4]; however, in type 2 diabetes mellitus (T2DM), the results are controversial.Early studies suggested that BMD in T2DM patients was lower than, equal to, or higher than that of the control groups [5][6][7][8].Interestingly, studies in recent years have shown that these subjects have signi cantly higher BMD than nondiabetic people.It is well-known that individuals with T2DM have a higher risk of fracture of the hip, spine, and peripheral sites than nondiabetic individuals [9][10][11][12].
Patients with T2DM have multiple chronic complications, such as retinopathy, kidney disease, neuropathy, coronary heart disease, stroke and dementia.Bone is also one of the organs that is affected by T2DM.It is estimated that patients with T2DM have a 1.7-fold increased risk of fracture compared with nondiabetics [13].Recent studies [14,15] suggest a complicated relationship between bone metabolism and glucose metabolism.Osteoporosis fracture is considered to be one of the complications of DM, and is called diabetic osteopathy, or "diabetic osteoporosis(DOP)".The Fracture Risk Assessment Tool (FRAX) model and BMD underestimate major osteoporotic and hip fracture risks.Bone turnover markers (BTMs) may be a useful exploratory method [16].BMD is mainly determined by the mineral composition in the bone, but cannot re ect the changes of organic components such as type I collagen in the bones and the changes in bone metabolism in the short term.The BTMs can not only sensitively re ect the dynamic changes of bone metabolism, but also can be used to monitor the e cacy of anti-OP drugs.BTMs are chemical compounds, a set of proteins and their derivatives, released during bone remodeling by osteoblasts or osteoclasts [17].Type 1 collagen N-terminal peptide (P1NP) is a speci c marker re ecting the activity of osteoblasts.BTMs can re ect the activity of osteoclast cells, such as Ctelopeptide of type l collagen (CTX), N-telopeptide of type l collagen (NTX), tartrate-resistant acid phosphatase (TRAP), and deoxypyridinoline [18].PINP and CTX are recommended as bone markers by the International Osteoporosis Foundation (IOF) and are widely used in clinical practice.
The current study aimed to identify the relationship between bone turnover markers and fasting blood glucose (FBG) in postmenopausal patients with type 2 diabetes in a Chinese population.The effect of hyperglycemia on bone metabolism was also determined.

Subjects and Methods
We performed a cross-sectional study, enrolling 1000 postmenopausal women aged 45-74 years from January to December 2017 from Danyang Lianhu Community Service Center in Jiangsu.All T2DM individuals had been diagnosed according to the 2010 criteria of the American Diabetes Association (ADA) and the 1999 World Health Organization de nition.We excluded individuals with diseases and those who were taking medicine known to affect BMD and bone metabolism, i.e., subjects who had hepatic, gastrointestinal, kidney, thyroid or malignant disorders causing low BMD and subjects who had been treated with hormone therapy, calcium supplements, vitamin D, bisphosphonates, antipsychotic drugs, diuretics or thiazolidinediones.Finally, a total of 612 participants (107 subjects with T2DM and 505 subjects without diabetes) were retained for further analysis.
Baseline data were recorded during a face-to-face interview by a trained professional using a structured questionnaire, which included questions about age, medical history, menopause age, physical activity, related treatment and family history.The participants were accurately measured for height, weight, and blood pressure.
BMD by DXA DXA (dual-energy X-ray absorptiometry) was performed on all participants on the lumbar spine (L1-L4) and femoral regions (femoral neck and total hip) with Hologicequipment (Danyang Lianhu Community Service Center; variation coe cient<1%).According to the World Health Organization (WHO) 1994 osteoporosis diagnostic criteria, in postmenopausal women, normal BMD is indicated by a T score ≥-1.0; osteopenia is indicated by -2.5 T score -1.0); and osteoporosis is indicated by a T score ≤ -2.5.

Biochemical parameters
In the morning, after approximately eight hours of fasting, blood samples from each participant were collected for total cholesterol, high-density lipoprotein and low-density lipoprotein cholesterol, triglycerides, fasting blood glucose, P1NP and CTX.Blood samples for fasting blood glucose were centrifuged at 1500 g for 10 minutes at room temperature and analyzed on the same day.Plasma glucose concentrations were measured by an Olympus AU2700 analyzer using the manufacturer's kits.
The bone formation marker P1NP and the bone resorption marker CTX were determined by electrochemiluminescence immunoassay (Roche).

Osteoporosis fracture ascertainment
In this study, the diagnostic criteria for osteoporosis fractures were residents' self-reported history of fragility fractures or resident-reported imaging fractures.A fragility fracture is de ned as a fracture that results from a fall to the ground or a similar degree of trauma from a standing position or lower than a standing position [19].

Ethical aspects
The study was approved by Danyang People's Government and Danyang Lianhu Community Service Center in Jiangsu.All participants provided written informed consent.

Statistical analyses
Baseline data were input into the computer with Epidata3.1.All statistical analyses were performed with IBM-SPSS version 24.0 for Windows (SPSS, Chicago, Illinois), based on the above two groups.Descriptive statistics, including means, frequencies, and percentages, were used to describe the study population and look at differences between groups.Continuous data were expressed as the mean and standard deviation (mean ± SD).The comparison of the means of the two groups was performed by independent sample t test.The categorical variables were analyzed by chi-squared test, and the regression analysis was peformed by multiple linear regression after adjustment for age, menopause age, weight and BMI.Pvalues 0.05 were considered statistically signi cant.

Basic characteristics of the participants
The clinical characteristics of the enrolled subjects are summarized in Table 1.All subjects included in this study were postmenopausal women.There were no statistically signi cant differences in age, menopause age, diastolic pressure, height total cholesterol , LDL cholesterol and weekly moderate physical activity between the two groups (p 0.05).However, the patients with T2DM had signi cantly higher systolic pressure, weight, body mass index, fasting blood glucose and triglycerides, and lower HDL cholesterol levels than the controls (p<0.05).75 of 107 patients received Oral anti-diabetic agents treatment ,18 patients used Insulin only, and 14 patients were using combination therapy.In the study, diabetic patients were more likely than nondiabetic patients to have complications with hypertension (57.9% vs. 40.6%,respectively, p=0.001) and cerebral apoplexy (5.9% vs. 1.0%, respectively, p=0.001).There was also no statistically signi cant difference in family history of fractures.However, postmenopausal women with diabetes had a higher prevalence of previous osteoporosis fracture than those without diabetes (27.1% vs. 17.4%,P=0.021).Continuous variables are expressed as the mean ± SD, and categorical variables are expressed as n (%) BMI body mass index, FBG fasting blood glucose, TC total cholesterol, TG triglycerides, LDL LDL cholesterol, HDL

HDL cholesterol
The postmenopausal women with T2DM had higher BMD (in the femoral regions) than the N-DM controls.

Discussion
To the best of our knowledge, this is a relatively large-sample study in a Chinese community with complete markers of bone metabolism and a large Chinese cross-sectional study that investigates the associations of T2DM and BMD, BTMs, and osteoporosis fracture in postmenopausal women.In our study, we found a negative correlation between FPG and BTMs and speculated that lower levels of P1NP and CTX may be associated with an increased fracture risk in patients with type 2 diabetes.We hypothesized that the underlying mechanism might be that hyperglycemia leads to bone marrow alterations and damaged differentiation potential, which changes bone metabolism, resulting in reduced bone turnover, poor bone quality, and ultimately fractures.
The effect of T2DM on BMD remains controversial, although fracture risk signi cantly increases in patients with T2DM [20,21].Epidemiological surveys show that in patients with type 1 diabetes, BMD is reduced, bone mass decreases and the incidence of osteoporosis ranges from 48% to 72% [22].BMD is increased, decreased or unchanged for patients with T2DM.These results have been reported in both domestic and foreign literature [23].In our study, there was a signi cant increase in BMD in patients with type 2 diabetes, which was consistent with other studies [9,24].This further con rms that BMD may be an inappropriate tool for assessing the risk of bone loss and fracture in patients with type 2 diabetes.For patients with diabetes, T value greater than -2.5 may be required as a cut-off value for osteoporosis diagnosis.
Recent studies have shown that osteoporosis fracture is one of the complications of diabetes in the skeletal system, but the speci c mechanism is not clear [20,25].The higher incidence of fractures in the diabetic population may be associated with speci c complications of diabetes, including peripheral neuropathy, impaired visual, postural hypotension, hypoglycemic attacks, and vascular disease.The de ciency of this article is that there is no additional detailed information about the speci c types of fracture observed.Therefore, it is di cult to further clarify the causal relationship between diabetes and osteoporosis fractures.
BTMs are considered independent diagnostic and prognostic indicators or as a supplementary index to BMD for osteoporosis fractures.Previous studies of bone turnover markers in diabetes have been limited and controversial, but most scholars have agreed that BTMs might be an important link between bone metabolism and glucose metabolism.Hussein RM [26]{Hussein, 2017 #133} and showed that patients with T2DM exhibited a signi cantly lower serum osteocalcin and calcium and a higher alkaline phosphatase compared to the controls.Moreover, osteocalcin was negatively correlated with FBG and DM duration.Wang J analyzed three BTMs in the Chinese population.The levels of N-terminal osteocalcin (N-MID), type 1 collagen N-terminal peptide (PINP) and type 1 collagen carboxyl-terminal peptide (CTX) were signi cantly decreased in the T2DM groups compared to the controls (P<0.01),revealing that BTMs are highly associated with T2DM, insulin sensitivity and beta cell function,which are compatible with ours.Another study concluded that bone formation was inhibited in postmenopausal women with T2DM, but that inconsistent with our research is that there was no signi cant difference in bone resorption [27].Reyes-García R[28] showed lower levels of bone resorption markers and PTH-i compared with the levels of controls.The lower the bone metabolism is, the higher the incidence of fracture.
In our study, women with T2DM showed lower bone turnover compared with N-DM controls.These results also supported the above ndings.Multiple regression analysis showed that both PINP and CTX were affected by fasting blood glucose.The higher the levels of FBG were, the lower the levels of PINP and CTX.This nding suggests that postmenopausal T2DM patients with increased FBG have decreased bone formation, bone resorption, and overall bone turnover.In this study, there was a lack of data on the course of diabetes, diabetes treatment plans, blood glucose control, glycosylated hemoglobin and other more accurate data.There are too many factors affecting FBG, which alone may be insu cient.
Advanced glycosylation end products (AGEs), stable cross-linking products, are formed by a series of nonenzymatic reactions between glucose and protein and are signi cantly increased in individuals with T2DM [29].AGEs affect attachment to the collagen matrix and interfere with the development of osteoblasts [30,31].AGEs may also decrease bone resorption by altering the structural integrity of matrix proteins and inhibiting osteoclastic differentiation [29].In postmenopausal T2DM patients with increased fasting glucose, both formation and bone resorption were decreased, and the level of overall bone transformation was decreased.RANKL is an agonist that regulates important aspects of osteoclasts, such as differentiation, fusion, survival, activation, and apoptosis [32].Hyperglycemia has been shown to inhibit the above pathway mediated by RANKL and could induce low bone turnover [33].Although there is no signi cant reduction in BMD in patients with T2DM, another factor that may contribute to the increase in fractures is microstructural abnormalities.Increased cortical porosity is a key determinant of bone fragility [34].In a recent community study, T2DM and elevated fasting glucose levels resulted in unfavorable cortical microarchitecture at the distal tibial cortex [35].This may indicate that the weakening of bone biomechanics is beyond the range of BMD measurements and that there may be signi cant microscopic contributors to osteoporosis fracture.Thus, BTMs may be a more sensitive alternative to BMD in assessing fracture risk in patients with diabetes.
Our study has some limitations.First, in our cross-sectional study, we only observed an association between BTMs and glucose metabolism, not a causal relationship.Further forward-looking and biomechanical studies are needed to con rm other potential associations.Second, FBG is only a temporary phenomenon, and there are too many factors in uencing it.We need a more stable longterm indicator, such as glycosylated hemoglobin or glycosylated plasma protein, to further explore the relationship between blood glucose and bone metabolism.Third, a few subjects with diabetes may have T1DM as we could not distinguish between type 1 and type 2 diabetes.Fourth, we lack data on the relationship between BTMs and glucose metabolism in premenopausal women, which may be different from the population included in this study.According to previous data, the level of sex hormone can affect bone metabolism [15].This means that we truly need to explore this issue further in different populations.
In conclusion, postmenopausal women with T2DM had higher BMD, but the risk of osteoporotic fracture was relatively high, and BMD underestimated the risk..The study suggested that actively controlling blood glucose may help maintain bone turnover balance and improve bone mass, thereby reducing the risk of fracture.The detection of BTMs in patients with type 2 diabetes can predict the risk of diabetes complicated with osteoporosis earlier than BMD, which is convenient for early intervention and treatment.

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Figure 1 Lower
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Table 4
Multiple linear regression analysis for association of P1NP and CTX as dependent variables with age, menopause age and above biochemical parameters