Plasma levels of adiponectin and chemerin are associated with early stage of atherosclerosis in older adults with type 2 diabetes mellitus

Abstract Objective Adipokines, such as adiponectin and chemerin, regulate fat metabolism and are critical for the development of atherosclerosis. Investigating the correlations of adiponectin and chemerin with atherosclerosis in older adults with type 2 diabetes mellitus (T2DM) will shed light on the search for new markers for early diagnosis of diabetic atherosclerosis. Methods A total of 120 older T2DM patients and nine healthy controls were enrolled in this study. The clinical parameters, such as brachial‐ankle pulse wave velocity (ba‐PWV), adiponectin, and chemerin, were examined and recorded. T2DM subjects were divided into three groups according to ba‐PWV level (high, medium, or low). The data were processed and analyzed by identical methods. Results Significantly higher chemerin and lower adiponectin levels were detected in the plasma of T2DM patients than in healthy controls. The plasma levels of chemerin and adiponectin were negatively correlated in T2DM patients. Moreover, chemerin and adiponectin were significantly correlated with body mass index, ankle‐brachial index, insulin, and ba‐PWV. Multiple linear regression analysis revealed that chemerin and adiponectin were independently affecting ba‐PWV. Conclusion Adiponectin and chemerin are potential markers for diagnosis and monitoring of early stage of atherosclerosis in older adults with T2DM. Further clinical investigations are required to confirm these markers.

the development of diabetes. 4,5 Obese adipose tissue secretes high amounts of chemerin and low levels of adiponectin, which leads to a pathogenic and pro-inflammatory environment for the development of atherosclerosis and diabetes. 3,4 Previous studies have suggested that serum adipokine levels may be helpful for the early diagnosis of atherosclerosis. 1,6 Pulse wave velocity (PWV), including carotid-femoral PWV and brachial-ankle PWV (ba-PWV), is the gold standard to assess arterial stiffness, as well as in identification of early stage of atherosclerosis and cardiovascular events, but its clinical application is limited due to the operation's complexity. 7,8 Still, whether the plasma levels of chemerin and adiponectin could reflect the degree of atherosclerosis in T2DM is still unknown. [9][10][11] In this study, we divided all patients with T2DM into three groups according to their ba-PWV values. The levels of adiponectin and chemerin together with other clinical parameters were measured and compared among all groups. Overall, our findings indicated that the plasma levels of adiponectin and chemerin correlated with the development of vascular stiffness, which may lead to atherosclerosis in T2DM.

| Patients and samples
This cross-sectional study included 129 subjects (aged over 65 years), including nine healthy older adults and 120 older patients with T2DM who had undergone medical treatment from June 1, 2015 to December 1, 2015

| Data collection and biochemical measurements
After enrollment, a serial medical examination was carried out, including age, history of T2DM, blood pressure (diastolic [DBP] and systolic [SBP]), height, weight, waist circumference, and hip circumference. Body mass index (BMI) was calculated by weight/height 2 (kg/m 2 ). We collected venous blood from all study subjects after 10 hours' fasting. A fully automatic biochemical analyzer (AU-640) was utilized to examine the liver function markers, total cholesterol, triglyceride, low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), albumin, glycosylated hemoglobin, C-reactive protein (CRP), renal function markers, and fasting glucose. Electro-chemiluminescence assays were used to detect the fasting insulin and C peptide levels. Meanwhile, the plasma was collected from 3-mL blood samples (centrifuged for 5 minutes at a speed of 1917 g) and stored at −80° for further analysis. Enzymelinked immunosorbent assay was used to examine adiponectin (No. ABIN364986, ACRIS) and chemerin (SEA945Hu, Cloud-Clone) levels in plasma. Samples were first incubated in microtitration wells for 60 minutes, then washed and incubated with biotin-labeled polyclonal anti-human chemerin or adiponectin antibody at 37°C for 90 minutes. After another washing, streptavidin-horseradish peroxidase conjugate was added and samples were incubated at 37°C for 30 minutes. The reaction was stopped with acidic solution (H2SO4). An enzyme standard instrument was used to detect the optical density (OD) value at 450 nm. Finally, we plotted a standard curve to calculate the chemerin and adiponectin concentrations of each sample. Meanwhile, the ba-PWV, bone mineral density T value (DPX-Bravo), and ankle-brachial index (ABI) were examined as in previous studies. 10,12

| Statistical analysis
All analyses were done with IBM SPSS Statistics Version 22.0. The data according to normal distribution are shown as mean ± SD, otherwise logarithmic transformation was carried out. Before further analysis, we first carried out an F test and a normality test. The Student's t test was utilized to assess the difference between different groups. Correlation analysis between two factors was analyzed with Pearson's test. P < .05 was considered as a statistically significant difference. Multiple linear stepwise regression analysis was applied to verify principal influence factors.

| Evaluation of the correlation of ba-PWV and several clinical parameters in T2DM patients
Several clinical parameters were collected and measured from all the enrolled patients as well as healthy controls (HCs). The insulin, insulin resistance index, and ba-PWV levels were significantly higher in T2DM patients than in HCs (P < .05), which is consistent with our diagnosis in the inpatient clinic. The 120 T2DM subjects were separated into three groups according to the ba-PWV level: the high-, moderate-, or low-ba-PWV groups. The clinical parameters were compared among the different groups. The high-ba-PWV group had elevated levels for SBP, BMI, glycosylated hemoglobin, CRP, brain natriuretic peptide (BNP), LDL-C, and fasting glucose insulin, while its levels for albumin, HDL-C, bone mineral density T value score, and ABI were significantly lower than those in the moderate-ba-PWV group (P < .05; Table 2). Compared with the low-ba-PWV group, the high-ba-PWV group showed higher levels for T2DM history, DBP, waist circumference, and insulin (P < .05; Table 2). Furthermore, lower levels for SBP, BMI, waist circumference, BNP, and LDL-C were observed in the low-ba-PWV group, along with higher levels for HDL-C, bone mineral density T value score, and ABI compared with the moderate-ba-PWV group (P < .05; Table 2). Interestingly, ABI, which protects generalized atherosclerosis at high levels, was negatively associated with ba-PWV levels, and their combination has been suggested for better diagnosis of atherosclerosis. 13 There were no differences in age, aspartate, aminotransferase, hip circumference, blood urea nitrogen, creatinine, uric acid triglycerides, total cholesterol, or C peptide among these groups (P < .05).

| Evaluation of the links between chemerin and adiponectin levels and ba-PWV in T2DM patients
First, power analysis was carried out according to Cohen's d by using G*Power software, 14 and support our experiment design with the sample size analyzed in each group in this study ( Figure 1).
We compared the chemerin and adiponectin levels between HC older adults (n = 9) and T2DM patients (n = 120 Next, the chemerin and adiponectin levels among these three groups were compared. Both the high-and moderate-ba-PWV groups showed higher levels of chemerin than the low-ba-PWV group ( Figure 2). Interestingly, inverted results were observed for adiponectin production in the low-ba-PWV group (Figure 3). Furthermore, the correlation analysis showed that chemerin and adiponectin levels were  TA B L E 2 Comparison of clinical parameters among low-, moderate-, and high-ba-PWV groups significantly related to ba-PWV levels (chemerin, r = 0.806, P < .001; adiponectin, r = −.554, P < .001; Table 3).

| Identification of the early diagnostic markers for vascular stiffness in T2DM patients
To investigate the potential markers for the prediction of atherosclerosis, we analyzed the correlation between chemerin levels and these measured clinical parameters in T2DM patients. As shown in Tables 4 and 5

| D ISCUSS I ON
Abnormal adipose tissue secretes chemerin and adiponectinpro-inflammatory and anti-inflammatory substances, which may link obesity to diabetes, atherosclerosis, and cardiovascular disease. 4,15,16 In the present study, we observed higher levels of In this study, all enrolled T2DM patients showed different degrees of atherosclerosis, which provides more evidence supporting the theory that diabetes promotes the development of atherosclerosis. 17 Similar results have been observed in previous reports: The ba-PWV level is significantly correlated with several clinical parameters that are risk factors for atherosclerosis, such as glycosylated hemoglobin and CRP. 10 PWV values represent a gold standard for measuring arterial stiffness, which is determined principally by age and blood pressure. 18 Arterial stiffening and atherosclerosis share some common pathophysiological mechanisms, such as endothelial dysfunction and insulin resistance. 19 In addition, insulin resistance has been connected to the constant arterial stiffening found in diabetic patients, 20 Recently, many studies have pointed out that adipose tissue is an endocrine organ, rather than just functioning as a fuel repository. 27,28 Adipose tissue secretes a series of adipokines, such as leptin, tumor necrosis factor, interleukin-6, angiotensin, chemerin, insulin-like growth factor, and adiponectin, which diversifies their functions in obesity patients. 4,28 Our results showed that chemerin and adiponectin levels were significantly related to a history of T2DM and to ba-PWV levels, suggesting that chemerin and adiponectin may play critical roles in the progression of T2DM and of vascular stiffness. Chemerin is one of the G-protein-coupled receptor (chemerin R23) ligands, which promote the function of mature fat cells, synthesize triglycerides, transport glucose, and inhibit lipid degradation, thus reducing blood sugar levels under normal circumstances. 29,30 Chemerin can promote cholesterol intake and foam-cell formation, which causes atherosclerotic plaque formation. 31,32 Furthermore, chemerin also enhances HDL-associated paraoxonase-1 activity and triggers high-grade inflammation and oxidative stress, which consequently increases the oxidized LDL level and  Abbreviations: ABI, ankle-brachial index; BMD T value, bone mineral density T value; BMI, body mass index; BNP, brain natriuretic peptide; CRP, C-reactive protein; DBP, diastolic blood pressure; FBG, fasting blood glucose; HDL-C, high-density lipoprotein cholesterol; IRI, insulin resistance index; LDL-C, low-density lipoprotein cholesterol; SBP, systolic blood pressure.

TA B L E 5 Correlation between plasma adiponectin level and clinical parameters
in patients with T2DM, 13,37 which should be considered for better prediction of atherosclerosis in T2DM patients. Thus, our data provide more support for utilizing chemerin and adiponectin levels as potential markers for vascular stiffness, together with ABI levels to better predict the early stage of atherosclerosis, and even for cardiovascular diseases.
In summary, chemerin and adiponectin levels in plasma are suggested as independent predictors for vascular stiffness, which promotes the development of subclinical atherosclerosis, and may be applied for the risk assessment of macrovascular diseases in clinical settings.

ACK N OWLED G EM ENTS
This study was supported by the National Natural Science

CO N FLI C T S O F I NTE R E S T
Nothing to disclose. Abbreviations: ba-PWV, brachial-ankle pulse wave velocity; BMD T value, bone mineral density T value.

O RCI D
TA B L E 6 Multiple regression analysis of chemerin with ba-PWV levels