We recruited a total of 162 elderly patients with different blood glucose levels at Geriatric Department of Ruijin hospital affiliated to Shanghai Jiao Tong University School of Medicine between December 2017 and May 2019. According to the plasma glucose criteria, patients were divided into three groups: 48 subjects with normal glucose tolerance (NGT) ,56 subjects with impaired glucose regulation (IGR, also known as prediabetes), including 28 subjects with impaired glucose tolerance (IGT) and 28 subjects with impaired fasting glucose (IFG), 58 subjects with type 2 diabetes mellitus (T2DM). Diagnoses were made according to the American Diabetes Association Criteria 15. A 75 g oral glucose tolerance test were performed in all subjects. Subjects with fasting plasma glucose(FPG) value <5.6 mmol/l and 2-h plasma glucose (2-h PG) value during a 75-g oral glucose tolerance test (OGTT) <7.8 mmol/l are considered as normal glucose tolerance (NGT); IFG is defined as FPG levels between 100 and 125 mg/dL (between 5.6 and 6.9 mmol/L) and IGT as 2-h post-load plasma glucose between 140 and 199 mg/dL (between 7.8 and 11.0mmol/L). Patients with IGR are defined by the presence of IFG and/or IGT and/or HbA1C 5.7–6.4%. The fasting plasma glucose (FPG)≥126 mg/dL (7.0 mmol/L) or the 2-h plasma glucose (2-h PG) value ≥200mg/dL (11.1mmol/L), or HbA1C criteria ≥6.5% are diagnosed as type 2 diabetes mellitus (T2DM). Patients with type 1 diabetes or other types of diabetes were not included in this study. The clinical characteristics of the three groups are shown in Table 1.
Exclusion criteria: Those with acute cardiovascular and cerebrovascular diseases, acute infectious diseases, chronic heart failure, liver and kidney dysfunction, valvular diseases, arrhythmias, autoimmune diseases, malignancies and hyperthyroidism were excluded in this study.
This study was approved by the Clinical Research Ethics Committee of Ruijin Hospital Shanghai Jiao Tong University School of Medicine. Informed consent was obtained from each participant after the purpose of this study was explained.
2.2 Anthropometric and biochemical measurements
The subjects’ age, gender, previous history and medication history were recorded by special personnel. Body weight and height were measured in all participators using standard protocols, BMI was calculated according to the following formula: BMI=weight/height squared (kg/m2). Blood pressure was measured on the right arm after a 10-minute rest for three times, values used in the analysis are the average of three readings taken at 5-minute intervals. Those who has a history of hypertension or whose systolic blood pressure (SBP)≥140mmHg and/or diastolic blood pressure (DBP)≥90mmHg were defined as hypertension.
Fasting elbow venous blood samples were collected in the morning after 12-h fasting period for biochemical test. Fasting plasma glucose was evaluated by the automated glucose oxidase method. HbA1c was measured by high-performance liquid chromatography. Serum total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) and serum triglyceride (TG) concentrations were measured by using enzymatic assays. Insulin resistance was calculated by the homeostasis model assessment of insulin resistance (HOMA-IR), which was automatically calculated by the following formula: HOMA-IR=fasting insulin (μU/ml) x fasting glucose (mmol/L)/22.5.
Serum concentrations of omentin, adiponectin, TNF-α, IL-6 were determined by an enzyme-linked immunosorbent assay (ELISA) kit (provided by Shanghai Senxiong Bio-Tech CO. Ltd). All standards are strictly handled according to the instructions of the kit.
2.3 The assessment of macrovascular function
All patients underwent assessment of arterial dilator function, following the International Brachial Artery Reactivity Task Force guidelines 5. Before testing, all subjects were in a fasting state for at least 6 hours, vasoactive drugs (such as angiotensin converting enzyme inhibitors, Calcium antagonist, beta receptor blockers), alcohol, caffeine, tea, cigarettes and nicotine were withheld for more than 12 hours. Subjects rested in the supine position for at least 10 minutes in a quiet and warm (temperature of 22-24℃) room, A 10-MHz multifrequency linear array probe attached to a high-resolution ultrasound machine (Toshiba Artida) was used to acquire images of the right brachial artery. The lumen diameter of the artery was defined as the distance from the echo front edge of the near wall-lumen interface to the echo front edge of the far wall-lumen interface. In brief, the basal diameter of the brachial artery was obtained in the cubital region before inducing forearm ischemia, recorded as D0 (mm). Subsequently, a forearm cuff, positioned 1 cm above the antecubital fossa, was inflated to 50 mmHg above systolic blood pressure for 5min and then released to elicit forearm reactive hyperemia. The diameter at the same point of the artery was recorded as D1 (the maximum dilatation after deflation). Flow mediated dilation of brachial artery (FMD), described as the maximum percent change of the brachial artery diameter during forearm hyperemia compared to the basal diameter, was calculated by the following formula: FMD (%) = (D1-D0)/D0*100. After 15 minutes, the brachial artery returned to the baseline. Brachial artery diameter was monitored continuously until achievement of maximal dilatation after administration of 0.5mg of sublingual nitroglycerin, which was measured as D2, and Nitrate-mediated dilation of brachial artery (NMD) was expressed as the percent maximal increase of the artery diameter after nitroglycerin compared to baseline [NMD(%)=(D2-D0)/D0*100]. We use flow mediated dilatation (FMD) to quantify vascular endothelial function, while apply nitrate-mediated dilation (NMD) to assess endothelium-independent dilator function. The value of FMD ＜10% indicates impaired endothelial function, and the lower the FMD value is, the worse the endothelial function is. The variability for this ultrasound determination of FMD and NMD showed a coefficient of variation of 1.2%-4.2% and 3.97±0.24% respectively, suggesting our method has good reproducibility 16. Throughout the measurement of each individual, the probe was maintained in a fixed position. All operations were performed by the same experienced sonographer who was blind to the subjects’ clinical and biochemical characteristics.
2.4 Statistical analysis
Date are expressed as the mean and standard deviations (SDs), or median (interquartile range), or as percentage according to the variables were normal distributing or not, which were examined by the Kolmogorov-Smirnov equality of distributions test. Skewed parameters, such as triglycerides levels, HDL-C, fasting glucose, fasting insulin, HOMA-IR，TNF-α, adiponectin and omentin levels were logarithmically transformed to normalize their distributions before regression analysis. Differences among the three groups were tested using ANOVA for normally variables while Kruskal-Wallis H test was used for non-normally distributed variables. Pearson’s correlation coefficient was used for calculation of associations between serum omentin levels and clinical factors, so as the FMD. While multiple stepwise linear regression analysis was used to determine significant confounding factors for circulating omentin levels and FMD separately. Clinically and statistically significant variables were added in the regression model. Data were analyzed using SPSS 22.0 software. We regarded P value of less than 0.05 as statistically meaningful.