Study design and participants
The study subjects were 30 people with type 2 diabetes without carotid plaque, 30 people with type 2 diabetes with carotid plaque, and 30 control participants with normal glucose tolerance. Diabetes was defined as a fasting plasma glucose value ≥ 7.0 mmol/L (126 mg/dL) or a previous diagnosis of diabetes. All were self-referred from the Department of Endocrinology and Metabolism at Korea University Guro Hospital. All subjects in the control group were administered a 75-g oral glucose loading test to exclude subjects with pre-diabetes or undiagnosed diabetes using the criteria of the American Diabetes Association . The study exclusion criteria included histories of cardiovascular diseases (myocardial infarction, unstable angina, stroke, peripheral artery disease, or cardiovascular revascularization), stage 2 hypertension (resting blood pressure ≥ 160/100 mmHg), uncontrolled diabetes mellitus (HaA1c > 10%), malignancy, or severe renal or hepatic disease. We also excluded patients with inflammatory conditions such as vasculitis or aortitis, or acute infectious disease. We investigated smoking and medication histories including use of insulin, oral hypoglycemic agents, statins, anti-platelet agents, angiotensin receptor blocker (ARB), angiotensin-converting enzyme (ACE) inhibitors or calcium channel blockers. All participants provided written informed consent, and the Korea University Institutional Review Board, in accordance with the Declaration of Helsinki of the World Medical Association, approved the study protocol.
Clinical and laboratory measurements
Body mass index (BMI) was calculated as weight/height2 (kg/m2) and waist circumference was measured at the midpoint between the lower border of the rib cage and the iliac crest. All blood samples were obtained the morning after a 12-hour overnight fast, and were immediately stored at -80°C. Serum triglycerides and high-density lipoprotein-cholesterol (HDL-C) were determined enzymatically using a chemical analyzer (Hitachi 747; Hitachi Inc., Tokyo, Japan). Low-density lipoprotein (LDL)-cholesterol concentration was estimated using the Friedewald formula . The glucose oxidase method was used to measure plasma glucose and an electrochemiluminescence immunoassay (Roche Diagnostics, Indianapolis, IN, USA) was used to measure insulin levels. Insulin resistance was calculated by the homeostasis model assessment of insulin resistance (HOMA-IR). hsCRP levels were measured by latex-enhanced turbidimetric immunoassay (HiSens hsCRP LTIA, HBI Co., Ltd.), with an interassay coefficient of variation of 7.2%. Serum concentrations of omentin-1 were determined by an enzyme-linked immunosorbent assay (ELISA) kit (Apotech, Axxora, Nottingham, UK); intra- and inter-assay variations were 3.6 ± 0.7% and 4.6 ± 0.3%, respectively.
Measurement of baPWV
After subjects rested in the supine position for 5 minutes, baPWV was measured using a volume-plethysmographic apparatus (model BP-203RPE II; Colin, Komaki, Japan) that simultaneously recorded baPWV and brachial and ankle blood pressures on the left and right sides. The details of this method, including validity and reproducibility, were described previously by Yamashina et al. . The intra- and inter-observer reproducibility of this method in the present study were 10.0% and 8.4%, respectively. The baPWV was calculated as the mean of the left and right baPWV values.
Measurement of carotid IMT
The IMT of the common carotid artery was determined using high-resolution B-mode ultrasonography (EnVisor; Philips Medical Systems, Andover, MA, USA) with a 5-12 MHz transducer. Carotid plaque was defined as a focal structure protruding into the arterial lumen with a thickness ≥ 1.3 mm . Measurements of the carotid IMT were made using IMT measurement software (Intimascope; Media Cross Co., Tokyo, Japan) at three levels of the lateral and medial walls of the carotid artery, 1-3 cm proximal to the carotid bifurcation. The maximal IMT was the value at the maximal point of the region. Carotid IMT was calculated as the mean of the maximal left and right IMT values. All measurements were recorded by a single trained technician who was blinded to the anthropometric and laboratory data.
Each variable was examined for normal distribution by the Kolmogorov-Smirnov equality of distributions test. Data are expressed as the mean with standard deviations (SDs) or median (interquartile range, 25%-75%), or as a percentage. Differences among the groups were tested using ANOVA for normally distributed variables or the Kruskal-Wallis H test for non-normally distributed variables, and Fisher's exact test or Pearson's chi-square test for categorical variables. Subsequent pair-wise comparisons were performed by Tukey's HSD post hoc analysis or the Wilcoxon rank-sum test. Analysis of covariance (ANCOVA) was used to compare the mean baPWV values according to tertile of serum omentin-1 level after adjusting for known covariates. Multiple linear stepwise regression analysis with baPWV as a dependent variable was performed to identify the risk factors that determined baPWV in type 2 diabetes. Subsequently, age, gender, BMI, systolic blood pressure, fasting blood glucose, smoking and medication history, including usage of statins and ARB or ACE-inhibitors, and circulating omentin-1 level, were used as independents variables. Unadjusted and adjusted odds ratios (ORs) with 95% confidence intervals (CIs) predicting carotid plaque in type 2 diabetes patients based on circulating omentin-1 level were generated using univariate and multivariate logistic regression analyses after controlling for other potential covariates. All statistical results were based on 2-sided tests. Data were analyzed using SPSS for Windows (version 12.0; SPSS Inc., Chicago, IL, USA). We regarded a P value of less than 0.05 as statistically meaningful.