2.1. Study population
A total of 84 subjects (all men) participated in this case-control study, including subjects with T2D (N=42) and subjects without T2D (N=42). All subjects were recruited from Shariati Hospital, Tehran, Iran. Outpatient clinic from March 2012 until November 2013. Informed written consent was obtained before the study, and the study was approved by the Ethics Committee of the Tehran University of Medical Sciences (TUMS). The participants were excluded if they had evidence of any chronic or acute systemic disease such as infectious disease, acute or chronic renal failure, malignancies, coronary artery diseases, and type 1 diabetes. It should be noted that none of the participants were current smoking.
To diagnosis, the T2DM, the basis of American Diabetes Association (ADA) criteria was used as fasting blood glucose (FBG) ≥126 mg/dl (7.0 mmol/l) or 2 hours plasma glucose ≥200 mg/dl (11.1 mmol/l) during an oral glucose tolerance test (OGTT) or random plasma glucose ≥200 mg/dl (11.1 mmol/l) (26). VAI was calculated as a novel sex-specific index, based on waist circumference (WC), BMI, triglycerides (TG), and high-density lipoprotein cholesterol (HDL-C), indirectly expressing visceral adipose function in all participants as described previously (27). We would like to stress that this study is reported in compliance with STROBE guidelines (Supplementary material).
2.2. Ultrasound methods
Ultrasound examinations for measurement of the cIMT and visceral adipose tissue thickness (VAT) were performed using an Accuvix XQ ultrasound unit (Medison, Seoul, Korea) equipped with a 3-7 MHZ curved-array and a 5-12 MHz linear-array transducer. The technique for measuring cIMT and VAT has been previously described (28, 29). In brief, cIMT measured at its thickest point on the distal wall of the carotid arteries, along a 1.5-2 cm proximal to the carotid bulb. cIMT on the left and right sides was evaluated and mean values of both sides were determined as carotid IMT. Also, VAT (in millimeter) was measured as the distance between the anterior wall of the aorta and the internal face of the rectus abdominis muscle perpendicular to the aorta.
2.3. Anthropometric and clinical characterization
Anthropometric indices of all participants including age, weight, height, BMI, WC, hip, WHR, and blood pressure were examined. BMI was measured based on the ratio of weight in kg divided by height in m2 to assess participants' obesity. WC using a flexible inch strip in the middle between the lowest rib and the iliac crest was calculated. Furthermore, the hip was measured at the maximum circumference of the buttocks. WHR based on the ratio of WC in centimeters divided by hip circumference in centimeters was measured. After a 15-minute rest in sitting position, systolic and diastolic blood pressures were measured by a manual sphygmomanometer. VAI, as a gender-specific mathematical index was calculated based on simple anthropometric [BMI and WC] and metabolic [TG and HDL Cholesterol (HDL)] parameters.
See formula 1 in the supplementary files.
2.4. Biochemical and laboratory measurements
Fresh venous blood samples were collected into sterile tubes containing the EDTA-K2 after overnight fasting, to biochemical analyses. Fasting blood glucose (FBG), urea, creatinine, TG, total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), HDL-C, aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), and gamma-glutamyl transferase (γ-GT) were measured by autoanalyzer using commercial kits (Pars Azmoon, Tehran, Iran). Additionally, fasting plasma insulin was calculated by enzyme-linked immunosorbent assay (ELISA) kit (Monobind Inc., USA). To examine the IR, homeostasis model assessment of IR (HOMA-IR) was calculated with the equation of fasting blood glucose (mg/dL) × fasting blood insulin (µU/mL) / 405.
2.5. Plasma adiponectin measurement
Plasma levels of adiponectin were determined by using the ELISA Kit (Elabscience, Wuhan, China) according to the manufacturer’s protocol. Intra-assay and inter-assay Coefficients of Variability (CV) were <10%.
2.6. Plasma CTRP5 and CTRP1 measurement
CTRP1 concentration was measured by ELISA kit (Biovendor research and diagnostic products) with a minimum detectable concentration of 0.016 ng/ml, Intra assay Coefficients of Variability (CV) was 2.7% and Inter-assay CV was 8.5%. Plasma levels of CTRP5 were measured by immunoassay using the Cayman system kit according to the manufacturer’s protocol. The inter-assay variability and intra-assay variabilities were 6.975 and 6.3 %, respectively.
2.7. Bias
Selection bias was addressed by closely matching cases to controls based on age. Moreover, all participants were men.
2.8. Statistical analysis
The sample size was calculated based on our previous studies. In detail, we estimated that considering alpha=0.05 and power=90%, a difference of 50% in the mean value of CTRP5 circulating levels between the two studied groups could be detected with a minimum of 30 subjects in each group.Here , we included 42 subjects in each groups.
Continuous variable with normal distribution is presented as mean ± standard deviation (SD) and variables with non-normal distribution are presented as median (interquartile ranges (IQR)). Descriptive analysis was applied and normality was tested for all quantitative variables using the Shapiro-Wilk test. For data with normal distribution, comparisons of anthropometric, biochemical, and laboratory parameters between two groups were done by the student’s t-test and by the Mann-Whitney U test when we have non-normal distribution. A p-value <0.05 was applied to interpret all achieved data from analysis. All data analysis was performed using SPSS 20 (SPSS, Chicago, IL, USA).