2.1. Study population
This study consisted of T2D patients (n=42) and control subjects (subjects without T2D) (n=42). All participants (T2D patients and controls) were men between the ages of 43 - 72 years. Informed written consent was obtained from all participants before the study, and the study was approved by the Ethics Committee of the Tehran University of Medical Sciences (TUMS).
Patients with T2D were consecutively recruited from the outpatient clinic of Shariati Hospital, affiliated with Tehran University of Medical Sciences, Tehran, Iran from March 2012 until November 2013. All patients in this study were clinically deﬁnite diagnosed with T2D based on the T2DM, the basis of American Diabetes Association (ADA) criteria which were 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) . T2D Patients with evidence of any (1) chronic or acute systemic diseases such as infectious disease, (2) acute or chronic renal failure, (3) malignancies, (4) congenital cardiac disease, and (5) type 1 diabetes (T1D) were excluded.
Subjects without T2D as the control group were selected from the same geographical areas and were selected among subjects attending the Shariati Hospital, Tehran, Iran for a routine check-up. The exclusion criteria for controls were as follows 1) T2D; 2) T1D;3) chronic or acute systemic diseases such as infectious disease;4) acute or chronic renal failure; 5) malignancies, and 6) congenital cardiac disease. It should be noted that none of the participants were current smokers and alcohol drinkers. All controls were received a regular medical check-up by a physician. Regarding the history of receiving medication, the current use of antidiabetic drugs was reported in 8 patients with T2D. Moreover, 10 patients with T2D and 2 individuals in controls were receiving antihypertensive drugs. 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 [25, 26]. 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 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 was measured based on the ratio of WC in centimeters divided by hip circumference in centimeters. After a 15-minute rest in a 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 .
2.4. Measurement of Biochemical and laboratory parameters
Fresh venous blood samples were collected into sterile tubes containing the EDTA-K2 after overnight fasting for measuring 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. Measurement of plasma level of adiponectin
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. Measurement of plasma levels of CTRP5 and CTRP1
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.
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 group.
Continuous variables with normal distribution were presented as mean ± standard deviation (SD) and variables with non-normal distribution were presented as median (interquartile ranges (IQR)). Descriptive analysis was applied and normality was tested for all quantitative variables using the Shapiro-Wilk test. The student’s t-test and the Mann-Whitney U test were used to compare continuous variables between two groups for data with normal and non-normal distribution, respectively. Pearson's correlation coefficient was used to determine the association of CTRP1, CTRP5, and CTRP1/ CTRP5 levels with anthropometric indices and biochemical parameters. It should be mentioned that variables with non-parametrical distribution were log-transformed before analysis. Moreover, linear regression modeling was used to assess the association between the association of CTRP1, CTRP5, and CTRP1/ CTRP5 levels with obesity indices and cIMT, adjusted for history of receiving medication. It should be noted that the linear regression model when we found a significant correlation between CTRP1, CTRP5, and CTRP1/ CTRP5 levels with obesity indices and cIMT.
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).