In this cross-sectional study, we enrolled 137 adults, aged range 18-84 years, who were hospitalized for abdominal surgery. According to their IR status, participants were divided into two groups, subjects with IR (n=46) and healthy (n=91). Participants who have cancer, diabetes mellitus, or received fat-reducing, anti-obesity, and blood sugar drugs were excluded. Moreover, individuals with pregnancy and lactation or hospitalization less than two days before surgery were also excluded.
People's general characteristics, including sex, age, medical history, and history of drug use (hypoglycemic drugs, lipid-lowering drugs, hypertension drugs, heart drugs, hormonal drugs, supplements), were asked and recorded.
Blood samples, anthropometric information, and demographic characteristics were obtained before surgery. About 100 mg of VAT and SAT was also collected during surgery. The study's protocol was approved by the Research Institute for Endocrine Sciences (RIES) of Shahid Beheshti University of Medical Sciences (NO: IR.SBMU.ENDOCRINE.REC.1395.279). All participants consciously signed the written consent form approved by the committee.
Measurement of anthropometric parameters and blood pressure
As described previously, the weight, height, waist circumference, and blood pressure of participants were assessed [23, 24]. Body mass index (BMI) was calculated as weight (kg) divided by the square of height (m2).
Assessment of Biochemical and glucose homeostasis
Preoperative and fasting venous blood samples were taken from the participants and poured into potassium-EDTA tubes. Fasting plasma glucose (FPG) was measured by the enzymatic method of glucose oxidase. The enzyme colorimetric method with glycerol phosphate oxidase was also used to measure triglycerides (TGs). FPG and TGs were measured using commercial kits (Pars Azmoon Inc., Tehran, Iran). Insulin levels were measured with Mercodia kits (Uppsala, Sweden) using enzyme-linked immunosorbent assay (ELISA). The intra- and inter-test CVs were 1.7 and 2.3%, respectively.
RNA extraction and quantitative real-time polymerase chain reaction (qRT-PCR)
Both adipose tissues were isolated by biopsy and collected in RNAlater solution. After transfer to the laboratory, the RNAlater solution was removed, and samples were placed in liquid nitrogen and then stored at -80° C. We extracted total RNA from both adipose tissues using the TRIzol reagent (Invitrogen U.S Cat. No. 15596-026) according to the manufacturer's protocol. The quantity and purity of RNA were evaluated by NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, USA), and the absorption ratio (260/280 nm) of all samples were within an acceptable range. In order to eliminate genomic traces and increase purity, total RNA was treated with DNase I before the synthesis of complementary DNA (cDNA). The cDNA synthesis kit (BioFact, Korea) was used according to the manufacturer's protocol. GAPDH gene was also used as a reference gene to normalize omentin gene expression. The sequence of primers is shown in Table 1.
The Quantitative Reverse Transcriptase-PCR (qRT-PCR) amplification was performed in a 20 μl reaction volume by the SYBR Green master mix (Biofact, South Korea) was done using the Rotor-Gene 6000 device (R Corbett Research, Sydney, Australia). The following thermal cycling included initial denaturation (5 min at 95°C), followed by 45 cycles of 30 s at 95 °C, 30 s at 60°C, and 30 s at 72 °C. For each gene, samples were run in duplicate for inter-assay control with the GAPDH reference gene and the non-template control (NTC). Relative expression of the omentin gene in each sample calculated by the 2–DCT method based on its threshold cycle (Ct), the reference gene was used to normalize CT . All qPCR laboratory steps were written according to the MIQE guidelines .
We used the following formulas to calculate HOMA-IR, HOMA-B, and QUICKI:
HOMA-IR= [fasting insulin µU/mL= fasting glucose mmol/1)]/22. HOMA-IR stands for the evaluation of the homeostatic model of IR. Participants with HOMA-IR ≥3.2 were categorized in the IR group.
HOMA-B= 20 × fasting insulin (μIU/ml)/fasting glucose (mmol/ml) − 3.5. HOMA-B stands for insulin sensitivity.
QUICKI= 1/(log [fasting insulin μU/l] + log [fasting glucose mg/dl]). QUICKI stands for quantitative insulin sensitivity check index.
The normal distribution of variables was evaluated by histogram and Kolmogorov-Smirnov test. Data analysis was cried out by Statistical Package for Social Sciences software (SPSS) (Chicago IL. Ver. 15). P<0.05 was considered statistically signiﬁcant. Continuous variables were reported as mean±standard deviation (SD). As plasma TGs and insulin were skewed, we reported median and inter-quartile ranges. T-test and x2 tests were used to compare demographic data, anthropometrical, and plasma biochemical parameters between IR and healthy subjects. Linear regression was performed to determine the association of glucose homeostasis and omentin expression in VAT and SAT, and standardized β (STZβ), after adjusting for BMI and sex.