Resistin

Before the end of the study, at weeks six and twelve, the mice were fasted for six hours in the morning. Under isoflurane anesthesia, blood was collected via cardiac punction, and euthanasia was performed by cervical dislocation. Blood samples were incubated for 30 min at room temperature and centrifuged for 15 min at 1500× g, and the obtained serum was immediately frozen and stored at −80 °C. Serum cholesterol and triglyceride levels as well as alanine aminotransferase (ALT) activity were measured by fluorometric assays (Abcam, Cambridge, UK). Serum insulin (Mercodia Ltd., Uppsala, Sweden), serum amyloid A (SAA), leptin, resistin, adipsin, and adiponectin (R&D Systems Europe Ltd., Abingdon, UK) were measured by enzyme-linked immunoassays (ELISAs). The detection limits were 33 pmol/L for insulin, 62.5 ng/L for SAA, 7.8 ng/L for leptin and resistin, 375 ng/L for adipsin, and 15.6 ng/L for adiponectin.

Blood morphology and serum C-reactive protein (CRP) levels were assessed. CRP concentrations were assessed by an automated system (Modular PPE, Roche Diagnostics GmbH, Mannheim, Germany). The inter-assay coefficient of variability was 5.7%.
The ELISA method was used for measurements of plasma and fluids leptin (TECOmedical AG Sissach, Switzerland), adiponectin (TECOmedical AG Sissach, Switzerland), omentin (DRG Instruments GmbH, Marburg, Germany), RBP4 levels (Phoenix Pharmaceuticals, Burlingame, USA), resistin (R&D, Minneapolis, MN, USA), and visfatin/NAMPT (BioVendor, Brno, The Czech Republic) with the LoQ of 0.08 ng/mL, 0.11 ng/mL, 0.2 ng/mL, 0.6 ng/mL and 0.5 ng/mL, 2.17 ng/mL, 0.05 ng/mL, and 30 pg/mL respectively; intra- and inter-assay coefficients of variations were 4.6% and 7% for leptin, 5% and 6% for adiponectin, 3.7% and 4.6% for omentin-1, 5.0% and <14.0% for RBP4, <5.5% and <9.2% for resistin, and 5.6% and 5.9% for visfatin/NAMPT.

Commercially available enzyme-linked immunosorbent assay (ELISA) kits were used to perform the biochemical measurements of adipokines and insulin according to the manufacturer’s instructions (visfatin kit was from BioVision; chemerin, PAI-1, resistin, CCL-2, IL-6, IL-8, IL-10 and TNF-α kits were from R&D; leptin, adiponectin and insulin kits were from Thermo Fisher Scientific). The coefficient of variability (CV) for the ELISA kits were shown as follows: visfatin (intra-assay: 4.4–8%; inter-assay: 8.2%), chemerin (intra-assay: 3.9%; inter-assay: 7.3%), PAI-1 (intra-assay: 6.8%; inter-assay: 7%), resistin (intra-assay: 4.7%; inter-assay: 8.4%), CCL-2 (intra-assay: 5%; inter-assay: 5.1%), IL-6 (intra-assay: 2.6%; inter-assay: 4.5%), IL-8 (intra-assay: 4.7–6.7%; inter-assay: 5.8–7.7%), IL-10 (intra-assay: 3.7%; inter-assay: 6.9%), TNF-α (intra-assay: 4.9–7.8%; inter-assay: 4.7%-5.8%), leptin (intra-assay: 3.9%; inter-assay: 5.3%), adiponectin (intra-assay: 3.8%; inter-assay: 5.5%), and insulin (intra-assay: 4.8%-6%; inter-assay: 8.1% to 9%). All measurements were performed in duplicates or triplicates. Seven controls (one blank, two at lower concentrations, two at medium concentrations, and two at higher concentrations) were also quantified in duplicates to check for the reproducibility of measurements and confirm acceptable reproducibility.