Resistin

The Enzyme Linked-Immunosorbent Assay (ELISA) tests were conducted for quantitative determination of resistin (BioVendor, Czech Republic), adiponectin (BioVendor, Czech Republic), leptin (BioVendor, Czech Republic), IL-6 (Gen-Probe, France), ADMA (Immunodiagnostic, Bensheim, Germany), and PIIINP (Cloud-Clone Corp, China) in human serum.

Primary porcine vascular smooth muscle cells (VSMC) were isolated and cultured from rest materials (porcine aortas) collected from a slaughter house as previously described^{42 (link)} and used up to passage five. Thus, no ethical approval was needed. The VSMC identity was verified by staining for smooth muscle α-Actin. VSMC were always seeded onto collagen G (10 µg/ml) (Biochrom) prior to any experiment. Cells were starved in DMEM containing 1% fetal calf serum (FCS), and 1% antibiotic antimycotic solution (APS, Sigma-Aldrich) containing streptomycin and amphotericin B 24 h before treatment with resistin (100 ng/ml) (BioVendor).
Human microvascular endothelial cells (HMEC) were provided by Ades et al.^{43 (link)} and cultured as previously described^{44 (link)}.

Glucose, triglycerides, total cholesterol, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in the plasma were estimated by kit method (Biosystems, Barcelona, Spain). Plasma insulin was measured using rat insulin ELISA kit (Mercodia AB, Uppsala, Sweden) according to the manufacturer’s instructions. Homeostasis model assessment of insulin resistance (HOMA-IR) index was calculated from fasting plasma glucose and insulin to estimate insulin resistance. The adiponectin (Invitrogen, California, USA), leptin (EMD Millipore, Billerica, MA, USA) and resistin (BioVendor, Czech Republic) levels in the plasma were estimated by commercial ELISA kits.

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.

Circulating levels of IL-17A (Enzo Life Sciences, Farmingdale, NY, USA) and several adipo/cytokines—IL-6 (Quantikine, R&D Systems, Minneapolis, MN, USA), adiponectin (EMD Millipore, St. Charles, MI, USA), resistin (BioVendor, Brno, Czech Republic), lipocalin-2 (R&D Systems, Minneapolis, MN, USA) and TNFRII (AssayPro, St. Charles, IL, USA)—were measured in duplicate using enzyme-linked immunosorbent assays (ELISA) following the manufacturer’s instructions. The IL-17A assay sensitivity was 0.201 pg/mL, and intra-assay and inter-assay coefficients of variation (CV) were 5.4% and 9.4%, respectively. IL-6 assay sensitivity was 0.039 pg/mL, and intra-assay and inter-assay CV were 7.4% and 7.8%, respectively. adiponectin assay sensitivity was 0.2 ng/mL, and intra-assay and inter-assay CV were 3.4% and 5.7%, respectively. resistin assay sensitivity was 0.012 ng/mL, and intra-assay and inter-assay CV were 5.9% and 7.6%, respectively. lipocalin-2 assay sensitivity was 0.012 ng/mL, and intra-assay and inter-assay CV were 3.7% and 6.5%, respectively. Finally, the TNFRII assay sensitivity was 0.1 ng/mL and the inter-assay and intra-assay coefficients of variation were less than 3.2% and 3.3%, respectively.

Serum levels of IL-6 (Invitrogen, CA, USA), TNF-α (Invitrogen, CA, USA), leptin (Linco Research, St. Charles, MO, USA), adiponectin (B-bridge International, USA), resistin (Biovendor, Czech Republic) were measured by the enzyme-linked immunoadsorbent assays (ELISA) methods.