Insulin

Total (intra-assay CV, 4·0 %, inter-assay CV, 7·8 %) and acylated ghrelin (intra-assay CV, 4·2 %, inter-assay CV, 11·3 %) (Bertin Pharma), PYY (intra-assay CV, 4·3 %, inter-assay CV, 11·1 %) and total glucagon-like peptide-1 (intra-assay CV, 4·8 %, inter-assay CV, 27·0 %) (Merck Millipore) assays were conducted using plasma, with leptin (intra-assay CV, 3·4 %, inter-assay CV, 6·4 %) (R&D Systems, Inc.) and insulin (intra-assay CV, 4·7 %, inter-assay CV, 12·5 %) (Mercodia AB) assays conducted using serum. All assays were done using commercially available ELISA, employed according to manufacturer's instructions, with all samples batches analysed at the conclusion of the study and samples from each participant included on the same plate. Analysis of blood samples for NEFA (intra-assay CV, < 5 %, inter-assay CV, < 5 %), glucose (intra-assay CV, < 5 %, inter-assay CV, < 6 %) and urea (intra-assay CV, < 5 %, inter-assay CV, < 3 %) were conducted using plasma on a Daytona (Randox Laboratories) automated analyser according to manufacturer's instructions using commercially available immunoassays (Randox Laboratories).

Glucagon, insulin (Mercodia AB, Uppsala Sweden) and somatostatin (Peninsula labs, San Carlos, CA, USA) concentrations in culture media and cell lysate samples were measured by ELISA. Hormone content was normalized to total protein content as measured by the DC protein assay (Bio-Rad Laboratories, Hercules, CA, USA) and then presented as fold of control treatment.

Blood samples were collected by cardiac puncture after a 5-hour fast using heparin anticoagulation tube. Enzyme-linked immunosorbent assay kit was used to detect plasma concentrations of insulin (Mercodia), adiponectin (AdipoGen), and ANP and BNP (Phoenix Pharmaceuticals) according to the respective manufacturer’s instructions.

ELISAs were used for determination of insulin, glucagon (both mercodia, Uppsala, Sweden), leptin (Crystal Chem Inc., IL, USA) and triglycerides (Wako, VA, USA) according to manufacturer’s instructions.

Blood was collected and stored as serum or plasma using standard methods, apart from samples for analysis of acylated ghrelin, for which blood was treated with proteases to prevent degradation as previously described 15. Total ghrelin (intra‐assay coefficient of variation [CV], 4.0%) and acylated ghrelin (intra‐assay CV, 4.2%; Bertin Pharma, Montigny le Bretonneux, France) and peptide YY (PYY) (intra‐assay CV, 4.3%) assays were conducted using plasma. Leptin (intra‐assay CV, 3.4%) (R&D Systems Inc., Abingdon, UK) and insulin (intra‐assay CV, 4.7%) (Mercodia AB, Uppsala, Sweden) assays were conducted using serum. All samples were batch analyzed upon study completion and samples from each participant assayed on the same plate. Plasma samples were analyzed for nonesterified fatty acids (intra‐assay CV, < 5 %), glucose (intra‐assay CV, < 5 %), and urea (intra‐assay CV, < 5 %) using a Daytona automated analyzer (Randox Laboratories, Crumlin, Northern Ireland) according to manufacturer guidelines.

The serum levels of triglycerides and cholesterol were measured by the colorimetric enzymatic method using commercial kits (SGM Italia, Rome, Italy, and Randox Laboratories Limited, Crumlin, UK). Glucose levels were determined by glucometer (Contour next, Ascensia, Switzerland). The serum levels of interleukin (IL)-1β, tumor necrosis factor-α (TNF-α; BioVendor, Brno, Czechia), insulin (Mercodia AB, Uppsala, Sweden), adiponectin and leptin (B-Bridge International, Mountain View, CA, USA) were measured using commercially available kits. As an index of insulin resistance (IR), HOmeostasis Model Assessment (HOMA)-IR was calculated using formula [HOMA = fasting glucose (mmol/L⁻¹) × fasting insulin (μU/mL⁻¹)/22.5].