Nycocard

Most clinical chemistry determinations were done in duplicate using colorimetric kits from Wiener Laboratory, Argentina, and Reflotron^® clinical chemistry test strips (Hoffman-La Roche, Switzerland). High-sensitivity C-reactive protein (hsCRP) was measured using an immunoturbidimetric latex assay from BioSystems (CAT# 22921, BioSystems, Spain). HbA1c and urinary albumin excretion were measured employing immunocolorimetric kits from NycoCard (AxisShield, Norway); catalog numbers of the individual and/or multiplex ELISA kits employed for the determinations of plasma concentrations of hormones are respectively: insulin: Abcam CAT# ab213815; GLP-1 and ghrelin: Millipore Magpix CAT# HMHEMAG34K05; FGF-21: Abcam CAT# ab125966 and irisin: Phoenix CAT# EK-067-29.

The intraperitoneal glucose tolerance test (IPGTT) was performed after a fasting period of 6 h. A fixed dose of 50 mg glucose was injected as a prewarmed sterile-filtered solution in a volume of 200 μl Krebs–Ringer buffer. These conditions have been described to yield reliable values.^{28 (link)} Blood was sampled at 0, 15, 30, 60 and 120 min by tail bleeding. Blood glucose was determined as described above. At 60 min, 30 μl blood was collected, immediately centrifuged (2000 g × 15 min) and the insulin content determined in 5 μl plasma using an ultrasensitive ELISA (Mercodia, Uppsala, Sweden). HbA1c was determined in 5 μl capillary blood using an immunoassay based on the boronate affinity technology (Nycocard, Axis-Shield, Heidelberg, Germany).