Nefac colorimetric assay

Samples of the medium were collected and measured for glycerol and FFA release. The glycerol assay kit (Sigma-Millipore, St. Louis, MO, USA) was used in accordance with the manufacturer’s instructions. FFA were measured using the NEFAC colorimetric assay in accordance with the manufacturer’s instructions (Wako Pure Chemical Industries, Richmond, VA, USA).

To determine insulin clearance at a steady state, mice were fasted from 1700 h until 1100 h the next day, and their retro-orbital venous blood was drawn into heparinized micro-hematocrit capillary tubes (Fisherbrand, Waltham, MA, USA), as previously optimized [6 (link)]. Plasma was processed and analyzed for insulin (80-INSMSU-E01, Alpco, Salem, NH, USA), C-peptide (80-CPTMS-E01, Alpco), adiponectin (ab108785, Abcam, Cambridge, MA, USA), non-esterified fatty acids (NEFA-C colorimetric assay; Wako, Richmond, VA, USA), tumor necrosis factor-alpha and interleukin-6 (ELISA Kits, Abcam), and nitric oxide (NO) (Nitrate/Nitrite fluorometric assay Kit; Cayman Chemical, Ann Arbor, MI, USA). To determine plasma leptin levels, mice were fasted up to 4 h in the morning before blood was drawn (Linco Research, Billerica, MA, USA).