80 cptms e01

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).

Serum insulin level obtained was determined using blood collected from the saphenous vein in 2 mL heparin-coated Eppendorf tubes and measured using an ultrasensitive mouse insulin ELISA Kit in a semi-fasted state (i.e., removal of food from the cage) (#80-INSMSU-E01/E10, ALPCO, Salem, New Hampshire, USA). Glycogen was measured using frozen liver samples according to the manufacturer’s instructions (Cayman Chemicals, Ann Arbor, MI, USA). Glucagon (Crystal Chemicals, Elk Grove Village, IL, USA) concentrations were determined using commercially available kits (Pro-insulin: #10–1,232-01, Mercodia, Winston Salem, NC, insulin-degrading enzyme (IDE): LS-F5702, LSBio, Seattle, WA, USA, and c-peptide:#80-CPTMS-E01, ALPCO, Salem, New Hampshire, USA). Serum samples were diluted per kit recommendation, if applicable. All samples were read at OD 450 nm using BioTek microplate reader (Santa Clara, CA, USA). Total serum concentration of Insulin-like growth factor-1 (IGF-1) was measured using Mouse IGF-1 ELISA Kit (#RAB (1)0229, MilliporeSigma, Oakville, ON, Canada). Samples were prepared following manufacturers’ standard procedures and read at OD 450 nm on the BioTek microplate reader (Santa Clara, CA, USA).