4.2.7. Measurements of Serum Insulin Level and Insulin Resistance

At Week 16, we collected blood from the tail vein of a 14 h fasting mouse to measure serum insulin concentration. Blood was collected in BD Microtainer serum separator tubes and centrifuged at 2000× g for 20 min to obtain serum. Serum insulin levels were measured using an ultrasensitive mouse insulin ELISA kit (Crystal Chem Inc., Chicago, USA). The samples and insulin standards were plated into 96-well antibody-coated microplates (5 μL each) and incubated for 2 h at 4 °C. After five washes, anti-insulin enzyme conjugate was added to each well and incubated at room temperature for 30 min. After seven washes, enzyme substrate solution was added to each well and incubated for 40 min. Finally, reaction stop solution was added, and after 10 min, absorbance was measured using an ELISA reader at 450 nm. From the fasting blood glucose (FBG) and insulin concentration, insulin resistance was evaluated using the homeostatic model assessment of insulin resistance (HOMA-IR). The HOMA-IR was calculated using the following equation: HOMA-IR = fasting blood glucose (mg/dL) × fasting insulin (ng/mL) × 0.0717225161669606.