2.5. Intraperitoneal Glucose Tolerance Testing (IPGTT)

Initial glucose tolerance testing was performed on 10 to 12-week-old mice (baseline) and repeated after 12–14 weeks of dietary intervention (continued chow feeding or HFD feeding). Mice were fasted overnight for approximately 16 hours by transferring mice to clean cages with no food but continued access to drinking water. The following day mice were weighed and an approximately 3 mm section of tail removed with sterile surgical scissors immediately prior to glucose tolerance testing. The first drop of tail blood was discarded on laboratory tissue and a second drop of tail blood was expressed and placed on an AimStrip® Plus Blood Glucose Test Strip (Germaine Laboratories, Inc., San Antonio, Texas) in combination with an AimStrip® Plus Blood Glucose Metre (20 to 600 mg/dL glucose range, Germaine Laboratories, Inc.). This data point was the baseline glucose level (t = 0 min). A 10% w/w solution of D-glucose was made by diluting stock D-glucose (45% w/w D-glucose in H₂O, G8769 Sigma-Aldrich, St. Louis, MO) in normal saline (114–055-721, Quality Biological, Gaithersburg, MD). The 10% glucose solution was warmed for 30 minutes in a 37°C bead bath prior to syringe loading (26 G tuberculin syringe, 1 mL, BD30962, Becton Dickinson, Franklin Lakes, NJ). Mice were injected intraperitoneally (IP) with the volume of 10% w/w glucose injected (μl) = 10 x body weight (g), which is equivalent to 1 g of glucose/kg of body weight. Tail blood glucose levels were measured at 15, 30, 60 and 120 minutes (t = 15, t = 30, t = 60 and t = 120) after glucose injection. Prior to each measurement a laboratory tissue was used to remove the blood clot and fresh blood expressed by gently massaging the mouse tail as necessary. Measurements that exceeded the upper limit of detection were recorded as the limit, 600 mg/dL. At the end of the glucose tolerance testing, mice were returned to clean cages with water and food available ad libitum and monitored carefully for health and well-being.