Onetouch ultra blood glucose monitoring system

Glucose tolerance tests (GTTs) and insulin tolerance tests (ITTs) were performed as described [37 (link)]. For GTTs, mice were fasted overnight and received an intraperitoneal injection of 2 g/kg body weight glucose (Sigma-Aldrich, St. Louis, Missouri). For ITTs, mice were injected intraperitoneally with 0.5 U/kg body weight insulin (Biosynthetic Human Insulin, 100 U/mL; Novo Nordisk, Bagsvaerd, Denmark) after a 4-hour fast. Tail blood glucose levels were measured at 0, 15, 30, 60, 90, and 120 minutes after challenge using the Onetouch Ultra blood glucose monitoring system (LifeScan, Shanghai, China). AUC was calculated using GraphPad Prism.

Blood was collected from mice fasted for 6 hours. For Ts65Dn, Dp16 and Ts1Rhr mice, blood was obtained from tail tips and glucose measured using a One-Touch Ultra Blood Glucose Monitoring System (LifeScan, Milpitas, CA, USA) or Contour Blood Glucose Monitoring System (Bayer HealthCare, Mishawaka, IN, USA). For Tc1 mice, blood was collected from the retro-orbital sinus and measured using an AU680 clinical chemistry analyser (Beckman Coulter, Brea CA, USA). After 12–16 hour overnight fasting, glucose tolerance was measured by administering intra-peritoneal glucose (2.0 g/kg).

Mice were housed in cages with woodchip bedding and deprived of food for 5–6 h. A OneTouch Ultra blood glucose monitoring system (LifeScan; Wayne, PA, USA) was used to measure glucose levels in blood that was acquired from the tip of the tail. Glucose tolerance tests were performed on age matched mice fasted 5–6 h (morning fast). A fixed glucose solution (50 µg) was administered via intraperitoneal injection (I.P). The glucose levels were measured through the acquisition of blood sample from the tail tip at indicated time points. Insulin levels were measured using the ultra-sensitive mouse insulin ELISA kit according to manufacturer’s instructions (Crystal Chem Inc; Elk Grove Village, IL, USA).

The Ajou University Animal Care and Use Committee approved all animal studies (IACUC2015-0001), and the experiment conformed to the Guide for the Care and Use of Laboratory Animals published by the United States National Institutes of Health. All experiments were performed in accordance with relevant guidelines and regulations. Six-week-old C57BLKS/J-Lepr^db/Lepr^db or wild type C57BL/6J male mice were purchased from Orient Bio, Inc. (Seongnam, Korea) and acclimatised for one week. Mice were randomly grouped and orally administered with vehicle, WY14643 (20 mg/kg), rosiglitazone (20 mg/kg), or MD001 (5 mg/kg or 20 mg/kg) once a day for two months. For the oral glucose tolerance test (OGTT) and intraperitoneal insulin tolerance test (IPITT), mice were fasted for 12 h and were treated with sterile glucose (1 g/kg, Sigma-Aldrich/Millipore) or human insulin (1 unit/kg, Eli Lilly and Company, Indianapolis, IN, USA). Blood glucose levels were measured at the indicated time point using an OneTouch Ultra Blood Glucose Monitoring System (LifeScan, Inc., Milpitas, CA, USA). For the toxicity study, wild type C57BL/6J male mice were randomly grouped and orally administered with vehicle and MD001 (50 mg/kg or 100 mg/kg) once a day for two month. Blood cells, body weight change, and blood and urine metabolites were analysed for toxicity.