STZ-Induced Diabetic Mouse Model

C57BL/6 mice were given STZ (Amresco, Solon, Ohio, USA) for a consecutive 5-day schedule (at 8∼12 a.m. every day) according to published methods and our previous protocol (Szkudelski, 2001 (link); Yu et al., 2016 (link)). Briefly, the compound was dissolved in a citrate buffer (pH 4.5), and injected intraperitoneally (60 mg/kg/d) within 15 min of dissolution. The control group received citrate buffer solution without STZ correspondingly. Three weeks post STZ stimulation, animals with random blood glucose value ≥300 mg/dL were defined as STZ-induced diabetic mice. The mice were then divided into three groups: (1) Control; (2) STZ; and (3) Metformin treated (STZ + Met), and housed in groups. The dose of Metformin administered to mice in this study was calculated according to clinically relevant human dose based on body surface area. Metformin (250 mg/kg/d; Sigma-Aldrich, St. Louis, MO, USA) dissolved in vehicle solution (5% sodium carboxymethylcellulose, CMC-Na; Sangon Biotech, Shanghai, China) was administered by gavage for 2 weeks (at 2∼4 p.m. every day). Mice in the Control and STZ groups were treated with vehicle correspondingly. On day 34, mice were used for ex vivo studies (Fig. 1A)