Animal experiments

All animals received humane care, and all animal-related protocols were approved by the Institutional Animal Care and Use Committees at Shanghai Institute of Materia Medica, Chinese Academy of Sciences. db/db and ob/ob male mice were obtained from the Jackson Laboratory (CA, USA) and were housed in a room maintained at 20–25 °C, 50% relative humidity and a 12-h light/12-h dark cycle with food and water ad libitum. Eight-week-old male mice were divided into three groups by fasting blood glucose and body weight. Vehicle or IVQ-HCl (23, 46 mg/kg) was orally administered by gavage needle daily for 5 weeks (n = 8). Fasting blood glucose levels from 6-h fasted mice were measured weekly. For the oral glucose tolerance test (OGTT)/pyruvate tolerance test (PTT), the mice were fasted for 16 h at the fourth/fifth week, and glucose (1.5 g/kg)/pyruvate (1.5 g/kg) was administered orally/intraperitoneally. The glucose levels were measured from tail vein blood samples at 0, 15, 30, 60, 90, and 120 min by ACCU-CHEK (Roche, Basel, Switzerland). At the termination of the assay, the mice were sacrificed, and liver tissues were stored at −80 °C for analysis.

For pharmacokinetic assay in vivo, the mice were administered IVQ-HCl (30 mg/kg, po.; 5 mg/kg, iv.), and rats were administered IVQ-HCl (20 mg/kg, po.; 10 mg/kg, iv.). The plasma samples were then collected to detect the concentrations of IVQ at 0.25, 0.5, 1, 2, 4, 8, and 24 h, finally fitting out the related pharmacokinetic parameters. The metabolite of IVQ was detected and confirmed by standard samples of QVO in mouse plasma after oral administration. The QVO concentration was determined in the liver tissues of the mice and rats at the peak time.