Streptozotocin-Induced Diabetic Rat Model and BFM B1 Treatment

Sprague–Dawley rats weighing 180–200 g (Charles River Laboratories, Shizuoka, Japan) had ad libitum access to tap water and standard rat chow. Diabetes was induced by a single tail vein injection of streptozotocin (STZ, 60 mg/kg body weight; Sigma Chemical, St. Louis, MO, USA) [diabetes mellitus (DM) rats]; the control rats were injected with an equal volume of citrate buffer. Three weeks after STZ injection, a group of DM rats was treated with BFM B1 (50, 100, 200 nmol/kg/day intraperitoneally; Enzo Life Sciences, Ann Arbor, MI, USA). Twenty-four-hour urine and blood samples were collected using a metabolic cage until day 7 morning under feeding condition with free access to water and food, and then under 24-h starvation conditions without food [16 (link)]. On day 8, the rats were anesthetized with pentobarbital (50 mg/kg body weight), and then their kidneys and liver were removed and used for western blotting or immunohistochemistry. Intravenous insulin tolerance tests (ITTs) were performed to assess the degree of insulin resistance, and the extent of insulin resistance was evaluated according to the K index of ITT (KITT) as described previously [16 (link)].
All the procedures were conducted in accordance with the Guidelines for Animal Experiment and Ethics Committee in The University of Tokyo (P10-079, 15-P-134), and Dokkyo Medical University (17-918).