Tofogliflozin

The animal committee of Juntendo University Faculty of Medicine approved all animal experiments conducted herein. Five‐week‐old male KK‐A^y and KK mice were purchased from CLEA Japan (Tokyo, Japan). Tofogliflozin was generously provided by Kowa Company, Ltd. (Tokyo, Japan). Starting at seven weeks of age, KK‐A^y mice were administered 0.015% Tofogliflozin, which was mixed in their diet for eight weeks. Fluid intake was measured every week. KK‐A^y mice were then divided into the following two groups: Tofogliflozin treatment (Tx group, n = 14) and nontreatment (DKD group, n = 15) groups. The nondiabetic KK mice were used as normal control (CTRL, n = 9). The mice were individually housed in plastic cages with free access to rodent pellet diet (CE‐2, CLEA Japan) and water throughout the experimental period. At 15 weeks of age, the mice were sacrificed for blood and tissue collection. All animals were housed under a 12‐h light/dark cycle (07:00–19:00), controlled room temperature (20–26 °C), and humidity. The mice were treated according to the guidelines for animal experimentation of Juntendo University, Tokyo, Japan (310147).

Four-week-old male db/db mice were obtained from Japan SLC Inc., (Shizuoka, Japan) and were humanely maintained at the Gifu University Life Science Research Center in accordance with the Institutional Animal Care Guidelines. DEN was purchased from Sigma-Aldrich Chemical Corp., (St. Louis, MO, USA) while tofogliflozin was kindly provided by Kowa Co., Ltd. (Tokyo, Japan).

Tofogliflozin was provided by Kowa Company, Ltd. (Tokyo, Japan). 0.005% (w/w) of Tofogliflozin were mixed with HFD (D12492, 60 kcal%fat, Research Diets, New Brunswick, NJ, USA) or NC (D12450J, 10 kcal%fat), and fed to mice about 4 mg/kg/day or 3 mg/kg/day, respectively. These were pharmacological doses of Tofogliflozin [16 (link)][17 (link)] in mice.

Tofogliflozin was kindly provided by Kowa Company, Ltd. The db/db mice were randomly allocated to either the untreated group (n = 6) or the treatment group (n = 6). The db/db mice in both groups had ad libitum access to solidified regular food. In the treatment group, the solidified food was mixed with 0.004% Tofogliflozin (5 mg/kg/day) at the same dose as reported in the previous study [35 (link)].

Tofogliflozin (Kowa Co. Ltd., Tokyo, Japan) was prepared at a final concentration of 0.5 mg·L⁻¹ in the tank containing the fish to be treated, as described previously 25; this concentration is the C_max of 500 ng·mL⁻¹ in humans treated with a standard Tofo dose of 20 mg. Briefly, Tofo was dissolved in dimethyl sulfoxide (Nacalai Tesque, Kyoto, Japan) to a concentration of 100 mg·mL⁻¹ and then added to the water of the plastic tank at a final concentration of 0.5 mg·L⁻¹. The same amount of dimethyl sulfoxide was administrated to the tank of the HFD group as vehicle control.