Empagliflozin Incorporation into High-Fat Diet

A subset of mice from each HFD group was subsequently placed on empagliflozin as described in Section 3. Mice were divided and housed in cages (2–3 cages per group) without a standardised randomisation procedure. Empagliflozin (Advanced ChemBlocks, Hayward, CA, USA) was incorporated into the HFD rodent chow at a concentration of 116.7 mg empagliflozin per 1 kg chow. This incorporation was accomplished by first pulverising the rodent chow with a mortar and pestle and spreading it over a large glass plate. The chow was then evenly sprayed with an empagliflozin solution that consisted of empagliflozin dissolved in 50% ethanol (11.67 mg/mL). The food was allowed to dry on the bench top for 30 min for ethanol evaporation and was then mixed with a 7% starch solution (tapioca starch in water; 2 mL solution per 100 g of chow) to reform the pellets. The control HFD chows used throughout this study were subjected to the same procedure, but empagliflozin was omitted from the 50% ethanol solution. The added starch (1.4 mg/g of chow) did not significantly change the caloric content (kcal/g) or the carbohydrate percentage of either HFD.
Mice were allowed to feed ad libitum. Each mouse consumed approximately 2.5–3 g of food per day delivering an approximate empagliflozin dose of 0.3–0.35 mg/day.