Pharmacological studies were conducted in compliance with FELASA protocols using 40 male mice including 10 wild-type (WT) and 30 db/db (BKS.Cg Dock7m+/+ Leprdb/J) mice. Starting from 3 weeks of age, all db/db mice were fed a high-fat diet (HFD) (S0372 E010, ssniff Spezialdiäten, Soest, Germany)
[18]. After 3 weeks of being on the HFD, the 6-week-old animals were treated for 2 weeks via gavage once a day between 5:00-6:00 p.m. before the onset of the dark phase (6:00 p.m.). The treatment groups consisted of 10 vehicle-gavaged (VG) diabetic mice that were vehicle-gavaged with a solution of 5% solutol and 95% hydroxyethylcellulose, 10 MET mice treated with 300 mg/kg metformin (Sigma Aldrich, Taufkirchen, Germany), and 10 SGLT2i + MET mice treated with 30 mg/kg SGLT2i (AVE2268, Sano AG, Frankfurt, Germany) and 300 mg/kg metformin (Sigma Aldrich, Taufkirchen, Germany) [19] .
After the completion of the treatment period, which lasted for 2 weeks, the 8-week-old mice (± 3 days) underwent a fasting period of four hours before being sacri ced. An overdose of iso urane was used for euthanasia, and immediate blood and organ collection were performed as previously reported [18, 20] . Murine plasma was prepared from whole blood by centrifugation at 4°C, and tissues were freezeclamped. All samples were stored at -80°C until further analyses.
[18]. After 3 weeks of being on the HFD, the 6-week-old animals were treated for 2 weeks via gavage once a day between 5:00-6:00 p.m. before the onset of the dark phase (6:00 p.m.). The treatment groups consisted of 10 vehicle-gavaged (VG) diabetic mice that were vehicle-gavaged with a solution of 5% solutol and 95% hydroxyethylcellulose, 10 MET mice treated with 300 mg/kg metformin (Sigma Aldrich, Taufkirchen, Germany), and 10 SGLT2i + MET mice treated with 30 mg/kg SGLT2i (AVE2268, Sano AG, Frankfurt, Germany) and 300 mg/kg metformin (Sigma Aldrich, Taufkirchen, Germany) [19] .
After the completion of the treatment period, which lasted for 2 weeks, the 8-week-old mice (± 3 days) underwent a fasting period of four hours before being sacri ced. An overdose of iso urane was used for euthanasia, and immediate blood and organ collection were performed as previously reported [18, 20] . Murine plasma was prepared from whole blood by centrifugation at 4°C, and tissues were freezeclamped. All samples were stored at -80°C until further analyses.
