Fig 1.
Effects of empagliflozin on glucose homeostasis.
(A) Blood glucose levels in vehicle- and empagliflozin-treated (3mg/kg for 8 days) mice during an intraperitoneal glucose tolerance test. (B) Total change in blood glucose levels over time, expressed as area under curve (AUC). (C) The effect of empagliflozin on random blood glucose levels (BGL) in T1DM mice after (A) 4-day and (D) 7-day drug administration (3mg/kg). Data are expressed as means±S.E.M. *P <0.05; **P < 0.01 vs vehicle group. (n = 9–11 for each group).
Fig 2.
Effects of empagliflozin on insulin expression and serum levels.
The effect of vehicle or empagliflozin treatment (3mg/kg) for 8 days on insulin expression (A) mRNA, and (B) serum levels. Data are expressed as mean±S.E.M. *P <0.05 vs vehicle group. (n = 10–11 for each group for serum insulin; n = 8 for each group for mRNA).
Fig 3.
Assessment of insulin and glucagon producing cells after empagliflozin treatment.
β-cell mass was examined from the whole pancreata isolated from T1DM mice after treatment with empagliflozin (3mg/kg) or vehicle for 8 days. (A) Double fluorescence labeling using glucagon (green) and insulin (red) in pancreas sections from vehicle- and empagliflozin-treated mice. (B) Bar graph shows the % of β-cell area/ total pancreatic area. (C) Bar graph shows the % of α-cell area/ total pancreatic area. (D) Bar graphs show the mean area of insulin/glucagon ratio per pancreatic cross-section. Data are expressed as mean±S.E.M. Scale bar = 100 μm. **P<0.01 vs vehicle group. (n = 10–12 for each group).
Fig 4.
Measurement of β-cell proliferation after empagliflozin treatment.
Immunohistochemical staining of Ki-67 of whole pancreata was performed from T1DM mice after treatment with empagliflozin (3mg/kg) or vehicle for 8 days. (A) Double fluorescence labeling using insulin (green) and Ki-67 (red) in pancreatic sections from vehicle- and empagliflozin-treated mice. Arrows indicate Ki-67-positive β-cells. (B) Bar graphs show the % of Ki-67 positive β-cell/ total number of β-cells. Data are expressed as mean±S.E.M. Scale bar = 100 μm. *P<0.05 vs vehicle group. (n = 8 for each group).
Fig 5.
Measurement of β-cell apoptosis after empagliflozin treatment.
TUNEL assay of whole pancreata was performed from T1DM mice after treatment with empagliflozin (3mg/kg) or vehicle for 8 days. (A) Double fluorescence labeling using insulin and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay in pancreas sections from vehicle- and empagliflozin-treated mice. (B) Bar graphs show the % of TUNEL positive β-cell/total number of β-cells. Data are expressed as mean±S.E.M. Scale bar = 100 μm.*P<0.05 vs vehicle group. (n = 10–12 for each group).
Fig 6.
Measurement of β-cell ROS levels after empagliflozin treatment.
DHE staining of whole pancreata was performed from T1DM mice after treatment with empagliflozin (3mg/kg) or vehicle for 8 days. (A) Double fluorescence labeling using insulin and dihydroethidium (DHE) staining in pancreas sections from vehicle- and empagliflozin-treated mice. (B) Bar graphs show the % of DHE positive β-cell/total number of β-cells. Data are expressed as mean±S.E.M. Scale bar = 100 μm. *P<0.05 vs vehicle group. (n = 10–12 for each group).