Figures Legends
Figure 1 . Effects of AST and CAG on plasma glucose and glucose tolerance in KM mice. 18-22 g male KM mice were gavage administrated with AST (5 mg/kg) and CAG (5 mg/kg) by for ten days. Fasted mice overnight and performed the OGTT experiment by orally administrated glucose at a dosage of 2 g/kg, determined plasma glucose at the time point of 0, 15, 30, 60, and 120 min. The curve and area under curve (AUC) were calculated by Origin 8 software. Dapagliflozin (1 mg/kg) and Metformin (100 mg/kg) were used as positive controls. (A) Chemical structures of AST andCAG . (B) The content of plasma glucose in the OGTT experiment. (C) The calculated AUC in the OGTT experiment.#p <0.05, compared with the lean group; *p <0.05, compared with the vehicle group. N = 8 mice/group.
Figure 2 . Effect of CAG on glucose uptake in cells. HEK293 cells with over-expression SGLT2 were treated with CAG(10 μM), Dapagliflozin (10 μM), and Metformin (1 mM) alone or combined with insulin (10 μg/mL) for 1 h, then cells were stained with 2-NBDG (120 μM) and Hoechst 33342 (1 μg/mL) at 37 ºC for 30 min. Representative images were captured and fluorescence were quantified. Image magnification, ×100. #p <0.05, compared with the vehicle cells; *p <0.05, compared with the insulin-treated cells. N = 5 independent experiments.
Figure 3. Determination of the effects of CAG on ameliorating diabetes related MetS in ZDF diabetes rats. ZDF diabetes rats were treated with Dapagliflozin (1 mg/kg/d) and CAG (1 or 5 mg/kg/d) for five weeks, respectively. Relative parameters were determined. (A) Change of plasma glucose level in ZDF diabetes rats; (B-C) OGTT test after one-week treatment of drugs; (D) Change of body weight of the ZDF rats; (E) Levels of glucose, HbA1c, and insulin in serum, respectively; (F-H) Levels of triglylceride, cholesterol, and cholesterol in serum, respectively; (I) Ratio of HDL-c/LDL-c in serum.#p <0.05 compared with lean group mice; *p <0.05 compared with vehicle group mice. N = 8 mice / group.
Figure 4. Determination of the protective effect ofCAG on coronary artery in ZDF diabetic rats. Rats were treated with Dapagliflozin (1 mg/kg/d) and CAG (1 or 5 mg/kg/d) for five weeks, the tissues coronary artery were collected and subjected to H&E and PAS staining. (A) Representative H&E and PAS staining image of coronary artery. Image magnification, ×200. (B) Determination of the thickness of coronary artery.#p <0.05, compared with lean group mice; *p <0.05, compared with vehicle group mice. N = 8 mice / group.
Figure 5. Determination of the anti-fibrosis effects ofCAG in the heart and the kidney of ZDF diabetic rats. Rats were treated with Dapagliflozin (1 mg/kg/d) and CAG (1 or 5 mg/kg/d) for five weeks, the tissues of heart and kidney were collected and subjected to pathological determination and immunohistochemistry assay for TGF-β1. (A-C) Sirius red and Masson staining of the heart and quantification. Image magnification, ×200. (D-F) Sirius red and Masson staining of the kidney and quantification. Image magnification, ×200. (G-I) Determination of the protein TGF-β1 in the heart and the kidney by using an immunohistochemistry assay and quantification.#p <0.05, compared with lean group mice; *p <0.05, compared with vehicle group mice. N = 8 mice / group.
Figure 6. Effects of CAG on glucose reabsorption in the kidney of ZDF rats. Rats were treated with Dapagliflozin (1 mg/kg/d) and CAG (1 or 5 mg/kg/d). After 1 week of treatment, the urinary glucose (A), urinary sodium (B) and urine efflux (C) within 24 hours were determined. After 5 week of treatment, the total urinary glucose (D), urinary sodium (E) and urine efflux (F) in 24 hours were determined. (G) Effect of CAG on the water intake after 1- or 5-week treatment. #p <0.05, compared with lean group mice; *p <0.05, compared with vehicle group mice. N = 8 mice / group.
Figure 7. Inhibition of CAG on SGLT2’s expression. (A) Effects of CAG and Dapaglifloin on the expression of SGLT2 in the kidney of the ZDF rats by using an immunohistochemistry assay. (B) Determination of the SGLT2 expression in HEK293 cell by using Western blot. NC indicates cells without transfection by any plasmid, SGLT2 indicates cells transfected with the plasmid plvx-puro-SGLT2, and Ctrl indicates cells transfected with the control plasmid. (C) The HEK293 cells with over-expression of SGLT2 were treated with Dapagliflozin andCAG for 24 hours, and cells were subjected to SGLT2 determination by using Western blot.
Figure 8. Proposed mechanism underlying the anti-diabetes effects of CAG in ZDF diabetic rat.