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.