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Tables
Table S1. Primers used for real-time PCR
Table S2. Antibodies used in the Western Blot assay
Figure legends
Figure 1. Effect of
puerarin on general indicators of STZ-induced type 1 diabetic rats. Rats
were treated with 100 mg kg-1 day-1puerarin intragastrically for two months. (a) Change in body weight was
detected every week. (b) Food intake at the 8th week.
(c) Fasting blood glucose at the 8th week. (d)
Blood-glucose curve of IPGTT. (e) AUC of IPGTT. (f) LA content in serum.
(g) PA content in serum. a, d, e, Data are represented as the means ±
SEM (control, n = 6; model, n = 10; puerarin, n = 8). Statistical
analysis tested by one-way ANOVA with Dunnett’s multiple comparisons
test analysis. b, f, g, Data are represented as the means ± SEM (n = 5).
Statistical analysis tested by one-way ANOVA with Dunnett’s multiple
comparisons test analysis. c, Data are represented as the means ± SEM
(control, n = 6; model, n = 10; puerarin, n = 10). Statistical analysis
tested by one-way ANOVA with Dunnett’s multiple comparisons test
analysis. # p < 0.05, ## p < 0.01 and ### p
< 0.001 vs. the control group. * p < 0.05 vs. the
model group.
Figure 2. Puerarin increased skeletal muscle strength and
weight in type 1 diabetic rats. a-c, Effect of 100 mg
kg-1 day-1 puerarin on muscle
strength. (a) Foreleg tensile force. (b) Time on inclined plane. (c)
Time on hanging wire. d-i, Muscle
weights and muscle tissue indexes (muscle weight/body weight).
(d) Soleus weight. (e) Soleus
tissue index. (f) TA weight. (g) TA tissue index. (h) Gastrocnemius
weight. (i) Gastrocnemius tissue index. Data presented are individual
values with means ± SEM from n=5 rats for each group. Statistical
analysis tested by one-way ANOVA with Dunnett’s multiple comparisons
test analysis. # p < 0.05, ## p < 0.01 and ###
p < 0.001 vs. the control group. * p < 0.05, ** p
< 0.01, *** p < 0.001 and vs. the model group.
Figure 3.Puerarin improved skeletal muscle
fiber area in type 1 diabetic rats. (a) Representative images of H&E
staining of soleus, TA and gastrocnemius muscle (400 x magnification).
(b) Cross-sectional area of soleus muscle. (c) Cross-sectional area of
TA muscle. (d) Cross-sectional area of gastrocnemius muscle. e-g,
Frequency distribution of cross-sectional area of three types of muscle
fibers in the (e) soleus, (f) TA, and (g) gastrocnemius muscles. b-d,
Values shown are the means ± SEM from 3 fields, n=5.
Statistical analysis tested by
one-way ANOVA with Dunnett’s multiple comparisons test. e-g, Statistical
analysis tested by Chi-square (and Fisher’s exact) test. ### p
< 0.001 vs. the control group. *** p < 0.001 and vs.
the model group.
Figure 4. Puerarin
downregulated the expression of
muscle atrophic markers in type 1
diabetic rats. a-c, Total mRNA extracted from the soleus, TA and
gastrocnemius muscles was used for real-time PCR analysis. (a)
Atrogin-1, Murf-1
and MyHC expression in the soleus
muscle. (b)
Atrogin-1, Murf-1 and MyHC
expression in the TA muscle. (c) Atrogin-1, Murf-1, MyHC and Mstn
expression in the gastrocnemius muscle. d-g, Total protein extracted
from the gastrocnemius muscle was used for Western Blot analysis. (d-g)
Immunoblot analysis of Atrogin-1,
Murf-1 and MyHC of the gastrocnemius muscle. Data are represented as the
means ± SEM (n=5). Statistical analysis tested by one-way ANOVA with
Dunnett’s multiple comparisons test analysis. # p < 0.05,
## p < 0.01 and ### p < 0.001 vs. the control
group. * p < 0.05, ** p < 0.01, *** p <
0.001 and vs. the model group.
Figure 5.Effect of puerarin on muscle fiber
phenotype in type 1 diabetic rats. Frozen sections of gastrocnemius
muscle samples were subject to SDH and COX staining. (a) Representative
images of SDH (above) and COX (below) staining in the gastrocnemius
muscle. (b) Integrated optical
density analysis of SDH staining. (c) Muscle fiber number ratio (type
I/type II) in SDH staining. (d) Integrate optical density analysis of
COX staining. (e) Muscle fiber number ratio (type I/type II) in COX
staining. (f) Analysis of muscle
fiber area of type I, type IIA and type IIB in SDH staining. (g)
Analysis of muscle fiber area of type I, type IIA and type IIB in COX
staining. (h) Myh1, Myh2, Myh4 and Myh7 expression in the gastrocnemius
muscle. b-e, Values shown are means ± SEM from n=5. Statistical analysis
tested by one-way ANOVA with Dunnett’s multiple comparisons test
analysis. f, g, Values shown are means ± SEM from 4 fields (n=6). h,
Values shown are means ± SEM from n=5. Statistical analysis tested by
one-way ANOVA with Dunnett’s multiple comparisons test analysis. # p
< 0.05, ## p < 0.01 and ### p < 0.001
vs. the control group. * p < 0.05, ** p < 0.01, ***
p < 0.001 and vs. the model group.
Figure 6.Puerarin decreased muscle atrophic
markers in L6 myotubes. a-d, Fully differentiated L6 rat myotubes were
treated with D-glucose. (a, b) Dose-response effect of high glucose (0,
50, 100 mM) for 48 h on the expression of muscle atrophic markers (a)
Atrogin-1 and (b) Murf-1 gene. (c,
d) Time-response effect of 100 mM glucose (0 h, 6 h, 12 h, 24 h, 48 h,
72 h) on (c) Atrogin-1 and (d)
Murf-1 gene expression. e-h, 10, 100, 1000 μM puerarin or 100 nM insulin
or 100 mM mannitol were used to treat fully differentiated L6 rat
myotubes with or without 100 mM glucose for 48 h, and total mRNA was
extracted for real-time PCR analysis. (e)
Expression of Atrogin-1. (f)
Expression of Murf-1. (g) Expression of Mstn. (h) Expression of MyHC.
Data are represented as the means ± SEM from n=3 experiments.
Statistical analysis tested by one-way ANOVA with Dunnett’s multiple
comparisons test analysis. # p < 0.05, ## p <
0.01 and ### p < 0.001 vs. DM group. * p < 0.05,
** p < 0.01, *** p < 0.001 and vs. HG group.
Figure 7.Puerarin ameliorated muscle atrophy
induced by high glucose in
L6 myotubes. a-d, Fully
differentiated L6 myotubes were treated with 10, 100, 1000 μM puerarin
or 100 nM insulin with or without 100 mM glucose for 48 h, and total
protein was extracted for Western Blot analysis. (a-d) Immunoblot
analysis of Atrogin-1, Murf-1 and MyHC. e-g, Immunofluorescence staining
for MyHC (100 x magnification) in L6 rat myotubes treated with 10, 100,
1000 μM puerarin or 100 nM insulin with or without 100 mM glucose for 48
h. (e) Representative images of immunofluorescence staining for MyHC.
(f) Myotube average area analysis
of L6 rat myotubes. (g) Frequency distribution of myotube area. b-d,
Data are represented as the means ± SEM from n=3 experiments.
Statistical analysis tested by one-way ANOVA with Dunnett’s multiple
comparisons test analysis. f, Values shown are means ± SEM from 5 fields
(n=3). Statistical analysis tested by one-way ANOVA with Dunnett’s
multiple comparisons test analysis. g, Statistical analysis tested by
Chi-square (and Fisher’s exact) test. ## p < 0.01 and ###
p < 0.001 vs. DM group. * p < 0.05, ** p <
0.01, and *** p < 0.001 vs. the HG group.
Figure 8. Puerarin
attenuated autophagy but upregulated Akt/mTOR signaling in L6 myotubes.
Fully differentiated L6 myotubes were treated with 10, 100, or 1000 μM
puerarin or 100 nM insulin with or without 100 mM glucose for 48 h, and
total protein was extracted for Western Blot analysis. (a-d)
Immunoblot analysis of
phosphorylation of ULK1, p62 and LC3II/I. (e-j) Immunoblot analysis of
phosphorylation of Akt, FoxO3a, mTOR, p70S6K and 4EBP1. Data are
represented as the means ± SEM from n=3 experiments. Statistical
analysis tested by one-way ANOVA with Dunnett’s multiple comparisons
test analysis. # p < 0.05, ## p < 0.01 and ###
p < 0.001 vs. DM group. * p < 0.05, ** p <
0.01, *** p < 0.001 and vs. HG group.