dugh@imm.ac.cn
Word count: 2956 words
Acknowledgements
This work was financially supported by the following foundations: the
CAMS Initiative for Innovative Medicine (CAMS-I2M, 2016-I2M-3-007 and
2017-I2M-1-010), National Major Research Development Program of China
(2018ZX09711001-012, 2018ZX09711001-003-005, and 2017YFG0112900) and the
National Natural Science Foundation of China (81470159 and 81770847).
Conflict of interest
The authors declare that there are no conflicts of interest.
Author contributions:
L.Y., G.F.Q., X.Y.Y. and G.H.D. participated experimental design; L.Y.
X.C. N.L. W.H.J. N.Q.W. B.Y.H. and X.Y.Y. performed the experiments;
L.Y., X.C., N.L. and X.Y.Y analyzed the data; L.Y., X.Y.Y. and G.H.D.
wrote the manuscript; and L.Y., L.Z., G.F.Q., X.Y.Y. and G.H.D. edited
the manuscript.
Abstract
Background and Purpose:Puerarin
is an important isoflavone component extracted from Pueraria
lobate in traditional Chinese medicine. It has a wide range of
pharmacological effects. Increasing evidence indicates that puerarin
alleviates hyperglycemia and numerous
related complications. In this study, we explored the effect of puerarin
on skeletal muscle atrophy caused by
type 1 diabetes in rats.
Experimental Approach: Male Sprague Dawley (SD) rats with
streptozotocin (STZ)-induced type 1
diabetes were used in this study. We measured skeletal muscle weight,
size and strength together with the transformation of skeletal muscle
types in type 1 diabetic rats. Skeletal muscle L6 cells were used forin vitro study.
Key Results: Puerarin
increased muscle tissue weights and improved muscle strength. An
enhanced skeletal muscle cross-sectional area was accompanied by reduced
mRNA expression of muscle atrophy marker genes, including
F-box only protein 32 (Atrogin-1)
and muscle-specific RING-finger 1(Murf-1), both in vitro andin vivo . The transformation
from
type I fibers (slow muscle) to type
II fibers (fast muscle) was also observed after puerarin administration.In vitro studies suggested
that puerarin upregulated Akt/mTOR but downregulated
the LC3/p62
signaling pathway, eventually
resulting in muscle hypertrophy.
Conclusions and Implications:Our study observed that puerarin
mitigated skeletal muscle atrophy in
type 1 diabetic rats. Subsequently,
we found that the related mechanisms closely involved the upregulation
of protein synthesis via the Akt/mTOR signaling pathway. Whether this
anti-diabetic muscle atrophy effect in mice applies to humans remains
unknown.
Keywords
Puerarin; type 1 diabetes; skeletal muscle atrophy; rats; protein
synthesis; muscle type transformation
Abbreviations
Atrogin-1, F-box only protein 32; AUC, area under the curve; COX,
cytochrome oxidase; DM, differentiation medium; FoxO3a, forkhead box
class O factor 3a; HG, high glucose; I, insulin; IPGTT, intraperitoneal
glucose tolerance test; LA, lactic acid; M, mannitol; Mstn, myostatin;
Murf-1, muscle-specific RING-finger 1; Myh1, myosin heavy chain IIx/d;
Myh2, myosin heavy chain IIa; Myh4, myosin heavy chain IIb; Myh7, myosin
heavy chain I; MyHC, myosin heavy chain; PA, pyruvic acid; SDH,
succinate dehydrogenase; STZ, streptozotocin; T1D, type 1 diabetes; TA,
tibialis anterior;
Introduction
Diabetes is one of the most
challenging public health problems and affects approximately 425 million
people worldwide, leading to poor health outcomes and high health care
costs (Arneth, et al.,2019). Type 1 diabetes (T1D) is a major subtype of
diabetes and is an autoimmune disease that is characterized by the
destruction of islet β cells in the pancreas triggered by genetic and
environmental factors (Zheng, et al.,2018).
T1D patients have many complications
involving heart, kidney, and other tissues. Type 1 diabetic subjects
display a dramatic loss of muscle, causing severe skeletal muscle
atrophy as the disease progresses (Sala and Zorzano,2015). Skeletal
muscle is a major target tissue of diabetic damage (Krause, et
al.,2011). Skeletal muscle atrophy is a pathological condition defined
as a reduction in muscle mass caused by excessive protein degradation
(Hong, et al.,2019). Skeletal muscle atrophy disrupts quality of life
and increases mortality and morbidity (Powers, et al.,2016).
An increasing number of studies
indicate that the balance between protein synthesis and degradation is
responsible for muscle atrophy (Hoffman and Nader,2004). Pathways
involved in this process include the mammalian target of rapamycin
(mTOR) signaling pathway (Laplante and Sabatini,2012), which is a
crucial component of the anabolic machinery for protein synthesis, and
the ubiquitin-proteasome pathway (Milan, et al.,2015), which is
responsible for the turnover of the majority of soluble and myofibrillar
muscle proteins, such as myosin heavy chain (MyHC), via transcriptional
activation of a set of E3 ligase-encoding genes, e.g., muscle
RING-finger 1 (Murf1) and F-box only protein 32 (Atrogin-1) (Chen, et
al.,2019). In addition, autophagy also exerts a vital role in the
degradation of skeletal muscle as a consequence of an ordered
transcriptional program involving a battery of genes, e.g., LC3 and p62
(Llano-Diez, et al.,2019). Although our understanding of the molecular
mechanisms involved in skeletal muscle atrophy has substantially
progressed in recent decades, no effective drug has come into the market
to date. Thus, there is an urgent unmet need for the development of
novel drugs to combat skeletal muscle atrophy.
In traditional Chinese medicine, Pueraria lobata is a medical and
edible plant that is widely distributed in eastern and southern Asia
(Chen, et al.,2018). Puerarin is one of its major bioactive components.
Puerarin is an isoflavone compound with a polyphenol structure. Puerarin
has documented therapeutic effects on diabetes and diabetic
complications (Hsu, et al.,2003; Wu, et al.,2013; Chen, et al.,2018;
Yang, et al.,2019; Yin, et al.,2019). Puerarin exerts a hypoglycemic
effect (Tanaka, et al.,2016), improves insulin resistance (Chen, et
al.,2018), and protects islet cells (Rojas, et al.,2018) in diabetes
patients. In addition, puerarin also has beneficial effects on diabetic
complications, especially diabetic cardiovascular complications (Pan, et
al.,2009; Li, et al.,2016), diabetic nephropathy (Li, et al.,2017), and
diabetic retinopathy (Ren, et al.,2000; Zhu, et al.,2014).
In this study, we hypothesis that
puerarin may exert protective effects on skeletal muscle atrophy
provoked by type 1 diabetes. We aim to contribute to the research and
development of drugs for diabetic skeletal muscle atrophy.
Methods
2.1 Materials
Puerarin (HPLC, 98%) was provided as a lyophilized powder by the
Institute of Materia Medica, Chinese Academy of Medical Sciences
(Beijing, China). Streptozotocin (STZ) was
purchased from Sigma (St. Louis, MO,
USA). Blood glucose testing was performed using an Accu-Chek Active
meter from Roche (Basel, Switzerland). Lactic acid (LA) and
pyruvic acid (PA) content kits as
well as succinate dehydrogenase
(SDH) and cytochrome oxidase (COX)
staining kits were purchased from
Solarbio
(Beijing, China). SuperScript III
reverse transcriptase and TRIzol isolation reagent were obtained from
Invitrogen (Carlsbad, CA, USA). Direct-zol RNA kits were obtained from
ZYMO research (Irvine, CA, USA). SsoFast™ EvaGreen® Supermix was
obtained from Bio-Rad (Hercules, CA,
USA). RIPA buffer, 5× loading buffer, and enhanced chemiluminescence
kits were purchased from Applygen
(Beijing, China). Protease inhibitor
cocktail and phosphatase inhibitor cocktail were obtained from CWbio
(Jiangsu, China). Protein concentrations were quantified using a BCA
assay kit purchased from Thermo Fisher Scientific (Rockford, IL, USA).
Antibodies used in this study are listed in Table 1.
2.2 Animal care
Male Sprague-Dawley (SD) rats (160-190 g) were obtained from Beijing
HuaFuKang Bioscience Co., Ltd. (Beijing, China). The rats were housed
under a 12-hour light/dark cycle at a temperature of 22 ± 3°C and a
humidity of 55 ± 5%. Rats were given free access to food and water for
seven days before the experiment. All animal procedures were approved by
the animal care and use committee of the Institute of Materia Medica,
Chinese Academy of Medical Sciences and were performed in strict
accordance with research guidelines for the care and use of laboratory
animals.
2.2 Type 1 diabetic animal modeling and drug
treatments
Briefly, 30 rats were fed adaptively for 1 week, and then 6 rats were
randomly divided into the normal control group (control). The remaining
rats were injected intraperitoneally with 65 mg kg-1streptozotocin (STZ). Fasting blood glucose was tested using blood
obtained from the tail tip after 1 week of STZ injection. In addition,
20 rats with fasting blood glucose levels greater than 16.5 mmol
L-1 were chosen as type 1 diabetic rats and randomly
divided into two groups: T1D control group (model, N=10) and puerarin
administration group (puerarin, N=10). The administration group was
treated with orally with 100 mg
kg-1day-1 puerarin, while the normal control group and the
diabetic model group were treated with a corresponding dose of normal
saline.
2.3 Animal muscle strength
detection
The grip strength test was employed
to determine foreleg tensile force using a grip strength meter provided
by Beijing Zhishuduobao Biological Technology (Beijing, China) per the
manufacturer’s instruction. In brief, rats were
positioned to grasp the metal bar
with forelegs only and then pulled horizontally until letting go. Pulls
were repeated thrice with three
rounds in sequence in rats. For repetitive pulls, rats were given a
30-second pause in between the pulls; between rounds, rats were given a
30-min rest period. The maximum grip
strength of the 9 pulls was used for analysis.
The inclined plane test was employed
using a modified Rivlin method (Rivlin and Tator,1977). The rats were
positioned on a 2-mm thick, 14-cm wide and 24-cm long plate with a
rubber mat loaded on a rectangular wooden board. The rat was placed on
the mat with its head up, and its body was parallel to the longitudinal
axis of the inclined plane. The angle between the sloping plate and the
ground was 90 degrees. The maximum
persistent duration on the sloping
plate was recorded, and the test was repeated thrice.
The wire-hanging test was performed with a 3 mm-diameter wire. The wire
was suspended at a height of 0.5 m. Rats hung from the center of the
wire by exclusively using forelegs to grab the wire. The time until the
rat fell from the wire was recorded. The test was performed thrice in
succession, and the average value was calculated. Values for voluntarily
jumps were not included in the calculation.
2.4 Histological analysis
Skeletal muscle tissues were fixed in 10% formalin. Transverse paraffin
sections of 5 μm in thickness were
subjected to hematoxylin-eosin (HE)
staining. For succinate dehydrogenase (SDH) and cytochrome oxidase (COX)
staining, gastrocnemius muscle
samples were obtained and flash frozen in liquid nitrogen. Then, 10-μm
thick frozen sections were subject to staining according to the
instructions of SDH and COX staining kits (Solarbio, Beijing, China).
2.5 Quantitative real-time
PCR
Total
RNA was isolated using TRIzol isolation reagent (Invitrogen, USA) and
then further purified with Direct-zol RNA kits (ZYMO Research, USA).
First-strand cDNA was synthesized using 1.5 μg of total RNA with a
reverse transcription reaction mix that included SuperScript III reverse
transcriptase (Invitrogen, USA)
and Oligo-dT17 as primers. Gene expression was detected using SsoFast™
EvaGreen® Supermix (Bio-Rad, USA) on a CFX-96
real-time PCR System (Bio-Rad, USA) with gene-specific primer pairs
(Table S1). The results were
quantified after normalization with TBP (Radonić, et al.,2004).
2.6 Cell culture and
differentiation
L6 rat myoblasts were purchased from ATCC (Manassas, Virginia, USA) and
cultured in DMEM with 10% FBS
serum, 100 I.U.
mL-1 penicillin and 100 μg mL-1streptomycin (Invitrogen, USA). The differentiation of myoblasts into
myotubes was achieved by incubating confluent myoblasts with
differentiating media (2% FBS serum, 100 I.U. mL-1penicillin and 100 μg mL-1 streptomycin) for six days.
2.7 Western Blot analysis
Protein samples from gastrocnemius
muscle and cells were lysed in ice-cold
RIPA
buffer (Applygen, Beijing, China) supplemented with
protease inhibitor cocktail and
phosphatase inhibitor cocktail (CWbio, Jiangsu, China). Lysates were
clarified by centrifugation (12,000 × g for 15 min at 4°C), and
the concentration was quantified
using a BCA assay kit (Thermo Fisher Scientific, Rockford, IL, USA).
Samples were mixed with 5 × loading buffer
(Applygen,
Beijing, China), and proteins were then denatured at 100°C for 10 min.
For immunoblotting, equal amounts of protein were fractionated by
SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes.
Then, membranes were blocked with 5% bovine serum albumin (BSA) for 2 h
at room temperature. Membranes were incubated with primary antibodies
(Table S2) followed by a horseradish peroxidase-conjugated secondary
antibody (Cell Signaling Technology, MA, USA). Immunoreactive bands were
detected by enhanced
chemiluminescence (Applygen, Beijing, China). Semi-quantitative
grayscale intensity was measured using Gel-pro 3.1 (Media Cybernetics,
Silver Spring, MD, USA).
2.8 Immunofluorescence
analysis
To determine the diameter of
myotubes in vitro, L6 myotubes were fixed with 4% paraformaldehyde for
30 min at room temperature, permeabilized with 0.5% Triton X-100 in PBS
for 15 min, and then blocked with 5% BSA in PBST for 1 h at room
temperature. Myotubes were incubated with an anti-MyHC antibody
(MF-20,
1:100, DSHB) diluted in 5% BSA
overnight at 4°C. After washing in wash buffer, myotubes were incubated
with Goat Anti-Mouse IgG H&L (Alexa Fluor® 488, ab150113, 1:1000,
Abcam) for 1 h at room temperature. Nuclei were stained with DAPI
(1:1000, Sigma). Images were captured by a high-content screening
machine (Cellomics, Thermo Fisher Scientific, USA), and the diameter of
the myotubes was measured by ImageJ software (NIH, USA).
2.9 Data and statistical
analysis
The exact group size (n) for each experimental group/condition is
provided, and “n” refers to independent values not replicates. Results
are expressed as the means ± SEM. Statistical analysis was performed
using GraphPad Prism 7.0 (GraphPad Software, Inc. CA, USA). For
statistical comparisons, unpaired two-tailed Student’s t test or one-way
ANOVA or Chi-square (or Fisher’s exact) test was used as appropriate.
Differences were considered significant at P≤0.05.
Results
Effect of puerarin on general indicators of type 1
diabetic rats induced by STZ
Body weights of rats were assessed every week during the 8-week
treatment (Figure 1a). Compared with
those in the normal control group, body weights in the diabetic control
group significantly decreased. However, 100 mg kg-1puerarin treatment had no pronounced effect on body weights of T1D rats
with the exception of values recorded in the 3rd week.
Puerarin also has no significant effect on food intake (Figure 1b).
Fasting blood glucose levels did not
change significantly under 100 mg kg-1 puerarin
treatment (Figure 1c). Glucose tolerance was assessed using the
intraperitoneal glucose tolerance test
(IPGTT) at week 8 of treatment. The
results showed that 100 mg kg-1 puerarin decreased
blood glucose levels after intraperitoneal injection of glucose at 60
min (Figure 1d). However, there was
no notable decrease in the area under the curve (AUC) of IPGTT in the
puerarin administration group
compared with the T1D control group (Figure 1e). T1D modeling increased
both blood lactic acid and pyruvic acid levels in rats, while 100 mg
kg-1 puerarin treatment did not show a significant
effect (Figure 1f, 1g).
3.2 Puerarin improved skeletal muscle weight, strength
and fiber area in type 1 diabetic
rats
Skeletal muscle atrophy is characterized by reductions in muscle mass
and fiber size; thus, we explored the effect of puerarin on skeletal
muscle function and structure. Skeletal muscle strength is a main
indicator for muscle function. After one month, foreleg tensile force
was reduced in type 1 diabetic rats (Figure. 2a). Conversely, no change
in time on the inclined plane and hanging wire was observed in the model
group, while puerarin treatment extended time both on the inclined plane
and hanging wire (Figure 2b, 2c). After two months of drug
administration, our results showed an impressive decrease in skeletal
muscle strength in type 1 diabetic rats, including foreleg tensile force
and time on the inclined plane and hanging wire. Simultaneously, this
change was reversed by puerarin treatment as revealed by a marked
improvement in foreleg tensile force (Figure 2a) and time on the
inclined plane (Figure 2b) and hanging wire (Figure 2c).
Our data showed that the muscle weights, including the soleus muscle
that predominantly contains slow-twitch fibers, the fast-twitch muscle
tibialis anterior (TA) and mixed-type gastrocnemius muscle, were
remarkably decreased in T1D rats compared with normal controls. After 8
weeks of puerarin administration, weights of the soleus, TA and
gastrocnemius muscles significantly increased (Figure 2d, 2f, 2h). In
addition, muscle tissue indexes were also increased after puerarin
treatment compared with those of diabetic controls (Figure 2e, 2g, 2i).
Then, hematoxylin-eosin (HE)
staining was also performed to
clarify the effect of puerarin on
muscle fiber
size (Figure 3a).
In the present study, we found that
the cross-sectional areas of the soleus, TA and gastrocnemius muscles
all significantly shrank; however, after two months of puerarin
treatment, the cross-sectional areas of the three muscle types
strikingly
increased (Figure 3b-d). Consistent
with the muscle fiber size results, the
frequency distributions of muscle
fiber areas for different muscle fibers yielded similar results (Figure
3e-g).
3.3 Puerarin downregulated muscle atrophic markers in T1D
rats
The occurrence and development of muscle atrophy were accompanied by
significant changes in muscle
atrophic factors, such as F-box only protein 32
(Atrogin-1) and muscle-specific
RING-finger 1 (Murf-1) (Cohen, et
al.,2015; Lee, et al.,2018). We measured the expression of Atrogin-1,
Murf-1 and myosin heavy chain
(MyHC) mRNA, which represents the
amount of muscle fibers. Atrogin-1
and Murf-1 were prominently upregulated, and MyHC was downregulated in
T1D rats. On the other hand, puerarin administration distinctly reversed
this trend as evidenced by the downregulation of Atrogin-1 and Murf-1
and the upregulation of MyHC (Figure
4a-c). In parallel with changes in
mRNA levels, changes in levels of Atrogin-1, Murf-1 and MyHC protein
also yielded the same results
(Figure 4d-g). Moreover, mRNA
expression of myostatin (Mstn), which specifically acts as a negative
regulator of skeletal muscle growth, was also decreased in the
gastrocnemius (Figure 4c).
3.4 Puerarin promoted the transformation from slow-twitch
muscle to fast-twitch muscle in T1D
rats
We investigated the effect of puerarin on the change in skeletal muscle
fiber types and the preferred metabolic types. Our study found that
T1D rats exhibited a tendency to
transform muscle from a glycolytic
type (fast or type II fibers) to
oxidative type (slow or type I
fibers). After 8 weeks of puerarin treatment, this trend was
attenuated (Figure 5a) as
demonstrated by a dramatic increase in integrate optical density and the
ratio of type I fibers to type II fibers in the model group. However,
these parameters decreased in the group administered
puerarin (Figure 5b-e). More
specifically, puerarin increased the muscle fiber area of fast-twitch
glycolytic type IIB fibers but reduced the area of slow oxidative
type I
fibers
(Figure 5f, 5g). In addition, the expression of Myh7, Myh2, Myh4 and
Myh1, which encode myosin isoforms MyHC-I, MyHC-IIa, MyHC-IIb and
MyHC-IIx/d, respectively, were also analyzed, and the results confirmed
that the production both of fast-twitch and slow-twitch fibers was
increased in the puerarin-treated group compared with the T1D model
group (Figure 5h).
3.5 Puerarin ameliorated muscle atrophy induced by high
glucose in L6
myotubes
We also explored the effect of puerarin in L6 myotubes under high
glucose stimulation in vitro . We used a
dose-response
(D-glucose, 0, 50, 100 mM) and time-response (D-glucose, 0, 6, 12, 24,
48, 72 h) model to investigate the effect of glucose concentration on
the expression of muscle atrophic
markers, including Atrogin-1 and Murf-1, in myotubes. As shown in Figure
6a, b, 100 mM glucose resulted in
a time-dependent increase the expression of muscle atrophic markers
(Atrogin-1 and Murf-1), and significant differences were noted at 24-72
h (Figure 6c, 6d). We then used 100
mM glucose and a 48-h incubation for subsequent experiments. Using an
equal concentration of mannitol as a hyperosmotic control and 100 nM
insulin as a protective control, we treated L6 myotubes with 10, 100,
1000 μM puerarin with or without 100 mM glucose for 48 h. Here, cells
incubated in 100 mM glucose for 48 h also demonstrated upregulated
myostatin (Mstn) expression and downregulated myosin heavy chain (MyHC)
expression. Puerarin did not affect the expression of muscle atrophic
markers in normal L6 myotubes but obviously downregulated the expression
of Atrogin-1, Murf-1 and Mstn
(Figure
6e-g). Puerarin enhanced MyHC expression (Figure 6h) in a dose-dependent
manner.
Consistent with the results of mRNA expression, Western Blot analysis
showed that puerarin markedly inhibited Atrogin-1 and Murf-1 and
improved MyHC expression (Figure
7a-d). To further determine the effect of puerarin on the phenotype of
L6 myotubes, MyHC immunofluorescence staining was performed (Figure 7e).
The results showed that 100 mM glucose reduced the myotube area. When
treated with puerarin, the area of myotubes under 100 mM glucose
stimulation increased significantly (Figure 7f). In addition, the
analysis of frequency distribution of myotube area yielded similar
results (Figure 7g).
3.6 Puerarin attenuated autophagy and upregulated
Akt/mTOR signaling in L6
myotubes
Muscle atrophy is characterized by an imbalance in protein synthesis and
degradation. To explore the mechanism of puerarin, we assessed the
autophagy pathway, which plays a
vital role in protein degradation (Llano-Diez, et al.,2019), and the
Akt/mTOR pathway, which is closely associated with protein synthesis
(Laplante and Sabatini,2012; Ogasawara, et al.,2016; Yoon,2017). Our
results revealed that 100 mM glucose promoted the autophagy signaling
pathway as determined by upregulated phosphorylation of ULK1, p62
expression and the ratio change of LC3II to LC3I. Administration of
puerarin remarkably restrained these changes, reflecting an inhibitive
effect on the autophagy pathway
(Figure 7a-d). In addition, phosphorylation of Akt, mTOR, and its
downstream targets, the 70 kDa ribosomal S6 kinase (p70S6K) and
eIF4E-binding protein 1 (4EBP1), was downregulated by 100 mM glucose in
L6 myotubes. Phosphorylation of Forkhead box class O factor 3a (FoxO3a),
a main transcription factor regulating Atrogin-1 expression (Sandri, et
al.,2004), was also significantly decreased under 100 mM glucose
incubation in L6 myotubes. Notably, phosphorylation of Akt,
mTOR, p70S6K, 4EBP1 and FoxO3a
was increased after puerarin
treatment (Figure 7e-j).
Discussion
In this paper, we demonstrated that
puerarin (100 mg
kg-1) administration could significantly improve
skeletal muscle atrophy in type 1 diabetic rats. This robust effect in
puerarin-treated rats was manifested by increased skeletal strength and
weights (soleus, TA and gastrocnemius), enlarged muscle fiber size,
downregulated muscle atrophic markers (Atrogin-1 and Murf-1) and
increased MyHC levels. Puerarin also promoted the transformation from
slow-twitch muscle to fast-twitch muscle. Subsequently, we found that
the anti-diabetic muscle atrophic effect of puerarin was closely related
to attenuation of autophagy and upregulation of Akt/mTOR signaling.
In this study, we first demonstrated that puerarin administration could
significantly ameliorate diabetic skeletal muscle atrophy. This effect
is consistent with a previous report that
the extraction of the plant from
which puerarin is derived, namely, Radix Pueraria lobata (RP),
prevented the skeletal muscle atrophy induced by a high-fat diet (HFD)
in mice (Jung, et al.,2017). Additionally, one study reported that
puerarin reduced triceps surae atrophy in mice with sciatic nerve injury
(Wu, et al.,2014). Our study did not find that puerarin (100 mg
kg-1) obviously changed the animal’s body weight
compared with the diabetic model group. Considering that puerarin can
promote the oxidation of fatty acids (Kim, et al.,2016; Cheung, et
al.,2017; Oh, et al.,2019) and prevent the accumulation of
intramyocellular lipids in diabetic rats (Chen, et al.,2018), we
hypothesize that puerarin increases the weights of skeletal muscle while
reducing fat content, resulting in almost no observed change in body
weight.
Muscle fiber types can be roughly
divided into fast and slow muscles, which represent glycolytic
metabolism and oxidative metabolism, respectively (Schiaffino,2018).
Muscle fiber types can transform from slow-twitch (oxidative type) to
fast-twitch (glycolytic type) or vice versa (Zhang, et al.,2019), which
represents a change in the type of muscle metabolism. There are few
reports about changes in skeletal muscle fiber types caused by type 1
diabetes. In this paper, we reveal a transformation in muscle fiber
types in type 1 diabetic rats from fast to slow type. Two months of
diabetic conditions in rat were necessary and sufficient to cause muscle
atrophy.
Studies have found that puerarin regulates activities of the
mitochondrial respiratory chain complex in diabetic nephropathy (Liang,
et al.,2019). In addition, puerarin could exert a role of mitochondrial
protection (Chen, et al.,2018; Wang, et al.,2018, 2018). Additionally,
puerarin has been reported to increase mitochondrial biogenesis in C2C12
cells (Jung, et al.,2017). This evidence indicated that puerarin could
regulate energy metabolism in many different cells and tissues.
Consistent with these reports, in our study, we stained for succinate
dehydrogenase (SDH) and cytochrome oxidase (COX), which are aerobic
metabolic enzymes that reflect aerobic metabolism levels in tissues
(Özkök, et al.,2015). Our results suggest that puerarin promotes muscle
metabolism in a glycolytic manner under diabetic conditions as evidenced
by promoting muscle fibers to transform from slow-twitch muscle to
fast-twitch muscle.
Previous studies reported that several factors are involved in skeletal
muscle mass regulation, including Akt/mTOR
signaling (Laplante and
Sabatini,2012; Ogasawara, et al.,2016; Yoon,2017) and autophagy
signaling (Cid-Díaz, et al.,2017; Jiao and Demontis,2017; Pratiwi, et
al.,2018). In addition, the forkhead box (FOX) protein family has been
implicated as a key regulator of muscle loss under various conditions,
such as diabetes and sepsis (Kang, et al.,2017). Other reports showed
that puerarin could upregulate phosphorylation of Akt and mTOR and
inhibit autophagy, exerting an antifibrotic effect in atrial fibroblasts
(Xu, et al.,2019). However, it was also reported that puerarin induced
apoptosis in HPV-positive HeLa cervical cancer cells by inhibiting
PI3K/Akt/mTOR signaling (Jia, et al.,2019). Moreover, puerarin
suppresses autophagy to protect the rat brain against
ischemia/reperfusion injury (Wang, et al.,2018), prevents the
progression of experimental hypoxia-induced pulmonary hypertension
(Zhang, et al.,2019), and provides a neuroprotective effect against
transient cerebral ischemia at the ischemic penumbra in neurons
(Hongyun, et al.,2017). In the present study, we clarified that puerarin
alleviated muscle atrophy by increasing protein synthesis via the
Akt/mTOR signaling pathway while
inhibiting the autophagy signaling pathway. In addition, the
phosphorylation of transcriptional factor FoxO3a was downregulated by
puerarin, which is consistent with a previous report that
FoxO3a played a regulatory role in
autophagy and the ubiquitin-proteasome system during muscle atrophy
(Sandri, et al.,2004).
Considering all the therapeutic effects we identified in our study, it
is theoretically possible that puerarin may serve as a potential
therapeutic agent targeting diabetic skeletal muscle atrophy. In
addition, whether puerarin can be utilized in other types of atrophy
remains unknown. Study on key regulatory factors and the target of
puerarin should be performed in the future.
Taken together, our study reports for the first time that puerarin can
ameliorate skeletal muscle atrophy in T1D animals. This finding not only
provides a therapeutic indication for puerarin but also facilitates drug
development for diabetic muscle
atrophy.