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Introduction
Type 2 diabetes mellitus (T2DM) is one of the toughest
cases of human diseases worldwide. The number of T2DM
patients is expected to affect 300 million people by the year
2025[1] due to an increased number of elderly people, a greater
prevalence of obesity, and sedentary lifestyles. Therefore,
it is very important to reinforce the effective prevention and
cure of this disease. Traditional Chinese medicines and their
extractions demonstrate the characteristics of economy and
effectiveness to cure diabetes and its complications. In
traditional Chinese medicine, Astragalus is commonly found in
mixtures with other herbs, and is used in the treatment of
numerous ailments, including heart, liver, and kidney
diseases, as well as cancer, viral infections, and immune
system disorders. Western herbalists began using Astragalus in the 1800s as an ingredient in various tonics. The use of Astragalus became popular in the 1980s based on theories
about anticancer properties, although these proposed
effects have not been clearly demonstrated in reliable
human studies. Astragalus polysaccharides (APS), the
polysaccharide component of the ethanol extract of
Astragalus roots is an important active component of
Astragalus. Apart from the actions of the antioxidant,
antihypertensive, and immunomodulatory
activities[2_4] of APS, we found that it also shows insulin-sensitizing and
hypoglycemic activity by decreasing the elevated
expression and activity of protein-tyrosine phosphatase 1B (PTP1B)
in the skeletal muscles of T2DM rats in our previous
research[5].
Among the 3 major insulin-responsive tissues (fat, muscle,
and liver), liver plays a central role in the control of glucose
homeostasis and is subject to complex regulation by insulin
and other hormones[6]. Many studies have proved that the
impaired regulation of hepatic glucose production is a
characteristic feature of the metabolic syndrome, which is also
known as "insulin resistance
syndrome"[7]. It has recently been discovered that endoplasmic reticulum (ER) stress
maybe a key link between obesity, insulin resistance, and
T2DM[8]. Hepatocytes have a well-developed ER structure
and ER stress is involved in the development of hepatic
insulin resistance[9]. In light of the important role of the liver
in glucose homeostasis and the pathogenesis of diabetes,
we sought to examine the potential effect of APS on the
insulin signal and ER stress response signal in hepatocytes
of diabetic animals and a high glucose-treated cell model.
In this study, we provide evidence that the mechanistic
link mentioned earlier can be exploited for therapeutic
purposes with the traditional oral Chinese herb APS, which
alleviates ER stress in high glucose-treated HepG2 cells and a
diabetic animal model. The treatment of obese and diabetic
KKAy mice, which is the yellow offspring of the KK mice,
expressed Ay gene, with APS resulted in the normalization of
hyperglycemia, restoration of systemic insulin sensitivity,
resolution of fatty liver disease, and the enhancement of
insulin action in the liver. Our results demonstrated that
APS can improve insulin sensitivity coupled with the
enhanced adaptive capacity of the ER and acts as potent
antidiabetic modalities with potential application in the
treatment of type 2 diabetes.
Materials and methods
Plant materials and preparation of APS Astragalus
membranaceus (Fisch) and Bunge var
mongholicus (Bunge) Hsiao were purchased from Shanghai Medicinal Materials
(Shanghai, China) and identified by the Department of
Authentication of Chinese Medicine, Hubei College of
Chinese Traditional Medicine (Wuhan, China). We used the
representative specimen which had been kept in our
laboratory by anterior researchers. In brief, APS was extracted
with optimized techniques using direct water decoction, as
described previously[10]. Three subtypes of APS are defined
by phytochemical screening: APSI, II, and III (1.47:1.21:1).
APSI consists of d-glucose, d-galactose, and
l-arabinose in molar ratios of 1.75:1.63:1 and has an average molecular
weight of 36 300 kDa. Both APSII and APSIII are dextrans,
the linkage mode of which is mainly α-(1>4) linkages, and in
which α-(1>6) linkages are exiguous. APS is a hazel-colored
and water-soluble powder. It was diluted to 12% in normal
saline before use.
Biochemical reagents GAPDH (ab9845), glycogen
synthase kinase 3 beta (GSK3β, 9332), p
(ser9)-GSK3β (9336), and p (ser641)-GS (3891) were purchased from
Abcam (Abcam, Cambridge, UK) and Cell Signaling
Technology (Danvers, MA,USA). DMSO (D098-100) and
tunicamycin (Tun, T-7765) were from Sigma (St Louis, MO,
USA). Dulbecco's modified Eagle's medium (1×), liquid
(no glucose) (11966025)without glucose and sodium
pyruvate containing L-glutamine were from Invitrogen-Gibco
(Frederick, MD, USA). Enhanced chemiluminescence (ECL)
was performed by using the protein detector Lumi-GLO
western blot kit from Kierkegaard and Perry Laboratories
(Gaither-sburg, MA, USA). The BCA (bicincho-ninic acid) protein
assay kit was from Pierce Biotechnology (Rockford, IL, USA).
The RevertAid first strand cDNA synthesis kit (K1622) and
reagents for PCR were from Fermentas (Glen Burnie, MD,
USA). The EZNA gel extraction kit was from Omega (Peqlab,
Erlangen, Germany). The DyNAmo SYBR green qPCR kit
was from Finnzymes (Ipswich, MA, USA). All other
chemicals and reagents were of analytical grade.
Mouse models and administration of APS Female KKAy
and C57BL/6J mice from the age of 8 weeks were obtained
from the Chinese Academy of Medical Sciences (Beijing,
China) and housed individually in plastic cages at 20
oC, with lighting on from 6:00_18:00. The C57BL/6J mice
were fed a normal chow diet consisting (as a percentage of
total kcal) of 12% fat, 60% carbohydrates, and 28% protein.
The KKAy mice were fed a high-fat diet consisting of 41%
fat, 41% carbohydrates, and 18% protein. The KKAy mice
were a cross between glucose-intolerant black KK female
mice and male, yellow, obese Ay mice and are known to
serve as excellent models of T2DM, while
C57BL/6J mice with normal diets are generally used as non-diabetic
controls[11_14]. All experimental procedures were approved and
carried out in compliance with the guidelines of the Wuhan
University School of Medicine Committee on Animals.
From 12 weeks of age the animals were given APS (700
mg·kg-1·d-1) orally or vehicle treatment (PBS,
phosphate-buffered saline) for 2 months at the same time. Plasma
glucose, insulin, glycogen[15], free fatty acid (FFA), and
triglyceride[16] were measured with commercial kits. Before
necropsy, saline- and APS-treated animals were
intraperitoneally administered insulin (10 U/kg body wt) in saline
or vehicle (saline) after overnight fasting. After 10 min, the
mice were killed and the liver was rapidly
excised and snap frozen in liquid nitrogen.
Insulin sensitivity (oral glucose tolerance test and
the homeostasis model assessment, HOMA-IR) Insulin
sensitivity was identified by the comprehensive analysis of the
oral glucose tolerance test (OGTT) and calculated
HOMA-IR (HOMA-IR index=FPG(Fasting plasma glucose)
[mmol/L]×FINS(fasting insulin)
[µU/mL]/22.5)[17]. The OGTT was
performed after a 16 h overnight fast. Glucose (2 g/kg) was
administered orally and blood was collected from the orbital
sinus at 0, 30, 60, and 120 min,
respectively[18]. The area under the curve (AUC) was calculated for glucose during
the OGTT (AUC=0.5× [Bg0+Bg30]/2+0.5× [Bg30+Bg60]/2+1×
[Bg60+Bg120]/2).
Liver histology The liver samples were embedded in
paraffin, and sections were cut into 6 mm slices. Neutral
lipid was stained with Sudan III on frozen sections. The
hepatocyte ultrastructure was presented by transmission
electron microscope (TEM). The total operative
procedures complied with the standard protocols.
Cell culture and pretreatment with APS The human
hepatocarcinoma cell line HepG2 cells were cultured in
minimal essential medium containing 10% fetal bovine serum and
0.5 mg/mL geneticin, supplemented with 100 U/mL penicillin,
100 µg/mL streptomycin, and 2 mmol/L
l-glutamine in a humidified atmosphere with 5%
CO2 at 37 oC. The cells were
plated at a density of 3×104
cells/cm2 and maintained in culture medium for 24 h before treatment. The HepG2 cells were
grown on coverslips and treated with tunicamycin (Tm, 10
µg/mL)[19] for 16 h as positive ER stress cell control and treated
with high glucose culture medium (30 and 45 mmol/L,
respectively) for 5 h to induce ER stress response
in vitro. The HepG2 cells were pretreated with APS (200 µg/mL) for 24
h.
Analysis of XBP1 (XhoI site-binding protein 1) mRNA
transcription and splicing by real-time RT-PCR RNA was
extracted from the liver samples and HepG2 cells using Trizol
reagent. Gene transcription was analyzed by semiquantity
RT_PCR and real-time PCR. The total RNA concentration
and purity were determined by measuring the
OD260 and
OD260/OD280 ratio. The specific primers for the mouse and
human samples are as follows: mXBP1 mRNA, sense:
5'-AAACAGAGTAGCAGCGCAGACTGC-3' and antisense: 5'-TCCTTCTGGGTAGACCTCTGGGAG-3';
mβ-actin mRNA, sense: 5'-TCATCACTATTGGCAACGAGC-3' and antisense:
5'-AACAGTCCGCCTAGAAGCAC-3'; hXBP1 mRNA, sense: 5'-AAACAGAGTAGCAGCTCAGACTGC-3' and antisense:
5'-TCCTTCTGGGTAGACCTCTGGGAG-3'; and hβ-actin mRNA, sense: 5'-CAGGGCGTGATGGTGGGCA-3' and
anti-sense: 5'-CAAACATCATCTGGGTCATCTTCTC-3'. In brief,
1 µg total RNA was used to prepare cDNA. Real-time PCR
reaction was performed by the following thermal cycling
contidions: 94 oC for 4 min, 94
oC for 10 s, 65 oC for 30 s,
repeated for 40 cycles, and 72 oC for 30 s. Digestion with
PstI (which cuts only in the unspliced cDNA) was used to
distinguish the unspliced from the spliced bands and then we ran
a carefully prepared 2% gel that would present the 2 PCR
products clearly. This protocol works for the human and
mouse genes. The quantity of specific mRNA was
normalized as a ratio to the amount of β-actin mRNA.
Analysis of protein expression and phosphorylation by
Western blotting The cell lysates from tissues or cells were
prepared in 1 mL lysis buffer (20 mmol/l Tris, pH 7.5, 5
mmol/L EDTA, 10 mmol/L
Na4P2O7, 100 mmol/L NaF, 2 mmol/L
Na3VO4, 1% Nonidet P-40, 1 mmol/L phenyl-methylsulfonyl fluoride,
and 10 µg/mL aprotinin) on ice in 1.5 mL microtubes. The
lysates were solubilized by continuous stirring for 1 h at 4
oC and centrifuged for 10_15 min at 14
000×g. The supernatants were collected and protein concentrations were measured
with BCA protein assay reagent and then stored at -80
oC until further analysis. The cell lysates were subjected to
SDS-PAGE and blotted onto a polyvinylidene difluoride
membrane followed by incubation with the primary
antibodies anti-GSK3β, anti-ser9GSK3β, and anti-ser641GS. The
proteins were detected with an ECL system.
Statistical analysis All values are expressed as mean±
SEM. Statistical significance was determined using ANOVA
followed by Turkey's test. P<0.05 was considered
statistically significant.
Results
Effect of APS treatment on systematic glucose
metabolism and insulin sensitivity in KKAy mice
Characteristics of experimental animals To
investigate the in vivo effects of APS, we employed obese and
diabetic (KKAy) mice, a model of severe obesity and
insulin resistance. The glucose levels of both fasting and fed
mice were significantly upregulated in KKAy mice (T2DM)
than those in normal C57BL/6J mice (control) (1.6-fold,
P<0.05; 3.6-fold, P<0.05, respectively; Figure 1), which
were significantly reduced after treatment with APS (700
mg·kg-1·d-1,
po) for 8 weeks. Notably, the insulin
concentrations in KKAy mice were higher than the values of the
control mice (6-fold, P<0.05), but treatment with APS did
not affect the insulin levels in both the control and type 2
diabetic mice, suggesting that the action of APS may not be
mediated by changing the insulin levels. The KKAy
diabetic mice weighed 20 g more than the normal chow-fed
C57BL/6J mice (20.4±0.4 vs 40.3±0.6,
P<0.05). APS treatment could significantly inhibit body weight gain in
diabetic mice (KA: 41.0±0.9 vs KK: 45.6±1.4,
P<0.05) and had no effect on that of the control mice (CA: 20.9±0.1; C:
21.1±0.1, P>0.05; Figures 2, 3). All data are expressed as
mean±SEM calculated from the results of 8_10 mice
Insulin sensitivity The KKAy mice did not show
significant reductions in insulin levels after APS treatment (KA
mouse group; Figure 1). To confirm whether APS improved
glucose intolerance in KKAy mice, we performed a OGTT
and observed that the mice treated with APS showed a lower
peak of plasma glucose concentration at 30 min after the
glucose load. The plasma glucose level declined more
rapidly compared with that in the vehicle-treated KKAy mice
(Figure 4). Accordingly, after APS therapy, the HOMA-IR
index in the KA mouse group was significantly lower than
that in the KK mouse group (10.0±0.51 vs
15.5±0.52, P<0.05). These results suggest that the blood glucose-lowering
effect of APS is due to increased systemic insulin sensitivity.
In addition, neither of these parameters, blood glucose and
insulin levels, were different the between APS-treated (C
group mice) and vehicle-treated lean control C57BL/6J mice
(CA group mice). Taken together, the impaired insulin
sensitivity in the T2DM rats was improved following APS
treat-ment.
Liver pathology and biochemistry Obesity and
diabetes in mice and humans is associated with alterations in
liver lipid metabolism and fatty liver
disease[19_21]. APS treatment resulted in the resolution of the obesity-induced
lipid accumulation and glycogen synthesis in the liver from
KKAy mice (Figure 5). There was a significant reduction in
liver triglyceride and FFA content in the APS-treated KKAy
mice compared to the control animals (P<0.05; Figure 5).
Consistent with this, liver Sudan III staining also showed a
significant alleviation of fatty degeneration in APS-treated
KKAy mice (Figure 6). Additionally, the dilated ER of
hepatocytes was observed by TEM, which can be ameliorated
with APS therapy (Figure 7).
Effect of APS treatment on hepatic insulin signal
transduction in KKAy mice In an attempt to understand the
ameliorating effect of APS on insulin signal transduction in the
liver tissue, we next examined whether APS affected the
expression and activity of hepatic GSK3β in the obese and
diabetic KKAy mouse model. APS treatment significantly
reduced GSK3β protein levels in KKAy mice
(P<0.05, Figure 8). Importantly, treatment with APS significantly induced
GSK3β phosphorylation at serine 9, which is the inactivated
form of this kinase (P<0.05, Figure 8). Hepatic glycogen
synthase (GS), which is a key enzyme in the regulation of
glycogen synthesis, is regulated by the phosphorylation of
the sites between Ser641 and
Ser653 targeted by GSK3[22]. To
support this, GSK3 inhibitors stimulate hepatic glycogen
synthase. Thus, we further examined the effect of APS on
the insulin-induced Ser641 phosphorylation of GS. APS
treatment significantly reduced GS phosphorylation at the
Ser641 site (P<0.05, Figure 9). These results indicate that APS has a
positive effect on hepatic insulin transduction.
Effect of APS treatment on markers of ER stress in KKAy
mice As metabolic demands increase, for example, in the
state of hyperglycemia, ER becomes over loaded, which can
perturb the protein folding in this protein factory. The
distressed ER may further contribute to impaired insulin action
in obesity and ER stress leads to the development of insulin
resistance and eventually type 2
diabetes[8]. To confirm whether APS can act as an agent to enhance the adaptive
capacity of the ER, XBP1 transcription and splicing in the
liver of the KK group mice was detected with a significant
increase compared with the controls (P<0.05), indicating that
high ER stress exists in this diabetic animal model (Figure
10). Notably, APS administration could reduce the level of
XBP1 (P<0.05) in KKAy mice. Spliced XBP1 levels in the
liver from the KK group were also significantly increased
compared with those of the KA group (P<0.05). XBP1
transcription and splicing in the liver of normal control mice was
not affected by APS treatment (Figure 10A, 10D).
Effect of APS treatment on the high glucose-treated
HepG2 cell model The transcription factor XBP1 is a basic
motif-leucine zipper protein. The spliced or processed form
of XBP1 (XBP1s) is a key factor in ER stress through the
transcriptional regulation of an array of genes, including
molecular chaperones[23_26]. The modulation of XBP1s in
cells could alter insulin action via its potential impact on the
magnitude of the ER stress
responses[27]. Hyperglycemia is a direct cause of insulin resistance, which is the hallmark of
T2DM. We next examined whether ER stress is increased in
high glucose conditions and whether APS can perform this
effect on ER stress in vitro by measuring XBP1 transcription
and splicing in the high glucose-treated cell model. High
glucose (30 mmol/L) is suitable to induce significant ER
stress. We observed that APS inhibited high
glucose-induced ER stress responses in cultured HepG2 cells (Figure
10), while it had no significant effect on Tm-induced ER stress.
These may indicate that APS is not an antagonist of
tunicamycin, and APS inhibition of ER stress is likely by
modification of metabolic disturbance and maintaining
glucose homeostasis.
Discussion
T2DM is one of the most prevalent and serious
metabolic diseases in the world. Hyperglycemia can directly cause
insulin resistance, which is associated with an imbalance
between endocrine pancreatic function and hepatic and
extrahepatic insulin sensitivity[28]. Among the 3 major
insulin-responsive tissues (fat, muscle, and liver), the liver plays
a central role in the control of glucose homeostasis; insulin
signaling in liver is critical in maintaining normal hepatic
function[6,7]. Many studies have proved that the impaired
regulation of hepatic glucose production is a
characteristic feature of the metabolic
syndrome[29], which is also known as
"insulin resistance syndrome", including obesity, insulin
resis-tance, type 2 diabetes, and other metabolic
disorders[20].
The ER is a membranous network that provides a
specialized environment for processing and folding newly
synthesized proteins. As metabolic demands increase, which
can perturb the protein folding in the ER, so does the
work-load of this protein factory, collectively called ER
stress[19]. Since hepatocytes have a well-developed ER structure, ER
stress is involved in liver-related
diseases[9]. Sustained ER stress, which appears to occur as a result of obesity and
diabetes, modulates insulin action in the
liver[8]. The development of hepatocellular ER stress as a result of
diabetes/obesity appears to be a major contributor to insulin
resistance[9]. Inhibiting ER stress in the liver or increasing
hepatic sensitivity to insulin might break the vicious circle
linking hyperinsulinemia and insulin resistance that leads to
elevated triglyceride and FFA concentrations,
progressive steatosis, ultrastructural mitochondrial
lesions in the hepatocytes, and ultimately hepatocyte death. Therefore,
we wonder whether the beneficial function of APS on the
insulin signal pathway is associated with suppressing ER
stress in the liver.
In this study, we adopted KKAy mice as a model of T2DM.
KKAy mice show hyperglycemia, hyperlipidemia, and hyperinsulinemia compared with C57BL/6J mice. Consistent
with our previous study[5], we prove that APS has
significant hypoglycemic activity and insulin-sensitizing effects in
the present study. The diabetic mouse model was
significantly obesity-resistant and showed alleviated hepatic fatty
degeneration in response to APS therapy. As the liver is the
hinge of nutritive material metabolism, hepatic insulin
resistance has been suggested to be a later factor in the
development of hyperglycemia. Increased hepatic glucose
production is tightly correlated with fasting hyperglycemia in type
2 diabetics[30]. The presence of fatty liver inT2DM and obese
patients, which is also known as non-alcohol fatty liver, has
long been reported. Obesity-related insulin resistance
might be partly responsible for liver fat
deposition[21], so it is important for us to detect whether APS can ameliorate hepatic
insulin sensitivity and its mechanism.
To further analyze the action of APS on hepatic glucose
metabolism and insulin action, we observed the expression
and activity of hepatic GSK3b, one of the important negative
regulators of insulin signal transduction in the liver.
Previous studies suggested that the inhibition of GSK3 (ser9 for
β subunit) in animal models of diabetes leads to the
normalization of blood glucose levels and improved hepatic and
peripheral insulin resistance, while high GSK3 activity has been
reported in T2DM, which is also involved in diminished
levels of the IPF1/PDX1 (islet transcription factor1, also known
as IPF-1, IDX-1, and STF-1) protein and β cell dysfunction
during the progression of
diabetes[31,32]. Our findings indicate that the hypoglycemic activity of APS is mediated by
insulin sensitivity improvement at least partly related to GSK3
inhibition. Further-more, it has been recently reported that
GSK3 may play a central role in signaling the downstream
effects of ER stress[33,34].Since ER stress-induced
lipid accumulation and cell death play a role in the
pathogenesis of disorders, diabetes mellitus, and hepatic
steatosis[34,35], one can speculate that the protection against ER stress-induced
cellular dysfunction occurs while GSK3 activity is inhibited.
Thus, GSK3 inhibition undoubtedly leads to insulin
sensitivity improvement which will promote a beneficial cycle
coupled with the enhancement of ER function to cope with
metabolic alterations.
To determine how APS performs its function on the
management of metabolic abnormalities associated with obesity
and diabetes, in the present study we hypothesize that the
reversal of hyperglycemia, increases glucose tolerance and
insulin sensitivity induced by APS is related to a decrease in
ER stress so APS-treated KKAy mice should display a
reduction in ER stress. Spliced XBP1 mRNA induced by
activated IRE1 (Inositol-Requiring Enzyme 1) is translated to
the protein, a potent transcription factor that induces BiP
expression[21,25]. XBP1 is also induced by activated ATF6
(Activating Transcription Factor 6)
[23]. It is thus thought to be an important marker reflecting both IRE1 and ATF6
signaling in response to ER
stress[24,36]. So it is important to distinguish between the spliced and non-spliced form of
XBP1 mRNA for the quantitative measurement of
XBP1 gene expression[37,38]. Our data indicated that in APS-treated
KKAy mice, the transcription and splicing of XBP1 in the
liver was markedly reduced in comparison with the controls.
Similar to these results, high glucose (30 mmol/L) was used
to induce ER stress in a cultured cell model, and increased
XBP1 gene expression and splicing was significantly
suppressed in HepG2 cells pretreated with APS. Therefore, APS
has a role in inhibiting hepatic ER stress in the state of
hyperglycemia. We concluded that APS promotes insulin
signal transduction, thus it enables insulin-sensitizing and
hypoglycemic activity, which is related to the enhanced
adaptive capacity of the ER. The alleviation of ER stress also
contributes to insulin signaling. This positive interaction
shows that APS has a promising application in the treatment
of type 2 diabetes.
Although our results in this study show a remarkable
pharmacological effect on hepatic insulin resistance, we
cannot directly extrapolate our results to humans. Hence, it is
necessary to reveal a more detailed mechanism of APS in the
improvement of insulin resistance in humans in our
future research. In conclusion, ER stress is a key link
between obesity, insulin resistance, and type 2 diabetes. Our
study provides new evidence that APS renders its
hypoglycemic action through decreasing liver insulin resistance
coupled with alleviating ER stress. In addition, the
treatment of obese and diabetic mice with APS resulted in a
significant alleviation of hyperglycemia, restoration of systemic
insulin sensitivity, resolution of fatty liver disease, and
enhancement of insulin action in liver tissue. Our research
demonstrates that APS can enhance the adaptive capacity
of the ER and act as potent antidiabetic modalities with
promising application in the treatment of type 2 diabetes.
Acknowledgment
We appreciate the help given by the Experimental Animal
Center of Wuhan University (No SCXK 2003-0003) and other
cooperative partners.
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