Astragalus polysaccharide reduces hepatic endoplasmic reticulum stress and restores glucose homeostasis in a diabetic KKAy mouse model.

Abstract

To examine the potential effects of Astragalus polysaccharide (APS) on hepatic endoplasmic reticulum (ER) stress in vivo and in vitro and its link with hypoglycemia activity, thus establishing the mechanism underlying the hypoglycemic action of APS.
The obese and type 2 diabetic KKAy mouse model, which is the yellow offspring of the KK mice expressed Ay gene (700 mg/kg-1/d-1, 8 weeks) and a high glucose-induced HepG2 cell model (200 microg/mL, 24 h) were treated with APS. The oral glucose tolerance test was measured to reflex insulin sensitivity with the calculated homeostasis model assessment (HOMA-IR) index. XBP1 (XhoI site-binding protein 1) transcription and splicing, an indicator of ER stress, was analyzed by RT-PCR and real-time PCR. The expression and activation of glycogen synthase kinase 3 beta (GSK3beta), an insulin signaling protein, was measured by Western blotting.
APS can alleviate ER stress in cultured cells in vivo. The hyperglycemia status, systemic insulin sensitivity, fatty liver disease, and insulin action in the liver of diabetic mice were partly normalized or improved in response to APS administration.
Our results indicate that APS enables insulin-sensitizing and hypoglycemic activity at least in part by enhancing the adaptive capacity of the ER, which can further promote insulin signal transduction. Thus, APS has promising application in the treatment of type 2 diabetes.

Full text

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Acta Pharmacol Sin 2007 Dec; 28 (12): 1947–1956
©2007 CPS and SIMM
Full-length article
Astragalus polysaccharide reduces hepatic endoplasmic reticulum stress
and restores glucose homeostasis in a diabetic KKAy mouse model1
Xian-qing MAO2, Yong WU3, Ke WU2, Ming LIU2, Jing-fang ZHANG4, Feng ZOU2, Jing-ping OU-YANG2,5
2Department of Pathophysiology, Medical College of Wuhan University, Hubei Provincial Key Laboratory of Allergy and Immune-Related
Diseases, Wuhan 430071, China; 3Division of Endocrinology and Diabetes, Department of Medicine, University of Oklahoma Health Science
Center, Oklahoma city, Oklahoma 73104, USA; 4Department of Pathology and Physiology, Jingmen Vocational College, Jingmen 448000,
China
Abstract
Aim: To examine the potential effects of Astragalus polysaccharide (APS) on
hepatic endoplasmic reticulum (ER) stress in vivo and in vitro and its link with
hypoglycemia activity, thus establishing the mechanism underlying the hypogly-
cemic action of APS. Methods: The obese and type 2 diabetic KKAy mouse
model, which is the yellow offspring of the KK mice expressed Ay gene (700
mg·kg-1·d-1, 8 weeks) and a high glucose-induced HepG2 cell model (200 µg/mL, 24 h)
were treated with APS. The oral glucose tolerance test was measured to reflex
insulin sensitivity with the calculated homeostasis model assessment (HOMA-
IR) index. XBP1 (XhoI site-binding protein 1) transcription and splicing, an indica-
tor of ER stress, was analyzed by RT–PCR and real-time PCR. The expression and
activation of glycogen synthase kinase 3 beta (GSK3β), an insulin signaling protein,
was measured by Western blotting. Results: APS can alleviate ER stress in cul-
tured cells in vivo. The hyperglycemia status, systemic insulin sensitivity, fatty
liver disease, and insulin action in the liver of diabetic mice were partly normalized
or improved in response to APS administration. Conclusion: Our results indicate
that APS enables insulin-sensitizing and hypoglycemic activity at least in part by
enhancing the adaptive capacity of the ER, which can further promote insulin
signal transduction. Thus, APS has promising application in the treatment of type
2 diabetes.
Key words
Astragalus membranaceus; polysaccharide;
type 2 diabetes mellitus; insulin resistance;
endoplasmic reticulum; stress
1 Project supported by grants from the National
Natural Science Foundation of China (No
30370673).
5 Correspondence to Dr Jing-ping OU-YANG.
Phn 86-27-8733-1146.
Fax 86-27-8733-1077.
E-mail jpoy@163.com
Received 2007-04-26
Accepted 2007-06-14
doi: 10.1111/j.1745-7254.2007.00674.x
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 tra-
ditional 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 sys-
tem 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

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Acta Pharmacologica Sinica ISSN 1671-4083Mao XQ et al
hypoglycemic activity by decreasing the elevated expres-
sion 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 char-
acteristic 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 pur-
poses with the traditional oral Chinese herb APS, which alle-
viates 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 en-
hanced adaptive capacity of the ER and acts as potent an-
tidiabetic modalities with potential application in the treat-
ment 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 Chi-
nese Traditional Medicine (Wuhan, China). We used the
representative specimen which had been kept in our labora-
tory 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 Tech-
nology (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 pyru-
vate containing L-glutamine were from Invitrogen-Gibco
(Frederick, MD, USA). Enhanced chemiluminescence (ECL)
was performed by using the protein detector Lumi-GLO west-
ern 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 chemi-
cals 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 con-
trols[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 trig-
lyceride[16] were measured with commercial kits. Before
necropsy, saline- and APS-treated animals were intraperi-
toneally administered insulin (10 U/kg body wt) in saline

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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 sen-
sitivity 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 per-
formed 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 proce-
dures complied with the standard protocols.
Cell culture and pretreatment with APS The human
hepatocarcinoma cell line HepG2 cells were cultured in mini-
mal 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 con-
trol and treated with high glucose culture medium (30 and
45 mmol/L, respectively) for 5 h to induce ER stress re-
sponse 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 distin-
guish 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 normal-
ized 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 antibod-
ies anti-GSK3β, anti-ser9GSK3β, and anti-ser641GS. The pro-
teins 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 statisti-
cally significant.
Results
Effect of APS treatment on systematic glucose metabo-
lism and insulin sensitivity in KKAy mice
Characteristics of experimental animals To investi-
gate the in vivo effects of APS, we employed obese and
diabetic (KKAy) mice, a model of severe obesity and insu-
lin 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 concen-
trations 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

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Acta Pharmacologica Sinica ISSN 1671-4083Mao XQ et al
mediated by changing the insulin levels. The KKAy dia-
betic 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 treat-
ment could significantly inhibit body weight gain in dia-
betic 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 sig-
nificant 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 rap-
idly 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 sensi-
tivity in the T2DM rats was improved following APS treat-
ment.
Liver pathology and biochemistry Obesity and diabe-
tes 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
Figure 2. Comparison of appearance in KKAy mice with Astragalus
polysaccharide therapy.
Figure 3. Growth curve of body weight in experimental animals.
Data are expressed as mean±SEM. bP<0.05 vs C group; eP<0.05 vs
KA group.
Figure 1. Characteristics of the experimental animals. Data are
expressed as mean±SEM. bP<0.05 vs C57BL/6J mice (the same age);
eP<0.05 vs C group mice; iP<0.05 vs KA group mice. n indicates the
number of animals studied, which is presented in the parenthesis of the
figure.

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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 hepa-
tocytes was observed by TEM, which can be ameliorated
with APS therapy (Figure 7).
Effect of APS treatment on hepatic insulin signal trans-
duction in KKAy mice In an attempt to understand the ame-
liorating effect of APS on insulin signal transduction in the
liver tissue, we next examined whether APS affected the ex-
pression 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 treat-
ment 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
Figure 4. Effect of APS on
OGTT in experimental
animals. Data are expressed
as mean±SEM. bP<0.05 vs
C57BL/6J mice; eP<0.05 vs
C group mice; iP<0.05 vs KA
group mice. n indicates the
number of animals studied,
which is presented in the
parenthesis of the figure.
APS, Astragalus polysac-
charide.
Figure 5. Biochemical pa-
rameters of experimental
animals. FFA indicates free
fatty acid. Data are expressed
as mean±SEM. bP<0.05 vs C
group; eP<0.05 vs KA group.
n indicates the number of ani-
mals studied,which is pre-
sented in the parenthesis of
the figure.