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GLUT4 pathway
Accepted Manuscript Baicalin against obesity and insulin resistance through activation of AKT/AS160/ GLUT4 pathway Penghua Fang, Mei Yu, Lei Zhang, Dan Wan, Mingyi Shi, Yan Zhu, Ping Bo, Zhenwen Zhang PII:
S0303-7207(17)30200-9
DOI:
10.1016/j.mce.2017.03.027
Reference:
MCE 9900
To appear in:
Molecular and Cellular Endocrinology
Received Date: 13 December 2016 Revised Date:
4 March 2017
Accepted Date: 26 March 2017
Please cite this article as: Fang, P., Yu, M., Zhang, L., Wan, D., Shi, M., Zhu, Y., Bo, P., Zhang, Z., Baicalin against obesity and insulin resistance through activation of AKT/AS160/GLUT4 pathway, Molecular and Cellular Endocrinology (2017), doi: 10.1016/j.mce.2017.03.027. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Baicalin against obesity and insulin resistance through activation of AKT/AS160/GLUT4 pathway Penghua Fanga,b, Mei Yub, Lei Zhanga, Dan Wana, Mingyi Shia, Yan Zhuc, Ping Boa,c,*,
a
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Zhenwen Zhangc*,
Jiangsu Key Laboratory of Integrated Traditional Chinese and Western Medicine for
Prevention and Treatment of Senile Diseases, Medical College, Yangzhou University,
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Yangzhou, China 225001; b
Taizhou, Jiangsu, China, 225300; c
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Department of Physiology, Nanjing University of Chinese Medicine Hanlin College,
Department of Endocrinology, Clinical Medical College, Yangzhou University,
Yangzhou, Jiangsu, China, 225001;
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Running head: Baicalin and obesity and insulin resistance
Acknowledgments: This work was supported by the National Natural Scientific Fund of China (No. 81673736) and the National Health and Family Planning Commission of
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China (No. W201309).
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Competing Interests: The authors declared no conflict of interest.
*Corresponding author: Department of Endocrinology, Clinical Medical College, Yangzhou University, Yangzhou, China 225001; Tel: +86-514-87978880; Fax: +86-514-87341733; Email: [email protected] (Bo P.) and [email protected] (Zhang Z.).
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Abstract Obesity may cause several metabolic complications, including insulin resistance and type 2 diabetes mellitus. Despite great advances in medicine, people still keep exploring novel
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and effective drugs for treatment of obesity and insulin resistance. The aim of this study was to survey if baicalin might ameliorate obesity-induced insulin resistance and to explore its signal mechanisms in skeletal muscles of mice. Diet-induced obese (DIO)
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mice were given 50mg/kg baicalin intraperitoneally (i.p.) once a day for 21 days, and C2C12 myotubes were treated with 100, 200, 400 µM baicalin for 12 h in this study.
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Then insulin resistance indexes and insulin signal protein levels in skeletal muscles were examined. We discovered that administration of baicalin decreased food intake, body weight, HOMA-IR and NT-PGC-1α levels, but enhanced GLUT4, PGC-1α, pP38MAPK, pAKT and pAS160 contents, as well as GLUT4 mRNA, PGC-1α mRNA, PPARγ mRNA,
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GLUT1 mRNA expression in skeletal muscles of obese mice and myotubes of C2C12 cells, and reversed high fat diet-induced glucose and insulin intolerance, hyperglycemia and insulin resistance in the mice. These results suggest that baicalin is a powerful and
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promising agent for treatment of obesity and insulin resistance via Akt/AS160/GLUT4 and P38MAPK/ PGC1α/ GLUT4 pathway.
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Key words: Baicalin; GLUT4; Insulin resistance; Obesity
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Introduction Obesity is an identified risk factor for developing insulin resistance and type 2 diabetes mellitus (Liu et al., 2009; Lazar, 2005). In obese subjects, chronic inflammation arises in
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adipose tissue, resulting in increased adipokine secretion and decreased glucose
transporter 4 (GLUT4) expression and translocation in skeletal muscles and adipose
tissues, which are an early step to develop insulin resistance and type 2 diabetes (Saltiel
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and Kahn, 2001).
Herbal medicines are characterized by comprehensive regulation of body homeostasis
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through multiple components existing together in a prescription. It is attractive to explore new medicines with less expenditure and side effects from Herbal extracts than synthetic hypoglycemic agents for treatment of insulin resistance. Scutellaria baicalensis Georgi (known as huáng qín) is a traditional Chinese herb, exhibiting antioxidant,
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anti-inflammatory, antidiabetic and antidyslipidemic properties (de Oliveira et al., 2015; Xi et al., 2015). Baicalin is one of the most potent and abundant polyphenolic extracts from Scutellaria baicalensis (Li and Chen, 2005). A few studies reported that baicalin
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possesses the anti-obesity characteristic via inhibition of adipogenesis in 3T3-L1 preadipocytes, and prevention of dyslipidemia to decrease epididymal fat, hyperlipidemia,
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liver steatosis and body weights in murinae fed with high fat diet (Lee et al., 2009; Yang et al., 2016; Zhu et al., 2016, Xi et al., 2015, 2016). Besides, baicalin was reported to have hepatoprotective effects through the Ca2+/CaM-dependent protein kinase kinase β (CaMKKβ)/AMP-activated protein kinase (AMPKα) /acetyl-CoA carboxylase (ACC) pathway in liver (Xi et al., 2015). However, the ameliorative effect of baicalin on insulin resistance has not been sufficiently explored in mice fed high-fat diet. The primary aim of
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this study was to investigate whether baicalin could attenuate obesity-induced insulin resistance and what is the relative signal pathway in skeletal muscles of mice and C2C12
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myotubes.
Materials and Methods Drugs and reagents
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Baicalin and 2-[N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl) amino]-2-deoxy-D-glucose
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(2-NBDG) were purchased from Sigma-Aldrich, USA. Antibodies against P38MAPK, pP38MAPK, Akt, pAkt, AS160 and pAS160 were acquired from Cell Signaling Technology Inc, USA. Antibodies against GLUT4, peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) and its splice variant N terminal (NT)-PGC-1α (NT-PGC-1α)
from
Merck
Millipore
Inc,
Germany.
Antibodies
against
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glyceraldehyde-phosphate dehydrogenase (GAPDH) from BOSTER Inc, China. Insulin ELISA kits from Uscn Life Science, Inc. Wuhan, China. RIPA from Bioteke Corporation, Beijing, China. BCA™ protein assay kit from Pierce Chemical Company, Rockford,
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USA. Trizol reagent from Gibco Invitrogen Inc, Invitrogen, USA.
Animals
Six-week-old male C57BL/6J mice were kept in a standard laboratory condition of temperature 21 ± 2°C, relative humidity 50 ± 15%, 12 hour light-dark cycles, with water and food available ad libitum. All animals used were closely monitored to ensure that none lived through stress and discomfort. The mice were fed a high fat diet (20%
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carbohydrates, 21% protein and 59% fat) for 16 weeks. Then the obese mice were divided into two groups: obese control group (n=8) and obese group with baicalin (n=8). Besides, a normal diet group (n=8) was set up. The mice in the obese group with baicalin
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were given 50mg/kg baicalin intraperitoneally (i.p.) once a day for continuous 21 days at 6:00 am (Chen et al., 2012). All controls were given vehicle (DMSO) i.p. The body
weight of all mice was recorded every day. The food intake of all mice was measured
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beginning and at the end of the experiment.
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once a week. The delta weight was calculated by weight deviations of animals at the
Glucose tolerance test and insulin tolerance test
After fasted for 6 h, all animals were subcutaneously injected with insulin (1 U/kg). The insulin levels in the tail vein blood were quantified at 0, 15, 30 and 60 min after the
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insulin challenge using competitive insulin ELISA kits according to the manufacturer's specification. The assay range for insulin was 123.5–10000 pg/ml, and intra-assay precision CV% < 10% and inter-assay precision CV% < 12%. At the day before the
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insulin tolerance test, 1.5 g/kg glucose dissolved in sterile water was i.p. injected into the mice after an overnight fast (12 h). The blood glucose levels in the tail vein blood were
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monitored at 0, 15, 30, 60, 90 and 120 min after the glucose challenge using a Glucometer (HMD Biomedical, Taiwan). HOMA-IR was calculated by fasting serum insulin concentration (mU/ml) ×fasting blood glucose level (mmol/L)/22.5. All measurements were performed in duplicate and the mean of two measurements was considered.
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Collection of blood sample and skeletal tissue After fasted for 24 h all animals were sacrificed on the second day after the insulin tolerance test. Then 1 ml blood and 5 g skeletal tissue were fast collected. The blood
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samples were centrifugated at 3500 r.p.m. for 10 min to obtain the plasma which was stored at –80°C until further analysis. The skeletal tissues were rinsed, weighed and
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Animal Studies Committee of Yangzhou University.
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frozen at −80°C too. This experiment was performed with the specific acceptance of the
Cell culture
C2C12 myoblasts (purchased from the cell bank of the Institute of Biochemistry and Cell Biology, Shanghai) were grown in DMEM supplemented with 10% FBS, 100 U/ml penicillin, 100 mg/ml streptomycin at 37 °C in an humidified atmosphere included 5%
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CO2. The cells were plated at 2×105/well in 6-well plates and used at subconfluence after 48 h preincubation, then transferred to DMEM containing 2% horse serum for 4-6 days. Differentiation was confirmed by visualization of myotube formation. After formation of
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elongated and multinucleate myotubes, these differentiated cells were starved for 4 h,
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then treated with 100, 200, 400 µM baicalin for 12 h (Kwak et al., 2014; Lee et al., 2010).
2-NBDG uptake in flow cytometry The experiments were performed in 6 culture plates of C2C12 myotubes by treatment with 100, 200, 400 µM baicalin respectively. Then all culture medium was replaced with 1000 µl of culture medium in the absence or presence of 100 µM fluorescent 2-NBDG, incubated at 37 °C with 5% CO2 for 30 min before flow cytometry analysis. The
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2-NBDG uptake was stopped by removing the incubation medium and washing twice with cold phosphate buffered saline (PBS). The cells were subsequently resuspended in 300 µl pre-cold PBS for later flow cytometry analysis within 30 min at 4 °C. For each
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flow cytometric measurement, data from 15000 single cell events were collected using a BD FACS calibur (Beckman Coulter FC500) flow cytometer within 20 s to analyze
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Total RNA extraction and real-time PCR
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2-NBDG fluorescence intensity.
Total RNA was extracted with Trizol from 100 mg frozen skeletal muscle or myotubes. The RNA concentration was calculated by spectrophotometric assays of 260/280 nm, and the RNA integrity was assessed by running samples on a 1% formaldehyde agarose gel in TAE buffer (40 mmol/L tris-acetic acid, 1 mmol/L EDTA). cDNA was synthesized from
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1 µg RNA using MMLV reverse transcriptase. Real-time quantitative PCR was performed for gene expression levels using real-time fluorescent detection in an Applied Biosystems 7500 real-time PCR instrument (ABI 7500, USA). The oligonucleotide
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primers were as follows: GLUT4 Forward Sequence 5’-
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GGCTTTGTGGCCTTCTTTGAG-3’, Reverse Sequence5’GACCCATAGCATCCGCAACAT-3’; PGC-1α Forward Sequence5’-ACCATGACTACTGTCAGTCACTC-3’, Reverse Sequence5’-GTCACAGGAGGCATCTTTGAAG-3’; PPAR-γ Forward Sequence5’-AAGGCGAGGGCGATCTTG-3’, Reverse Sequence5’-ATCATTAAGGAATTCATGTCGTAGATGAC -3’; GLUT1 Forward Sequence 5’-CCAGCTGGGAATCGTCGTT-3’, Reverse
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Sequence5’-CAAGTCTGCATTGCCCATGAT-3’; GAPDH Forward Sequence 5’AGAACATCATCCCTGCATCC -3’, Reverse Sequence5’TCCACCACCCTGTTGCTGTA -3’. Amplification condition was: an initial denaturation
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at 95°C for 10 min; 95°C for 15 s, 62°C for 60 s, 40 cycles. The 2−∆CT method was used
Subcellular fractionation of skeletal muscle
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to analyze the PCR data. GAPDH was used as an endogenous housekeeping gene.
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Membrane fractions from muscle homogenates and myotubes were separated by sucrose-gradient centrifugation, as described previously (Klip et al., 1987; Kristiansen et al., 1998). In brief, skeletal muscle or C2C12 myotubes were washed, minced and homogenized in cold buffer (20 mmol/L NaHCO3, 250 mmol/L sucrose, 5 mmol/L NaNO3, 100 µmol/L PMSF, 1 µmol/L leupeptin, 1 µmol/L pepstatin A, 2 mmol/L
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orthovanadate in ddH2O, pH 7.4, 4 °C). The homogenates were centrifuged at 1200 g for 10 min (4 °C). The supernatants were recentrifuged at 8000 g for 10 min (4 °C). Then the supernatants were layered on a 25% and 50% sucrose gradient and centrifuged at 210,000
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membranes.
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g for 2 h (4 °C) with L7-55Ul ultracentrifuge (Beckman, USA) to yield the plasma
Western blot analysis
Total proteins of skeletal muscle or myotubes were extracted using RIPA agents and quantified with BCA protein assay kit to determine protein levels. Briefly, fifty micrograms of samples were separated by a 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride filter membranes. 8
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Membranes were blocked in Tris-buffered saline (pH 7.5) containing 0.05% Tween-20 (1×TBST) and 5% skimmed milk for 2 h, then probed overnight at 4°C with an antibody against GLUT4, GAPDH, P38MAPK, AKT, AS160, pP38MAPK, pAKT and pAS160,
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respectively. Membranes were washed with 1×TBST for 10 min and incubated for 2 h with horseradish peroxidase-conjugated secondary antibody. Lastly, immunoreactive
Immunofluorescence staining
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Quantity One Analysis Software (Bio-Rad).
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bands were visualized by chemiluminescence and quantified by densitometry using a
Cultured cells in 6-well plates were fixed with 4% paraformaldehyde for 30 min and permeabilized with 0.5% Triton-X 100 (Solabio, Beijing, China) for 10 min at 37°C.
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After wash, the cells were blocked in 5% BSA for 2 h and incubated with primary rabbit antibody against GLUT4 (1:50, Merck Millipore Inc, Germany) at 4 °C overnight. After incubation and wash, cells were blocked with the fluorescent secondary antibody (1:200,
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IgG-FITC) for 1 hour. After washes with PBS, the nuclei were stained with 4’, 6-diamidino-2-phenylindole (DAPI) (Beyotime, Jiangsu, China) for 5 min. Photo capture
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was performed by a Nikon TE2000 microscope (Nikon, Tokyo, Japan). Randomly selected the perspective GLUT4 positive areas and DAPI cells were under the microscope.
Statistical analysis SPSS 17.0 for Windows was used for statistical analysis. Comparisons between the means of three groups were analyzed by one-way ANOVA with Duncan’s tests. Data 9
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were presented as mean ± SEM with P < 0.05 as the limit for statistical significance.
Results
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Body weight and food intake
All of the six-week-old mice used in this experiment exhibited similar body weight and food intake at the beginning of the experiment. Feeding of a high fat diet for 16 weeks
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enhanced body weight of the mice in obese control and baicalin groups compared with the normal diet group before the administration of baicalin (Fig.1). As shown in Fig. 1
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and Fig. 2 the body weight and delta weight at 21 days were significantly decreased by 33.9% (P < 0.01) and 423.94% (P < 0.001) in the baicalin group compared with the obese controls, but increased by 71.3% (P< 0.01) and 4.4% (P> 0.05) in the obese control group compared with the normal controls, respectively. As shown in Fig. 3, the food intake of
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mice was significantly decreased by 36.2% (P< 0.01) , 42% (P< 0.01) and 26.1% (P< 0.01) in the baicalin group compared with the obese controls, and by 5.1% (P> 0.05), 9.3% (P> 0.05) and 9.1% (P> 0.05) in the obese control group compared with the normal
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controls in the first, second and third week, respectively.
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(Fig. 1, Fig. 2 and Fig.3 were located here)
Glucose tolerance and insulin tolerance test During the glucose tolerance test, as shown in Fig. 4, the circulating glucose levels at 120 min were markedly decreased by 24% (P < 0.05) in the baicalin group compared with obese controls, but increased by 69.9% (P< 0.01) in the obese control group compared with normal controls. As shown in Fig. 5, the circulating glucose levels were significantly decreased at 0 min (P < 0.01), 15 min (P < 0.001), 30 min (P < 0.01), 45 min (P < 0.01)
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and 60 min (P < 0.001) after administration of insulin in the baicalin group compared with the obese controls during the insulin tolerance test.
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(Fig. 4 and Fig. 5 were located here)
HOMA-IR index
As shown in Fig. 6A, HOMA-IR index of mice was significantly decreased by 34.4% (P
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< 0.01) in the baicalin group compared with obese controls, but increased by 278.4% (P< 0.01) in the obese control group compared with normal controls. The blood insulin and
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glucose levels was decreased by 16.2 % (P < 0.01) and 13.09% (P < 0.01) in the baicalin group compared with the obese controls, but elevated by 51.9 % (P < 0.05) and 102.2% (P < 0.01) in the obese control group compared with the normal controls (Fig. 6B and Fig. 6C).
2-NBDG uptake
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(Fig.6 was located here)
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As shown in Fig. 7, the 2-NBDG uptake in the baicalin group compared with the controls was significantly elevated by 30.9% (P < 0.01), 46.1% (P < 0.01), 52.8% (P < 0.01) after
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treatment with 100, 200, 400 µM baicalin respectively. (Fig.7 was located here)
GLUT4 mRNA, PGC-1α mRNA, PPAR- γ mRNA and GLUT1 mRNA expression levels As shown in Fig. 8, the GLUT4 mRNA, PGC-1α mRNA, PPAR-γ mRNA and GLUT1 mRNA expression levels were increased by 83.4% (P < 0.01), 169.4% (P < 0.05),
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587.6% (P < 0.01) and 83.4% (P < 0.01) in baicalin group compared with obese controls, but decreased by 67.6% (P < 0.01), 74.7% (P < 0.01), 87.7% (P < 0.01) and 65.4% (P < 0.01) in the obese control group compared with the normal controls, respectively.
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After treatment with 100, 200, 400 µM baicalin, the GLUT4 mRNA expression compared with the controls was increased by 224.3% (P < 0.05), 408.1% (P < 0.01) and 724.8% (P < 0.01), the PGC-1α mRNA expression by 168.6% (P < 0.05), 277.9% (P < 0.01) and
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206.1% (P < 0.01), the PPAR-γ mRNA expression by 13.4% (P > 0.05), 49.4% (P < 0.01) and 44.3% (P < 0.05), and the GLUT1 mRNA expression by 43.6% (P > 0.05), 269.3%
(Fig.8 and Fig.9 were located here)
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(P < 0.01) and 302.9% (P < 0.01), respectively (Fig. 9).
GLUT4 contents in plasma membranes of C2C12 cells and skeletal muscle
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Compared with controls, GLUT4 immunoreactivitie in plasma membranes of C2C12 cells was elevated by 142.9 % (P < 0.01), 248.3% (P < 0.01) and 317.4% (P < 0.01) after treatment with 100, 200, 400 µM baicalin, respectively (Fig. 10A). Also, the
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immunoreactivitie was elevated by 36.4 % (P < 0.05) in the skeletal muscle of the baicalin group compared with the obese controls, but decreased by 47% (P< 0.01) in the
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obese control group compared with the normal controls (Fig 10B). As shown in Fig 11, the GLUT4 immunofluorescence in the plasma membrane of C2C12 cells after treatment of 100, 200, 400 uM baicalin was higher than the controls. (Fig. 10 and Fig. 11 were located here)
The ratios of pAKT/AKT, pAS160/AS160, pP38MAPK/P38MAPK, PGC-1α and
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NT-PGC-1α levels in skeletal muscle As shown in Fig 12A-E, after treatment with 100, 200, 400 µM baicalin in C2C12 cells, the ratios of pAKT/AKT were enhanced by 180.6% (P < 0.01), 244.3% (P < 0.01) and
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848.9% (P < 0.01), the ratios of pAS160/AS160 by 95.3% (P < 0.01), 155.8% (P < 0.01) and 409.1% (P < 0.01), the ratios of pP38MAPK/P38MAPK by 51.2% (P < 0.05), 73% (P < 0.01) and 241.9% (P < 0.01), and the PGC-1α contents by 156.4% (P < 0.05),
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200.7% (P < 0.01) and 580.6% (P < 0.01) compared with the controls, respectively. As shown in Fig 13A-F, the ratios of pAKT/AKT, pAS160/AS160,
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pP38MAPK/P38MAPK and PGC-1α contents were enhanced by 53.5% (P < 0.05), 68.4% (P < 0.01), 28.8% (P < 0.01) and 59% (P < 0.01) in baicalin group compared with obese controls, but decreased by 50.6% (P < 0.01), 53.5% (P < 0.01), 24.9% (P < 0.01) and 50.1% (P < 0.01) in obese control group compared with the normal controls,
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respectively. However, the NT-PGC-1α contents were reduced by 32.7% (P < 0.05) in baicalin group compared with obese controls, but increased by 167.3% (P < 0.01) in the obese control group compared with the normal controls.
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Discussion
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(Fig. 12 and Fig.13 were located here)
Baicalin is a bioactive flavonoid isolated from the radix of Scutellaria baicalensis, which contains several bioactive compounds, such as baicalein, baicalin, β-sitosterol, norwogonin, oroxylin A, and wogonin (Li and Chen, 2005, de Oliveira et al., 2015). Recent research indicated that administration of baicalin could alleviate obesity, hyperglycemia and insulin resistance.
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First, a few studies reported that baicalin might play an anti-obesity role (Lee et al., 2009; Xi et al., 2015; Yang et al., 2016). Treatment of 3T3-L1 preadipocytes with baicalin inhibited triglyceride accumulation and lipid droplet formation during adipogenesis (Lee
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et al., 2009; Yang et al., 2016; Zhu et al., 2016). Besides, oral treatment
with baicalin (100, 200 and 400 mg/kg/d) significantly decreased epididymal fat,
hyperlipidemia, liver steatosis and body weights in mice fed with high fat diet (Xi et al.,
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2015). Furthermore, i.p. injection with baicalin significantly ameliorated the elevated
serum cholesterol and free fatty acid concentrations in rats fed with high fat diet (Guo et
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al., 2009; Waisundara et al., 2011). Consistent with these, the current results revealed that i.p. injection of baicalin decreased food intake and body weight of diet-induced obese (DIO) mice, further demonstrating that baicalin had the anti-obesity effects. Next, baicalin was reported to have antihyperglycemic effects (Li et al., 2011; Shi et al.,
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2016; Xi et al., 2016). I.p. administration of baicalin significantly decreased plasma glucose levels in a dose-dependent manner in streptozotocin-nicotinamide induced diabetic rats (Li et al., 2011; Shi et al., 2016). In accordance with these, we found that i.p.
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injection of baicalin reduced circulating glucose levels in DIO mice too, further manifesting that baicalin had the antihyperglycemic effects.
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Furthermore, baicalin possessed the potency to ameliorate insulin resistance. In this study, 2-NBDG, a fluorescent deoxyglucose analog, was used to investigate the effect of the baicalin on glucose uptake into C2C12 myotubes. 2-NBDG with deoxyglucose can be transported into cells through the help of glucose transporters (Lanner et al., 2006; Zou et al., 2005). The current results showed that baicalin significantly promoted 2-NBDG uptake into C2C12 myotubes in a dose-dependent manner and decreased glucose levels
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during the insulin tolerance test in obese mice. More interestingly, treatment with baicalin in obese mice significantly decreased HOMA-IR values compared to the obese controls. These results implicate that baicalin can increase glucose uptake and insulin sensitivity.
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Last, baicalin can regulate the expression of PPAR-γ, PGC-1α, GLUT1 and GLUT4 in muscles of DIO mice and C2C12 myotubes. First, PPAR-γ is a specific target of the anti-diabetic drug, such as rosiglitazone. Second, PGC-1α is a critical regulator of
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mitochondrial biogenesis in skeletal muscle to maintain an energy balance (Puigserver et al., 1999; Wu et al., 1999). PGC-1α can enhance the expression of GLUT4 and PPAR-γ
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to increase glucose uptake (Wright, 2007, Summermatter et al., 2003; Wu et al., 2016). Overexpressed PGC-1α increased glucose uptake in both C2C12 and L6 muscle cells (Michael et al., 2001), while reduced PGC-1α expression in muscle was relative to the pathogenesis of type 2 diabetes (Mootha et al., 2003; Patti et al., 2003). Third,
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NT-PGC-1α is a short isoform of PGC-1α produced by alternative splicing of the PGC-1α gene. It can increase insulin resistance in DIO mice (Jun et al, 2014). Fourth, p38MAPK is involved in PGC-1α expression to reduce free fatty acid content in skeletal
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muscles (Akimoto et al., 2005; Crunkhorn et al., 2007; Hong et al., 2001; Wright et al., 2007). Blockade of p38MAPK may prevent the increase in expression of PGC-1α. Fifth,
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GLUT1 belongs to the major facilitator superfamily, and is responsible for the constant and basic glucose uptake. Last, GLUT4 is a particularly important glucose transporter for maintaining glucose homeostasis and insulin sensitivity in response to insulin stimuli. Its translocation from intracellular membrane compartments to plasma membranes is a rate-limiting step of glucose uptake and is closely associated with insulin resistance in skeletal muscle (Leto and Saltiel, 2012). The current study found that administration of
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baicalin could upregulate PPAR-γ, GLUT4, PGC-1α and pP38MAPK levels, but inhibit NT-PGC-1α levels in muscle of DIO mice and C2C12 myotubes, suggesting that administration of baicalin decreased NT-PGC-1α or enhanced PGC-1α levels via
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upregulation of pP38MAPK to boost GLUT4 and PPAR-γ expression, resulting in the
increase in glucose uptake (Summermatter et al., 2003; Wu et al., 2016, Wright, 2007). More interestingly, we found that the GLUT4 contents in the plasma membranes were
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markedly increased in muscles of baicalin-treated DIO mice or C2C12 myotubes,
implicating that baicalin not only enhances GLUT4 and GLUT4 mRNA expression levels,
plasma membranes in muscle.
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but also accelerates GLUT4 translocation from intracellular membrane compartments to
To understand the signal mechanism that baicalin promotes GLUT4 translocation, we examined the levels of insulin signaling proteins, including Akt and pAS160 in skeletal
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muscle of DIO mice and C2C12 myotubes. The Akt/AS160 pathway mediates the effect of insulin on surface recruitment of GLUT4 (Samovski et al., 2012; Mikłosz et al., 2016). Activation of Akt mediates the phosphorylation of Akt substrate of 160 kDa (AS160) to
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trigger GLUT4 translocation from intracellular membrane compartments to plasma membranes to accelerate glucose uptake (Sakamoto and Holman, 2008). The increase in
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pAkt and pAS160 contents may boost GLUT4 translocation in skeletal muscle (Oak et al., 2006; Thong et al., 2007). The results in the present study showed that treatment with baicalin enhanced pAkt and pAS160 contents to boost GLUT4 translocation and glucose uptake, suggesting that baicalin increased glucose uptake via the Akt/ AS160/GLUT4 pathway. In summary, the results of the current study revealed that administration of baicalin can
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decreased food intake, body weight, HOMA-IR, NT-PGC-1α content, and circulating glucose and insulin levels, but enhanced GLUT4, PGC-1α, pP38MAPK, pAKT and pAS160 expression, reversed high fat diet-induced glucose and insulin intolerance,
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hyperglycemia and insulin resistance in skeletal muscle of DIO mice. Also, treatment with baicalin significantly elevated the GLUT4 mRNA, PGC-1α mRNA, PPAR-γ mRNA and GLUT1 mRNA expression in skeletal muscles and C2C12 cells. These results
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suggested that Akt/AS160/GLUT4 and P38MAPK/ PGC-1α/ GLUT4 pathway, at least in part, mediated the beneficial effect of baicalin on high fat diet-induced obesity and
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insulin resistance in the skeletal muscle. This study contributes to our understanding of the anti-obesity and anti-diabetic role of baicalin, and provides a possibility of using baicalin to treat obesity and insulin resistance in clinic.
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Acknowledgments
This work was supported by the National Natural Scientific Fund of China (No. 81673736) and in part by the National Health and Family Planning Commission of China
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(Grant No. W201309).
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Competing Interests
The authors declared no conflict of interest.
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Methods. 2005;64:207-15.
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a fluorescent indicator for direct glucose uptake measurement. J Biochem Biophys
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Fig. 1. The i.p. administration of baicalin for 21 days decreased the body weight of high fat diet-induced obese mice (n=8). The body weight was lower in the obese group with baicalin than the obese controls (OC), but higher in OC than the normal controls (NC).
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All data shown are the means ± SEM. ** P < 0.01 vs. OC; ++P < 0.01 vs. NC.
Fig. 2. The i.p. administration of baicalin for 21 days decreased the delta weight (delta
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weight was calculated by weight deviations of animals at the beginning and at the end of the experiment) of high fat diet-induced obese mice (n=8). The delta weight was lower in
± SEM. ** P < 0.01 vs. OC.
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the obese group with baicalin than the obese controls (OC). All data shown are the means
Fig. 3. The i.p. injection of baicalin significantly decreased the food intake of animals
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(n=8). The food intake of mice in the baicalin group compared with obese controls (OC) was significantly decreased in the first, second and third week respectively. All data
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shown are the means ± SEM. ** P < 0.01 vs. OC.
Fig. 4. The i.p. administration of baicalin significantly decreased circulating glucose
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levels during the glucose tolerance test (n=8). The circulating glucose levels were markedly decreased at 0 and 120 min in the obese group with baicalin compared with the obese controls (OC), but increased at 0 and 120 min in the obese control group compared with the normal controls (NC). All data shown are the means ± SEM. * P < 0.05 vs. OC ; ++P < 0.01 vs. NC.
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Fig. 5. The i.p. administration of baicalin significantly decreased circulating glucose levels during the insulin tolerance test (n=8). The circulating glucose levels were significantly decreased at 0, 15, 30, 45, 60 min in the obese group with baicalin compared
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with obese controls (OC), but increased at 0, 15, 30, 45, 60 min in the obese control group compared with normal controls (NC). All data shown are the means ± SEM. ** P <
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0.01 vs. OC; ++P < 0.01 vs. NC.
Fig. 6. The i.p. injection of baicalin significantly decreased the HOMA-IR index in
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animals (n=8). Fig. 6A, HOMA-IR index in the baicalin group was significantly decreased compared with obese the controls (OC), but that increased in OC compared with normal controls (NC). Fig. 6B and Fig. 6C showed that blood insulin and glucose levels were lower in the baicalin group than OC, but higher in OC than NC. All data
++P < 0.01 vs. NC.
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shown are the means ± SEM. * P < 0.05 vs. OC; ** P < 0.01 vs. OC; +P < 0.05 vs. NC;
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Fig. 7. The administration of baicalin significantly increased the fluorescence 2-NBDG intensity of cells (n=3). The 2-NBDG uptake of cells in the baicalin group compared with
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controls was significantly elevated after treatment with 100, 200, 400 µM baicalin respectively. All data shown are the means ± SEM. ** P < 0.01 vs. controls.
Fig. 8. The i.p. injection of baicalin significantly increased GLUT4 mRNA, PGC1α mRNA, PPAR-γ mRNA and GLUT1 mRNA expression levels (n=6). The GLUT4 mRNA, PGC1α mRNA, PPAR-γ mRNA and GLUT1 mRNA expression levels were
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increased in the baicalin group compared with the obese controls (OC), but decreased in OC compared with the normal controls (NC) in the skeletal muscle. A, GLUT4; B,
** P < 0.01 vs. OC; ++P < 0.01 vs. NC.
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PGC1α; C, PPAR-γ; D, GLUT1. All data shown are the means ± SEM. * P < 0.05 vs. OC;
Fig. 9. The treatment with elevated GLUT4 mRNA, PGC1α mRNA, PPAR-γ mRNA and
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GLUT1 mRNA expression (n=3). The treatment with 100, 200, 400 µM baicalin significantly elevated the GLUT4 mRNA, PGC1α mRNA, PPAR-γ mRNA and GLUT1
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mRNA expression levels in C2C12 cells. A, GLUT4; B, PGC1α; C, PPAR-γ; D, GLUT1. All data shown are the means ± SEM. * P < 0.05 vs. OC; ** P < 0.01 vs. OC.
Fig. 10. The administration of baicalin significantly elevated GLUT4 protein levels in
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plasma membranes of C2C12 cells (n=3) and plasma membranes of skeletal muscle (n=3). As shown in Fig.10A, the GLUT4 immunoreactivities in plasma membranes of C2C12 cells were elevated in the baicalin group compared with the controls after
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treatment with 100, 200, 400 µM, respectively. The sequence of representative Western blot lines in each panel is Control, 100 µM, 200 µM and 400 µM. As shown in Fig 10B,
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the GLUT4 immunoreactivities in the skeletal muscle was increased in the baicalin group compared with the obese controls (OC), but decreased in OC compared with the normal controls (NC). The sequence of representative Western blot lines in each panel is NC, OC and Baicalin group. All data shown are the means ± SEM. ** P < 0.01 vs. OC; ++P < 0.01 vs. NC.
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Fig. 11. GLUT4-immunofluorescence in C2C12 cells after treatment with baicalin (n=3). The photomicrographs of GLUT4- immunofluorescence in the plasma membrane of
respectively. A, Control; B, 100 µM; C, 200 µM; D, 400 µM.
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C2C12 cells were higher than the controls after treatment of 100, 200, 400 µM baicalin
Fig. 12. Treatment with baicalin significantly elevated pAKT, pAS160, pP38MAPK and
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PGC1α levels of C2C12 cells (n=3). As shown in Fig 12A-D, treatment of 100, 200, 400 µM baicalin significantly elevated pAKT, pAS160, pP38MAPK and PGC1αlevels of
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C2C12 cells compared with the controls respectively. As shown in Fig 12E, the sequence of representative Western blot lines in each panel is Control, 100 µM, 200 µM and 400 µM. All data shown are the means ± SEM. ** P < 0.01 vs. Control.
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Fig. 13. I.p. injection of baicalin elevated pAKT, pAS160, pP38MAPK and PGC1α levels, but reduced NT-PGC1α levels of skeletal muscle (n=3). As shown in Fig 13A-E, the pAkt, pAS160, pP38MAPK and PGC1α contents were enhanced, but the NT-PGC1α
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contents were reduced in the baicalin group compared with the obese controls (OC) respectively. The pAkt, pAS160, pP38MAPK and PGC1α contents were decreased, but
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the NT-PGC1α contents were increased in OC compared with normal controls (NC) respectively. As shown in Fig 13F, the sequence of representative Western blot lines in each panel is NC, OC and Baicalin group. All data shown are the means ± SEM. * P < 0.05 vs. OC; ** P < 0.01 vs. OC; ++P < 0.01 vs. NC.
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ACCEPTED MANUSCRIPT ► I.p injection of baicalin decreases body weight and food intake of obese mice. ► I.p injection of baicalin reduces blood glucose concentration and insulin resistance. ► I.p injection of baicalin increases GLUT4 expression levels in skeletal muscle.
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► I.p injection of baicalin improves GLUT4 translocation level in skeletal muscle. ►Baicalin attenuates skeletal muscle insulin resistance via the Akt/AS160/GLUT4
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pathway.
S0303-7207(17)30200-9
DOI:
10.1016/j.mce.2017.03.027
Reference:
MCE 9900
To appear in:
Molecular and Cellular Endocrinology
Received Date: 13 December 2016 Revised Date:
4 March 2017
Accepted Date: 26 March 2017
Please cite this article as: Fang, P., Yu, M., Zhang, L., Wan, D., Shi, M., Zhu, Y., Bo, P., Zhang, Z., Baicalin against obesity and insulin resistance through activation of AKT/AS160/GLUT4 pathway, Molecular and Cellular Endocrinology (2017), doi: 10.1016/j.mce.2017.03.027. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Baicalin against obesity and insulin resistance through activation of AKT/AS160/GLUT4 pathway Penghua Fanga,b, Mei Yub, Lei Zhanga, Dan Wana, Mingyi Shia, Yan Zhuc, Ping Boa,c,*,
a
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Zhenwen Zhangc*,
Jiangsu Key Laboratory of Integrated Traditional Chinese and Western Medicine for
Prevention and Treatment of Senile Diseases, Medical College, Yangzhou University,
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Yangzhou, China 225001; b
Taizhou, Jiangsu, China, 225300; c
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Department of Physiology, Nanjing University of Chinese Medicine Hanlin College,
Department of Endocrinology, Clinical Medical College, Yangzhou University,
Yangzhou, Jiangsu, China, 225001;
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Running head: Baicalin and obesity and insulin resistance
Acknowledgments: This work was supported by the National Natural Scientific Fund of China (No. 81673736) and the National Health and Family Planning Commission of
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China (No. W201309).
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Competing Interests: The authors declared no conflict of interest.
*Corresponding author: Department of Endocrinology, Clinical Medical College, Yangzhou University, Yangzhou, China 225001; Tel: +86-514-87978880; Fax: +86-514-87341733; Email: [email protected] (Bo P.) and [email protected] (Zhang Z.).
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Abstract Obesity may cause several metabolic complications, including insulin resistance and type 2 diabetes mellitus. Despite great advances in medicine, people still keep exploring novel
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and effective drugs for treatment of obesity and insulin resistance. The aim of this study was to survey if baicalin might ameliorate obesity-induced insulin resistance and to explore its signal mechanisms in skeletal muscles of mice. Diet-induced obese (DIO)
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mice were given 50mg/kg baicalin intraperitoneally (i.p.) once a day for 21 days, and C2C12 myotubes were treated with 100, 200, 400 µM baicalin for 12 h in this study.
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Then insulin resistance indexes and insulin signal protein levels in skeletal muscles were examined. We discovered that administration of baicalin decreased food intake, body weight, HOMA-IR and NT-PGC-1α levels, but enhanced GLUT4, PGC-1α, pP38MAPK, pAKT and pAS160 contents, as well as GLUT4 mRNA, PGC-1α mRNA, PPARγ mRNA,
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GLUT1 mRNA expression in skeletal muscles of obese mice and myotubes of C2C12 cells, and reversed high fat diet-induced glucose and insulin intolerance, hyperglycemia and insulin resistance in the mice. These results suggest that baicalin is a powerful and
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promising agent for treatment of obesity and insulin resistance via Akt/AS160/GLUT4 and P38MAPK/ PGC1α/ GLUT4 pathway.
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Key words: Baicalin; GLUT4; Insulin resistance; Obesity
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Introduction Obesity is an identified risk factor for developing insulin resistance and type 2 diabetes mellitus (Liu et al., 2009; Lazar, 2005). In obese subjects, chronic inflammation arises in
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adipose tissue, resulting in increased adipokine secretion and decreased glucose
transporter 4 (GLUT4) expression and translocation in skeletal muscles and adipose
tissues, which are an early step to develop insulin resistance and type 2 diabetes (Saltiel
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and Kahn, 2001).
Herbal medicines are characterized by comprehensive regulation of body homeostasis
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through multiple components existing together in a prescription. It is attractive to explore new medicines with less expenditure and side effects from Herbal extracts than synthetic hypoglycemic agents for treatment of insulin resistance. Scutellaria baicalensis Georgi (known as huáng qín) is a traditional Chinese herb, exhibiting antioxidant,
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anti-inflammatory, antidiabetic and antidyslipidemic properties (de Oliveira et al., 2015; Xi et al., 2015). Baicalin is one of the most potent and abundant polyphenolic extracts from Scutellaria baicalensis (Li and Chen, 2005). A few studies reported that baicalin
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possesses the anti-obesity characteristic via inhibition of adipogenesis in 3T3-L1 preadipocytes, and prevention of dyslipidemia to decrease epididymal fat, hyperlipidemia,
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liver steatosis and body weights in murinae fed with high fat diet (Lee et al., 2009; Yang et al., 2016; Zhu et al., 2016, Xi et al., 2015, 2016). Besides, baicalin was reported to have hepatoprotective effects through the Ca2+/CaM-dependent protein kinase kinase β (CaMKKβ)/AMP-activated protein kinase (AMPKα) /acetyl-CoA carboxylase (ACC) pathway in liver (Xi et al., 2015). However, the ameliorative effect of baicalin on insulin resistance has not been sufficiently explored in mice fed high-fat diet. The primary aim of
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this study was to investigate whether baicalin could attenuate obesity-induced insulin resistance and what is the relative signal pathway in skeletal muscles of mice and C2C12
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myotubes.
Materials and Methods Drugs and reagents
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Baicalin and 2-[N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl) amino]-2-deoxy-D-glucose
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(2-NBDG) were purchased from Sigma-Aldrich, USA. Antibodies against P38MAPK, pP38MAPK, Akt, pAkt, AS160 and pAS160 were acquired from Cell Signaling Technology Inc, USA. Antibodies against GLUT4, peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) and its splice variant N terminal (NT)-PGC-1α (NT-PGC-1α)
from
Merck
Millipore
Inc,
Germany.
Antibodies
against
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glyceraldehyde-phosphate dehydrogenase (GAPDH) from BOSTER Inc, China. Insulin ELISA kits from Uscn Life Science, Inc. Wuhan, China. RIPA from Bioteke Corporation, Beijing, China. BCA™ protein assay kit from Pierce Chemical Company, Rockford,
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USA. Trizol reagent from Gibco Invitrogen Inc, Invitrogen, USA.
Animals
Six-week-old male C57BL/6J mice were kept in a standard laboratory condition of temperature 21 ± 2°C, relative humidity 50 ± 15%, 12 hour light-dark cycles, with water and food available ad libitum. All animals used were closely monitored to ensure that none lived through stress and discomfort. The mice were fed a high fat diet (20%
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carbohydrates, 21% protein and 59% fat) for 16 weeks. Then the obese mice were divided into two groups: obese control group (n=8) and obese group with baicalin (n=8). Besides, a normal diet group (n=8) was set up. The mice in the obese group with baicalin
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were given 50mg/kg baicalin intraperitoneally (i.p.) once a day for continuous 21 days at 6:00 am (Chen et al., 2012). All controls were given vehicle (DMSO) i.p. The body
weight of all mice was recorded every day. The food intake of all mice was measured
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beginning and at the end of the experiment.
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once a week. The delta weight was calculated by weight deviations of animals at the
Glucose tolerance test and insulin tolerance test
After fasted for 6 h, all animals were subcutaneously injected with insulin (1 U/kg). The insulin levels in the tail vein blood were quantified at 0, 15, 30 and 60 min after the
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insulin challenge using competitive insulin ELISA kits according to the manufacturer's specification. The assay range for insulin was 123.5–10000 pg/ml, and intra-assay precision CV% < 10% and inter-assay precision CV% < 12%. At the day before the
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insulin tolerance test, 1.5 g/kg glucose dissolved in sterile water was i.p. injected into the mice after an overnight fast (12 h). The blood glucose levels in the tail vein blood were
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monitored at 0, 15, 30, 60, 90 and 120 min after the glucose challenge using a Glucometer (HMD Biomedical, Taiwan). HOMA-IR was calculated by fasting serum insulin concentration (mU/ml) ×fasting blood glucose level (mmol/L)/22.5. All measurements were performed in duplicate and the mean of two measurements was considered.
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Collection of blood sample and skeletal tissue After fasted for 24 h all animals were sacrificed on the second day after the insulin tolerance test. Then 1 ml blood and 5 g skeletal tissue were fast collected. The blood
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samples were centrifugated at 3500 r.p.m. for 10 min to obtain the plasma which was stored at –80°C until further analysis. The skeletal tissues were rinsed, weighed and
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Animal Studies Committee of Yangzhou University.
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frozen at −80°C too. This experiment was performed with the specific acceptance of the
Cell culture
C2C12 myoblasts (purchased from the cell bank of the Institute of Biochemistry and Cell Biology, Shanghai) were grown in DMEM supplemented with 10% FBS, 100 U/ml penicillin, 100 mg/ml streptomycin at 37 °C in an humidified atmosphere included 5%
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CO2. The cells were plated at 2×105/well in 6-well plates and used at subconfluence after 48 h preincubation, then transferred to DMEM containing 2% horse serum for 4-6 days. Differentiation was confirmed by visualization of myotube formation. After formation of
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elongated and multinucleate myotubes, these differentiated cells were starved for 4 h,
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then treated with 100, 200, 400 µM baicalin for 12 h (Kwak et al., 2014; Lee et al., 2010).
2-NBDG uptake in flow cytometry The experiments were performed in 6 culture plates of C2C12 myotubes by treatment with 100, 200, 400 µM baicalin respectively. Then all culture medium was replaced with 1000 µl of culture medium in the absence or presence of 100 µM fluorescent 2-NBDG, incubated at 37 °C with 5% CO2 for 30 min before flow cytometry analysis. The
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2-NBDG uptake was stopped by removing the incubation medium and washing twice with cold phosphate buffered saline (PBS). The cells were subsequently resuspended in 300 µl pre-cold PBS for later flow cytometry analysis within 30 min at 4 °C. For each
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flow cytometric measurement, data from 15000 single cell events were collected using a BD FACS calibur (Beckman Coulter FC500) flow cytometer within 20 s to analyze
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Total RNA extraction and real-time PCR
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2-NBDG fluorescence intensity.
Total RNA was extracted with Trizol from 100 mg frozen skeletal muscle or myotubes. The RNA concentration was calculated by spectrophotometric assays of 260/280 nm, and the RNA integrity was assessed by running samples on a 1% formaldehyde agarose gel in TAE buffer (40 mmol/L tris-acetic acid, 1 mmol/L EDTA). cDNA was synthesized from
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1 µg RNA using MMLV reverse transcriptase. Real-time quantitative PCR was performed for gene expression levels using real-time fluorescent detection in an Applied Biosystems 7500 real-time PCR instrument (ABI 7500, USA). The oligonucleotide
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primers were as follows: GLUT4 Forward Sequence 5’-
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GGCTTTGTGGCCTTCTTTGAG-3’, Reverse Sequence5’GACCCATAGCATCCGCAACAT-3’; PGC-1α Forward Sequence5’-ACCATGACTACTGTCAGTCACTC-3’, Reverse Sequence5’-GTCACAGGAGGCATCTTTGAAG-3’; PPAR-γ Forward Sequence5’-AAGGCGAGGGCGATCTTG-3’, Reverse Sequence5’-ATCATTAAGGAATTCATGTCGTAGATGAC -3’; GLUT1 Forward Sequence 5’-CCAGCTGGGAATCGTCGTT-3’, Reverse
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Sequence5’-CAAGTCTGCATTGCCCATGAT-3’; GAPDH Forward Sequence 5’AGAACATCATCCCTGCATCC -3’, Reverse Sequence5’TCCACCACCCTGTTGCTGTA -3’. Amplification condition was: an initial denaturation
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at 95°C for 10 min; 95°C for 15 s, 62°C for 60 s, 40 cycles. The 2−∆CT method was used
Subcellular fractionation of skeletal muscle
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to analyze the PCR data. GAPDH was used as an endogenous housekeeping gene.
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Membrane fractions from muscle homogenates and myotubes were separated by sucrose-gradient centrifugation, as described previously (Klip et al., 1987; Kristiansen et al., 1998). In brief, skeletal muscle or C2C12 myotubes were washed, minced and homogenized in cold buffer (20 mmol/L NaHCO3, 250 mmol/L sucrose, 5 mmol/L NaNO3, 100 µmol/L PMSF, 1 µmol/L leupeptin, 1 µmol/L pepstatin A, 2 mmol/L
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orthovanadate in ddH2O, pH 7.4, 4 °C). The homogenates were centrifuged at 1200 g for 10 min (4 °C). The supernatants were recentrifuged at 8000 g for 10 min (4 °C). Then the supernatants were layered on a 25% and 50% sucrose gradient and centrifuged at 210,000
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membranes.
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g for 2 h (4 °C) with L7-55Ul ultracentrifuge (Beckman, USA) to yield the plasma
Western blot analysis
Total proteins of skeletal muscle or myotubes were extracted using RIPA agents and quantified with BCA protein assay kit to determine protein levels. Briefly, fifty micrograms of samples were separated by a 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride filter membranes. 8
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Membranes were blocked in Tris-buffered saline (pH 7.5) containing 0.05% Tween-20 (1×TBST) and 5% skimmed milk for 2 h, then probed overnight at 4°C with an antibody against GLUT4, GAPDH, P38MAPK, AKT, AS160, pP38MAPK, pAKT and pAS160,
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respectively. Membranes were washed with 1×TBST for 10 min and incubated for 2 h with horseradish peroxidase-conjugated secondary antibody. Lastly, immunoreactive
Immunofluorescence staining
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Quantity One Analysis Software (Bio-Rad).
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bands were visualized by chemiluminescence and quantified by densitometry using a
Cultured cells in 6-well plates were fixed with 4% paraformaldehyde for 30 min and permeabilized with 0.5% Triton-X 100 (Solabio, Beijing, China) for 10 min at 37°C.
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After wash, the cells were blocked in 5% BSA for 2 h and incubated with primary rabbit antibody against GLUT4 (1:50, Merck Millipore Inc, Germany) at 4 °C overnight. After incubation and wash, cells were blocked with the fluorescent secondary antibody (1:200,
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IgG-FITC) for 1 hour. After washes with PBS, the nuclei were stained with 4’, 6-diamidino-2-phenylindole (DAPI) (Beyotime, Jiangsu, China) for 5 min. Photo capture
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was performed by a Nikon TE2000 microscope (Nikon, Tokyo, Japan). Randomly selected the perspective GLUT4 positive areas and DAPI cells were under the microscope.
Statistical analysis SPSS 17.0 for Windows was used for statistical analysis. Comparisons between the means of three groups were analyzed by one-way ANOVA with Duncan’s tests. Data 9
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were presented as mean ± SEM with P < 0.05 as the limit for statistical significance.
Results
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Body weight and food intake
All of the six-week-old mice used in this experiment exhibited similar body weight and food intake at the beginning of the experiment. Feeding of a high fat diet for 16 weeks
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enhanced body weight of the mice in obese control and baicalin groups compared with the normal diet group before the administration of baicalin (Fig.1). As shown in Fig. 1
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and Fig. 2 the body weight and delta weight at 21 days were significantly decreased by 33.9% (P < 0.01) and 423.94% (P < 0.001) in the baicalin group compared with the obese controls, but increased by 71.3% (P< 0.01) and 4.4% (P> 0.05) in the obese control group compared with the normal controls, respectively. As shown in Fig. 3, the food intake of
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mice was significantly decreased by 36.2% (P< 0.01) , 42% (P< 0.01) and 26.1% (P< 0.01) in the baicalin group compared with the obese controls, and by 5.1% (P> 0.05), 9.3% (P> 0.05) and 9.1% (P> 0.05) in the obese control group compared with the normal
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controls in the first, second and third week, respectively.
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(Fig. 1, Fig. 2 and Fig.3 were located here)
Glucose tolerance and insulin tolerance test During the glucose tolerance test, as shown in Fig. 4, the circulating glucose levels at 120 min were markedly decreased by 24% (P < 0.05) in the baicalin group compared with obese controls, but increased by 69.9% (P< 0.01) in the obese control group compared with normal controls. As shown in Fig. 5, the circulating glucose levels were significantly decreased at 0 min (P < 0.01), 15 min (P < 0.001), 30 min (P < 0.01), 45 min (P < 0.01)
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and 60 min (P < 0.001) after administration of insulin in the baicalin group compared with the obese controls during the insulin tolerance test.
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(Fig. 4 and Fig. 5 were located here)
HOMA-IR index
As shown in Fig. 6A, HOMA-IR index of mice was significantly decreased by 34.4% (P
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< 0.01) in the baicalin group compared with obese controls, but increased by 278.4% (P< 0.01) in the obese control group compared with normal controls. The blood insulin and
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glucose levels was decreased by 16.2 % (P < 0.01) and 13.09% (P < 0.01) in the baicalin group compared with the obese controls, but elevated by 51.9 % (P < 0.05) and 102.2% (P < 0.01) in the obese control group compared with the normal controls (Fig. 6B and Fig. 6C).
2-NBDG uptake
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(Fig.6 was located here)
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As shown in Fig. 7, the 2-NBDG uptake in the baicalin group compared with the controls was significantly elevated by 30.9% (P < 0.01), 46.1% (P < 0.01), 52.8% (P < 0.01) after
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treatment with 100, 200, 400 µM baicalin respectively. (Fig.7 was located here)
GLUT4 mRNA, PGC-1α mRNA, PPAR- γ mRNA and GLUT1 mRNA expression levels As shown in Fig. 8, the GLUT4 mRNA, PGC-1α mRNA, PPAR-γ mRNA and GLUT1 mRNA expression levels were increased by 83.4% (P < 0.01), 169.4% (P < 0.05),
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587.6% (P < 0.01) and 83.4% (P < 0.01) in baicalin group compared with obese controls, but decreased by 67.6% (P < 0.01), 74.7% (P < 0.01), 87.7% (P < 0.01) and 65.4% (P < 0.01) in the obese control group compared with the normal controls, respectively.
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After treatment with 100, 200, 400 µM baicalin, the GLUT4 mRNA expression compared with the controls was increased by 224.3% (P < 0.05), 408.1% (P < 0.01) and 724.8% (P < 0.01), the PGC-1α mRNA expression by 168.6% (P < 0.05), 277.9% (P < 0.01) and
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206.1% (P < 0.01), the PPAR-γ mRNA expression by 13.4% (P > 0.05), 49.4% (P < 0.01) and 44.3% (P < 0.05), and the GLUT1 mRNA expression by 43.6% (P > 0.05), 269.3%
(Fig.8 and Fig.9 were located here)
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(P < 0.01) and 302.9% (P < 0.01), respectively (Fig. 9).
GLUT4 contents in plasma membranes of C2C12 cells and skeletal muscle
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Compared with controls, GLUT4 immunoreactivitie in plasma membranes of C2C12 cells was elevated by 142.9 % (P < 0.01), 248.3% (P < 0.01) and 317.4% (P < 0.01) after treatment with 100, 200, 400 µM baicalin, respectively (Fig. 10A). Also, the
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immunoreactivitie was elevated by 36.4 % (P < 0.05) in the skeletal muscle of the baicalin group compared with the obese controls, but decreased by 47% (P< 0.01) in the
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obese control group compared with the normal controls (Fig 10B). As shown in Fig 11, the GLUT4 immunofluorescence in the plasma membrane of C2C12 cells after treatment of 100, 200, 400 uM baicalin was higher than the controls. (Fig. 10 and Fig. 11 were located here)
The ratios of pAKT/AKT, pAS160/AS160, pP38MAPK/P38MAPK, PGC-1α and
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NT-PGC-1α levels in skeletal muscle As shown in Fig 12A-E, after treatment with 100, 200, 400 µM baicalin in C2C12 cells, the ratios of pAKT/AKT were enhanced by 180.6% (P < 0.01), 244.3% (P < 0.01) and
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848.9% (P < 0.01), the ratios of pAS160/AS160 by 95.3% (P < 0.01), 155.8% (P < 0.01) and 409.1% (P < 0.01), the ratios of pP38MAPK/P38MAPK by 51.2% (P < 0.05), 73% (P < 0.01) and 241.9% (P < 0.01), and the PGC-1α contents by 156.4% (P < 0.05),
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200.7% (P < 0.01) and 580.6% (P < 0.01) compared with the controls, respectively. As shown in Fig 13A-F, the ratios of pAKT/AKT, pAS160/AS160,
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pP38MAPK/P38MAPK and PGC-1α contents were enhanced by 53.5% (P < 0.05), 68.4% (P < 0.01), 28.8% (P < 0.01) and 59% (P < 0.01) in baicalin group compared with obese controls, but decreased by 50.6% (P < 0.01), 53.5% (P < 0.01), 24.9% (P < 0.01) and 50.1% (P < 0.01) in obese control group compared with the normal controls,
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respectively. However, the NT-PGC-1α contents were reduced by 32.7% (P < 0.05) in baicalin group compared with obese controls, but increased by 167.3% (P < 0.01) in the obese control group compared with the normal controls.
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Discussion
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(Fig. 12 and Fig.13 were located here)
Baicalin is a bioactive flavonoid isolated from the radix of Scutellaria baicalensis, which contains several bioactive compounds, such as baicalein, baicalin, β-sitosterol, norwogonin, oroxylin A, and wogonin (Li and Chen, 2005, de Oliveira et al., 2015). Recent research indicated that administration of baicalin could alleviate obesity, hyperglycemia and insulin resistance.
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First, a few studies reported that baicalin might play an anti-obesity role (Lee et al., 2009; Xi et al., 2015; Yang et al., 2016). Treatment of 3T3-L1 preadipocytes with baicalin inhibited triglyceride accumulation and lipid droplet formation during adipogenesis (Lee
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et al., 2009; Yang et al., 2016; Zhu et al., 2016). Besides, oral treatment
with baicalin (100, 200 and 400 mg/kg/d) significantly decreased epididymal fat,
hyperlipidemia, liver steatosis and body weights in mice fed with high fat diet (Xi et al.,
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2015). Furthermore, i.p. injection with baicalin significantly ameliorated the elevated
serum cholesterol and free fatty acid concentrations in rats fed with high fat diet (Guo et
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al., 2009; Waisundara et al., 2011). Consistent with these, the current results revealed that i.p. injection of baicalin decreased food intake and body weight of diet-induced obese (DIO) mice, further demonstrating that baicalin had the anti-obesity effects. Next, baicalin was reported to have antihyperglycemic effects (Li et al., 2011; Shi et al.,
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2016; Xi et al., 2016). I.p. administration of baicalin significantly decreased plasma glucose levels in a dose-dependent manner in streptozotocin-nicotinamide induced diabetic rats (Li et al., 2011; Shi et al., 2016). In accordance with these, we found that i.p.
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injection of baicalin reduced circulating glucose levels in DIO mice too, further manifesting that baicalin had the antihyperglycemic effects.
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Furthermore, baicalin possessed the potency to ameliorate insulin resistance. In this study, 2-NBDG, a fluorescent deoxyglucose analog, was used to investigate the effect of the baicalin on glucose uptake into C2C12 myotubes. 2-NBDG with deoxyglucose can be transported into cells through the help of glucose transporters (Lanner et al., 2006; Zou et al., 2005). The current results showed that baicalin significantly promoted 2-NBDG uptake into C2C12 myotubes in a dose-dependent manner and decreased glucose levels
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during the insulin tolerance test in obese mice. More interestingly, treatment with baicalin in obese mice significantly decreased HOMA-IR values compared to the obese controls. These results implicate that baicalin can increase glucose uptake and insulin sensitivity.
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Last, baicalin can regulate the expression of PPAR-γ, PGC-1α, GLUT1 and GLUT4 in muscles of DIO mice and C2C12 myotubes. First, PPAR-γ is a specific target of the anti-diabetic drug, such as rosiglitazone. Second, PGC-1α is a critical regulator of
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mitochondrial biogenesis in skeletal muscle to maintain an energy balance (Puigserver et al., 1999; Wu et al., 1999). PGC-1α can enhance the expression of GLUT4 and PPAR-γ
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to increase glucose uptake (Wright, 2007, Summermatter et al., 2003; Wu et al., 2016). Overexpressed PGC-1α increased glucose uptake in both C2C12 and L6 muscle cells (Michael et al., 2001), while reduced PGC-1α expression in muscle was relative to the pathogenesis of type 2 diabetes (Mootha et al., 2003; Patti et al., 2003). Third,
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NT-PGC-1α is a short isoform of PGC-1α produced by alternative splicing of the PGC-1α gene. It can increase insulin resistance in DIO mice (Jun et al, 2014). Fourth, p38MAPK is involved in PGC-1α expression to reduce free fatty acid content in skeletal
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muscles (Akimoto et al., 2005; Crunkhorn et al., 2007; Hong et al., 2001; Wright et al., 2007). Blockade of p38MAPK may prevent the increase in expression of PGC-1α. Fifth,
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GLUT1 belongs to the major facilitator superfamily, and is responsible for the constant and basic glucose uptake. Last, GLUT4 is a particularly important glucose transporter for maintaining glucose homeostasis and insulin sensitivity in response to insulin stimuli. Its translocation from intracellular membrane compartments to plasma membranes is a rate-limiting step of glucose uptake and is closely associated with insulin resistance in skeletal muscle (Leto and Saltiel, 2012). The current study found that administration of
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baicalin could upregulate PPAR-γ, GLUT4, PGC-1α and pP38MAPK levels, but inhibit NT-PGC-1α levels in muscle of DIO mice and C2C12 myotubes, suggesting that administration of baicalin decreased NT-PGC-1α or enhanced PGC-1α levels via
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upregulation of pP38MAPK to boost GLUT4 and PPAR-γ expression, resulting in the
increase in glucose uptake (Summermatter et al., 2003; Wu et al., 2016, Wright, 2007). More interestingly, we found that the GLUT4 contents in the plasma membranes were
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markedly increased in muscles of baicalin-treated DIO mice or C2C12 myotubes,
implicating that baicalin not only enhances GLUT4 and GLUT4 mRNA expression levels,
plasma membranes in muscle.
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but also accelerates GLUT4 translocation from intracellular membrane compartments to
To understand the signal mechanism that baicalin promotes GLUT4 translocation, we examined the levels of insulin signaling proteins, including Akt and pAS160 in skeletal
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muscle of DIO mice and C2C12 myotubes. The Akt/AS160 pathway mediates the effect of insulin on surface recruitment of GLUT4 (Samovski et al., 2012; Mikłosz et al., 2016). Activation of Akt mediates the phosphorylation of Akt substrate of 160 kDa (AS160) to
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trigger GLUT4 translocation from intracellular membrane compartments to plasma membranes to accelerate glucose uptake (Sakamoto and Holman, 2008). The increase in
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pAkt and pAS160 contents may boost GLUT4 translocation in skeletal muscle (Oak et al., 2006; Thong et al., 2007). The results in the present study showed that treatment with baicalin enhanced pAkt and pAS160 contents to boost GLUT4 translocation and glucose uptake, suggesting that baicalin increased glucose uptake via the Akt/ AS160/GLUT4 pathway. In summary, the results of the current study revealed that administration of baicalin can
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decreased food intake, body weight, HOMA-IR, NT-PGC-1α content, and circulating glucose and insulin levels, but enhanced GLUT4, PGC-1α, pP38MAPK, pAKT and pAS160 expression, reversed high fat diet-induced glucose and insulin intolerance,
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hyperglycemia and insulin resistance in skeletal muscle of DIO mice. Also, treatment with baicalin significantly elevated the GLUT4 mRNA, PGC-1α mRNA, PPAR-γ mRNA and GLUT1 mRNA expression in skeletal muscles and C2C12 cells. These results
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suggested that Akt/AS160/GLUT4 and P38MAPK/ PGC-1α/ GLUT4 pathway, at least in part, mediated the beneficial effect of baicalin on high fat diet-induced obesity and
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insulin resistance in the skeletal muscle. This study contributes to our understanding of the anti-obesity and anti-diabetic role of baicalin, and provides a possibility of using baicalin to treat obesity and insulin resistance in clinic.
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Acknowledgments
This work was supported by the National Natural Scientific Fund of China (No. 81673736) and in part by the National Health and Family Planning Commission of China
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(Grant No. W201309).
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Competing Interests
The authors declared no conflict of interest.
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a fluorescent indicator for direct glucose uptake measurement. J Biochem Biophys
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Fig. 1. The i.p. administration of baicalin for 21 days decreased the body weight of high fat diet-induced obese mice (n=8). The body weight was lower in the obese group with baicalin than the obese controls (OC), but higher in OC than the normal controls (NC).
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All data shown are the means ± SEM. ** P < 0.01 vs. OC; ++P < 0.01 vs. NC.
Fig. 2. The i.p. administration of baicalin for 21 days decreased the delta weight (delta
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weight was calculated by weight deviations of animals at the beginning and at the end of the experiment) of high fat diet-induced obese mice (n=8). The delta weight was lower in
± SEM. ** P < 0.01 vs. OC.
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the obese group with baicalin than the obese controls (OC). All data shown are the means
Fig. 3. The i.p. injection of baicalin significantly decreased the food intake of animals
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(n=8). The food intake of mice in the baicalin group compared with obese controls (OC) was significantly decreased in the first, second and third week respectively. All data
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shown are the means ± SEM. ** P < 0.01 vs. OC.
Fig. 4. The i.p. administration of baicalin significantly decreased circulating glucose
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levels during the glucose tolerance test (n=8). The circulating glucose levels were markedly decreased at 0 and 120 min in the obese group with baicalin compared with the obese controls (OC), but increased at 0 and 120 min in the obese control group compared with the normal controls (NC). All data shown are the means ± SEM. * P < 0.05 vs. OC ; ++P < 0.01 vs. NC.
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Fig. 5. The i.p. administration of baicalin significantly decreased circulating glucose levels during the insulin tolerance test (n=8). The circulating glucose levels were significantly decreased at 0, 15, 30, 45, 60 min in the obese group with baicalin compared
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with obese controls (OC), but increased at 0, 15, 30, 45, 60 min in the obese control group compared with normal controls (NC). All data shown are the means ± SEM. ** P <
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0.01 vs. OC; ++P < 0.01 vs. NC.
Fig. 6. The i.p. injection of baicalin significantly decreased the HOMA-IR index in
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animals (n=8). Fig. 6A, HOMA-IR index in the baicalin group was significantly decreased compared with obese the controls (OC), but that increased in OC compared with normal controls (NC). Fig. 6B and Fig. 6C showed that blood insulin and glucose levels were lower in the baicalin group than OC, but higher in OC than NC. All data
++P < 0.01 vs. NC.
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shown are the means ± SEM. * P < 0.05 vs. OC; ** P < 0.01 vs. OC; +P < 0.05 vs. NC;
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Fig. 7. The administration of baicalin significantly increased the fluorescence 2-NBDG intensity of cells (n=3). The 2-NBDG uptake of cells in the baicalin group compared with
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controls was significantly elevated after treatment with 100, 200, 400 µM baicalin respectively. All data shown are the means ± SEM. ** P < 0.01 vs. controls.
Fig. 8. The i.p. injection of baicalin significantly increased GLUT4 mRNA, PGC1α mRNA, PPAR-γ mRNA and GLUT1 mRNA expression levels (n=6). The GLUT4 mRNA, PGC1α mRNA, PPAR-γ mRNA and GLUT1 mRNA expression levels were
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increased in the baicalin group compared with the obese controls (OC), but decreased in OC compared with the normal controls (NC) in the skeletal muscle. A, GLUT4; B,
** P < 0.01 vs. OC; ++P < 0.01 vs. NC.
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PGC1α; C, PPAR-γ; D, GLUT1. All data shown are the means ± SEM. * P < 0.05 vs. OC;
Fig. 9. The treatment with elevated GLUT4 mRNA, PGC1α mRNA, PPAR-γ mRNA and
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GLUT1 mRNA expression (n=3). The treatment with 100, 200, 400 µM baicalin significantly elevated the GLUT4 mRNA, PGC1α mRNA, PPAR-γ mRNA and GLUT1
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mRNA expression levels in C2C12 cells. A, GLUT4; B, PGC1α; C, PPAR-γ; D, GLUT1. All data shown are the means ± SEM. * P < 0.05 vs. OC; ** P < 0.01 vs. OC.
Fig. 10. The administration of baicalin significantly elevated GLUT4 protein levels in
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plasma membranes of C2C12 cells (n=3) and plasma membranes of skeletal muscle (n=3). As shown in Fig.10A, the GLUT4 immunoreactivities in plasma membranes of C2C12 cells were elevated in the baicalin group compared with the controls after
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treatment with 100, 200, 400 µM, respectively. The sequence of representative Western blot lines in each panel is Control, 100 µM, 200 µM and 400 µM. As shown in Fig 10B,
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the GLUT4 immunoreactivities in the skeletal muscle was increased in the baicalin group compared with the obese controls (OC), but decreased in OC compared with the normal controls (NC). The sequence of representative Western blot lines in each panel is NC, OC and Baicalin group. All data shown are the means ± SEM. ** P < 0.01 vs. OC; ++P < 0.01 vs. NC.
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Fig. 11. GLUT4-immunofluorescence in C2C12 cells after treatment with baicalin (n=3). The photomicrographs of GLUT4- immunofluorescence in the plasma membrane of
respectively. A, Control; B, 100 µM; C, 200 µM; D, 400 µM.
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C2C12 cells were higher than the controls after treatment of 100, 200, 400 µM baicalin
Fig. 12. Treatment with baicalin significantly elevated pAKT, pAS160, pP38MAPK and
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PGC1α levels of C2C12 cells (n=3). As shown in Fig 12A-D, treatment of 100, 200, 400 µM baicalin significantly elevated pAKT, pAS160, pP38MAPK and PGC1αlevels of
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C2C12 cells compared with the controls respectively. As shown in Fig 12E, the sequence of representative Western blot lines in each panel is Control, 100 µM, 200 µM and 400 µM. All data shown are the means ± SEM. ** P < 0.01 vs. Control.
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Fig. 13. I.p. injection of baicalin elevated pAKT, pAS160, pP38MAPK and PGC1α levels, but reduced NT-PGC1α levels of skeletal muscle (n=3). As shown in Fig 13A-E, the pAkt, pAS160, pP38MAPK and PGC1α contents were enhanced, but the NT-PGC1α
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contents were reduced in the baicalin group compared with the obese controls (OC) respectively. The pAkt, pAS160, pP38MAPK and PGC1α contents were decreased, but
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the NT-PGC1α contents were increased in OC compared with normal controls (NC) respectively. As shown in Fig 13F, the sequence of representative Western blot lines in each panel is NC, OC and Baicalin group. All data shown are the means ± SEM. * P < 0.05 vs. OC; ** P < 0.01 vs. OC; ++P < 0.01 vs. NC.
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Baicalin
ACCEPTED MANUSCRIPT ► I.p injection of baicalin decreases body weight and food intake of obese mice. ► I.p injection of baicalin reduces blood glucose concentration and insulin resistance. ► I.p injection of baicalin increases GLUT4 expression levels in skeletal muscle.
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► I.p injection of baicalin improves GLUT4 translocation level in skeletal muscle. ►Baicalin attenuates skeletal muscle insulin resistance via the Akt/AS160/GLUT4
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pathway.