Research Article Astragalus Polysaccharide Suppresses...

Hindawi Publishing CorporationOxidative Medicine and Cellular LongevityVolume 2013 Article ID 782497 10 pageshttpdxdoiorg1011552013782497

Research ArticleAstragalus Polysaccharide Suppresses SkeletalMuscle Myostatin Expression in Diabetes Involvement ofROS-ERK and NF-120581B Pathways

Min Liu12 Jian Qin2 Yarong Hao3 Min Liu4 Jun Luo5 Tao Luo4 and Lei Wei1

1 Department of Pathology and Pathophysiology School of Medicine Wuhan University Wuhan 430071 China2 Central Laboratory Renmin Hospital of Wuhan University Wuhan 430060 China3Department of Cardiology Renmin Hospital of Wuhan University Wuhan 430060 China4Department of Anesthesiology Renmin Hospital of Wuhan University Wuhan 430060 China5 Department of Pathology Zhongnan Hospital of Wuhan University Wuhan 430071 China

Correspondence should be addressed to Lei Wei leiweifrhotmailcom

Received 7 August 2013 Revised 24 October 2013 Accepted 27 October 2013

Academic Editor Zhengyuan Xia

Copyright copy 2013 Min Liu et al This is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Objective The antidiabetes drug astragalus polysaccharide (APS) is capable of increasing insulin sensitivity in skeletal muscle andimproving whole-body glucose homeostasis Recent studies suggest that skeletal muscle secreted growth factor myostatin plays animportant role in regulating insulin signaling and insulin resistance We hypothesized that regulation of skeletal muscle myostatinexpression may be involved in the improvement of insulin sensitivity by APS Methods APS was administered to 13-week-olddiabetic KKAy and nondiabetic C57BL6J mice for 8 weeks Complementary studies examined APS effects on the saturated acidpalmitate-induced insulin resistance and myostatin expression in C2C12 cells Results APS treatment ameliorated hyperglycemiahyperlipidemia and insulin resistance and decreased the elevation of myostatin expression and malondialdehyde production inskeletal muscle of noninsulin-dependent diabetic KKAy mice In C2C12 cells in vitro saturated acid palmitate-induced impairedglucose uptake overproduction of ROS activation of extracellular regulated protein kinases (ERK) and NF-120581B were partiallyrestored by APS treatment The protective effects of APS were mimicked by ERK and NF-120581B inhibitors respectively ConclusionOur study demonstrates elevated myostatin expression in skeletal muscle of type 2 diabetic KKAy mice and in cultured C2C12cells exposed to palmitate APS is capable of improving insulin sensitivity and decreasing myostatin expression in skeletal musclethrough downregulating ROS-ERK-NF-120581B pathway

1 Introduction

Skeletal muscle comprises 40 to 50 of the total body massand is the major tissue responsible for insulin-dependentglucose utilization [1] Impaired insulin-stimulated muscleglycogen synthesis plays a significant role in insulin resistanceand noninsulin-dependent diabetes mellitus [2] Several fac-tors including elevated oxidative stress inflammation andfree fatty acids have been implicated as the major defectsresponsible for causing muscle insulin resistance in patientswith type 2 diabetes

Myostatin is a growth factor produced by skeletal muscleand secreted into the circulation that negatively regulates

muscle mass [3] Recent studies suggest that myostatin playsa major role in regulating insulin signaling and insulinresistance [4] Patients with type 2 diabetes had higher levelsof muscle myostatin mRNA content than the control subjects[5] A loss-of-function mutation in either one or both allelesof themyostatin genewas able to protectmice against obesity-induced insulin resistance [6] Therefore myostatin couldbe a potent target for the treatment of diabetes and insulinresistance

The dry roots of Astragalus membranaceus (Fisch) Bge(Leguminosae) also known as Huang Qi in China havelong been used as an important component of herbal pre-scriptions to treat diabetes in traditional Chinese medicine

2 Oxidative Medicine and Cellular Longevity

[7ndash9] Studies from our group and others have previouslyshown that astragalus polysaccharide (APS) the extracts ofAstragalus membranaceus can effectively alleviate diabetesand diabetes related complications such as cardiovascular andkidney disease [10ndash12] One of the mechanisms of the APSrsquosantidiabetic effect is to improve whole-body glucose home-ostasis and increase insulin sensitivity in skeletal muscle [13]We hypothesized that regulation of skeletal muscle myostatinexpression may be involved in the improvement of insulinsensitivity by APS In the current study we investigated theeffects of APS on myostatin expression in skeletal muscle oftype 2 diabetic KKAymice in vivo and culture skeletal musclemyocyte in vitro

2 Materials and Methods

21 Chemicals and Reagents Astragalus membranaceus(Fisch) Bunge var mongholicus (Bunge) Hsiao was pur-chased from Shanghai Medicinal Materials (ShanghaiChina) Identification was carried out by the Departmentof Authentication of Chinese Medicine Hubei Universityof Chinese Traditional Medicine (Wuhan China) APSswere extracted with optimized techniques using direct waterdecoction as previously described [10] C2C12 myoblast cellline was obtained from CCTCC (Wuhan China) Insulin2-deoxyglucose fatty acid-free bovine serum albumin (BSA)palmitate (PA) PD98059 and parthenolide were purchasedfrom Sigma-Aldrich (Shanghai China) High glucose-DMEM fetal bovine serum (FBS) and horse serumwere from GIBCO (Shanghai China) NF-120581Bp65 I120581B120572ERK (4244) phospho-ERK (4244) p38 phospho-p38SAPKJNK phospho-SAPKJNK and GAPDH antibodieswere purchased from Cell Signal Technology (USA) Myo-statin (GDF-8) antibody was purchased from Santa CruzFluorescent-conjugated secondary antibodies were fromLI-COR Biosciences

22 In Vivo Experiments The experimental procedures andprotocols used in this investigation were approved by theEthical Committee for the Experimental Use of Animalsat Renmin Hospital of Wuhan University (Wuhan China)Eight-week-old KKAymale mice and age-matched C57BL6Jmale mice were purchased from Beijing HFK Bioscience CoLtd (Beijing China) Mice were housed under a 12 h light-dark cycle and an ambient temperature of 22∘C The KKAymice were fed with a purified high-fat diet consisting of apercentage of total kcal of 41 fat 41 carbohydrates and18 protein The C57BL6J mice were fed with a normalchow diet consisting of 12 fat 60 carbohydrates and 28protein KKAy mice were treated with either vehicle (KKAy119899 = 8) or APS (KKAy + APS 119899 = 8) starting at 13 weeksof age Age-matched C57BL6J mice were also dosed withvehicle (C57BL6J 119899 = 8) or APS (C57BL6J + APS 119899 = 8) ashealthy nondiabetic control animals The APS was deliveredslowly into the animal stomach through a stainless steel ball-tipped gavage needle at a dose of 700mg kgminus1 dayminus1 for 8weeksThe control groups received an equal volumeof vehicle(saline)

23 Blood Chemistry Assay Blood glucose and insulin levelswere determined before (age of 12 weeks) and after APS treat-ment (age of 20 weeks) Blood glucose levels were assessedusing blood collected from the tail vein with a One-TouchUltra blood glucose meter (LifeScan Milpitas CA USA)Plasma insulin concentrations were determined using bloodcollected from the orbita of anesthetized animal followinga 12 h overnight fasting period with mouse high rangeinsulin ELISA kit (ALPCO Diagnostics USA) The index ofhomeostasismodel assessment of insulin resistance (HOMA)was calculated as fasting plasma glucose [mmolL] times fast-ing plasma insulin [mUL]225 Blood plasma FFAs weremeasured using the spectrophotometric NEFA C kit (WakoChemicals Neuss Germany)

24 Malondialdehyde (MDA) Analysis in Skeletal MuscleMDA level in the skeletal muscle was determined by the thio-barbituric acid (TBA) method with an assay kit according tomanufactory guidance (Jiancheng Bioengineering InstituteNanjing China) Briefly samples were incubated with TBAand SDS at 95∘C for 1 h followed by a centrifugation at 800timesgfor 10min Supernatants were transferred to a 96-well plateand the absorbance was measured at 532 nmThe MDA levelafter the calculation was further corrected by sample proteinconcentration (mmolmg protein)

25 Cell Culture and Treatment C2C12 myoblasts were cul-tured in Dulbeccorsquos modified Eaglersquos medium (DMEM) sup-plementedwith 10FBS 2mMglutamine 100 unitmL peni-cillin and 100 120583gmL streptomycin Cells were maintainedat 37∘C under 5 CO

2in a humidified incubator Differen-

tiation of myoblasts into myotubes was induced when thecells had achieved 70 confluence by replacing the mediawith DMEM containing 2 horse serum Six days later thedifferentiated C2C12 cells had fused into myotubes and wereused for the experiments

Palmitate (C160 Sigma) stock solutions of 20mmolLwere dissolved in ethanol Before application to the cellspalmitate was conjugated to bovine serum albumin (BSA) bydiluting the palmitate solution with differentiation mediumcontaining 5 (wv) palmitate-free BSA (Sigma) Myotubeswere incubated for 24 h in DMEM containing 5 BSA ineither the presence (palmitate-treated cells) or absence (con-trol cells) of 05mmolL palmitate Cells treated with APSwere incubatedwith additional APS at the final concentrationof 200120583gmL

26 In Vitro Glucose Uptake Assay Glucose uptake wasassayed using [3H]2-deoxyglucose according to previousreport After 24 h of treatment cells were incubated in thepresence or in the absence of 100 nm insulin for 30min andthen washed two times with wash buffer [20mmHEPES (pH74) 140mm NaCl 5mm KCl 25mm MgSO

4 and 1mm

CaCl2] Cells were then incubated in buffer transport solution

(wash buffer containing 10120583M of unlabeled 2-deoxy-D-glucose and 05 120583CimL [3H]2-deoxy-D-glucose) for 10minNonspecific uptake was determined incubating the cells inthe presence or in the absence of 5 120583Mcytochalasin B Uptake

Oxidative Medicine and Cellular Longevity 3

was terminated by aspiration of the solution Cells were thenwashed three times and radioactivity associatedwith the cellswas determined by cell lysis in 005m NaOH followed byscintillation counting

27 Measurement of ROS Production C2C12 cells (2 times105 cellswell) in a 96-well plate were treated with or withoutAPS for 1 h followed by incubation with palmitate for 24 hROS generation was measured by incubation of the cellswith 10mM DCFH2-DA for 45min The fluorescence cor-responding to intracellular ROS was measured on a Victor31420 Multilabel Counter (PerkinElmer Turku Finland) at485 nm excitation and 530 nm emission wavelengths

28 Measurements of mRNA Total RNA was extracted fromsoleus muscle biopsies of both KKAy and C57BL6J miceusing TRIZOL reagent (Invitrogen) following the manufac-turersquos instruction Levels of myostatin mRNA were exam-ined using semiquantitative reverse transcriptase (RT) PCROne microgram of total RNA was reverse-transcribed withFirst Strand cDNA Synthesis Kit (Thermo Scientific) Thesequences of primers used for amplification were 51015840-GGT-CTGCTGAGTTAGGAGGGT-31015840 and 51015840-TGTGTGTGT-GGAGATGCACCT-31015840 Amplification of gene yielded a sin-gle band of the expected size 339 bp Thermal amplificationswere carried out with PCR Master Mix (Thermo Scientific)PCR was performed within a linear range of amplificationwhich was determined in a preliminary experiment All PCRdata were normalized to beta-actin gene expression

29 Western Blot Analysis For the preparation of total pro-tein extract frozen tissues (50mg) from soleus mus-cles or cells (1 times 106) were homogenized in 05mL or02mL ice-cold lysis buffer (50mM Tris-HCl pH 74 1NP-40 025 sodium deoxycholate 150mM NaCl 1mMEGTA) containing protease inhibitor cocktail (1mM phenyl-methylsulfonyl fluoride 1 120583gmL aprotinin 5120583gmL leu-peptin 1mM Na

3VO4 and 1mM NaF) Tissue or cell

lysates were centrifuged at 12000timesg for 15min at 4∘CThe supernatant was collected and stored at minus70∘C astotal protein samples The preparation of cytoplasmic andnuclear extracts was performed using a commercial kit(Pierce USA) according to the manufacturerrsquos instructionsProtein concentration was then determined using BCAprotein assay kit Samples of the lysates were separatedby 10 SDS-PAGE and then transferred onto PVDF mem-branes After being placed in blocking buffer the mem-branes were incubated with the following primary antibodies(1 1000 dilutions) anti-GDF-8 anti-NF-120581Bp65 anti-I120581B120572anti-p-P38 anti-P38 anti-p-ERK and anti-ERK anti-p-SARPJNK anti-SARPJNK and GAPDH Then secondaryantibody was conjugated to a fluorescent entity IRDye800-conjugated goat anti-rabbit IgG andor Alexa Fluor680-conjugated goat anti-mouse IgG (dilution 1 10000) in10mL LI-COR blocking buffer with gentle agitation for1 h at room temperature The membrane was scanned andanalyzed on the Odyssey Infrared Imaging System (LI-CORBiosciences)

210 Statistical Analysis Data are expressed as means plusmnSD Statistical significance was determined using analysis ofvariance (ANOVA) followed by Tukeyrsquos test A 119875 value of lessthan 005 was considered statistically significant

3 Results

31 Effect of APS onBodyWeight BloodGlucoseHOMAScoreand Plasma FFAs To test whether APS improves glucosemetabolism and lowers insulin resistance in KKAymice APSwas administered for 8 weeks starting at 13 weeks of ageDuring this period blood was collected from the tail veinweekly and plasma glucose levels were measured Consistentwith our previous finding plasma glucose was significantlyreduced in the APS + KKAy group compared with the KKAygroup In particular at the end of 8 weeks of APS treatmentthe glucose level was 30 plusmn 363mM in the KKAy group andapproximately 1705 plusmn 369mM in the APS + KKAy group(119875 lt 0001 119899 = 8 Figure 1(a))

At the beginning of APS treatment the index of home-ostasismodel assessment of insulin resistance (HOMAScore)was not different between the APS + KKAy group andthe KKAy group (data not shown) However after 8 weekstreatment the HOMA score (Figure 1(b)) was significantlylower in the APS + KKAy (841 plusmn 212) group than in theKKAy group (1372 plusmn 384 119875 lt 0001 119899 = 8) SimilarlyAPS treatment resulted in a significant decrease in plasmaFFA levels (Figure 1(c)) and weight gain (Figure 1(d)) for theKKAymice (119875 lt 005 KKAy +APS versus KKAy at the age of20 weeks) whereas APS by itself had no effect on body weightand glucose metabolism in nondiabetic C57BL6J mice

32 Effect of APS on MDA Production and Myostatin Expres-sion in KKAy Mice Oxidative stress plays a causal role in thedevelopment of insulin resistance We tested whether APStreatment alters skeletal muscle redox balance by assessingMDA levels a stable indicator of oxidative stress As shownin Figure 2(a) MDA level in the skeletal muscle of KKAymice was increased approximately 2-fold when comparedwith that in the C57BL6C mice (119875 lt 0001 119899 = 6KKAy versus C57BL6J) APS administration significantlydecreased muscular MDA content (119875 lt 001 119899 = 6 KKAy +APS versus KKAy)

Since myostatin is expressed predominantly in skeletalmuscle we therefore set out to determine the effects of theAPS on both protein and mRNA levels of myostatin in theskeletal muscle tissues of KKAy mice Immunoblotting assayshowed that there was a 33-fold increase in the levels ofmyostatin in KKAy mice (119875 lt 0001 119899 = 6 KKAy versusC57BL6J) APS administration for 8 weeks resulted in 37decrease in myostatin as compared to vehicle treated KKAygroup (119875 lt 0001 119899 = 6 KKAy + APS versus KKAy)(Figure 2(b))

The decreased levels of myostatin could be due to thedecrease in generation or increase in degradation of myo-statin We then assessed the effects of the APS on the mRNAlevels of myostatin (Figure 2(c)) Compared with normalC57BL6J mice diabetic KKAy mice exhibited prominent


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Figure 1 Effect of APS on blood glucose level (a) insulin resistance index HOMA score (b) plasma FFAs level (c) and body weight (d)Changes in blood glucose and body weight were evaluated before and after APS treatment for 8 weeks Insulin resistance HOMA score andplasma FFAs were examined at the end of 8-week APS administration Results are expressed as means plusmn SD for 8 animals in each group119875 lt 0001 versus C57BL6J at the same age lowast119875 lt 005 lowastlowastlowast119875 lt 001 versus KKAy at the same age HOMA homeostasis model assessment

FFA free fatty acids

upregulated myostatin gene expression (119875 lt 0001 119899 = 6KKAy versus C57BL6J) in the skeletal muscle tissues Theincreases in myostatin mRNA were significantly attenuatedby APS treatment (119875 lt 0001 119899 = 6 KKAy + APS versusKKAy)

33 APS Attenuates Insulin Resistance in C2C12 Cells In VitroElevated lipids can cause insulin resistance and exposureof C2C12 skeletal muscle cells to the FFA palmitate hasbeen widely used as an in vitro model of insulin resis-tance To extend these observations from KKAy mice toa cellular model of insulin resistance C2C12 cells weretreated with palmitate to induce insulin resistance asassessed by their decreased intracellular glucose uptakeC2C12 cells exposed to 05mmolL palmitate showed a23 (119875 lt 005) reduction in insulin-stimulated deoxyglu-cose uptake compared with untreated control skeletal

muscle cells (Figure 3(a)) This palmitate-induced insulinresistance coincided with a 33-fold increase in myo-statin accumulation (Figure 3(b)) As expectedAPS adminis-tration resulted in significant reduction of myostatin expres-sion and improvement in insulin-stimulated deoxyglucoseuptake

34 APS Attenuates Palmitate-Induced Generation of ROSin C2C12 Cells Oxidative stress has been implicated inthe pathogenesis of insulin resistance It is suggested thatincreased ROS levels are an important trigger for insulinresistance We next investigated whether ROS levels werealtered by palmitate and APS treatment As shown inFigure 4 ROS levels were increased in C2C12 cells byexposure to palmitate (05mmolL 24 h) APS treatmentsignificantly inhibited generation of ROS in response topalmitate


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Figure 2 Effect of APS on MDA production and myostatin expression in skeletal muscle At the end of 8-week APS administration skeletalmuscle MDA level (a) myostatin protein (b) and mRNA expression (c) were assessed respectively Results are expressed as means plusmn SD for6 animals in each group 119875 lt 0001 versus C57BL6J lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 versus KKAy MDA malondialdehyde MSTN myostatin

35 APS Inhibits ERKNF-120581B Pathway in C2C12 Cells ROShave been shown to induce various signaling pathwaysincluding the p38 MAPK ERK and JNK pathways Wethen investigated the downstream pathways involved inthe upregulation of myostatin following palmitate exposureImmunoblotting detection of total and phosphorylatedERK12 (Figure 5(a)) revealed that palmitate treatmentinduced activation of phosphorylated ERK12 (34-fold

induction 119875 lt 0001) The upregulation of phospho-rylated ERK12 levels achieved by palmitate was abro-gated when the cells were coincubated with APS (119875 lt0001 versus palmitate-treated cells Figure 5(a)) Althoughpalmitate treatment also induced phosphorylation of P38and JNK APS showed no significant effect on P38 andJNK activation (Figures 5(b)-5(c)) These results indi-cate that inhibition of the MAPK-ERK cascade might


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Figure 3 Effect of APS on glucose uptake and myostatin expression in C2C12 cells exposure to palmitate C2C12 cells were incubated for24 h in DMEM containing 5 BSA in either the presence (FFA-treated cells) or absence (control cells) of 05mmolL FFAs Cells treated withAPS were incubated with additional APS at the final concentration of 200120583gmL (a) Glucose uptake was assayed using [3H]2-deoxyglucosein the presence or in the absence of 100 nM insulin 119875 lt 005 versus vehicle lowast119875 lt 005 versus palmitate (b) Myostatin protein expressionwas assessed by western blot Data are expressed as means plusmn SD of 6 different experiments 119875 lt 0001 versus vehicle lowastlowastlowast119875 lt 0001 versuspalmitate PAL palmitate MSTN myostatin

be involved in the decrease of myostatin following APStreatment

It is reported that C2C12 exposure to palmitate acti-vates NF-120581B and activation of the MAPK-ERK cascademay influence NF-120581B activation in skeletal muscle We nextdetermined whether activation of this transcription factorwas involved in palmitate-mediated myostatin upregulationPalmitate treatment resulted in a 19-fold increase in nuclearNF-120581Bp65 protein whereas this activation was preventedin cells coincubated with palmitate and APS (Figure 6(a))Since NF-120581B is located in the cytosol bound to the inhibitorI120581B we next assessed whether palmitate resulted in changesin the content of I120581Ba (Figure 6(b)) Palmitate addition tocells caused a 51 decrease (119875 lt 0001) in the abundanceof I120581Ba whereas APS significantly blocked the palmitate-induced I120581Ba degradation (Figure 6(b)) thereby inhibitingactivation and translocation of NF-120581B

To clearly demonstrate whether ERK12NF-120581B path-way was involved in APS downregulating palmitate-inducedmyostatin expression we used pharmacological inhibitorsPD98059 and parthenolide to block ERK and NF-120581B respec-tively Coincubation of the cells with palmitate in eitherthe presence of PD98059 or parthenolide prevented theupregulation of myostatin (Figure 7) Overall these resultssuggest that myostatin downregulation in skeletal musclecells followingAPS exposure is regulated by the ERK-MAPK-NF-120581B pathway

4 Discussion

We showed that APS treatment ameliorated hyperglycemiahyperlipidemia and insulin resistance whichwere associatedwith the decreased MDA and myostatin level in the skeletalmuscle of KKAy mice Myostatin is an important negativeregulator of skeletal muscle growth In our study increasedmyostatin expression is inversely related to insulin sensitivityin diabetes The finding suggests the concept that myostatinmay play a role in the development of insulin resistance[14] Previous report by Palsgaard et al performed a genechip analysis of skeletal muscle biopsies from human subjectand demonstrated that the levels of myostatin mRNA wereincreased in type 2 diabetics [5] The elevation of muscleand plasma myostatin protein contents in insulin-resistantpatients can be decreased by aerobic exercise training [15]Notably preclinical study showed that administration ofa neutralizing antibody to myostatin for six weeks caneffectively reduce the blood glucose level in obob mice[16] while injection of recombinant myostatin decreasedinsulin sensitivity in healthymale mice [15]Themechanismsby which myostatin achieves this suppression of insulinsensitivity have not been clearly defined Myostatin is knownto increase glucose uptake and glycolysis and inhibit glycogensynthesis in cultured skeletal muscle cells in vitro via anAMP kinase-dependent mechanism [17] Myostatin may alsoaffect glucose uptake indirectly through its effects on TNF-120572


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Figure 4 Effects of APS on the production of ROS in palmitate-stimulated C2C12 cells C2C12 cells were incubated for 24 h inDMEMcontaining 5BSA in either the presence (FFA-treated cells)or absence (control cells) of 05mmolL FFAs Cells treated withAPS were incubated with additional APS at the final concentrationof 200 120583gmL The intracellular levels of ROS were determined byfluorescence using a fluorescence microplate reader with excita-tionemission set to 485530 nm Cells were plated at 1times 106 cellsmLand pretreated with APS for 05 h followed by incubation withpalmitate for 24 h Data are expressed as means plusmn SD of 6 differentexperiments

119875 lt 0001 versus vehicle lowastlowastlowast119875 lt 0001 versuspalmitate ROS reactive oxygen species PAL palmitate

expression which can antagonize the effects of insulin onglucose uptake [18]

Since elevated plasma FFA is a major cause of insulinresistance in type 2 diabetes [19] we therefore used an in vitroFFA-induced insulin resistance cell culture model to furthercharacterize the mechanisms by which APS attenuatingmyostatin expression In this in vitro model FFA palmitateinhibited insulin-stimulated glucose uptake accompaniedwith a strong induction of the myostatin expression proteinin skeletal muscle C2C12 cells which was significantly atten-uated by APS Because an inverse relationship between theinsulin resistance and muscle myostatin has been reported[14] we hypothesized that reducing skeletalmusclemyostatinexpression might be one of the mechanisms by which APSenables insulinsensitizing and hypoglycemic activity

Oxidative stress which may be precipitated by hyper-glycemia and hyperlipidemia plays a pivotal role in thedevelopment of diabetes [20] ROS overproduction can leadto impairment of intracellular signaling pathways and devel-opment of insulin resistance [21] Oxidative stress has beenproposed as a link between FFA and skeletal muscle insulinresistance [22] Consequently reducing oxidative stress bylowering ROS production is crucial in the managementof insulin resistance We showed that palmitate stimulatesROS formation and APS presented effective suppressionon palmitate-induced ROS overproduction and facilitatedinsulin action on the C2C12 cells well demonstrating itsantioxidant potency against FFA insult Consistent with

our in vitro finding the decreased MDA level in skeletalmuscle of KKAy mice after APS treatment also supportedan antioxidant action of APS There are several pathwaysinvolved in ROS production under the conditions of insulinresistance including NADPH oxidase xanthine oxidase andmitochondria-mediated pathways [23] APS is reported toprotect mitochondria by scavenging ROS inhibiting mito-chondrial permeability transition and increasing the activi-ties of antioxidases [24] Future researchwill therefore involveinvestigations of the mechanisms behind the antioxidanteffect of APS in response to palmitate stimulation and otherpathophysiological conditions

We studied several different signal transduction pathwaysknown to be activated by ROS MAPKs are importantmediators involved in a variety of cell signalling functionsincluding insulin resistance The MAPK family includesp38 ERK and JNK [25] Our data indicate that palmitateinduced marked phosphorylation of ERK in C2C12 cellswhile APS downregulated the expression of the phospho-ERK following palmitate stimulation The transcription fac-tor NF-120581B has been proposed as a critical mediator betweenoxidant stress and gene expression Activation of the NF-120581B has been suggested to participate in diabetes and itscomplications [26] We therefore explored whether NF-120581Bparticipates in APS attenuating palmitate-induced myostatinexpression and insulin resistance Exposure of C2C12 cells topalmitate resulted in degradation of I120581B120572 and the subsequentrelease and translocation of NF-120581B into the nucleus APSadministration however leads to the upregulation of I120581B120572and decreased NF-120581B translocation We propose that APSmay reduce FFA palmitate-stimulated myostatin expressionin skeletal muscle cells through a mechanism involvingactivation of the ROS-ERK-NF-120581B pathway since the ERKinhibitor PD98059 as well as NF-120581B inhibitor parthenolidepartially reversed the effect of palmitate-stimulated myo-statin expression

Several other lines of evidences also suggest an effectof APS on ROS-ERKNF-120581B signaling pathway For exam-ple Astragalus membranaceus can inhibit inflammation viaphospho-P38 MAPK and NF-120581B pathways in advanced gly-cation end product-stimulated macrophages [18] Astragalusmembranaceus has been shown to inhibit mRNA expressionsof NF-120581B and I120581B in renal cortex of streptozotoxin-induceddiabetic rats [27] Similar to our finding a recent studysuggests that astragalus polysaccharide inhibits palmitate-induced insulin resistance in C2C12 myotubes by inhibitingexpression of PTP1B and regulating NF-120581B [28] In previousstudies from our colleagues APS has been shown to increaseinsulin-induced tyrosine phosphorylation of the insulinreceptor and IRS-1 in the skeletal muscles of fat-fed diabeticrats with parallel reduces in protein levels and activityof protein tyrosine phosphatase-1B [10] Furthermore ourrecent finding indicated that the antihyperglycemic activityof APS ismediated by insulin sensitivity improvement relatedto GSK3 inhibition in the liver [11] All these results suggestthat APS might regulate insulin sensitivity at multiple sites invarious diabetic animal models

In conclusion the present study demonstrated elevatedmyostatin expression in skeletal muscle of type 2 diabetic


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Figure 5 Effects of APS on the MAPK expression in palmitate-stimulated C2C12 cells C2C12 cells were incubated for 24 h in DMEMcontaining 5 BSA in either the presence (FFA-treated cells) or absence (control cells) of 05mmolL FFAs Cells treated with APS wereincubated with additional APS at the final concentration of 200120583gmL The expression of ERK p38 and JNK and phosphorylated ERKp38 and JNK were determined by western blot Data are expressed as means plusmn SD of 6 different experiments 119875 lt 0001 versus vehiclelowastlowastlowast

119875 lt 0001 versus palmitate ERK extracellular signal-regulated kinase JNK c-Jun amino-terminal kinases


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Figure 6 Effects of APS on NF-120581B activation in palmitate-stimulated C2C12 cells C2C12 cells were incubated for 24 h in DMEM containing5 BSA in either the presence (FFA-treated cells) or absence (control cells) of 05mmolL FFAs Cells treated with APS were incubated withadditional APS at the final concentration of 200120583gmL The expression of nuclear NF-120581B and cytoplasmic I120581Ba was determined by westernblot Data are expressed as means plusmn SD of 6 different experiments 119875 lt 0001 versus vehicle lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 versus palmitateNF-120581B nuclear factor-kappa B

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MSTN

lowastlowastlowast lowastlowastlowast

PAL +PD98059

PAL +parthenolide

Figure 7TheERK-MAPK cascade is involved in palmitate-inducedmyostatin expression in skeletal muscle cells C2C12 myotubes wereincubated with either 05mM palmitate in the presence or in theabsence of 100120583M PD98059 or with 10120583M parthenolide Analysisof the protein levels of myostatin was analyzed by western blotassay Data are expressed as means plusmn SD of 6 different experiments119875 lt 0001 versus vehicle lowastlowastlowast119875 lt 0001 versus palmitate MSTN

myostatin

KKAymice and in cultured C2C12 cells exposed to palmitateAPS is capable of improving insulin sensitivity and decreasingmyostatin expression in skeletal muscle through downreg-ulating ROS-ERK-NF-120581B pathway This study provides newinsight into the molecular mechanisms of antidiabetes actionof APS

Acknowledgment

This work is supported by NSFC Grant nos 8110286381271205 and 81172043

References

[1] D R Sinacore and E A Gulve ldquoThe role of skeletal muscle inglucose transport glucose homeostasis and insulin resistanceimplications for physical therapyrdquo Physical Therapy vol 73 no12 pp 878ndash891 1993

[2] K Eckardt A Taube and J Eckel ldquoObesity-associated insulinresistance in skeletal muscle role of lipid accumulation andphysical inactivityrdquo Reviews in Endocrine and Metabolic Disor-ders vol 12 no 3 pp 163ndash172 2011

[3] B Elliott D Renshaw S Getting and R Mackenzie ldquoThecentral role of myostatin in skeletal muscle and whole bodyhomeostasisrdquo Acta Physiologica vol 205 no 3 pp 324ndash3402012

[4] T Guo W Jou T Chanturiya J Portas O Gavrilova and A CMcPherron ldquoMyostatin inhibition in muscle but not adiposetissue decreases fatmass and improves insulin sensitivityrdquoPLoSONE vol 4 no 3 Article ID e4937 2009


[5] C Brandt A R Nielsen C P Fischer J Hansen B K Pedersenand P Plomgaard ldquoPlasma and muscle myostatin in relation totype 2 diabetesrdquo PLoSONE vol 7 no 5 Article ID e37236 2012

[6] J JWilkes D J Lloyd andNGekakis ldquoLoss-of-functionmuta-tion in myostatin reduces tumor necrosis factor a productionand protects liver against obesity-induced insulin resistancerdquoDiabetes vol 58 no 5 pp 1133ndash1143 2009

[7] Y Tong and H Hou ldquoEffects of Huangqi Guizhi Wuwu Tangon diabetic peripheral neuropathyrdquo Journal of Alternative andComplementary Medicine vol 12 no 6 pp 506ndash509 2006

[8] Z Sang L Zhou X Fan and R J McCrimmon ldquoRadixz astra-gali (Huangqi) as a treatment for defective hypoglycemiacounterregulation in diabetesrdquoTheAmerican Journal of ChineseMedicine vol 38 no 6 pp 1027ndash1038 2010

[9] Y W Zhang D Xie B Xia R T Zhen I M Liu and J TCheng ldquoSuppression of transforming growth factor-1205731 geneexpression by danggui buxue tang a traditional Chinese herbalpreparation in retarding the progress of renal damage instreptozotocin-induced diabetic ratsrdquo Hormone and MetabolicResearch vol 38 no 2 pp 82ndash88 2006

[10] YWu J P Ou-Yang KWu YWang Y F Zhou and C YWenldquoHypoglycemic effect of Astragalus polysaccharide and its effecton PTP1Brdquo Acta Pharmacologica Sinica vol 26 no 3 pp 345ndash352 2005

[11] X Q Mao Y Wu K Wu et al ldquoAstragalus polysaccharidereduces hepatic endoplasmic reticulum stress and restoresglucose homeostasis in a diabetic KKAy mouse modelrdquo ActaPharmacologica Sinica vol 28 no 12 pp 1947ndash1956 2007

[12] Y X Chen Q Y Zhang and J Wang ldquoChanges of periph-eral blood dendritic cell functions in children with Henoch-Schonlein purpura and in vitro effect of Astragalus mem-branaceusrdquo Zhonghua Er Ke Za Zhi vol 46 no 9 pp 708ndash7092008 (Chinese)

[13] M Liu K Wu X Mao Y Wu and J Ouyang ldquoAstragaluspolysaccharide improves insulin sensitivity in KKAy miceregulation of PKBGLUT4 signaling in skeletal musclerdquo Journalof Ethnopharmacology vol 127 no 1 pp 32ndash37 2010

[14] D L Allen D S Hittel and A C McPherron ldquoExpression andfunction of myostatin in obesity diabetes and exercise adapta-tionrdquo Medicine and Science in Sports and Exercise vol 43 no10 pp 1828ndash1835 2011

[15] D S Hittel M Axelson N Sarna J Shearer K M HuffmanandW E Kraus ldquoMyostatin decreases with aerobic exercise andassociates with insulin resistancerdquo Medicine and Science inSports and Exercise vol 42 no 11 pp 2023ndash2029 2010

[16] B L Bernardo T S Wachtmann P G Cosgrove et al ldquoPostna-tal PPARdelta activation andmyostatin inhibition exert distinctyet complimentary effects on the metabolic profile of obeseinsulin-resistant micerdquo PLoS ONE vol 5 no 6 Article IDe11307 2010

[17] C Zhang C McFarlane S Lokireddy et al ldquoMyostatin-defi-cient mice exhibit reduced insulin resistance through activatingthe AMP-activated protein kinase signalling pathwayrdquo Dia-betologia vol 54 no 6 pp 1491ndash1501 2011

[18] Q Qin J Niu Z Wang W Xu Z Qiao and Y Gu ldquoAstra-galus membranaceus inhibits inflammation via phospho-P38mitogen-activated protein kinase (MAPK) and nuclear factor(NF)-120581B pathways in advanced glycation end product-stimu-lated macrophagesrdquo International Journal of Molecular Sciencesvol 13 no 7 pp 8379ndash8387 2012

[19] G Boden ldquoEffects of free fatty acids (FFA) on glucosemetabolism significance for insulin resistance and type 2 dia-betesrdquo Experimental and Clinical Endocrinology and Diabetesvol 111 no 3 pp 121ndash124 2003

[20] K Stadler ldquoOxidative stress in diabetesrdquo Advances in Experi-mental Medicine and Biology vol 771 pp 272ndash287 2012

[21] F Giacco and M Brownlee ldquoOxidative stress and diabeticcomplicationsrdquo Circulation Research vol 107 no 9 pp 1058ndash1070 2010

[22] A R Martins R T Nachbar R Gorjao et al ldquoMechanismsunderlying skeletal muscle insulin resistance induced by fattyacids importance of the mitochondrial functionrdquo Lipids inHealth and Disease vol 11 article 30 2012

[23] D H Endemann and E L Schiffrin ldquoNitric oxide oxidativeexcess and vascular complications of diabetesmellitusrdquoCurrentHypertension Reports vol 6 no 2 pp 85ndash89 2004

[24] X T Li Y K Zhang H X Kuang et al ldquoMitochondrial protec-tion and anti-aging activity of Astragalus polysaccharides andtheir potential mechanismrdquo International Journal of MolecularSciences vol 13 no 2 pp 1747ndash1761 2012

[25] E K Kim and E J Choi ldquoPathological roles of MAPK signalingpathways in humandiseasesrdquoBiochimica et Biophysica Acta vol1802 no 4 pp 396ndash405 2010

[26] A Bierhaus S Schiekofer M Schwaninger et al ldquoDiabetes-associated sustained activation of the transcription factornuclear factor-120581BrdquoDiabetes vol 50 no 12 pp 2792ndash2808 2001

[27] Y W Zhang C Y Wu and J T Cheng ldquoMerit of Astragaluspolysaccharide in the improvement of early diabetic nephropa-thy with an effect on mRNA expressions of NF-120581B and I120581B inrenal cortex of streptozotoxin-induced diabetic ratsrdquo Journal ofEthnopharmacology vol 114 no 3 pp 387ndash392 2007

[28] M Zhao Z F Zhang Y Ding J B Wang and Y Li ldquoAstragaluspolysaccharide improves palmitate-induced insulin resistanceby inhibiting PTP1B andNF-120581B inC2C12myotubesrdquoMoleculesvol 17 no 6 pp 7083ndash7092 2012

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


[7ndash9] Studies from our group and others have previouslyshown that astragalus polysaccharide (APS) the extracts ofAstragalus membranaceus can effectively alleviate diabetesand diabetes related complications such as cardiovascular andkidney disease [10ndash12] One of the mechanisms of the APSrsquosantidiabetic effect is to improve whole-body glucose home-ostasis and increase insulin sensitivity in skeletal muscle [13]We hypothesized that regulation of skeletal muscle myostatinexpression may be involved in the improvement of insulinsensitivity by APS In the current study we investigated theeffects of APS on myostatin expression in skeletal muscle oftype 2 diabetic KKAymice in vivo and culture skeletal musclemyocyte in vitro

2 Materials and Methods

21 Chemicals and Reagents Astragalus membranaceus(Fisch) Bunge var mongholicus (Bunge) Hsiao was pur-chased from Shanghai Medicinal Materials (ShanghaiChina) Identification was carried out by the Departmentof Authentication of Chinese Medicine Hubei Universityof Chinese Traditional Medicine (Wuhan China) APSswere extracted with optimized techniques using direct waterdecoction as previously described [10] C2C12 myoblast cellline was obtained from CCTCC (Wuhan China) Insulin2-deoxyglucose fatty acid-free bovine serum albumin (BSA)palmitate (PA) PD98059 and parthenolide were purchasedfrom Sigma-Aldrich (Shanghai China) High glucose-DMEM fetal bovine serum (FBS) and horse serumwere from GIBCO (Shanghai China) NF-120581Bp65 I120581B120572ERK (4244) phospho-ERK (4244) p38 phospho-p38SAPKJNK phospho-SAPKJNK and GAPDH antibodieswere purchased from Cell Signal Technology (USA) Myo-statin (GDF-8) antibody was purchased from Santa CruzFluorescent-conjugated secondary antibodies were fromLI-COR Biosciences

22 In Vivo Experiments The experimental procedures andprotocols used in this investigation were approved by theEthical Committee for the Experimental Use of Animalsat Renmin Hospital of Wuhan University (Wuhan China)Eight-week-old KKAymale mice and age-matched C57BL6Jmale mice were purchased from Beijing HFK Bioscience CoLtd (Beijing China) Mice were housed under a 12 h light-dark cycle and an ambient temperature of 22∘C The KKAymice were fed with a purified high-fat diet consisting of apercentage of total kcal of 41 fat 41 carbohydrates and18 protein The C57BL6J mice were fed with a normalchow diet consisting of 12 fat 60 carbohydrates and 28protein KKAy mice were treated with either vehicle (KKAy119899 = 8) or APS (KKAy + APS 119899 = 8) starting at 13 weeksof age Age-matched C57BL6J mice were also dosed withvehicle (C57BL6J 119899 = 8) or APS (C57BL6J + APS 119899 = 8) ashealthy nondiabetic control animals The APS was deliveredslowly into the animal stomach through a stainless steel ball-tipped gavage needle at a dose of 700mg kgminus1 dayminus1 for 8weeksThe control groups received an equal volumeof vehicle(saline)

23 Blood Chemistry Assay Blood glucose and insulin levelswere determined before (age of 12 weeks) and after APS treat-ment (age of 20 weeks) Blood glucose levels were assessedusing blood collected from the tail vein with a One-TouchUltra blood glucose meter (LifeScan Milpitas CA USA)Plasma insulin concentrations were determined using bloodcollected from the orbita of anesthetized animal followinga 12 h overnight fasting period with mouse high rangeinsulin ELISA kit (ALPCO Diagnostics USA) The index ofhomeostasismodel assessment of insulin resistance (HOMA)was calculated as fasting plasma glucose [mmolL] times fast-ing plasma insulin [mUL]225 Blood plasma FFAs weremeasured using the spectrophotometric NEFA C kit (WakoChemicals Neuss Germany)

24 Malondialdehyde (MDA) Analysis in Skeletal MuscleMDA level in the skeletal muscle was determined by the thio-barbituric acid (TBA) method with an assay kit according tomanufactory guidance (Jiancheng Bioengineering InstituteNanjing China) Briefly samples were incubated with TBAand SDS at 95∘C for 1 h followed by a centrifugation at 800timesgfor 10min Supernatants were transferred to a 96-well plateand the absorbance was measured at 532 nmThe MDA levelafter the calculation was further corrected by sample proteinconcentration (mmolmg protein)

25 Cell Culture and Treatment C2C12 myoblasts were cul-tured in Dulbeccorsquos modified Eaglersquos medium (DMEM) sup-plementedwith 10FBS 2mMglutamine 100 unitmL peni-cillin and 100 120583gmL streptomycin Cells were maintainedat 37∘C under 5 CO

2in a humidified incubator Differen-

tiation of myoblasts into myotubes was induced when thecells had achieved 70 confluence by replacing the mediawith DMEM containing 2 horse serum Six days later thedifferentiated C2C12 cells had fused into myotubes and wereused for the experiments

Palmitate (C160 Sigma) stock solutions of 20mmolLwere dissolved in ethanol Before application to the cellspalmitate was conjugated to bovine serum albumin (BSA) bydiluting the palmitate solution with differentiation mediumcontaining 5 (wv) palmitate-free BSA (Sigma) Myotubeswere incubated for 24 h in DMEM containing 5 BSA ineither the presence (palmitate-treated cells) or absence (con-trol cells) of 05mmolL palmitate Cells treated with APSwere incubatedwith additional APS at the final concentrationof 200120583gmL

26 In Vitro Glucose Uptake Assay Glucose uptake wasassayed using [3H]2-deoxyglucose according to previousreport After 24 h of treatment cells were incubated in thepresence or in the absence of 100 nm insulin for 30min andthen washed two times with wash buffer [20mmHEPES (pH74) 140mm NaCl 5mm KCl 25mm MgSO

4 and 1mm

CaCl2] Cells were then incubated in buffer transport solution

(wash buffer containing 10120583M of unlabeled 2-deoxy-D-glucose and 05 120583CimL [3H]2-deoxy-D-glucose) for 10minNonspecific uptake was determined incubating the cells inthe presence or in the absence of 5 120583Mcytochalasin B Uptake









3 Results







0

10

20

30

40

KKAY C57BL

Bloo

d gl

ucos

e (m

mol

L)



13 W 14 W 15 W 16 W 17 W 18 W 19 W 20 W

(a)

C57BL6J KKAy0

5

10

15

20

VehicleAPS

HO

MA

scor

e

lowastlowastlowast

(b)

C57BL6J KKAy00

01

02

03

04

Bloo

d FF

As (

mm

olL

)

lowast

VehicleAPS

(c)


10

20

30

40

50

Before treatment

After treatment

lowast

Body

wei

ght (

g)

(d)








C57BL6J KKAy00

05

10

15

MD

A le

vel

lowastlowast


(a)

C57BL6J KKAy00

05

10

15

Myo

stat

in p

rote

in ex

pres

sion

MSTN

GAPDH


lowastlowastlowast

(b)

C57BL6J KKAy0

2

4

6

8

Myo

stat

in m

RNA

MSTN


120573-Actin

lowastlowastlowast

(c)





Vehicle PAL0

50

100

150

200

Glu

cose

upt

ake (

o

f bas

al)

PAL + APS

Noninsulin

Insulin

lowast

(a)

Vehicle PAL00

02

04

06

08

10

12

14

Myo

stat

in p

rote

in ex

pres

sion

MSTN

GAPDH


lowastlowastlowast

(b)





4 Discussion



Non-PAL PAL0

500

1000

1500

2000

Vehicle

APS

ROS

(abs

orba












Vehicle PAL00

02

04

06

08

10

p-ER

K(1

2) p

rote

in ex

pres

sion

GAPDH

ERK

P-ERK(12)

Vehicle +APS

PAL + APS

lowastlowastlowast

(a)

00

02

04

06

08

10

p-P3

8 pr

otei

n ex

pres

sion

p38

GAPDH


PAL + APS

P-p38

(b)

Vehicle PAL00

02

04

06

08

10

12

p-JN

K pr

otei

n ex

pres

sion

JNK

GAPDH

P-JNK

Vehicle +APS

PAL + APS

(c)




Vehicle PAL00

02

04

06

08

10

12

14

NF-120581

B pr

otei

n ex

pres

sion

NF-120581B

GAPDH


lowastlowastlowast

(a)

00

02

04

06

08

I120581Ba

pro

tein

expr

essio

n

GAPDH


I120581B120572

lowastlowast

PAL + APS

(b)


Vehicle PAL00

02

04

06

08

Myo

stat

in p

rote

in ex

pres

sion

GAPDH

MSTN


PAL +PD98059

PAL +parthenolide


myostatin


Acknowledgment


References



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
























3 Results







0

10

20

30

40

KKAY C57BL

Bloo

d gl

ucos

e (m

mol

L)



13 W 14 W 15 W 16 W 17 W 18 W 19 W 20 W

(a)

C57BL6J KKAy0

5

10

15

20

VehicleAPS

HO

MA

scor

e

lowastlowastlowast

(b)

C57BL6J KKAy00

01

02

03

04

Bloo

d FF

As (

mm

olL

)

lowast

VehicleAPS

(c)


10

20

30

40

50

Before treatment

After treatment

lowast

Body

wei

ght (

g)

(d)








C57BL6J KKAy00

05

10

15

MD

A le

vel

lowastlowast


(a)

C57BL6J KKAy00

05

10

15

Myo

stat

in p

rote

in ex

pres

sion

MSTN

GAPDH


lowastlowastlowast

(b)

C57BL6J KKAy0

2

4

6

8

Myo

stat

in m

RNA

MSTN


120573-Actin

lowastlowastlowast

(c)





Vehicle PAL0

50

100

150

200

Glu

cose

upt

ake (

o

f bas

al)

PAL + APS

Noninsulin

Insulin

lowast

(a)

Vehicle PAL00

02

04

06

08

10

12

14

Myo

stat

in p

rote

in ex

pres

sion

MSTN

GAPDH


lowastlowastlowast

(b)





4 Discussion



Non-PAL PAL0

500

1000

1500

2000

Vehicle

APS

ROS

(abs

orba












Vehicle PAL00

02

04

06

08

10

p-ER

K(1

2) p

rote

in ex

pres

sion

GAPDH

ERK

P-ERK(12)

Vehicle +APS

PAL + APS

lowastlowastlowast

(a)

00

02

04

06

08

10

p-P3

8 pr

otei

n ex

pres

sion

p38

GAPDH


PAL + APS

P-p38

(b)

Vehicle PAL00

02

04

06

08

10

12

p-JN

K pr

otei

n ex

pres

sion

JNK

GAPDH

P-JNK

Vehicle +APS

PAL + APS

(c)




Vehicle PAL00

02

04

06

08

10

12

14

NF-120581

B pr

otei

n ex

pres

sion

NF-120581B

GAPDH


lowastlowastlowast

(a)

00

02

04

06

08

I120581Ba

pro

tein

expr

essio

n

GAPDH


I120581B120572

lowastlowast

PAL + APS

(b)


Vehicle PAL00

02

04

06

08

Myo

stat

in p

rote

in ex

pres

sion

GAPDH

MSTN


PAL +PD98059

PAL +parthenolide


myostatin


Acknowledgment


References



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

10

20

30

40

KKAY C57BL

Bloo

d gl

ucos

e (m

mol

L)



13 W 14 W 15 W 16 W 17 W 18 W 19 W 20 W

(a)

C57BL6J KKAy0

5

10

15

20

VehicleAPS

HO

MA

scor

e

lowastlowastlowast

(b)

C57BL6J KKAy00

01

02

03

04

Bloo

d FF

As (

mm

olL

)

lowast

VehicleAPS

(c)


10

20

30

40

50

Before treatment

After treatment

lowast

Body

wei

ght (

g)

(d)








C57BL6J KKAy00

05

10

15

MD

A le

vel

lowastlowast


(a)

C57BL6J KKAy00

05

10

15

Myo

stat

in p

rote

in ex

pres

sion

MSTN

GAPDH


lowastlowastlowast

(b)

C57BL6J KKAy0

2

4

6

8

Myo

stat

in m

RNA

MSTN


120573-Actin

lowastlowastlowast

(c)





Vehicle PAL0

50

100

150

200

Glu

cose

upt

ake (

o

f bas

al)

PAL + APS

Noninsulin

Insulin

lowast

(a)

Vehicle PAL00

02

04

06

08

10

12

14

Myo

stat

in p

rote

in ex

pres

sion

MSTN

GAPDH


lowastlowastlowast

(b)





4 Discussion



Non-PAL PAL0

500

1000

1500

2000

Vehicle

APS

ROS

(abs

orba












Vehicle PAL00

02

04

06

08

10

p-ER

K(1

2) p

rote

in ex

pres

sion

GAPDH

ERK

P-ERK(12)

Vehicle +APS

PAL + APS

lowastlowastlowast

(a)

00

02

04

06

08

10

p-P3

8 pr

otei

n ex

pres

sion

p38

GAPDH


PAL + APS

P-p38

(b)

Vehicle PAL00

02

04

06

08

10

12

p-JN

K pr

otei

n ex

pres

sion

JNK

GAPDH

P-JNK

Vehicle +APS

PAL + APS

(c)




Vehicle PAL00

02

04

06

08

10

12

14

NF-120581

B pr

otei

n ex

pres

sion

NF-120581B

GAPDH


lowastlowastlowast

(a)

00

02

04

06

08

I120581Ba

pro

tein

expr

essio

n

GAPDH


I120581B120572

lowastlowast

PAL + APS

(b)


Vehicle PAL00

02

04

06

08

Myo

stat

in p

rote

in ex

pres

sion

GAPDH

MSTN


PAL +PD98059

PAL +parthenolide


myostatin


Acknowledgment


References



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















C57BL6J KKAy00

05

10

15

MD

A le

vel

lowastlowast


(a)

C57BL6J KKAy00

05

10

15

Myo

stat

in p

rote

in ex

pres

sion

MSTN

GAPDH


lowastlowastlowast

(b)

C57BL6J KKAy0

2

4

6

8

Myo

stat

in m

RNA

MSTN


120573-Actin

lowastlowastlowast

(c)





Vehicle PAL0

50

100

150

200

Glu

cose

upt

ake (

o

f bas

al)

PAL + APS

Noninsulin

Insulin

lowast

(a)

Vehicle PAL00

02

04

06

08

10

12

14

Myo

stat

in p

rote

in ex

pres

sion

MSTN

GAPDH


lowastlowastlowast

(b)





4 Discussion



Non-PAL PAL0

500

1000

1500

2000

Vehicle

APS

ROS

(abs

orba












Vehicle PAL00

02

04

06

08

10

p-ER

K(1

2) p

rote

in ex

pres

sion

GAPDH

ERK

P-ERK(12)

Vehicle +APS

PAL + APS

lowastlowastlowast

(a)

00

02

04

06

08

10

p-P3

8 pr

otei

n ex

pres

sion

p38

GAPDH


PAL + APS

P-p38

(b)

Vehicle PAL00

02

04

06

08

10

12

p-JN

K pr

otei

n ex

pres

sion

JNK

GAPDH

P-JNK

Vehicle +APS

PAL + APS

(c)




Vehicle PAL00

02

04

06

08

10

12

14

NF-120581

B pr

otei

n ex

pres

sion

NF-120581B

GAPDH


lowastlowastlowast

(a)

00

02

04

06

08

I120581Ba

pro

tein

expr

essio

n

GAPDH


I120581B120572

lowastlowast

PAL + APS

(b)


Vehicle PAL00

02

04

06

08

Myo

stat

in p

rote

in ex

pres

sion

GAPDH

MSTN


PAL +PD98059

PAL +parthenolide


myostatin


Acknowledgment


References



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Vehicle PAL0

50

100

150

200

Glu

cose

upt

ake (

o

f bas

al)

PAL + APS

Noninsulin

Insulin

lowast

(a)

Vehicle PAL00

02

04

06

08

10

12

14

Myo

stat

in p

rote

in ex

pres

sion

MSTN

GAPDH


lowastlowastlowast

(b)





4 Discussion



Non-PAL PAL0

500

1000

1500

2000

Vehicle

APS

ROS

(abs

orba












Vehicle PAL00

02

04

06

08

10

p-ER

K(1

2) p

rote

in ex

pres

sion

GAPDH

ERK

P-ERK(12)

Vehicle +APS

PAL + APS

lowastlowastlowast

(a)

00

02

04

06

08

10

p-P3

8 pr

otei

n ex

pres

sion

p38

GAPDH


PAL + APS

P-p38

(b)

Vehicle PAL00

02

04

06

08

10

12

p-JN

K pr

otei

n ex

pres

sion

JNK

GAPDH

P-JNK

Vehicle +APS

PAL + APS

(c)




Vehicle PAL00

02

04

06

08

10

12

14

NF-120581

B pr

otei

n ex

pres

sion

NF-120581B

GAPDH


lowastlowastlowast

(a)

00

02

04

06

08

I120581Ba

pro

tein

expr

essio

n

GAPDH


I120581B120572

lowastlowast

PAL + APS

(b)


Vehicle PAL00

02

04

06

08

Myo

stat

in p

rote

in ex

pres

sion

GAPDH

MSTN


PAL +PD98059

PAL +parthenolide


myostatin


Acknowledgment


References



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Non-PAL PAL0

500

1000

1500

2000

Vehicle

APS

ROS

(abs

orba












Vehicle PAL00

02

04

06

08

10

p-ER

K(1

2) p

rote

in ex

pres

sion

GAPDH

ERK

P-ERK(12)

Vehicle +APS

PAL + APS

lowastlowastlowast

(a)

00

02

04

06

08

10

p-P3

8 pr

otei

n ex

pres

sion

p38

GAPDH


PAL + APS

P-p38

(b)

Vehicle PAL00

02

04

06

08

10

12

p-JN

K pr

otei

n ex

pres

sion

JNK

GAPDH

P-JNK

Vehicle +APS

PAL + APS

(c)




Vehicle PAL00

02

04

06

08

10

12

14

NF-120581

B pr

otei

n ex

pres

sion

NF-120581B

GAPDH


lowastlowastlowast

(a)

00

02

04

06

08

I120581Ba

pro

tein

expr

essio

n

GAPDH


I120581B120572

lowastlowast

PAL + APS

(b)


Vehicle PAL00

02

04

06

08

Myo

stat

in p

rote

in ex

pres

sion

GAPDH

MSTN


PAL +PD98059

PAL +parthenolide


myostatin


Acknowledgment


References



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Vehicle PAL00

02

04

06

08

10

p-ER

K(1

2) p

rote

in ex

pres

sion

GAPDH

ERK

P-ERK(12)

Vehicle +APS

PAL + APS

lowastlowastlowast

(a)

00

02

04

06

08

10

p-P3

8 pr

otei

n ex

pres

sion

p38

GAPDH


PAL + APS

P-p38

(b)

Vehicle PAL00

02

04

06

08

10

12

p-JN

K pr

otei

n ex

pres

sion

JNK

GAPDH

P-JNK

Vehicle +APS

PAL + APS

(c)




Vehicle PAL00

02

04

06

08

10

12

14

NF-120581

B pr

otei

n ex

pres

sion

NF-120581B

GAPDH


lowastlowastlowast

(a)

00

02

04

06

08

I120581Ba

pro

tein

expr

essio

n

GAPDH


I120581B120572

lowastlowast

PAL + APS

(b)


Vehicle PAL00

02

04

06

08

Myo

stat

in p

rote

in ex

pres

sion

GAPDH

MSTN


PAL +PD98059

PAL +parthenolide


myostatin


Acknowledgment


References



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Vehicle PAL00

02

04

06

08

10

12

14

NF-120581

B pr

otei

n ex

pres

sion

NF-120581B

GAPDH


lowastlowastlowast

(a)

00

02

04

06

08

I120581Ba

pro

tein

expr

essio

n

GAPDH


I120581B120572

lowastlowast

PAL + APS

(b)


Vehicle PAL00

02

04

06

08

Myo

stat

in p

rote

in ex

pres

sion

GAPDH

MSTN


PAL +PD98059

PAL +parthenolide


myostatin


Acknowledgment


References



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Research Article Astragalus Polysaccharide Suppresses...

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