Research Article Soy Glycinin Contains a Functional...

Hindawi Publishing CorporationInternational Journal of EndocrinologyVolume 2013 Article ID 907565 11 pageshttpdxdoiorg1011552013907565

Research ArticleSoy Glycinin Contains a Functional Inhibitory Sequence againstMuscle-Atrophy-Associated Ubiquitin Ligase Cbl-b

Tomoki Abe1 Shohei Kohno1 Tomonari Yama1 Arisa Ochi1 Takuro Suto1

Katsuya Hirasaka1 Ayako Ohno1 Shigetada Teshima-Kondo1 Yuushi Okumura1

Motoko Oarada2 Inho Choi3 Rie Mukai4 Junji Terao4 and Takeshi Nikawa1

1 Department of Nutritional Physiology Institute of Health Biosciences The University of Tokushima Graduate School3-18-15 Kuramoto-cho Tokushima 770-8503 Japan

2Medical Mycology Research Center The University of Chiba Chiba 260-8673 Japan3Division of Biological Science and Technology College of Science and Technology Institute of Biomaterials The University of YonseiWonju 220-710 Republic of Korea

4Department of Food Science Institute of Health Biosciences The University of Tokushima Tokushima 770-8503 Japan

Correspondence should be addressed to Takeshi Nikawa nikawanutrmedtokushima-uacjp

Received 22 December 2012 Revised 7 April 2013 Accepted 22 April 2013

Academic Editor Maria Antonietta Pellegrino

Copyright copy 2013 Tomoki Abe et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Background Unloading stress induces skeletal muscle atrophy We have reported that Cbl-b ubiquitin ligase is a master regulatorof unloading-associated muscle atrophyThe present study was designed to elucidate whether dietary soy glycinin protein preventsdenervation-mediated muscle atrophy based on the presence of inhibitory peptides against Cbl-b ubiquitin ligase in soy glycininprotein Methods Mice were fed either 20 casein diet 20 soy protein isolate diet 10 glycinin diet containing 10 casein or20 glycinin diet One week later the right sciatic nerve was cut The wet weight cross sectional area (CSA) IGF-1 signaling andatrogene expression in hindlimb muscles were examined at 1 3 35 or 4 days after denervation Results 20 soy glycinin dietsignificantly prevented denervation-induced decreases in muscle wet weight and myofiber CSA Furthermore dietary soy proteininhibited denervation-induced ubiquitination and degradation of IRS-1 in tibialis anterior muscle Dietary soy glycinin partiallysuppressed the denervation-mediated expression of atrogenes such as MAFbxatrogin-1 and MuRF-1 through the protectionof IGF-1 signaling estimated by phosphorylation of Akt-1 Conclusions Soy glycinin contains a functional inhibitory sequenceagainst muscle-atrophy-associated ubiquitin ligase Cbl-b Dietary soy glycinin protein significantly prevented muscle atrophy afterdenervation in mice

1 Introduction

Skeletal muscle atrophy caused by unloading is characterizedby both decreased responsiveness to myogenic growth fac-tors such as insulin-like growth factor (IGF-1) and insulinand increased proteolysis [1ndash3] We reported previously thatunloading stress resulted in skeletal muscle atrophy throughthe induction and activation of a ubiquitin ligase Casitas B-cell lymphoma-b (Cbl-b) [4] Cbl-b induced ubiquitinationand degradation of insulin receptor substrate-1 (IRS-1) theIGF-1 signaling intermediate In turn the loss of IRS-1 caused

the forkhead transcription factor-3- (FOXO3-) dependentinduction ofmuscle atrophy F-box protein (MAFbx)atrogin-1 a dominant mediator of proteolysis in atrophic muscle Wealso demonstrated that Cbl-b-deficient mice were resistant tounloading-induced atrophy and loss of muscle function [4]These results indicate that the Cbl-b-dependent degradationof IRS-1 is a criticalmediator of increased protein degradationand reduced protein synthesis in unloading-induced muscleatrophy

We have also reported that a pentapeptide mimeticof tyrosine608-phosphorylated IRS-1 DGpYMP which we

2 International Journal of Endocrinology

named Cblin (Cbl-b inhibitor) prevented denervation-in-duced muscle atrophy in mice [4] We also found thatCblin inhibited Cbl-b-mediated IRS-1 ubiquitination and ex-pression of MAFbxatrogin-1 [4]These results indicated thatthe inhibition of Cbl-b-mediated IRS-1 ubiquitination couldbe therapeutically beneficial in unloading-induced muscleatrophy

Soy protein and derived peptides are effective for mitiga-tion of muscle damage [5] promotion of protein synthesisand translation initiation after exercise [6] In addition wedemonstrated previously that diet containing soy proteinprevents exercise-induced protein degradation in skeletalmuscle through the suppression of calpain-mediated prote-olysis [7] Thus dietary soy protein affects protein turnoverin skeletal muscle However to date there is no informationon the effects of soy protein on unloading-induced muscleatrophy

In a preliminary study we found that the sequence ofsoy glycinin a major component of soy protein is similarto that of Cblin Based on this finding we explored whethersoy glycinin and its derived peptides prevent denervation-associated muscle atrophy in mice Treatment of HEK293cells with soy-glycinin-derived peptides suppressed Cbl-b-mediated IRS-1 ubiquitination Furthermore mice fed withsoy glycinin showed inhibition of denervation-associatedmuscle atrophy and this effect was mediated through theconservation of IGF-1 signalingThese findings indicated thatsoy glycinin is a useful dietary protein for muscle atrophyassociated with muscle unloading

2 Materials and Methods

21 Multiple Alignment of Amino Acid Sequence We analyz-ed the primary structure of glycinin precursor with ClustalXsoftware (httpwww-igbmcu-strasbgfrBioInfoClustalXTophtml)

22 Cell Culture HEK293 cells were maintained in Dulbec-corsquos modified Eagle medium (DMEM) containing 10 fetalbovine serum 100UmL penicillin and 100 120583gmL strepto-mycin at 37∘C under 5 CO

2-195 air gas mixture

23 Transfection HEK293 cells (40ndash60 of confluence) weretransfected with plasmid vectors using Hily Max lipofec-tion reagent (DOJINDO Kumamoto Japan) as describedpreviously [8] The expression plasmids used in this studywere pCEFL-human Cbl-b-HA pcDNA31-rat IRS-1-V5 andpcDNA3-FLAG-Ubiquitin

24 Immunoblotting and Immunoprecipitation Immunoblotand immunoprecipitation analyses were performed as de-scribed previously [9] The following antibodies were usedanti-actin (Calbiochem La Jolla CA USA) anti-HA11(BabCo Richmond CA USA) anti-V5 (Invitrogen Carls-bad CA USA) anti-FLAG M2 (Sigma St Louis MOUSA) anti-IRS-1 (Upstate Biotechnology Lake Placid NYUSA) anti-Akt-1 (PharMingen International Tokyo) and

anti-phospho-S473-Akt-1 (Cell Signaling Technology BeverlyMA USA)

25 Cell-Free Ubiquitination Assay The cell-free ubiquitina-tion assay was performed as described previously [4] Weprepared immunoprecipitated Cbl-b (IP-Cbl-b) and IRS-1(IP-IRS-1) respectively bound to anti-HA- and anti-IRS-1-IgG-linked protein A beads from extracts (1mg protein)of HEK293 cells transfected with pCEFL-Cbl-b-HA andpcDNA31-IRS-1-V5 (5 120583g) Slurries of IP-Cbl-b and IP-IRS-1 were incubated at 37∘C for 4 hours in reaction buffer(50120583L) containing ATP-regenerating system recombinantmouse E1 (500 ng) UbcH7 (5 120583g E2) GST-tagged ubiquitin(10 120583g) andor ubiquitin-aldehyde (Ubc-CHO) (1 120583g a deu-biquitinase inhibitor) which were purchased from BostonBiochem Inc (Cambridge MA USA) Then 20 120583L of boiledsupernatant was separated by SDS-PAGE and visualized byimmunoblotting with an anti-IRS-1 antibody

26 Preparation of Soy-Derived Peptides and Synthetic Pep-tides We used the following pentapeptides or hexapep-tides derived from soy soy-glycinin-derived peptide soy-120573-conglycinin-derived peptide lipoprotein- (LP-) derivedpeptide and soy protein isolate (SPI) These peptides werekindly provided by Fuji Oil Co (Osaka Japan) In additionwe used the following synthetic peptides Cblin DGpYMPwhich exhibited inhibitory activity on Cbl-b-mediated IRS-1ubiquitination in our previous study [4] A patent applicationwas lodged for the sequence of this peptide (Pat no 5113346)Negative control was a peptide with VGpYLR sequencewhich had no inhibitory activity against Cbl-b-mediated IRS-1 ubiquitination [4] These peptides were kindly provided byOtsuka Pharmaceutical Co (Tokushima Japan)We also syn-thesized a pentapeptide with Cblin-like amino acid sequencein soy glycinin termed it Cblin-like peptide or DIpYNP andchecked its inhibitory activity using cell-free ubiquitinationassay as described above Since soy-glycinin-derived peptidesshowed inhibitory activity on IRS-1 ubiquitination in the cell-free system (pat no 4963044) it was subjected to furtheranalysis in cell cultures

27 Animal Models Experiments were conducted in C57BL6J mice (Japan SLC Shizuoka Japan) In anesthetized 6-week-old mice the sciatic nerve of the right leg was cut anda 10mm piece was excised The left leg remained innervatedand was used as a control Mice were sacrificed at 1 3 35 or 4days after operation Mice were fed one of the following fourdiets from 1 week before denervation till sacrifice a mixeddiet containing 20 casein protein (Control diet) a mixeddiet containing 20 SPI (SPI diet) a mixed diet containing20 soy glycinin protein (20glycinin diet) and amixed dietcontaining 10 casein protein plus 10 soy glycinin protein(10 glycinin diet) These proteins were kindly provided byFuji Oil Co (Osaka Japan) The macronutrient compositionsof the different diets are listed in Table 1 The Committee forthe Care and Use of Laboratory Animals of The Universityof Tokushima approved all protocols of this study whichwere conducted according to the Guide for the Care and

International Journal of Endocrinology 3

Table 1 Composition of the different experimental diets used in the present study

Product Control diet SPIlowast diet 10 soy glycinin diet 20 soy glycinin diet (g100 g)Casein 2260 0 1128 0SPIlowast 0 2240 0 0Glycinin 0 0 1147 2289120572-Starch 4444 4455 4436 4427Sucrose 2223 2229 2219 2215Lard 423 424 422 421Cellulose 200 201 199 199Mineral mix 350 351 349 349Vitamin mix 100 100 100 100lowastSPI soy protein isolate

Table 2 Primers used for real-time RT-PCR

Target gene Sequence Length (bp)

MAFbxatrogin-1 SAS

51015840-GGCGGACGGCTGGAA-3101584051015840-CAGATTCTCCTTACTGTATACCTCCTTGT-31015840 100

MuRF-1 SAS

51015840-ACGAGAAGAAGAGCGAGCTG-3101584051015840-CTTGGCACTTGAGAGAGGAAGG-31015840 179

GAPDH SAS

51015840-CGTGTTCCTACCCCCAATGT-3101584051015840-ATGTCATCATACTTGGCAGGTTTCT-31015840 74

AS antisense primer S sense primer MAFbxatrogin-1 muscle atrophy F-box protein MuRF-1 muscle-specific RING finger protein 1 GAPDH glycer-aldehyde-3-phosphate dehydrogenase

Use of Laboratory Animals at The University of Tokushima(httpcmsdbtokushima-uacjpDAVorganization10998anex-HPanexaigo-ttokusima-u doubutukannrikisokupdf)

28 Histochemical Analysis The tibialis anterior (TA)musclewas isolated after sacrifice and immediately frozen in chilledisopentane and liquid nitrogen and stored at minus80∘ asdescribed previously [10] Sections were stained with hema-toxylin and eosin (HampE) TA muscle sections were digitallycaptured using bright field with BZ-II Analyzer (KEYENCEOsaka Japan) Digital imaging was performed at 10x finalmagnification Image processing was performed using AdobePhotoshop CS (Adobe Japan Tokyo) The circumference ofeach fiber was outlined using an ImageJ software (NationalInstitute for Health) to generate CSA of myofibers Criteriaused in the selection of muscle fibers to measure for CSA ofmyofibers included an intact distinct cell membrane withoutsignificant signs of distortion or folding Elongated fibersindicating an oblique section were excluded Image analyseswere performed by two coauthors (TY and TS)

29 Real-Time RT-PCR To measure the amount of mRNAin small samples real-time reverse transcription-polymerasechain reaction (RT-PCR) was performed with SYBR Greendye using an ABI 7300 real-time PCR system (Applied Bio-systems Foster City CA USA) as described previously [11]The oligonucleotide primers used for amplification are listedin Table 2

210 Statistical Analysis All data were statistically evaluatedby multiple comparison using Excel Statistic ver 60 (Esumi

Co Tokyo Japan) and were expressed as mean plusmn SD Differ-ences between two groups were assessed with Duncanrsquos mul-tiple range test or Shirley-Williamsrsquo multiple comparison testDifferences were considered significant at 119875 lt 005

3 Results

31 Identification of Cblin-Like Sequence in Soy Glycinin Wereported previously that Cblin DGpYMP inhibited Cbl-b-mediated ubiquitination and degradation of IRS-1 both invitro and in vivo [4] We searched proteins containing Cblin-like sequence using the Basic Local Alignment Search Tool(httpblastncbinlmnihgovBlastcgi) and found that soyglycinin in soy protein contains a sequence similar to thatof Cblin peptide DIFYNP Multiple sequence alignments offive soy glycinin homologues G1 G2 G3 G4 and G indicat-ed that Cblin-like sequence DIYNP is conserved amongthese glycinin homologues (Figure 1)

32 Cblin-Like Peptide in SoyGlycinin Inhibits Cbl-b-MediatedIRS-1 Ubiquitination in HEK293 Cells First we synthesizedpentapeptides of the above sequence including Cblin-likesynthetic peptideDIpYNP andDIYNPWest andTowers [12]reported that food-derived phosphopeptides were absorbedat least in part without dephosphorylation Immunoblotanalysis showed that soy glycinin contained more phospho-rylated tyrosine than egg white (Figure 2) Therefore weused the phosphorylated peptide DIpYNP in the followingexperiments Treatment with IGF-1 induced ubiquitinationof IRS-1 in HEK293 cells transfected with Cbl-b IRS-1and ubiquitin (Figure 3) As reported previously treatment


IAVPTGVAWWMYNNEDTPVVAVSIIDTNSLENQLDQMPRRFYLAGNQEQEFLKYQ--QEQGGHQS--QKGKHQQEEENEGGSILSGFTLEFLEHAFSVDKQIAKNLQGENEGEDKGAIVTVKGGLSVIKPPTDEQQ-------------- 273IAVPTGVAWWMYNNEDTPVVAVSIIDTNSLENQLDQMPRRFYLAGNQEQEFLKYQ-QQQQGGSQS--QKGK-QQEEENEGSNILSGFAPEFLKEAFGVNMQIVRNLQGENEEEDSGAIVTVKGGLRVTAPAMRKPQ-------------- 270IAVPTGFAYWMYNNEDTPVVAVSLIDTNSFQNQLDQMPRRFYLAGNQEQEFLQYQPQKQQGGTQS--QKGKRQQEEENEGGSILSGFAPEFLEHAFVVDRQIVRKLQGENEEEEKGAIVTVKGGLSVISPPTEEQQ-------------- 272LVIPPSVPYWTYNTGDEPVVAISLLDTSNFNNQLDQTPRVFYLAGNPDIEYPETMQQQQQQKSHGGRKQGQHQQEEEEEGGSVLSGFSKHFLAQSFNTNEDIAEKLESPDDERKQ--IVTVEGGLSVISPKWQEQQDEDEDEDEDDEDEQ 294LVIPLGVPYWTYNTGDEPVVAISPLDTSNFNNQLDQNPRVFYLAGNPDIEHPETMQQQQQQKSHGGRKQGQHRQQEEE-GGSVLSGFSKHFLAQSFNTNEDTAEKLRSPDDERKQ--IVTVEGGLSVISPKWQEQ--EDEDEDEDEEYGR 291

180210240270300

------------QRPQEEEEEEEDEKPQCK----------------------------------GKDKHCQRPRGSQSKSRRNGIDETICTMRLRHNIGQTSSPDIYNPQAGSVTTATSLDFPALSWLRLSAEFGSLRKNAMFVPHYNLN 377------------OEE---DDDDEEEQPQCV----------------------------------ETDKGCQ----RQSKRSRNGIDETICTMRLRQNIGQNSSPDIYNPQAGSITTATSLDFPALWLLKLSAQYGSLRKNAMFVPHYTLN 367------------QRP------EEEEKPDCD----------------------------------EKDKHCQ----SQS---RNGIDETICTMRLRHNIGQTSSPDIFNPQAGSITTATSLDFPALSWLKLSAQFGSLRKNAMFVPHYNLN 363IPSHPPRRPSHGKREQDEDEDEDEDKPRPSRPSQGKRNKTGQDEDEDEDEDQPRKSREWRSKKTQPRRPRQEEPRERGCETRNGVEENICTLKLHENIARPSRADFYNPKAGRISTLNSLTLPALRQFQLSAQYVVLYKNGIYSPHWNLN 444TPSYPPRRPSHGKHEDDEDEDEEEDQPRPDHPPQ------------------------------RPSRPEQQEPRGRGCQTRNGVEENICTMKLHENIARPSRADFYNPKAGRISTLNSLTLPALRQFGLSAQYVVLYRNGIYSPDWNLN 411

330360390420450

ANSIIYALNGRALIQVVNCNGERVFDGELQEGRVLIVPQNFVVAARSQSDNFEYVSFKTNDTPMIGTLAGANSLLNALPEEVIQHTFNLKSQQARQIKNNNPFKFLVPPQESQKRAVA--- 495ANSIIYALNGRALVQVVNCNGERVFDGELQEGGVLIVPQNFAVAAKSQSDNFEYVSFKTNDRPSIGNLAGANSLLNALPEEVIQHTFNLKSQQARQVKNNNPFSFLVPPQESQRRAVA--- 495ANSIIYALNGRALVQVVNCNGERVFDGELQEGQVLIVPQNFAVAARSQSDNFEYVSFKTNDRPSIGNLAGANSLLNALPEEVIQQTFNLRRQQARQVKNNNPFSFLVPPKESQRRVVA--- 491ANSVIYVTRGQGKVRVVNCQGNAVFDGELRRGQLLVVPQNFVVAEQAGEQGFEYIVFKTHHNAVTSYLK---DVFRAIPSEVLAHSYNLRQSQVSELKYEGNWGPLVNPESQQGSPRVKVA 562ANSVT-MTRGKGRVRVVNCQGNAVFDGELRRGQLLVVPQNPAVAEQGGEQGLEYVVFKTHHNAVSSYIK---DVFRVIPSEVLSNSYNLGQSQVRQLKYQGNSGPLVNP------------ 516

480510540570

G1 -----MAKLVFSLCFLLFSGCCFAFSSREQPQQNECQIQKLNALKPDNRIESEGGLIETWNPNNKPFQCAGVALSRCTLNRNALRRPSYTNGPQEIYIQQGKGIFGMIYPGCPSTFEEPQQPQQRGQSSRP----QDRHQKIYNFREGDL 141G2 -----MAKLVLSLCFLLFSGC---FALREQAQQNECQIQKLNALKPDNRIESEGGFIETWNPNNKPFQCAGVALSRCTLNRNALRRPSYTNGPQEIYIQQGNGIFGMIFPGCPSTYQEPQESQQRGRSQRP----QDRHQKVHRFREGDL 138G3 -----MAKLVLSLCFLLFSGCCFAFSFREQPQQNECQIQRLNALKPDNRIESEGGFIETWNPNNKPFQCAGVALSRCTLNRNALRRPSYTNAPQEIYIQQGSGIFGMIFPGCPSTFEEPQQ---KGQSSRP----QDRHQKIYHFREGDL 138G4 MGKP-FTLSLSSLCLLLLSSACFAISSS---KLNECQLNNLNALEPDHRVESEGGLIQTWNSQHPELKCAGVTVSKLTLNRNGLHSPSYSPYPRMIIIAQGKGALGVAIPGCPETFEEPQEQSNRRGSRSQKQQLQDSHQKIRHFNEGDV 146

G MGKPFFTLSLSSLCLLLLSSACFAITSS---KFNECQLNNLNALEPDHRVESEGGLIETWNSQHPELQCAGVTVSKRTLNRNGSHLPSYLPYPQMIIVVQGKGAIGFAFPGCPETFEKPQQQSSRRGSRSQ-QQLQDSHQKIRHFNEGDV 146

Ruler

G1G2G3G4

G

Ruler

G1G2G3G4

G

Ruler

G1G2G3G4

G

Ruler

1306090120150

Figure 1 Alignment of glycinin precursor protein sequences The sequence of soy glycinin was similar to that of Cblin peptide indicated bythe box The sequences were retrieved from the UniProt database (httpwwwuniprotorg) G1 Glycinin G1 G2 Glycinin G2 G3 GlycininG3 G Glycinin G G4 Glycinin G4

IB P-tyrosine

97

21

BSA

Egg

whi

te

Gly

cini

n

MMSTD(kDa)

Figure 2 Identification of phosphorylated tyrosine in soy glycininBovine serumalbumin eggwhite and soy glycininwere subjected toimmunoblot analysis for phosphorylated tyrosine MMSTDmolec-ular mass standards Representative findings of three experimentswith matching results

with 120120583M Cblin inhibited IRS-1 ubiquitination and inter-action between Cbl-b and IRS-1 (Figure 3) InterestinglyCblin-like synthetic peptide significantly inhibited Cbl-b-mediated ubiquitination of IRS-1 in a dose-dependent man-ner (Figure 3) Furthermore Cblin-like synthetic peptideprevented the interaction betweenCbl-b and IRS-1 (Figure 3)Densitometric analysis demonstrated that the IC

50value of

this peptide was approximately 350 120583M (Figure 3) while theIC50

value of Cblin was approximately 120120583M [4] Theseresults suggest that the Cblin-like peptide functioned as aCbl-b inhibitor

Since soy glycinin contains Cblin-like peptide we exam-ined the inhibitory activities of various peptides (averagelength= 5-6 amino acids) derived from soy glycinin onCbl-b-mediated ubiquitination of IRS-1 In our cell-free ubiquitina-tion system the interaction between Cbl-b and IRS-1 resultedin Cbl-b-dependent IRS-1 ubiquitination together with theexpected electrophoretic pattern of ubiquitinated products ofIRS-1 (Figure 4(a)) Among the various peptides employedsoy-glycinin-derived peptides significantly inhibited Cbl-b-mediated IRS-1 ubiquitination in the cell-free system butsoy-120573-conglycinin- LP- and SPI-derived peptides did not(Figure 4(a)) We also examined the inhibitory effects ofsoy-glycinin-derived peptides using the cultured cell sys-tem Consistent with the result of cell-free ubiquitinationassay soy-glycinin-derived peptides inhibited IRS-1 ubiq-uitination in Cbl-b-transfected HEK293 cells (Figure 4(b))Furthermore anti-IRS-1 immunoprecipitates from HEK293cells treated with soy-glycinin-derived peptides containedsmaller amounts of Cbl-b compared to treatment withcontrol peptide (Figure 4(b)) These results suggest thatsoy-glycinin-derived peptides significantly prevented Cbl-b-mediated IRS-1 ubiquitination through the suppression ofCbl-b and IRS-1 interaction

33 Dietary Soy Glycinin Protein Prevents Denervation-Induc-edMuscleWetWeight Loss To examine the inhibitory effectsof soy glycinin on Cbl-b-mediated muscle atrophy in vivothe right leg muscles of C57BL6J mice were denervated bysectioning the sciatic nerve while the left leg muscles of mice


00 0 0 200100 400 600

00 1200 00 00

00 0600 00 00

180

MMSTD(kDa)

100

50

00

200 400 600 800

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

++

+

++

minus

119910 = minus01689119909 + 11037

1198772= 09552

(120583M)

FLAG

-Ub

IRS-1-V5 (substrate)

Cbl-b-HA (E3)

Ubiquitin-FLAG

Control peptide (120583M)

Cblin (120583M)

Cblin-like peptide (120583M)

IP IRS-1-V5

IB Ub-FLAG

IB Cbl-b-HA

IB IRS-1-V5

Figure 3 Inhibitory effect of Cblin-like synthetic peptide on Cbl-b-mediated degradation and ubiquitination of IRS-1 HEK293 cellswere transfected with mock vectorpCEFL-Cbl-b-HA pcDNA31-rat IRS-1-V5 and pcDNA3-FLAG-Ubiquitin and then treated with100 nM epoxomicin at 2 hours before treatment with each peptideat the indicated concentrations One hour later these cells weretreated with 10 ngmL IGF-1 for 1 hour Immunoprecipitates (IP)from whole cell lysates incubated with an anti-IRS-1 antibody weresubjected to immunoblot (IB) analysis for the indicated proteinsThe densitometric measurement of FLAG-Ub (the area indicatedby right parenthesis) was performed with an ImageJ software(National Institute for Health) MMSTDmolecular mass standardsRepresentative findings of three experiments with matching results

were sham operated The denervated mice were divided intofour groups based on the type of diet used 20 casein dietgroup (119899 = 5) 20 SPI diet group (119899 = 5) 10 soy glycinin+ 10 casein diet group (119899 = 5) and 20 soy glycinin dietgroup (119899 = 5) There were no significant differences in dailyintake of food protein and energy among the four groups of

mice (Table 3) In the denervated mice fed with 20 caseindiet thewetweight of denervatedTAmuscle decreased by 9on 4 days after denervation compared with sham-operatedTAmuscle (Figure 5(a)) In contrast the SPI 10 soy glycinin+ 10 casein diet and 20 soy glycinin diet preventeddenervation-induced decrease in wet weight of TA muscle(Figure 5(a)) Interestingly we also noted a dose-dependentinhibitory action of soy glycinin protein on denervation-induced wet TA muscle weight loss (Figure 5(a)) Similarinhibitory effects of soy glycinin on TA muscle were notedin gastrocnemius and EDL muscles (Figure 5(a)) Howeverchanges in these muscles besides TA muscle were notstatistically significant Dietary soy proteins did not show aninhibitory effect on unloading-mediated muscle mass lossin soleus muscle (Figure 5(a)) Based on these results wenext examined the effects of dietary soy glycinin protein ondenervation-mediated decrease in cross sectional area (CSA)of TA muscle In mice of the 20 casein diet group 4-day-denervation decreased the CSA of TA muscle by 10compared with sham operation (Figure 5(b)) SPI diet or 10soy glycinin + 10 casein diet failed to restore the decreasein the CSA of denervated TA muscle (Figure 5(b)) whereasfeeding with 20 soy glycinin diet significantly restored theCSA of denervated TA muscle to that of sham-operated TAmuscle Feeding with 20 soy glycinin diet also shifted theCSA distribution broader than that of mice of the SPI dietand 10 soy glycinin + 10 casein diet groups (Figure 5(c))

34 Soy Glycinin Prevents Attenuation of IGF-1 Signaling inDenervated Muscle To address whether soy glycinin pre-vents Cbl-b-mediated degradation of IRS-1 IGF-1 signalingwas estimated by immunoblotting in TA muscle of dener-vated mice of the control and 20 glycinin diet groupsThe amount of IRS-1 in denervated TA muscle decreasedin a time-dependent manner (Figure 6(a)) Furthermore thedegradation of IRS-1 in denervated TA muscle appeared atday 3 after denervation and was completed by day 4 There-fore we examined the inhibitory effect of dietary soy glycininprotein on IRS-1 degradation using TA muscle after day 35following denervation The degradation and ubiquitinationof IRS-1 were significantly prevented in mice of the 20glycinin group compared with the control group (Figures6(b) and 6(c)) Based on these results we also examinedphosphorylation of Akt-1 in IGF-1 signaling Consistent withIRS-1 protein level the decrease in Akt-1 phosphorylationwas smaller in the denervated TAmuscle of the 20 glyciningroup than in the control group (Figure 6(d)) Furthermoredenervation-induced expression of MAFbxatrogin-1 andMuRF-1 but not Cbl-b was abrogated in the denervated TAmuscle of the 20 glycinin group compared with the controlgroup (Figure 6(e)) These results suggest that soy glycininameliorates denervation-induced decrease in IGF-1 signalingby suppressing IRS-1 degradation

4 Discussion

We reported previously that Cbl-b interacts with and de-grades IRS-1 an IGF-1 signaling intermediate in skeletal


MMSTD(kDa)

200

180

SPI LP C G

IRS-1 (substrate)Cbl-b (E3)

E1UbcH7 (E2)

GST-UbPeptides

IRS-1

Ub-IRS-1

IB IRS-1

++++++

++++++

++++++

+++++

minusminus

minus

+++++minus

(a)

200

120

180

6060 00

00 2010

MMSTD(kDa)

++++

++++

+++minus


Ubiquitin-FLAG

IP IRS-1-V5

IB Ub-FLAG

IB IRS-1-V5

IB Cbl-b-HA

Casein-derivedpeptides (120583gmL)Glycinin-derived

peptides (120583gmL)

(b)

Figure 4 Inhibitory effect of soy-glycinin-derived peptides on Cbl-b-mediated IRS-1 ubiquitination (a) Purified soy protein isolate (SPI)lipoprotein (LP) soy glycinin (G) and soy 120573-conglycinin (C) were digested with trypsin then 20120583gmL of each of the hydrolysates wassubjected to cell-free ubiquitination assay to elucidate their inhibitory effects on Cbl-b-mediated IRS-1 ubiquitination (b) HEK293 cellstransfected with mock vectorpCEFL-Cbl-b-HA pcDNA31-rat IRS-1-V5 and pcDNA3-FLAG-Ubiquitin were treated with the indicatedconcentration of casein-derived (control) or soy-glycinin-derived peptides for 2 hours in the presence of 100 nM epoxomicin and 10 ngmLIGF-1 Cell lysates from these cells were immunoprecipitatedwith an anti-V5 antibodyThe immunoprecipitateswere subjected to immunoblot(IB) analysis for the indicated proteins MMSTD molecular mass standards Representative findings of three experiments with matchingresults

Table 3 Daily intake of food protein and energy in mice

Control SPI 10 glycinin 20 glycininFood intake (gday) 478 431 420 417Energy intake (kcalday) 1814 1647 1598 158Protein intake (gday) 091 082 080 079

muscles during unloading conditions [4] suggesting thatCbl-b is a key enzyme in unloading-related muscle atrophyWe also reported that the pentapeptide Cblin inhibited Cbl-b-mediated degradation of IRS-1 resulting in suppressionof denervation-mediated muscle atrophy [4] Therefore wehypothesized that foodstuffs that contain Cblin-like aminoacid sequence can be used to prevent unloading-inducedmuscle atrophy The present study showed that the pep-tide ldquoDIpYNPrdquo contained in soy glycinin inhibited Cbl-b-mediated IRS-1 ubiquitination in vitro Furthermore dietarysoy glycinin protein inhibited denervation-induced ubiqui-tination and degradation of IRS-1 in vivo and significantlyprevented losses of wet weight and CSA of denervated TAmuscle Considered together the present study suggests thatsoy glycinin is an effective protein source against unloading-related muscle atrophy possibly through diet-mediated

inhibition of muscle ubiquitin ligase Cbl-b Soy protein mod-ulates protein turnover in skeletal muscles [5ndash7] althoughthe mechanism of this beneficial effect is still unknown TheCbl-bs inhibition of glycinin-derived peptide contributes atleast in part to the beneficial effects of soy protein on muscleturnover

Cbl-b conserves tyrosine kinase binding (TKB) domainwhich is a modified Src homology 2 (SH2) domain andmediates binding in Cbl-b target molecules Therefore thedephosphorylated form of Cblin weakly interacted with theTKB domain of Cbl-b In fact the dephosphorylated Cblinpeptide had less inhibitory activity on Cbl-b-mediated ubiq-uitination of IRS-1 than the phosphorylated one the IC

50val-

ues of Cblin and dephosphorylated Cblin were approximately120120583Mand 1mM respectively [4] In the present study phos-phorylated Cblin-like peptide DIpYNP effectively inhibited


10

gly

cini

n

20

gly

cini

n

20

case

in

20

SPI

10

gly

cini

n

20

gly

cini

n

20

case

in

20

SPI

10

gly

cini

n

20

gly

cini

n

20

case

in

20

SPI

10

gly

cini

n

20

gly

cini

n

20

case

in

20

SPI

TA Gast

0

100

50

75

25

0

100

50

75

25

EDL Sol

75

0

100

50

25

0

100

50

75

25

lowast

lowast lowast lowast

lowast

lowast

Den

erva

ted

mus

cle w

eigh

t (

)

(a)

10 glycinin 20 glycinin20 casein 20 SPI


Sham

Den

erva

tion

CSA

()

0

50

100lowast

(b)

Figure 5 Continued


10 glycinin

20 SPI

0

10

20

30

40

50

0

10

20

30

40

50 20 glycinin

20 casein

0

10

20

30

40

50

CSA

()

CSA

()

CSA

()

CSA

()

0

10

20

30

40

50

ShamDenervation

lowast

0sim500

501sim1000

1001sim1500

1501sim2000

2001sim2500

2501sim3000

3001sim3500

3501sim4000

4001

0sim500

501sim1000

1001sim1500

1501sim2000

2001sim2500

2501sim3000

3001sim3500

3501sim4000

4001

Myofiber CSA (120583m2)

0sim500

501sim1000

1001sim1500

1501sim2000

2001sim2500

2501sim3000

3001sim3500

3501sim4000

4001



0sim500

501sim1000

1001sim1500

1501sim2000

2001sim2500

2501sim3000

3001sim3500

3501sim4000

4001


ShamDenervation

lowast

(c)

Figure 5 Effects of dietary soy glycinin protein on denervation-related decrease in muscle wet weight and muscle cross sectional area Theright and left legs of C57BL6 mice were subjected to denervation and sham operation respectively Mice were divided at random into oneof the following four diet groups 20 casein group 20 soy protein isolate group 10 soy glycinin + 10 casein group (10 glycinin) and20 soy glycinin group (20 glycinin) Each diet was started at 1 week before denervation and continued for the duration of the experimentperiod Their hindlimb muscles were isolated at 4 days after denervation (a) Wet weight of hindlimb muscles Percent of denervated muscleweight was defined as the ratio of denervated hindlimb muscle wet weight to sham-operated hindlimb muscle wet weight in the mice TAtibialis anterior muscle Gast gastrocnemiusmuscle EDL extensor digitorum longusmuscle Sol soleusmuscle Data aremean plusmn SD (119899 = 5)lowast

119875 lt 005 versus sham operated 119875 lt 005 versus 20 casein diet by the Shirley-Williamsrsquo multiple comparison test (b c) Frozen sections

of TAmuscle were stained with HampE (b) Scale = 100 120583mThe cross sectional area (CSA) of myofibers was measured as described inMaterialsand Methods The distribution of CSA in TA muscle was also shown (c) Percent of CSA indicates the ratio of the number of myofibers withthe indicated area to the number of total myofibers in the section The mean value from five individual sections was shown Arrow indicatesthe most common value of CSA of denervated TAmuscle Solid bars denervation group Open bars sham-operated group Data are mean plusmnSD (119899 = 5) lowast119875 lt 005 versus sham-operated lowast119875 lt 005 versus sham-operated

119875 lt 005 versus 20 casein diet by the Shirley-Williamsrsquomultiple comparison test

Cbl-b-mediated ubiquitination and degradation of IRS-1although we could not determine the IC

50value of dephos-

phorylated Cblin-like peptide However casein phosphopep-tide (CPP) is a phosphorylated protein and resists dephos-phorylation during digestion [12] Furthermore we notedthat glycininwas remarkably phosphorylated comparedwithegg white and bovine serum albumin proteins Based onthese findings we suggest that Cblin-like sequence of dietarysoy glycinin may be phosphorylated and absorbed withoutdephosphorylation Further studies are necessary to elucidatethe mechanism of the inhibitory effect of dietary soy glycininin vivo

We suggest that Cbl-b is a negative regulator of IGF-1signaling during unloading [4] Disturbed IGF-1 signaling

such as degradation of IRS-1 induces expression of MAFbxatrogin-1 and MuRF-1 through dephosphorylation of AktSince unloading induces expression of Cbl-b at the mRNAlevel to ubiquitinate and degrade IRS-1 protein it is consistentthat expression of MAFbxatrogin-1 and MuRF-1 was nega-tively regulated by dietary soy glycinin but expression of Cbl-b was not in our in vivo experiment

Functional peptides are food-derived peptides that exertbeyond their nutritional value physiological or hormone-like effect in humans Functional peptides are inactive withinthe sequence of their parent protein and can be released byenzymatic hydrolysis either during gastrointestinal digestionor food processing They are found in milk egg meat andvarious kinds of fish as well as in many plants Since amino


IB actin

Denervation

0 1 3 4(Day)

IB IRS-1

(a)

IB IRS-1

IB actin

Sham DenervationControl 20 glycinin

Sham Denervation

Control 20 glycinin0

1

05

15

lowast

lowast

ShamDenervation

IRS-

1ac

tin

(b)

Sham Denervation

Control 20 glycinin

Sham Denervation

IB ubiquitin

IP IRS-1

180

MMSTD(kDa)

Ub-

IRS-1

(c)

IB P-Akt-1

IB Akt-1

Sham Denervation Sham Denervation

Control 20 glycinin


1

05lowast

lowast

P-A

kt-1

Akt

-1

ShamDenervation

(d)

Figure 6 Continued


Control 20 glycinin Control 20 glycinin Control 20 glycinin0

2

4

6

0

2

4

6

8

0

1

2

3

ShamDenervation

MA

Fbx

atro

gin-

1G

APD

Hlowast

lowast

lowastlowast

lowast

lowast

MuR

F-1

GA

PDH

Cbl-b

GA

PDH

ShamDenervation

ShamDenervation

(e)

Figure 6 Effects of dietary soy glycinin protein on IGF-1 signaling in denervated muscle The right and left legs of C57BL6 mice weresubjected to denervation and sham operation respectivelyThemice were fed with 20 casein (control) or 20 soy glycinin diet from 1 weekbefore denervation till the end of the experiment TA muscles were isolated at days 1 3 35 and 4 after denervation (a) Homogenates of TAmuscles isolated were subjected to immunoblotting (IB) for IRS-1 and actin on the indicated days after denervation (bndashd) Homogenates ofTA muscles isolated from mice fed with control or 20 glycinin diet at 35 days after denervation were subjected to IB for IRS-1 and actin(b) and phosphorylated Akt-1 and total Akt-1 (d) The densitometric analysis of these images was performed with ImageJ software Data aremean plusmn SD (119899 = 3) lowast119875 lt 005 versus sham operation

119875 lt 005 versus control diet in denervated muscle Immunoprecipitates (IP) fromthe TAmuscles with an anti-IRS-1 antibody were subjected to IB for ubiquitin (c) Representative findings of five experiments with matchingresults (e) Expression levels of MAFbxatrogin-1 MuRF-1 Cbl-b and GAPDH (internal control) transcripts in TA muscle analyzed by real-timeRT-PCRThe level of each transcript was normalized to sham-operatedmice fedwith control diet Data aremeanplusmn SD (119899 = 5) lowast119875 lt 005versus sham operation

119875 lt 005 versus control diet in denervated muscle

acid sequence similar to Cblin in soy glycinin inhibiteddenervation-mediated ubiquitination of IRS-1 this sequenceof soy is a potent functional peptide against muscle atrophyUnlike various types of medicines food-derived peptideshave little side effects Based on this property dietary soyprotein is a suitable protein source for efficient control ofcatabolism of muscle protein In addition to soy proteincertain plants for example papaya and plum contain pro-teins with amino acid sequence similar to Cblin Unfortu-nately they have only one Cblin-like sequence per moleculeTherefore a high dose of functional foods is necessary toproduce the inhibitory effect of ubiquitination in vivo Globalsearch for such food is necessary to develop more effectivefunctional foods against muscle atrophy

There is a controversy on the detection of food-derivedbioactive peptides The potential biological activities ofpeptides in food protein hydrolysates have frequently beenscreened by in vitro assays as shown in the present studyUnlike other functional substances such as polyphenols andpolyamines some peptides in foods with in vitro biologicalactivity might be further degraded by peptidases duringthe process of ingestion digestion and absorption Con-sequently these peptides might lose their potential activitydetecting using in biological activity Therefore the amount

of the peptides should be measured in their target organsthrough a feeding experiment However there are few papersshowing the detection of food-derived peptides in targetorgans mainly due to the low concentration and interactionwith plasma proteins They suggest only that food-derivedpeptides could be accumulated in target organs since theyhave a chronic rather than an acute effect on health In thepresent study we also failed tomeasure the quantity of Cblin-like peptide Therefore it is the next important subject Weare feeding mice a gene-modified protein which containshigh amount of the Cblin-like sequences and are detectingthe functional peptide in mouse portal veinThe detection ofdiet-derived Cblin-like peptides in muscle will be reported inour next paper

5 Conclusion

The present study identified Cblin-like sequence DIFYNPin soy glycinin Similar to Cblin treatment with soy glycininprevented muscle atrophy in mice through the suppressionof Cbl-b-induced IRS-1 degradation The results also showedthat 20 soy glycinin diet prevented denervation-related TAmuscle wet weight loss in mice suggesting that intake of soyglycinin could prevent muscle atrophy


Abbreviations

ATP Adenosine triphosphateAtrogenes Muscle atrophy-related genesCbl Casitus b-lineage lymphomaCSA Cross sectional areaDMEM Dulbeccorsquos modified Eagle mediumEDL Extensor digitorum longus muscleFOXO3 Forkhead transcription factor-3GAPDH Glyceraldehyde-3-phosphate

dehydrogenaseHampE Hematoxylin-eosin stainingIB ImmunoblottingIC50 50 inhibitory concentration

IGF-1 Insulin-like growth factor-1IRS-1 Insulin receptor substrate-1LP LipoproteinMAFbxatrogin-1 Muscle atrophy F-box proteinMuRF-1 Muscle-specific RING finger protein 1PCR Polymerase chain reactionSH2 domain Src homology 2 domainSPI Soy protein isolateTA muscle Tibialis anterior muscleTKB domain Tyrosine kinase binding domain

Conflict of Interests

The authors declare no conflict of interests

Acknowledgments

The authors thank Dr Lipkowitz for pCEFL-Cbl-b-HA andDr Suzuki for pcDNA3-FLAG-Ubiquitin They also thankFuji Oil Co for providing SPI glycinin protein and soy-derived peptides This study was supported by a grant fromFuji Foundation for Protein Research This study was alsocarried out as part of ldquoPromotion of Basic Research Activi-ties for Innovative Biosciencesrdquo from Bio-oriented Technol-ogy Research Advancement Institution Japan (to TakeshiNikawa)

References

[1] D S Criswell F W Booth F DeMayo R J Schwartz S EGordon andM L Fiorotto ldquoOverexpression of IGF-I in skeletalmuscle of transgenic mice does not prevent unloading-inducedatrophyrdquo American Journal of Physiology Endocrinology andMetabolism vol 275 no 3 pp E373ndashE379 1998

[2] S M Harman and M R Blackman ldquoThe effects of growthhormone and sex steroid on lean body mass fat mass mus-cle strength cardiovascular endurance and adverse events inhealthy elderly women and menrdquo Hormone Research vol 60no 1 pp 121ndash124 2003

[3] M J Rennie ldquoClaims for the anabolic effects of growth hor-mone a case of the emperorrsquos new clothesrdquo British Journal ofSports Medicine vol 37 no 2 pp 100ndash105 2003

[4] R Nakao K Hirasaka J Goto et al ldquoUbiquitin ligase Cbl-bis a negative regulator for insulin-like growth factor 1 signalingduring muscle atrophy caused by unloadingrdquo Molecular andCellular Biology vol 29 no 17 pp 4798ndash4811 2009

[5] K Masuda M Maebuchi M Samoto et al ldquoEffect of soy-pep-tide intake on exercise-induced muscle damagerdquo Japan Societyof Clinical Sports Medicine vol 15 pp 228ndash235 2007

[6] T G Anthony B J McDaniel P Knoll P Bunpo G LPaul and M A McNurlan ldquoFeeding meals containing soy orwhey protein after exercise stimulates protein synthesis andtranslation initiation in the skeletal muscle ofmale ratsrdquo Journalof Nutrition vol 137 no 2 pp 357ndash362 2007

[7] T Nikawa M Ikemoto T Sakai et al ldquoEffects of a soy proteindiet on exercise-induced muscle protein catabolism in ratsrdquoNutrition vol 18 no 6 pp 490ndash495 2002

[8] K Hirasaka S Kohno J Goto et al ldquoDeficiency of Cbl-bgene enhances infiltration and activation of macrophages inadipose tissue and causes peripheral insulin resistance in micerdquoDiabetes vol 56 no 10 pp 2511ndash2522 2007

[9] K Hirasaka T Nikawa L Yuge et al ldquoClinorotation preventsdifferentiation of rat myoblastic L6 cells in association withreduced NF-120581B signalingrdquo Biochimica et Biophysica Acta vol1743 no 1-2 pp 130ndash140 2005

[10] S Kohno T Ueji T Abe et al ldquoRantes secreted from macro-phages disturbs skeletal muscle regeneration after cardiotoxininjection inCbl-b-deficientmicerdquoMuscle andNerve vol 43 no2 pp 223ndash229 2011

[11] TOgawaH FurochiMMameoka et al ldquoUbiquitin ligase geneexpression in healthy volunteers with 20-day bedrestrdquo Muscleand Nerve vol 34 no 4 pp 463ndash469 2006

[12] D W West and G E Towers ldquoA study of the enzymic de-phosphorylation of 120573 casein and a derived phosphopeptiderdquoBiochimica et Biophysica Acta vol 453 no 2 pp 383ndash390 1976

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


named Cblin (Cbl-b inhibitor) prevented denervation-in-duced muscle atrophy in mice [4] We also found thatCblin inhibited Cbl-b-mediated IRS-1 ubiquitination and ex-pression of MAFbxatrogin-1 [4]These results indicated thatthe inhibition of Cbl-b-mediated IRS-1 ubiquitination couldbe therapeutically beneficial in unloading-induced muscleatrophy

Soy protein and derived peptides are effective for mitiga-tion of muscle damage [5] promotion of protein synthesisand translation initiation after exercise [6] In addition wedemonstrated previously that diet containing soy proteinprevents exercise-induced protein degradation in skeletalmuscle through the suppression of calpain-mediated prote-olysis [7] Thus dietary soy protein affects protein turnoverin skeletal muscle However to date there is no informationon the effects of soy protein on unloading-induced muscleatrophy

In a preliminary study we found that the sequence ofsoy glycinin a major component of soy protein is similarto that of Cblin Based on this finding we explored whethersoy glycinin and its derived peptides prevent denervation-associated muscle atrophy in mice Treatment of HEK293cells with soy-glycinin-derived peptides suppressed Cbl-b-mediated IRS-1 ubiquitination Furthermore mice fed withsoy glycinin showed inhibition of denervation-associatedmuscle atrophy and this effect was mediated through theconservation of IGF-1 signalingThese findings indicated thatsoy glycinin is a useful dietary protein for muscle atrophyassociated with muscle unloading

2 Materials and Methods

21 Multiple Alignment of Amino Acid Sequence We analyz-ed the primary structure of glycinin precursor with ClustalXsoftware (httpwww-igbmcu-strasbgfrBioInfoClustalXTophtml)

22 Cell Culture HEK293 cells were maintained in Dulbec-corsquos modified Eagle medium (DMEM) containing 10 fetalbovine serum 100UmL penicillin and 100 120583gmL strepto-mycin at 37∘C under 5 CO

2-195 air gas mixture

23 Transfection HEK293 cells (40ndash60 of confluence) weretransfected with plasmid vectors using Hily Max lipofec-tion reagent (DOJINDO Kumamoto Japan) as describedpreviously [8] The expression plasmids used in this studywere pCEFL-human Cbl-b-HA pcDNA31-rat IRS-1-V5 andpcDNA3-FLAG-Ubiquitin

24 Immunoblotting and Immunoprecipitation Immunoblotand immunoprecipitation analyses were performed as de-scribed previously [9] The following antibodies were usedanti-actin (Calbiochem La Jolla CA USA) anti-HA11(BabCo Richmond CA USA) anti-V5 (Invitrogen Carls-bad CA USA) anti-FLAG M2 (Sigma St Louis MOUSA) anti-IRS-1 (Upstate Biotechnology Lake Placid NYUSA) anti-Akt-1 (PharMingen International Tokyo) and

anti-phospho-S473-Akt-1 (Cell Signaling Technology BeverlyMA USA)

25 Cell-Free Ubiquitination Assay The cell-free ubiquitina-tion assay was performed as described previously [4] Weprepared immunoprecipitated Cbl-b (IP-Cbl-b) and IRS-1(IP-IRS-1) respectively bound to anti-HA- and anti-IRS-1-IgG-linked protein A beads from extracts (1mg protein)of HEK293 cells transfected with pCEFL-Cbl-b-HA andpcDNA31-IRS-1-V5 (5 120583g) Slurries of IP-Cbl-b and IP-IRS-1 were incubated at 37∘C for 4 hours in reaction buffer(50120583L) containing ATP-regenerating system recombinantmouse E1 (500 ng) UbcH7 (5 120583g E2) GST-tagged ubiquitin(10 120583g) andor ubiquitin-aldehyde (Ubc-CHO) (1 120583g a deu-biquitinase inhibitor) which were purchased from BostonBiochem Inc (Cambridge MA USA) Then 20 120583L of boiledsupernatant was separated by SDS-PAGE and visualized byimmunoblotting with an anti-IRS-1 antibody

26 Preparation of Soy-Derived Peptides and Synthetic Pep-tides We used the following pentapeptides or hexapep-tides derived from soy soy-glycinin-derived peptide soy-120573-conglycinin-derived peptide lipoprotein- (LP-) derivedpeptide and soy protein isolate (SPI) These peptides werekindly provided by Fuji Oil Co (Osaka Japan) In additionwe used the following synthetic peptides Cblin DGpYMPwhich exhibited inhibitory activity on Cbl-b-mediated IRS-1ubiquitination in our previous study [4] A patent applicationwas lodged for the sequence of this peptide (Pat no 5113346)Negative control was a peptide with VGpYLR sequencewhich had no inhibitory activity against Cbl-b-mediated IRS-1 ubiquitination [4] These peptides were kindly provided byOtsuka Pharmaceutical Co (Tokushima Japan)We also syn-thesized a pentapeptide with Cblin-like amino acid sequencein soy glycinin termed it Cblin-like peptide or DIpYNP andchecked its inhibitory activity using cell-free ubiquitinationassay as described above Since soy-glycinin-derived peptidesshowed inhibitory activity on IRS-1 ubiquitination in the cell-free system (pat no 4963044) it was subjected to furtheranalysis in cell cultures

27 Animal Models Experiments were conducted in C57BL6J mice (Japan SLC Shizuoka Japan) In anesthetized 6-week-old mice the sciatic nerve of the right leg was cut anda 10mm piece was excised The left leg remained innervatedand was used as a control Mice were sacrificed at 1 3 35 or 4days after operation Mice were fed one of the following fourdiets from 1 week before denervation till sacrifice a mixeddiet containing 20 casein protein (Control diet) a mixeddiet containing 20 SPI (SPI diet) a mixed diet containing20 soy glycinin protein (20glycinin diet) and amixed dietcontaining 10 casein protein plus 10 soy glycinin protein(10 glycinin diet) These proteins were kindly provided byFuji Oil Co (Osaka Japan) The macronutrient compositionsof the different diets are listed in Table 1 The Committee forthe Care and Use of Laboratory Animals of The Universityof Tokushima approved all protocols of this study whichwere conducted according to the Guide for the Care and






MAFbxatrogin-1 SAS


MuRF-1 SAS


GAPDH SAS








3 Results





180210240270300


330360390420450


480510540570



Ruler

G1G2G3G4

G

Ruler

G1G2G3G4

G

Ruler

G1G2G3G4

G

Ruler

1306090120150


IB P-tyrosine

97

21

BSA

Egg

whi

te

Gly

cini

n

MMSTD(kDa)



50value of






00 0 0 200100 400 600

00 1200 00 00

00 0600 00 00

180

MMSTD(kDa)

100

50

00

200 400 600 800

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

++

+

++

minus

119910 = minus01689119909 + 11037

1198772= 09552

(120583M)

FLAG

-Ub


Cbl-b-HA (E3)

Ubiquitin-FLAG


Cblin (120583M)


IP IRS-1-V5

IB Ub-FLAG

IB Cbl-b-HA

IB IRS-1-V5





4 Discussion



MMSTD(kDa)

200

180

SPI LP C G


E1UbcH7 (E2)

GST-UbPeptides

IRS-1

Ub-IRS-1

IB IRS-1

++++++

++++++

++++++

+++++

minusminus

minus

+++++minus

(a)

200

120

180

6060 00

00 2010

MMSTD(kDa)

++++

++++

+++minus


Ubiquitin-FLAG

IP IRS-1-V5

IB Ub-FLAG

IB IRS-1-V5

IB Cbl-b-HA



(b)







50val-



10

gly

cini

n

20

gly

cini

n

20

case

in

20

SPI

10

gly

cini

n

20

gly

cini

n

20

case

in

20

SPI

10

gly

cini

n

20

gly

cini

n

20

case

in

20

SPI

10

gly

cini

n

20

gly

cini

n

20

case

in

20

SPI

TA Gast

0

100

50

75

25

0

100

50

75

25

EDL Sol

75

0

100

50

25

0

100

50

75

25

lowast


lowast

lowast

Den

erva

ted

mus

cle w

eigh

t (

)

(a)



Sham

Den

erva

tion

CSA

()

0

50

100lowast

(b)

Figure 5 Continued


10 glycinin

20 SPI

0

10

20

30

40

50

0

10

20

30

40

50 20 glycinin

20 casein

0

10

20

30

40

50

CSA

()

CSA

()

CSA

()

CSA

()

0

10

20

30

40

50

ShamDenervation

lowast

0sim500

501sim1000

1001sim1500

1501sim2000

2001sim2500

2501sim3000

3001sim3500

3501sim4000

4001

0sim500

501sim1000

1001sim1500

1501sim2000

2001sim2500

2501sim3000

3001sim3500

3501sim4000

4001


0sim500

501sim1000

1001sim1500

1501sim2000

2001sim2500

2501sim3000

3001sim3500

3501sim4000

4001



0sim500

501sim1000

1001sim1500

1501sim2000

2001sim2500

2501sim3000

3001sim3500

3501sim4000

4001


ShamDenervation

lowast

(c)






50value of dephos-






IB actin

Denervation

0 1 3 4(Day)

IB IRS-1

(a)

IB IRS-1

IB actin


Sham Denervation


1

05

15

lowast

lowast

ShamDenervation

IRS-

1ac

tin

(b)

Sham Denervation

Control 20 glycinin

Sham Denervation

IB ubiquitin

IP IRS-1

180

MMSTD(kDa)

Ub-

IRS-1

(c)

IB P-Akt-1

IB Akt-1


Control 20 glycinin


1

05lowast

lowast

P-A

kt-1

Akt

-1

ShamDenervation

(d)

Figure 6 Continued



2

4

6

0

2

4

6

8

0

1

2

3

ShamDenervation

MA

Fbx

atro

gin-

1G

APD

Hlowast

lowast

lowastlowast

lowast

lowast

MuR

F-1

GA

PDH

Cbl-b

GA

PDH

ShamDenervation

ShamDenervation

(e)







5 Conclusion



Abbreviations






Acknowledgments


References


















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















MAFbxatrogin-1 SAS


MuRF-1 SAS


GAPDH SAS








3 Results





180210240270300


330360390420450


480510540570



Ruler

G1G2G3G4

G

Ruler

G1G2G3G4

G

Ruler

G1G2G3G4

G

Ruler

1306090120150


IB P-tyrosine

97

21

BSA

Egg

whi

te

Gly

cini

n

MMSTD(kDa)



50value of






00 0 0 200100 400 600

00 1200 00 00

00 0600 00 00

180

MMSTD(kDa)

100

50

00

200 400 600 800

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

++

+

++

minus

119910 = minus01689119909 + 11037

1198772= 09552

(120583M)

FLAG

-Ub


Cbl-b-HA (E3)

Ubiquitin-FLAG


Cblin (120583M)


IP IRS-1-V5

IB Ub-FLAG

IB Cbl-b-HA

IB IRS-1-V5





4 Discussion



MMSTD(kDa)

200

180

SPI LP C G


E1UbcH7 (E2)

GST-UbPeptides

IRS-1

Ub-IRS-1

IB IRS-1

++++++

++++++

++++++

+++++

minusminus

minus

+++++minus

(a)

200

120

180

6060 00

00 2010

MMSTD(kDa)

++++

++++

+++minus


Ubiquitin-FLAG

IP IRS-1-V5

IB Ub-FLAG

IB IRS-1-V5

IB Cbl-b-HA



(b)







50val-



10

gly

cini

n

20

gly

cini

n

20

case

in

20

SPI

10

gly

cini

n

20

gly

cini

n

20

case

in

20

SPI

10

gly

cini

n

20

gly

cini

n

20

case

in

20

SPI

10

gly

cini

n

20

gly

cini

n

20

case

in

20

SPI

TA Gast

0

100

50

75

25

0

100

50

75

25

EDL Sol

75

0

100

50

25

0

100

50

75

25

lowast


lowast

lowast

Den

erva

ted

mus

cle w

eigh

t (

)

(a)



Sham

Den

erva

tion

CSA

()

0

50

100lowast

(b)

Figure 5 Continued


10 glycinin

20 SPI

0

10

20

30

40

50

0

10

20

30

40

50 20 glycinin

20 casein

0

10

20

30

40

50

CSA

()

CSA

()

CSA

()

CSA

()

0

10

20

30

40

50

ShamDenervation

lowast

0sim500

501sim1000

1001sim1500

1501sim2000

2001sim2500

2501sim3000

3001sim3500

3501sim4000

4001

0sim500

501sim1000

1001sim1500

1501sim2000

2001sim2500

2501sim3000

3001sim3500

3501sim4000

4001


0sim500

501sim1000

1001sim1500

1501sim2000

2001sim2500

2501sim3000

3001sim3500

3501sim4000

4001



0sim500

501sim1000

1001sim1500

1501sim2000

2001sim2500

2501sim3000

3001sim3500

3501sim4000

4001


ShamDenervation

lowast

(c)






50value of dephos-






IB actin

Denervation

0 1 3 4(Day)

IB IRS-1

(a)

IB IRS-1

IB actin


Sham Denervation


1

05

15

lowast

lowast

ShamDenervation

IRS-

1ac

tin

(b)

Sham Denervation

Control 20 glycinin

Sham Denervation

IB ubiquitin

IP IRS-1

180

MMSTD(kDa)

Ub-

IRS-1

(c)

IB P-Akt-1

IB Akt-1


Control 20 glycinin


1

05lowast

lowast

P-A

kt-1

Akt

-1

ShamDenervation

(d)

Figure 6 Continued



2

4

6

0

2

4

6

8

0

1

2

3

ShamDenervation

MA

Fbx

atro

gin-

1G

APD

Hlowast

lowast

lowastlowast

lowast

lowast

MuR

F-1

GA

PDH

Cbl-b

GA

PDH

ShamDenervation

ShamDenervation

(e)







5 Conclusion



Abbreviations






Acknowledgments


References


















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















180210240270300


330360390420450


480510540570



Ruler

G1G2G3G4

G

Ruler

G1G2G3G4

G

Ruler

G1G2G3G4

G

Ruler

1306090120150


IB P-tyrosine

97

21

BSA

Egg

whi

te

Gly

cini

n

MMSTD(kDa)



50value of






00 0 0 200100 400 600

00 1200 00 00

00 0600 00 00

180

MMSTD(kDa)

100

50

00

200 400 600 800

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

++

+

++

minus

119910 = minus01689119909 + 11037

1198772= 09552

(120583M)

FLAG

-Ub


Cbl-b-HA (E3)

Ubiquitin-FLAG


Cblin (120583M)


IP IRS-1-V5

IB Ub-FLAG

IB Cbl-b-HA

IB IRS-1-V5





4 Discussion



MMSTD(kDa)

200

180

SPI LP C G


E1UbcH7 (E2)

GST-UbPeptides

IRS-1

Ub-IRS-1

IB IRS-1

++++++

++++++

++++++

+++++

minusminus

minus

+++++minus

(a)

200

120

180

6060 00

00 2010

MMSTD(kDa)

++++

++++

+++minus


Ubiquitin-FLAG

IP IRS-1-V5

IB Ub-FLAG

IB IRS-1-V5

IB Cbl-b-HA



(b)







50val-



10

gly

cini

n

20

gly

cini

n

20

case

in

20

SPI

10

gly

cini

n

20

gly

cini

n

20

case

in

20

SPI

10

gly

cini

n

20

gly

cini

n

20

case

in

20

SPI

10

gly

cini

n

20

gly

cini

n

20

case

in

20

SPI

TA Gast

0

100

50

75

25

0

100

50

75

25

EDL Sol

75

0

100

50

25

0

100

50

75

25

lowast


lowast

lowast

Den

erva

ted

mus

cle w

eigh

t (

)

(a)



Sham

Den

erva

tion

CSA

()

0

50

100lowast

(b)

Figure 5 Continued


10 glycinin

20 SPI

0

10

20

30

40

50

0

10

20

30

40

50 20 glycinin

20 casein

0

10

20

30

40

50

CSA

()

CSA

()

CSA

()

CSA

()

0

10

20

30

40

50

ShamDenervation

lowast

0sim500

501sim1000

1001sim1500

1501sim2000

2001sim2500

2501sim3000

3001sim3500

3501sim4000

4001

0sim500

501sim1000

1001sim1500

1501sim2000

2001sim2500

2501sim3000

3001sim3500

3501sim4000

4001


0sim500

501sim1000

1001sim1500

1501sim2000

2001sim2500

2501sim3000

3001sim3500

3501sim4000

4001



0sim500

501sim1000

1001sim1500

1501sim2000

2001sim2500

2501sim3000

3001sim3500

3501sim4000

4001


ShamDenervation

lowast

(c)






50value of dephos-






IB actin

Denervation

0 1 3 4(Day)

IB IRS-1

(a)

IB IRS-1

IB actin


Sham Denervation


1

05

15

lowast

lowast

ShamDenervation

IRS-

1ac

tin

(b)

Sham Denervation

Control 20 glycinin

Sham Denervation

IB ubiquitin

IP IRS-1

180

MMSTD(kDa)

Ub-

IRS-1

(c)

IB P-Akt-1

IB Akt-1


Control 20 glycinin


1

05lowast

lowast

P-A

kt-1

Akt

-1

ShamDenervation

(d)

Figure 6 Continued



2

4

6

0

2

4

6

8

0

1

2

3

ShamDenervation

MA

Fbx

atro

gin-

1G

APD

Hlowast

lowast

lowastlowast

lowast

lowast

MuR

F-1

GA

PDH

Cbl-b

GA

PDH

ShamDenervation

ShamDenervation

(e)







5 Conclusion



Abbreviations






Acknowledgments


References


















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















00 0 0 200100 400 600

00 1200 00 00

00 0600 00 00

180

MMSTD(kDa)

100

50

00

200 400 600 800

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

++

+

++

minus

119910 = minus01689119909 + 11037

1198772= 09552

(120583M)

FLAG

-Ub


Cbl-b-HA (E3)

Ubiquitin-FLAG


Cblin (120583M)


IP IRS-1-V5

IB Ub-FLAG

IB Cbl-b-HA

IB IRS-1-V5





4 Discussion



MMSTD(kDa)

200

180

SPI LP C G


E1UbcH7 (E2)

GST-UbPeptides

IRS-1

Ub-IRS-1

IB IRS-1

++++++

++++++

++++++

+++++

minusminus

minus

+++++minus

(a)

200

120

180

6060 00

00 2010

MMSTD(kDa)

++++

++++

+++minus


Ubiquitin-FLAG

IP IRS-1-V5

IB Ub-FLAG

IB IRS-1-V5

IB Cbl-b-HA



(b)







50val-



10

gly

cini

n

20

gly

cini

n

20

case

in

20

SPI

10

gly

cini

n

20

gly

cini

n

20

case

in

20

SPI

10

gly

cini

n

20

gly

cini

n

20

case

in

20

SPI

10

gly

cini

n

20

gly

cini

n

20

case

in

20

SPI

TA Gast

0

100

50

75

25

0

100

50

75

25

EDL Sol

75

0

100

50

25

0

100

50

75

25

lowast


lowast

lowast

Den

erva

ted

mus

cle w

eigh

t (

)

(a)



Sham

Den

erva

tion

CSA

()

0

50

100lowast

(b)

Figure 5 Continued


10 glycinin

20 SPI

0

10

20

30

40

50

0

10

20

30

40

50 20 glycinin

20 casein

0

10

20

30

40

50

CSA

()

CSA

()

CSA

()

CSA

()

0

10

20

30

40

50

ShamDenervation

lowast

0sim500

501sim1000

1001sim1500

1501sim2000

2001sim2500

2501sim3000

3001sim3500

3501sim4000

4001

0sim500

501sim1000

1001sim1500

1501sim2000

2001sim2500

2501sim3000

3001sim3500

3501sim4000

4001


0sim500

501sim1000

1001sim1500

1501sim2000

2001sim2500

2501sim3000

3001sim3500

3501sim4000

4001



0sim500

501sim1000

1001sim1500

1501sim2000

2001sim2500

2501sim3000

3001sim3500

3501sim4000

4001


ShamDenervation

lowast

(c)






50value of dephos-






IB actin

Denervation

0 1 3 4(Day)

IB IRS-1

(a)

IB IRS-1

IB actin


Sham Denervation


1

05

15

lowast

lowast

ShamDenervation

IRS-

1ac

tin

(b)

Sham Denervation

Control 20 glycinin

Sham Denervation

IB ubiquitin

IP IRS-1

180

MMSTD(kDa)

Ub-

IRS-1

(c)

IB P-Akt-1

IB Akt-1


Control 20 glycinin


1

05lowast

lowast

P-A

kt-1

Akt

-1

ShamDenervation

(d)

Figure 6 Continued



2

4

6

0

2

4

6

8

0

1

2

3

ShamDenervation

MA

Fbx

atro

gin-

1G

APD

Hlowast

lowast

lowastlowast

lowast

lowast

MuR

F-1

GA

PDH

Cbl-b

GA

PDH

ShamDenervation

ShamDenervation

(e)







5 Conclusion



Abbreviations






Acknowledgments


References


















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















MMSTD(kDa)

200

180

SPI LP C G


E1UbcH7 (E2)

GST-UbPeptides

IRS-1

Ub-IRS-1

IB IRS-1

++++++

++++++

++++++

+++++

minusminus

minus

+++++minus

(a)

200

120

180

6060 00

00 2010

MMSTD(kDa)

++++

++++

+++minus


Ubiquitin-FLAG

IP IRS-1-V5

IB Ub-FLAG

IB IRS-1-V5

IB Cbl-b-HA



(b)







50val-



10

gly

cini

n

20

gly

cini

n

20

case

in

20

SPI

10

gly

cini

n

20

gly

cini

n

20

case

in

20

SPI

10

gly

cini

n

20

gly

cini

n

20

case

in

20

SPI

10

gly

cini

n

20

gly

cini

n

20

case

in

20

SPI

TA Gast

0

100

50

75

25

0

100

50

75

25

EDL Sol

75

0

100

50

25

0

100

50

75

25

lowast


lowast

lowast

Den

erva

ted

mus

cle w

eigh

t (

)

(a)



Sham

Den

erva

tion

CSA

()

0

50

100lowast

(b)

Figure 5 Continued


10 glycinin

20 SPI

0

10

20

30

40

50

0

10

20

30

40

50 20 glycinin

20 casein

0

10

20

30

40

50

CSA

()

CSA

()

CSA

()

CSA

()

0

10

20

30

40

50

ShamDenervation

lowast

0sim500

501sim1000

1001sim1500

1501sim2000

2001sim2500

2501sim3000

3001sim3500

3501sim4000

4001

0sim500

501sim1000

1001sim1500

1501sim2000

2001sim2500

2501sim3000

3001sim3500

3501sim4000

4001


0sim500

501sim1000

1001sim1500

1501sim2000

2001sim2500

2501sim3000

3001sim3500

3501sim4000

4001



0sim500

501sim1000

1001sim1500

1501sim2000

2001sim2500

2501sim3000

3001sim3500

3501sim4000

4001


ShamDenervation

lowast

(c)






50value of dephos-






IB actin

Denervation

0 1 3 4(Day)

IB IRS-1

(a)

IB IRS-1

IB actin


Sham Denervation


1

05

15

lowast

lowast

ShamDenervation

IRS-

1ac

tin

(b)

Sham Denervation

Control 20 glycinin

Sham Denervation

IB ubiquitin

IP IRS-1

180

MMSTD(kDa)

Ub-

IRS-1

(c)

IB P-Akt-1

IB Akt-1


Control 20 glycinin


1

05lowast

lowast

P-A

kt-1

Akt

-1

ShamDenervation

(d)

Figure 6 Continued



2

4

6

0

2

4

6

8

0

1

2

3

ShamDenervation

MA

Fbx

atro

gin-

1G

APD

Hlowast

lowast

lowastlowast

lowast

lowast

MuR

F-1

GA

PDH

Cbl-b

GA

PDH

ShamDenervation

ShamDenervation

(e)







5 Conclusion



Abbreviations






Acknowledgments


References


















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















10

gly

cini

n

20

gly

cini

n

20

case

in

20

SPI

10

gly

cini

n

20

gly

cini

n

20

case

in

20

SPI

10

gly

cini

n

20

gly

cini

n

20

case

in

20

SPI

10

gly

cini

n

20

gly

cini

n

20

case

in

20

SPI

TA Gast

0

100

50

75

25

0

100

50

75

25

EDL Sol

75

0

100

50

25

0

100

50

75

25

lowast


lowast

lowast

Den

erva

ted

mus

cle w

eigh

t (

)

(a)



Sham

Den

erva

tion

CSA

()

0

50

100lowast

(b)

Figure 5 Continued


10 glycinin

20 SPI

0

10

20

30

40

50

0

10

20

30

40

50 20 glycinin

20 casein

0

10

20

30

40

50

CSA

()

CSA

()

CSA

()

CSA

()

0

10

20

30

40

50

ShamDenervation

lowast

0sim500

501sim1000

1001sim1500

1501sim2000

2001sim2500

2501sim3000

3001sim3500

3501sim4000

4001

0sim500

501sim1000

1001sim1500

1501sim2000

2001sim2500

2501sim3000

3001sim3500

3501sim4000

4001


0sim500

501sim1000

1001sim1500

1501sim2000

2001sim2500

2501sim3000

3001sim3500

3501sim4000

4001



0sim500

501sim1000

1001sim1500

1501sim2000

2001sim2500

2501sim3000

3001sim3500

3501sim4000

4001


ShamDenervation

lowast

(c)






50value of dephos-






IB actin

Denervation

0 1 3 4(Day)

IB IRS-1

(a)

IB IRS-1

IB actin


Sham Denervation


1

05

15

lowast

lowast

ShamDenervation

IRS-

1ac

tin

(b)

Sham Denervation

Control 20 glycinin

Sham Denervation

IB ubiquitin

IP IRS-1

180

MMSTD(kDa)

Ub-

IRS-1

(c)

IB P-Akt-1

IB Akt-1


Control 20 glycinin


1

05lowast

lowast

P-A

kt-1

Akt

-1

ShamDenervation

(d)

Figure 6 Continued



2

4

6

0

2

4

6

8

0

1

2

3

ShamDenervation

MA

Fbx

atro

gin-

1G

APD

Hlowast

lowast

lowastlowast

lowast

lowast

MuR

F-1

GA

PDH

Cbl-b

GA

PDH

ShamDenervation

ShamDenervation

(e)







5 Conclusion



Abbreviations






Acknowledgments


References


















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















10 glycinin

20 SPI

0

10

20

30

40

50

0

10

20

30

40

50 20 glycinin

20 casein

0

10

20

30

40

50

CSA

()

CSA

()

CSA

()

CSA

()

0

10

20

30

40

50

ShamDenervation

lowast

0sim500

501sim1000

1001sim1500

1501sim2000

2001sim2500

2501sim3000

3001sim3500

3501sim4000

4001

0sim500

501sim1000

1001sim1500

1501sim2000

2001sim2500

2501sim3000

3001sim3500

3501sim4000

4001


0sim500

501sim1000

1001sim1500

1501sim2000

2001sim2500

2501sim3000

3001sim3500

3501sim4000

4001



0sim500

501sim1000

1001sim1500

1501sim2000

2001sim2500

2501sim3000

3001sim3500

3501sim4000

4001


ShamDenervation

lowast

(c)






50value of dephos-






IB actin

Denervation

0 1 3 4(Day)

IB IRS-1

(a)

IB IRS-1

IB actin


Sham Denervation


1

05

15

lowast

lowast

ShamDenervation

IRS-

1ac

tin

(b)

Sham Denervation

Control 20 glycinin

Sham Denervation

IB ubiquitin

IP IRS-1

180

MMSTD(kDa)

Ub-

IRS-1

(c)

IB P-Akt-1

IB Akt-1


Control 20 glycinin


1

05lowast

lowast

P-A

kt-1

Akt

-1

ShamDenervation

(d)

Figure 6 Continued



2

4

6

0

2

4

6

8

0

1

2

3

ShamDenervation

MA

Fbx

atro

gin-

1G

APD

Hlowast

lowast

lowastlowast

lowast

lowast

MuR

F-1

GA

PDH

Cbl-b

GA

PDH

ShamDenervation

ShamDenervation

(e)







5 Conclusion



Abbreviations






Acknowledgments


References


















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















IB actin

Denervation

0 1 3 4(Day)

IB IRS-1

(a)

IB IRS-1

IB actin


Sham Denervation


1

05

15

lowast

lowast

ShamDenervation

IRS-

1ac

tin

(b)

Sham Denervation

Control 20 glycinin

Sham Denervation

IB ubiquitin

IP IRS-1

180

MMSTD(kDa)

Ub-

IRS-1

(c)

IB P-Akt-1

IB Akt-1


Control 20 glycinin


1

05lowast

lowast

P-A

kt-1

Akt

-1

ShamDenervation

(d)

Figure 6 Continued



2

4

6

0

2

4

6

8

0

1

2

3

ShamDenervation

MA

Fbx

atro

gin-

1G

APD

Hlowast

lowast

lowastlowast

lowast

lowast

MuR

F-1

GA

PDH

Cbl-b

GA

PDH

ShamDenervation

ShamDenervation

(e)







5 Conclusion



Abbreviations






Acknowledgments


References


















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















2

4

6

0

2

4

6

8

0

1

2

3

ShamDenervation

MA

Fbx

atro

gin-

1G

APD

Hlowast

lowast

lowastlowast

lowast

lowast

MuR

F-1

GA

PDH

Cbl-b

GA

PDH

ShamDenervation

ShamDenervation

(e)







5 Conclusion



Abbreviations






Acknowledgments


References


















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Abbreviations






Acknowledgments


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 Soy Glycinin Contains a Functional...

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