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Ingestion of 10 grams of whey protein prior to a single bout of resistance exercise does not augment Akt/mTOR pathway si...
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Title: Ingestion of 10 grams of whey protein prior to a single bout of resistance exercise does not augment Akt/mTOR pathway signaling compared to carbohydrate
Series Title: Journal of the International Society of Sports Nutrition
Physical Description: Archival
Language: English
Creator: Cooke, Matthew B.
La Bounty, Paul
Buford, Thomas
Shelmadine, Brian
Redd, Liz
Hudson, Geoffrey
Willoughby, Darryn S.
Publisher: BioMed Central
Publication Date: 2011
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Abstract: Background: This study examined the effects of a whey protein supplement in conjunction with an acute bout of lower body resistance exercise, in recreationally-active males, on serum insulin and insulin like growth factor 1 (IGF-1) and Akt/mTOR signaling markers indicative of muscle protein synthesis: insulin receptor substrate 1 (IRS-1), AKT, mammalian target of rapamycin (mTOR), p70S6 kinase (p70S6K) and 4E-binding protein 1 (4E-BP1). Methods: In a randomized, double-blind, cross-over design, 10 males ingested 1 week apart, either 10 g of whey protein (5.25 g EAAs) or carbohydrate (maltodextrose), 30 min prior to a lower-body resistance exercise bout. The resistance exercise bout consisted of 4 sets of 8-10 reps at 80% of the one repetition maximum (RM) on the angled leg press and knee extension exercises. Blood and muscle samples were obtained prior to, and 30 min following supplement ingestion and 15 min and 120 min post-exercise. Serum and muscle data were analyzed using two-way ANOVA. Results: No significant differences were observed for IGF-1 (p > 0.05). A significant main effect for Test was observed for serum insulin (p < 0.01) at 30 min post-ingestion and 15 and 120 min post-exercise, with no Supplement × Test interaction (p > 0.05). For the Akt/MTOR signaling intermediates, no significant Supplement × Test interactions were observed (p > 0.05). However, significant main effects for Test were observed for phosphorylated concentrations of IRS, mTOR, and p70S6K, as all were elevated at 15 min post-exercise (p < 0.05). Additionally, a significant main effect for Test was noted for 4E-BP1 (p < 0.05), as it was decreased at 15 min postexercise. Conclusion: Ingestion of 10 g of whey protein prior to an acute bout of lower body resistance exercise had no significant preferential effect compared to carbohydrate on systemic and cellular signaling markers indicative of muscle protein synthesis in untrained individuals.
General Note: Publication of this article was funded in part by the University of Florida Open-Access publishing Fund. In addition, requestors receiving funding through the UFOAP project are expected to submit a post-review, final draft of the article to UF's institutional repository, IR@UF, (www.uflib.ufl.edu/ufir) at the time of funding. The Institutional Repository at the University of Florida (IR@UF) is the digital archive for the intellectual output of the University of Florida community, with research, news, outreach and educational materials
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Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution.
Resource Identifier: doi - 10.1186-1550-2783-8-18
System ID: AA00008945:00001

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RESEARCHARTICLE OpenAccessIngestionof10gramsofwheyproteinpriortoa singleboutofresistanceexercisedoesnot augmentAkt/mTORpathwaysignalingcompared tocarbohydrateMatthewBCooke1,3,PaulLaBounty1,ThomasBuford1,4,BrianShelmadine1,LizRedd1,GeoffreyHudson1,5and DarrynSWilloughby1,2*AbstractBackground: Thisstudyexaminedtheeffectsofawheyproteinsupplementinconjunctionwithanacuteboutof lowerbodyresistanceexercise,inrecreationally-activemales,onseruminsulinandinsulinlikegrowthfactor1(IGF-1) andAkt/mTORsignalingmarkersindicativeofmuscleproteinsynthesis:insulinreceptorsubstrate1(IRS-1),AKT, mammaliantargetofrapamycin(mTOR),p70S6kinase(p70S6K)and4E-bindingprotein1(4E-BP1). Methods: Inarandomized,double-blind,cross-overdesign,10malesingested1weekapart,either10gofwhey protein(5.25gEAAs)orcarbohydrate(maltodextrose),30minpriortoalower-bodyresistanceexercisebout.The resistanceexerciseboutconsistedof4setsof8-10reps at80%oftheonerepetitionmaximum(RM)ontheangledleg pressandkneeextensionexercises.Bloodandmusclesampleswereobtainedpriorto,and30minfollowingsupplement ingestionand15minand120minpost-exercise.Serumandmuscledatawereanalyzedusingtwo-wayANOVA. Results: NosignificantdifferenceswereobservedforIGF-1(p>0.05).AsignificantmaineffectforTestwas observedforseruminsulin(p<0.01)at30minpost-ingestionand15and120minpost-exercise,withno SupplementTestinteraction(p>0.05).FortheAkt/MTORsignalingintermediates,nosignificantSupplement Testinteractionswereobserved(p>0.05).However,significantmaineffectsforTestwereobservedfor phosphorylatedconcentrationsofIRS,mTOR,andp70S6K,asallwereelevatedat15minpost-exercise(p<0.05). Additionally,asignificantmaineffectforTestwasnotedfor4E-BP1(p<0.05),asitwasdecreasedat15minpostexercise. Conclusion: Ingestionof10gofwheyproteinpriortoanacuteboutoflowerbodyresistanceexercisehadno significantpreferentialeffectcomparedtocarbohydrateonsystemicandcellularsignalingmarkersindicativeof muscleproteinsynthesisinuntrainedindividuals.IntroductionThemaintenanceofskeletalmusclemassisdeterminedby thelong-termnetbalanceofskeletalmuscleprotein synthesis(MPS)andmuscleproteinbreakdown,defined bynetproteinbalance.ThoughthebalancebetweenMPS andmuscleproteinbreakdownisdependentuponfeeding state[1-6]aswellastrainingstatus[7,8],changesinnet proteinbalancearethoughttooccurpredominantly throughchangesinMPS,whichisresponsivetoboth resistanceexerciseandaminoacidprovision[9,10].Resistanceexerciseleadstoacuteup-regulationoftheinward aminoacidtransport[11]tothemuscleresultinginan elevatedfractionalsyntheticrateofmuscleproteinforas manyas48hoursfollowingeachexercisebout[12]. Someoftheprincipleintracellularsignalingpathways involvedinMPSarebecomingmoredefinedintheliterature[13].Asaresult,determiningtheactivityofthevariouspathways,specificallyth eirintermediates,areoften *Correspondence:darryn_willoughby@baylor.edu1DepartmentofHealth,HumanPerformanceandRecreation,Baylor University,Waco,TX,USA FulllistofauthorinformationisavailableattheendofthearticleCooke etal JournaloftheInternationalSocietyofSportsNutrition 2011, 8 :18 http://www.jissn.com/content/8/1/18 2011Cookeetal;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommons AttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,andreproductionin anymedium,providedtheoriginalworkisproperlycited.

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usedasmarkersofMPS.MPSisstimulated,atleastin part,bytheAkt/mTORpathway,inwhichpathwayintermediateactivityisaffectedbythelevelofphosphorylation atdifferentaminoacidsites[14].Specifically,theregulationoftranslationinitiationviatheAkt/mTORpathwayis recognizedasasignificantregulatorofMPS[15].Key downstreamtargetsofthekinasemTORincludethe eukaryoticinitiationfactor4E(eIF4E)bindingprotein(4EBP1),whichuponphosphorylationreleasesitsinhibition overeIF4Etopromote5 -methylguanosinecap-dependent translationinitiationandp70S6kinase(p70S6K)[16]. Phosphorylationof4E-BP1isimportantduetothefact thatitpreventstheinteractionandinhibitionof4E-BP1 witheIF4Eandhence,increasestranslationandMPS[16]. Conversely,p70S6KinfluencesMPSpartiallythrough ribosomalproteinS6(rpS6)aswellasthroughsomeother proteinssuchaseukaryoticelongationfactor2(eEF2)[17]. Ingestionofsupplementaryprotein(wholeorasindividualaminoacids),eitherbeforeorimmediatelyfollowing resistanceexercisetraining,enhancesAkt/mTORpathway activityandMPS[13,14].Notwithstanding,ingestionof proteinoressentialaminoacids(EAA)withorwithout carbohydratepriorto,during,andintheearlyrecovery phasefollowingaboutofresistanceexercisecanleadto increasedphosphorylationofmTOR[15,18],p70S6K [19-21],andrpS6[22,23]withinthefirst4hrpost-exercise inbothrodentandhumanmodels.Theseresultsalsosuggestthattimingofingestionisimportant,withincreased circulationandnutrienttransporttotheskeletalmuscle followingexerciseoccurringconcomitantlywithinthe timeperiodwhenMPShasthegreatestelevationin responsetoexercise[12,24,25].Inaddition,proteinsource and/ordosageappeartoplayakeyroleinpre-andpostexercisemuscleproteinkinetics[26,27].Aslittleas10gof protein(4.2gEAA)hasbeenshowntostimulateMPSfollowingresistanceexercise[27],whileacuteingestionof between20-40gofintactprotein[28],or9-10gofEAA [25],seemstoinduceaplateauinMPSindependentof exercise. Albuminproteinintakeatadoseof10g(4.3gEAAs) hasbeenshowntosignificantlyincreaseMPS,buthadno effectontheactivitiesoftheAkt/mTORpathwayintermediatesS6K1(Thr389),rps6(Ser240/244),oreIF2B (Ser539)afterresistanceexercise[10].Asaresult,we soughttodetermineif10gofwheyprotein,butwith5.25 gofEAAs,wouldproduceincreasesinotherkeyAkt/ mTORsignallingintermediatesfollowingresistanceexercise.Therefore,theprimarypurposeofthisstudywasto determinetheconsumptionofawheyproteinsupplement priortoanacuteboutoflowerbodyresistanceexercisein recreationallyactivemalesonseruminsulinandIGF-1 andtheAkt/mTORsignalingmarkersindicativeofMPS: IRS-1,AKT,mTOR,p70S6Kand4E-BP1.MethodsParticipantsTenapparentlyhealthy,recreationally-active(exercise23timesperweek),butnotresistance-trained(noregular resistancetrainingforatleastoneyear)maleparticipants (20.11.4yrs,1748.7cm,78.512kg,)participated inthisstudy.AllsubjectssignedinformedconsentdocumentsandthestudywasapprovedbytheBaylorUniversityInstitutionalReviewBoardfortheProtectionof HumanSubjectspriortoanydatacollection.Subjects werenotallowedtoparticipateinthisstudyifthey reportedanyofthefollowing:1)currentorpasthistory ofanabolicsteroiduse;2)anymetabolicdisordersortakinganythyroid,hyperlipidmeic,hypoglycemic,antihypertensive,orandrogenicmedications;3)ingestedany ergogeniclevelsofcreatine, HMB,thermogenics,ribose, pro-hormones(i.e.,DHEA,androstendione,etc.)orother purportedanabolicorergogenicnutritionalsupplements within2monthspriortobeginningthestudy;4)nottakinganyadditionalnutritionalsupplementorcontraindicatedprescriptionmedicationduringtheprotocol.ExperimentaldesignThestudywasconductedinacross-over,randomized, double-blinded,andplacebo-controlledmanner.Participantsexpressinginterestinthestudywereinterviewedon thephone/orviaemailtodeterminewhethertheyappear toqualifytoparticipateinthisstudy.Participantsbelieved tomeeteligibilitycriteriaweretheninvitedtoattendan entry/familiarizationsessi on.Oncereportingtothelab, participantscompletedamedicalhistoryquestionnaire andunderwentageneralphysicalexaminationtodeterminewhethertheymeteligibilitycriteria.Oncecleared, participantswerefamiliarizedtothestudyprotocolviaa verbalandwrittenexplanationoutliningthestudydesign. Alleligibleparticipantswhoagreedtoparticipateinthe studyreadandsignedtheuniversity-approvedinformed consentdocuments.Participa ntswerefamiliarizedwith theangledlegpressandlegextensionmachines,thecorrecttechniqueinperformingeachoftheexercises,and thenperformedtwolow-re sistance(30%ofbodymass) practice/warm-upsetsof10repetitionsoneachexercise tofamiliarizethemwiththeprotocolandtoalsoinsure thattheywereabletocompletetheprotocolbeforebeing formallyadmittedtothestudy.Participantsthencompletedaninitialstrengthtesttoassesstheironerepetition maximum(1-RM)foreachlegontheangledlegpress (NebulaFitness,Inc.,Versailles,OH),andlegextension (BodyMasters,Inc.,Rayne,LA)exercisesusingstandard guidelinesroutinelyemployedourlaboratory[29].Followingthepracticetrials,participantswerescheduledto return48hourslaterfortesting.Participantswereasked tonotchangetheirdietaryhabitsinanywaythroughoutCooke etal JournaloftheInternationalSocietyofSportsNutrition 2011, 8 :18 http://www.jissn.com/content/8/1/18 Page2of9

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thestudy.Thiswasmonitoredbyhavingeachparticipant documentdietaryintakefortwodaysbeforeeachtesting session.Inaddition,eachparticipantwasinstructedtofast foreighthoursandnottoperformanyphysicalactivity forthe48hoursproceedingeachtestingsession.ResistanceexerciseprotocolAtthebeginningofeachtestingsession,participants hadtheirbodymassmeasuredaccordingtostandard proceduresusingaself-calibratingdigitalscale(HealthO-Meter,Bridgeview,IL,USA)withanaccuracyof 0.02kg.Participantsperformedtwoseparateboutsof resistanceexercise,eachsessioninvolvingonlyoneleg, eachseparatedbytwoweeks.Thesupplementandleg utilizedforthefirstexerciseboutwasrandomly assigned.Usingonlyoneleg,participantsperformed4 setsof8-10repetitionsat75%-80%1-RMontheangled legpress(NebulaFitness,Inc.,Versailles,OH)andknee extension(BodyMasters,Inc.,Rayne,LA)exercises. Eachsetwasperformedoverthecourseof25-30secondsandfollowedby120secondsofrest,while150secondsofrest(1:5,work:restratio)wereallowedbetween thetwoexercises.Trainingvolumeforeachexercise wascalculatedbymultiplyingtotalnumberofrepsby thetotalamountofweightliftedoverthefoursets.SupplementationprotocolParticipantswereassignedinadouble-blindandrandomizedmannertoorallyingest10gramsofmaltodextrose placebo(CHO)orwheyprotein(WP)containing5.25gof EAAs,mixedwith500mlofwater.Supplementswere ingested30minutesbefore eachexercisesession.Both supplementswereisocaloricandindependentlyprepared inindividuallyblindedpackag es(GlanbiaNutritionals, TwinFalls,ID,USA).Theaminoacidcompositionofthe WPsupplementisdisplayedinTable1.DietaryinventoriesFortwodaysimmediatelypriortoeachtestingsession, participantswereinstructedtorecordallfoodandfluid intake,whichwasreflectiveoftheirnormaldietaryintake. Dietaryinventorieswerethenanalyzedforaverageenergy andmacronutrientintakeusingtheESHAFoodProcessor NutritionalAnalysissoftware(Salem,OR,USA).BloodandmusclecollectionproceduresApproximately20mlofvenousbloodwasobtainedfrom anantecubitalveinusingstandardphlebotomyprocedures onfourseparateoccasionsateachofthetworesistance exercisesessions;1)30minpriortoexerciseandingestion ofthesupplement,2)immediatelybeforeexercisefollowingingestionofthesupplement,3)15minpost-exercise, and4)120minpost-exercise.Bloodanalyzedforserum IGFandinsulinwereplacedintotwoserumseparation tubesandimmediatelycentrifugedat1,100gfor15min. Serumwasseparatedandstoredat-80Cinpolypropylene cryovialsforlateranalysis. Approximately50-75mgofmusclewasobtainedfrom thelateralportionofthe vastuslateralis midwaybetween thepatellaandiliaccrestofthelegusinga5-mmBergstromstylebiopsyneedle.Musclesamplesweretakenon 3separateoccasionsateachofthetworesistanceexercise sessions;1)30minpriortoexerciseandingestionofthe supplement,2)15minpost-exercise,and3)120minpostexercise.Participantswereinstructedtorefrainfromexercise48hrpriortoeachmusclebiopsy.Afterremoval,adiposetissuewastrimmedfromthemusclespecimensand immediatelyfrozeninliquidnitrogenandthenstoredat -80Cforlateranalysis.SerumIGFandinsulinTheconcentrationsofseruminsulinandIGF-1were determinedinduplicateandtheaverageconcentrations reportedusingcommerciallyavailableenzyme-linked immunoabsorbentassay(ELISA)kits(DiagnosticSystems Laboratories,Webster,TX;Biosource,Camarillo,CA). Standardcurvesweregeneratedusingspecificcontrolpeptides.Concentrationsweredeterminedatanopticaldensityof450nmwithamicroplatereader(WallacVictor Table1Aminoacidcompositionofthewheyprotein (WP)supplement(g/500ml)EssentialAminoAcids(EAAs) Concentration(g) Isoleucine 0.61 Leucine 1.55 Lysine 0.76 Threonine 0.85 Valine 0.63 Methionine 0.32 Tryptophan 0.18 Phenylalanine 0.35 TotalEAAs 5.25 Non-EssentialAminoAcids(NEAAs)Concentration(g) AsparticAcid 0.94 Serine 0.45 GlutamicAcid 1.47 Glycine 0.14 Alanine 0.59 Tyrosine 0.27 Histidine 0.16 Arginine 0.14 Proline 0.44 Cystine 0.15 TotalNEAAs 4.75 TotalAminoAcids 10.00 Cooke etal JournaloftheInternationalSocietyofSportsNutrition 2011, 8 :18 http://www.jissn.com/content/8/1/18 Page3of9

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1420,PerkinElmer,Boston,MA,USA).Theoverallintraassaypercentcoefficientofvariationwas4.6%and2.9% forinsulinandIGF-1,respectively.IRS-1andAkt/mTORsignalingpathwayprotein expressionApproximately20mgofeachmusclesamplewashomogenizedusingacommercialc ellextractionbuffer(Biosource,Camarillo,CA,USA)andatissuehomogenizer. Thecellextractionbufferwassupplementedwith1mM phenylmethanesulphonylfluoride(PMSF)andaprotease inhibitorcocktail(SigmaChemicalCompany,St.Louis, MO,USA)withbroadspecificityfortheinhibitionofserine,cysteine,andmetallo-proteases.Musclehomogenates wereanalyzedforphosphorylatedIRS-1(Ser312),Akt (Ser473),4E-BP1(Thr46)andp70S6K(Thr389)using commercially-availablephosphoELISAkits(Invitrogen, Carlsbad,CA,USA).Thissensitivityoftheseparticular assaysisreportedbythemanufacturertobelessthan 1U/mL.Theabsorbances,whicharedirectlyproportional totheconcentrationinthesamples,weredeterminedat 450nmwithamicroplatereader(WallacVictor1420, PerkinElmer,BostonMA,US A).Asetofstandardsof knownconcentrationsforeachphosphorylatedmuscle variablewereutilizedtoconstructstandardcurvesbyplottingthenetabsorbancevaluesofthestandardsagainst theirrespectiveproteinconcentrations.Byapplyingafour partparametercurveusingMikroWinmicroplatedata reductionsoftware(MicrotekLabSystems,Germany),the concentrationsinthemusclesampleswereappropriately calculated.Proteinconcentrationswereexpressedrelative tomusclewet-weight.Theoverallintra-assaypercent coefficientofvariationforallassayswaslessthan7% PhosphorylatedmTORwasassessedthroughtheuse ofELISAusedbymethodspreviouslydescribed[29].A polyclonalantibodyspecificforphosphorylatedmTOR (Ser2448),wheretargetantigenspecificitieshavebeen verifiedthroughWesternblottingbythemanufacturer, waspurchasedfromSantaCruzBiotech(SantaCruz, CA).Initially,theantibodywasdilutedto0.5 g/mlin coatingbuffer(Na2CO3,NaHCO3,andddH2O,pH9.6) andallowedtoincubateatroomtemperatureovernight. Followingincubation,theplateswerewashed(1phosphatebufferedsaline,Tween-20),blocked(10phosphatebufferedsaline,bovineserumalbumin,ddH2O), washed,andthenincubatedwithasecondaryantibody (IgGconjugatedtoHRP)dilutedto0.5 g/mlindilution buffer(10phosphatebufferedsaline,Tween-20, bovineserumalbumin,ddH2O).Afterwashing,astabilizedTMBchromogenwasaddedandtheplateswere coveredandplacedinthedarkforthelast30-minprior tobeingstoppedwith0.2Msulphuricacid.Thesubsequentabsorbances,whicharedirectlyproportionalto theconcentrationofthephosphorlyatedmTORinthe samples,weremeasuredatawavelengthof450nm. TherewerenostandardsusedinthisELISA,thusno standardcurvewascreated.Therefore,theabsorbances relativetomuscleweightwereassessed.Theoverall intra-assaypercentcoefficientofvariationwas7.12%.StatisticalanalysesDataarepresentedinalltablesandthroughoutthetextas meanSD.SerumIGFandinsulinwereanalyzedusing 24[Supplement(CHO,WP)Test(pre,30minpost supp,15minpost-ex,and120minpost-ex)]factorialanalysesofvariance(ANOVA)withrepeatedmeasuresonthe Testfactor.Muscleproteinlevelswereanalyzedusing2 3[Supplement(CHO,WP)Test(pre,15minpost-ex, and120minpost-ex)]factorialANOVAwithrepeated measuresontheTestfactor.Furtheranalysisofthemain effectswasperformedbyseparateone-wayANOVAs.Significantbetween-groupdifferencesweredeterminedusing BonferroniPost-HocTest.Participantcharacteristics, resistanceexercisevolume,and1-RMsfortheangledleg pressandlegextensionexercisesforeachtestingsession wereanalyzedusingapairedsamplet-test.Allstatistical procedureswereperformedusingSPSS16.0softwareand aprobabilitylevelofp<0.05wasadoptedthroughout.ResultsParticipantcharacteristicsandsupplementsideeffectsTherewerenosignificantdifferencesinthebodyweight, restingbloodpressure,orheartratebetweenthetwotestingsessions(datanotshown).Inapost-studyquestionnaireadministeredinablindedmanner,noadverseevents werereportedconcerningthesupplementationorstudy protocol.DietaryanalysisAnalysisofdietaryintake(excludingsupplementation) fortwodaysimmediatelypriortoeachtestingsession revealednodifferences(p>0.05)intotalcaloric,protein,fat,orcarbohydrateintakebetweentestingsession duringthecourseofthestudy(Table2).MusclestrengthandresistanceexercisevolumeTherewerenosignificantdifferencesinthe1-RMvalues betweenlegsateachtestingsessionfortheangledleg press(p=0.35)andlegextension(p=0.42)exercises. The1-RMforthelegpresswas156.0518.86kgforthe rightlegand154.2925.52kgfortheleftleg,andthe1RMforthelegextensionwas44.943.91kgforthe rightlegand44.695.11kgfortheleftleg.Additionally, therewerenosignificantdifferencesintheresistance exercisevolumebetweenthetwotestingsessions.The volumeforlegpresswas4744.5960.4kgforWPandCooke etal JournaloftheInternationalSocietyofSportsNutrition 2011, 8 :18 http://www.jissn.com/content/8/1/18 Page4of9

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4841.61212.9kgforCHO(p=0.89),andthevolume forlegextensionwas1187.5267.6kgforWPand 1285.2180.1kgforCHO(p=0.35).SerumIGF-1andinsulinForIGF-1,nosignificantmaineffectsforSupplement andTestortheSupplementTestinteractionwere observed(p>0.05)(Table3). Forinsulin,nosignificant maineffectforSupplementortheSupplementTest interactionwasobserved(p>0.05);although,asignificantmaineffectforTest(p<0.001)wasobserved.Posthocanalysisshowedsignificantdifferencesbetweenbaseline,30minpost-supplementingestion,15minpostexercise,and120minpost-exercise(Table3).Akt/mTORsignalingintermediatesWhilenosignificantmaineffectsforSupplementorthe SupplementTestinteractionwereobservedforanyof thevariables(p>0.05),asignificantmaineffectforTest (p<0.05)wasobservedforIRS-1(p=0.040),mTOR (p=0.002),p70S6K(p=0.046),and4E-BP1(p=0.001). NosignificantmaineffectsforTestwasobservedforAkt (p=0.359).Subsequentanalysesrevealedasignificant increasefrombaselineinIRS-1at15and120mpostexercise,anincreaseinmTORandp70S6Kat15min post-exercise,andasignificantdecreasein4E-BP1at15 minpost-exercise(Table4).DiscussionInthepresentstudy,wechosetoassesschangesinthe activityofAkt/mTORpathwayintermediatesasmarkers ofMPSinresponsetoresistanceexerciseafteringesting 10gofwheyprotein.Asaresult,weobservedresistance exercisetoeffectivelyactivatesignalingintermediatesof theAkt/mTORpathway.Specifically,wedemonstrated increasedphosphorylationofIRS-1,AKT,andmTOR. Relativetotheirdownstreamtargets,p70S6Kwashyperphosphorylatedat15minpost-exercise,whereas4E-BP1 washypo-phosphorylatedat15minpost-exercise.Conversely,wealsoobservedthatingesting10gofwheyprotein wasunabletoinduceagreaterresponseinsuchkinase phosphorylationwhencompa redtoingestingcarbohydrate.Therefore,ourresultssuggestthatingestionof10g ofwheyprotein(5.25gEAAs)isnodifferentthanan equalamountofcarbohydrateatenhancingtheactivityof systemicandcellularsignalingmarkersindicativeofMPS followingresistanceexercise. ResistanceexerciseandaminoacidseffectivelystimulateMPS[30].Basedonpreviousstudies,therolethat nutrientingestionplaysinactivatingtheAkt/mTOR pathway[15,18-20]isnotcompletelyunderstood,and maylikelyberelatedtotheamountofaminoacidsavailableorwhetherco-ingestedwithcarbohydrate.Previous studieshavedemonstratedthat20gofwheyprotein(8.6 gEAAs)[10]and10gEAAs[26]maximallystimulated MPS,butthatMPSwasalsoincreasedevenatwheyproteindosesof5g(2.2gEAAs)and10g(4.3gEAAs)[10] andanEAAdoseof5g[26].Whensmalleramountsof EAAs(3-6g)wereingested,with[31]andwithout[32] carbohydrate,thepost-exe rciseincreaseinMPSwas similar,butgreaterthanbasalorpost-exercisefasted levels.Inthepresentstudy,ratherthanassessingMPS, ourinterestwasprimarilyfocusedontheextentwith which10gofwheyproteincomprisedof5.25EAAs wouldaffecttheactivityoftheAkt/mTORpathwayafter resistanceexercisewhencomparedtocarbohydratealone Table2Dietaryanalysesperformedtwodays immediatelypriortoeachtestingsessionDietaryVariable WPCHOp-value TotalCalories(kcal/kg/day)31.147.330.435.10.84 Protein(g/kg/day) 0.830.20.860.10.73 Fat(g/kg/day) 0.930.10.960.10.22 Carbohydrate(g/kg/day)4.400.94.221.320.13Dataaremeansstandarddeviations.SIunitconversionfactor:1kcal=4.2 kJ.Valuesexcludesupplementationdose. Table3SerumIGF-1andinsulinlevelsforWPandCHOVariable TimePoint WP CHO p-value IGF-1(ng/ml) Baseline 0.460.4 0.390.3 Supplement(S)=0.64 30minpost-ingestion 0.470.4 0.450.4 Test(T)=0.34 15minpost-exercise 0.440.5 0.390.3 ST=0.89 120minpost-exercise 0.500.4 0.440.3 Insulin( IU/ml) Baseline 12.836.1 14.057.1 Supplement(S)=0.95 30minpost-ingestion 51.9025.3 50.5934.9 Test(T)=0.001 # 15minpost-exercise 23.6014.1 14.628.9 ST=0.76 120minpost-exercise 10.086.5 9.335.5Dataaremeansstandarddeviations. representssignificantdifferencefrombaselineat30minpost-ingestion. representssignificantdifferencefrombaselineat15minpost-exercise. #representssignificantdifferencefrombaselineat120minpost-exercise.Cooke etal JournaloftheInternationalSocietyofSportsNutrition 2011, 8 :18 http://www.jissn.com/content/8/1/18 Page5of9

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andifthisactivitymightalsobesystemicallyaffectedby eitherinsulinorIGF-1.Thereasonforourinterestwas anattempttodiscernifthe5.25gofEAAscontained within10gofwheyprotein,withoutcarbohydrate,was adequatetoactivatetheAkt/mTORcomparedtocarbohydrateinresponsetoasing leboutofresistanceexercise.Ourinterestwasheigh tenedbyapreviousstudyin whichalbuminproteinintakeat10g(4.3gEAAs)significantlyincreasedMPS,andmaximallywhen20g(8.6g EAAs)and40g(16.4gEAAs)wereingested,yetnoneof thethreeconcentrationshadanyaffectontheactivities oftheAkt/mTORpathwayintermediatesS6K1(Thr389), rps6(Ser240/244),oreIF2B (Ser539)at60and240min post-exercise[10].Despitepr eviousevidenceindicating otherwise[10],wewerecurioustodetermineif10gof wheyproteinwouldproduceincreasesinotherkeyAkt/ mTORsignallingintermediatesfollowingresistance exercise. Itisevidentthatacuteresistanceexerciseresultsina significantincreaseintherateofinitiationofprotein synthesiscomparedwithrestingmuscle[33].Itissuggestedthatsignaltransductionpathwayscontroltherate ofinitiationofMPS,andappeartobethekeyfactorsin thehypertrophicprocess[34,35].Ofparticularimportanceisthecomplexmyriadof signalingproteins,with Aktsuggestedtobeakeyregulator.Maximalactivation ofAktoccursthroughphosphorylationofSer473andit appearsthatAktmayhavearelativelyshortperiodof activationafteranacuteboutofresistanceexercise[36]. ResearchintotheregulationofAktsignallingbyexercise hasproducedconflictingresults.Aseriesofstudieshave demonstratedthatcontractileactivityeitherpositivelyor negativelyregulatesAktactivity[15,37-39],whileothers failedtofindanychange[40-42].Inthecurrentstudy, wefoundthatresistanceexerciseandnutrie ntingestion failedtoinduceasignificantchangeinthephosphorylationofAkt. StimulioftheAktpathwayincludeshormonesandmusclecontraction.Insulin[43]andIGF-I[44]bindtotheir respectivemembrane-boundreceptorsandsubsequently activatephosphatidylinosi tol-3kinase(PI-3K),an upstreamactivatorforAktphosphorylation.QuantificationofcirculatingIGF-Ilevelshasyieldedinconsistent results,withlevelsbeingreportedtodecline[45],increase [46],orremainunchanged[47]aftertheonsetofexercise. Furthermore,circulatingIGF-1hasbeenshowntohave nodirecteffectonmusclehypertrophy[48].Inthecurrent study,weobservednochangesinserumIGF-1following theexerciseboutorduetonutrientingestion,therebysuggestinghepatically-derivedIGF-1tohavenoappreciable effectonAktpathwayactivation. Seruminsulinwasincreasedinbothgroups.ItisevidentastowhyinsulinincreasedintheCHOgroupas 10gofcarbohydratewereingested.Inaddition,theWP groupalsounderwentasimilarincreaseininsulininthe absenceofingestedcarbohydrate,whichisinagreement withtheinsulinresponsepreviouslydemonstratedwith 20gofwheyprotein(10gEAAs)[49].TheAkt/mTOR Table4PhosphorylatedlevelsofAkt/mTORpathwayintermediatesforWPandCHOVariable TimePoint WP CHO p-value IRS-1 Baseline 15.689.6 19.526.4 Supplement(S)=0.88 15minpost-exercise 29.046.6 22.2811.2 Test(T)=0.04 # 120minpost-exercise 25.406.0 19.659.2 ST=0.44 Akt Baseline 5.041.9 6.881.1 Supplement(S)=0.21 15minpost-exercise 6.042.6 5.614.1 Test(T)=0.35 120minpost-exercise 4.781.4 4.582.1 ST=0.82 mTOR Baseline 3.340.34 3.620.19 Supplement(S)=0.93 15minpost-exercise 3.750.62 3.660.27 Test(T)=0.002 120minpost-exercise 3.330.19 3.520.28 ST=0.34 P70S6K Baseline 8.513.2 10.413.2 Supplement(S)=0.96 15minpost-exercise 14.146.6 11.182.9 Test(T)=0.04 120minpost-exercise 13.326.1 11.245.0 ST=0.74 4E-BP1 Baseline 4.302.4 5.331.7 Supplement(S)=0.28 15minpost-exercise 2.661.3 2.281.0 Test(T)=0.001 120minpost-exercise 4.071.9# 4.901.8 ST=0.64Dataaremeansstandarddeviations. p70S6K,eIF4E-BP1,AKTandIRS-1areexpressedasU/ml/mg. mTORisexpressedasabsorbanceunitsat450nm/mg. representssignificantdifferencefrombaselineat15minpost-exercise. #representssignificantdifferencefrombaselineat120minpost-exercise.Cooke etal JournaloftheInternationalSocietyofSportsNutrition 2011, 8 :18 http://www.jissn.com/content/8/1/18 Page6of9

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signallingpathwayisactivatedbyinsulin.Insulinbinds withitsreceptorandleadstoanincreaseintyrosine phosphorylationofIRS-1andeventuallymTORactivation.Inthepresentstudy,insulinsignificantlyincreased inbothgroups30minpost-supplementingestionand15 minpost-exercise,whichwasmirroredbysignificant increasesinIRS-1activationat15minpost-exercise. EventhoughAktphosphorylationwasnotsignificantly increased,activationofIRS-1likelycontributedtothe observedincreasesinmTORactivation;however,this activitywasnotpreferentiallycontingenton10gofwhey proteiningestion. mTORisa289kDaserine/threoninekinasedownstreamofAktandstimulatesproteinsynthesisthrough downstreamactivationofp70S6Kand4E-BP1,providinga keypointofconvergenceforbothresistanceexerciseand aminoacids[14].Aminoacidingestionhasbeenshownto significantlyenhancemTORsignalling[25,50].Inthepresentstudy,theacuteboutsofresistanceexercisesignificantlyincreasedmTORandp70S6Kactivationat15min post-exercise,whileamarkeddecreasein4E-BP1activationwasalsoobservedat15minpost-exercise.Whilewe observedmTORactivationtobeenhancedbyresistance exercise,theAkt/mTORpathwaysignallingintermediates weassessedwereunaffectedbytheprovisionof10gof wheyproteincomprisedof5.25gEAAs. Previousworkhassuggestedthataminimalamountof 20gisneededtostimulateMPS[10];however,othershave demonstratedpositiveeffectsutilizingadosageaslowas 6gEAAs[51].IncreasesinMPSfollowingresistanceexercisehavebeenobservedwhenutilizing10gofwheyprotein;however,theproteinsupplementwasco-ingested with21gofcarbohydrate[26].However,ithasrecently beenshownthatapproximately5g(2.2gEAAs)and10g (4.2gEAAs)ofwheyproteinwithoutcarbohydratesignificantlyincreasedMPS37%and56 %,respectively,overbaseline.Inthisstudy,itwasalsoshownthat20g(8.6gEAAs) maximallystimulatedMPSfollowingresistanceexercise [27].Although,ourresultsaresupportedbypreviousdata whichdemonstratedthat20g ofalbuminprotein(8.6g EAAs)enhancedMPSafterresistanceexercise,yethadno effectsonactivationofthemTORpathwayintermediates, S6K1,rps6,andeIF2B post-exercise[27],thedosageused inthecurrentstudy(10gwheyprotein,5.25gEAA)was apparentlyinsufficienttoreachanaminoaciddosagecapableofstimulatingAkt/mTORpathwayactivity. Anumberoflimitationsexistinthecurrentstudy. Firstly,weonlyassessedtherelativechangesinthephosphorlatedlevelsofvariousAkt/mTORpathwayintermediates.Thus,thesecanonlybeusedasmarkersindicativeof MPS.Wedidnotmeasureproteinsynthesisdirectlyand thuscautionneedstobetakenwheninterpretingchanges inphosphorylationstatusofsignalingpathwayintermediatestoimplychangesinhumanMPS,asthisdoesnot alwaysdeterminefunctionalchanges.Secondly,nocontrol wasusedandthusnodirectcomparisonbetweenisoenergeticcarbohydrateandwheyproteinandresistanceexercisecouldbemade.However,previousresearchhas clearlyindicatedthatresistanceexerciserobustlyactivates Akt/mTORsignalling.Thirdly,onlyonedosagewasused (10g)andthusanycomparisonbetweenotherdosages cannotbemadedirectly.Finally,ourstudyfocusedonthe earlypost-exerciserecoveryresponseinsignallingand, therefore,weacknowledgethepossibilitythatlong-term activationofAkt/mTORsignallinganditsdownstream targetssuchasat6,24,or48hrpost-exercisemaybebetterindicatorsofmuscleMPSoverthecourseofaresistancetrainingprogram. Inconclusion,thepresentstudyshowsthatingestionof 10gwheyprotein(5.25gEAAs)priortoasingleboutof lowerbodyresistanceexercisehadnosignificanteffecton activatingsystemicandcellularsignalingintermediatesof theAkt/mTORpathway,otherwiseindicativeofMPS,in untrainedmen.Futureresearchshouldexaminetheeffects ofdoseresponseandtimingofproteiningestionandcomparetheeffectsofvariousforms/fractionsofproteinson post-exercisecellsignallingresponsestoresistance exercise.Acknowledgements Theauthorswouldliketothankthestudyparticipantsfortheirhardwork andwillingnesstodonatebloodandmusclebiopsysamples.Thisworkwas supportedbyGlanbiaNutritionals,TwinFalls,ID,USAandtheExerciseand BiochemicalNutritionLaboratoryatBaylorUniversity. Authordetails1DepartmentofHealth,HumanPerformanceandRecreation,Baylor University,Waco,TX,USA.2InstituteforBiomedicalStudies,BaylorUniversity, Waco,TX,USA.3SchoolsofMedicineandHumanMovementStudies,The UniversityofQueensland,Brisbane,QLD,Australia.4DepartmentofAging andGeriatricResearch,UniversityofFlorida,Gainesville,FL,USA.5Schoolof HumanPerformanceandRecreation,UniversityofSouthernMississippi, Hattiesburg. Authors contributions MCcoordinatedthestudy,carriedouttheexercisesessionsandallanalyses, anddraftedthemanuscript.PLBcarriedouttheexercisesessionsand helpedwithanalysis.TBhelpedwiththebiochemicalanalysisLRhelped withexercisetestingsessionsBShelpedwithexercisesessionsbiochemical analysisGHhelpedwithexercisesessionsbiochemicalanalysis.DSW conceivedthestudy,developedthestudydesign,securedthefundingfor theproject,assistedandprovidedoversightforalldataacquisitionand statisticalanalysis,assistedandprovidedoversightindraftingthe manuscript,andservedasthefacultymentorandprincipalinvestigatorfor theproject.Allauthorsreadandapprovedthefinalmanuscript. Competinginterests Allresearchersinvolvedindependentlycollected,analyzed,andinterpreted theresultsfromthisstudyandhavenofinancialinterestsconcerningthe outcomeofthisinvestigation. Received:14September2011Accepted:8November2011 Published:8November2011Cooke etal JournaloftheInternationalSocietyofSportsNutrition 2011, 8 :18 http://www.jissn.com/content/8/1/18 Page7of9

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