Rev. Bras. Ciênc. Esporte vol.39 número4

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www.rbceonline.org.br

Revista

Brasileira

de

CIÊNCIAS

DO

ESPORTE

ORIGINAL

ARTICLE

Effects

of

supplementation

with

L-glutamine

and

L-alanine

in

the

body

composition

of

rats

submitted

to

resistance

exercise

Audrey

Yule

Coqueiro

∗

_,

_Raquel

_Raizel,

_Thaís

_Menezes

_Hypólito,

_Julio

_Tirapegui

UniversidadedeSãoPaulo,FaculdadedeCiênciasFarmacêuticas,DepartamentodeAlimentoseNutric¸ãoExperimental, SãoPaulo,SP,Brazil

Received26January2016;accepted13June2017 Availableonline9August2017

KEYWORDS Glutamine; Alanine;

Resistanceexercise; Bodycomposition

Abstract We investigatedthe effects ofglutamine and alanine supplementation on body compositionofratssubmittedtoresistanceexercise.Wistarratsweresubmittedtoeight-week ofresistanceexercise,whichconsistedofclimbingaladderwithprogressiveloads(25---100%of bodyweight).Inthelast21daysoftraining,animalsweresupplementedwithL-glutamineand L-alanine,asadipeptideorintheirfreeform(DIP,GLN+ALAandALAgroups),orwater(SED andCTRLgroups).REattenuatedbodyweightgainandlipidcontentsofCTRLgroup(p<0.05 vs.SED)andDIPsupplementationpromotedanincreaseintibialismuscleweight,aswellasin proteincontent(p<0.05vs.CTRL).Takentogether,ourdataindicatedthatresistanceexercise improvesbodycompositionanddipeptidepotentiatedthemusclehypertrophiceffect. ©2017Col´egioBrasileirodeCiˆenciasdoEsporte.PublishedbyElsevierEditoraLtda.Thisisan openaccessarticleundertheCCBY-NC-NDlicense( http://creativecommons.org/licenses/by-nc-nd/4.0/).

PALAVRAS-CHAVE Glutamina;

Alanina;

Exercícioresistido; Composic¸ãocorporal

Efeitosdasuplementac¸ãocomL-glutaminaeL-alaninanacomposic¸ãocorporalderatos submetidosaoexercícioresistido

Resumo Foram investigados os efeitos da suplementac¸ão com glutamina e alanina na composic¸ão corporal de ratos submetidos a exercício resistido. Ratos Wistar foram sub-metidos, durante oito semanas, ao exercício resistido, que consistia em subiruma escada com cargas progressivas (25 a 100% do peso corporal). Nos últimos 21 dias de treina-mento, os animais foram suplementados com L-glutamina e L-alanina, como dipeptídeo ou em sua forma livre (DIP, GLN+ALA ALA e grupos) ou água (grupos SED e CTRL). Exercício resistido atenuou o ganho de peso corporal e conteúdo lipídico do CTRL (p < 0,05 vs. SED) e o DIP promoveu aumento no peso do músculo tibial, bem como no

∗_{Corresponding}_author.

E-mail:audreycoqueiro@hotmail.com(A.Y.Coqueiro). http://dx.doi.org/10.1016/j.rbce.2017.06.003

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teordeproteína(p<0,05vs.CTRL).Osnossosdadosindicamqueoexercícioresistido,melhora acomposic¸ãocorporaledipeptídeopotencializaoefeitohipertróficomuscular.

PALABRASCLAVE

Glutamina; Alanina; Ejerciciode resistencia;

Composicióncorporal

EfectosdelasuplementaciónconL-glutaminayL-alaninaenlacomposicióncorporal deratonessometidosaejercicioderesistencia

Resumen Seinvestigaronlosefectosdelaglutaminaylaalaninaenlacomposición corpo-ralderatonessometidosaejercicioderesistencia.AlgunosratonesWistarfueronsometidos a8semanasdeejercicioderesistencia,queconsistía ensubirunaescalera concargas pro-gresivas(del25al100%delamasacorporal).Enlosúltimos21días,losanimalesrecibieron unsuplementodeL-glutaminayL-alanina,enformadedipéptidooensuformalibre(grupos DIP,GLN+ALAyALA)oagua(gruposSEDyCTRL).Elejercicioderesistenciaredujoelaumento demasacorporalylaconcentracióndelípidosdelCTRL(p<0,05vs.SED).Lasuplementación conDIPpromovióunaumentodepesodelmúsculotibial,asícomoenelcontenidodeproteína (p<0,05frenteaCTRL).Nuestrosresultadosindicanqueelejercicioderesistenciamejorala composicióncorporalyelDIPpotenciaelefectohipertrófico.

Introduction

Body composition is influenced by geneticfactors, physi-calactivity,nutrition,diseasesandothers.Changesinbody composition,suchasraisinginfatandreductionofmuscle tissue,areassociatedwiththedevelopmentofpathologies (Miller et al., 2013). Fat accumulation, characteristic in overweight andobesity, increases the risk of chronic dis-eases, such as diabetes, hypertension and cardiopathies. Similarly,decreased levelsofmusclemass interfere nega-tivelyintherestingmetabolicrate,reducephysicalcapacity andlifequality(Dalyetal.,2014).

Thesedentarylifestyleandhighcaloriefoodintake con-tributetotheincreaseinbodyfat,throughpositiveenergy balance(Ekelundetal.,2014)anddisturbancein homeosta-sisofhormonesinvolvedinappetiteandweightregulation (StieglerandCunliffe,2006).Musclemass gainis compro-mised in asedentary lifestyle due reducedstimulation of muscle protein synthesis (Shahar et al., 2013). Similarly, low-caloriedietspromotereductioninmusclemass, indicat-ingtheimportanceof balanceddietsfor weightreduction programs(Milleretal.,2013).Dietaryprogramsandphysical exercisearethemaininterventionsusedforpreventionand treatmentofmetabolicdisordersrelatedtobody composi-tiondisturbance(Kreideretal.,2010).

Studies demonstratethe effectivenessofaerobic exer-ciseinreducingbodyweightandadiposity(Sillanpaaetal., 2009;Aadland etal., 2014;Miller etal.,2014). However, thereareevidencesthatresistanceexercisepromotes sim-ilareffects(Arnarsonetal.,2014;Villanuevaetal.,2014; Youngetal.,2014),andthemainhypothesisisrelatedtoan increaseinmusclemass,increaseinrestingmetabolicrate andenergyexpenditure(StieglerandCunliffe,2006), reduc-tion in appetite and energy consumption (Rogero et al., 2005), aswell increasedlipid oxidation after an exercise session(DeFeo,2013).

Muscle hypertrophy, induced by resistance exercise, is promoted by myofibrilsreplication intothemuscle fibers. Thismechanismpromotesgrowthofthefibersandincrease in the production of muscle strength (Villanueva et al., 2014). The protein synthesis and muscle hypertrophy are enhanced by supplemental protein and essential amino acids.Thesenutrientsprovidesubstratesforanabolic reac-tions and repair after a physical exercise session.Hence, aminoacids supplementationisaverypopularalternative (Hartmanetal.,2007).

Glutamine,themostabundantaminoacidinthebody,is apotentiallyusefulsupplementforathletes(Cruzatetal., 2010).Besidesitsimportantroleforimmunefunction, glu-tamine has been related to hypertrophy. The hypothesis thatglutamineoptimizeproteinsynthesisandhypertrophy induced by exercise is based on the increase in cell vol-ume(Kreideretal.,2010),aswellasthroughoptimizingthe energystores,preventingproteolysis(Fontanaetal.,2003) andmodulationoftheimmunesystem,reducingtherelease of cytokines,which can impair muscle mass gain (Cruzat etal.,2010).However,theeffectivenessofthehypertrophic potentialofglutamineisnotwellelucidated(Kreideretal., 2010).

Oral or enteral supplementation with glutamine has reduced influence on glutaminemia and tissue glutamine, due to the high metabolism of this nutrient in the intes-tine. Inthis sense,administration withdipeptideL-alanyl L-glutaminehasbeenconsideredanalternativetoimprove glutamine absorption through to intestinal oligopeptide transporter (Pept-1), which is more efficient in transport dipeptidethanfreeaminoacids(Cruzatetal.,2014).

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betweentheinterventionscouldimprovethebody composi-tion,reducinglipidcontent,increasingfat-freemassaswell asmuscleproteincontent.

Materials

and

methods

Animals

Forty adultmaleWistar ratswereprovidedby theanimal houseoftheUniversityofSãoPauloforuseinthisstudy. Ani-malswerehousedthreepercageinacontrolenvironment at 22±2◦_C_and_relative_air _humidity_of₅₅_±_10%, _under_a 12-hlight/12-hdarkcycle(lightson4PM,lightsoff4AM)for aperiodofeightweeks.Animalsweredistributedintofive groups:sedentary(SED)andtrainedcontrol(CTRL);trained and supplemented withL-alanine (ALA), L-glutamineplus L-alanine in their free form (GLN+ALA), and the dipep-tide L-alanyl-L-glutamine (DIP). Rats had free access to waterandstandardchow(NUVILABCR1,NuvitalNutrients, Curitiba,Brazil). Foodintakeand bodyweightwere mea-suredthreetimesperweekandwaterintakewasregistered daily.AllprocedureswereapprovedbytheEthics Commit-teeonAnimalUseoftheUniversityofSãoPaulo(protocol: CEUA/FCF/428).

Resistanceexerciseprotocol

The exercise protocol has been adapted from Hornberger andFarrar(2004)andSchefferetal.(2012).Physicaltraining wasperformed duringeightweeksandconsistedof climb-ingaverticalladder(1.1m×0.18m,2cmgrid,80◦_inclined) withweightaffixedtothebaseofrattail.Setsconsistedof eightladderclimbsandrest of 2min. Thisprocedure was repeatedonceat thefirsttwoweeks(adaptationperiod), withloadequalto5%ofbodyweight(BW).Afterthe adap-tationperiod,theexercisetrainingstartedwithloadof25% ofBW,withthreesets,duringtwoweeks.Subsequent exer-cisesessionswereincreasedtofoursets(50%ofBW)forone week,fivesets(75%ofBW)foroneandahalfweekand,in thelastsessions,animalsperformedsixsetscarrying100% of BW. Each day of exercise was considered one session, conductedevery48h(Fig.1).

Supplementation

Supplementswereadministeredinthelast21daysof exper-iment, diluted to 4% in drinking water and provided ad

libitum.Thesupplementintakewasdailyassessed.Amino acids amount wascalculated based in commercial dipep-tideconcentration(Dipeptiven® solutionconsistsof 20mg ofL-alanyl-L-glutaminedissolvedin100mLofwater,which equals8.2gofL-alanineand13.46gof L-glutamine).Free L-glutamine and free L-alanine were manufactured and supplied by Labsynth (Synth, São Paulo, SP, Brazil) and L-alanyl-L-glutaminewas manufactured by Fresenius Kabi S.A.(BadHomburg,HE,Germany).

Tissuemeasurements

Ratsweresacrificedbydecapitation1hafterthelastsession ofresistance exercise. Epididymal adiposetissue and tib-ialismuscleweresurgicallyexcisedaftersacrifice,weighted andaliquotswerestoredat−80◦_C_for_further_analysis. Mus-clesamples(1g)werehomogenizedin2mLof lysisbuffer andproteinextractioncontaining20mMTris---HCl(pH7.4), 150mMNaCl,2% NonidetP-40, 1mM EDTA(pH 8.00),10% glycerol,20mMsodium fluoride, 30mM sodium pyrophos-phate,0.2% SDS,0.5% sodiumdeoxycholate andultrapure water.Was addedtothebuffer 1mMPMSF, manufactured bySigma---Aldrich.

Forhomogenizationwasusedelectrictypehomogenizer Polytron(IKAT10basic),keepingsampleschilledtoreduce theactivityofproteolyticenzymesandphosphatases.After this process, the samples were centrifuged for 15min at 14,000rpm at −4◦_C _temperature. _The _supernatant _was dilutedtentimesindemineralizedwaterandhomogenized byvortexing.

Protein contentwasquantifiedin tibialis skeletal mus-cleusing BCA Protein Assaykit (Thermo Fisher Scientific, Massachusetts,USA, BCAProtein AssayKit). Briefly, 10␮L ofeach standard curve point and10␮L ofdiluted sample werepipettedintriplicateina96wellsplate,then200␮L ofworkingsolutionwasaddedintoeachwell.Theplatewas incubatedat37◦_C_for₃₀_min_and_absorbance_was_measured in450nmbyMicroplateReader340---750nmUV/vis(Biorad Benchmark--- California,EUA).

Bodyfat

Accordingtoanalyticalstandards ofthe AdolfoLuts Insti-tute (1985), after drying of carcasses, the lipid fraction was extracted with ethyl ether for 48h in extractor of soxhlet(InfratecMultiTE188).Afterextraction,the mate-rialremainedintheincubatorat105◦_C_for_an_hour_and_then

Physical training initiation

Supplementation initiation

Adaptation load 5%

1 sets

Load 25% 3 sets

Load 50% 4 sets

Load 75% 5 sets

Load 100% 5 sets

Sacrifice

1* 2* 3* 4* 5* 6* 7* 8*

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Table1 Foodintake,bodyweightgain,bodyfatandpercentageofepididymaladiposetissueinratssubmittedtoresistance exerciseandsupplementationwithL-glutamineandL-alanine.

SED CTRL ALA GLN+ALA DIP

Foodintake(g/day) 62.08±4.39 54.13±1.63a _54.00_±_1.56 _53.96_±_2.29 _54.13_±_0.91 Bodyweightgain(%) 43.48±2.47 35.89±1.14a _35.91_±_1.88 _35.94_±_1.70 _36.98_±_0.85 Bodyfat(%) 24.83±2.28 14.53±2.41a _19.21_±_2.48 _18.86_±_2.70 _16.41_±_2.93 Epididymaladiposetissue(%) 1.34±0.19 0.94±0.20a _0.69_±_0.18 _1.00_±_0.08 _0.97_±_0.13

SED,sedentarygroupreceivedwater;CTRL,controlgroupreceivedwater;ALA,supplementedwithL-alanine;GLN+ALA,supplemented withL-alanine plusL-glutamine;DIP, supplementedwithL-alanyl-L-glutamine. Supplementsweredilutedindrinking waterina4% solutionandofferedadlibituminthelast21daysoftheexperiment.Eight-weekexerciseprotocolconsistedofclimbingaladderwith progressiveloadincrease.Theweightoftheepididymaladiposetissuewasdeterminedbythepercentageoftotalbodyweight.Data arepresentedasmean±SDM(n=8pergroup).

a_p_<_0.05_vs._SED₍_t_test).

wascooled in the desiccator prior tofinal weighing. The resultwasobtainedbycalculating:

Calculation: _B

−A C

×100=%Lipids

Aistheweightoftheemptyballoon;Bistheweightof theballoonwithsample;CistheCarcassweight.

Statisticalanalysis

To test normality was used Kolmogorov---Smirnov test. To parametric data was used Student’s T-test for compari-son between SED and CTRL groups and One-way ANOVA, with post-test Tukey HSD (Honestly Significant Differ-ences), was used to compare CTRL and supplemented groups. Mann---Whitney test and One-way ANOVA followed byKruskal---Walliswereperformedtoanalyzenonparametric data.Differenceswithpvalues<0.05wereconsidered sta-tisticallysignificant. Analyseswereperformed usingGraph PadPrism5.0anddatawereexpressedasmean±standard deviationofthemean(SDM).

Results

Foodintakeandbodyweight

Food intake was statistically higher in sedentary rats (62.08±4.39g/day) compared to animals submitted to resistance training (54.13±1.63g/day). Resistance exer-cise promoted reduced body weight in CTRL group (35.89±1.14%) compared to sedentary (43.48±2.47%) (p<0.05vs.CTRL).ThedifferencesbetweenSEDandCTRL groups,concerningfoodintakeandbodyweightgain, con-sistof 15% and 21%,respectively. These data supportthe efficacyofprogressiveresistancetraininginreducingfood intakeandbodyweightgain.However,supplementsdidnot influencetheseparameters(Table1).

Bodyfat

AsshowedinTable1,resistanceexercisewasabletoreduce the lipid content of trained animals. Both body fat and

SED CTRL ALA GLN+ALA DIP

0.00 0.05 0.10 0.15 0.20 0.25

*

# #

% o

f BW

Figure2 Percentageoftibialismuscleweightrelativetothe totalfinalWistarratsweightsubmittedtoresistancetraining, exceptSEDgroup.Animalssupplementedfor21 dayswith4% solution,containingdipeptide(DIP),L-glutamineandL-alanine intheirsfreeforms(GLN+ALA)orL-alanine(ALA).SEDandCTRL receivedfilteredwater.Dataarepresentedasmean±SDM(n=8 pergroup).*p<0.05vs.SED(ttest);#_p_<_0.05_vs._CTRL_(ANOVA, Tukey’sHSD).

weightofepididymaladiposetissuewerehigherin seden-taryanimals(24.83±2.28%and1.34±0.19%,respectively), comparedtotrainedgroup(14.53±2.41%and0.94±0.20%) (p<0.05vs.SED).Thesevaluesrepresentdifferenceof71% and 43% amongsedentary and trainedgroups, concerning bodyfat andweightofepididymaladipose tissue, respec-tively.Nodifferencewasfoundamonggroupssupplemented withaminoacids.

Musclemassgainandintramuscularprotein

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SED CTRL ALA GLN+ALA DIP 0

10 20 30 40

*

#

ug

/u

L

Figure3 Intramuscular protein intibialismuscleof Wistar ratssubmittedtoresistancetraining,exceptSEDgroup.Animals were supplemented for 21 days with 4% solution,containing dipeptide(DIP),L-glutamineandL-alanineintheirsfreeforms (GLN+ALA)orL-alanine(ALA).SEDandCTRLreceivedfiltered water. Data are presented as mean±SDM (n=8 per group). *p<0.05vs.SED(ttest);#_p_<_0.05_vs._CTRL,_ALA_and_GLN₊_ALA (ANOVA,Tukey’sHSD).

Discussion

Progressiveresistanceexercisereducedfoodconsumption, body weight gain and lipid content in trained rats, con-firmingtheeffectivenesstochangethebodycomposition. Interestingly, L-glutamine administrationin the free form alongwithL-alanineorasdipeptideimprovedtheREeffects enhancingtibialismuscleweightintrainedanimals.Similar effectwasobservedinintramuscularproteincontent,which wasenhancedbyprogressiveresistanceexercise,however improvedonlybydipeptideadministration.

Intensephysicalexercisesareknowntoreduceappetite and food intake (Kreher and Schwartz, 2012) by increas-ingcytokinesreleaseandactivationofhypothalamicnuclei (Rogeroetal.,2005).Thedecreaseinenergyconsumption and increase in energy expenditure promoted by physi-cal exercise originate the negative energy balance state (Ekelundetal.,2014;StieglerandCunliffe,2006).Themain contributingfactoroftotalenergyexpenditureisthe exer-cise,correspondingto30---40%.Musclemassincreaseinduces high energy demands and contributes to the energy bal-anceandincreasedmetabolicrate,consequently reducing fatmass(StieglerandCunliffe,2006).

Thelipidcontentscanbemodifiedbytheresistance exer-cisethroughvariousmechanisms.Subsequenttoanexercise session,lipidoxidationisoptimizedassubstratetoprovide energy(DeFeo,2013).Theoptimizationofmetabolicrate, energyexpenditureandreducedfoodconsumptionarealso responsiblefor increaseduseof lipidsasenergysubstrate (Stiegler andCunliffe, 2006; Kreher andSchwartz, 2012). Furthermore,studiesindicatethatresistanceexercise pro-moteschangesin theprofileofplasmaticlipoproteinsand these findings were consistent with the reduction of adi-posetissueandimprovementofbodycomposition(Arnarson etal.,2014).

Resistance exercises can promote muscle hypertrophy, which is understood as increasingin the thicknessof the fibres, both due to the myofibrils accumulation, by the contractileproteins(actinandmyosin),asforaccumulation of non-contractilestructures,such aswaterand glycogen

(Buccietal.,2005).Themusclehypertrophycausedby exer-cise occurswhen there aresuccessive periods of positive muscleproteinbalance, which indicates proteinsynthesis higher than degradation (Hartman et al., 2007). Myofib-rilsreplicationwithinthemusclefibres,resultingfromthe hypertrophy,promotesgrowthoffibresandincreasein pro-ductionofmusclestrength(Villanuevaetal.,2014).

In thisstudy, therewasan increase of protein synthe-sis,evidenced byhigherlevelsofintramuscularproteinin animals subjected to progressive resistance exercise and supplementedwith dipeptide.This result,combined with theincrease oftheweightofmuscletissue,alsohigherin theexercisingandsupplementedgroup(GLN+ALAandDIP), correspondstotheaforementionedhypertrophyconcept.

Arnarson et al. (2014) subjected individuals to resis-tance exercise and found increased body weight, due to the increased muscle mass, as well decrease of approxi-mately1%ofbodyfat(Arnarsonetal.,2014).Theresultsof bodyweightdifferfromthepresentstudy,possiblydueto intensityoftheappliedexercise,sinceexhaustiveexercise reducesappetite,foodintakeand,therefore,bodyweight (Rogeroetal.,2005).

Aadland etal.(2014) subjected individualsto predom-inantly aerobic exercises. There was reduction of body weight and body fat of 10% and 16%, respectively. How-ever, the authorsalso observed reductionin muscle mass (Aadlandetal.,2014).Contrarytoaforementionedstudies, Youngetal. (2014) submittedrowers tosessionsof resis-tanceand aerobic exercise.There was reductionof body weightandbodyfat,aswellasanincreaseinmusclemass (Youngetal.,2014).

As a result of the benefits promoted by aerobic and anaerobicexercises, physical trainingcovering both types can provide better results on body composition parame-ters, regarding thereduction in body fat throughaerobic exercise, and potent elevation of the muscle mass, due toresistance exercise (Young et al.,2014). However,the exerciseprotocolappliedinthisstudywaseffectivein pro-motingimprovement in body composition, especially due toitsintensity,whichallowedbothbodyfatreductionand musclemassincrease.Musclehypertrophycausedby exer-ciseisenhancedbysupplementalproteinandaminoacids, sinceitprovidesthe substratesneeded for anabolic reac-tions (Hartman et al., 2007). In addition, protein intake contributestothepositiveproteinbalanceandinhibits pro-teindegradation(Villanuevaetal.,2014).

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thecell andis an anabolic signal for hypertrophy (Cruzat etal.,2010).

Candow et al. (2001) subjected young adults to glu-taminesupplementationandresistanceexercise,however, differentfromtheresults presentedin thisstudy, supple-mentation didnot attenuate muscleprotein degradation, andtherewasnosignificantdifferenceinperformanceand bodycomposition(Candowetal.,2001).Thesefindingscan beexplainedby thesupplementationprotocol used,since thefree glutamine administrationhas reduced effects on glutaminemiaandtissueglutamine.

Supplementation with glutamine through dipeptide L-alanyl L-glutamine or a solution containing these amino acids in their free form, asapplied in the present study, havebeenprioritized,sinceitsabsorptionismoreefficient than absorptionof independentamino acids in free form (Cruzatetal.,2014).Previousstudiescorroboratethis find-ing(Cruzat andTirapegui,2009,2010; Petryetal.,2014). In this study, we observed a higher influence of supple-mentation withdipeptide L-alanyl L-glutamine onmuscle protein,comparedtothegroupssupplementedwitha solu-tion containing L-glutamine and L-alanine in their free forms.

Conclusion

We conclude that eight weeks of progressive resistance training resulted in improvements on body composition evidencedbyreductionofbodyweightandbodyfat,aswell asenhanced intramuscular proteincontent and increased musclemass.Supplementationwiththedipeptide L-alanyl-L-glutaminewasable tooptimizethe hypertrophic effect promotedbyexercise.

Funding

The present workwasfunded by SãoPaulo State Founda-tionforResearchSupport(FAPESP,Process:2012/21087-4). ThestudentshipswereprovidedbyBrazilianNational Coun-cilforScientificandTechnologicalDevelopment(CNPq)and theHigherEducationandTrainingCoordination(CAPES).

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

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