Rev. Bras. Ciênc. Esporte vol.37 número1

www.rbceonline.org.br

Revista

Brasileira

de

CIÊNCIAS

DO

ESPORTE

ORIGINAL

ARTICLE

Influence

of

glutamine

on

the

effect

of

resistance

exercise

on

cardiac

ANP

in

rats

Romeu

Rodrigues

de

Souza

,

Cleomara

Angélica

Vieira

Caldeira

,

Patrícia

Oliva

Carbone

_,

_Eduardo

_Victor

_Pianca

a_{DepartmentofAnatomy,UniversidadeSãoJudasTadeu,SãoPaulo,SP,Brazil}

b_{PostgraduatePrograminAnatomyofDomesticandWildAnimals,FacultyofVeterinaryMedicineandZootechny,Universidade}

deSãoPaulo,SãoPaulo,SP,Brazil

c_{UniversidadeSãoJudasTadeu,SãoPaulo,SP,Brazil}

d_{DepartmentofAnatomy,CentroUniversitárioSant’Anna,SãoPaulo,SP,Brazil}

Received29August2011;accepted7March2013 Availableonline27January2015

KEYWORDS

Glutamine;

Resistanceexercise; ANP;

Rat

Abstract Various nutritional supplements (herbs, vitamins, and micronutrients) improve

responses andadaptationsto resistanceexercise.ANP isaheart hormone thatcontributes tofluid,electrolyteandbloodpressurehomeostasisthroughitsnatriureticandvasodilative actions.In thepresent study,the adaptationofANPinresponsetoresistanceexercise was investigated inrats supplemented with glutamine for fiveweeks. The results showed that supplementationwithglutaminedidnotinfluencethenumberofANPgranulesperatrial cardio-cyteinsedentaryanimals.Inexercised-trainedrats,thenumberanddiameterofthegranules was significantlyhigherincomparisonwiththecontrolgroupandinexercised animals sup-plementedwithglutaminetherewassignificantincreaseinthenumberanddiameterofANP granulescomparedwithcontrols.Altogether,thesedataindicatedthatinresistanceexercise rats, glutaminesignificantlyenhancescardiacANPthusimplicatingthebeneficialeffectsof glutaminesupplementationtotheANPsystem.

©2015ColégioBrasileirodeCiênciasdoEsporte.PublishedbyElsevierEditoraLtda.Allrights reserved.

PALAVRAS-CHAVE

Glutamina; Treinamento resistido; ANP; Rato

InfluênciadaglutaminanosefeitosdotreinamentoresistidonoANPcardíaco emratos

Resumo Váriossuplementos nutricionais(ervas, vitaminasemicronutrientes) melhoramas

respostaseadaptac¸õesao exercícioresistido.OANPéum hormôniocardíaco quecontribui paraahomeostasedelíquidos,eletrólitosecontroledapressãoarterialatravésdesuasac¸ões natriuréticaevasodilatadora.Nopresenteestudo,aadaptac¸ãodoANPemrespostaao treina-mentoresistidofoiinvestigadaemratossuplementadoscomglutaminadurantecincosemanas.

∗_{Correspondingauthor.}

E-mail:souzarrd@uol.com.br(R.R.deSouza). http://dx.doi.org/10.1016/j.rbce.2013.03.001

Osresultados mostraram queasuplementac¸ãocomglutaminanão influenciouonúmerode grânulosdeANPporcardiomiócitoemanimaissedentários.Emratostreinados,onúmeroeo diâmetrodosgrânulosfoisignificativamentemaioremcomparac¸ãocomogrupocontroleenos animaistreinadosesuplementadoscomglutaminahouveumaumentosignificativononúmero ediâmetrodosgrânulosdeANPcomparativamentecomoscontroles.Emconjunto,estesdados indicamque,nosratossubmetidosaotreinamentoresistido,aglutaminaaumentou significa-tivamenteosníveisdeANPcardíaco,mostrandoassim,osefeitosbenéficosdasuplementac¸ão comglutaminaparaosistemadeANP.

©2015ColégioBrasileirodeCiênciasdoEsporte.PublicadoporElsevierEditoraLtda.Todosos direitosreservados.

PALABRASCLAVE

Glutamina; Entrenamiento deresistencia; ANP;

Ratón

Influenciadelaglutaminasobrelosefectosdelentrenamientoderesistenciasobrela ANPcardíacaenratas

Resumen Variossuplementosnutricionales(hierbas,vitaminas y micronutrientes) mejoran

lasrespuestasyadaptacionesalejercicioderesistencia.ElANPesunahormonacardíacaque contribuyealahomeostasisdefluidos,electrolitosydecontroldelapresiónarterialatravés desusaccionesvasodilatadoraynatriurética.Enelpresenteestudio,seinvestigólaadaptación delaANPenrespuestaalentrenamientoderesistenciaenratonessuplementadoscon glutam-inadurantecincosemanas.Losresultadosmostraronquelasuplementacióndeglutaminano influyóenelnúmerodegránulosdeANPporcardiomiocitoenanimalessedentarios.Enratas entrenadas,elnúmeroyeldiámetrodelosgránulosfuesignificativamentemayoren compara-ciónconelgrupocontrolyenlosanimalesentrenadosysuplementadosconglutaminahubo unaumentosignificativoenelnúmeroydiámetrodelosgránulosdeANPencomparacióncon loscontroles.Enconjunto,estosdatosindicanqueenratonessometidosaentrenamientode resistencia,la glutaminaaumentósignificativamenteelnivel deANPcardiacomostrandode estamaneralosefectosbeneficiososdelaadministracióndeglutaminaparaelsistemadeANP. ©2015ColégioBrasileirodeCiênciasdoEsporte.PublicadoporElsevierEditoraLtda.Todoslos derechosreservados.

Introduction

Severalfactorscontributetothebeneficialeffectsof exer-ciseinmaintainingcardiovascularhomeostasis(Gutkowska etal.,2007;Agarwal,2012;RowlandandUnnithan,2013). ANPisahearthormonethatcontributestofluid,electrolyte, andbloodpressurehomeostasisthroughitsnatriureticand vasodilativeactions. It wasshown in humansand animals thatexerciseprovokesincreasedsynthesisofANPthus main-taining adequate levels for the optimal control of blood pressure (Tanaka et al., 1986; Guezennec et al., 1989; Barletta et al., 1998; Ohba et al., 2001; Edwards, 2012; Gutkowskaetal.,2007;Wiesneretal.,2010;Endlichetal., 2011).

Because of the critical functions of hormones, researchershaveinvestigatedvariousmethods toenhance the exercise---endocrine interaction. Forinstance, feeding subjectsbeforeand/orimmediatelyafterresistance exer-cise alters hormone response (Kraemer and Volek, 1998; Kraemer et al., 2007; Gulli et al., 2012). In addition to feeding, variousnutritionalsupplements (herbs,vitamins, and micronutrients) improve responses and adaptations to resistance exercise. Nutritional supplements such as creatine, l-carnitine and l-glutamine positively affect

strength development and resistance exercise recovery (Williamsetal.,2002;Kraemerand Volek,1998; Kraemer

etal.,2007; Rawson andVolek, 2003; Volek andRawson, 2002;Cermaketal.,2012;Waxetal.,2012).Oneofthese nutritionalsupplements,glutaminehavebeenwidely used by athletes (Mero et al., 2009). However, the effects of this supplement in ANP levels in practitioners of resis-tance exercise are not known. Therefore, utilizing the rat as an animal model the following study was under-taken to test the hypothesis that glutamine stimulates the cardiac hormone ANP in chronic resistance exercise practitioners.

Materials

and

methods

Animals

Orall-glutaminesupplementation

An aqueous solution of l-glutamine was given to rats by

gavage (1gkg−1 _{body weight in 3ml saline) 1h before} the exercise session according to Shewchuk et al. (1997) and Lagranha et al. (2004). Glutamine solution was freshlypreparedbeforeadministrationtoavoidglutamine hydrolysis.

Exercisetraining

RatsintheEandEGgroupsweretrainedtoclimba1.1m vertical(80◦ _{incline) ladderwithweightstiedtotheirtail} (Hornberger and Farrar, 2004), five days per week, for five weeks. Each training session consisted of six climbs. The weight carried during each session was progressively increased. Over the course of five weeks, the maximal weightcarriedbytheratswas50%oftheirbodyweight.This loadwasmaintainedthroughout the rest ofthe periodof experiment.TheratsintheSandSGgroupswereplacedon astationarytreadmillfor10mindaily.Thebodyweight(BW) wasmeasuredatthebeginningandattheendofthe exper-iment.HandlingofanimalswasapprovedbytheUniversity Ethics Committee, in accordance with the International Guiding Principles for Biomedical Research involving Ani-mals.

Preparationforelectronmicroscopy

Attheendoftheexperiment,eachanimalwasanesthetized withintraperitonealPentobarbitalsodium(3mg/100gbody weight)andthenkilled.Theanimalswereheparinizedprior tofixation to optimizeperfusion-fixation. The atriawere perfused through the left and right ventricles at a cons-tant pressure of 80mmHg, using 0.1M cacodylate buffer (3min) followed by 2.5% glutaraldehyde solution diluted in cacodylate buffer. Next, the heart was isolated and weighed. The right atrium was isolated and divided into slicesapproximately3mm wideand5mmlong.These tis-sue slices were post-fixed in osmium tetroxide in sodium cacodylate buffer for 1h. The tissue was dehydrated in graded alcohols, embedded in Epon resin, and sectioned so that the cardiocytes were cut in longitudinal section. Thin sections for transmission electron microscopy were stainedwithuranylacetate,andleadcitrate(Mifuneetal., 2004).

Ultrastructuralmorphometry

Tworandomlychosenblocksfromeachatrium,inwhichthe cardiocyteswerecut inlongitudinalsectionwereusedfor quantitativeanalysis.The ultrathin sectionswereplaced onacopper grid and10 randomlychosen fields perblock wereselectedformicrographs takenwithaJeol transmis-sionelectronmicroscope.Thenumberandsizesofsecretory granuleswereobtainedaccordingtothemethodofCantin etal. (1979). Fiveelectron micrographs per animal, cho-senbysystematicrandomsamplingofsquaresweretaken atafinalmagnificationof 7500×andthenumberof gran-ules/cardiocytewasdetermined.Diametersofallgranules

*

_*

2.0

1.5

1.0

0.5

0.0

S SG E EG

Groups

Heart weight (g)

Figure1 HeartweightofS,SG,EandEGrats.*Significantvs.

SandSG.

presentineachfieldweredeterminedinanotherfive micro-graphs per animal at a final magnification of 15,000×. In bothcases,acomputerizedprogram(AxioVision,Zeiss)was used.

Statisticalanalysis

Allresultsaremeans±SE.Datawasperformedusing one-wayanalysisofvariance(ANOVA)andmultiplecomparison procedurewasperformedusingTukey’stestwithp<0.05as thelevelofsignificance.

Results

Heartweight

AsshowninFig.1,nodifferenceinheartweightwasseen betweenthegroupsS(1.1±0.2g)andSG(1.1±0.1g)and betweenthegroupsE(1.5±0.2g)andEG(1.5±0.1g). How-ever,inEandEGtheheartshowedsignificanthypertrophy comparedtoS(p<0.05).

Ultrastructure

The ANP granules weremainly located in the perinuclear regionandwerevariableinnumber(Fig.1)andsize(Fig.2). ThenumberofgranuleswasincreasedinEandEGcompared withSandSGrats(Figs.2and3).

Ultrastructuralmorphometry

N

N

_N

N

a

b

d

c

Figure2 ElectronmicrographsoftherightatrialcardiocytesinS(a),SG(b),E(c)andEG(d)rats.Inatrialcardiocytes, ANP-granules(arrows)arevariableinnumberandaremainlylocatedintheperinuclearregion.ThenumberofgranulesinE(c)andEG (d)ratsishigherthaninS(a)andSG(b).Bar:2␮m.N---Nucleus.

thenumberofgranules/cardiocytebetweenSGandS rats (p>0.05).Thediameterofgranuleswassignificantlyhigher inEG(320±24nm)andE(260±10nm)ratscomparedtoS rats(212±12nm) andSG(218±8nm) rats (p<0.05).The diameterofgranuleswassignificantlyhigherinEGthaninE rats(p<0.05).

Discussion

Therearethreemajorfindingsinthiswork.First,glutamine supplementationforfiveweekshadnoinfluenceonthe lev-elsofANPinatrialcardiocytes.Second,chronicresistance exerciseincreased significantlythe levelsof ANPin atrial cardiocytes and third, glutamine enhances the effects of resistance exercise on the levels of ANP in atrial cardio-cytes.

Glutamine is an important energy source (nitrogen and carbon) for synthesis of other molecules such as nucleotides,adenosinetriphosphate(ATP)andotheramino acids(Fontanaetal.,2003).Glutamineisalsonecessaryfor theabsorptionoffluidsandelectrolytesandforthe regula-tionofnitrogenbalance(Walshetal.,1998).Inthepresent study,animalsreceivingglutaminedidnotexhibitany sig-nificantenhancementofcardiacANPlevelscomparedwith sedentarycontrols,indicatingthatglutaminehadnoeffect onthenumberofANPgranulesbycardiocytes.

Thepresentstudydemonstratedasignificantincreasein thenumberof cardiacANPgranules inratswith exercise-trainingcomparedtotheSgroup.Thisresultisinagreement withthatofGutkowskaetal.(2007)inshowingthatchronic exerciseaugmentedANPexpression intheright atrium.It is possible that resistance exercise promotes an increase in levels of ANP in cardiomyocytes due to the necessity

of greater release of this hormone to the plasma during exercise.According toGutkowska etal. (2007) the bene-ficial effectsof exercise may bedue, at least in part, to activationofcardiacoxytocinpeptidereceptorsand subse-quentenhancementofANPsynthesisandreleasealthough adirecteffectofexerciseontheANPcannotbeexcluded. Thisconclusionemergedfromstudiesshowingthat activa-tionofcardiacoxytocinreceptoriscoupledwithANPrelease (Gutkoswskaetal.,1997).ThephysiologicalactionofANP isconveyedbybindingtoparticulateGC-coupledcell sur-face functional receptor GC-A which activation promotes theintracellular generation of cGMP(Potter etal., 2006; Gutkowskaetal.,2007).

N

N

N

_N

a

b

d

c

Figure3 ElectronmicrographsoftherightatrialcardiocytesinS(a),SG(b),E(c)andEG(d)rats.Itcanbeseenthatinallgroups ANP-granules(arrows)arevariableinsize.InEandEGrats,thesizesofthegranulesarehigherthaninSandSG.Bar:1␮m.N

---Nucleus.

*

**

S SG E EG

Groups

80

60

40

20

0

No. of granules/cardiocyte

Figure 4 Number of ANP-granules/cardiocyte in the right atriafromS,SG,EandEGrats.*Significantvs.S,SGandEG groups(p<0.05).**Significantvs.SandSGgroups(p<0.05).

*

**

S SG E EG

Groups

400

300

200

100

0

Diameter (nm)

Conclusion

GlutaminesupplementationdidnotimproveANPproduction by the cardiocytes but when associated with resistance-training it potentiates the increased cardiac ANP levels promotedbyexercise.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

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