The Combined Intervention with Germinated Vigna radiata and Aerobic Interval Training Protocol Is

22 Pages·2017·2.42 MB·English

nutrients
Article
The Combined Intervention with Germinated
Vigna radiata and Aerobic Interval Training Protocol
Is an Effective Strategy for the Treatment of
Non-Alcoholic Fatty Liver Disease (NAFLD) and
Other Alterations Related to the Metabolic Syndrome
in Zucker Rats
GaryfalliaKapravelou1,RosarioMartínez1,ElenaNebot1 ID,MaríaLópez-Jurado1,
PilarAranda1,FranciscoArrebola2 ID,SamuelCantarero3,MilagrosGalisteo4
andJesusM.Porres1,*
1 DepartmentofPhysiology,InstituteofNutritionandFoodTechnology,CentreforBiomedicalResearch,
InstituteofHealthandSport,UniversityofGranada,18071Granada,Spain;[email protected](G.K.);
[email protected](R.M.);[email protected](E.N.);[email protected](M.L.-J.);[email protected](P.A.)
2 DepartmentofHistology,InstituteofNeurosciences,CentreforBiomedicalResearch,UniversityofGranada,
18071Granada,Spain;[email protected]
3 CentrodeInstrumentaciónCientífica,UniversityofGranada,CampusUniversitariodeFuentenuevas/n,
18071Granada,Spain;[email protected]
4 DepartmentofPharmacology,SchoolofPharmacy,UniversityofGranada,CampusUniversitariodeCartuja
s/n,18071Granada,Spain;[email protected]
* Correspondence:[email protected];Tel.:[phone]
Received:24April2017;Accepted:14July2017;Published:19July2017
Abstract: Metabolicsyndrome(MetS)isagroupofrelatedmetabolicalterationsthatincreasetherisk
ofdevelopingnon-alcoholicfattyliverdisease(NAFLD).Severallifestyleinterventionsbasedon
dietarytreatmentwithfunctionalingredientsandphysicalactivityarebeingstudiedasalternative
orreinforcementtreatmentstothepharmacologicalonesactuallyinuse. Inthepresentexperiment,
the combined treatment with mung bean (Vigna radiata), a widely used legume with promising
nutritionalandhealthbenefitsthatwasincludedintheexperimentaldietasrawor4day-germinated
seed flour, and aerobic interval training protocol (65–85% VO max) has been tested in lean and
2
obeseZuckerratsfollowinga2×2×2(2phenotypes,2dietaryinterventions,2lifestyles)factorial
ANOVA(AnalysisofVariance)statisticalanalysis.GerminationofV.radiataoveraperiodoffourdays
originatedasignificantproteinhydrolysisleadingtotheappearanceoflowmolecularweightpeptides.
The combination of 4 day-germinated V. radiata and aerobic interval training was more efficient
compared to raw V. radiata at improving the aerobic capacity and physical performance, hepatic
histologyandfunctionality,andplasmalipidparametersaswellasrevertingtheinsulinresistance
characteristicoftheobeseZuckerratmodel. Inconclusion,thejointinterventionwithlegumesprouts
andaerobicintervaltrainingprotocolisanefficienttreatmenttoimprovethealterationsofglucose
andlipidmetabolismaswellashepatichistologyandfunctionalityrelatedtothedevelopmentof
NAFLDandtheMetS.
Keywords: non-alcoholic fatty liver disease; metabolic syndrome; Vigna radiata; legume protein
hydrolyzate;aerobicintervaltraining;diet;germination;obesity;exercise;liverhistology
Nutrients2017,9,774;doi:10.3390/nu9070774 www.mdpi.com/journal/nutrients
Nutrients2017,9,774 2of22
1. Introduction
Metabolicsyndrome(MetS)isagroupofmetabolicalterationscharacterizedbycentralobesity,
dyslipidemia,elevatedplasmaglucose,insulinresistance,elevatedbloodpressure,pro-thromboticand
pro-inflammatorystate[1]. PatientswithMetSarealsomoresusceptibletodeveloptype2diabetes
mellitus and suffer increased risk of developing cardiovascular disease. In addition, their hepatic
morphologyandfunctionalitycanbeadverselyaffectedleadingtothedevelopmentofNonAlcoholic
FattyLiverDisease(NAFLD).Suchdiseasehasbeenhistoricallyregardedasthelivermanifestationof
MetSalthoughrecentstudieshaveconcludedthatisoneofthemajorriskfactorsthatstronglydetermines
itsprogression[2,3]. HepaticmorphologyandfunctionalityinNAFLDisadverselyaffectedandcan
becategorizedinfourdifferentstagesstartingfromasimplesteatosis(liverfatdepositionandmild
inflammation),thedevelopmentofNonalcoholicsteatohepatitis(NASH)thatincludessteatosisplus
inflammationandhepatocyte“ballooning”,theappearanceoffibrosisand,insomecases,canleadtothe
laststageofhepatocellularcarcinoma(HCC)[4].AlthoughtheexactmechanismsleadingtoNAFLDare
notyetcompletelyunderstood,insulinresistance,hormonessecretedfromtheadiposetissue,nutritional
factors,gutmicrobiotaandgeneticandepigeneticfactorshavebeendescribedtoplayamajorrole[5].
The development of strategies for the prevention and treatment of MetS includes changes in
lifestyle such as caloric restriction, low fat and low glycemic index diets, consumption of foods
rich inbeneficial bioactiveingredients, andregularphysical activity[1,6]. In this regard, legumes
exhibit a variety of health effects related to their antioxidant, hypoglycemic, and hypolipidemic
properties [7,8] due to the specific characteristics of legume proteins, carbohydrates and lipids,
and also from several non-nutritional compounds like polyphenols, phytic acid or α-galactoside
oligosaccharides. Germination and fermentation are alternative biotechnological treatments that
render protein preparations with important degree of hydrolysis and bioactive compounds with
beneficialhealthactions[9,10]. Theresultingbioactivepeptidesmayactaspotentialphysiological
modulators of metabolism, given that they inhibit the activity of angiotensin converting enzyme
and show antioxidant and bile acid-binding properties [11], thus showing promising potential as
functionalingredients. Vignaradiataisawidelyusedlegumeforhumanconsumptionthatexhibits
aninterestingcompositionofnutrientsandbioactivecompounds[12,13]. Suchnutritivevaluecan
besignificantlyimprovedbytechnologicalprocessinglikesprouting[14]. Infact,V.radiatasprouts
areusuallycommercializedtotakepartinnumeroushealthydishesthatarebecomingincreasingly
popularforthegeneralconsumer.
TheeffectsofdifferenttypesofexerciseonthetreatmentofMetShavebeenstudiedconcerning
the cardiovascular risk, and high-intensity aerobic interval training has been reported to be more
effectiveatreducingsuchriskinratswithMetSthanmoderate-intensitycontinuoustrainingdid[15].
Withregardtolivermetabolism,severalauthorshavestudiedthebeneficialeffectsofmoderateor
vigorousintensityexerciseondifferentaspectsofNAFLD[6,16].
Differentinvivoexperimentalmodelshavebeenusedtotestthebeneficialeffectsofdietand
exercise on several parameters of MetS. Among them, the obese Zucker rat is a widely accepted
animalexperimentalmodeltostudythemultifactorialeffectsofdietandexerciseonMetSassociated
conditions. This rat strain is known to present a genetic defect in leptin receptor that causes the
development of hyperphagia leading to obese phenotype [17], and shares many similarities with
humansaffectedbyMetS.[18]. Wehavepreviouslydemonstratedthebeneficialeffectsofanaerobic
interval training (AIT) protocol on NAFLD, insulin resistance and other features related to the
developmentofMetSintheobeseZuckerratmodel[6].
The aim of this study was to assess the effects on glucose and lipid metabolism parameters,
aswellasonliverhistologyandfunctionality,exhibitedbyanoveljointinterventioncombiningthe
consumptionof4day-germinatedV.radiataasdietarysourceofbioactivecompoundsandhydrolyzed
protein obtained using natural methods, together with an AIT protocol. Such joint intervention
(dietplusexercise)intheabovementionedexperimentalanimalmodeloftheobeseZuckerratwasan
efficientstrategytoimprovetheNAFLDconditionrelatedtothedevelopmentofMetS.
Nutrients2017,9,774 3of22
2. MaterialsandMethods
2.1. VignaradiataSeedsandGerminationProcess
VignaradiataL.(LaAsturiana,León)seedswereobtainedfromacommercialestablishmentand
powdered(0.18mmsieve)forsampleanalysis. Sproutingwasdoneinseedspreviouslysterilized
by immersion in sodium hypochlorite for 3 min and washed out with sterilized, type-2 water to
eliminateanytracesofsodiumhypochlorite. Theseedswerethenleftsoakinginwaterduring8h
anddistributedintraysoversheetsoffilterpaperwheretheywereleftcoveredindarkness,at30◦C,
during4days. Afterthisperiod,sproutedV.radiataseedswerecollected,milledandlyophilized. Once
lyophilized,thesproutsweremilledagaintoafinepowder(0.18mmsieve)forsampleanalysisand
dietpreparation.
2.2. CompositionAnalysisofRawandGerminatedV.radiataFloursandExperimentalDiets
Moisturecontentoftheraw(RVR)and4day-germinated(GVR)V.radiatafloursandexperimental
dietswasdeterminedbydryingtoconstantweightinanovenat105±1◦C.TotalNwasdetermined
accordingtoKjeldahl’smethod. CrudeproteinwascalculatedasN×6.25. InsolubleNandsoluble
proteinandnon-proteinNofthelegumefloursweremeasuredasdescribedbyKapravelouetal.[7].
Samples(0.5g)werethriceextractedwith10mLof0.02NNaOHduring45minandthethreeextracts
pooled. Insolublematerialwasremovedbycentrifugationat3000rpmfor20min. Thesupernatant
wasmixedwith20mLof30%trichloroaceticacidandthemixturestirredfor15minat4◦C.Protein
was removed by centrifugation at 3000 rpm for 15 min. Total N was measured in the insoluble
material(insolubleN),proteinpellet(proteinN)andsupernatant(nonproteinN).Totalfatcontent
wasdeterminedbygravimetryoftheetherextractafteracidhydrolysisofthesample. Ashcontent
wasmeasuredbycalcinationat450◦Ctoaconstantweight.
2.3. SodiumDodecylSulfate-PolyacrylamideGelElectrophoresis(SDS-PAGE)
WasdoneaccordingtothemethodofLaemmli[19]. Thefinalconcentrationofacrylamideinthe
runninggelwas14%. EqualamountsofN(2.0µg)wereloadedineachlane. Thegelswerefixedand
stainedwithCoomassiebrilliantblueR-250(EZBlue, Sigma, St. Louis, MO,USA).Themixtureof
molecularweightmarkersranged8–220KDa(ColorBurstTM,Sigma,St. Louis,MO,USA).
2.4. MassSpectrophotometry
AnUltraPerformanceLiquidChromatography(UPLC)(AcquityHClass,WatersCorporation,
Manchester, UK) coupled by quadrupole-time-of-flight mass spectrometry (Synap G2, Waters
Corporation, Manchester, UK) was employed for all high-resolution mass spectrometry analysis.
Priortomassspectrometryanalysis,allpowderedsamples(100mg)wereextractedwith10mLof
acidifiedwater(pH2),andfilteredthrough0.22µmnylondiskfilters(Millipore,Billerica,MA,USA).
10µLofthefinalsolutionwereinjectedintothechromatograph. Analyticalseparationofpeptides
andproteinswasperformedonanAcquityBEH300C4analyticalcolumn(100mm×2.1mminternal
diameter, 1.7 µm; Waters Corporation, Manchester, UK). A mobile phase consisting in a gradient
programcombiningdeionizedwaterwith0.1%ofacidformicassolventAandacetonitrilewith0.1%
ofacidformicassolventBwasused. Theinitialconditionswere95%Aand5%B.Alineargradient
wasthenestablishedtoreach95%(v/v)ofBat15min. Totalruntimewas25minandpost-delaytime
5min. Mobilephaseflowratewas0.4mL/min.
Afterchromatographicseparation,ahigh-resolutionmassspectrometryanalysiswascarriedout
inpositiveelectrosprayionization(ESI+ve). Thegasusedfordesolvation(800L/h)andCone(25L/h)
washigh-puritynitrogen. Spectrawererecordedoverthemass/charge(m/z)range50–1200.
Nutrients2017,9,774 4of22
2.5. AnimalsandExperimentalDesign
Forty male obese homozygous (fa/fa) (O) and 40 lean heterozygous (fa/+) (L) Zucker rats
(aged5weeks) with an initial mean body weight of 137 ± 2.5 and 120 ± 1.6 g, respectively, were
allocatedtoeightdifferentexperimentalgroups(fourobeseandfourleangroups,n=10ratseach)
within2differentexperimentsRVRandGVR(rawvs.4day-germinatedV.radiatadietaryinterventions).
Two of the experimental groups in each experiment (an obese and a lean one) performed aerobic
intervalexercise(E)accordingtoanestablishedtrainingprotocol(OE,LE)whilethetworemaining
wereconsideredassedentary(S)groups(OS,LS).Theexperimentlastedfor8weeks,duringwhichthe
animalswerehousedinawellventilated,thermostaticallycontrolledroom(21±2◦C).Areversed12:12
light/darkcyclewasimplementedsotheanimalswouldperformthetrainingprotocolindarkness.
Throughoutthetrial,animalshadfreeaccesstotype2waterandconsumedtheexperimentaldiet
(Table1)adlibitum. Foodintakewasrecordeddailywhereasbodyweightwasmeasuredonceaweek.
Attheendofexperimentalperiod,aglucosetolerancetestwasperformed48hafterthelasttraining
sessionbyoralglucoseoverload. Theanimalswereallowedtorecoverfor48hpriorbeingfastedfora
further8h,anesthetizedwithxylazine/ketamineandsacrificed. Bloodwascollectedbypunctureof
theabdominalaorta(withheparinasanticoagulant)andcentrifugedat1458×gfor15mintoseparate
plasmathatwassubsequentlyfrozeninliquidnitrogenandstoredat−80◦C.Theliverwasextracted,
weighed,photographedformacroscopicstudies,dividedintovariousportionsandimmediatelyfrozen
inliquidnitrogenandstoredat−80◦C.AllexperimentswereundertakenaccordingtoDirectional
GuidesRelatedtoAnimalHousingandCare[20]andallprocedureswereapprovedbytheAnimal
ExperimentationEthicsCommitteeoftheUniversityofGranada,Spain(ProjectReferenceAGR4658).
2.6. ExperimentalDiet
TheexperimentaldietswereformulatedfollowingtheguidelinesoftheAmericanInstituteof
Nutrition(AIN-93M)[21],inordertomeetthenutritionalrecommendationsofadultrats[22],with
whey(30%)andRVRorGVR(70%)asproteinsources,toreacha12%proteinlevel(Table1). A0.5%
methioninewasaddedtofulfilthespecificneedsthatthelaboratoryratshaverespectingthisspecific
aminoacid. Thepercentageofdietaryfibreaddedinthedietwasestablishedat10%andwasprovided
mainlyfromRVRorGVRflourwhileasmallamountofcellulosewasaddedinordertoachievethe
10%level. Thepercentageoffatwassetat4%,usingsunfloweroilwithoutanyadditionofsaturated
fatorothercholesterolsource.
Table1.Compositionofrawand4day-germinatedV.radiatafloursandexperimentaldietformulation.
V.radiataFlour RVR GVR
Moisture(g/kg) 83.0 88.4
TotalN(g/kgDM) 34.4 42.0
InsolubleN(%) 12.9 9.4
SolubleProteinN(%) 81.6 50.9
SolubleNonProteinN(%) 5.5 39.7
Totalfat(g/Kg) 17.0 14.6
Ash(g/kgDM) 37.1 43.4
DietFormulation(g/kg) RVR GVR
RawV.radiata 367 -
GerminatedV.radiata - 333
WheyProtein 56 56
Sucrose 100 100
Cellulose 11 21
Methionine 5 5
SunflowerOil 40 40
Mineralpremix 35 35
Vitaminpremix 10 10
Cholinebitartrate 2.5 2.5
Starch 427 411
RVR,RawV.radiata;GVR,4day-germinatedV.radiata;DM,drymatter.
Nutrients2017,9,774 5of22
2.7. ExerciseProtocol
TheexercisegroupsfollowedaprotocolofAITfivedaysaweekduringtheeightweeksofthe
experimentalperiod. ThetrainingprotocolhasbeenpreviouslydescribedbyKapravelouetal.[6],and
wasperformedinamotorizedtreadmillspeciallydesignedforrats(PanlabTreadmillsforfiverats,LE
8710R).Allsessionswereperformedduringthedarkcycleoftheanimals(activeperiod). Oneweek
before the start of the study, the animals were adapted to the training procedures through a low
intensityrunningprotocoleverydayfor20mininthetreadmillat18m/min. Therunningsessionsof
1hstartedwitha10minwarmupat40%VO max,andconsistedofsuccessive4minexerciseperiods
2
at65–80%ofVO max,followedby3minrecoveryperiodsat50–65%ofVO max. Theintensitiesand
2 2
lengthofthetrainingweregraduallyincrementedeveryweek(Table2). Toestablishthevelocitythat
wouldcorrespondtotheVO maxofeachrat,amaximalincrementaltestwasperformedatthestart
2
ofthestudy. Afinalincrementaltestwasperformed96hpriortheendofthestudytotestthemaximal
aerobiccapacityandphysicalperformanceachievedbytheanimalsasaresultoftheintervention.
ThemaximalincrementaltestwascarriedoutfollowingtheprotocoldescribedbyWisløffetal.[23]
andClementeetal.[24]withslightmodifications. Lactateconcentrationsweremeasuredattheendof
theincrementaltestinbloodobtainedfromtheanimals’tail(LactatePro,Arkray,TheNetherlands).
Table2.Detailsoftheaerobicintervaltraining(AIT)protocol.
Week(5Days/Week) WorkTime(min/Day) %VO max
2
50%→3min
1 45(cid:48)
65%→4min
55%→3min
2 50(cid:48)
70%→4min
60%→3min
3 50(cid:48)
75%→4min
60%→3min
4 55(cid:48)
75%→4min
65%→3min
5–8 60(cid:48)
80%→4min
VO2max,maximaloxygenconsumption.
2.8. Blood,PlasmaandLiverBiochemicalAnalysis
Fasting blood glucose and glucose tolerance test were performed by oral glucose overload
followingtheprotocoldescribedbyPrietoetal.[25]. Bloodglucoseconcentrationfromtheanimals’
tailwasrecordedatperiods0,15,30,90and120minaftertheglucoseoverloadingestion(BREEZE®2,
BayerHealthcareAG,Leverkusen,Germany),andtheareaunderthecurve(AUC)wasdetermined.
Biochemicalparametersoflipidmetabolism,andliverfunctionalityweremeasuredinplasmausing
aShenzhenMindrayBS-200ChemistryAnalyzer(ShenzhenMindrayBio-MedicalElectronics,CO.,
LTD. Nanshan, Shenzhen, China) at the Bioanalysis Unit of the Scientific Instrumentation Centre
(BiomedicalResearchPark,UniversityofGranada,Granda,Spain). Aportionofliverwaslyophilized
inordertodeterminethepercentageofwater. Hepaticlipidswereextractedfromthefreeze-driedliver
portionusingthemethoddescribedbyFolchetal.[26]withslightmodifications[7]. Theextracted
lipidsweredissolvedin1mLof96%hexanetomeasuretriglycerideandtotal-cholesterolcontent
(Spinreact,S.A.,Girona,Spain).
2.9. MacroscropicandMicroscopicLiverStudy
Liverareaofthemacroscopicimagewasestimatedinalltheliverimagesoftheeightexperimental
groupsassayedbymorphometricstudyusingthesoftwareImageProPlus6.0(MediaCybernetics,Inc.,
Rockville,MD,USA).Aportionofliverwasfixedin10%phosphate-bufferedformalin,dehydratedin
ethanol,embeddedinparaffin,andsectionedforhistologicalexaminationusinghematoxylin-eosin
Nutrients2017,9,774 6of22
(HE), and Masson’s trichrome (MT) staining for general microscopy morphology and fibrosis
development,respectively. Fourdifferentpreparationsofeachstainingwereanalyzedforeachanimal,
and10animalswereevaluatedineachexperimentalgroup(n=40samplespergroup). Histological
alterations were evaluated according to the following grading score: -, non-existent; +, mild; ++,
mild/moderate;+++,moderate;++++,abundant;+++++,severe.
2.10. LiverProteinExpressionAnalyses
Liver samples were homogenized in 20 mM Tris HCl buffer containing 0.1% Igepal, 100 mM
Ethyleneglycol-bis(2-aminoethylether)-N,N,N(cid:48),N(cid:48)-tetraaceticacid,andacocktailofproteaseinhibitors
(Sigma,St. Louis,MO,USA).Liverhomogenateswerecentrifugedat13,000×gfor45min,at4◦Cand
supernatantswerealiquotedandstoredat−80◦C,untilfurtheruseforwesternblotanalysis. Protein
concentrationwasmeasuredbythemethodofLowryetal.[27]. Equalamountsoftotalproteinfor
eachsamplewereloadedperwell,subjectedto12%SDS-PAGE,andelectrophoreticallytransferredto
nitrocellulosemembranes(Schleicher&Schuell,Dassel,Germany)bywettransferat100Vfor1h
using a Mini Trans-Blot cell system (Bio-Rad Laboratories, Hercules, CA, USA). Membranes were
blockedusing5%non-fatdrypoweredmilkdissolvedinTris-BufferedsalineTween-20(TBST)for
90minatroomtemperature(RT).Theprimaryantibodiesfor5(cid:48)-AMP-activatedproteinkinase(AMPK)
and phosphorylated-AMPK (pAMPK) (Cell Signaling Technology, Inc., Danvers, MA, USA) were
usedaccordingtothemanufacturerrecommendeddilutions(1:1000)andwereincubatedovernight
at4◦C.Themembraneswerethenwashedthreetimesfor10minwithTBST,beforeincubationfor
2 h at RT with secondary peroxidase conjugated goat anti-rabbit antibody (Sigma, St. Louis, MO,
USA)dilutedat1:2000in5%skim-milkpowder–TBST.Membraneswerewashedasbefore,andthe
boundantibodieswerevisualizedbyanECLProsystem(PerkinElmer,Boston,MA,USA)usinga
FujifilmLuminescentAnalyzerLAS-4000miniSystem(Fujifilm,Tokyo,Japan). pAMPKexpression
wasdeterminedinrelationtoAMPKexpression. Resultswereexpressedinrelativedensityunits.
2.11. StatisticalAnalyses
Time-repeatedmeasurementanalysiswasappliedtoweeklyfoodintakeandbodyweightgain
data as well as to blood glucose content after an oral glucose overload in order to analyze within
subjecteffects(time)orwithingroupeffects(phenotypeorAITprotocol)ontheaboveparameters.
The effect of phenotype (lean vs. obese corresponding to a fa/+ or fa/fa genotype), AIT protocol,
and dietary intervention with RVR or GVR on final body weight, aerobic capacity and physical
performance, plasma and liver biochemical parameters, hepatic antioxidant enzyme activity, and
proteinexpressionwasanalyzedby2×2×2factorialANOVAwithphenotype(leanvs. obese),AIT
protocol(sedentaryvs. exercise)anddietaryintervention(RVRvs. GVR)asmaintreatments. Theuse
ofa2×2×2factorialANOVAisbasedonthepotentialinteractionsamongthethreeinterventions
assayed(phenotype,exercise,anddiet)beingsignificantinourstatisticalmodelinadditiontosingle
effectsofexerciseordietarytreatmentineachofthetwophenotypestested. Toreinforcethepotential
integrativestrengthofthestatisticalmodelimplemented,theR2statistichasbeenincludedinthetables
asameasureofgoodnessoffitofthemodel,giventhatthecoefficientofdeterminationindicatesthe
proportionofvariabilityinadatasetthatcanbeaccountedbythestatisticalmodel. Resultsaregiven
asmeanvaluesandpooledstandarderrorofthemean. Bonferroni’stestwasusedtodetectdifferences
betweentreatmentmeans. TheanalyseswereperformedwithSAS,version9.0(SASInstituteInc.,
Cary,NC,USA),andthelevelofsignificancewassetatp<0.05.
3. Results
3.1. ChemicalAnalysis
Thecompositionintotal,insoluble,solubleproteinandnon-proteinN,fat,andashcontentofRVR
andGVRflours,togetherwiththeformulationandproximatecompositionofthedifferentexperimental
Nutrients 2017, 9, 774 7 of 21
3. Results
3.1. Chemical Analysis
The composition in total, insoluble, soluble protein and non-protein N, fat, and ash content of
Nutrients2017,9,774 7of22
RVR and GVR flours, together with the formulation and proximate composition of the different
experimental diets is presented in Table 1. With regard to RVR flour, 81.6% of the total N content
cdoierrtessipsopnrdeseedn ttoe dsoinluTbaleb lper1o.teWini tNh,r weghaerrdeatos R5.V5%R flcoorurre,s8p1o.6n%deodf ttoh esotloutballeN noconn-pternottecionr Nre,s apnodn d1e2d.9%to
csoorlurebslpeopnrdoetedi ntoN i,nwsohluerbelaes N5.. 5G%ercmorirneastpioonn dperdocteosss oclauubsleedn ao n7--pforoldte iinncNre,aasen din1 t2h.9e% cocnotrernets poof nsodleudbtleo
ninosno-lpurboleteNin. GNe, rmanidn aati o1n.4p raoncde s1s.c6a-fuosledd dae7c-rfeoaldsei nincr etahsee lienvtehles coofn itnensotloufbsloel uabnlde nsoonlu-pblreo tpeirnotNei,na nNd
rae1sp.4eactnidve1l.y6.- fGoledrmdeincraetaiosne incatuhseelde vae ls1.o2f-fionlsdo luinbclreeaansed sino luabshle cporontteeinnt Nwriethspouect tiavfefelyc.tiGnegr mtoitnaal tifoant
ccoamuspeodsaiti1o.2n-.f oldincreaseinashcontentwithoutaffectingtotalfatcomposition.
FFiigguurree 11 sshhoowwss tthhee cchhaannggeess iinn tthhee SSDDSS--PPAAGGEE ppaatttteerrnn ooff pprrootteeiinnss eexxttrraacctteedd ffrroomm RRVVRR aanndd GGVVRR
fflloouurrss.. GGeerrmmiinnaattiioonn pprorocceessssc acuasuesdeda rae dreudctuioctnioinn dine ndseitnysoitryt hoer dthisea pdpiseaaprapnecaeraonfcme aoinf pmraoitnei nprboatnedins
baannddtsh eansdu bthseeq suuebnsteaqpupeenat raapnpceeaorfaancsem oefa ar somfleoawr omf ololewcu mlaorlewceuilgahr twperiogtheitn pbroantedins. bands.

FFiigguurree 11.. DDeetetecctitoionn pprorocecesssesse sofo fVV. r.ardaidaitaat aprportoetieni ndidgiegsetisotnio nbyb gyegrmerimnaintiaotnio pnropcreoscse.Bssa.nBdan pdatpteartnte rinn
SinDSSD-PSA-PGAEG. EM.,M m,omleoclueclaurl awrewigehigt hmtmarakrekrse;r s1;, 1r,arwaw VV. .rardadiaitaat apprrooteteinin eexxttrraacct;t ;22,, 44 ddaayy--ggeerrmmiinnaatteedd
pprrootteeiinn eexxttrraacctt.. TThhee aammoouunntt ooff KKjjeellddaahhll--NN llooaaddeeddp peerrl alanneew waass2 2.0.0µ µgg..T Thheefi fgiguurerei sisr erpeprerseesnetnattaivtieveo fo3f
3in idnedpeepnednednetnat naanlaylsyesse.s.
Figure 2A shows a chromatogram and a mass spectrum (Retention time 13.27 min) of RVR
Figure2Ashowsachromatogramandamassspectrum(Retentiontime13.27min)ofRVRprotein.
protein. Consistent with the complexity of the sample, a wide range of chromatographic peaks were
Consistentwiththecomplexityofthesample,awiderangeofchromatographicpeaksweredetected.
detected. The presence of proteins was proved by high-resolution mass spectrometry as it is
Thepresenceofproteinswasprovedbyhigh-resolutionmassspectrometryasitisindicatedinthe
indicated in the mass spectrum example at 13.27 min. A typical protein profile for mass
mass spectrum example at 13.27 min. A typical protein profile for mass spectrometry was clearly
spectrometry was clearly obtained, due to the detection of multiple-charged ions provided by electro
obtained,duetothedetectionofmultiple-chargedionsprovidedbyelectrosprayionizationofproteins
spray ionization of proteins present in the sample. In comparison to the former results, a
presentinthesample. Incomparisontotheformerresults,achromatogramandamassspectrumdata
chromatogram and a mass spectrum data of GVR protein are presented in Figure 2B. A simpler
ofGVRproteinarepresentedinFigure2B.Asimplerchromatogramwasachieved,showinga36%
chromatogram was achieved, showing a 36% reduction in the area under the chromatogram that
reductionintheareaunderthechromatogramthatmatchedthedisappearanceorconsistentdecrease
matched the disappearance or consistent decrease in the area of major peaks, and the minor
intheareaofmajorpeaks,andtheminorappearanceofnewchromatographicpeaksat0.985,1.202,
appearance of new chromatographic peaks at 0.985, 1.202, and 1.883 min, respectively. The typical
and1.883min,respectively. Thetypicalproteinprofileformassspectrometrywasobtainedinthe
protein profile for mass spectrometry was obtained in the major peak (13.03 min), whereas the
majorpeak(13.03min),whereastheappearanceofmono-chargedionsthatinvolvedthepresenceof
appearance of mono-charged ions that involved the presence of smaller molecules such as amino
smallermoleculessuchasaminoacidsorpeptidescharacterizedthesmallerpeaks(0.985,1.202,and
acids or peptides characterized the smaller peaks (0.985, 1.202, and 1.883 min) that ranged
1.883min)thatrangedmass/charge(m/z)of93–893.
mass/charge (m/z) of 93–893.
Nutrients2017,9,774 8of22
Nutrients 2017, 9, 774 8 of 21
_12_2014_Radiata Cruda centroide 1: TOF MS ES+
13.71 TIC
100
CHROMATOGRAM 12.96 3.45e6
%
9.189.46 14.30
6.40 7.21 14.98
0.74 7.77 11.26
0 Time
2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00
122014Radiata4Dcentroide 1:TOFMSES+
01_12_2014_Radiata Cruda centroide 1731 (13.270) 1: TOF MS ES+
923.9736 4.68e4
860.3442
831.6954 891.0739 MASS SPECTRUM
805.0110 924.0551
924.1409
779.8365
756.1319
% 997.9050
756.0447 997.9890
738.3999 1084.5829
725.4885 1133.7701
1187.6343
1247.1577 1312.6609
0 m/z
550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 1250 1300 1350 1400 1450 1500
(A)
2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00
01_12_2014_Radiata 4D centroide 1: TOF MS ES+
100 13.34 TIC
4.10e6
CHROMATOGRAM
13.78
%
14.77
0.72 6.40 9.20 11.59
0 Time
2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00
01122014Rdit Hid li d t id 1TOFMSES+
01_12_2014_Radiata 4D centroide 127 (0.985) 1: TOF MS ES+
120.0801 1.40e5
MASS SPECTRUM0.985 min
%
166.0858
121.0831
93.0692 103.0539 136.0638149.0591 167.0886 255.9820 268.1024 310.1294
0 m/z
80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320
01_12_2014_Radiata 4D centroide 155 (1.202) 1: TOF MS ES+
188.0697 1.22e5
MASS SPECTRUM1.202 min
%
0 113.9623118.0656 144.0810466.0592159.0911 170.0596 18199.007.037180205.0966 m/z
70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300
01_12_2014_Radiata 4D centroide 244 (1.883) 1: TOF MS ES+
364.1235 2.44e4
311.0760
MASS SPECTRUM1.885 min
189.0525
%
369.0791 747.1093
0 161.0588 20273.10.604571226299.30.7096242 312.0793 409.1111451.1223 539.1160547.1054407.6014 651.1074667.0817 720.1509 774580..11101127 845.0733 893.20m1/2z
150 200 250 300 350 400 450 500 550 600 650 700 750 800 850
(B)
Figure 2. (A) Chromatogram of raw V. radiata protein and Mass Spectrum of major peak (13.270 min)
Figure2.(A)ChromatogramofrawV.radiataproteinandMassSpectrumofmajorpeak(13.270min)
of chromatogram; (B) Chromatogram of 4 day-germinated V. radiata protein after hydrolysis and
ofchromatogram;(B)Chromatogramof4day-germinatedV.radiataproteinafterhydrolysisandMass
Mass Spectrum of hydrolysis-derived peaks (0.985, 1.202, and 1.885 min, respectively) of
Spectrumofhydrolysis-derivedpeaks(0.985,1.202,and1.885min,respectively)ofchromatogram.
chromatogram.
Nutrients2017,9,774 9of22
Nutrients 2017, 9, 774 9 of 21
33..22.. BBiioollooggiiccaall AAnnaallyyssiiss
33..22..11.. FFoooodd IInnttaakkee aanndd BBooddyy WWeeiigghhtt GGaaiinn
TThhee eeffffeeccttss ooff pphheennootytyppe,e ,AAITI Tprportootcooclo alnadn ddiedtaiertya rtryeatrtmeaetmnte wntithw RitVhRR VorR GoVrRG oVnR woenekwlye feokoldy
fionotadkein atankde wanedekwlye ebkoldyyb wodeyigwhte iggahint gaarien parreespenretseedn itne dFiinguFrieg u3r. eB3o.thB optahrpamareatmerest ewrserwe earfefeacftfeedct ebdy
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((FFiigguurree 33AA)) aanndd bbooddyy wweeiigghhtt ggaaiinn ((FFiigguurree 33BB)) iinn tthhee ggrroouuppss tthhaatt uunnddeerrwweenntt tthhee eexxeerrcciissee ttrraaiinniinngg
pprroottooccooll wwhheenn ccoommppaarreedd ttoo tthheeiirr sseeddeennttaarryy ccoouunntteerrppaarrttss.. SSuucchh rreedduucciinngg eeffffeeccttss ooff eexxeerrcciissee oonn ffoooodd
iinnttaakkee aanndd wweeiigghhtt ggaaiinn wweerree mmoorree pprroonnoouunncceedd inin oobbeessee ththaann inin lelaeann pphhenenotoytpype,e t,htuhsu srerseuslutilntign gini na
astasttaisttiisctaicl ailnitnertearcaticotnio nbebtweteweene tnheth eexeexrceirscei sientienrtveervnetinotnio anndan tdhet hpehpenhoetnyoptey poef roaftsra (tps =( p0.=0205.0 a2n5da np d<
p0.<0000.10,0 r0e1s,preecsptievcetliyv)e. ly).
26 26
24 24
ke (g/d)2202 ke (g /d)2202
nta nta
kly food i1168 kly food i1168
Wee14 Wee14
12 12
10 10
W1 W2 W3 W4 W5 W6 W7 W1 W2 W3 W4 W5 W6 W7
RVRLS RVRLE RVROS RVROE GVRLS GVRLE GVROS GVROE
Phenotype Effect: P<0.001
Exercise Effect: P=0.007
Diet Effect: P<0.001
Phenotype Exercise: P=0.025
(A)
8 8
7 7
Weekly body weight gain (g/d) 23456 Weekly body weight gain (g/d)23456
1 1
0 0
W1 W2 W3 W4 W5 W6 W7 W8 W1 W2 W3 W4 W5 W6 W7 W8
RVRLS RVRLE RVROS RVROE GVRLS GVRLE GVROS GVROE
Phenotype Effect: P<0.001
ExerciseEffect: P=0.006
DietEffect: P<0.001
Phenotype×Exercise: P<0.001
Phenotype×Diet: P=0.001
(B)
Figure 3. Effect of dietary treatment with raw or 4 day-germinated V. radiata and aerobic interval
Figure3. Effectofdietarytreatmentwithrawor4day-germinatedV.radiataandaerobicinterval
training protocol on food intake (A) and body weight gain (B) of lean and obese Zucker rats. (A)
trainingprotocolonfoodintake(A)andbodyweightgain(B)ofleanandobeseZuckerrats.(A)Weekly
Weekly food intake (g DM/day); (B) Weekly body weight gain (g/day). DM, dry matter, RVR, raw V.
foodintake(gDM/day);(B)Weeklybodyweightgain(g/day).DM,drymatter,RVR,rawV.radiata,
radiata, GVR, 4 day-germinated V. radiata. Groups: LS, Lean (fa/+) sedentary rats, LE, Lean (fa/+) rats
GVR, 4 day-germinated V. radiata. Groups: LS, Lean (fa/+) sedentary rats, LE, Lean (fa/+) rats
performing aerobic interval exercise, OS, Obese (fa/fa) sedentary rats, OE, Obese (fa/fa) rats
performingaerobicintervalexercise,OS,Obese(fa/fa)sedentaryrats,OE,Obese(fa/fa)ratsperforming
performing aerobic interval exercise. W, week. Values are means ± SEM depicted by vertical bars (n =
aerobicintervalexercise.W,week.Valuesaremeans±SEMdepictedbyverticalbars(n=10).
10).
Nutrients2017,9,774 10of22
Withregardtothedietarytreatment,GVRledtolowerfoodintakeandbodyweightgainwhen
comparedtoRVRdiet,althoughtheeffectsonweightgainweremorepronouncedintheobesewhen
comparedtotheleananimals,thusgivingrisetoasignificantphenotype×dietinteraction(p=0.001).
3.2.2. AerobicCapacityandPhysicalPerformance
Theeffectsofphenotype,AITprotocol,anddietarytreatmentonaerobiccapacityandphysical
performanceofZuckerratsundergoingamaximaloxygenconsumptionincrementaltest,together
with the effects on hematic parameters related to aerobic capacity are shown in Table 3. With the
exceptionofVO max,allparametersrelatedtoaerobiccapacityandphysicalperformancederived
2
fromamaximaloxygenconsumptionincrementaltestweresignificantlyaffectedbyphenotypeand
exercise. Bloodlactatecontentwas2-foldhigherinsedentaryobeseratscomparedtotheleangroups,
whileitwasonly1.3–1.5-foldhigherinobesetrainedratsfedGVRandRVR,respectively(p<0.001for
bothphenotypeandexerciseeffect). Significanteffectsofphenotypeandexercisewerealsoobserved
inrunningtime,maximalspeedanddistancerunthatwerelowerinobesecomparedtoleananimals
andimprovedsubstantiallyinbothgroupsasaresultoftheAITprotocol(allp<0.001)Furthermore,
asignificanteffectofexercisewasobservedonVO maxthatincreasedinbothphenotypes(p<0.001),
2
although the effects were more pronounced in lean compared to obese animals, thus resulting in
significant phenotype × exercise interaction (p < 0.001). Regarding the inclusion of RVR or GVR
in the experimental diets, running time, maximal speed and distance run were enhanced by the
GVRcomparedtoRVRdiet,withmorepronouncedeffectsinleananimals. Allthese,gaveriseto
significantdieteffect(p<0.001)anddiet×phenotypeinteractions(p=0.001andp<0.001forrunning
time and distance run, respectively). In addition, the parameters related to physical performance
weredifferentiallyaffectedbyexercisedependingonthetypeofdietconsumed. Inthisregard,the
enhancingeffectofexerciseinbothleanandobeseanimalswasgreatestinratsfedGVR,thusresulting
insignificantdiet×exerciseinteraction(allp<0.001). TheAITprotocolassayedtendedtoincreaseall
thehematicparametersrelatedtoaerobiccapacity(Table3). Suchenhancingeffectvariedbetween
leanandobeseratsinmostoftheparametersstudied,andgaverisetosignificanteffectofexerciseand
phenotype×exerciseinteraction.
3.2.3. PlasmaParametersofGlucoseandLipidMetabolism
Theeffectsofphenotype,AITprotocol,anddietarytreatmentonbloodandplasmabiochemical
parametersrelatedtoglucoseandlipidmetabolismareshowninFigure4andTable4. Asignificant
effect of phenotype was observed on all the parameters analyzed that were higher in obese when
comparedtoleanrats. TheAITprotocolassayedcausedasignificantreductioninbloodglucoseand
AUC,aswellasinplasmalipidparameters,withamorepronouncedeffectinobeseanimals,giving
risetoasignificantphenotype × exerciseinteraction. AUCvalueswerealsolowerinanimalsfed
theGVRwhencomparedtoRVRdiet,andsuchdietaryeffectwasmodulatedbyphenotype,thus
resultinginsignificantphenotype×dietinteraction(p<0.001). Likewise,dietsignificantlyaffected
theinfluenceofexerciseonAUC,withalessevidenteffectinGVRcomparedtoRVR,resultingina
significantdiet×exerciseinteraction. Alltheseeffectswerereflectedindifferenttrendsofevolutionin
bloodglucosevaluesafteranoralglucoseoverloadinGVRcomparedtoRVR(Figure4),withlower
levels of peak blood glucose and time to return to basal levels by the obese animals in the former
dietarytreatment.