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Neuregulin 1 affects leptin levels, food intake and weight
gain in normal-weight, but not obese, db/db mice
Gaël Ennequin, Nathalie Boisseau, Kévin Caillaud, Vivien Chavanelle, Michel
Etienne, Xiaojian Li, Christophe Montaurier, Pascal Sirvent
To cite this version:
Gaël Ennequin, Nathalie Boisseau, Kévin Caillaud, Vivien Chavanelle, Michel Etienne, et al..
Neureg-ulin 1 affects leptin levels, food intake and weight gain in normal-weight, but not obese, db/db mice.
Diabetes and Metabolism, Elsevier Masson, 2015, 41 (2), pp.168-172. �10.1016/j.diabet.2014.12.002�.
�hal-01216265v2�
Availableonlineat
ScienceDirect
www.sciencedirect.comDiabetes&Metabolism41(2015)168–172
Short
Report
Neuregulin
1
affects
leptin
levels,
food
intake
and
weight
gain
in
normal-weight,
but
not
obese,
db/db
mice
G.
Ennequin
a,b,
N.
Boisseau
a,b,
K.
Caillaud
a,b,
V.
Chavanelle
a,b,
M.
Etienne
a,b,
X.
Li
e,
C.
Montaurier
b,c,d,
P.
Sirvent
a,b,∗aEA3533,LaboratoryofMetabolicAdaptationstoExerciseinPhysiologicalandPathologicalConditions,ClermontUniversity,BlaisePascalUniversity,
BP10448,63000Clermont-Ferrand,France
bClermontUniversity,AuvergneUniversity,HumanNutritionUnit,BP10448,63000Clermont-Ferrandcedex1,France cFrenchNationalInstituteforAgriculturalResearch(INRA),UMR1019,UNH,CRNHAuvergne,63000Clermont-Ferrand,France
dUFRMédecine,UniversityClermont1,63001Clermont-Ferrand,France eZensunSciandTechLtd.,Shanghai,China
Received27September2014;receivedinrevisedform24November2014;accepted2December2014 Availableonline5January2015
Abstract
Aim.–Studiesinvitrohavehighlightedthepotentialinvolvementofneuregulin1(NRG1)intheregulationofenergymetabolism.Thiseffecthas alsobeensuggestedinvivo,asintracerebroventricularinjectionofNRG1reducesfoodintakesandweightgaininrodents.Thus,itwashypothesised thatNRG1mightaffectserumleptinlevelsinmice.
Methods.–Weight,foodintakes,energyexpenditure,spontaneousphysicalactivityandserumleptinlevelswereevaluatedinnormal-weight C57BL/6JRJmicefollowingintraperitonealadministrationofNRG1(50g/kg,threetimes/week)orsalinefor8weeks.Basedontheresultsof thisfirstexperiment,leptin-resistantobesedb/dbmicewerethengivenNRG1for8weeks.
Results.–LeptinserumconcentrationsweresixtimeshigherinC57BL/6JRJmicetreatedwithNRG1thanintheanimalsgivensaline.NRG1 treatmentalsoreducedweightgainby10%andfoodintakesby15%comparedwithsalinetreatment,whileenergyexpenditureremainedunchanged. Indb/dbmice,serumleptinconcentrations,weightgain,foodintakes,energyexpenditureandspontaneousphysicalactivitywerenotalteredby NRG1treatment.
Conclusion.–ThedecreaseinfoodintakesandweightgainassociatedwithNRG1treatmentinC57BL/6JRJmicemaybepartlyexplainedby increasedleptinlevels,whereasdb/dbmicewerenotaffectedbythetreatment,suggestingresistancetoNRG1inthispathologicalstate. ©2014ElsevierMassonSAS.Allrightsreserved.
Keywords: NRG1;Obesity;Energyexpenditure;Weightloss
Abbreviations: NRG1,Neuregulin1;ErbB,Erythroblasticleukaemiaviral oncogenehomologue;RQ,Respiratoryquotient;VHL,Vehicle(saline).
∗Corresponding author. Laboratoire des Adaptations Métaboliques à
l’ExerciceenConditionsPhysiologiquesetPathologiques(AME2P), Univer-sitéBlaise-Pascal,BâtimentBiologieB,24,avenuedesLandais,BP80026, 63171Aubièrecedex,France.Tel.:+33473407133;
fax:+0033473405062.
E-mailaddress:pascal.sirvent@univ-bpclermont.fr(P.Sirvent).
1. Introduction
Neuregulin1(NRG1)belongstotheepidermalgrowth fac-torfamily.Itsbiologicalactivityismediatedbyerythroblastic leukaemiaviraloncogenehomologue(ErbB)receptors.NRG1 playsafundamentalroleinbothdevelopingandmatureheart tis-sue,andNRG1treatmenthaspositiveeffectsincardiovascular diseases[1].AlteredNRG1/ErbBsignallinghasbeen demon-stratedduringthedevelopmentandprogressionofmanytumours
[2]. Complex crosstalk between ErbB2 receptors and leptin appearstotakeplaceincancer[3],andErbB2inducesleptin transcriptionalupregulationinhumanbreastepithelialcells[4].
http://dx.doi.org/10.1016/j.diabet.2014.12.002
G.Ennequinetal./Diabetes&Metabolism41(2015)168–172 169 Asleptinplaysamajorroleinenergybalanceregulation,and
giventhelinkbetweenleptinandNRG1/ErbBsignallingin can-cer,itwashypothesizedthatNRG1treatmentmightalterleptin levels.
Therefore,theeffectofchronic(8-week)intraperitoneal(i.p.) NRG1treatmentonleptin,weightstatus,fatmass,foodintakes, physicalactivityandenergyexpenditurewasassessedin normal-weightmiceand,basedontheresultsofthisfirstexperiment,in leptin-resistantdb/dbmicealso.
2. Methods
2.1. Animals
A total of 16C57BL/6JRJ and 16 BKS(D)-Leprdb/JOrlRj
mice (10weeks old), provided by CERJ Janvier(Le Genest-Saint-Isle, France), were kept in temperature-controlled (20–22◦C) cages (single-housed) with free access to water andfood (A04diet;Scientific AnimalFoodandEngineering, France). All animal husbandry and experimental procedures wereinaccordancewiththecurrentlegislationonanimal exper-imentation,andwereapproved bythe localethicscommittee (CEMEAAuvergne,CE97-12).
2.2. NRG1treatment
Recombinant NRG1 (beta-2a; Zensun Sci and Tech, Shanghai, China) [5] at 50g/kgbody weight or an equiva-lentvolumeof0.9%NaCl(sodiumchloride)wasadministered byi.p.injectionthreetimesaweekfor8weeks(n=8foreach group).Micewereeuthanised3daysaftertheendoftreatment.
2.3. Foodintakes,bodymassandbodycomposition
Body weight and food intakes were recorded every week duringthe treatment period.Oneweekbefore sacrifice,body compositionwasevaluatedbyquantitativenuclearmagnetic res-onanceimaging(EchoMRI;EchoMedicalSystems,Houston, TX,USA).
2.4. Dailyenergyexpenditureandphysicalactivity
VO2, VCO2 and physicalactivity were measured inmice using a PhenoMaster/LabMaster home cage system (TSE Systems, Bad Homburg, Germany). Energy expenditure was calculatedusing theWeirequation [6]frommeasurements of gas exchanges computed for each cage from data sampled every5min.Respiratoryquotient (RQ)was determinedfrom VCO2/VO2. Spontaneous activity was measured using three-dimensionalmeshesoflightbeams.Foradaptation,micewere placedinindividualcalorimetriccages(22◦C)for24hpriorto datacollection.Dailyenergyexpenditureandphysicalactivity werecomputedfora24-hperiod.
2.5. Leptinmeasurement
Serum leptin was evaluated with an ELISA kit (Abcam, Cambridge,MA,USA),andsampleswerereadat450nmusing aPowerWavespectrophotometer(USBioTek,NorthShoreline, WA,USA).
2.6. Statisticalanalysis
SPSSAdvancedStatisticssoftwarewasusedforthe statisti-calanalysis(IBM,Armonk,NY,USA).Dataarepresentedas themean±SEM.One-wayanalysisofvariance(ANOVA)with repeated measureswas performed to assessweight andfood intakechangesfollowingNRG1orsalinetreatment.Unpaired Student’sttestwasusedwhereappropriate.Statistical signifi-cancewassetatP<0.05.
3. Results
3.1. NRG1increasesserumleptinanddecreasesweight
gain,foodintakesandspontaneousphysicalactivityin
C57BL/6JRJmice
OnevaluatingNRG1effectsinC57BL/6JRJmice(Fig.1), serum leptin was strongly increased compared with the salinevehicle(VHL)-treatedanimals(183.4±46.6pg.mL−1vs.
29.5±5.1pg.mL−1,respectively;P<0.05;Fig.1A).TheVHL group alsogained 9.6% inbodymass (from 25.1±0.95g to 27.6±0.85g)whereas,inNRG1-treatedmice,weightremained stable (from 26.6±0.52g to 26.1±0.43g) with treatment (Fig.1B).Similarly,foodintakesincreasedby15.3%intheVHL group (from 27.1±0.59g.week−1 to 31.5±1.5g.week−1;
P<0.05; Fig.1C),but didnot changein NRG1-treatedmice (from 29.3±0.57g.week−1 to 29.3±0.69g.wk−1; Fig. 1C). Attheendofthe treatmentperiod,bodyfatpercentageswere lowerintheNRG1thaninthe VHLgroup(12.5±0.67%vs.
16.3±0.91%, respectively; P<0.05; Fig. 1D). Daily energy expenditure(Fig.1E)andRQ(notshown)didnotdifferbetween the groups. Spontaneous activity over 24h and at night was lowerintheNRG1thanintheVHLgroup(288.2±31.5mvs.
502.6±102.3mand227.7±24.9vs. 373.6±56.1m, respec-tively;P<0.05),whereasdiurnal activity wassimilarinboth groups(Fig.1F).
3.2. NRG1hasnoeffectonserumleptin,weightgain,food
intakesorspontaneousphysicalactivityindb/dbmice
As NRG1 treatment increased serum leptin levels in C57BL/6JRJmice,itwaspostulatedthatNRG1effectsmightbe bluntedinleptin-resistantdb/dbmice.Serumleptin concentra-tionswerecomparableinbothNRG1-andVHL-treateddb/db mice (Fig.2A).Similarly, weight(Fig.2B)and food intakes (Fig.2C)remainedstableinbothgroupsofdb/dbmice,andthe percentageofbodyfatwasalsocomparableinbothgroupsat theendoftreatment (Fig.2D).Energyexpenditure (Fig.2E),
Fig.1.Effectsof8-weekneuregulin1(NRG1)andsaline(VHL)intraperitonealtreatmentinnormal-weightC57BL/6JRJmice.(A)Serumleptinconcentrationsat theendofNRG1(50g.kg−1)andsalinetreatment;variations(aspercentages)in(B)bodyweightand(C)foodintakeduringNRG1andsalinetreatment;and(D) bodyfat,(E)energyexpenditureand(F)spontaneousphysicalactivityattheendoftreatment.Valuesaremeans±SEM(n=8/condition).*P<0.05vs.VHL-treated
mice.
RQ(notshown)andspontaneousphysicalactivity(Fig.2F)did notsignificantlydifferbetweengroups.
4. Discussion
Ourresultsshowthat,inC57BL/6JRJmice,NRG1treatment increasesserumleptinconcentrations,preventsweightgainand lowers foodintakes comparedwithsaline-treatedcontrols.In contrast,NRG1hadnosignificanteffectsindb/dbmice.
ThestrongleptinincreaseinC57BL/JJRJmiceafter8weeks ofNRG1treatmentmightexplainthedecreasesinfoodintakes, weightgainandbodyfatpercentages,asshownpreviouslywith
leptintreatment[7].Thecauseoftheleptinincrease,however,is unknown.Leptinproductionisindirectlyunderinsulincontrol viathePI3K/AKTsignallingpathway[8],andNRG1stimulates thispathwayinmusclecells[9].ErbB2activationupregulates leptin inhuman breastepithelialcells [4].However,it is not knownwhetherNRG1administrationcanaffectwhiteadipose tissue,themainleptinproducer[8].ThefactthatErbBreceptors areexpressedinhumanpreadipocytes[10]allowsthe assump-tionthatadiposetissuemaybeinvolvedintheNRG1-mediated increaseinleptin.
Alternatively, leptinlevels mightberegulated centrally, as thebrainhasbeenshowntoexpressleptinandtoreleaseitinto
G.Ennequinetal./Diabetes&Metabolism41(2015)168–172 171
Fig.2.Effectsof8-weekintraperitonealtreatmentwithneuregulin1(NRG1)andsaline(VHL)inobesedb/dbmice.(A)Serumleptinconcentrationsattheendof NRG1(50g.kg−1)andsalinetreatment;variations(aspercentages)in(B)bodyweightand(C)foodintakebetweenweeks1and8;and(D)bodyfat,(E)energy expenditureand(F)spontaneousphysicalactivityattheendoftreatment.Valuesaremeans±SEM(n=8/condition).
thebloodstream[11].Indeed,intracerebroventricularinjection ofNRG1 inhamstershadeffectson foodintakes andweight gainsimilartothosereportedinourstudy[12],suggestingthat thebrainmaybeparticipatingintheNRG1-mediatedincrease incirculatingleptin,althoughitcannotbeexcludedthatNRG1 effects mayalso beindependent of leptin.Also, thereis evi-dencetosuggestthatNRG1couldalsohaveleptin-independent effects.Spontaneousphysicalactivitywasmarkedlydecreased innormal-weightmice,aspreviouslyreported[12],whiletotal energyexpenditurewasnotaffectedbyNRG1treatment.
Theseresultssuggestthattherestingmetabolicrate,which representsamajorcomponentoftotalenergyexpenditure(about 86%inmice[13]),couldcompensateforthedecreasedenergy
expenditurefromphysicalactivity,resultinginunchangedtotal daily energy expenditure. Nevertheless, leptin is known to increasebothspontaneousphysicalactivityandenergy expen-diture [14].Therefore,our present results suggesta complex action of NRG1 whereby food intakes and weight gain are both decreased possibly through an increase in leptin, while physical activity andenergy expenditure are limited through unknown pathways. However, the possibility of a side-effect inducedbyNRG1treatment thatmightpromoteadecreasein mouselocomotioncannotberuledout,althoughhumanstudies using comparable dose ranges have shown no such signifi-cant side-effectinduced bychronic NRG1treatment [15,16]. More studies are now needed to elucidate the origin of the
NRG1-inducedincreaseincirculatingleptinandthe contribu-tionofincreasedcirculatingleptintoalteredfoodintakesand weightgain.
Unexpectedly,noneoftheeffectsobservedinC57BL/6JRJ micefollowingNRG1treatmentwereseenintheleptin-resistant obese db/db mice, and serum leptin concentrations did not increase.Thisresult suggeststhat the NRG1/ErbB signalling pathwaymightbeimpairedinthesemice.AsNRG1activates thePI3K/Aktpathway[9],insulinresistanceindb/dbmicecould have inhibited leptin production, whichis triggered by these kinases[10].Moreover,palmitateimpairsNRG1signallingin ratcardiacmyocytes[17].Thissuggeststhatthedyslipidaemia commonlyseeninmetabolicdiseases,andparticularlyindb/db mice,mightperturbNRG1signalling.SuchanNRG1-resistant statecould,inturn,resultinsilencedleptinupregulation.
In conclusion, our present study has demonstrated that chronicNRG1 administration increasesserum leptin concen-trationinnormal-weight mice,andthisincreasemight partly explain the reduction in food intakes, body weight gain and bodyfatpercentageinthesemice.Onthe otherhand,NGR1 treatmentappearedtohavenosucheffectsinobesedb/dbmice, suggesting‘NRG1resistance’inthispathologicalstate.
Disclosureofinterest
XinyanLiisemployedbyZensunSciandTechLtd.
Acknowledgements
WethankZensunSciandTechLtd.fortheiradviceandgift ofrhNRG1.
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