Opposing Activity Changes in AMP Deaminase and AMP-Activated Protein Kinase in the

19 Pages·2015·0.87 MB·English

RESEARCHARTICLE
Opposing Activity Changes in AMP
Deaminase and AMP-Activated Protein
Kinase in the Hibernating Ground Squirrel
MiguelA.Lanaspa1☯*,L.ElaineEpperson2☯,NanxingLi1☯,ChristinaCicerchi1,Gabriela
E.Garcia1,CarlosA.Roncal-Jimenez1,JessicaTrostel1,SwatiJain1,ColinT.Mant3,
ChristopherJ.Rivard1,TakujiIshimoto1,MichikoShimada1,LauraGabrielaSanchez-
Lozada1,4,TakahikoNakagawa1,AlkeshJani1,PeterStenvinkel5,SandraL.Martin2,
RichardJ.Johnson1,6
1 DivisionofRenalDiseasesandHypertension,UniversityofColoradoDenver,Aurora,CO,80045,United
StatesofAmerica,2 DepartmentofCellandDevelopmentalBiology,Aurora,CO,80045,UnitedStatesof
a11111
America,3 DepartmentofBiochemistryandMolecularGenetics,Aurora,CO,80045,UnitedStatesof
America,4 LaboratoryofRenalPhysiopathologyandNephrologyDept,INCIgnacioChavez,MexicoCity,
Mexico,5 DivisionofRenalMedicine,DepartmentofClinicalInterventionandTechnology,Karolinska
Institutet,Stockholm,Sweden,6 DivisionofNephrology,EasternColoradoHealthSystem,Departmentof
VeteranAffairs,Denver,CO,UnitedStatesofAmerica
☯Theseauthorscontributedequallytothiswork.
* [email protected]
OPENACCESS
Citation:LanaspaMA,EppersonLE,LiN,Cicerchi
C,GarciaGE,Roncal-JimenezCA,etal.(2015) Abstract
OpposingActivityChangesinAMPDeaminaseand
AMP-ActivatedProteinKinaseintheHibernating Hibernatinganimalsdevelopfattyliverwhenactiveinsummertimeandundergoaswitchto
GroundSquirrel.PLoSONE10(4):e0123509.
afatoxidationstateinthewinter.Wehypothesizedthatthisswitchmightbedeterminedby
doi:10.1371/journal.pone.0123509
AMPandthedominanceofopposingeffects:metabolismthroughAMPdeaminase
AcademicEditor:MiguelLópez,Universityof
(AMPD2)(summer)andactivationofAMP-activatedproteinkinase(AMPK)(winter).Liver
SantiagodeCompostelaSchoolofMedicine-
CIMUS,SPAIN sampleswereobtainedfrom13-linedgroundsquirrelsatdifferenttimesduringtheyear,in-
cludingsummerandmultiplesstagesofwinterhibernation,andfatsynthesisandβ-fatty
Received:May19,2014
acidoxidationwereevaluated.Changesinfatmetabolismwerecorrelatedwithchangesin
Accepted:March4,2015
AMPD2activityandintrahepaticuricacid(downstreamproductofAMPD2),aswellas
Published:April9,2015 changesinAMPKandintrahepaticβ-hydroxybutyrate(amarkeroffatoxidation).Hepatic
Copyright:©2015Lanaspaetal.Thisisanopen fataccumulationoccurredduringthesummerwithrelativelyincreasedenzymesassociated
accessarticledistributedunderthetermsofthe withfatsynthesis(FAS,ACLandACC)anddecreasedenoylCoAhydratase(ECH1)and
CreativeCommonsAttributionLicense,whichpermits
carnitinepalmitoyltransferase1A(CPT1A),ratelimitingenzymesoffatoxidation.Insum-
unrestricteduse,distribution,andreproductioninany
medium,providedtheoriginalauthorandsourceare mer,AMPD2activityandintrahepaticuricacidlevelswerehighandhepaticAMPKactivity
credited. waslow.Incontrast,theactivephosphorylatedformofAMPKandβ-hydroxybutyrateboth
DataAvailabilityStatement:Allrelevantdataare increasedduringwinterhibernation.Therefore,changesinAMPD2andAMPKactivitywere
withinthepaper. paralleledwithchangesinfatsynthesisandfatoxidationratesduringthesummer-winter
Funding:ThisworkissupportedbyNIHgrants cycle.ThesedatailluminatetheopposingforcesofmetabolismofAMPbyAMPD2andits
HL68607,RC4DK90859andstartupfundsfromthe availabilitytoactivateAMPKasaswitchthatgovernsfatmetabolismintheliverofhibernat-
UniversityofColoradotoRJJandgrantHL089049to
inggroundsquirrel.
SLMforsupportoftheanimalsamples.Thefunders
hadnoroleinstudydesign,datacollectionand
analysis,decisiontopublish,orpreparationofthe
manuscript.
PLOSONE|DOI:10.1371/journal.pone.0123509 April9,2015 1/19
AMPDeaminase,AMPKinaseandHibernation
CompetingInterests:DrsMAL,CJR,GEGandRJJ Introduction
arelistedasinventorsonapatentfortheuseof
Bodyweightistightlyregulatedinmostspecies.Forexample,Keeseyetalhaveshownthat
AMPD2inhibitorstotreatobesityandinsulin
resistance(US8,697,628).MAL,CJR,LGSL,CARJ ratsreturntotheirbaselineweightaftereitherforcefeedingorforcefasting[1].Migrating
andRJJarealsomembersofColoradoResearch songbirdsthatfastduringthesummerregaintheirweightduringtherecoveryphase[2].In
PartnersLLCthatisinterestedindevelopingnovel bothsituationstheanimalsreturntotheirbaselineweightfortheirageandthetimeofthe
treatmentsforobesity.Therearenofurtherpatents,
year.Manyspeciesalsogainweightinpreparationforperiodsoffoodshortage.Forexample,
productsindevelopmentormarketedproductsto
theEmperorpenguin(Aptenodytesforsteri)doublesitsbodyweightinfattosurviveforupto
declare.Thisdoesnotaltertheauthors'adherenceto
allthePLOSONEpoliciesonsharingdataand 4monthsontheAntarcticiceduringbrooding[3].Hibernatingmammalsalsoprovideexcel-
materials,asdetailedonlineintheguideforauthors. lentexamplesofanimalsthatgainlargeamountsoffatandthenloseitwhilefastingthrough-
outwinter[4].
Groundsquirrelsandothersciuridrodenthibernatorsactivelyaccumulatefatinthefall,
andthenliveofftheirfatstoresthroughoutthemanymonthsofwinterhibernation[5,6].In
thefall,weightgainmaybeenhancedbyreducedmetabolicratesandfatstoresincrease
throughoutthebodyincludingabdominaland,toalesserextent,hepaticfat[7].Hibernating
mammalssuchastheyellow-belliedmarmot(Marmotaflaviventris)and13-linedground
squirrel(Ictidomystridecemlineatus)alsobecomeinsulinresistant[7,8].Throughatriggering
mechanismthatremainsunclear,theanimalstheninitiatefastingandareductioninmetabo-
lismfollowedbyadropinbodytemperatureandentryintotorpor[9,10].Duringtorporthe
animalsgenerateenergyprimarilybyfatoxidation[6].Hibernating13-linedsquirrelsremain
indeeptorporfor1to2weeks,withmetabolicratesof1to5percentofthesummeractive
level,accompaniedbyseverereductionsinheartrate(to5–10beats/minute),respiration
(5times/minute)andbodytemperature(toaslowas-2.9°C)[11]Animalsthenrewarmin
about2hoursandremainat~37°Cforapproximately10hours(interboutarousal)beforecool-
ingagainintoanotherboutoftorpor.Thiscyclerepeatsthroughoutthewinteruntiltheanimal
finallyemergesfromhibernationinthespring[5].
Theoverallresponsetotemperaturechangesinacircannualhibernatorinvolvelocaladap-
tationsfrommultiplecentralandperipheralorgansincludingtheskeletalmuscle[12],adipose
tissue-brownandwhite-[13,14,15],heart[16],kidney[17]andliver[18,19,20].Inthisregard,in
bothdailytorporandhibernation,thereisageneraldecreaseinmetabolicrateallowingani-
malstocopewithcoldenvironmentsand/orlimitedfood.Amongtheseorgans,theliverplays
aspecificroleinbodyadaptationtotorpor.Forexample,previousstudiesinhibernatingmam-
malsindicateanimportantreductioninlivermitochondrialrespiration[19,20],probablyasa
waystoreducethermogenesisandinduceenergysavings.
Oneofthemostimportantenzymesinvolvedincontrollingfatoxidationisadenosinemo-
nophosphate-activatedproteinkinase(AMPK).InresponsetoanincreasedratioofAMPto
totalATP,AMPKisactivatedbyphosphorylation[21].Therefore,instatesofenergydeficiency
theratiobetweenATPandAMPorADPlevelsisdecreased,thereforefacilitatingtheactivation
ofAMPK.ActivatedAMPK,inturn,stimulatescatabolicpathways(fatoxidation,glycolysis
andglycogenolysis)whilesimultaneouslyinhibitinganabolicpathways(fatsynthesis,gluco-
neogenesisandglycogenesis)[21].Duringhibernation,tissuesrequireATPastheenergysource
tosurvivestarvation.Asaconsequence,AMPmayaccumulatethusactingasanAMPKactiva-
tortostimulateβ-oxidationoffattyacids.Ofinterest,AMPcanalsoactasametabolicsub-
strateforAMPD2(AMPD2)whichconvertsAMPtoinosinemonophosphate(IMP).IMPis
eventuallymetabolizedbydifferentenzymesintodownstreamproductssuchasuricacid.Re-
centstudiessuggestthatthespecificactivationofAMPD2decreasestheavailabilityofAMP
forAMPKactivation;henceAMPD2activationmaylimittheabilityofAMPKtobecomeacti-
vated,counteringitseffects[22,23,24,25].
PLOSONE|DOI:10.1371/journal.pone.0123509 April9,2015 2/19
AMPDeaminase,AMPKinaseandHibernation
WethereforetestedthehypothesisthathepaticAMPD2andAMPKhaveopposingpatterns
ofactivityduringperiodsoffataccumulation(summer)andfatoxidation(winterhibernation)
in13-linedgroundsquirrelstraversingtheiryearlycycleofweightgainandloss.
Methods
Ethicsstatement
AllAnimalexperimentswereperformedaccordingtoprotocolsapprovedbytheUniversityof
ColoradoAnimalCareandUseCommittee.
Animals
GroundsquirrelswereobtainedfromtheUniversityofWisconsin13-linedgroundsquirrel
captivebreedingprogramatOshkosh.GroundsquirrelswerehousedattheUniversityofColo-
radoandmaintainedunder14hlight-10hdarkconditionswithcatchowadlibitumuntillate
SeptemberorearlyOctoberwhentheyweretransferredtoahibernaculuminwhichtempera-
turesweremaintainedat4°Cindarkness;foodandwaterwereremovedaftertheanimalsen-
teredtorporuntiltheybegantoemergefromhibernationinspring.Toidentifythevarious
stagesofhibernationbasedonbodytemperature(Tb),eachgroundsquirrelwassurgicallyim-
plantedintraabdominallywitharadiotelemeterandadatalogger(VM-FHdisks,Minimitter,
iButton,EmbeddedDataSystems)[26].
TissueCollection
Groundsquirrels(n=3to6foreachofthe7timepoints)wereanesthetizedwithisoflurane,
euthanizedbycardiacexsanguination,andthelivertissuesnapfrozenwithliquidnitrogen.
LiverswereexcisedenblocandsampleswereusedforimmunohistochemistryandoilredO
staining(fromsamplesembeddedinOCTandfrozen)orsnapfrozenforproteinandfat
determination.
Thetimepointswereasfollows:Summeractive(SA),obtainedinJulyandearlyAugust;Fall
transition(FT),obtainedinSeptemberandOctober;InterboutArousal(IBA),approximately
3hafterreachingTbof35–37°Cfollowingaperiodoftorpor;Entrance(Ent),Tbdecreasingto
between27and23°Casanimalsenteranewboutoftorpor;EarlyTorpor(ET),Tbof4°Cfor
lessthan10%ofprevioustorporboutlength;LateTorpor(LT),Tb4°Cfor80–95%ofthetime
oftheprevioustorporbout;Arousing(Ar),Tbbetween7–12°Cduringspontaneousarousal
fromtorpor;SpringActive(Sp),afteremergencefromhibernation,thegroundsquirrelwasho-
meothermicfor11–20daysandhadresumedeating,butthehibernaculumwasstilldarkand
cold(4°C).
Proteinextractionandwesternblotting
ProteinlysateswerepreparedfromliversemployingMAPKinaselysisbufferaspreviouslyde-
scribed[27].Sixanimalspercondition(SA,FT,IBA,Ent,ET,LT,ArandSp)wereanalyzedby
westernblotintwosetsofthreeanimals.Allproteinsanalyzedweredetectedusingtwosepa-
rateblots(10%acrylamide)accordingtotheirdifferentmolecularweights.Blot1(dilutions
employedinTTBS)wasemployedfordetectionofFAS(250Kda),AMPD2(90kDa),
P-AMPK(65kDa)andactin(42kDa)whileinblot2ACC(250kDa),ACL(125kDa),AMPK
(65kDa)andECH1(33kDa)weredetected.Blot2wassubsequentlystrippedandreprobedfor
actintoassureequalproteinloadingintheblotandtonormalizeproteinabundance.Blotsde-
pictedcorrespondwithrepresentativewesternblotsobtainedforeachprotein.Antibodyto
AMPD2wasobtainedfromAbnova(1:1000)whileantibodytoECH1wasobtainedfrom
PLOSONE|DOI:10.1371/journal.pone.0123509 April9,2015 3/19
AMPDeaminase,AMPKinaseandHibernation
proteintech(1:500).SampleproteincontentwasdeterminedbytheBCAproteinassay
(Pierce).Approximately50mgoftissuewashomogenizedin350μlofMAPKlysisbuffercon-
tainingproteasesandphosphatasesinhibitors(Roche),sampleswerekeptonicefor20min-
utes,centrifugedatfullspeedat4°Candsupernatantcollected.40μgoftotalproteinwere
loadedperlaneforSDS-PAGE(10%w/v)andthentransferredtoPVDFmembranes.Mem-
braneswereincubatedwithprimaryantibodiesandvisualizedusingahorseradishperoxidase
secondaryantibodyandtheHRPImmunstardetectionkit(Bio-Rad,Hercules,CA).Chemilu-
minescencewasrecordedwithanImageStation440CFandresultsanalyzedwiththe1D
ImageSoftware(KodakDigitalScience,Rochester,NY).Rabbitpolyclonalantibodyto
AMPD2(1:1000dilutioninTTBS,H00000271)waspurchasedfromNovus(Littleton,CO),
antibodiestoβ-actin(3700),totalACC(3676),totalACL(4332),totalAMPK(2532)andphos-
phorylatedAMPK(Thr172,2535),toLKB1,P-LKB1,CPT1A,AMPKalpha1,AMPKalpha2,
wereemployedata1:1000dilutioninTTBS.andobtainedfromCellSignaling(Danvers,MA)
whiletheantibodytoECH1(11305-1-AP)waspurchasedfromProteintech(Chicago,IL).Sec-
ondaryantibodiesconjugatedwithhorseradishperoxidasewerefromCellSignaling.
AMPD2activityassay
AMPD2activitywasdeterminedbyestimatingtheproductionofammoniabyamodification
ofthemethoddescribedbyChaneyandMarbach[28]fromcellscollectedinabuffercontain-
ing150mMKCl,20mMTris-HCl,pH7.51mMEDTA,and1mMdithiothreitol.Briefly,the
reactionmixtureconsistedof25mMsodiumcitrate,pH6.0,50mMpotassiumchloride,and
theindicatedconcentrationsofAMP.Theenzymereactionwasinitiatedbytheadditionofthe
enzymesolutionandincubatedat37°Cfor15minforallsamplescollected.Inaddition,we
measuredAMPD2activityfor15minattheapproximatephysiologicaltemperatureforeach
animalwithdepressedTb,i.e.,25°Cforarousalandenteringtorporand4°Cforearlyandlate
torporsamples,respectively.Thereactionswerestoppedwiththeadditionofthephenol/hypo-
chloritereagents:ReagentA(100mMphenoland0.050g/Lsodiumnitroprussideinwater)
wasadded,followedbyreagentB(125mMsodiumhydroxide,200mMdibasicsodiumphos-
phate,and0.1%sodiumhypochloriteinH O).Afterincubationfor30minat25°C,theabsor-
2
banceofthesampleswasmeasuredat625nmwithaspectrophotometer.Todeterminethe
absolutespecificactivityofammoniaproduction(micromolesammonia/min),acalibration
curvewasdeterminedintherangeof5μMto1mMofammonia.
LiverOilRedOstaining
LivertissuewasembeddedinOptimalCuttingTemperaturegel(OCT;SakuraFinetek,Tor-
rance,CA)andfrozeninliquidnitrogen.Air-driedcryostattissuesections(8μm)weredipped
informalin,washedwithrunningtapwater,rinsedwith60%isopropanol,stainedforlipids
withOilRedOandcounterstainedwithhematoxylin.OilredOwasthenextractedfromthe
slideswithisopropanolcontaining4%NonidetP-40,transferredtoplasticcuvettes,andoptical
density(OD)wasthenmeasuredatawavelengthof520nm.
Determinationofintrahepatictriglycerides,β-hydroxybutyrate,inosine,
uricacidandphosphate
Fortriglyceride(TG)determinationinliver,fatwassolubilizedbyhomogenizationin1mlso-
lutioncontaining5%nonidetP40(NP-40)inwater,slowlysampleswereexposedto80–100°C
inawaterbathfor5minutesuntiltheNP-40becamecloudy,thencooleddowntoroomtem-
perature.Sampleswerethencentrifugedfor2mintoremoveanyinsolublematerial.Triglycer-
idedeterminationwiththeVetAceautoanalyzerconsistedintheirinitialbreakdownintofatty
PLOSONE|DOI:10.1371/journal.pone.0123509 April9,2015 4/19
AMPDeaminase,AMPKinaseandHibernation
acidsandglycerol.Glyceroloxidationgeneratesaproductthatreactswiththeprobetoproduce
colorat570nm.Similarly,uricaciddeterminationisbasedintheconversionofuricacidtoal-
lantoin,hydrogenperoxide(H O )andcarbondioxidebyuricase.TheH O then,isdeter-
2 2 2 2
minedbyitsreactionwiththeprobetogeneratecoloratapproximately571nm.Values
obtainedwerenormalizedpermgofsolubleproteininthelysates.Hepaticβ-hydroxybutyrate,
inosineandphosphatelevelsinMAPKlysatesweredeterminedbyenzymatickits(K632,K712
andK410,Biovision,Milpitas,CA).
DeterminationofIMPbyHPLC
Liversampleswerepreparedasdescribed[29,30].Inbrief,squirrelliverswerehomogenized
with6volumesofice-cold0.6Mperchloricacid.Extractswerethencentrifugedat14,000gfor
5minandthesupernatantwasneutralizedbyaddingtwovolumesof0.5Mtri-N-octylamine
in1,1,2-trichlorotriflouroethaneandmixedfor1minonavortexmixer.Aftercentrifugation,
analiquotwasinjectedintotheHPLCsystem.ThechromatographicseparationofIMPwas
performedusingaZORBAXEclipseXDB-C18columnwithameanparticularsizeof5mm
(AgilentTechnologies,SantaClara,CA).Theeffluentwasmonitoredat254nmandpeaks
werequantifiedusingpeakheightsandstandardsolutionsofIMP.
Plasmametabolites
Acetoacetateand3-hydroyxbutryate(ketonesgeneratedduringfatoxidation),uricacidandal-
lantoinwereidentifiedandquantifiedingroundsquirrelplasmasamplesbyMetabolonby
massspectrometryasdescribedpreviously[26].
Dataanalysis
Alldataarepresentedasthemean±standarddeviation(SD).Datagraphicsandstatistical
analysiswereperformedusingInstat(version3.0)andPrism5(bothGraphPadSoftware,San
Diego,CA).DatawereanalyzedusingANOVAfollowedbytheTukey-Kramermultiplecom-
parisontestwithα=0.05.Inallcasesexperimentswereperformed3timeswithindependent
replicates.Totaldatapoints(n,numberofgroundsquirrelspertimepoint)areidentifiedin
Figurelegends.
Results
Increasedhepaticlipogenesisanddevelopmentofhepaticsteatosisin
summeractive13-linedgroundsquirrels
Wefirstevaluatedthegroundsquirrelliversforfatbythepresenceofoilred-Ostainingand
triglyceridecontent(Fig1A–1C).Thesestudiesshowedthatfattyliver(hepaticsteatosis)was
presentintheanimalsduringthesummerandfall,anddecreasedmarkedlyduringthehiber-
nationperiod,withthelowestlevelsinthespring.Duringhibernationwecouldnotobserve
significantdifferencesbetweendifferentstagesalthoughwefoundatendencytobelowerin
IBAandEnteringtorporcomparedwiththerestofgroups.Wealsodeterminedthehepatic
abundanceofvariousenzymesinvolvedinfatsynthesisbywesternblotting,includingfatty
acidsynthase(FAS),acetylCoAcarboxylase(ACC)andATPcitratelyase(ACL).Allofthese
enzymeswerehighestduringsummer,begantodecreaseduringthefalltransitionperiod,were
lowthroughoutwinterhibernation,andthenincreasedagaininthespring(Fig2).
PLOSONE|DOI:10.1371/journal.pone.0123509 April9,2015 5/19
AMPDeaminase,AMPKinaseandHibernation
Fig1.13-linedgroundsquirrelsdevelophepaticsteatosiswhenactiveinsummer.(A)Representative
imagesofneutrallipidstaining(oilred-O)inliversfromanimalsinspring,summer,earlytorporandfall
transition.B)Oilred-Oquantitationfromliversfromspring,summer,falltransition,andearlytorporanimals
demonstratesignificantincreaseinhepaticlipidaccumulationinsummertimethatremainshighuntiltorpor.
C)HepaticTGquantitationfromliversofgroundsquirrelsinsummeractive(SA),falltransition(FT),interbout
arousal(IBA),enteringtorpor(Ent),earlytorpor(ET),latetorpor(LT),arousingfromtorpor(Ar),andSpring
(Sp)(n(cid:1)6animalsperphysiologicalstage,smalllettersindicatesignificantlydifferentgroups).
doi:10.1371/journal.pone.0123509.g001
PLOSONE|DOI:10.1371/journal.pone.0123509 April9,2015 6/19
AMPDeaminase,AMPKinaseandHibernation
Fig2.Hepaticlipogenicenzymesaresignificantlydown-regulatedasgroundsquirrelsenterinto
hibernation.A)Representativewesternblotoflipogenicgenesfattyacidsynthase(FAS),acetyl-CoA
carboxylase(ACC)andATP-citratelyase(ACL)inliversfromsummeractive(SA),falltransition(FT),
interboutarousal(IBA),enteringtorpor(Ent),earlytorpor(ET),latetorpor(LT),arousingfromtorpor(Ar),and
Spring(Sp)animals.B-D)Westernblotdensitometryfromallgroundsquirrelsanalyzed,n(cid:1)6animalsper
physiologicalstage,smalllettersindicatesignificantlydifferentgroups.
doi:10.1371/journal.pone.0123509.g002
PLOSONE|DOI:10.1371/journal.pone.0123509 April9,2015 7/19
AMPDeaminase,AMPKinaseandHibernation
Fig3.Fatoxidationisactivatedinliverduringhibernation.A)RepresentativewesternblotofactivatedandtotalAMP-activatedproteinkinase(P-AMPK,
AMPK),inactivatedandtotalACC(P-ACC,ACC),phosphorylatedandtotalLKB1(P-LKB1andLKB1),alpha1andalpha2subunitsofAMPK,carnitine
palmitoyltransferase1A(CPT1A),andenoyl-CoAhydratase1(ECH1)inliversfromanimalsineightcircannualstages(seeFig2legend).B-F)Westernblot
densitometryandP-AMPK/AMPK,P-ACC/ACCratiosfromallgroundsquirrelsanalyzed.G)Intrahepaticβ-hydroxybutyrate(amarkeroffatoxidation)levels
fromallgroundsquirrelsanalyzed,n(cid:1)6animalsperphysiologicalstage,smalllettersindicatesignificantlydifferentgroups).
doi:10.1371/journal.pone.0123509.g003
FatoxidationandAMPKareactivatedinliversofgroundsquirrels
enteringhibernation
WealsoexaminedtheabundanceofbothtotalandactivatedAMPkinase.TheactiveAMPK,
phosphorylatedatthreonine172(P-AMPK),isimportantinoxidationoffattyacids.ECH1
(enoylCoAhydratase-1)andCPT1A(carnitinepalmitoyiltransferase1A)-rate-limitingen-
zymesinβ-fattyacidoxidation-andβ-hydroxybutyrate(aketonegeneratedduringfatoxida-
tion)levelswerealsoassessedforamorecompletepictureoffattyacidoxidationactivity.As
showninFig3A–3F,activated(phosphorylated)AMPKwaslowinsummer,inassociation
withlowlevelsofECH1,CPT1Aandβ-hydroxybutyratelevels,providingevidencethatfatoxi-
dationisminimalatthistime.Thisisconsistentwiththeobservationthathepatictriglyceride
levelsarethehighestinsummerandfalltransition(Fig1C).Duringthewintertorpor-arousal
cycle,however,aninterestingpatternemerged.ActivatedP-AMPKwasmaximallyincreased
duringtheinterboutarousalwhentheanimalhadwarmedto37°C.Asanimalsenteredinto
PLOSONE|DOI:10.1371/journal.pone.0123509 April9,2015 8/19
AMPDeaminase,AMPKinaseandHibernation
torportheP-AMPKlevelbegantofall,andbylatetorporhadreacheditslowestwinterlevel,
whereitremaineduntiltheanimalwarmedagainto37°C(IBA).TotalAMPKalsoreflecteda
similarpatternasP-AMPKwithapredominantexpressionofthealpha1subunit(α1)overthe
alpha2(α2)whichwasobservedonlyinsummer,fallandspringstages.Itisimportanttonote
thattotalAMPKexpressionbegantoincreaseinspring(Sp)evenwhenP-AMPKremained
low(Fig3A).Ofinterest,P-AMPKexpressioncorrelateswiththephosphorylativestateof
ACC.P-ACC(inactive)toACCratio,asshowninFig3D,islowinsummer,fallandspringin-
dicatingthatlivermetabolismisdirectedtowardlipogenesisinthesestages.Incontrast,during
hibernation,P-ACClevelspeakatenteringandinterboutarousalcorrespondingwiththehigh-
estP-AMPKtoAMPKratiosuggestiveofACCphosphorylationbyAMPK.Oneparticulardif-
ferenceisobservedatarousalandlatetorporinwhichP-ACClevelsremainhighdespite
P-AMPKlevelsbeinglow.ThiswouldsuggestthateitherACCcanbephosphorylatedbyan
AMPK-independentmechanismorthatthephosphorylationatACCismorestablethanthe
oneonAMPK.WehypothesizethatthemechanismwherebyAMPD2andAMPKcounter-
regulateismediatedbythepoolofAMPavailabletostimulateeachenzyme.AMPisknownto
activateAMPKallostericallyorthroughliverkinaseB1(LKB1)[31].AsshowninFig3,total
expressionofLKB1doesnotsubstantiallychangeatanystage.However,wefoundthatLKB1
wasphosphorylatedatserine334(P-LKB1)inapatternsimilartotheonefoundforP-AMPK
thussuggestingthepossibilitythatLKB1couldbeanimportantmediatorinAMPKactivation
duringarousalandininterboutarousalstages.ECH1andCPT1aexpressionsincreasedinwin-
tercomparedtosummer,falltransitionandspring.However,withinthewintercycle,both
ECH1andCPT1Aweresignificantlydecreasedatarousalcomparedtotheotherstages.β-
hydroxybutyrate,reflectingfattyacidoxidationthatiscontrolledbyP-AMPK,remainedhigh
throughoutthewinterhibernationstages.Consistently,triglyceridelevelsatIBAandentering
werefoundtobethelowestamongthehibernationstates(Fig1C).Theoverallpatternsuggests
thattheactivationofP-AMPKisdependentonwarmingto37°C;thismayallowthestimula-
tionoffattyacidoxidationtocontinuethroughoutthetorporperiod.
AMPDeaminase2(AMPD2)ShowsanOpposingActivityPattern
ComparedtoAMPKinase
AMPactivatesAMPkinasebutisasubstratefordegradationbyAMPD2(AMPD2)inthe
liver.AMPD2proteinabundancetendedtobehighestinsummer(SA)andduringthefall
transition(FT),withlowerlevelsduringthehibernationperiod,butthedifferenceswerenot
significant(Fig4A).AMPD2enzymaticactivitywasalsomeasured(Fig4BandTable1),and
foundtovaryamongsamplegroupswhenmeasuredat37°C(ANOVAp=0.03).Althoughno
significantpairwisedifferenceswereidentified(Tukeyp>0.05),AMPD2activitytendedtobe
elevatedinSAandFTcomparedtotheothergroups(Table1).WhenthegroupswithTb
~37°C(i.e.,Sp,SA,FTandIBA)werecompared,IBAandSpwerefoundtohavesignificantly
reducedAMPD2activitycomparedtoSAandFT.Moreover,APMD2activitywasseverelyre-
ducedatthephysiologicaltemperaturesoftorpor,andhadintermediateactivityat25°C
(Table1),consistentwithQ effects(ameasureofhowchangesintemperatureaffectsenzyme
10
activity)andindicatingthattheenzymaticactivityofAMPD2isimmeasurablylowwhileani-
malsareintorpor(ETandLT).Hibernatinganimalswarmbrieflyto37°Cduringeachinterb-
outarousal(IBA).TheratioofAMPD2activitytoP-AMPK(reflectingAMPKactivity)when
theanimal’sbodytemperatureisat37°C(SA,FT,IBAandSp)islowestduringIBA(ratio
15.71),comparedtosummeractive(SA,ratio100.25),falltransition(FTratio71.42),and
spring(Spratio61.11).Thus,therelativeactivityofAMPD2toAMPKishigherduringthe
PLOSONE|DOI:10.1371/journal.pone.0123509 April9,2015 9/19
AMPDeaminase,AMPKinaseandHibernation
Fig4.HepaticabundanceandactivityofAMPD22(AMPD2)isreducedduringhibernation.A)Representativewesternblotanddensitometryoftotal
hepaticAMPD2.B)PlotemphasizingreciprocalrelationshipbetweenAMPD2activityandactivatedP-AMPK(formwithphosphorylationatThr172)/AMPK
ratio.AMPD2activityasmeasuredattherelevantphysiologicaltemperatureforthatstate(Table1,e.g.,4°CforETandLT,25°CforEntandArand37°Cfor
SA,FT,IBAandSp).GroupsareasdefinedinFig2.
doi:10.1371/journal.pone.0123509.g004
PLOSONE|DOI:10.1371/journal.pone.0123509 April9,2015 10/19