Metformin Activates AMP Kinase through Inhibition of AMP Deaminase

12 Pages·2010·1.29 MB·English

THEJOURNALOFBIOLOGICALCHEMISTRYVOL.286,NO.1,pp.1–11,January7,2011
©2011byTheAmericanSocietyforBiochemistryandMolecularBiology,Inc. PrintedintheU.S.A.
Metformin Activates AMP Kinase through Inhibition of AMP
Deaminase
Receivedforpublication,March9,2010,andinrevisedform,November5,2010 Published,JBCPapersinPress,November8,2010,DOI10.1074/jbc.M110.121806
JiangyongOuyang‡,RahulkumarA.Parakhia§,andRaymondS.Ochs§1
Fromthe‡DepartmentofPathology,NewYorkUniversitySchoolofMedicine,NewYork,NewYork10010andthe§Departmentof
PharmaceuticalSciences,SchoolofPharmacy,St.John’sUniversity,Queens,NewYork11439
ThemechanismforhowmetforminactivatesAMPK(AMP- olism.Theenzymeisbroadlydistributed,andestablishedasa
activatedkinase)wasinvestigatedinisolatedskeletalmuscle keyregulatorofskeletalmusclemetabolism(4–6).
L6cells.Awidelyheldnotionisthatinhibitionofthemito- TheregulationofAMPKisillustratedinFig.1.AMPKacti-
chondrialrespiratorychainiscentraltothemechanism.We vationrequiresphosphorylationataspecificthreonineresi-
alsoconsideredotherproposalsformetforminaction.Asmet- due(7).Inmuscle,themajorkinasecatalyzingthisreaction
abolicpathwaymarkers,wefocusedonglucosetransportand hasbeensuggestedtobeLKB1(8).Thus,theupstreamkinase
fattyacidoxidation.Wealsoconfirmedmetforminactionson ofAMPKisapotentialregulatorysite(7).Infact,ithasbeen
othermetabolicprocessesinL6cells.Metforminstimulated proposedthatinlivercellsLKB1activationisthemechanism
bothglucosetransportandfattyacidoxidation.Themito- formetforminactivationofAMPK,basedontheobservation
chondrialComplexIinhibitorrotenonealsostimulated thatgeneticablationoftheLKB1eliminatedtheabilityof
glucosetransportbutitinhibitedfattyacidoxidation,inde- metformintoactivateAMPKinvivo(9).However,evidence
pendentlyofmetformin.Theperoxynitritegenerator3-mor- alsoexistsagainstthepossibilitythatLKB1isthetargetof
pholinosydnoniminestimulatedglucosetransport,butinhib- metforminaction(10,11).
itedfattyacidoxidation.Additionofthenitricoxideprecursor Thereareotherproposalsforhowmetforminmightacti-
argininetocellsdidnotaffectglucosetransport.Thesestudies vateAMPK,andalloftheseinvolveincreasedproductionof
differentiatemetforminfrominhibitionofmitochondrialres- AMP.Thisnucleotide,whereaspresentinnearmillimolar
pirationandfromactivenitrogenspecies.Knockdownofad- concentrationsincells,ispresentinitsfreestateinthecy-
enylatekinasealsofailedtoaffectmetforminstimulationof tosolatmicromolarconcentrations(12).IncreasingAMP
glucosetransport.Hence,anymeansofincreaseinADPap- concentrationsfavoritsbindingtoAMPK,bothpromoting
pearsnottobeinvolvedinthemetforminmechanism.Knock- andmaintainingtheactivephosphorylatedform(13).
downofLKB1,anupstreamkinaseandAMPKactivator,did Acommonlyproposedmechanismformetforminactionis
notaffectmetforminaction.Havingruledoutexistingpropos- aninhibitionofmitochondrialrespirationatComplexI.This
als,wesuggestanewone:metforminmightincreaseAMP interruptionofenergyproductionwoulddecreaseATPpro-
throughinhibitionofAMPdeaminase(AMPD).Wefoundthat ductionandhenceelevatetheconcentrationofADP.Thisin
metformininhibitedpurifiedAMPdeaminaseactivity.Fur- turnwouldelevatetheconcentrationofAMPthroughthe
thermore,aknowninhibitorofAMPDstimulatedglucoseup- actionofadenylatekinase(14–17).Thisisconsistentwithan
takeandfattyacidoxidation.BothmetforminandtheAMPD increaseinglucosetransportthatoccurswithmitochondrial
inhibitorsuppressedammoniaaccumulationbythecells. respiratoryinhibition(18).
KnockdownofAMPDobviatedmetforminstimulationofglu- Avariantonthemitochondrialrespirationinhibitionisthe
cosetransport.WeconcludethatAMPDinhibitionisthe possibilitythatareactivenitrogen(19)and/oroxygenspecies
mechanismofmetforminaction. (20)isresponsibleforactivationofAMPK.Thusnitricoxide
ortheadductperoxinitrateofnitricoxideplussuperoxide
(whichisformedwhenmitochondrialrespirationisblocked),
MetforminiscurrentlythemostwidelyuseddrugforType havebeenshowntoactivateAMPK,andcouldalsoaccount
IIdiabetes,anditsuseisindicatedforotherdiseasesrelated forincreasedglucoseuptake(19).
tometabolicsyndrome(1).Althoughthedrughasbeenused However,itisalsoknownthatinbothliverandmuscleother
forover50years(2),itsmechanismremainsunclear.Re- metabolicprocessesareaffectedbymetformin.Inbothofthese
cently,itwasdiscoveredthatmetformincausesanactivation tissues,metforminstimulatesfattyacidoxidation(21–24).Itis
oftheenzymeAMP-activatedkinase(AMPK)2(3),whichis difficulttorationalizeaninhibitionofrespirationwithastimula-
nowestablishedasacentralregulatorofintermediarymetab- tionoffattyacidoxidation.Wethereforeassessedcurrentlypro-
posedmechanismsofactionformetforminfortheirabilityto
stimulatebothglucoseuptakeandfattyacidoxidation.Asnone
1Towhomcorrespondenceshouldbeaddressed.Tel.:[phone];Fax:
ofthecurrentproposalsmettheseconditions,weformedanew
718-990-1936;E-mail:[email protected].
2Theabbreviationsusedare:AMPK,AMP-activatedproteinkinase;ACC, hypothesis:AMPdeaminase(representingAMPbreakdown)
acetyl-CoAcarboxylase;AK1,adenylatekinase1;AMPD,AMPdeaminase;
EHNA,erythro-9-(2-hydroxy-3-nonyl)-adenine;KHB,Krebs-Henseleit
buffer;MCD,malonyl-CoAdecarboxylase;(cid:1)-MEM,(cid:1)-minimumessential SIN-1,3-morpholinosydnonimine;BisTris,2-[bis(2-hydroxyethyl)amino]-2-
medium;MTT,3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide; (hydroxymethyl)propane-1,3-diol.
JANUARY7,2011•VOLUME286•NUMBER1 JOURNALOFBIOLOGICALCHEMISTRY 1
This is an Open Access article under the CC BY license.
ActivatingAMPKinasethroughAMPDeaminase
takenforenzymaticendpointassay(26)usingtheHTS7000
BioPlateReader.
GlycogenContentMeasurement—Glycogencontentwas
assessedbyusingmodifiedamyloglucosidaseassayasprevi-
ouslydescribed(27,28).Afteraspirationofthemedium,ad-
herentcellmonolayerswereextractedwith400(cid:2)lof30%
(w/v)KOHandboiledfor15min.Aliquotswerespottedonto
Whatman3Tpaper,whichwasthenimmersedinice-cold
66%(v/v)ethanol.Thepaperswerewashedtwicewith66%
FIGURE1.AMPKinmuscle.Theproposedmechanismsfortheactionof
ethanol,anddriedpaperscontainingprecipitatedglycogen
metformininstimulatingAMPKcanbemappedtothisdiagram.Theen-
zymeneedstobephosphorylated(1),whichiscatalyzedprincipallybyLBK1 wereincubatedin2mlof0.4Macetatebuffer(pH4.8)con-
inliverandmuscle.Dephosphorylation(2)inactivatestheenzyme;how- taining25units/mlof(cid:1)-amyloglucosidasefor90minat37°C.
ever,bindingtoAMPleadstoactivation(3)bymakingAMPKapoorersub-
Glucosereleasedfromglycogenwasmeasuredbyusingan
strateforthephosphatase.Normally,energydemand(4)canincreaseADP
andconsequentlyAMPconcentrationbymeansofincreasedflowthrough enzymaticendpointassayforglucoseusinghexokinase.
adenylatekinase(5).Alternatively,AMPmayarisebyanincreasedADPdue GlucoseUptakeAssay—Glucoseuptakewasdeterminedas
tointerruptionofmitochondrialenergysupply,whichwouldalsoincrease
AMPconcentrationinvolvingflow-throughadenylatekinase(5).Thepres- therateof2-deoxy-D-[2,6-3H]glucoseuptake,usingamodifi-
entworksupportsanewsiteofactionformetformin,theAMPdeaminase cationofapreviousmethod(29).Thecellswerefirstincu-
(6).Alsoshownareseveralmetabolicprocessesknowntobeaffectedby
batedfor15minwithKHB,glucose,andotheragentsasindi-
AMPKactivationviametformin:glucoseuptake,fattyacidoxidation,glyco-
gensynthesis,andlactateformation. cated.Atthispoint,thelabeleddeoxyglucose(0.6(cid:2)Ci)was
addedtoeachwellandtheincubationcontinuedfor45min.
Themediumwasaspirated,andthewellswerewashedthree
mightbeinhibitedbymetformin.Wepresentevidenceconsist-
timeswithice-coldKHBtoremoveexogenouslabel.Thecells
entwiththishypothesis.
werelysedbytheadditionof0.1%TritonX-100(1ml).Sam-
plesofeachwellweremixedwithaqueousscintillationfluid
EXPERIMENTALPROCEDURES
andmeasuredbyliquidscintillationcounting.
Materials—Metformin,andmostchemicals,includingBSA PalmitateOxidationAssay—Palmitateoxidationwasdeter-
(bovineserumalbumin,essentiallyfattyacidfree),wereob- minedbyamodificationofquantitativemeasuringtherateof
tainedfromSigma.Priortouse,BSAwasdialyzedextensively 14CO productionfrom14C-labeledpalmiticacidasdescribed
2
againstKrebs-Henseleitbicarbonatebuffer(KHB).Palmitic previously(30).TheKHBfortheseincubationswassupple-
acidwasfromNu-ChekPrepInc.(Elysian,MN).Dulbecco’s mentedwithfattyacid-pooralbumindialyzedagainstthe
modifiedEagle’smedium(DMEM),(cid:1)-minimumessential samebuffer(threechanges).Thefinalalbuminconcentration
medium((cid:1)-MEM),Hanks’balancedsaltsolution,fetalbovine was1%.Afteraninitial10minofincubation,allsamplesre-
serum(FBS),horseserum,trypsin,andNuPAGE4–12%Bis- ceived2mMcarnitineand[1-14C]palmiticacid(1(cid:2)Ci/mmol),
Trisgel,andLipofectaminetransfectionreagentswerefrom andotheradditionsasnoted,andincubationswerecontinued
Invitrogen.ScintillationvialswereobtainedfromWheaton for3h.Aliquotsof0.8mlweretakenfromeachwelltoan
(Millville,NJ)andScintisafe(30%advancedsafetyLSCmix- Eppendorftube.Eachtubehadacircularpieceoffilterpaper,
ture)fromFischerScientific(Fairlawn,NJ).Cellcultureplates producedwithapaperpunch,attachedtotheinsideofthelid,
andflaskswereobtainedfromCorningInc.(Corning,NY). towhich15(cid:2)lof2MNaOHwasadded.200(cid:2)lof3Mper-
2-Deoxy-D-[2,6-3H]glucose,[1-14C]palmiticacid,and chloricacidwascarefullyaddedtothe0.8mltoensureno
D-[U-14C]glucosewerefromAmershamBiosciences.Nitro- acidwasdepositedonthesideofthetube,andthelidwas
cellulosemembraneswereobtainedfromBio-Rad.Adenylate quicklyclosed.Thetubeswereincubatedovernighttoallow
kinase1siRNA(siGenomeduplexAK1),DharmaFECT3 the[14C]CO tobeabsorbedintothewick.Thecapswere
2
siRNAtransfectionreagent,negativecontrolsiRNA detachedandplacedin10mlofliquidscintillationfluidfor
(siCONTROLNon-TargetingsiRNA),5(cid:1)siRNAbuffer,and counting.
transfectionmediumwereobtainedfromDharmaconInc. GlycogenSynthesis—Glycogensynthesiswasdeterminedby
(Chicago,IL).AK1antibody,LKB1siRNA,andAMPDsiRNA amodificationoftheincorporationofD-[U-14C]glucoseinto
werefromSantaCruzBiotechnology.Allotherantibodies glycogenaspreviouslydescribed(31,32).Afterincubation,
includingAMPK(cid:1)antibodyandP-AMPK(cid:1)antibody(phos- cellswerewashedwithice-coldKHBandlysedin0.5mlof
phor-Thr172specific)werefromCellSignalingTechnology KOH(30%).Aliquotsof0.2mlwerespottedontoWhatman
(Danvers,MA). 3Tpaper,andglycogenwasprecipitatedbyimmersingthe
CellCulture—L6ratskeletalmusclecellswereculturedin papersinice-cold66%(v/v)ethanolovernight.Whatman3T
DMEMsupplementedwith10%FBS.Cellswereconvertedto papercontainingprecipitatedglycogenwastransferredto
myotubesbyswitchingto(cid:1)-MEMwith2%horseserumfor1 scintillationvialsforradioactivitycounting.
day,andthento(cid:1)-MEMwith1%horseserumfor5–8days TransfectionofsiRNA—L6cellsweretransfectedwith100
priortouseforexperiments.Detailsofthecellpreparation nMAK1/LKB1/AMPDsiRNAfor3daysbythefollowingpro-
areprovidedinapriorpublication(25). cedure:104cells/wellwereseededin12-wellplatesthatwere
LactateFormation—Forlactateformation,cellswereincu- incubatedwithDMEMwith10%FBSfor2hbeforetransfec-
batedfor4h,afterwhich125(cid:2)lofincubationmediumwas tionwith2(cid:2)MsiRNAsolutionin1(cid:1)siRNAbufferorcontrol
2 JOURNALOFBIOLOGICALCHEMISTRY VOLUME286•NUMBER1•JANUARY7,2011
ActivatingAMPKinasethroughAMPDeaminase
RNase-freesolution.siRNAandtransfectionreagentwere imidazole-HCl(pH7.0),2mMAMP,and150mMKCl.The
dilutedwithtransfectionmedium,addedtocellsinDMEM enzymeactivityisexpressedasmicromolesofIMPproduced
and10%FBS,andincubatedat37°Cin5%CO for2days. perminpermgofsolubleprotein.
2
Themediumwaschangedto(cid:1)-MEMwith1%horseserum AmmoniaAssay—Ammoniawasmeasuredasdescribedby
withtransfectionsolutionfor1day.Finally,themediumwas Kun(35).Anendpointanalysiswasperformedmeasuring
switchedto(cid:1)-MEMwith1%horseserumwithouttransfec- (cid:1)-ketoglutaricacid-dependentNADHoxidationat340nm,
tionsolutionforanother4days.Forexperiments,cellswere drivenbyglutamatedehydrogenase.
incubatedwithserum-freemediumfor2h,treatedwithor StatisticalAnalysis—Statisticalevaluationwasperformed
without10mMmetformininthepresenceof5mMglucosefor usingStudent’sttestorone-wayanalysisofvariancefollowed
2h.Expressedproteincontent(AK1,LKB1,andAMPD)was bytheStudent-Newman-Keulsposthoctestasappropriate.
visualizedbyWesternblot. Dataareexpressedasmean(cid:2)S.E.(errorbars),andthelevel
WesternBlot—Toextracttotalprotein,cellswerelysed ofsignificancewassetatp(cid:3)0.05.
with100(cid:2)lofbuffercontaining2%(w/v)SDS,62.5mMTris
RESULTS
(pH6.8),10%glycerol,50mMDTT,and0.01%(w/v)brom-
phenolblue.Theprocedureofthecommercialsourceofthe MetabolicCharacterizationofMetformininL6Skeletal
antibody(CellSignalingTechnology)wasessentiallyfollowed. MuscleCellCultures—Todemonstratecharacteristicmet-
Thelysateswereheatedat95–100°Cfor5minandcooledon forminactioninourmodel,wemeasuredanumberofparam-
ice.Aliquotsof20(cid:2)gofproteinwereloadedontoa10%SDS- eterslinkedtometformin.Theseincluded:glucoseuptake,
PAGEgel,andsubsequentlyelectroblottedontonitrocellulose fattyacidoxidation,lactateformation,andthephosphoryla-
membranes.Membraneswereincubatedatroomtempera- tionofAMPK.Forthekeyprocessesofglucoseuptakeand
turefor1hinblockingbuffercontainingTris-bufferedsaline, fattyacidoxidation,weadditionallyincubatedcellswithsub-
0.1%Tween20,and5%nonfatdrymilk.Membraneswere strateconcentrationsrepresentingsubmaximalandmaximal
thenincubatedwith1:1000dilutionofprimaryantibodyin10 concentrationstoassessthisvariableinmetforminaction.
mlofblockingbufferovernightat4°C.Thenmembranes Theknownabilityofmetformintocausephosphorylation
wereincubatedwithhorseradishperoxidase-conjugatedsec- ofAMPKwasconfirmed(Fig.2A).Wehaveconfirmedand
ondaryantibody(1:2000)andhorseradishperoxidase-conju- extendedmetforminstimulationofglucosetransportforL6
gatedanti-biotinantibody(1:1000)todetectbiotinylatedpro- cells,usingtwoseparateconcentrationsofmediumglucose
teinmarkersin10mlofblockingbufferfor1hatroom (Fig.2B).Metforminalsostimulatedpalmitateoxidationat
temperature.Immunoreactiveproteinswererevealedusing twolevelsofthissubstrate:submaximal(60(cid:2)M)andnearsat-
enhancedchemiluminescencedetection(LumiGLOTM,Cell urating(120(cid:2)M)palmitateconcentration,asevidentinFig.
Signaling). 2C.Wealsoexploredthemetabolicconsequencesofthe
AMPDeaminasePurification—RabbitskeletalmuscleAMP knownglycogensynthaseinhibitionbyAMPKactivation(36,
deaminasewaspurifiedbyusingcellulosephosphateasde- 37).AsevidentfromFig.2D,metformininhibitedglycogen
scribedbySmileyetal.(33).Briefly,frozenrabbitmusclewas synthesis,althoughnochangeintotalglycogencontentwas
ground,mixedwith3.3timesitsweightofextractionbuffer observed(Fig.2E).Wealsoconfirmedanincreaseinlactate
(0.18MKCl,0.054MKH PO ,0.035MK HPO ,pH6.5),and productionbymetformin(Fig.2F),onceagainatdifferent
2 4 2 4
homogenizedfor15sinaWaringBlender.Theslurrywas concentrationsofmediumglucose.
stirredatroomtemperaturefor1handcentrifugedat Thepercentstimulationofbothglucosetransportandfatty
14,000(cid:1)gfor15min.Thesupernatantfractionwaspoured acidoxidationinthisstudywasusuallybetween30and50%;
throughtwolayersofcheesecloth,afterwhichdrycellulose lactateformationwasstimulatedsomewhatmore(about
phosphate,whichwaspreviouslywashedandequilibrated 60%).Thechangeswereunaffectedbysubstrateconcentra-
withtheextractionbuffer,wasadded.Afterstirringatleast10 tions(glucose,palmitate).
min,thecellulosephosphatewasallowedtosettle.Thecellu- ComparisonofMetformintoRotenone—Severalreports
losephosphateslurrywastransferredtoasinteredglassfilter, suggestthatmetforminactsthroughinhibitionofmitochon-
andwashedrepeatedlywithextractionbufferfollowedbya drialrespirationbyinhibitingcomplexIoftheelectrontrans-
solutioncontaining0.45MKCladjustedtopH7with1M portchain(38–40).Toinvestigatethismechanismweused
K HPO .Whennomoreproteinwasdetectedinthewash, rotenone,amitochondrialrespirationinhibitorknowntoact
2 4
thecellulosephosphatewaselutedwith1MKCl(pH7).The atComplexI.Itiswellknownthatrotenonecanstimulate
A :A ratioforthefinalelutewas1.8orhighershowing bothglucosetransportandAMPKphosphorylation(41).
280 260
littleornocontaminationbynucleicacids.Theenzymewas However,rotenonedidnotreplicatemetforminactionson
precipitatedbytheadditionof0.26gofsolidK HPO for fattyacidoxidation.Whereasmetforminstimulatedfattyacid
2 4
eachmilliliterofsolution.Aftercentrifugation,theprecipitate oxidation,rotenoneinhibitedthisprocess,atanyconcentra-
wasdissolvedinaminimalvolumeof0.45MKC1atpH7to tionupto250(cid:2)M(Fig.3A).Althoughthestimulationoffatty
formanearsaturatedsolutionatroomtemperature. acidoxidationbymetforminiswellestablished,andtheinhi-
EnzymeAssay—AMPdeaminaseactivitywasmeasuredas bitionofmitochondrialrespirationbyrotenoneisalsowell
describedbyAshbyandFrieden(34).Theassaymonitored known,thisisthefirstreporttodemonstratebothwithinthe
theabsorbancechangeat285nmasaresultofAMPconver- contextofmetforminaction.Thisstronglybringsintoques-
siontoIMPinareactionvolumeof2mlcontaining50mM tionthepossibilitythatmetforminactsasaninhibitorofmi-
JANUARY7,2011•VOLUME286•NUMBER1 JOURNALOFBIOLOGICALCHEMISTRY 3
ActivatingAMPKinasethroughAMPDeaminase
4 JOURNALOFBIOLOGICALCHEMISTRY VOLUME286•NUMBER1•JANUARY7,2011
ActivatingAMPKinasethroughAMPDeaminase
FIGURE3.Effectofrotenoneonfattyacidoxidationandglucoseuptake.A,effectofdifferentconcentrationsofrotenoneonmetformin-inducedpalmi-
tateoxidationinL6myotubes.L6cellswereincubatedwith120(cid:2)Mpalmiticacidwithorwithoutdifferentconcentrationsofrotenoneand10mMmet-
forminfor3h,thenpalmitateoxidationwasmeasuredasdescribedunder“ExperimentalProcedures.”Control(darkbar)orwithmetformin(lightbar);*,p(cid:3)
0.05.B,effectofrotenoneonglucoseuptakeinL6cells.L6cellswereincubatedwith5mMglucosewithorwithout250(cid:2)Mrotenone.Dataareexpressedas
permgofcellularprotein.
tochondrialrespiration.Weconfirmedtherotenonestimula- factstimulateglucosetransport(Fig.4C),itinhibitedfatty
tionofglucosetransportatthehighestlevelofrotenoneused acidoxidation(Fig.4D).Thus,itsbehaviormimickedrote-
forfattyacidoxidation(Fig.3B).Thepercentstimulationof none,ratherthanmetformin.
palmitateoxidationbymetforminwasabout30%inthisseries ExplorationofaRoleforLKB1—LKB1isanupstreamki-
ofexperiments,andwasunaffectedbythevariousconcentra- naseofAMPK,proposedasthemediatorofmetforminaction
tionsofrotenone. inliver(9)andestablishedastheprincipalkinaseforconvert-
PossibleRoleofReactiveNitrogenSpeciesinMetformin ingAMPKtotheactiveforminmuscle(10).Todetermineits
Mechanism—Analternativeproposalforthemechanismof possibleroleinmetforminactioninL6cells,aknockdownof
metforministheinvolvementofnitricoxide(42).Asonetest LKB1proteinwasperformedusingaselectivesiRNA.As
ofthispossibility,weincubatedL6cellswitharginine,apre- showninFig.5A,thesiRNAforLKB1,butnotthecontrol
cursorofnitricoxideandmeasuredeffectsonglucosetrans- siRNAsubstantiallyreducedLKB1proteinlevels.Asshownin
port.However,evenatlevelsupto40mM,argininehadno Fig.5B,ablationofLKB1hadnoeffectontheabilityofmet-
effectonglucosetransport(Fig.4A).Asaconverseexperi- formintostimulateglucoseuptake.
ment,wedeterminedifmetforminstimulationcouldbean- PossibleInvolvementofAdenylateKinaseinMetformin
tagonizedbyinhibitionofarginase.However,asshowninFig. Action—Adenylatekinasecatalyzesthenearequilibriumreac-
4B,additionoftwodifferentselectivearginaseinhibitorsdid tionofADPwithATPandAMP;theprincipalmuscleiso-
notaffectmetforminstimulationofglucosetransport. formisAK1(45).IfmetforminactioncausesincreasedATP
Arelatedpossibilityisthatthereactivenitrogenspecies turnoverbyanymeans,adenylatekinasefluxwouldbere-
peroxynitrite(ONOO(cid:4)),theadductofnitricoxidewithsu- quiredfortheincreasedAMPtoactivateAMPK.Totestthis
peroxide,istheintermediateformetforminactivationof possiblerole,aknockdownofAK1wasperformedwithase-
AMPK(19).Totestthis,weusedthecompoundSIN-1, lectivesiRNA.Fig.6AshowsthatthesiRNAforAK1reduced
knownasageneratorofONOO(cid:4)(43).AlthoughSIN-1didin proteinexpressiontoverylowlevels.Asadenylatekinaseisin
FIGURE2.MetaboliceffectsofmetformininL6myotubes.A,effectofmetforminonAMPK(cid:1)phosphorylationinL6myotubes.L6cellswereincubated
withorwithout10mMmetforminfor2h,andthenAMPK(cid:1)phosphorylationatThr172wasmeasuredbyWesternblot;AMPK(cid:1)wasusedasacontrol.
P-AMPK(cid:1),phosphorylatedAMPK(cid:1).AMPK(cid:1)andP-AMPK(cid:1)wereprobedwithseparateselectiveantibodies.Con,control;Met,metformin.B,effectofmet-
forminonglucoseuptakeinL6myotubes.L6cellswereincubatedwithlow(0.5mM)orhigh(5mM)glucosewithorwithout10mMmetforminfor2h,and
thenglucoseuptakewasmeasuredasdescribedunder“ExperimentalProcedures.”*,p(cid:3)0.05versus5mMglucose(5mMGlu);*,p(cid:3)0.05versus0.5mM
glucose(0.5mMGlu);Met,metformin.C,effectofmetforminonpalmitateoxidationinL6myotubes.L6cellswereincubatedwith60or120(cid:2)Mpalmiticacid
withorwithout10mMmetforminfor3h,andthenpalmitateoxidationwasmeasuredasdescribedunder“ExperimentalProcedures.”*,p(cid:3)0.05versus60
(cid:2)Mpalmiticacid(60(cid:2)MPA)and120(cid:2)Mpalmiticacid(120(cid:2)MPA),respectively;Met,metformin.D,effectofmetforminonglycogensynthesisinL6myo-
tubes.L6cellswereincubatedwith0.5or5mMglucosewithorwithout10mMmetforminfor2h,andthenglycogensynthesiswasmeasuredasdescribed
under“ExperimentalProcedures.”*,p(cid:3)0.05versus0.5mMglucose(0.5mMGlu)and5mMglucose(5mMGlu),respectively;Met,metformin.E,effectofmet-
forminonglycogencontentinL6myotubes.L6cellswereincubatedwith0.5or5mMglucosewithorwithout10mMmetforminfor4h,andthentotalgly-
cogencontentwasmeasuredasdescribedunder“ExperimentalProcedures.”Glu,glucose;Met,metformin.F,effectofmetforminonlactateformationinL6
myotubes.L6cellswereincubatedwith0.5or5mMglucosewithorwithout10mMmetforminfor4h,andthenlactateformationwasmeasuredasde-
scribedunder“ExperimentalProcedures.”*,p(cid:3)0.05versus0.5mMglucose(0.5mMGlu)and5mMglucose(5mMGlu),respectively.Dataareexpressedas
permgofcellularprotein.
JANUARY7,2011•VOLUME286•NUMBER1 JOURNALOFBIOLOGICALCHEMISTRY 5
ActivatingAMPKinasethroughAMPDeaminase
FIGURE4.Effectofarginineandreactivenitrogenspeciesonmetforminaction.A,effectofdifferentconcentrationsofarginineonglucoseuptakeinL6
myotubes.L6cellswereincubatedwith5mMglucosewithorwithoutdifferentconcentrationsofargininefor2h.B,effectofarginaseinhibitorsonmet-
formin-inducedglucoseuptakeinL6myotubes.L6cellswereincubatedwith5mMglucosewithorwithout10mMmetformin,andcombinedwith10
(cid:2)MNOHAor1(cid:2)MBECfor2h,andthenglucoseuptakewasmeasured.Met,metformin;NOHA,N(cid:3)-hydroxy-nor-L-arginine;BEC,(S)-(2-boronoethyl)-L-
cysteine,HCl.*,p(cid:3)0.05versuscontrol.C,effectofSIN-1onglucoseuptakeinL6myotubes.L6cellswereincubatedwith5mMglucosewithorwith-
out1mMSIN-1for2h,andthenglucoseuptakewasmeasuredasdescribedunder“ExperimentalProcedures.”*,p(cid:3)0.05versuscontrol.D,effectof
SIN-1onpalmitateoxidationinL6myotubes.L6cellswereincubatedwith120(cid:2)Mpalmiticacidwithorwithout1mMSIN-1for3h,andthenpalmi-
tateoxidationwasmeasuredasdescribedunder“ExperimentalProcedures.”*,p(cid:3)0.05versuscontrol.Dataareexpressedaspermgofcellular
protein.
veryhighconcentrationsinmuscle,weperformedafurther
controltoassessthefunctionalknockdownofAK1,shownin
Fig.6B.Weusedpotassiumdepolarizationasameanstoin-
creaseATPturnover,whichshouldthenincreaseAMPby
fluxthroughadenylatekinase.Fig.6Bshowsthattheknock-
downitselfdidnotaffectglucoseuptake,nordidsub-thresh-
oldlevelsofpotassium.However,80mMorhigherlevelsof
potassiumdidincreaseglucosetransport,andunderthese
circumstances,concomitantknockdownofadenylatekinase
ledtoinhibitionofglucosetransportbelowcontrollevels.
Thisestablishedthevalidityofdepolarizationasacontrolfor
adenylatekinaseinvolvement.
WethenexaminedtheeffectoftheAK1knockdownonmet-
forminstimulationofglucosetransport.Wefoundthat,unlikeK
depolarization,metforminstimulationofglucoseuptakewas
unaffectedbytheablationofadenylatekinase(Fig.6C).
AMPDeaminaseInhibitionasaLocusofMetforminAction—
Havingruledoutexistingproposalsfortheactionofmet-
formin,weconsideredanewpossibility:ratherthan
metforminincreasingtheformationofAMP,perhapsit
attenuatesitsremoval.BecausethefirststepofAMPre-
FIGURE5.EffectofLKB1onmetforminaction.A,LKB1proteinexpres- movalisdeaminationtoIMP,weexaminedthepossibility
siontreatedwithLKB1siRNA.B,effectofLKB1siRNAonmetformin-in-
thatAMPdeaminase(AMPD)mightbeinhibitedby
ducedglucoseuptakeinL6myotubes.AfterLKB1siRNAtransfection,L6
cellswereincubatedwith5mMglucosewithorwithout10mMmet- metformin.
forminfor2h,andthenglucoseuptakewasmeasuredasdescribedun-
Fig.7Ashowsthatmetformininhibitedtheactivityofpuri-
der“ExperimentalProcedures.”Dataareexpressedaspermgofcellular
protein.*,p(cid:3)0.05versusnometformin. fiedAMPD,obtainedfromrabbitmuscle.Wehavefoundin-
6 JOURNALOFBIOLOGICALCHEMISTRY VOLUME286•NUMBER1•JANUARY7,2011
ActivatingAMPKinasethroughAMPDeaminase
FIGURE6.Effectofadenylatekinaseonmetforminaction.A,AK1proteinexpressionafterablationwithAK1siRNA.B,effectofadenylatekinaseknock-
downonpotassiumgluconateactionsonglucoseuptake.Potassiumgluconatewaspresentat40mM(40K),80mM(80K),and120mM(120K).C,control;
A,AK1siRNA;K,potassiumgluconate.#,p(cid:3)0.05comparedwithcontrol.*,p(cid:3)0.05comparewith80and120KwithoutAK1siRNA.C,metformin-induced
glucoseuptakeinL6myotubes.AftertransfectedbyAK1siRNA,L6cellswereincubatedwith5mMglucosewithorwithout10mMmetforminfor2h,and
thenglucoseuptakewasmeasuredasdescribedunder“ExperimentalProcedures.”Met,metformin.*,p(cid:3)0.05comparedwithnometformin.Dataareex-
pressedaspermgofcellularprotein.
FIGURE7.EffectofmetforminonAMPdeaminaseactivity.A,theeffectof10mMmetforminonenzymeactivityofisolatedAMPdeaminaseatvarious
substrate(AMP)concentrations.*,p(cid:3)0.05versuscontrolatdifferentconcentrationofAMP,respectively.Dataareexpressedaspermgofsolubleprotein.
B,effectofmetforminonammoniaproductioninL6myotubes.L6cellswereincubatedwith5mMglucosewithorwithout10mMmetforminfor2h,and
ammoniaproductionwasmeasuredasdescribedunder“ExperimentalProcedures.”*,p(cid:3)0.05versuscontrol.Dataareexpressedaspermgofcellu-
larprotein.
distinguishableresultsusingcommerciallyobtainedadeno- 7B)thatmetformindecreasedammoniaaccumulationinthe
sinedeaminase(datanotshown).Thisisprobablyduetothe medium.
factthattheenzymemechanismsareextremelysimilar;in Asaseparatetest,wecomparedtheactionofaknown
factbothenzymesareequallysensitivetothesameinhibitors AMPDinhibitorerythro-9-(2-hydroxy-3-nonyl)-adenine
(46). (EHNA)withmetformin.AsshowninFig.8,AandB,EHNA
Afurtherpredictionbasedonthehypothesisthatmet- didinfactstimulatebothglucosetransportandfattyacidoxi-
forminactsthroughAMPDinhibitionisthatammoniafor- dationlikemetformin,andthattheactionsofthetwoagents
mationshouldbereducedwhencellsareincubatedwithmet- werenotadditive.Fig.8Cshowsthat,likemetformin,EHNA
formin.Inagreementwiththisexpectation,wefound(Fig. alsoledtoanincreasedphosphorylationofAMPK.EHNA
JANUARY7,2011•VOLUME286•NUMBER1 JOURNALOFBIOLOGICALCHEMISTRY 7
ActivatingAMPKinasethroughAMPDeaminase
FIGURE8.EffectofEHNA,metformin,orbothonglucoseuptakeinL6myotubes.A,L6cellswereincubatedwith5mMglucosewithorwithout10mM
metformin,100(cid:2)MEHNA,orbothfor2h,andthenglucoseuptakewasmeasuredasdescribedunder“ExperimentalProcedures.”*,p(cid:3)0.05versuscontrol.
B,effectofEHNA,metformin,orbothonpalmitateoxidationinL6myotubes.L6cellswereincubatedwith120(cid:2)Mpalmiticacidwithorwithout10mMmet-
formin,100(cid:2)MEHNA,orbothfor3h,andthenpalmitateoxidationwasmeasuredasdescribedunder“ExperimentalProcedures.”*,p(cid:3)0.05versuscontrol.
C,effectofEHNAonAMPK(cid:1)phosphorylationinL6myotubes.L6cellswereincubatedwithorwithout100(cid:2)MEHNAfor2h,andthenAMPK(cid:1)phosphoryla-
tionatThr172wasmeasuredbyWesternblot;AMPK(cid:1)wasusedasacontrol.P-AMPK(cid:1),phosphorylatedAMPK(cid:1).AMPK(cid:1)andP-AMPK(cid:1)wereprobedwith
separateselectiveantibodies.D,effectofEHNAonammoniaproductioninL6myotubes.L6cellswereincubatedwith5mMglucosewithorwithout100(cid:2)M
EHNAfor2h,andthenammoniaproductionwasmeasuredasdescribedunder“ExperimentalProcedures.”*,p(cid:3)0.05versuscontrol.Dataareexpressedas
permgofcellularprotein.
alsoreplicatedthemetformin-inducedinhibitionofammonia
formationbyL6cells,andthisactionwasnotadditivetothat
ofmetformin(Fig.8D).
Finally,weconductedknockdownofAMPD1usingsiRNA
inL6cells.Ifourhypothesisiscorrect,thistreatmentshould
preventmetforminstimulationofglucosetransport.Asis
evidentinFig.9,theknockdownexperimentgreatlyreduced
AMPDprotein,andobviatedthemetforminstimulationof
glucoseuptakebythecells.
DISCUSSION
Boththeinterruptionofmitochondrialenergyformation
andthepresenceofmetforminleadtothesameendpoint:the
activationofAMPK(3,20).Itisthereforenotsurprisingthat
thehypothesisemergedthatmetformincausesrespiratory
inhibition,andtherebyactivatesAMPK(40).Allthreeevents:
mitochondrialinhibition,thepresenceofmetformin,andthe
activationofAMPK,alsocorrelatewithastimulationofglu-
coseuptake(7,48).
Ifweconsideronlyglucoseuptakeandmitochondrialalter-
ations,theeffectsseemconsistentwithfundamentalcellregu-
latoryproperties.Thenotionthatadecreasedmitochondrial FIGURE9.EffectofAMPDonmetforminaction.A,AMPdeaminasepro-
teinexpressiontreatedwithAMPDsiRNA.B,effectofAMPDsiRNAonmet-
activityincreasesglycolysisisknownastheCrabtreeeffect formin-inducedglucoseuptakeinL6myotubes.AfterAMPDsiRNAtransfec-
(49,50),theoppositeofthePasteureffect(decreasedglucose tion,L6cellswereincubatedwith5mMglucosewithorwithout10mM
metforminfor2h,andthenglucoseuptakewasmeasuredasdescribed
utilizationuponadmissionofoxygentocells).However,the
under“ExperimentalProcedures.”Dataareexpressedaspermgofcellular
widerconsiderationoffattyacidoxidationdevelopedinthis protein.*,p(cid:3)0.05versusnometformin.
8 JOURNALOFBIOLOGICALCHEMISTRY VOLUME286•NUMBER1•JANUARY7,2011
ActivatingAMPKinasethroughAMPDeaminase
studyunderscoresthecomplicationofdrawingtheconclu- notmetformin,anditbecomesclearthataseparatemecha-
sionthatmetforminactsbymitochondrialrespiratoryinhibi- nismformetforminactionisneeded.
tion.Itislikelythattheresponseofthecelltoenergyinter- Weconcludethattherearetwodistinctmeansofactivating
ruptionisadistinctmeansofAMPKactivationfromthe AMPK.Oneoftheseisrepresentedbyinterruptionofmito-
responsetometformin. chondria,andphysiologicallyrepresentedbyanaerobicme-
Arecentdebatehighlightedthepositionsfor(11)and tabolism.Becausefattyacidoxidationisnotpossibleinthis
against(51)thenotionthatmetformininhibitsmitochondrial condition,theonlyenergypathwayavailable,glycolysis,is
energyproduction.Akeyargumentagainstaninvivorolefor enhancedbytheAMPK-directedstimulationofglucosetrans-
metforminactingasamitochondrialinhibitoristhefactthat port.Metforminalsostimulatesglucoseuptakebutaddition-
theextentofinhibitionismodest.Afurthercomplicationis allystimulatesfattyacidoxidationandcanthereforeoperate
thefactthatinsulinresistanceofdiabetescanbeameliorated aerobically.TheabilitytoinhibitAMPDpresentsaseparate
bysuppressingtheoxidativedamagethatarisesfrommito- meansofincreasingAMPK,leadingtoincreasedutilizationof
chondrialinhibition(52).Thismakesmitochondrialinhibi- bothmajorenergyfuels,glucoseandfat.
tionanunlikelysiteofactionformetformin,adrugthatim- Wewereunabletofindevidencethatreactivenitrogenspe-
provesratherthanexacerbatesinsulinsensitivity. cieswereinvolvedinthemechanismofmetforminaction.
Thestimulationoffattyacidoxidationisanestablished Nitricoxideitselfappearedtohavenoeffectoneitherglucose
actionofmetformin(24).Byusingthispathwayinadditionto uptakeorfattyacidoxidation.Theperoxynitrategenerator
glucosetransport,wefoundthatnoneofthecurrentlypro- SIN-1wasabletostimulateglucoseuptake,butinhibitedfatty
posedhypothesesformetforminweremetabolicallyconsis- acidoxidation,suggestingthatittoomayactinamanner
tent.Moreover,thefindingthatablationofadenylatekinase similartorotenone,thatis,inhibitionofrespiration.This
doesnotaffectmetforminactionsuggeststhatanymecha- wouldimplythatanyagent,whichsuppressesmitochondrial
nismthatinvolvesincreasedAMPformationdrivenbyATP respiration,mightalsostimulateglucoseuptakeandactivate
turnover(andhence,increasedADPformation)isunlikely. AMPK,buttheyarenotreplicatingtheactionofmetformin,
Afunctionalcontrolfortheadenylatekinaseknockdownis butrathermitochondrialenergysupplyinterruption.One
importantbecausethisenzymeisabundantinskeletalmuscle. recentstudy(55)didfindthatSIN-1couldincreaseinsulin
Infact,itwasfirstdiscoveredinthistissue,asisevidentfrom sensitivityandfattyacidoxidation,usingtheC2C12muscle
itsoriginalname,myokinase(53).Itisofinteresttonotealso cellculture.However,thosefindingswerenotcomparable,as
thatdepolarizingconcentrationsofpotassiumthatstimulate theSIN-1wasnotanacutetreatment,butincubatedseveral
glucoseuptakecausedaninhibitionofthisprocesswhenad- hours.
enylatekinasewasattenuated.Thisimpliesthatadenylate AseparatesiteofAMPKactionproposedrecentlyisthe
kinaseisneededtomaintainthephosphorylationpotentialof pyruvatedehydrogenasecomplex(56).Thiswouldbeconsis-
thecelltosupportglucoseuptake. tentwiththemetabolicalterationsobservedhereformet-
Inonestudy(16)itwassuggestedthatmethylsuccinate formin;inparticular,anincreaseinfattyacidoxidationwould
couldantagonizemetformininculturedisletcells.Thisim- beexpectedtodepressPDCactivity.Futureexperimentsex-
pliesthatmetforminindeedinhibitsrespirationatcomplexI, aminingmoremetabolictargetswilladdweighttothecon-
andthatoncesuccinatecouldbeintroduced,itovercomesthe nectionsbetweenmetformin,AMPD,andAMPK.
inhibitionbyprovidingelectronstoubiquinone.However,the ThepossibilitythatmetforminactsthroughLKB1,theup-
actualevidenceinthatstudywasindirect.Theinvestigators streamkinaseandaseparateactivatorofAMPK,wasraised
usedatetrazoliumdye(3-(4,5-dimethylthiazol-2-yl)-2,5-di- forliverby(9).However,ithassubsequentlybeenshownthat
phenyltetrazoliumbromide),commonlyknownasMTT. metformindoesnotactthroughLKB1(10,11);wehavecon-
Priorstudiesdidsuggestthatincreased(MTT)reductioncor- firmedinthisstudythatsiRNAknockdownofLKB1hadno
relatestoglucoseavailability(54).However,therearesome effectonmetforminstimulationofglucoseuptake.
difficultiesinacceptingtheconclusionsofthemethylsucci- ItisestablishedthatactivationofAMPKrequiresphosphor-
natestudy(16).First,thedyeacceptselectronsfromubiqui- ylation(13),andLKB1isamajorAMPKkinase(57).Thusour
none,butisnotasteadystatemeasureofrespiration.More- findingthatablationofLKB1didnotpreventtheactionof
over,intheactualdataofthatstudy,MTTreductionwas metforminissurprising.However,thereareotherupstream
inhibitedbymetforminwhethermethylsuccinatewaspresent AMPKkinases,suchastheCa2(cid:5)-calmodulinproteinkinase
ornot.Infact,therewasmerelymorereductioninthepres- (58).Moreover,itisknownthatelevationinAMPconcentra-
enceofmethylsuccinate. tionsalonecanactivateandphosphorylateAMPK,evenwith-
AsitiswellknownthatAMPKcaninfactbeactivatedby outalterationsintheupstreamkinases,asAMPbindingto
aninhibitionofrespiration(18),itmayseemirresistibleto AMPKmakesitbothabetterphosphorylationsubstrateanda
concludethatmetforminmustworkinthesameway.How- poorerproteinphosphatasesubstrate(59).
ever,theknownstimulationoffattyacidoxidationbymet- ThenotionthatAMPDinhibitioncouldincreaseAMPand
formininthisstudyandothers(21–24)isincommensurate activateAMPKwasraisedpreviouslybyBorkowskietal.(60)
withmitochondrialrespiratoryinhibition.Addtothatthe inthecontextoftheknowncorrelationbetweenAMPDdefi-
currentfindingthatdirectinhibitionbyrotenonedidnot ciencyandcardioprotection(seealsoRef.61).Althoughthe
stimulaterespirationatanylevel,andthefactthatadenylate authorsraisethenotionbasedlargelyontheprotectivefunc-
kinaseknockdownpreventsstimulationbydepolarizationbut tionofdeaminaseinhibitors,itisconsistentwiththeproposal
JANUARY7,2011•VOLUME286•NUMBER1 JOURNALOFBIOLOGICALCHEMISTRY 9
ActivatingAMPKinasethroughAMPDeaminase
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