Emodin Regulates Glucose Utilization by ActivatingAMPactivated Protein Kinase2.docx

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Emodin Regulates Glucose Utilization by ActivatingAMPactivated Protein Kinase2.docx

EmodinRegulatesGlucoseUtilizationbyActivatingAMPactivatedProteinKinase2

EmodinRegulatesGlucoseUtilizationbyActivatingAMP-activatedProteinKinase*

ParkyongSong,‡JongHyunKim,‡JaewangGhim,‡JongHyukYoon,§AreumLee,‡YonghoonKwon,‡HyunjungHyun,¶Hyo-YoulMoon,‖Hueng-SikChoi,**Per-OlofBerggren,¶‡‡Pann-GhillSuh,‖andSungHoRyu‡¶,1

Fromthe‡DivisionofMolecularandLifeSciences,PohangUniversityofScienceandTechnology,Pohang,Kyungbuk790-784,RepublicofKorea,

§NovaCellTechnologyInc.,Pohang,Kyungbuk790-784,RepublicofKorea,

the¶DivisionofIntegrativeBiosciencesandBiotechnology,PohangUniversityofScienceandTechnology,Pohang790-784,RepublicofKorea,

the‖SchoolofNano-BiotechnologyandChemicalEngineering,UlsanNationalInstituteofScienceandTechnology,Ulsan689-805,RepublicofKorea,

the**HormoneResearchCenter,SchoolofBiologicalSciencesandTechnology,ChonnamNationalUniversity,Gwangju,RepublicofKorea,and

the‡‡RolfLuftResearchCenterforDiabetesandEndocrinology,KarolinskaInstitutet,SE-17177Stockholm,Sweden

1Towhomcorrespondenceshouldbeaddressed:

POSTECHBiotechCenter,San31Hyojadong,Pohang790-784,RepublicofKorea.,Tel.:

Phone:

82-54-279-2292;Fax:

82-54-279-0645;E-mail:

rk.ca.hcetsop@ohgnus.

Authorinformation►Articlenotes►CopyrightandLicenseinformation►

ReceivedDecember1,2012;RevisedJanuary5,2013

Copyright©2013byTheAmericanSocietyforBiochemistryandMolecularBiology,Inc.

ThisarticlehasbeencitedbyotherarticlesinPMC.

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Abstract

AMP-activatedproteinkinasehasbeendescribedasakeysignalingproteinthatcanregulateenergyhomeostasis.Here,weaimedtocharacterizenovelAMP-activatedkinase(AMPK)-activatingcompoundsthathaveamuchlowereffectiveconcentrationthanmetformin.Asaresult,emodin,anaturalanthraquinonederivative,wasshowntostimulateAMPKactivityinskeletalmuscleandlivercells.EmodinenhancedGLUT4translocationand[14C]glucoseuptakeintothemyotubeinanAMPK-dependentmanner.Also,emodininhibitedglucoseproductionbysuppressingtheexpressionofkeygluconeogenicgenes,suchasphosphoenolpyruvatecarboxykinaseandglucose-6-phosphatase,inhepatocytes.Furthermore,wefoundthatemodincanactivateAMPKbyinhibitingmitochondrialrespiratorycomplexIactivity,leadingtoincreasedreactiveoxygenspeciesandCa2+/calmodulin-dependentproteinkinasekinaseactivity.Finally,weconfirmedthatasingledoseadministrationofemodinsignificantlydecreasedthefastingplasmaglucoselevelsandimprovedglucosetoleranceinC57Bl/6Jmice.Increasedinsulinsensitivitywasalsoconfirmedafterdailyinjectionofemodinfor8daysusinganinsulintolerancetestandinsulin-stimulatedPI3Kphosphorylationinwildtypeandhighfatdiet-induceddiabeticmousemodels.OurstudysuggeststhatemodinregulatesglucosehomeostasisinvivobyAMPKactivationandthatthismayrepresentanoveltherapeuticprincipleinthetreatmentoftype2diabeticmodels.

Keywords:

AMP-activatedKinase(AMPK),Calcium,GlucoseMetabolism,InsulinResistance,MetabolicSyndrome

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Introduction

AMPK2isahighlyconservedmammalianserine/threonineproteinkinaseandactsasamasterenergysensorthatisresponsibleforregulatingenergyhomeostasis(1,2).Theseheterotrimericenzymesareactivatedundercellularstressconditionssuchasstarvation,exercise,oxidativedamage,andhypoxia.LiverkinaseB1(LKB1)andCa2+/calmodulin-dependentproteinkinasekinase(CaMKK)aretworegulatoryupstreamkinasesthatstimulateAMPKphosphorylation(3).AMPKcontrolswholebodyenergyhomeostasisbyregulatingglucoseandlipidmetabolisminmultipleperipheraltissues.Forexample,AMPKactivationby5-aminoimidazole-4-carboxamide-1-β-ribofuranosidestimulatesglucoseuptakeviaPI3K-independentGLUT4translocationinskeletalmuscle(4,5)andinhibitshepaticglucoseproduction(6).

Recently,manytypesofAMPKactivators,includingcytokines,drugs,andnaturalcompounds,havebeenidentified(3).Amongthem,metformin(1,1-dimethylbiguanidehydrochloride)andthiazolidinedionesarewidelyusedforthetreatmentoftype2diabetes.Themainfunctionofmetforministodecreasebloodglucoselevelsbyinhibitinghepaticgluconeogenesisandincreasingglucoseuptakeinskeletalmuscle(7).Interestingly,themitochondrialrespiratorycomplexisthecommontargetofmetformin,whichindirectlyactivatesAMPK(8).InhibitionofcomplexIactivitycancausechangesinthecellularnucleotideratio(9)andincreasereactiveoxygenspecies(10),whichareimportantintermediatestoincreaseAMPKactivity.Despitetheirbeneficialeffectstoglucoseutilization,metforminmustbeadministeredathighconcentrations,andtheyhaveadverseeffects,suchasgastrointestinalsymptomsandrelativelyrarelacticacidosisinvivo(11).Furthermore,somehumanstudieshaveshownthattheglucose-loweringeffectsofmetforminaresecondarytoreductioninhepaticgluconeogenesis(11–13).Thus,thereisagrowingdemandtoidentifymoreeffectiveAMPKactivators,whichhavelowdosageandaffectvariousmetabolicorgans.

Inthisstudy,wescreenedadruglibrarytoidentifynovelAMPKactivators.Asaresultofthisscreening,emodin(1,3,8-trihydroxy-6-methylanthraquinone),anaturalactivecompoundintheherbRheumpalmatumL.wasshowntostimulatetheAMPKpathway.Somepreviousstudieshavesuggestedthatemodinhasanbeneficialeffectonenergymetabolism,includingadipocytedifferentiation(14),anti-fibroticeffectonpancreaticfibrosis(15),andliver(16).However,validationofthemolecularmechanismandtheanti-diabeticeffectsofthiscompoundhasnotbeenfullyexamined.Here,weshowthatemodincanincreaseglucoseuptakeinskeletalmuscleviainhibitionofmitochondrialcomplexI,whichthenleadstoactivationofAMPK.Inaddition,emodininhibitedhepaticgluconeogenesisthroughAMPK.Importantly,acuteintravenousadministrationofemodininbothwildtypeandhighfatdiet-induceddiabeticmicesignificantlydecreasedbloodglucoselevelsassociatedwithincreasedglucoseutilizationinskeletalmuscleandliver.Basedontheimprovedglucosemetabolismandinsulinsensitivity,thiscompoundholdsgreatpromiseforeventualuseatherapeuticagenttotreattype2diabetes.

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EXPERIMENTALPROCEDURES

Materials

5-Aminoimidazole-4-carboxamide-1-β-ribofuranosidewaspurchasedfromTorontoResearchChemicalInc.(Toronto,Ontario,Canada).Emodin,dichlorodihydrofluoresceindiacetate,o-phenylenediamine,resveratrol,andmetforminwerepurchasedfromSigma,and2-deoxy[14C]glucosewasfromAmericanRadiolabeledChemicalsInc.Compoundc,anAMPKinhibitor,andSB203580,ap38MAPKinhibitor,wereprovidedbyMerck(RY70-100).

CellCulture

L6myoblastsweregrowninα-minimalessentialmedium,andmousehepatomaHepa1c1c7cellsweremaintainedinDMEMcontaining10%fetalbovineserum(FBS),50unitsofpenicillin/ml,and50μgofstreptomycin/mlat37°Cunderhumidifiedairatmospherecontaining5%CO2.Differentiationofmyoblastwasinducedinmediumsupplementedwith2%FBSwithin7days.

Immunoblotting

Topreparetotalcelllysates,myotubewaswashedwithcoldPBSandthenlysedincoldlysisbuffer(inmmol/liter:

40HEPES,pH7.5,120NaCl,1EDTA,10pyrophosphate,10glycerophosphate,50NaF,1.5Na3VO4,1PMSF,5MgCl2,0.5%TritonX-100,andproteaseinhibitormixture).Transferredmembranewasincubatedwithprimaryantibodies(1:

1000)overnightat4°C.Thefollowingwereused:

p-AMPK(Thr-172),ACC,p-Akt(Ser-473)(CellSignalingTechnology),AMPK(Upstate),p-ACC(Ser-79)(Millipore),phosphoenolpyruvatecarboxykinase(Cayman),PGC1α(Abcam,UK),andmonoclonalanti-Mycantibody(MilliporeCorp.,Billerica,MA).

AMPKActivityAssay

Cellswereserum-fastedfor3handthentreatedwithemodinfor5min.Afterprecipitation,AMPKactivitywasevaluatedasincorporationof32PintoasyntheticSAMSpeptideinkinasereactionbuffer(inmmol/liter:

40HEPES,pH7.5,80NaCl,1DTT,0.2AMP,0.2ATP,5MgCl2,0.1SAMS,0.25[γ-32P]ATP)at30°Cfor20min.

Determinationof2-Deoxy[14C]glucoseUptakeinCells

2-Deoxyglucoseuptakewasdeterminedasdescribedpreviously(17).Briefly,differentiatedL6cellswereserum-starvedfor2hpriortotheassay.CellswerethenwashedtwicewithKrebs-Henseleitbufferandwerestimulatedwithorwithouttheindicatedagentsfor60min.Glucoseuptakewasmeasuredbyincubationwith0.1mCi/ml2-deoxy[14C]glucoseatroomtemperaturefor10min.

MitochondrialComplexIActivity

Cellsweredisruptedwithnondenaturingdetergentlaurylmaltosidetomaintainenzymaticactivityandequilibratedwiththeindicatedconcentrationofmetforminandemodinfor10min.ReactionwasinitiatedbyaddingthereactionsubstrateNADH(500μmol/liter)andubiquinone-1(50μmol/liter).Adecreasein340nmabsorbancewasrecordedover10minusingaUVspectrophotometer.

FattyAcidOxidation

Serum-fastedmyotubewasincubatedwithemodinfor2hinoxidationmedia(0.1%lipid-freeBSA,100μm[3H]palmitate,vaporizedfor30minwithN2gastosaturatepalmitate).Afteroxidation,therestofthefreefattyacidwasremovedwith10%TCAprecipitation.Supernatantinacaplessmicrotubewasvaporizedinascintillationtubewithdistilledwateraddedat50°Cfor12h.

MitochondrialComplexIActivity

Cellsweredisruptedwithnondenaturingdetergentlaurylmaltosidetomaintainenzymaticactivityandequilibratedwiththeindicatedconcentrationofmetforminandemodinfor10min.ReactionwasinitiatedbyaddingthereactionsubstrateNADH(500μmol/liter)andubiquinone-1(50μmol/liter).Adecreasein340nmabsorbancewasrecordedover10minusing

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