Yangpresentationdiabetesandobesity.ppt

CytoplasmicATM anemergingtherapeutictargetfortype2diabetesandobesity Da QingYang Ph D TheMasonicCancerCenterandTheHormelInstituteUniversityofMinnesota ATAXIA TELANGIECTASIA ClinicalPhenotype CerebellarAtaxiaTelangiectasiasCancerPredispositionPrematureAgingGrowthRetardationInsulinResistanceGlucoseIntolerance ATAXIA TELANGIECTASIA CellularPhenotype GeneticInstabilityHypersensitivitytoIonizingRadiationG1 S G2CellCycleCheckpointDefectsEarlysenescenceCytoskeletaldefectsPoorGrowthinCulture higherrequirementsforserumgrowthfactors ATM amemberofthePI 3kinasesuperfamily PI 3Kdomain Rad3domain ATR S pombe Rad3 PI 3kinase Mec1 S cerevisiae Tel1 S cerevisiae TOR1 2 S cerevisiae FRAP mTOR mammalian DNAPK mammalian Mei 41 mammalian ATM mammalian D melanogaster PI 3Kdomain FKBP 12rapamycinbindingdomain IR ATM p53 chk2 mdm2 nbs1 G1CheckpointS phaseCheckpoint 4E BP1 S15 S343 S20 Rad50 Mre11 Brca1 Insulin TranslationInitiation adaptin TranslocationofCytoplasmicProteins NuclearATMProtein CytoplasmicATMProtein Anti ATM 32P 4EBP 1 4EBP 1staining Insulin A HEK 293B 3T3 L1 1 02 01 03 0 Yanget al NatureCellBiology 2000 Type2diabetesandA T Ahighincidence 59 ofType2diabetesinA Tpatientshasbeenreported A Tpatientswithtype2diabetesexhibitsymptomsofglucoseintoleranceandinsulinresistance Insulinresistanceiscausedbydefectiveglucoseuptakemediatedbyglucosetransporter4 GLUT4 GLUT 4translocationisregulatedbyInsulinthroughPI 3K AKTpathway Hypothesis ATMmayparticipateinGLUT4translocationinresponsetoinsulinsignaling Type1andtype2diabetes IRS I GlucoseTransporter4 GLUCOSE ENERGY MUSCLE STORAGE FAT PI3 Kinase PKB AKT Insulin P P IRS I PI3 Kinase PDK1 PKB AKT ATM Insulin Cellsurvival P P Proteintranslation Glucoseuptake Cellmembrane Ser473 Thr308 Viniegraetal J Biol Chem 2005 Halabyetal CellSignalling 2008 A38 A T A29 NL Insulin ATM Akt S473 P Akt Total Ratsonahigh fatdietControlratspvalues ATM Tubulin ratsonahigh fatdiet controlrats A AKT S473 P TotalAKT Insulin Ratsonahigh fatdiet Controlrats B Insulin KU 55933 TheATMinhibitorKU 55933inhibitsAktphosphorylationatbothSer473andThr308inL6myoblasts ATM AKT S473 P TotalAKT AKT T308 P TreatmentwiththeATMinhibitorKU 55933leadstoasignificantdecreaseinglucoseuptakeinL6myoblasts A C D B Indirectimmunofluorescense insulin insulin Insulin PI3 kinase Akt Thr308 Ser473 PDK1 ATM GLUT4translocationandGlucoseuptake Synip VAMP2 Chloroquine CaffeineKU 55933 RoleofATMininsulin mediatedAktphosphorylationandglucoseuptake p Akt473 ControlInsulinChloro Ins Chloro t Akt A B 2 DGuptake pmol mg min P Akt T Akt Chloroquine Insulin Fat fedChow fed Conclusions OurresultsfurtherdemonstratedthatATMmediatestheSer 473phosphorylationandthefullactivationofAKTinresponsetoinsulinInmuscletissues ATMmayregulateinsulin mediatedGLUT4translocationprocessthroughregulationofAKTactivityChloroquineisapromisingdrugcandidatethatcouldbeusedtotreattype2diabetes possiblythroughup regulationofAkt mediatedglucoseuptake FutureDirections Characterizetheupstreamkinase PI3 kinase orothers thatregulatesATMactivityinresponsetoinsulinIdentifyandcharacterizedownstreamsubstratesofATM mTOC2 PDK1 ATR thatmaydirectlyphosphorylateSer473ofAktinresponsetoinsulinFurtherdeterminethemechanismusedbyATMtoregulatetheinsulin mediatedglucoseuptakeprocess ATM mTORC2 Akt AS 160 GLUT4 orATM VAMP2 GLUT4 ExaminehowATMregulateAkt mediatedglycogensynthesisprocessinresponsetoinsulin ATMandbetacellfunction Atm micedevelopdiabeteswithageastheirbloodglucoseincreasedandtheirplasmainsulinlevelsdecreased C Peptidelevels whichareindicativeofinsulinsecretion weresignificantlydecreasedinagingA Tmice 27weeksorolder ascomparedtowild typemice ItwashypothesizedthatAtm micemayexhibitincreased cellapoptosisandreduced cellmassastheyage Milesetal AmJPhysiolEndocrinolMetab2007 Currentmodeloftype2diabetesmellitusisdefinedbyaninitialsymptomofinsulinresistance reducedglucoseuptakeandasubsequentdecreaseininsulinsecretionduetoreducedpancreatic cellmassorfunction IRS I PI3 Kinase PDK1 PKB AKT ATM Insulin Cellsurvival P P CellProliferation Glucoseuptake Cellmembrane Ser473 Thr308 Viniegraetal J Biol Chem 2005 Halabyetal CellSignalling 2008 ATM Elghazietal Regulationof cellmassandfunctionbytheAkt proteinkinaseBsignallingpathway Diabetes obesity andmetabolism 2007 Chloroquine Hypothesis Chloroquine byactivatingATMandAkt mayplayapositiveroleinpromotingbeta cellmassandfunction ConKUchloConKUchlo Insulin INS 1E rat cells p Akt Thr308 Total Akt beta actin p Akt Ser473 Chloroquine apromisingdrugcandidatefor cellregeneration INS 1Ecellswereseededina48wellplateandtreatedwithdifferentchemicalsfor48hoursunder0 5 FBS MTTassaywasperformed fourduplicateseachtreatment Componentsofcytokines cyto IFN gamma IL 1 beta Chloroquine chlo GLP 1 GLP INS 1Ecellswereseededina24wellplateandtreatedwithdifferentchemicalsfor48hours CelldeathELISAwasperformed fourduplicateseachtreatment DissociatedisletscellsfromATM ATM andATM micewerepretreatedwithchloroquineorGLP 1for1hrandthentreatedwithcytokines IL 1 IFN for24hrs ThecellproliferationrateineachwellwasdeterminedbyMTTassay P 0 005 IsletsfromATM orATM micewereisolatedfollowingavideoprotocolbySzotGL etal JoVE 2007 Isletsweredissociatedtosinglecellswithtrypsin Isletcellswerethenpretreatedwithchloroquine chlo orGLP 1 GLP for1hourbeforetheadditionofcytokines cyto IL 1 plusIFN foranother24hours AcelldeathELISAassay measuringDNAfragmentation wasthenperformed p 0 05 p 0 001fromfourduplicatesoftheexperiments ATM Elghazietal Regulationof cellmassandfunctionbytheAkt proteinkinaseBsignallingpathway Diabetes obesity andmetabolism 2007 Chloroquine Conclusions ResultsfrommygroupandothershavedemonstratedthatATMisanactivatorofAkt akeyregulatorofbetacellmassandfunctionWehaveshownthatchloroquine aknownactivatorofATM stimulatesactivityofAktanditsdownstreamsubstratesinINS1EcellsWealsoobservedthatchloroquinecanpreventcytokine mediatedcelldeathofINS 1EandprimarymouseisletcellsinanATM dependentmannerOurresultssuggestthatcholoroquinemaybeapromisingdrugcandidateforbetacellregenerationandfunction Evidencesupportingtheclinicalusageofcholoroquine Chloroquinehasbeentestedinmouseandratmodelsoftype2diabetesandinmultiplepilot scalehumanclinicaltrialsfortype2diabeticpatientsshowingsignificantimprovementofinsulinsensitivityandglucosetoleranceChloroquinehaslongbeenusedasadrugfortreatingmalariaanditisingeneralsafeforhumanuseAsananti rheumaticdrug chloroquinecanalsoinhibitthereleaseofcytokines suchasIL 1betaandTNF alpha OurpreliminarydatausingNODmiceandSTZ inducedmiceshowedthatchloroquinemayincreasebetacellmassTherefore inadditiontotype2diabetes chloroquinemaybeapromisingtherapeuticagentfortype1diabetesaswell Otherrelatedstudies ChloroquinehasalsobeenshowntoplayaroleinbodyweightreductionOurrecentresultssuggestthismayberelatedtoATM sroleinregulatinglipidmetabolismUnderserumstarvationorenergydeficientconditions ATMalsoregulatesLKB1andAMPKpathwayactivityWehaverecentlystarttocollaboratewi