A Novel High-Throughput Assay for Islet Respiration Reveals Uncoupling of Rodent and Human Islets 英文参考文献.docVIP

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A Novel High-Throughput Assay for Islet Respiration Reveals Uncoupling of Rodent and Human Islets 英文参考文献.doc

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A Novel High-Throughput Assay for Islet Respiration Reveals Uncoupling of Rodent and Human Islets 英文参考文献

ANovelHigh-ThroughputAssayforIsletRespiration RevealsUncouplingofRodentandHumanIslets JakobD.Wikstrom1,2,SamuelB.Sereda1,LinseyStiles6,AlvaroElorza1,3,EmmaM.Allister4, AndyNeilson5,DavidA.Ferrick5,MichaelB.Wheeler4,OrianS.Shirihai1* 1DepartmentofMedicine,BostonUniversitySchoolofMedicine,Boston,Massachusetts,UnitedStatesofAmerica,2TheWenner-GrenInstitute,StockholmUniversity, Stockholm,Sweden,3DepartamentodeBiologia,FacultaddeCienciasdelaSalud,UniversidadAndresBello,Santiago,Chile,4DepartmentsofPhysiologyandMedicine, UniversityofToronto,Toronto,Canada,5SeahorseBioscience,NorthBillerica,Massachusetts,UnitedStatesofAmerica, 6TuftsUniversitySchoolofMedicine,Boston, Massachusetts,UnitedStatesofAmerica Abstract Background:Thepancreaticbetacellisuniqueinitsresponsetonutrientbyincreasedfueloxidation.Recentstudieshave demonstrated that oxygen consumption rate (OCR) may be a valuable predictor of islet quality and long term nutrient responsiveness.Todate,high-throughputanduser-friendlyassaysforisletrespirationarelacking.Theaimofthisstudywas todevelopsuchanassayandtoexaminebioenergeticefficiencyofrodentandhumanislets. Methodology/PrincipalFindings:TheXF24respirometerplatformwasadaptedtoisletsbythedevelopmentofa24-well platespecificallydesignedtoconfineislets.Theisletplategenerateddatawithlowinter-wellvariabilityandenabledstable measurementofoxygenconsumptionforhours.TheF1F0ATPsynthaseblockeroligomycinwasusedtoassessuncoupling whilerotenonetogetherwithmyxothiazol/antimycinwasusedtomeasurethelevelofnon-mitochondrialrespiration.The use of oligomycin in islets was validated by reversing its effect in the presence of the uncoupler FCCP. Respiratory leak averagedto59%and49%ofbasalOCRinisletsfromC57Bl6/JandFVB/Nmice,respectively.Incomparison,respiratoryleak of INS-1 cells and C2C12 myotubes was measured to 38% and 23% respectively. Islets from a cohort of human donors showedarespiratoryleakof38%,significantlylowerthanmouseislets. Conclusions/Significance:Theassayforisletrespi