Increased Hexosamine Biosynthesis Pathway Activity to Membrane Cholesterol ToxicityWord文件下载.docx
- 文档编号:18182111
- 上传时间:2022-12-14
- 格式:DOCX
- 页数:24
- 大小:1.04MB
Increased Hexosamine Biosynthesis Pathway Activity to Membrane Cholesterol ToxicityWord文件下载.docx
《Increased Hexosamine Biosynthesis Pathway Activity to Membrane Cholesterol ToxicityWord文件下载.docx》由会员分享,可在线阅读,更多相关《Increased Hexosamine Biosynthesis Pathway Activity to Membrane Cholesterol ToxicityWord文件下载.docx(24页珍藏版)》请在冰豆网上搜索。
10.1210/en.2011-1295
PMCID:
PMC3159786
EvidenceCouplingIncreasedHexosamineBiosynthesisPathwayActivitytoMembraneCholesterolToxicityandCorticalFilamentousActinDerangementContributingtoCellularInsulinResistance†
PadmaBhonagiri,*
GuruprasadR.Pattar,*
KirkM.Habegger,
AliciaM.McCarthy,
LixuanTackett,and
JeffreyS.Elmendorf
DepartmentsofCellularandIntegrativePhysiology(P.B.,G.R.P.,A.M.M.,L.T.,J.S.E.)andBiochemistryandMolecularBiology(K.M.H.,J.S.E.)andCentersforDiabetesResearch(P.B.,G.R.P.,K.M.H.,A.M.M.,L.T.,J.S.E.),MembraneBiosciences(P.B.,G.R.P.,K.M.H.,L.T.,J.S.E.),andVascularBiologyandMedicine(J.S.E.),IndianaUniversitySchoolofMedicine,Indianapolis,Indiana46202
Correspondingauthor.
Addressallcorrespondenceandrequestsforreprintsto:
JeffreyS.Elmendorf,Ph.D.,DepartmentofCellularandIntegrativePhysiology,IndianaUniversitySchoolofMedicine,VanNuysMedicalScienceBuilding,Room308A,Indianapolis,Indiana46202.,E-mail:
jelmendo@iupui.edu.
†ThismanuscriptisacorrectedandupdatedversionofanarticlepreviouslypublishedinEndocrinology150(4):
1636–1645,2009.Inthatwork,valueswereincorrectlyreportedfortheglucoseuptakeassaypresentedin
Fig.2Candthearticlewasretracted(seeEndocrinology151(6):
2967,2010;
FederalRegister75(70):
18836–18837,2010forexpandeddetail).CorrectvaluesarenowpresentedinFig.2Bthatshowssimilarchanges,yetofslightlydifferentmagnitudes.Alsopresentedherearenewfindingssuggestingplasmamembranecholesterolaccrualasacomponentofhexosaminebiosynthesispathway-inducedinsulinresistance.
*P.B.andG.R.P.madesubstantialandequalcontributionstothiswork.
ReceivedMay24,2011;
AcceptedJune8,2011.
Copyright
©
2011byTheEndocrineSociety
Abstract
Hyperinsulinemiaisknowntopromotetheprogression/worseningofinsulinresistance.Evidencerevealsahiddencostofhyperinsulinemiaonplasmamembrane(PM)phosphatidylinositol4,5-bisphosphate(PIP2)-regulatedfilamentousactin(F-actin)structure,componentscriticaltothenormaloperationoftheinsulin-regulatedglucosetransportsystem.Herewedelineatedwhetherincreasedglucosefluxthroughthehexosaminebiosynthesispathway(HBP)causesPIP2/F-actindysregulationandsubsequentinsulinresistance.Increasedglycosylationeventsweredetectedin3T3-L1adipocytesculturedunderconditionscloselyresemblingphysiologicalhyperinsulinemia(5nm
insulin;
12h)andincellsinwhichHBPactivitywasamplifiedby2mm
glucosamine(GlcN).BoththephysiologicalhyperinsulinemiaandexperimentalGlcNchallengeinducedcomparablelossesofPIP2
andF-actin.Inadditiontoprotectingagainsttheinsulin-inducedmembrane/cytoskeletalabnormalityandinsulin-resistantstate,exogenousPIP2
correctedtheGlcN-inducedinsultontheseparameters.Moreover,inaccordancewithHBPfluxdirectlyweakeningPIP2/F-actinstructure,pharmacologicalinhibitionoftherate-limitingHBPenzyme[glutamine-fructose-6-phosphateamidotransferase(GFAT)]restoredPIP2-regulatedF-actinstructureandinsulinresponsiveness.Conversely,overexpressionofGFATwasassociatedwithalossofdetectablePMPIP2
andinsulinsensitivity.Evenlessinvasivechallengeswithglucose,intheabsenceofinsulin,alsoledtoPIP2/F-actindysregulation.MechanisticallywefoundthatincreasedHBPactivityincreasedPMcholesterol,theremovalofwhichnormalizedPIP2/F-actinlevels.Accordingly,thesedatasuggestthatglucosetransporter-4functionality,dependentonPIP2
and/orF-actinstatus,canbecriticallycompromisedbyinappropriateHBPactivity.Furthermore,thesedataareconsistentwiththePMcholesterolaccrual/toxicityasamechanisticbasisoftheHBP-induceddefectsinPIP2/F-actinstructureandimpairedglucosetransporter-4regulation.
Decodingtheharmfulcellularbasisofglucose-inducedinsulinresistancehasbeenanimportantresearchinitiativesincetheearly1980s.Atthattime,theconceptofglucosetoxicityemergedfromhumanandanimalobservationsshowingthathyperglycemiaimpairsnormalglucoseuptake(1,
2).Sincethen,aconcertedresearchefforthassoughtmechanisticinsightintothedesensitizationofglucosetransportintomuscleandfatcells.Inthesecellsexceedinglyintricateassembliesofproteinsregulateglucosetransporterglucosetransporter(GLUT)-4-mediatedglucosetransport(3).Itisappreciatedthatinsulinreceptoractivationpropagatesasignal(s)thatmobilizesintracellularGLUT4-containingvesiclestotheplasmamembrane(PM)wheresubsequentmembranefusionincreasesPMGLUT4contentandglucosetransport.
Marshall
etal.
(4),usingamodelsystemofprimaryadipocytes,firstproposedthatexcessglucosefluxthroughthehexosaminebiosynthesispathway(HBP)mayplayaroleinthedevelopmentofinsulinresistance.GlucoseentryintotheHBPiscatalyzedbythefirstandrate-limitingenzymeglutamine-fructose-6-phosphateamidotransferase(GFAT).Thisenzymeconvertsfructose-6-phosphateandglutamineintoglucosamine6-phosphate(GlcN-6-P)andglutamate.GlcN-6-Pisfurthermetabolizedtouridinediphosphate-N-acetylglucosamine(UDP-GlcNAc),themajorproductofthepathway(4).UDP-GlcNAcandotheraminosugarsgeneratedbythepathwayprovidebuildingblocksofglycosylsidechainsforproteinsandlipids.UDP-GlcNAcisalsotheobligatorysubstrateof
O-linked
N-acetylglucosamine(O-GlcNAc)transferase(OGT),acytosolicandnuclearenzymethatmodifiesSer/Thrresiduesofcertainproteinsbyattachingsingle
N-acetylglucosamine(GlcNAc)moietiesin
O-linkage(5,
6).
CellcultureandrodentstudiesroutinelymodelincreasedglucosefluxthroughHBPbyhighglucose(25mm)inthepresenceofinsulinor,alternatively,treatwithglucosamine(GlcN),whichenterstheHBPbypassingGFAT.In1991,Garvey
(7)firstdemonstratedthesynergisticeffectsofhighglucoseandinsulinonthedevelopmentofinsulinresistance.Duringthecontinualdissectionofinsulinsignalingpathways,severallaboratorieshaveusedmodifiedglucose/insulinandGlcNmodelstodefineapossiblemode(s)ofHBP-mediatedalterations.However,aconcernwithmostGlcNmodelsisthattheyresultintremendouscellularaccumulationofGlcN-6-PO4,whereasthiscompoundisnearlyundetectableincellsexposedtoglucosealone(8).
Importantly,GlcN-6-PO4
accumulationdepletesintracellularATP,whichwasclearlydemonstratedbyHresko
(9)in3T3-L1adipocytesincubatedwithhighconcentrationsofGlcN,inthepresenceofinsulinandnoglucose.However,undermilderconditionslackinginsulinand/orprovisionofanenergysource,ATPdepletionduetoGlcNtreatmentbecomestrivialandinsulintyrosinephosphorylation-basedsignalingabnormalitiesdonotoccur,yetGlcN-inducedinsulinresistancepersists(10).Interestingly,workhassuggestedanewmolecularframeworkaccountingforinsulin-inducedinsulinresistanceinvolvingdefectsinPMphosphatidylinositol4,5-bisphosphate(PIP2)-regulatedcorticalfilamentousactin(F-actin)(11,
12).Thebasisforthisinsulin-induceddefectcausinginsulinresistanceisnotknown.HerewetestedwhetherincreasedHBPactivitycoupledhyperinsulinemiatoPIP2/F-actindefectsandinsulinresistance.AdditionalstudyassessedwhetherincreasedHBPactivityand/orPIP2/F-actindefectsresultedfrommembranecholesterolaccrual,recentlyobservedininsulin-induced,insulin-resistant3T3-L1adipocytes(13).
MaterialsandMethods
Materials
Murine3T3-L1preadipocyteswerepurchasedfromAmericanTypeCultureCollection(Manassas,VA)andfromDr.HowardGreen(HarvardMedicalSchool,Boston,MA).DMEMwasfromInvitrogen(GrandIsland,NY).Fetalbovineserum(FBS)andbovinecalfserumwereobtainedfromHycloneLaboratoriesInc.(Logan,UT).Phosphatidylinositol4,5-bisphosphateandhistonecarrierwereobtainedfromEchelon(SaltLakeCity,UT).Monoclonalmousephosphatidylinositol4,5-bisphosphateantibodywaspurchasedfromAssayDesignsInc.(AnnArbor,MI).PolyclonalrabbitGLUT4antibodywasfromSantaCruzBiotechnology(SantaCruz,CA).β-Actin-specificmouseIgMantihumanF-actinantibodywasfromSerotec(Oxford,UK).Rhodaminered-X-conjugateddonkeyantirabbitorgoatantimouseantibodieswerefromJacksonImmunoResearchInc.(WestGrove,PA).Fluoresceinisothiocyanate(FITC)-conjugatedphalloidin,insulin,6-diazo-5-oxo-l-norleucine(DON)andallotherchemicalswerefromSigma(St.Louis,MO).
Cellculture
PreadipocyteswereculturedinDMEMcontaining25mm
glucoseand10%bovinecalfserumat37Cinan8%CO2
atmosphere.Confluentcultureswereinducedtodifferentiateintoadipocytesaspreviouslydescribed(11,
14,
15).Allstudieswereperformedonadipocytesthatwerebetween8and12dafterdifferentiation.
Inductionofinsulinresistance
Adipocytescultured/differentiatedunderthestandard25mm
glucoseprotocolwereswitchedintoDMEM/5.5mm
glucosemediumcontaining10%FBSfor2dbeforeinducinginsulinresistance.Threeexperimentalconditionswereusedroutinely:
1)control,2)5nm
insulin,and3)2mm
GlcN.Ineachoftheseconditions,theculturemediumforovernightinductionofinsulinresistancecontained1mm
pyruvateasanadditionalenergysource.Overnightincubationswerelimitedto12htominimizecomplicationsduetoeffectsofglucosedeprivationonglucosetransport(17).Thethreeexperimentalconditionsarelistedbelow.
Control
Adipocyteswereincubatedfor12hinserum-freeDMEM/5.5mm
glucosemediumintheabsenceofinsulin.Theconcentrationofglutamineintheincubationmediumwas2mm
(hereaft
- 配套讲稿:
如PPT文件的首页显示word图标,表示该PPT已包含配套word讲稿。双击word图标可打开word文档。
- 特殊限制:
部分文档作品中含有的国旗、国徽等图片,仅作为作品整体效果示例展示,禁止商用。设计者仅对作品中独创性部分享有著作权。
- 关 键 词:
- Increased Hexosamine Biosynthesis Pathway Activity to Membrane Cholesterol Toxicity
![提示](https://static.bdocx.com/images/bang_tan.gif)
链接地址:https://www.bdocx.com/doc/18182111.html