Introduction

Obesityisasignificantclinicalproblemthatcontributestolife-threateningdiseasessuchasdiabetesandatherosclerosis.Withanincreasingincidenceofobesityworldwide,rationalstrategiesareneededtocontrolADIpogenesis.Cellsthatundergodeterminationtotheadiposelineagecalledadipoblastsandthisprocesscalledadipogenesis.Adipogenesishasbeenextensivelystudiedusinginvitromodelsystemsconsistingeitherofestablishedadipogeniccelllines(clonallines3T3-L1,3T3-F442A,Ob17,BFC-1,ST13,A31T,...)andprimarycultureofadipocyteprecursorsandpre-adipocytes.GrowtharrestofadipoblastsattheG1/SphaseofthecellcycleappearstoberequiredforthecommitmentofadipoblaststopreadiposecellswhichisaccompaniedbytheemergenceofearlyMarkersofadipgenesis.Usingcellculturesystemscultivatedeitherinthepresenceofserumorindefinedmedium,ithasbeenpossIBLetoidentifyfactorsregulatingeitherpositivelyornegativelyadiposedifferentiation.Thepositiveregulatorsincludeseveralhormonessuchasinsulin,growthhormone,andtriiodothronine.Inaddition,serum,partiallypurifiedserumfactors,andconditionedmediumfromseveraladipogeniccelllineshavebeenshowntostimulateadiposedifferentiationinvitroandassumedtocontainadipogenicregulatorsdistinctfromthehormones.Mammalian3T3-L1cellsdifferentiateintoadipocytesaftercontinuousexposuretopharmacologicaldosesofinsulinorphysiologicaldosesofinsulin-likegrowthfactorI.

Principle

Whenadipoblaststreatedwithacombinationofdexamethasone,isobutylmethylxanthine(IBMXorMIX)andinsulin,cellsstarttoadoptaroundedphenotypeandwithin5-8daysbegintoaccumulatelipidsintracellularlyintheformoflipiddroplets.Theinductioncondtionsandmediavaryaccordingtothecelllines.TreatmentofcellswithdexamethasoneactivatesthetranscriptionfactorCCAAT/enhancer-bindingproteinb(C/EBPb).IBMXinhibitssolublecyclicnucleotidephosphodiesterasesandresultsinincreasedintracellularcAMPlevels.Atthenuclearlevel,treatmentwithIBMXresultsinactivationoftherelatedtranscriptionfactorC/EBPd.C/EBPbanddinturninducetranscriptionofC/EBPaandPPAR.Within3daysofexposuretoinducers,thecellsundergotworoundsofmitosis,termedmitoticclonalexpansion,whicharerequiredfordifferentiation.Insulinorinsulin-likegrowthfactor-1promoteadipocytedifferentiationbyactivatingPI3-kinaseandAktactivity.ModulationoftheactivityoftheforkheadtranscriptionfactorFoxo1appearstobenecessaryforinsulintopromoteadipocytedifferentiation.C/EBPaandPPARdirectthefinalphaseofadipogenesisbyactivatingexpressionofadipocyte-specificgenes,suchasfattyacidsynthetase,fattyacidbindingprotein,leptinandadiponectin.

Reagentsandrequirements

Materialandsources:

• DMEM(GibcoBRL-Cat#11965-084)
• CalfSerum(GibcoBRL-Cat#16170-078)
• FetalBovineSerum(GibcoBRL-Cat#10437-028)
• Isobutylmethylxanthine(IBMX;SigmaI-7018)
• Dexamethasone(SigmaD-4902)
• Insulin(Bovine;SigmaI-5500)
• MEMSodiumPyruvate(100mM;GibcoBRLCat#11360-070)
• Pen/Strep/Glutamine(100xP/S/G;GibcoBRLCat#10378-016)
• Sterilizedpetriplates
• Leminarairhood
• CO2incubator
• SterilizedPipettes

Preparationofsolutions

1. 10%CalfSerum/DMEM60mL:CalfSerum6mL:100mMMEMSodiumPyruvate6mL:100xP/S/G500mL:DMEM
2. 10%FBS/DMEM60mL:FetalBovineSerum(FilterSterilized)6mL:100mMMEMSodiumPyruvate6mL:100xP/S/G500mL:DMEM
3. IBMXSolution(makefresh)DissolveIBMXinasolutionmadeof0.5NKOHtoafinalconcentrationof0.0115g/mL.Filtersterilizethrougha0.22mmsyringefilter.
4. InsulinStockSolution167mM(1mg/mL)in0.02MHClFiltersterilizedthrough0.22mmfilterCanstoreat-20°Cforlongterm,4°Cshortterm.
5. DexamethasoneStockSolutionsFreezerStock:10mMofDexin100%ethanol(storeat-20°C)WorkingStock:DiluteFreezerstockto1mMinPBSFiltersterilizeandstoreat4°C.
6. MDIInductionMedia(10mL/10cmplate;5mL/6cmplate)Torequiredvolumeof10%FBS/DMEMadd:1:100IBMX1:1000Insulin1:1000Dexamethasoneworkingstock
7. InsulinMedia(10mL/10cmplate;5mL/6cmplate)Torequiredvolumeof10%FBS/DMEMadd:1:1000Insulin
8. OilredOstocksolution(0.5g/100mlisopropanol)Justbeforestaining:mix60mlofstockwith40mlofH₂0,letitsitfor1hratRTandfilterthroughwhatmanpaper3MM.

Procedures

Preadipocytemaintenanceandpassage:

Platethecellsin10%CS/FBS-DMEMonculturedishesandincubatethemat37°Cin10%CO₂.Itisimportanttofeedthepreadipocyteseverycoupleofdaysandavoidlettingthemgettooconfluent(>70%),ifyouwanttocontinuetopassagethemanddifferentiatethematalaterdate.So,takecaretosplitthemappropriately.Theycanbesplitasfaras1:15,thoughweusuallydo1:10orlessdependingonneed.

AdipocyteDifferentiationProtocol

1. Growpreadipocytes/fibrobalsttoconfluencyin10%FBS-DMEM.
2. Aftertwodaysofpostconfluency(whichiscountedasday0),stimulatethecellswithMDIinductionmedia.Youwillnoticeadistinctchangeinthemorphologyofthecells(becomemorespindly)inthenext2days.
3. AftertwodaysofMDIaninductionmedium(whichiscalledasday2)replacetheMDIinductionmediawithInsulinMedia.Themediawillbegintogetmoreviscousasfreefattyacidsareproducedbythecellsandsecretedintothemedia.
4. Twodayslater(whichiscalledasday4)changemediato10%FBS-DMEM.Feedcellswith10%FBS-DMEMeverytwodays.Fulldifferentiationisusuallyachievedbyday8.

Stainingprocedure:

1. Aspiratemedia,addformaldehydeslowlyandletsitfor30min.
2. AspirateformaldehydeandaddOilredOsolutiontocoverthewell,leave1hratRT.
3. Removethestainandwashwithdistilledwatertwice.
4. Letitdryforpictures.

Precautions:

1. DexamethasoneSolution,IBMXSolutionandInsulinSolutionshouldbestoredat-20ºC.
2. OilRedOSolution,WashSolution,andDyeExtractionSolutionshouldbestoredatroomtemperature.StorageofOilRedOSolutionandDyeExtractionSolutionat-20ºCmayresultinformationofinsolubleprecipitatesandisnotrecommended.IfOilRedOsolutionformsaprecipitate,removeparticulatesbypassagethrougha0.22or0.45micronfilter.OilRedOstainsskinandclothing.
3. IBMXanddexamethasoneareirritantsandpotentiallytoxic.DMSOisreadilyabsorbedthroughtheskin.Wearalabcoatandgloveswhenhandlingthesesolutions.
4. Isopropanolisflammable.Keepsolutionscontainingisopropanol(OilRedOSolution,WashSolutionandDyeExtractionSolution)awayfromopenflames.