Metabolic Signaling: Methods and Protocols

298 Pages·2019·9.131 MB·English

by  Sarah-Maria Fendt, Sophia Y. Lunt

Methods in
Molecular Biology 1862
Sarah-Maria Fendt
Sophia Y. Lunt Editors
Metabolic
Signaling
Methods and Protocols
M M B
ETHODS IN OLECULAR IO LO GY
SeriesEditor
JohnM.Walker
School of Lifeand MedicalSciences
University ofHertfordshire
Hatfield, Hertfordshire,AL109AB,UK
Forfurther volumes:
http://www.springer.com/series/7651
Metabolic Signaling
Methods and Protocols
Edited by
Sarah-Maria Fendt
Laboratory of Cellular Metabolism and Metabolic Regulation, VIB-KU Leuven Center for Cancer
Biology, Leuven, Belgium
Sophia Y. Lunt
DepartmentofBiochemistryandMolecularBiology,MichiganStateUniversity,EastLansing,MI,USA
Editors
Sarah-MariaFendt SophiaY.Lunt
LaboratoryofCellularMetabolism DepartmentofBiochemistry
andMetabolicRegulation andMolecularBiology
VIB-KULeuvenCenter MichiganStateUniversity
forCancerBiology EastLansing,MI,USA
Leuven,Belgium
ISSN1064-3745 ISSN1940-6029 (electronic)
MethodsinMolecularBiology
ISBN978-1-4939-8768-9 ISBN978-1-4939-8769-6 (eBook)
https://doi.org/10.1007/978-1-4939-8769-6
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Preface
Metabolism is the biochemical reaction network that allows cells to convert nutrients into
small molecules, called metabolites. Through these metabolite conversions, essential com-
ponents needed for cell survival and proliferation are generated. For example, metabolite
conversions allow the production of ATP, the energy currency of cells, as well as the
production of amino acids, fatty acids, and nucleotides—the building blocks for proteins,
membranes, and DNA/RNA. Moreover, dynamics in metabolite concentrations facilitate
the crosstalk between cell signaling and the biochemical reaction network of metabolism.
This bidirectional crosstalk is necessary to enable the response of cells to stimuli such as
nutrient availability/limitations and growth factors. The central role of metabolism in
integrating directly or indirectly (via cell signaling) various external and internal stimuli
definesmetabolicsignaling.
Inthisbook,weprovideprotocolstoquantifymetabolism(Chapters1–10),toidentify
metaboliccrosstalk(Chapters11–14),andtosetupanddeveloptoolsandmodelstogaina
systems-levelinsightintometabolicsignaling(Chapters15–20).Quantifyingmetabolismis
essentialtodescribeandunderstandmetabolicsignaling.Metaboliteconcentrations,meta-
bolic pathway activities, and metabolic fluxes constitute different functional readouts of
metabolism. Different methods are required to determine these readouts of metabolism.
Dependingonthecellularsystemandtheresolutionlevel,variantsofthesemethodsshould
beapplied.Themostglobalreadoutofmetabolismaremetaboliteconcentrations.Changes
inmetaboliteconcentrationspinpointthemetabolitenodeswithinthebiochemicalreaction
network that respond to a certain stimuli or perturbations. In Chapter 1, Wang and
colleagues describe a protocol to determine the most suitable metabolomics method for
broadmetabolitecoverage,andinChapter2,Langerborgandcolleaguesprovideamethod
to specifically assess bioactive lipids. The activity of metabolic pathways and how they are
fueled by nutrients is an important aspect of understanding the metabolic requirements of
cells. In Chapters 3 and 4, Ogrodzinski et al. and van Gorsel et al. provide workflows for
applying nutrients labeled with a stable isotope of carbon to determine metabolic pathway
activityandnutrientcontributionsinvitroinadherent2Dand3Dspheroidalcellcultures,
respectively.InChapters5and6,BroekaertandFendtandPinnicketal.provideprotocols
toextendtheuseofmetaboliteslabeledwithstableisotopestoanalyzeinvivoglucoseand
lipidmetabolisminmiceandhumans,respectively.Metabolicfluxesarethemostquantita-
tive readout of metabolism, and several different and complementary approaches exist to
measureabsolutefluxesinculturedcells.Birdetal.andNewmanandMaddocksdescribein
Chapters7and 8methodsto estimateATPand aminoacidsynthesisrates,respectively, by
estimatingthesteadystatefluxwithfluxinhibition.VeysandcolleaguesapplyinChapter9
radioactive tracers to determine fluxes in central carbon metabolism of endothelial cells,
while Nonnenmacher and colleagues estimate compartment-resolved metabolic fluxes in
Chapter10.
Metabolic signaling describes the bidirectional crosstalk between external stimuli and
cell signaling pathways with metabolism. Chapters 11 and 12 from Guillaume et al. and
Pu¨schelandMunoz-Pinedoidentifycrosstalkbetweennutrientmetabolismwithautophagy
and cell death pathways, respectively. Often, cellular functionality is directly linked to
v
vi Preface
metabolism.InChapters13and14,LiuandHoandFernandezGarciaandFendtprovide
protocols to assess the regulation exerted by nutrient availability on macrophage polariza-
tionandT-lymphocytemetabolism,respectively.
To further understand and exploit metabolism in systems medicine, different model
systemsandcellularcontextscanbeconsidered.Animportantmodelsystemusedincancer
researcharepatient-derivedxenografts.AnnibaliandcolleaguesdescribeinChapter15the
setupofpatient-derivedxenografts,whichcanbeusedtodetermineinvivotumormetabo-
lism as described in Chapter 5. Moreover, it has been established that tumor metabolism
changes during cancer progression. Therefore, it is important to closely follow cancer
progression, as described in Chapter 16 by Stanchi and colleagues using microscopy.
Further, external signals and stimuli are in crosstalk with metabolism. An important con-
tributor hereby is visceral adipose tissue (VAT), an active endocrine organ producing
hormones. To study the impact of VAT-produced hormones on metabolism, VATcan be
surgically removed. A protocol to do so is described in Chapter 17 by Chakraborty and
Bernard.Similarly,thesecretomeofotherorganssuchastheboneisafunctionofhealthand
disease. Potential interaction of this secretome with metabolism therefore requires quanti-
tativemethodstodefinebonestatusanditssecretome.AmethodhereofisdescribedbyLie
and colleagues in Chapter 18. Moving from a whole-body physiology level to the cellular
level, the relevance of organelle organization emerges, and it is tempting to speculate that
there might be a crosstalk between organelle organization and metabolic fluxes within
organelles(seeChapter10).AprotocolprovidedbyLatgeandSchauerinChapter19allows
determinationofintracellularorganelleorganization.Finally,large-scaledataoftenobtained
when studying metabolism can be effectively displayed using heat maps. Fundamentals of
constructingandinterpretingheatmapsareprovidedbyVacantiinChapter20.
With this book, we aim to provide researchers with methods to study, perturb and
functionally interpret metabolic signaling from the subcellular to the whole-body level.
Given the emerging importance of metabolism in sustaining health and metabolic deregu-
lation in disease, we believe that applying methods described in this book will foster
mechanistic understanding of metabolism and, in the long-term, prospectively support
thedevelopmentofinnovativediseasetreatmentstrategies.
Leuven,Belgium Sarah-MariaFendt
EastLansing,MI,USA SophiaY.Lunt
Contents
Preface ..................................................................... v
Contributors................................................................. ix
1 AProtocoltoCompareMethodsforUntargetedMetabolomics.............. 1
LingjueWang,FuadJ.Naser,JonathanL.Spalding,andGaryJ.Patti
2 High-ThroughputMeasureofBioactiveLipidsUsingNon-targeted
MassSpectrometry...................................................... 17
KimA.Lagerborg,JeramieD.Watrous,SusanCheng,andMohitJain
3 MeasuringtheNutrientMetabolismofAdherentCellsinCulture ............ 37
MartinP.Ogrodzinski,ShaoThingTeoh,LeiYu,DeannaBroadwater,
ElliotEnsink,andSophiaY.Lunt
4 13CTracerAnalysisandMetabolomicsin3DCulturedCancerCells .......... 53
MaritvanGorsel,IlariaElia,andSarah-MariaFendt
5 MeasuringInVivoTissueMetabolismUsing13CGlucoseInfusions
inMice ................................................................ 67
DorienBroekaertandSarah-MariaFendt
6 MeasuringHumanLipidMetabolismUsingDeuteriumLabeling:
InVivoandInVitroProtocols ........................................... 83
KatherineE.Pinnick,PippaJ.Gunn,andLeanneHodson
7 MeasuringRatesofATPSynthesis ........................................ 97
MatthewJ.Bird,SilviaRadenkovic,PieterVermeersch,
andDavidCassiman
8 DirectEstimationofMetabolicFluxbyHeavyIsotopeLabeling
SimultaneouswithPathwayInhibition:MetabolicFluxInhibitionAssay....... 109
TongZhang,ChristiaanF.Labuschagne,KarenH.Vousden,
andOliverD.K.Maddocks
9 MeasuringGlycolyticandMitochondrialFluxesinEndothelialCells
UsingRadioactiveTracers................................................ 121
KoenVeys,AbdielAlvarado-Diaz,andKatrienDeBock
10 DeterminingCompartment-SpecificMetabolicFluxes....................... 137
YannicNonnenmacher,RobertaPalorini,andKarstenHiller
11 DeterminingtheImpactofMetabolicNutrientsonAutophagy .............. 151
JessicaD.Guillaume,StephanieL.Celano,KatieR.Martin,
andJeffreyP.MacKeigan
12 MeasuringtheActivationofCellDeathPathwaysuponInhibition
ofMetabolism.......................................................... 163
FranziskaPu¨schelandCristinaMun˜oz-Pinedo
13 DeterminingMacrophagePolarizationuponMetabolicPerturbation ......... 173
Pu-SteLiuandPing-ChihHo
vii
viii Contents
14 AssessingtheImpactoftheNutrientMicroenvironment
ontheMetabolismofEffectorCD8+TCells............................... 187
JuanFerna´ndezGarcı´aandSarah-MariaFendt
15 DevelopmentofPatient-DerivedTumorXenograftModels.................. 217
DanielaAnnibali,EleonoraLeucci,ElsHermans,
andFre´de´ricAmant
16 ImagingGliomaProgressionbyIntravitalMicroscopy....................... 227
FabioStanchi,KenMatsumoto,andHolgerGerhardt
17 LipectomizingMiceforApplicationsinMetabolism......................... 245
DebrupChakrabortyandJamieJ.Bernard
18 QuantitativeMultiplexImmunoassayforProfilingBone
TurnoverBiomarkersinHumanBoneTissueCultureSupernatants........... 251
Wen-RongLie,DerekF.Amanatullah,andBonnieL.King
19 DeterminingtheIntracellularOrganizationofOrganelles ................... 263
BrunoLatge´andKristineSchauer
20 TheFundamentalsofConstructingandInterpretingHeatMaps ............. 279
NathanielM.Vacanti
Index ...................................................................... 293
Contributors
ABDIELALVARADO-DIAZ (cid:1) LaboratoryofExerciseandHealth,DepartmentofHealthSciences
andTechnology,ETHZu¨rich,Zu¨rich,Switzerland
DEREKF.AMANATULLAH (cid:1) DepartmentofOrthopaedicSurgery,StanfordUniversitySchoolof
Medicine,RedwoodCity,CA,USA
FRE´DE´RIC AMANT (cid:1) GynecologicalOncology,OncologyDepartment,LKILeuvenCancer
InstituteKULeuven-UniversityofLeuven,Leuven,Belgium;CentreforGynecologic
OncologyAmsterdam(CGOA),AntoniVanLeeuwenhoek-NetherladsCancerInstitute
(AvL-NKI)andUniversityMedicalCentra(UMC),Amsterdam,TheNetherlands
DANIELA ANNIBALI (cid:1) GynecologicalOncology,OncologyDepartment,LKILeuvenCancer
InstituteKULeuven-UniversityofLeuven,Leuven,Belgium
JAMIEJ.BERNARD (cid:1) DepartmentofPharmacologyandToxicology,MichiganStateUniversity,
EastLansing,MI,USA
MATTHEWJ.BIRD (cid:1) LaboratoryMedicine,UniversityHospitalsLeuven,Leuven,Belgium;
HepatologyLaboratory,DepartmentofChronicDiseases,MetabolismandAgeing,
KULeuven,Leuven,Belgium
DEANNA BROADWATER (cid:1) DepartmentofBiochemistryandMolecularBiology,MichiganState
University,EastLansing,MI,USA
DORIEN BROEKAERT (cid:1) LaboratoryofCellularMetabolismandMetabolicRegulation,
VIBCenterforCancerBiology,VIB,Leuven,Belgium;LaboratoryofCellularMetabolism
andMetabolicRegulation,DepartmentofOncology,LeuvenCancerInstitute(LKI),
KULeuven,Leuven,Belgium
DAVIDCASSIMAN (cid:1) HepatologyLaboratory,DepartmentofChronicDiseases,Metabolismand
Ageing,KULeuven,Leuven,Belgium;MetabolicCenter,UniversityofLeuven,Leuven,
Belgium
STEPHANIEL.CELANO (cid:1) CollegeofHumanMedicine,MichiganStateUniversity,Grand
Rapids,MI,USA
DEBRUPCHAKRABORTY (cid:1) DepartmentofPharmacologyandToxicology,MichiganState
University,EastLansing,MI,USA
SUSAN CHENG (cid:1) CardiovascularDivision,DepartmentofMedicine,BrighamandWomen’s
Hospital,HarvardMedicalSchool,Boston,MA,USA;FraminghamHeartStudy,
Framingham,MA,USA
KATRIENDEBOCK (cid:1) LaboratoryofExerciseandHealth,DepartmentofHealthSciencesand
Technology,ETHZu¨rich,Zu¨rich,Switzerland
ILARIAELIA (cid:1) LaboratoryofCellularMetabolismandMetabolicRegulation,VIBCenter for
CancerBiology,VIB,Leuven,Belgium;LaboratoryofCellularMetabolismandMetabolic
Regulation,DepartmentofOncology,LeuvenCancerInstitute(LKI),KULeuven,
Leuven,Belgium
ELLIOTENSINK (cid:1) DepartmentofBiochemistryandMolecularBiology,MichiganState
University,EastLansing,MI,USA
SARAH-MARIA FENDT (cid:1) LaboratoryofCellularMetabolismandMetabolicRegulation,
DepartmentofOncology,VIB-KULeuvenCenter forCancerBiology,Leuven,Belgium
ix
x Contributors
JUANFERNA´NDEZ-GARCI´A (cid:1) LaboratoryofCellularMetabolismandMetabolicRegulation,
VIBCenterforCancerBiology,VIB,Leuven,Belgium;LaboratoryofCellularMetabolism
andMetabolicRegulation,DepartmentofOncology,LeuvenCancerInstitute(LKI),
KULeuven,Leuven,Belgium
HOLGERGERHARDT (cid:1) VIB-KULeuvenCenter forCancerBiology(CCB),Leuven,Belgium;
MaxDelbru¨ckCenter forMolecularMedicine,Berlin,Germany
JESSICAD.GUILLAUME (cid:1) CollegeofHumanMedicine,MichiganStateUniversity,Grand
Rapids,MI,USA
PIPPAJ.GUNN (cid:1) OxfordCentreforDiabetes,EndocrinologyandMetabolism,Churchill
Hospital,UniversityofOxford,Oxford,UK
ELSHERMANS (cid:1) Trace,DepartmentofOncology,LKILeuvenCancerInstituteKULeuven-
UniversityofLeuven,Leuven,Belgium;VIB-KULeuvenCenter forCancerBiology,
Leuven,Belgium
KARSTEN HILLER (cid:1) DepartmentofBioinformaticsandBiochemistry,Braunschweig
IntegratedCenterofSystemsBiology(BRICS),TechnischeUniversita€tBraunschweig,
Braunschweig,Germany;ComputationalBiologyofInfectionResearch,HelmholtzCentre
forInfectionResearch,Braunschweig,Germany
PING-CHIHHO (cid:1) DepartmentofFundamentalOncology,FacultyofBiologyandMedicine,
UniversityofLausanne,Epalinges,Vaud,Switzerland;LudwigLausanneBranch,
Epalinges,Vaud,Switzerland
LEANNEHODSON (cid:1) OxfordCentreforDiabetes,EndocrinologyandMetabolism,Churchill
Hospital,UniversityofOxford,Oxford,UK
MOHITJAIN (cid:1) DepartmentofMedicine,UniversityofCalifornia,SanDiego,LaJolla,CA,
USA;DepartmentofPharmacology,UniversityofCalifornia,SanDiego,LaJolla,CA,
USA
BONNIEL.KING (cid:1) DepartmentofPediatrics,StanfordUniversitySchoolofMedicine,
Stanford,CA,USA
CHRISTIAANF.LABUSCHAGNE (cid:1) TheFrancisCrickInstitute,London,UK
KIMA.LAGERBORG (cid:1) DepartmentofMedicine,UniversityofCalifornia,SanDiego,LaJolla,
CA,USA;DepartmentofPharmacology,UniversityofCalifornia,SanDiego,LaJolla,
CA,USA
BRUNO LATGE´ (cid:1) MolecularMechanismsofIntracellularTransportGroup,InstitutCurie,
PSLResearchUniversity,ParisCedex05,France;CentreNationaldelaRecherche
Scientifique,Unite´MixtedeRecherche144,Paris,France
ELEONORALEUCCI (cid:1) Trace,DepartmentofOncology,LKILeuvenCancerInstitute
KULeuven-UniversityofLeuven,Leuven,Belgium;LaboratoryofRNACancerBiology,
DepartmentofOncology,LKILeuvenCancerInstituteKULeuven-UniversityofLeuven,
Leuven,Belgium
WEN-RONG LIE (cid:1) MilliporeSigmaCorporation,StLouis,MO,USA
PU-STELIU (cid:1) DepartmentofFundamentalOncology,FacultyofBiologyandMedicine,
UniversityofLausanne,Epalinges,Vaud,Switzerland;LudwigLausanneBranch,
Epalinges,Vaud,Switzerland;InstituteofCellularandSystemMedicine,NationalHealth
ResearchInstitutes,MiaoliCounty,Taiwan
SOPHIAY.LUNT (cid:1) DepartmentofBiochemistryandMolecularBiology,MichiganState
University,EastLansing,MI,USA;DepartmentofChemicalEngineeringandMaterials
Science,MichiganStateUniversity,EastLansing,MI,USA
JEFFREYP.MACKEIGAN (cid:1) CollegeofHumanMedicine,MichiganStateUniversity,Grand
Rapids,MI,USA