skeletal muscle metabolic adaptations in response … muscle metabolic adaptations in response to an...
TRANSCRIPT
Skeletalmusclemetabolicadaptationsinresponsetoanacutehighfatdiet
SuzanneMaeBowser
Dissertation submitted to the faculty of Virginia Polytechnic Institute and State University in partial fulfillment of the requirements for the degree of
Doctor of Philosophy
In Human Nutrition, Foods and Exercise
Matthew W. Hulver, Chair Brenda M. Davy Kevin P. Davy
Madlyn I. Frisard Andrew P. Neilson
Oct 6th, 2017 Blacksburg, Virginia
Keywords:skeletalmuscle,substrateoxidation,metabolicflexibility,
highfatdiet,metabolicadaptations
Skeletalmusclemetabolicadaptationsinresponsetoanacutehighfatdiet
SuzanneMaeBowser
ABSTRACT
Macronutrientmetabolismplaysanessentialroleintheoverallhealthofan
individual.Dependingonanumberofvariables,forexample,diet,fitnesslevel,or
metabolicdiseasestate,protein,carbohydrateandfathavevaryingcapacitiestobe
oxidizedandbalanced.Further,whenanalyzingtheoxidationofcarbohydrateand
fatintheskeletalmusclespecifically,carbohydratebalancehappensquiterapidly,
whilefatbalancedoesnot.Theabilityofskeletalmuscletoadaptandrespondto
variousnutrientstatesiscriticaltomaintaininghealthymetabolicfunction.Habitual
highfatintakehasbeenassociatedwithreducedoxidativecapacity,insulin
resistance,increasedgutpermeability,inflammation,andotherriskfactorsoften
precedingmetabolicdiseasestates.Thedisruptionofgutfunctionleadstogut
permeabilityandincreasesendotoxinsreleasedintocirculation.Endotoxinshave
beenshowntoplayanimportantroleinobesity-relatedwholebodyandtissue
specificmetabolicperturbations.Eachofthesedisruptedmetabolicprocessesis
knowntoassociatewithobesity,metabolicsyndromeanddiabetes.Todate,limited
researchhasinvestigatedtheroleofhighfatdietonskeletalmusclesubstrate
oxidationanditsrelationshiptogutpermeabilityandendotoxins.Thepurposeof
thisstudywastodeterminetheeffectsofanacute,five-day,isocalorichighfatdiet
(HFD)onskeletalmusclesubstratemetabolisminhealthynon-obesehumans.An
additionalpurposewastodeterminetheeffectsofaHFDongutpermeabilityand
bloodendotoxinsonhealthy,non-obese,sedentaryhumans.Thirteencollegeage
maleswerefedacontroldietfortwoweeks,followedbyfivedaysofanisocaloric
HFD.ToassesstheeffectsofaHFDonskeletalmusclemetabolicadaptabilityand
postprandialendotoxinlevels,subjectsunderwentahighfatmealchallengebefore
andafteraHFD.Musclebiopsieswereobtained;bloodwascollected;insulin
sensitivitywasassessedviaintravenousglucosetolerancetest;andintestinal
permeabilitywasassessedviathefour-sugarprobetestbeforeandaftertheHFD.
Postprandialglucoseoxidationandfattyacidoxidationinskeletalmuscleincreased
beforetheHFDinterventionbutwasdecreasedafter.Skeletalmuscleinvitroassay
ofmetabolicflexibilitywassignificantlybluntedfollowingtheHFD.Insulin
sensitivityandintestinalpermeabilitywerenotaffectedbyHFD,butfasting
endotoxinwassignificantlyhigherfollowingtheHFD.Thesefindingsdemonstrate
thatinyoung,healthymales,followingfivedaysofanisocalorichighfatdiet,
skeletalmusclemetabolicadaptationisrobust.Additionally,increasedfasting
endotoxinindependentofgutpermeabilitychangesarepotentiallyacontributorto
theinflammatorystatethatdisruptssubstrateoxidation.Thesefindingssuggestthat
evenshort-termchangesindietaryfatconsumptionhaveprofoundeffectson
skeletalmusclesubstratemetabolismandfastingendotoxinlevels,independentof
positiveenergybalanceandwhole-bodyinsulinsensitivity.
Skeletalmusclemetabolicadaptationsinresponsetoanacutehighfatdiet
SuzanneMaeBowser
GENERALABSTRACT
Macronutrients,namelycarbohydrates,fatsandprotein,andthewaytheyare
utilizedplayanimportantroleintheoverallhealthofanindividual.Manyvariables
comeintoplaywhenconsideringtheoxidization(orutilization)ofeach
macronutrient,including,butnotlimitedtodiet,fitnesslevel,andmetabolicdisease
state.Skeletalmuscleanditsroleintheseprocessesisofspecialinterestasitisthe
largestinsulinsensitiveorganinthebody.Itsabilitytoadaptandrespondto
variousnutrientstatesiscriticaltomaintaininghealthymetabolicfunction.Habitual
highfatintakehasbeenassociatedwithinsulinresistance,increasedgut
permeability(increasingendotoxins,whicharetoxinsreleasedintocirculationfrom
theintestines),reducedoxidativecapacity(abilitytoutilizemacronutrientsfor
energy),andinflammation,allofwhichareriskfactorsthatprecedemetabolic
diseasestates.Todate,limitedresearchhasinvestigatedtheroleofhighfatdieton
skeletalmuscleoxidationofmacronutrientsanditsrelationshiptowhatisgoingon
inthegut,orintestines.Thepurposeofthestudywastodeterminetheeffectsofa
shorttermhighfatdiet(fivedays)onskeletalmuscleinhealthy,non-obesehumans,
andtodeterminetheeffectsofthisdietongutpermeabilityandendotoxins.
Thirteencollege-agemaleswerefedacontroldietfortwoweeksfollowedbyfive
daysofahighfatdiet.Eachdiethadthesamecaloriccontent.Subjectsunderwenta
highfatmealchallengebeforeandafterthediettoassesstheeffectsofthedieton
skeletalmuscleadaptabilityandpostmealendotoxinlevels.Beforeandafterthe
highfatdiet,musclebiopsieswereobtained,bloodwascollected,insulinsensitivity
wasassessedandgutpermeabilitywasmeasured.Wefoundthatskeletalmuscle
metabolicadaptationisrobust.Additionally,increasedfastingendotoxinchanges
areapossiblecontributortotheinflammatorystatethatdisruptsmacronutrient
oxidation.Therefore,evenshort-termchangesindietaryfatconsumptionhave
profoundeffectsonskeletalmusclemetabolismandfastingendotoxinlevels,
independentofpositiveenergybalanceandwhole-bodyinsulinsensitivity.
vi
ACKNOWLEDGEMENTS
Matt:Thankyou,thankyou,thankyou!WhenIbeganmyjourneytoworktowarda
PhD,Icontactedmanyprofessorsofotheruniversities,wholookedatmyresume
anddidn’tseethescientificbackgroundnecessarytobesuccessful.WhenImetwith
you,yousawme,yousawpotentialandyousawexperienceinmylifethatcould
contributetoasuccessfulobtainingofaPhD.Forthat,Iamsograteful.Also,attimes
IcursedyournameforencouragingmetopursuetheRDcredentials,butIam
gratefulthatyourecommendedthatpath,asithasopenedupdoorsand
experiencesthathaveenrichedmyeducation.Thankyouforyourmentorshipand
yourconfidenceinme.
Madlyn:Yourguidanceanddirectionwithmy7zillionquestionshasbeengreatly
appreciated.Thankyoufortakingyourtimetotalkmethroughsomuchofthe
processesnecessarytobeasuccessfulPhDstudentatTech.Thankyouforyour
constantsupportandforalwaysmakingmefeellikeIamimportant.
Ryan:HowcanIeverthankyouenoughforthecountlesshoursyouhavetakento
helpmeunderstandconceptsandassaysandevenhowtoworkwithotherpeople
moreeffectively.Yourpatienceandconfidenceinme,andthetimeyouhavetaken
awayfromyourotherdutiestoeithersimplylistenortoread/editapaper,writea
recommendationortodirectmeinsomewaywassohelpfulandsovery
appreciated.Thankyou.
Drs.Davy:KevinandBrenda,thankyouforyoursupportandthetimeyouhave
takentoguidemetobecomingasuccessfulPhDcandidate,dieteticinternandPost
Docfellow.Thankyouforyourchallengingquestionsandforgivingmevaluable
vii
feedbackthroughoutmytime.IhavealsoappreciatedthewayIhavefeltwelcomein
yourhome.Kevin,thankyouforhelpingmegetscholarshipsandforyour
willingnesstowriterecommendationsforme.
Andrew:Thankyouforgivingmetheopportunitytoworkwithyouandyourgroup
tocollaborateandwritemyfirst–firstauthorpaper.JIvaluedyouradvicein
writingaswellasalloftheotherdetailsIhadtolearnbyjustattemptingitforthe
firsttime.Thankyouforyourpatiencewiththatprocessandwithteachingmethe
quickanddirtyversionofgutpermeability…youravailabilitytoassistmewhen
neededwasappreciated.
KrisOsterbergandNabilBoutagy:Thankyoufordoingsomuchoftheground
workoftheADAstudy,recruitingparticipants,screeningthem,andschedulingthe
appointments–yourwork,timeandeffortdidnotgounnoticed!!
Pastandpresentlabmates:Thankyounotonlyforyourfriendshipandmaking
thelabafunworkplace,butalso,Ihavelearnedsomuchfromeachofyou.Thank
youfortakingmeunderyourwingandofferingassistanceandadviceinthe
multipletimesIhaveneededit.
Mom,family,friends(myfamilyawayfromhome):Noneofyouhaveever
doubtedmeormyabilitytoaccomplishhardthings.Icouldneverdescribehow
gratefulIamforyourrelentlesssupportandencouragement.Mom,whenIhave
evenhalfasmuchconfidenceinmyselfasyouhaveinme,Iwillmovemountains.
Katie,thankyoufortakingtheroleofbigsisterthesepastfewyears,eventhough
youarethebaby!!Thankeachoneofyou,andespeciallymyniecesandnephews
whohaveawayofmakingmesmileandlaughandfeellikeamillionbucks!
viii
ATTRIBUTIONS
CHAPTER2:LITERATUREREVIEW
MatthewHulver,PhDMadlynFrisard,PhDandSuzanneBowserconceivedand
designedthereview;Ms.Bowserwrotethereview;Dr.HulverandDr.Frisard
editedthedocument.
CHAPTER5:SKELETALMUSCLEMETABOLICADAPTATIONSINREPONSETOAN
ACUTEHIGHFATDIET
MatthewHulver,PhDwastheprincipalinvestigatoronthegrantthatfundedthe
research.Heoversawtheentirestudy.KevinDavy,PhD,aco-investigatoronthe
project,wasresponsiblefordaytodayoperationsintheclinicallaboratory.Brenda
Davy,PhD,RDN,aco-investigatorontheproject,wasresponsibleforallaspectsof
dietarycontrol.AndrewNielson,PhDaco-investigatorontheproject,was
responsibleformeasuresofgutpermeability.RyanMcMillan,PhD,thestudy
coordinator,managedallaspectsofscheduling,testing,sampling,anddata
collectionandoversawallaspectsofmeasurementofskeletalmuscle.Madlyn
Frisard,PhDandtheabovementionedpersonnelcontributedtothedesignofthe
studyandwillco-authorthemanuscript.SuzanneBowserwrotethemanuscriptand
assistedDr.McMillaninscheduling,testing,sampling,anddatacollectionaswellas
inmeasurementsofskeletalmuscle.
ix
TABLEOFCONTENTS
ABSTRACT..................................................................................................................................ii
GENERALABSTRACT.............................................................................................................iv
ACKNOWLEDGEMENTS.........................................................................................................viATTRIBUTIONS.....................................................................................................................viii
TABLEOFCONTENTS............................................................................................................ix
LISTOFFIGURES.....................................................................................................................xiLISTOFTABLES......................................................................................................................xii
CHAPTER1:INTRODUCTION...............................................................................................1CHAPTER2:LITERATUREREVIEW....................................................................................6INTRODUCTION.................................................................................................................................6BACKGROUND....................................................................................................................................8MACRONUTRIENTMETABOLISM...............................................................................................11Proteinbalanceandoxidation.................................................................................................................11CHObalanceandoxidation.......................................................................................................................12Fatbalanceandoxidation.........................................................................................................................12Fatoxidationandfatbalanceinskeletalmuscle.............................................................................16
METABOLICFLEXIBILITY.............................................................................................................18GUTPERMEABILITY.......................................................................................................................22CONCLUSION.....................................................................................................................................25REFERENCES.....................................................................................................................................26
CHAPTER3:SPECIFICAIMS...............................................................................................34
CHAPTER4:RESEARCHDESIGN......................................................................................36CHAPTER5:SKELETALMUSCLEMETABOLICADAPTATIONSINREPONSETOANACUTEHIGHFATDIET.................................................................................................39ABSTRACT.........................................................................................................................................39INTRODUCTION...............................................................................................................................41METHODS...........................................................................................................................................43Participants.....................................................................................................................................................43Experimentaldesign...................................................................................................................................43ControlledFeedingProcedures.............................................................................................................44HighFatMealChallenge............................................................................................................................45MeasurementsandProcedures.............................................................................................................46Statistics...........................................................................................................................................................54
RESULTS.............................................................................................................................................55Participantcharacteristics.......................................................................................................................55Diet.....................................................................................................................................................................55Wholebodymeasurements.....................................................................................................................56Substratemetabolism................................................................................................................................57Pyruvatedehydrogenasecomplex.......................................................................................................59AdaptersandNon-AdaptersinFAOandGO.....................................................................................60
DISCUSSION.......................................................................................................................................63
x
Furtherdirections........................................................................................................................................69Conclusion.......................................................................................................................................................69
FIGURELEGENDS.............................................................................................................................70REFERENCES.....................................................................................................................................71
CHAPTER6:CONCLUSIONS/FUTUREDIRECTIONS...................................................75
xi
LISTOFFIGURES
CHAPTER2:LITERATUREREVIEW
Figure1:Schematicofmetabolicallyhealthyindividual……………………………………..….2
Figure2:Schematicofmetabolicallydiseasedindividual……………………………………….3
Figure3:Metabolicflexibility………………………………………………………………………….….19
CHAPTER5:SKELETALMUSCLEMETABOLICADAPTATIONSINRESPONSETOANACUTEHIGHFATDIETFigure1:Schematicofresearchdesign………………………………………………………………..44
Figure2:Mealchallengebloodmeasures…………………………………………………………….57
Figure3:Substrateoxidation……………………………………………………………………………...59
Figure4:Pyruvatedehydrogenasecomplex………………………………………………………...60
Figure5:Fattyacidoxidationadaptation………………………………………………….…………61
Figure6:Glucoseoxidationadaptation……………………………………………………………….62
xii
LISTOFTABLES
CHAPTER5:SKELETALMUSCLEMETABOLICADAPTATIONSINRESPONSETOANACUTEHIGHFATDIETTable1:MS/MSTransitionsfordetectionofsugarprobes…………………………………..49
Table2:Participantcharacteristics…………………………………………………………………….55
Table3:Dietmeanenergyandmacronutrientcontent………………………………………...55
Table4:Wholebodyfastingmeasures………………………………………………………………...56
Table5:SubstrateMetabolism……………………………………………………………………………58
1
CHAPTER1:INTRODUCTION
Obesityandothermetabolicdiseasesaremajorcontributorstoserioushealth
conditionsamongAmericans.TheprevalenceofobesityintheUnitedStatesandglobally
hasgrownrapidlyinthelastthreedecades.In2014morethanone-third(27.9%)ofUS
adultsmetthedefinitionofobesity(BodyMassIndexofgreaterthan30kg/m2)1.Likewise,
accordingtothe2014NationalDiabetesStatisticsReport,theprevalenceofType2
Diabetesmellitus(T2DM)isontherise.In2012,9.3%ofthepopulationhadT2DM,
accountingfor29.1millionpeople.Theprevalenceforadultsage20andolderin2012was
12.3%.Diabetesisthe7thleadingcauseofdeathwithintheUnitedStatesin20132.Inorder
tobetterunderstandT2DM,obesityandothermetabolicdiseases,researchintothe
mechanismscontributingtoorprimingthebodyfortheseconditionsisimperative.
Whileoverallhealthismulti-factorial,anumberofcharacteristicsofmetabolic
healthandlikewise,metabolicdisease,havebeenelucidated.Belowaretwosimplified
diagramsillustratinginFigure1,ametabolicallyhealthyindividualandinFigure2,a
metabolicallydiseasedindividual.Thesearecertainlynotexhaustiveinnature;however
provideanexemplaryofdisturbancesthatoccurasaresultofconsumingahabitualhigh
fatdiet.
2
Figure1.SchematicofMetabolicallyHealthyIndividual
Figure1,depictstheprocessesinametabolicallyhealthyindividual.When
consumingawell-balanceddiet,thegutmaintainsintegrityandproperfunctionofits
barrier,releasinglittletonoendotoxinsintocirculation.Theskeletalmusclerespondsto
substratesavailableandoxidationofthemostpredominantmacronutrientisupregulated.
Skeletalmuscleismetabolicallyflexible,andtheprocessesarehighlyfunctioning.
However,infigure2,whichdepictsametabolicallydiseasedindividual,these
processesaredisrupted.Ahighfatdietdisruptsgutbarrierfunction,increasinggut
permeability,leadingtoendotoxinsbeingreleasedintocirculation.Low-gradeelevationof
plasmaendotoxins,metabolicendotoxemia,activatestoll-likereceptor-4(TLR4),whichin
3
turncausesanincreaseinTLR4expressioninskeletalmuscle.AnincreasedTLR4presence
inskeletalmusclefavorsglucoseoxidation(GO)regardlessofthesubstratethatis
available.Likewise,thisfavoringofGO,inhibitsfattyacidoxidation(FAO).Thesedisrupted
processesleadtoaproinflammatorystateanddysregulatedmetabolismasseeninobesity,
Type2Diabetesandinsulinresistance.
Figure2.SchematicofMetabolicallyDiseasedIndividual
Thecomplexityofsubstrateoxidationinthepresenceofdifferentdietary
compositionshasbeenconnectedtometabolicdiseasestatesincludingobesity,T2DMand
4
metabolicsyndrome3–7.Whileproteinoxidationremainsrelativelystableregardlessofthe
compositionofthemeal,carbohydrateandfatoxidationareshowntofluctuategiven
differentpercentagesofmacronutrientsinthediet8.Theconsequencesofthealterable
oxidationandutilizationofthesesubstrateshasbeenasubjectofresearchasthegrowing
epidemicofobesityandT2DMcontinuestoplaguepeopleoftheworld.
Skeletalmuscleisnotonlyaprimarysiteofglucoseoxidation9,butalsomakes
substantialcontributionstowholebodyfatoxidation10.Habitualaswellasacutedietare
associatedwithvaryingdegreesofglucoseandfatoxidationwithintheskeletalmuscle.The
abilityofskeletalmuscletoutilizeandadapttoavailablesubstratesistermedmetabolic
flexibility11.Linkedtothevariableoxidationratesamongdifferentdietcompositions,
metabolicflexibility(orinflexibility)intheskeletalmusclehasbeenassociatedwith
diseasestates,suchasinsulinresistanceandobesity12.Whatisunknownisifmetabolic
inflexibilityinskeletalmuscleprecedesdiseasestatesorifdiseasestatescausemetabolic
inflexibility.Furtherresearchisneededtofurtherelucidatethisquestionandto
understanddisruptionsinsubstrateoxidationandmetabolicinflexibilitywhenparticipants
aresubjectedtoahighfatdiet.
Gutpermeability,whichisthecontrolofsubstancespassingthroughtheintestinal
wall,hasbeenassociatedwithdiseasestatesmentionedabove.Dieteticfactorshavebeen
showntoincreasegutpermeability13.Diethasalsobeenlinkedtoanincreasedpresenceof
endotoxinsintheblood14.Theassociationofhighfatdietandendotoxemiaoriginating
fromtheguthasbeenatopicofgreatinterest.Furtherresearchisneededinorderto
understandthecontributingfactorsofmetabolicendotoxemia.
5
Avarietyoffactorsmustbeconsideredwhendeterminingsubstratemetabolismin
skeletalmuscleanditsassociationtodiseasestates.Anadditionaltoolthatcanprove
valuableiscategorizingmetabolicphenotypesbyclassifyinggroupsofadaptersversus
non-adapters;adaptationtowhichvariabledependsontheresearchquestiontobe
answered.Forexample,whenanalyzingfattyacidoxidation,theadaptersareinreference
tothosewhoadaptedtohighfatfeedingbyincreasingfattyacidoxidation,whereasthe
non-adaptersarethosewhodidnot.Bycharacterizing,wemaybeabletopotentially
identifyfactorsthatcontributetotheonsetand/orprogressionofmetabolicdiseaseinthe
contextofhighfatfeeding.
6
CHAPTER2:LITERATUREREVIEW
INTRODUCTION Onlywithinthelast60yearshasobesitybecomeawidespreadissueofpublic
concern.WhiletherearehistoricartifactsofStoneAgeVenusandpaintingsofChinese
emperorswhowouldbeconsideredobese,andancientscholarsanddoctorswhotied
obesitytohealth(orlackthereof),thewidespreadprevalenceandresultingepidemicof
obesityisfairlyrecent.Accordingtothemostrecent(2011-2014)UnitedStatesNational
HealthandNutritionExaminationSurvey(NHANES)data,nearly40%ofAmericansare
obese(BMIgreaterthanorequalto30kg/m2)1,spanningacrosssocioeconomicclasses,
age,race,andgender.Annually,theestimatedmedicalcostsofobesityarenearly$150
billion15.Becauseoftheconsiderableeffectofobesityonchronicdisease,animmense
amountofresearchhasgoneintounderstandingitsimpact.Researchshowsthatlife
expectancycandecreaseanywherefrom3to14yearsforobeseindividuals,notingthatas
BMIincreases,relativeriskofmortalityincreases16,17.Trendsshowthepotentialfor
childrenborninthisgenerationtohaveashorterlifeexpectancythanthoseoftheir
parents;thefirsttimethiseffectisrealized18.RiskofT2DM,cardiovasculardisease,cancer,
becomingandremainingdisabled,andpsychologicaldisorderseachhaveapositive
correlationwithobesity19–21.Obesityisariskfactorfor7ofthe10leadingcausesofdeath
intheUnitedStates22.Obesityhasnotonlybecomemedicalizeditself,butitsclose
associationwithotherriskfactorsandchronicdiseasesmakeitasignificantissueofpublic
concern.
Althoughearlierresearchexistsonobesityanditsrelationshiptothedevelopment
ofchronicdisease,inthe1960’sand70’s,therebegantobeaconcentratedefforttodefine
7
thecauses,risks,mechanismsandanythingmorethatcouldbeacontributortoobesity.
Muchoftheresearchwasfocusedondeterminingbodyweightregulationandits
connectiontothedevelopmentofchronicdisease.Macronutrientshavebeenaprimary
focusofthisdiscussion.
Anextensiveamountofresourceshavebeencommittedtounderstandingobesity
andchronicdisease,butwhatdowereallyknowabouttheeffectsofmacronutrient
metabolismonhealth?Researchisprevalent,butaconcreteunderstandingand
comprehensiveknowledgeislackinginmanyareasofthisimportantissue.Therearemany
schoolsofthoughtinthehighlydebatedandcontroversialtopicoftheprimarydietary
factorsaffectingcardiovasculardisease,T2DMandobesity.However,inthe1950s-1960s,
thereweretwomainareasoffocus,1)fatwasthemaindietaryinfluenceofcoronaryheart
disease(CHD)or2)sugarwasamoresignificantcontributortotheassociatedrisksofCHD.
Studiesexaminingtheroleoffatoxidationandbalanceonmetabolismandthe
regulationofbodyweightareinterspersedintheliterature,butduetoobserved
associationsbetweensugarintakeandtheriseinobesity,thestudyonCHOloadandits
effectsonobesityhasbeenquitepopular.RecommendationsfromtheUnitedStates
DepartmentofAgriculture,asearlyasthe1980s,weremadetodecreasefatconsumption,
whichresultedinanunintendedincreasedrefinedsugarandCHOconsumption23.The
guidelines,evenfrom1980,suggestanincreaseincomplexcarbohydrates,meaning
vegetables,fruitsandwholegrains.However,thefoodindustry’smarketingresponsewas
thelowfatcraze,whichincidentallyincreasedintakeofrefinedsugarandsimple
carbohydrates.Bodyweight,T2DM,andotherchronicdiseasesamongAmericans
continuedtorise.
8
Thisreviewisintendedtoexaminewhatisknownaboutmacronutrientmetabolism
anditseffectsonhealth.TheRandlecycleandsubstratemetabolismanditsinrelationto
obesityandchronicdiseasewithaconcentrationonskeletalmusclewillbediscussed.More
specifically,wholebodyandskeletalmusclemetabolicflexibility,inthecontextofhighfat
feeding,willbeexamined,furtherexploringfatbalanceandfatoxidationinskeletalmuscle.
BACKGROUND
Theglucosefattyacidcycle,orRandlecycle,namedforSirPhilipRandle(1963),is
foundationaltoourunderstandingofmacronutrientmetabolismandenergyhomeostasis.
Inhiswork,heandhiscolleaguesdetailedthemechanismsbehindtheabilityofcardiac
andskeletalmuscletoshiftbetweencarbohydrate(CHO)andfatuseandstorage,
dependingonsubstrateavailability.Asthetheorywasconceived,Randleandhisgroup
usedthelong-standingideasthatsubstratescompeteforrespiration.Forexample,early
researchinthe1930sindicatedcompetitionbetweenaminoacidsandglucosewhenthe
deaminationofaminoacidsinkidneytissuewasinhibitedbyoxidizablesubstrates24,and
intheperfusateofdogheart-lungpreparation,thepresenceofcarbohydratesinhibit
ketoneutilization25.Furtherworkintheearly1960sreportedinhibitionofglucose
utilizationandoxidationbyacetoacetateandpalmitate26,27.Theseandotherstudiesled
Randleandhisgrouptodevisethetheoryoftheglucosefattyacidcycle.Thetheory
includedafewkeycomponents;thefirstofthosecomponents,simplystated,isthatthe
relationshipofglucoseandfattyacidmetabolismisreciprocal,andnotdependent,meaning
thatelevatedglucoseconcentrationsstimulateinsulinsecretionandsuppressfattyacid
releasefromadiposetissue.Secondly,fattyacidsandketonebodiesthatarereleasedinto
9
circulationintimesofdiseaseorstarvationinhibitthebreakdownofglucoseinmuscle.
Elevatedfattyacidconcentrationsincirculationareusuallyindicativeoflowglucoseand
insulin,therebybecomingtheprimaryfuelsourceofskeletalmuscle,whichreduces
glucoseuptakeandoxidation.Thepurposeoftheglucose-fattyacidcycletheory,whichis
nota“cycle”atall,wastoexplainthebiochemicalmechanismofthe
competition/interactionofglucoseandfattyacidoxidation.
ResearchershavecontinuallyinvestigatedtheRandlecycleanditsconstituentsto
furtherunderstandmechanismsresponsibleforthedevelopmentofinsulinresistance,
T2DM,andobesity,whichareclearlyassociatedwithalteredmacronutrientmetabolism.In
ordertoobtainaclearerunderstandingoftheirmechanismsofaction,methodsof
measuringmacronutrientsandspecifichormones,suchasinsulin,havebeendeveloped,
improvedandreinvented.ReubinAndresandhisgroupwerefirsttodescribethemethods
ofthehyperglycemicandeuglycemicclampsandtheiruseformeasuringglucoseand
insulinsensitivity28.Theuseofthesemethodsimprovedtheassessmentof2variables:
beta-cellresponsetoglucoseandsensitivityofbodytissuestoinsulin.Previously,ratiosof
insulinandglucoseconcentrationswereusedtocalculatethesevariables,however,the
resultswereofteninaccurategivenneithervaluestaysconstant,andtherelationshipisnot
linear.Additionally,thehyperglycemicportionofthemethodquantifiesthetimecourseof
theamountofglucosemetabolized.Theeuglycemicportionalleviatestheneuroendocrine
responseofhypoglycemiaandthepotentialhazardofhypoglycemicreactionsthatthe
insulintolerancetestinduced28.
RavussinandBogardus’sworkofputtingtogetherthemethodsfortheuseofthe
euglycemicclampandindirectcalorimetrywasmonumentalinourfurtherunderstanding
10
ofthefatesofglucoseandfattyacids29,30.Combiningthedataforthesetwotestingmethods
hasenabledscientiststonotonlyhaveaclearerpictureofmacronutrientmetabolism,but
alsoamoredependablemeasure.Previousestimationswerecalculatedbyratiosandother
equationsandwereinconsistent.Additionally,Ravussin’sgroupdidearlyresearchonuse
ofthehumanrespiratorychamberfordeterminingmetabolicratewhichenabledthemto
identifyphysiologicaldeterminantsofenergymetabolisminhumans31.Useofthechamber
isstillagoldstandardinmeasuringmetabolicrate.Studyofrespiratoryexchangeratio
(RER)whichistheratioofcarbondioxideproducedtooxygenconsumed,continuesto
revealfactorsotherthandietcompositionthatcontributetothefattoCHOoxidationratio.
Factorsworthmentioning(thatcontributetomacronutrientmetabolism)aregender32,33,
familymembership33,totalenergyexpenditure34,musclefibertype35,musclemass36,
trainingstatus4,37,habitualphysicalactivitylevel37,leanorobesebodycomposition32,37,38,
andofcourse,thepresenceofinsulinresistance/T2DM39–41.
Inareviewwrittenin1998,Randleacknowledgednewdevelopmentsontheeffects
offattyacidoxidationonglucosemetabolism,citingworkfromanumberofscientistsover
theperiodof35years,recognizingtheimportanceofongoingresearchandthecomplexity
ofthesemetabolicprocesses42.Oneofthemainconclusionsdrawnfromthisreview
involvedthemoreextensiveroleoffattyacidsinglucosemetabolism.Afewexamples
includefattyacidoxidation’sinhibitionofglucosecatabolismandstimulationof
gluconeogenesis,theroleoffattyacidsintheinsulinsecretoryresponseofisletbetacellsto
glucose,fattyacidoxidationimpairmentofglucoseoxidationindiseasestatessuchas
T2DM,andelevatedserumfattyacidsinhibitingglycogensynthesis.Manyfoundational
principlesareaccepted,butresearchersareconstantlychallengingthemfurtherinorderto
11
betterunderstanddiseasestatesthatareaffectingpeopleallovertheworld(obesity,
insulinresistance,T2DM).
MACRONUTRIENTMETABOLISM
CHObalanceistightlyregulated,substantiallymorethanfatbalance,duetoits
limitedstoragecapacityandthebody’sobligatoryuseofglucoseasafuelsource3–8.Protein
offersaverysmallandconstantsupplyofenergy,thereforetheintakeandutilizationof
CHOandfatareofprimaryinterestwhendeterminingmacronutrientmetabolism.
Proteinbalanceandoxidation
Proteinintake,aslongasitisadequate,haslittletonobearingonproteinbalance.
Thehealthybodyinstinctivelymaintainsaproteinbalancebyadjustingaminoacid
oxidationtoaminoacidintake.Recentresearchhasshowninaninsulinresistantstate,
increasedserumBCAAconcentrationdetrimentallyaffectsmitochondrialfunction43,44.
AdditionalresearchisneededtofurtherunderstandtheroleofBCAAsininsulinresistance.
Positiveenergybalance,aconditionoftenassociatedwithmetabolicdiseasestatesis
relatedtoadisruptionintheefficiencyofproteindegradationandstorage45.Also,high
proteinintakeshowsareducedenergyefficiency46.However,incomparisontoCHOand
fat,thefractionofdietaryenergyfromproteinisrelativelysmall.Therefore,regulationof
bodyweight,whenadiethassufficientamountsofprotein,isnotdeterminantonprotein
balance5,7,8.Proteolysisisessential,however,duringthebeginningstagesofstarvation(24-
48hours).Afterliverglycogenisdepleted,bloodglucosehomeostasisismaintained
throughgluconeogenesis.Proteolysisistheprimarysourceofenergyuntilketone
12
production,after~48hours,becomesthemainenergysourceinordertopreserve
protein47.Inthepostprandialperiod(1-5hours),proteinbalanceisaffectedverylittleby
proteinintake.
CHObalanceandoxidation
Postprandially,CHOoxidationhappenswithinminutes,andbalancewithinhours.
Glucoseoxidationinthepostprandialstatehappensattherateof~10g/hr5.Toputthat
amountintocontext,a500-caloriemealthatis50%CHOwouldcontainabout65gramsof
carbohydrates.SomeoftheingestedCHO(glucose)isconvertedintoglycogen,thestorage
formofglucose,primarilyintheskeletalmuscleandtheliver.Thebody’sglycogenstores
arefairlysmall(approximately120gintheliverand200-500ginskeletalmuscle)5,6,48
comparedtothedailyCHOturnover,soglucoseoxidationandstoragemustbefine-tuned
tomatchintake.Inanefforttomaintainbloodglucoseconcentrationswithinaspecific
range,thehormonesinsulin,intheeventofhyperglycemia,andglucagon,intheeventof
hypoglycemiaarereleased.Thesehormoneseitherpromotestorageofglucose(insulin)or
elicitabreakdownofglycogentoglucose(glucagon).Theseprocessesaretightlycontrolled
inordertomaintainCHObalance,inturnfacilitatingphysiologicalhomeostasisinthe
contextofbloodglucose.
Fatbalanceandoxidation
Fatdoesnothavethedirectregulatoryinteractionsinresponsetodietcomposition
thatisfoundinproteinandCHOmetabolism.Fatbalancecantakeuptoseveraldays,ifit
balancesatall–consideringdiseasestatesandhabitualdiet4,32,49.Theingestionoffatdoes
notautomaticallystimulatefattyacidoxidation49,unlikethepresenceofCHOstimulating
13
glucoseoxidation.Ithasbeensuggestedthatthecorrelationofintaketofatbalanceismore
pertinenttotheamountofCHOintakeratherthanfatintake3–5.Toexpandonthisidea,
someresearcherssuggestthatfatoxidationoccursafterCHOoxidation,notonlybecauseof
thelongertimeperiodneededforfattobedigested,butalsoduetothehighprioritygiven
toCHObalance.Ithasalsobeensuggestedthatwhenglycogenstoresarelowandahighfat
dietisconsumed,thebodytendstooxidizefatinordertopreserveglycogen7,8.
Flattandhisgroupfoundthatfatoxidationdidnotchangewhencomparingalow
fatmealtoamealsupplementedwithlong-chaintriglycerides(LCT)ormedium-chain
triglycerides(MCT)6.Respiratoryexchangemeasurementsweretakenusingaventilated
hoodsystem(indirectcalorimetry)afterparticipantsateoneofthethreemeals.
Carbohydrate,protein,andfatoxidationwerecalculatedusingtherespiratoryquotient
(RQ),andnodifferencesinoxidationwerefoundacrossthemeals.Whiletheoxidationwas
notdifferent,thechangesofRQovertimeweredifferent,showingthatparticipants’fat
balancewasnegativeafterbeingfedalowfatmeal,suggestingimportanceoffatintaketo
shorttermenergybalance.Inaddition,theparticipant’senergybalancewasessentially
equaltotheirfatbalance.Thissuggeststhatwhendeterminingenergybalance,importance
mustbeplacedonfatintake,eventhoughfatcontentinamealdoesnotinfluenceCHOor
fatoxidation.
Whenbloodglucoseconcentrationsrise,insulinsecretionisstimulated,whichin
turn,increasescarbohydrateoxidation,anddecreasesfatoxidation5.Glycogenstoresare
alsoadeterminantoffatoxidation.Whenglycogenstoresaredepleted,andthemealishigh
infat,postprandially,thebodyisprimedtofirstutilizetheCHOavailableinthemeal,but
betweenmeals,duetothelowglycogenavailable,fatoxidationwillbeincreased.Thiswas
14
observedinhumansubjectswhoconsumedMCTaspartofamixedmealincomparisonto
thosewhoconsumedLCToralowfatmeal6.TheingestionofMCTpromotedfatoxidation
inthepostprandialperiod,thereforemoreglycogenwasspared,evidencedbytheRQ
stayinghigheraftertheMCTmealcomparedtoaftertheothertwomeals.Conversely,if
glycogenstoresareatmaximumcapacity,dietaryfatisoftenconvertedtochylomicronsin
thegutandtargetedforlipogenesis.
In1996,Sidossis,etal.foundthatglucoseand/orinsulindeterminestherateoffat
oxidationandtermeditthe“Randlecyclereversed”50.TheratiooffattoCHOoxidation
determinestheRQ.HighRQindicatesmoreCHOoxidation,andlessfatoxidation,whereas
lowRQislessCHOoxidationandmorefatoxidation.Thisvaluerangesfrom0.70,whichis
consideredtobeprimarilyfatoxidation,to1.0,whichisconsideredtobeprimarilyCHO
oxidation.Fatandcarbohydrateoxidationratesaredependentonanumberofvariables,as
mentionedpreviously.Nomatterthecompositionofthemixedmeal,ifCHOispresent,CHO
oxidationwillbeapartofthepostprandialperiod(1-5hourspostmeal)duetothetight
regulationofthissubstrate;however,fatoxidationmaynotbeasactivelyengaged.Fat
oxidationoccursaftertheaminoacidandCHOoxidationratesadjustthemselvestothe
amountconsumedinthemeal5,7.
Ingestionoffoodatlevelssufficientenoughtomaintainglycogenstoresmaycause
fataccumulationinadiposetissue,whichcanstoreanenormousamountoffatenergy.
However,theprocessoflipolysisiscomplex.Eventhoughfatstoragecapacitymaybe
muchgreaterthanthatofCHOstorage(CHOstorageis~5%offatstorage.),fatenergy
storesmaynotbeasreadilyavailableoraccessible.Endocrinehormones,suchas
catecholaminesandglucagoninadditiontootherproteinsthroughoutthegastrointestinal
15
tract,blood,adiposetissueandskeletalmuscle(adiposetriglyceridelipase–ATGL,
monoacylglycerollipase–MGL,hormonesensitivelipase–HSL),worktogethertopromote
mobilizationoffatasanenergysourcewhenneeded.
Theprocessoflipolysis,briefly,involvescatecholaminesand/orglucagonsignaling
theneedforenergyfromtriglycerides.Hydrolysisoftriglyceridesreleasesfattyacidsand
glycerolintothecirculationtobeusedasenergy.TheprocessinvolvesATGL,MGLandHSL,
lipasesthathydrolyzetriglycerides,diacylglycerol(DAG)andmonoacylglycerol(MAG),
intofreefattyacidsandglycerol.Fattyacidsformedfromtheseprocessescanbeoxidized
andutilizedforenergythroughbeta-oxidation.Theamountofactivity(oramountof
energyneededfromlipolysis)isdeterminedbytheallostericorcovalentmodificationsof
specificstepsintheprocess.Whenasufficientamountoffreefattyacidshavemetthe
energydemand,insulinincreasesorcatecholamineandglucagondecrease,whichinhibits
lipolysis.Anexceptiontothisisfoundinstatesoffasting,starvationorextendedexercise,
whenlipolysisisactive.
Oxidationratesarehighlyvariable.Eveninacaseofenergybalance,theoxidationof
asubstrateforoneindividualdoesnotnecessarilyequaltheoxidationrateofanother,
giventhesamemealordiet.Adaptationsinmacronutrientmetabolismareextensivein
differentconditions.Differencesinfatoxidationareobservedduringexercisebetween
endurancetrainedanduntrainedindividuals,regardlessofthecompositionofthepre
exercisemeal51.Furtherevaluationshowedendurancetrainedindividualshaveahigher
rateoffatoxidationatahigherexerciseintensitywhencomparedtountrainedindividuals,
likelyduetodifferencesinintramusculartriacylglycerolstores,potentiallygreater
oxidativecapacity,andrecentresearchshowsincreasedvasculatureinskeletalmuscleof
16
trainedindividuals52.Inanotherstudy,highlytrainedindividualshadahighergene
expressionofspecificfattyacidbindingproteins,whichisobservedinconditionsof
increasedfattyacidutilization53.AgroupattheNationalInstitutesofHealthusedmice
deficientinmyostatin,andthereforewithgreaterskeletalmusclehypertrophythanwild
typemice,toshowthatanimalswithmoreleanmasscanoxidizefatataratesimilartothat
ofCHO36.Inobesemice,fast/glycolyticmusclefibertypewasassociatedwith
improvementsinfattyacidoxidation54.Familialmembershipandgenderwasfoundtobe
associatedwithmetabolicdifferences,specificallylowerfatutilization,infemalePima
Indians33.Theseandothercharacteristicsshowindividualvarianceinenergybalanceand
oxidationsofsubstrates.
Becausefatoxidationandbalanceisdependentonsomanydifferentvariables,
improvingourunderstandingoftheeffectsofdietcomposition,diseasestates,andthe
myriadofinter-participantdifferencesremainsanimportantaspectofdeveloping
expertiseinthemetabolicperturbationsassociatedwithlipidscontributingtodisease.
Fatoxidationandfatbalanceinskeletalmuscle
Fattyacidoxidationaffectsglucosemetabolismnotonlyatthewholebodylevel,but
alsowithinmuscle50,55–57.Aoncehighlydebatedtopic,denovolipogenesis(DNL)orthe
enzymaticpathwayresponsibleforturningdietarycarbohydratesintofat58,isstillunder
reviewandisfarfromunderstood.Researchminimallyshowsthatitisfunctionally
important58–60.Additionally,itsrelationtoCHOandfatintakeaffectsmetabolic
homeostasis61,62.DNLoccursprimarilyinhepatictissue,especiallyafterahighCHOload
whenglycogenstoresarefullandexcessCHOisconvertedtofattyacidsand
triacylglycerols(TAG)58.However,DNL,inthemuscle,isacontributingfactortoinsulin
17
sensitivityandmuscularstrength63aswellasapotentialmarkerfordisease64.Anexample
ofitsimpactwasaninvestigationdoneinrodents,whichrevealedskeletalmusclespecific
inactivationoffattyacidsynthaseprotectedmicefrominsulinresistance,butinduced
muscleweakness63.Furtherresearchisneededtounderstanddenovolipogenesisandits
impactonfatmetabolism,specifically.
Acommonthemeintheliteratureaddressingfatoxidationandbalanceisthe
alteredfatmetabolismthatoccursinskeletalmuscleinthepresenceofinsulinresistance
and/orT2DM12,65–71.Itiswell-knownthatinsulinresistantmusclehasanimpairedability
tooxidizefatduringconditionsofincreasedfattyacidsupply,suchasintimesoffastingor
exercise68,72.Additionally,fatoxidationhasbeenshowntobeimpairedinthepostprandial
stateinT2Dandobesity68,73.Researchhasalsoshownthroughstudyofinvitromyotubes
thatwhenextracellularfattyacidsareelevated,fattyacidoxidationisalsoelevated,which
inturnsuppressestheoxidationofintramyocellularlipids74.Theyalsofoundthatthe
oxidationrateoftheselipidsweredependentuponmitochondrialfunction,ratherthan
mass,observedthroughthestainingandlivecellimagingofmitochondria.The
accumulationofintramusculartriglyceridesovertimeisassociatedwithreducedoxidative
capacity68,75anddevelopmentofinsulinresistance76.Mechanismsarenotclearlydefined,
butmitochondrialfunctionisalikelycontributor.Clearly,fatoxidationandbalanceatthe
leveloftheskeletalmuscleplaysacriticalroleinhealthofthewholebody.
Furtherinvestigationoffatoxidationandbalance,especiallyinskeletalmuscle,will
leadtomoreanswersaboutwhatmaybehappeningbeforetheonsetofobesity,insulin
resistance,orT2DM.Infact,in2008,Galgani,Moro,andRavussinrecognizedthelackof
studiesinvestigatingskeletalmuscleresponsetohighfatdiets77.Recently,Saponaro,etal.
18
concludedtheirreviewfocusedonlipolysisandlipogenesis,withacalltoidentifyearly
biomarkersofcardio-metabolicdisease62.Intheauthor’sview,analysisofthemechanisms
behindchangeinfatoxidation(andlikewisemetabolicflexibility)inhighfatfeeding
studiesmayofferfurtherunderstandinginthoseareas.
METABOLICFLEXIBILITY
Theabilityofthebodytoutilizeandadapttothefuelsourcesavailableismetabolic
flexibility,atermintroducedbyKelleyandMandarino11.Whenexploringthecapacityof
wholebodyorskeletalmuscletoswitchbetweensubstrates,CHOandfatoxidationand
uptakeareanalyzed.Inlean,healthymodels(animalandhuman),glucoseuptakeand
oxidationistheprimarysourceofenergyuntilthefastedstate,atwhichtimefattyacid
oxidationrampsupinordertopreserveglucose.Adysfunctionintheseprocessesis
termedmetabolicinflexibility,occurringwheneithersubstrateisinefficientlyoxidized
whileit’stheprimaryfuelsource,asseeninFigure3.Thecomplexityofthisinflexibilityis
seeninmetabolicdiseasestates12.
Figure3:MetabolicFlexibilityfromKelley,JClinInvest.2005;115(7):1934-1931.
19
Metabolicflexibilityhasprimarilybeenmeasuredandanalyzedatthewholebody
level.Well-known,establishedapproachesofmeasuringmetabolicflexibilityatthewhole
bodylevelincludedifferenttypesofmethodsofindirectcalorimetryandrarelyuseddirect
calorimetry.Usingahoodsystemormetaboliccart,indirectcalorimetrymeasuresthe
amountofheatbygatheringtheoxygenconsumptiontocarbondioxideproductionratio
andcalculatingtheRQduringacertaintimeperiod.TheexcretionofCO2isusedto
determinethedominantfuelthatwasutilizedduringthesettimeperiod.Metabolic
flexibilityistypicallyevaluatedbythechangeinRQ(insulin-stimulatedRQ–fastingRQ).
UsingO2andCO2,indirectcalorimetrycanalsobeusedtomeasureRQinametabolic
chamberandacrossthearterialandvenousbloodacrossextremities.Onrareoccasion,
directcalorimetrygathersthesameinformation,oxygenandcarbondioxide,butusesheat
20
productionfromtheindividualtodeterminesubstrateutilizationinametabolicchamber.
Additionally,thehyperglycemic,euglycemic,andhyperinsulinemicclampmethodshave
beenusedtoquantifythechangeinRQinresponsetoinfusionsofglucoseandinsulin.
Substrateutilizationcanalsobemeasuredinskeletalmuscle.Specificskeletal
muscleanalysisisvaluablebecauseitisthetissueresponsibleforthemajorityofinsulin
stimulatedglucoseuptakeinthebody.Freefattyacidactivityinskeletalmusclecanbe
measuredbythelegbalancetechnique.Bloodsamplingisdonebeforeandafterasubstrate
isinfusedtodeterminetheactivityoftheradiolabeledsubstrate78,79.Glucoseandlipid
metabolismarethenestimatedbylegindirectcalorimetry;fromtheblood,RQcanbe
analyzed.Frequently,thismethodisaccompaniedbymusclebiopsies,oftenforpurposesof
determiningpyruvatedehydrogenaseandcitratesynthaseactivity.Themostwidelyused
methodtoobtainskeletalmuscleisthemodifiedBergströmbiopsymethod80.Muscleis
obtainedandpreparedaccordingtotheprotocolutilizedtodeterminesubstrateoxidation.
Anarrayofmetabolomicscanbeanalyzedinthesesamplesusingmassspectrometry81.
Metabolicflexibilitycanalsobemeasuredusingradiolabeledsubstrates-theratioof
radiolabeledpyruvateoxidationtopyruvateoxidationandpalmitate(methodsnotyet
published,MatthewHulverlaboratory,VirginiaTech).
Anotherrecentlyexaminedmethodofdeterminingmetabolicflexibilityisin
peripheralbloodmononuclearcells.Recently,Baigetal.showedthatobesityrelated
metabolicinflexibilitycanbeseeninmononuclearcells,afterahighCHOmeal,by
measuringpostprandialexpressionofvariousgenesinfattyacidandglucosemetabolic
pathways82.Evidencemaynotbestrongenoughtosupportusinggeneexpressionalone,
butthisgroupfoundtheevidencecompellingwhencomparedtoRQdataandsuggested
21
thismethodasanalternativetoskeletalmusclebiopsies.However,theydidnotcompare
thedatatoskeletalmusclebiopsiestodetermineiftheinformationisdirectlytranslatable
orspecifictoskeletalmusclemetabolicflexibility.
Limitedresearchhasexploredskeletalmusclemetabolicflexibility.Anincreased
understandingmayfurtherelucidatedifferencesinindividualswithandwithoutmetabolic
diseases.Explorationofthevariablesthatcontributetoinflexibility,likewise,canbe
beneficial.Metabolicflexibilityisimpairedindiseasestates68,77,82,83andafterhighCHOor
highfatmeals71,84–86.Whilesomeofthisresearchisspecifictoskeletalmuscle,thelargest
bodyofresearchhasbeendoneanalyzingwholebodymetabolicflexibility.
Galgani,MoroandRavussinreviewedmetabolicflexibilityandinsulinresistance
anddeterminedthatwiththeresearchavailable,impairedmetabolicflexibilitywasnot
responsibleforinsulinresistanceandimpairedintramyocellularlipid77.Differencesseenin
metabolicflexibilityduringtheclampisaconsequenceofglucosedisposalrate,andwhen
corrected,metabolicflexibilityisnotimpaired87.Inregardstolipidsandmetabolic
flexibility,theypointedoutthatmuchoftheresearchisdoneusingRQunderfastingand
restingconditions,whicharenotidealbecausefatoxidationisunlikelytoshowadefectin
thoseconditions.Duetothevariabletimeforfatbalanceasdiscussedpreviously,the
authorsaddemphasisontheimportanceofunderstandingtheadaptationsinfatoxidation,
pointingoutthatthetimetoadaptationisrelevanttofatgain.Skeletalmuscle
mitochondrialcharacteristics,suchassize,activity,andnumberofferapotentialreasonfor
thevariationsinmetabolicflexibilitytolipids88.
Researchin2011byChomentowski,etal.,foundthatlowermitochondrialcontent
inskeletalmuscleofinsulinresistantindividualsisassociatedwithalteredpatternsoffuel
22
oxidation(metabolicinflexibility).Theysuggestedthelowermitochondrialcontentmaybe
associatedwithintramyocellularlipidoverloadandassociatedmitochondrial
adaptations89.Likewise,Boushel,etal.foundmitochondrialfunctioninT2DMpatientsis
normalbutsuggestedlowermitochondrialcontentmaybethereasonfortheblunted
oxidativephosphorylationandelectrontransportcapacity90.In2013,vandeWeijer,etal.
concludedfromtheirinvestigationofT2DMpatients,thatdefectsinskeletalmuscle
mitochondrialfunctionareonlyreflectedinbasalsubstratehandling85.Thesefindings
suggestmitochondriaasapotentialtargetindiseasedmodels,butinordertofurther
understandifmitochondrianumber,function,sizeoracombinationofthese,effects
metabolicflexibility,skeletalmusclemetabolismmustbemorethoroughlyexamined.
Theeffectofdietonskeletalmusclesubstrateoxidationandmetabolicflexibilityin
lean,healthyhumanparticipantsislacking,atbest.Researcheffortshavebeenmadeina
varietyofdiseasedconditionsandevenhealthyskeletalmusclecells.However,controlled
feedingexaminingahealthypopulation’sskeletalmuscleresponsetoamealandanacute
diet,toourknowledge,hasnotbeendone.Thisresearchwillbroadenourunderstandingof
theeffectsofdiet,specificallyahighfatdiet,onwhat?beforeothercomplicationsareseen
atthewholebodylevel.Aretherechangesinflexibilityattheskeletalmusclelevelpriorto
insulinresistanceorbodyweightchange?Andifso,arethesechangesprimingthebodyfor
metabolicdisease?Investigatingdisruptionsinskeletalmusclemetabolisminresponsetoa
meal,andfurther,ahighfatdietwillhelpustounderstandbaselinecharacteristicsof
diseasestates.
GUTPERMEABILITY
23
Theeffectofgutmicrobiotaonobesity,T2DMandotherdiseasestateshasbeena
subjectofgreatinterestinthepastseveralyears.Dietdirectlyplaysaroleingut
microbiota,whichdirectlyinfluencesmetabolism.Severalbodiesofresearchhave
investigateddietanditsroleinthehealthofthemicrobiota,butfewerhaveextendedthat
researchtoincludeitsinfluenceonmetabolicperturbations.
Howdoesmetabolismrelatetogutmicrobiota?First,wemustunderstandtherole
endotoxinsplay.Endotoxins,complexlipopolysaccharides(LPS),arepotentiallytoxic
compoundscausedbygram-negativebacteriainthegut.Whentheseendotoxinsarefound
inhigherlevelsthannormalintheblood,causingendotoxemia,amalfunctioninthegutis
evident.Thismalfunctionisduetogutpermeabilitybeingcompromisedbylifestylefactors
(orothertraumaunrelatedtolifestyle)suchasdietandexercise.Gutpermeabilityis
definedasafunctionalfeatureoftheintestinalbarrier.Theinteractionandproper
functionoftheexternal,physicalbarrierandtheinner,functionalbarrieroftheintestinal
wallenablesequilibriumtobemaintained.Disruptionsinthisequilibriumand
consequently,itsdysfunction,leadstoalossofintestinalfunction,homeostasis,andcan
leadtodisease91,92.Whenthegutisunhealthy,includingthephysicalbarriers,featuresand
activeculturesthatdwellthereoranycomponentsofthese,thecontrolofsubstances
passingthroughiscompromised,leadingtotoxicityinthebloodandinflammatory
responsefromotherorgans91.
Metabolicendotoxemia,asdescribedbyCani,etal,isatwotothreetimeschronic
increaseinplasmaLPSconcentration,asystemiclow-levelelevation.Thiselevationissaid
tocontributetothelow-gradeinflammationseeninobesityandcardio-metabolicdisease
fromobesity14.Additionally,metabolicendotoxemiahasbeentiedtodisruptedsubstrate
24
oxidation,leadingtodecreasedmetabolicflexibility.Itiswell-knownthatdietaryfactors
contributetoweightgainseeninobesityandothermetabolicdiseases;theseinvestigations
addtothebodyofliteraturededicatedtothecauseofobesityandothermetabolicdisease
suggestingthatalteredgutmicrobiotaisacontributortothesediseases.Thespecific
mechanismsneedfurtherresearch,butliteraturesupportsthisthought.
Severalstudieshaveassociatedhighfatdietswithgutmicrobiotaalteration,gut
permeabilityandmetabolicendotoxemia13,14,93,94.Inonestudyoveraone-monthperiod,
researchersfoundhigherendotoxinlevelsinthewesternstylediet(40%fat,40%
carbohydrates)thaninthe“prudent”diet(20%fat,60%carbohydrate),concludingthata
higherfatdietmaycontributetoendotoxemia93.Ahighfatdiethasalsobeenshownto
inducechangesinthegutmicrobiota,andtheratioofgram-negativeandgram-positive
bacteria,thereforecausingadetrimentalincreaseingutpermeability13.Throughaseriesof
mouseandhumanstudiesonmetabolicendotoxemia,anothergroupfoundevidencethat
plasmaLPSconcentrationsmaytriggerhigh-fatdietinducedmetabolicdiseases14.
Therolethatthedetrimentaleffectsofincreasedgutpermeabilityhaveon
metabolismneedsfurtherresearch,buttheindicationsforunintentionalmetabolic
consequencesofanunhealthygutarefar-reaching.Furtherresearchisneeded,especially
inhumansexposedtovaryingdietarycompositions,tomoreclearlyunderstandnotonly
theinfluenceofthegutmicrobiotaonmetabolism,butalsotheinter-relationshipofthediet
andplasmaendotoxinlevels.
25
CONCLUSION
Furtherinvestigationabouthowsubstrateoxidationinskeletalmuscleisaffectedby
ahighfatfeeding,andfurther,howitisaffectedbyashorttermhighfatdietwillimprove
ourlimitedunderstandingofitseffectsonmetabolicheath.Addinggutpermeability
researchtothebodyofliteratureinthecontextofahighfatfeedingmayalsoprove
beneficialtounderstandingtheroleofthegut-endotoxin-metabolicdiseaserelationship.
Combiningthesevariablesandusingahealthy,non-obesehumanmodelmayimproveour
understandingofwhenmetabolicinflexibilitycanbedetected–priortodiagnoseddisease
orasaresultofdiseasestates.Lastly,phenotypingindividualsdependingontheirresponse
tospecificvariablesmayinformresearchersandhealthprofessionalsofcharacteristics
thatprecede,primethebodyfor,orinfluenceprogressionofdiseasestates.Thiswilladdto
thebodyofliteraturebyadvancingourknowledgeofskeletalmusclemetabolismandgut
permeabilityandtheirinfluenceondiseasestates.
26
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34
CHAPTER3:SPECIFICAIMS
SPECIFICAIM1:Testthehypothesisthatacutehighfatfeedingdisruptsmetabolic
adaptationinskeletalmuscleofhealthy,non-obese,sedentaryhumans.
Preliminaryevidenceshowsadisruptionintheadaptiveresponseinskeletalmuscle
toamealattheleveloftranscriptionandsubstratemetabolism.Studiesareproposedusing
wholemusclehomogenatesandisolatedmitochondriatoassesssubstratehandling,
metabolicflexibility,andbioenergetics.
Hypothesis:Substrateoxidationwillbesuppressedinresponsetothehighfatmeal
challengeafterthehighfatdiet.
Objective:Determinationoffastingandpostprandialmetabolicadaptationinskeletal
muscleinresponsetoahighfatmealchallengebeforeandafterahighfatdiet.
SPECIFICAIM2:Testthehypothesisthatacutehighfatfeedingresultsinincreasedgut
permeabilityandbloodendotoxinlevelsinhealthy,non-obese,sedentaryhumans.
Preliminaryevidenceshowsasignificantincreaseinfastingbloodendotoxinlevels
after5daysofhighfatfeedinginhealthyhumans.Asincreasedgutpermeabilityisalikely
mechanismforbloodendotoxin,studiesareproposedtoassessintestinalandcolonic
permeability.Serumendotoxinwillbeassessedunderfastingandfedconditions.
Hypothesis:Gutpermeabilityandserumendotoxinwillbeincreasedinresponsetofive
daysofhighfatfeeding.Thesechangeswillbecloselyrelatedtoskeletalmusclepro-
inflammatorysignalinganddecreasedmetabolicadaptability.
Objective1:Determinationofchangeingutpermeabilitybeforeandafterahighfatdiet.
35
Objective2:Determinationofbloodendotoxinlevelsatfastingandduringthe
postprandialresponsebeforeandafterthehighfatdiet.
36
CHAPTER4:RESEARCHDESIGN
Thedesignofthestudywillbeacontrolledfeedingwheretheparticipantswillserve
astheirowncontrols.Wewillrecruit24youngmaleswhoarehealthybutsedentary.Our
exclusioncriteriawillincludeaBMIgreaterthan25,familyhistoryofT2DM,anyknown
cardiovascularcondition,smokers,moderatetoheavydrinkersandthosewithahighfat
habitualdiet(determinedbydietaryfoodrecords).Eachmorningtheparticipantswill
reporttothemetabolickitchenwheretheywillweighin,havebreakfastandtakethe
remainderoftheirmealsfortheday.Allmealswillbepreparedinthemetabolickitchen
anddailymeasurementswillbekepttoensureweightmaintenanceaswellasadherenceto
thediet(s).Theparticipantswillundergoatwo-weeklead-inperiodwheretheywill
consumeanormal,healthycontroldiet.Energyneedswillbecalculatedforeachindividual
usingtheInstituteofMedicineestimatedenergyrequirementsequation.Afterthislead-in
period,theparticipantswillcometothelabfastedforapreHFDmusclebiopsy/meal
challengeday.TheBergströmbiopsymethodwillbeusedtoobtainmusclefromthevastus
lateralis.Afterthefirstbiopsy,theywillbefedthemealandfourhourslater,thesecond
biopsywillbeobtainedfromtheoppositeleg.Theparticipantswillthenbeplacedonthe
five-dayhighfatdiet,whichwillbeisocalorictothecontroldiet–remaininginenergy
balancethroughouttheentirestudy.Afterthefivedaysofhighfatfeeding,theywillrepeat
thebiopsy/mealchallengeday.
SPECIFICAIM1:Testthehypothesisthatacutehighfatfeedingdisruptsthemetabolic
adaptationinskeletalmuscleofhealthy,non-obese,sedentaryhumans.
37
Objective:Determinationofmetabolicadaptationinskeletalmuscleinresponsetoahigh
fatmealchallengebeforeandafterahighfatdiet.
ExperimentalStrategy:
Skeletalmusclesubstratemetabolismwillbeassessedthroughtheanalysisofglucose,fatty
acidandpyruvateoxidationinwholemusclehomogenatesthatwillbeprepared
immediatelyaftersamplecollection.Additionalmeasuresoftheenzymekineticsofcitrate
synthase,malatedehydrogenase,andbetahydroxylacyl-CoAwillbeperformedtofurther
understandinfluenceofTCAcycle,betaoxidationandelectrontransportchaininthe
adaptationsofsubstrateoxidation.Transcriptionofproteinsimportanttometabolic
regulationwillbeassessedusingqRT-PCRandwesternblotting.Thesemeasureswillbe
performedinisolatedmRNAand/orproteinextractedfromsamplesthatwereflashfrozen
attimeofcollection.
SPECIFICAIM2:Testthehypothesisthatacutehighfatfeedingresultsinincreasedgut
permeabilityandbloodendotoxinlevelsinhealthy,non-obese,sedentaryhumans.
Objective1:Determinationofchangeingutpermeabilitybeforeandafterahighfatdiet.
ExperimentalStrategy:
Thefour-sugarprobeurinetestwillbeperformedtoassesschangesingutpermeability.
Thisurinewillbecollectedemployedbeforeandafterthehighfatfeedinginorderto
determinedifferences.
Objective2:Determinationofbloodendotoxinlevelsatfastingandduringthe
postprandialresponsebeforeandafterthehighfatdiet.
38
ExperimentalStrategy:
Bloodwillbesampledduringthefastedstateandthroughoutthepostprandialperiodto
detectthechangeincirculatingendotoxinconcentrations.
39
CHAPTER5:SKELETALMUSCLEMETABOLICADAPTATIONSINREPONSETOANACUTEHIGHFATDIET
ABSTRACTTheabilityofskeletalmuscletoadaptandrespondtovariousnutrientstatesiscriticalto
maintaininghealthymetabolicfunction.Habitualhighfatintakehasbeenassociatedwith
reducedoxidativecapacity,insulinresistance,increasedgutpermeability,inflammation,
andotherriskfactorsoftenprecedingmetabolicdiseasestates.Todate,limitedresearch
hasinvestigatedtheroleofhighfatdietonskeletalmusclesubstrateoxidationandits
relationshiptogutpermeabilityandendotoxins.Thepurposesofthisstudywereto
determinetheeffectsofanacute,five-day,isocalorichighfatdiet(HFD)onskeletalmuscle
postprandialsubstratemetabolisminhealthynon-obese,humansandtodeterminethe
relationshipbetweenmetabolicadaptations,gutpermeabilityandcirculatingendotoxin.
Thirteencollegeagemaleswerefedacontroldietfortwoweeks,followedbyfivedaysof
anisocaloricHFD.ToassesstheeffectsofaHFDonskeletalmusclemetabolicadaptability
andpostprandialendotoxinlevels,subjectsunderwentahighfatmealchallengebeforeand
afteraHFD.Afteranovernightfast,musclebiopsieswereobtainedpriortoandfourhours
followingthemealandbloodwascollectedpriortoandeveryhourthroughfourhours
followingthesamemeal.InsulinsensitivitywasassessedpriortoandfollowingtheHFD
viaintravenousglucosetolerancetest.Intestinalpermeabilitywasassessedinthesame
mannerviasugarprobetest.Postprandialglucoseoxidationandfattyacidoxidationin
skeletalmuscleincreasedbeforetheHFDinterventionbutwasdecreasedafter.Skeletal
musclemetabolicflexibilitywassignificantlybluntedfollowingtheHFD.Insulinsensitivity
andintestinalpermeabilitywerenotaffectedbyHFD,butfastingendotoxinwas
significantlyhigherfollowingtheHFD.Thesefindingsdemonstratethatinyoung,healthy
40
males,followingfivedaysofanisocalorichighfatdiet,skeletalmusclemetabolic
adaptationisrobustandincreasedfastingendotoxinindependentofgutpermeability
changesarepotentiallyacontributortotheinflammatorystatethatdisruptssubstrate
oxidation.Thesefindingssuggestthatevenshort-termchangesindietaryfatconsumption
haveprofoundeffectsonskeletalmusclesubstratemetabolismandfastingendotoxin
levels,independentofpositiveenergybalanceandwhole-bodyinsulinsensitivity.
41
INTRODUCTION
Metabolism,thegeneraltermforthebiochemicalprocessesthatcontributetothe
conversionoffoodtoenergy,iswidelystudiedduetotheworldwideepidemicofobesity,
theconsistentriseinType2Diabetesandthewidespreadcomplicationsofcardiovascular
diseases.Someconditionsassociatedwiththesediseases,suchaschronicinflammation,
metabolicinflexibility,insulinresistance,highbodymassindex(BMI),andpoorlifestyle
behaviors,includingdietandphysicalactivityareofgreatinterestduetotheirdirect
correlationwithmetabolicprocesses.
Whileoverallhealthismulti-factorial,anumberofcharacteristicsofmetabolic
healthandlikewise,metabolicdisease,havebeenelucidated.Metabolicallyhealthy
individuals,andthosewhoconsumeawell-balanceddiethaveahighlyfunctioninggut
barrier.Inturn,thecirculatingbloodiswithoutendotoxins.Skeletalmuscleis
metabolicallyflexible,oxidizingthemostprominentcirculatingsubstrate,mostofteneither
fattyacidsorglucose.Metabolicdiseasestatesareuncommonunderthesecircumstances.
However,asaresultofhighfatdiet(HFD),multiplestepsimportanttometabolic
regulationaredisrupted,oftenresultinginmetabolicdiseasestates,suchasobesity,insulin
resistance,anddiabetes.HFDhasbeenshowntodysregulatenotonlytheprocesses
discussedhere,butothersthroughoutthebody,suchasadiposetissue,gutmicrobiota,and
functionsintheliver,tonameafew1–3.Skeletalmuscleanditssubstrate
oxidation/metabolicflexibilityandadaptationsareofgreatinterest,duetoskeletalmuscle
beingthelargestinsulinsensitivetissueinthebody.Therefore,adetailedanalysisofmajor
pointsofdysregulation,includingspecificskeletalmuscleexaminationishelpfulin
understandingthemechanismscontributingtometabolicdiseasestates.
42
Asmentioned,aHFDcontributestothedetrimentalchangesthatresultinmetabolic
disease.IncreasedgutpermeabilitycausedbyaHFDreleasesagreaternumberof
endotoxins,whichcirculateintheblood,causingmetabolicendotoxemia4–6.Theeffectof
thisspecificstateisyettobefullyunderstood,butitscontributiontochronicinflammation
andmetabolicderangements,havebeenreviewedandshowntoberelevant7–11.The
disruptedprocessesseenasaresultofHFDleadtoaproinflammatorystateand
dysregulatedmetabolismseeninobesity,diabetesandinsulinresistance.Thepurposeof
thepresentstudywastoinvestigatethemetabolicadaptationsinskeletalmusclethat
occurasaresultofanacuteHFDandtoexaminetheeffectsofaHFDongutpermeability
andbloodendotoxinsonhealthy,non-obese,sedentaryhumanparticipants.
43
METHODS
Participants
Thirteenhealthy,non-obese,sedentary(<2days,20min/dayoflow-intensity
physicalactivity)males,age22.2±1.6years,BMI22.3±2.8kg/m2servedasparticipants
forthestudy.Inclusioncriteriaincluded:weightstable(<±2.5kg)forsixmonthspriorto
enrollment,non-smokerswithnohistoryorfamilyhistoryofcardiometabolicdisease,
habitualcalorieintakecomposedof<40%totalfatand15%saturatedfat,BMIbetween20
and25kg/m2,nottakingmedicationsknowntoaffectstudymeasures,bloodpressure<
140/90mmHg,fastingglucose<100mg/dL,LDLcholesterol<130mg/dL,total
cholesterol<200mg/dL,andtriglycerides<250mg/dL.TheVirginiaPolytechnicInstitute
andStateUniversityInstitutionalReviewBoardapprovedallstudyprocedures.
Participantswereinformedofallprocedures,benefitsandanypotentialrisksassociated
withthestudybeforewrittenconsentwasobtained.
Experimentaldesign
Followingsuccessfulcompletionofscreeningprocedures,participantsbeganatwo-
weeklead-incontrolledfeedingperiod(controldiet).Thepreparedmealsconsistedof55%
CHO,30%fat,and15%protein.Followingthecontroldiet,participantsconsumedafive-
dayhigh-fatdiet(HFD),isocalorictothelead-indiet,consistingof50%fat(45%ofwhich
wassaturatedfat),35%CHO,and15%protein.AnacuteHFDwasemployedinorderto
eliminateconfoundingfactorsthatareoftenseenwithlongerexposuretoHFDs,suchas
increasedinsulinresistance,bodyweight,andincreasedbloodglucose,amongothers.
Participantscompletedahigh-fatmeal(HFM)challenge[820kcal(~30%kcal/d),52gCHO
44
(25%),24gprotein(12%),58gfat(63%,~26%saturatedfat)],beforeandafterthe5-day
HFD.Afteranovernightfast,musclebiopsiesweretakenimmediatelypriorto,andfour
hoursaftertheHFMforassessmentofskeletalmusclemetabolicresponseandadaptation
(seeFigure1).
Figure1:Schematicofresearchdesign
ControlledFeedingProcedures
Four-dayfoodintakerecordswereusedtoconfirmthathabitualdietscontained
lessthan40%oftotalcaloriesfromfat.Afterbeingtrainedonproperreportingtechniques
(usingfoodmodelsandmeasurementdevices)byaresearchdietitian,participants
recordedfoodintakeforthreeweekdaysandoneweekendday.Theresearchdietitian
usingthethree-passmethodreviewedhabitualdietrecordswiththeparticipant12.The
foodintakewasanalyzedusingNutritionDataSystemforResearch(NDS-R)software
version2012(UniversityofMinnesota)byatraineddiettechnician.Inordertoestimate
appropriateenergyrequirementsforeachparticipant,theInstituteofMedicineequation
wasusedbasedonheight,weight,age,andactivitylevel13.BoththecontroldietandHFD
45
wereadministeredonaseven-daycycleofmenusconsistingofmealsandsnackswithtwo
optionalsnackmodules(±250kcals).Dietswereplannedbyaregistereddietitianusing
NDS-Rsoftware.Thetwo-weeklead-incontrolledfeedingandfive-dayHFDperiod
requiredparticipantstoconsumeplannedmeals.Dietsaimedtoprovide3goffiberper
500kcal(±5g).AllmealswerepreparedintheDiningLaboratoryforEatingBehaviorand
WeightManagement.Participantsatebreakfastinthelaboratoryeverydayandcarriedout
acoolercontainingtheremainingfoodfortheday.Participantsweighedineachdayatthe
labpriortobreakfasttoensuretheyremainedweightstable.Atrendof>1.0kgweightloss
orgainwasoffsetbyaddingorsubtracting250kcalfoodmoduleswiththesame
macronutrientcompositionastheoveralldiet.Alluneatenitemsandunwashedcontainers
werereturnedtothemetabolickitchenwheretrainedresearchstaffmonitored
compliance.Participantswerenotpermittedtoconsumeanyadditionalfood,caffeineor
alcoholforthedurationofthestudy.Theywerealsoinstructedtoreportconsumptionof
allnon-studyfoods.
HighFatMealChallenge
ThepurposeofaHFMchallengethatwasperformedbeforeandafterthedietwasto
studythefastedtofedtransitionperiodaswellaspostprandialresponsetothediet.
Participantsarrivedatthelaboratoryfollowinga12-hourovernightfast.Uponarrival,they
wereinterviewedtoensureprotocolcomplianceafterwhichtheirfirstbiopsywastaken
fromthevastuslateralismuscle.BiopsiesweretakenbeforeandfourhoursafteraHFM.
ParticipantswererequiredtoconsumetheHFMwithintenminutes.Followingtheinitial
biopsy,participantswerefittedwithanintravenouscatheterintheantecubitalveinfor
46
baselineandhourlybloodsampling.Participantsremainedseatedandawakeforthe
durationofthemealchallenge;movies,reading,andhomeworkweretheactivitiesthat
werepermitted.Pre-andpostbiopsiesweretakenfromseparatelegs.
MeasurementsandProcedures
Bodymassandcomposition
Bodyweightwasmeasuredtothenearest±0.1kgonadigitalscale(Model5002,
Scale-Tronix,WhitePlains,NY).Heightwasmeasuredtothenearest±0.1cmusinga
stadiometer(Model5002,Scale-Tronix,WhitePlains,NY).Bodycomposition(totalfatand
fat-freemass)wasanalyzedbydual-energyx-rayabsorptiometry(GeneralElectric,Lunar
DigitalProdigyAdvance,softwareversion8.10eMadison,WI).
Intravenous-glucose-tolerancetest
Aninsulin-augmentedfrequentlysampledintravenous-glucose-tolerancetest
(IVGTT)wasusedtoassesswhole-bodyinsulinsensitivity,whichwasadministeredto
subjectsatbaselineandaftertheinterventionpost12hovernightfast14.Thetestwas
performedwhilethesubjectswereinaseatedposition,aftera30-minrelaxationperiod.
Anintravenouscatheterwasplacedineachantecubitalvein,onefortheadministrationof
insulinandglucoseandoneforcollectingbloodsamples.Bloodsamplesforthe
measurementofbaselineinsulinandglucoseconcentrationswasobtainedtenminutesand
thenagainfiveminutesbeforetheinfusionofabolusofglucose(0.3g/kgina50%
dextrosesolutioninfusedover90s).Twentyminutesaftertheglucoseinfusion,abolusof
47
insulin(0.03U/kg)wasinfused.Bloodsampleswereobtained2,3,4,5,6,8,10,12,14,16,
18,22,25,30,40,50,60,70,80,90,100,120,140,160,and180minaftertheinitial
glucoseinfusion.Theywerethencentrifugedat4°Cfor20minat2500×gandanalyzed
forglucoseconcentrationswiththeglucoseoxidasemethodbyusingaglucose
autoanalyzer(YellowSpringsInstruments,YellowSprings,OH).Asampleofserumwas
storedat−20°Cforlatermeasurementofinsulinconcentrationsbytheimmunoassay
analyzer,Immulite1000(SiemensCorporation,Washington,D.C.).Insulinandglucose
valuesfromtheIVGTTwereenteredintotheMINMODMillennialSoftwareprogram
(version3.0;R.Bergman,UniversityofSouthernCalifornia)fordeterminationofinsulin
sensitivity(SI),acuteinsulinresponsetoglucose(AIRG),andglucoseeffectiveness(SG).This
modelusedmeasurementsofplasmaglucoseandinsulinconcentrationsovera3-hperiod
toderiveinvivowhole-bodySI.
Intestinalpermeability,clinicalprocedure
Foursugarprobeswereemployedtoassessgutpermeability15.Sucroseisrapidly
degradedbyepithelialsucrose-isomaltaseactivityuponenteringtheduodenum,andisan
idealprobeofgastro-duodenalpermeabilityonly15.Lactuloseandmannitolare
metabolizedbythecolonicmicrofloraandaresuitableasprobesofsmallintestinal
permeability16.Sucraloseisaccumulatedinthecolonbutresistsmicrobialdegradation,and
isanidealprobeofcolonicpermeability17.Therefore,thisprobesystemwasemployedto
assesspermeabilityinallregionsofthegut.Forpermeabilityassessment,subjectsfasted
overnight(12h)withonlywaterallowed.Subjectsevacuatedtheirbladderspriorto
beginningthetest,followedimmediatelybyconsumptionofUSP-gradesaccharideprobes
48
40gsucrose,1gmannitol,1gsucralose(SpectrumChemicals,NewBrunswick,NJ)and5g
lactulose(TheCoghlanGroup,St.Paul,MN)in250mLbottledwater18–20.Subjectsthen
consumed500mLwaterwithin30mintostimulateurineproduction.Urinewascollected
intwopooledsamples:a0-5hsamplerepresentativeofgastricandsmallintestinal
permeability(collectedduringthevisit),anda6-24hsamplerepresentativeofcolonic
permeability(collectedafterthevisit)18,21.Urinewascollectedin24hcollectioncontainers
with5mL10%thymolinmethanol(w/v)toinhibitbacterialgrowth.
Intestinalpermeabilitycalculations
Urinesugarconcentrationswereconvertedtototalsugarexcretedusingurine
volume.Excretionwascalculatedasa%oftotalsugardoserecoveredinurinefor0-5and
6-24hsamples.Thelactulose/mannitolratio(LMR)wascalculatedforboth0-5and6-24h
samplesastheratiooflactuloseexcretiontomannitolexcretion22,asmannitolaconstant
measureofepithelialsurfacearea15.Gastro-duodenalpermeabilitywasdefinedas%
sucroseexcretionaswellassucrose/mannitolratio(SMR)(0-5h)19,23.Smallintestinal
permeabilitywasdefinedasthe0-5hand6-24hLMRs,andcolonicpermeabilitywas
definedas6-24hsucraloseexcretionandsucralose/mannitolratio(SMR)17,23.For
extractionandquantificationofsugarprobe,totalurinevolumewasmeasured,and
aliquotswerefrozenat−80°C.UrinarysugarsweremeasuredasdescribedbyCamilleriet
al22.50μLurinewascombinedwith50μLinternalstandard[20mg/mL13C6-glucosein
water/acetonitrile(98:2)],dilutedto4mLwithwaterandvortexedwith4mL
dichloromethane.Following30minincubationandcentrifugation(10min,3500xg),100
μLsupernatantwasdilutedwith900μLacetonitrile/water(85:15)andanalyzedbyUPLC-
49
MS/MS.UPLCseparationwasperformedonaWatersAcquityH-class(Milford,MA)
equippedwithanAcquityUPLCBEHAmidecolumn(2.1mm×50mm,1.7µmparticle
size).Isocraticelutionwasperformedat0.7mL/minusingacetonitrile:water(65:35)with
0.2%v/vtriethylamine(TEA).Columnandsampletemperatureswere35and10°C,
respectively.DetectionbyMS/MSwasperformedonaWatersAcquityTripleQuadrupole
Detector(TQD).Negative-modeelectrosprayionization[(−)-ESI]wasperformedwith
capillaryvoltageof−4kV,andsourceanddesolvationtemperaturesof150and450°C,
respectively.DesolvationandconegasseswereN2atflowratesof900and1L/hr,
respectively.ForMS/MS,thecollisiongaswasAr.Theconevoltages,collisionenergy,and
MultipleReactionMonitoring(MRM)transitionsforeachcompoundarelistedinTable1.
Peakwidthswere~4s,andAutoDwellwasemployedwithrequiredpoints-per-peaksetat
12.Theinterscandelaytimewas0.02s.Dataacquisition,processing,andquantification
wasperformedusingWatersMassLynxv4.1software.
Table 1. MS/MS transitions for detection of sugar probes compound retention time
(min) MW (g mol-1)
parent [M–H]– (m/z)
daughter (m/z)
cone voltage (V)
collison energy (eV)
sucralose 0.24 396.238 395.238 358.9705 42 10 mannitol 0.38 182.1748 181.1748 88.8979 28 14 surose 0.43 342.3319 341.3319 178.959 38 12 lactulose 0.46 342.3319 341.3319 160.934 12 8 13C6-glucose 0.39 186.2596 185.2596 91.8909 18 8
Bloodmeasures
SerumfreefattyacidconcentrationsweredeterminedusingtheFreefattyacids
half-microtestassay(RocheDiagnostics,Penzberg,Germany).Serumtriglyceride
concentrationsweredeterminedusingtheTriglyceride-GPOreagentsetassay(Teco
Diagnostics,Anaheim,CA)perthemanufacturer’sinstructions.Serumendotoxin
50
concentrationsweredeterminedusingthePyroGeneRecombinantFactorCendotoxin
detectionassay(Lonza,Basel,Switzerland)perthemanufacturer’sinstructions.
Musclebiopsies
Biopsiesweretakenfromthevastuslateralismuscleusingasuction-modified
Bergström-typeneedle(Cadence,Staunton,VA)technique24,25.Anareaofskinintheregion
ofthevastuslateraliswasshavenandcleansedwithapovidine-iodinesolution.Theskin,
adiposetissueandskeletalmusclefasciawasanesthetizedusing10mLlidocaine(1%).An
incision(0.75cm)wasmadeintheskinwitha#10scalpel,andthefasciafiberswere
separatedwiththebluntedgeofthescalpel.TheBergströmneedle(5mm)wasinserted
intothevastuslateralisandsuctionapplied.Themuscletissuewaspulledintotheneedle,
snippedandextracted.TissuesampleswereimmediatelyplacedinicecoldPBStoremove
bloodandconnectivetissue.Muscletissueusedtoassesssubstrateoxidationwas
immediatelyplacedin200uLofSETbuffer(0.25MSucrose,1mMEDTA,0.01MTris-HCl
and2mMATP)andstoredoniceuntilhomogenization(~25min).Muscletissueusedto
assessmitochondrialfunctionwereimmediatelyplacedinicecoldbuffer1for
mitochondrialisolation(IBM1)(67mMsucrose,50nMTris/HCl,50mMKcl,10mM
EDT/Trisand0.2%BSA)andstoreduntilisolation(~25min).Muscletissueusedfor
westernblottingwasplacedinice-coldcelllysisbuffer(50mMTris-HCl,EDTA1mM,NaCl
150mM,SDS0.1%,sodiumdeoxycholate0.5%,igepelCa6301%,pH7.5)withhalt
proteaseandphosphataseinhibitorcocktail(ThermoScientific,Pittsburgh,PA),thensnap-
frozeninliquidnitrogen.Samplescollectedforwesternblottingwerestoredat-80ºCfor
lateranalysis.
51
Musclehomogenization
Musclesamplesforsubstrateoxidation(~75mg)werecollectedandmincedwith
scissorsfollowedbytheadditionofSETBuffertoproduceafinal20-folddilution(wt:vol),
aspreviouslydescribed26.ThesampleswerethenhomogenizedinaPotter-Elvehjemglass
homogenizer(ThomasScientific,Swedesboro,NJ)attenpassesacross30secondsat150
RPMwithamotor-drivenTeflonpestle.
SubstrateMetabolism
Aspreviouslydescribed26,substrateoxidationinvastuslateralismusclewas
measuredusingradio-labeledfattyacid([1-14C]-palmiticacid)fromPerkinElmer
(Waltham,MA),specificallymeasuring14CO2productionand14C-labeledacid-soluble
metabolites(ASM).Sampleswereincubatedin0.5μCi/mLof[1-14C]-palmiticacidforone
hourafterwhichthemediawasacidifiedwith200μL45%perchloricacidforonehourto
liberate14CO2.The14CO2wastrappedinatubecontaining1MNaOH,andthesamplewas
thenplacedintoascintillationvialwith5mLscintillationfluid.Thevial’s14C
concentrationsweremeasuredona4500BeckmanCoulterscintillationcounter
(Indianapolis,IN).ASMweredeterminedbycollectingtheacidifiedmediaandmeasuring
14Clevels.Glucoseoxidation(GO)andpyruvateoxidation(PO)weremeasuredwith
methodssimilartothatoffattyacidoxidation(FAO)withtheexceptionofasubstitutionof
[U-14C]-glucoseand[1-14C]-pyruvatefor[1-14C]-palmiticacid,respectively.Metabolic
flexibilitywasassessedbymeasuring[1-14C]-POinthepresenceorabsenceofnon-labeled
BSA(0.5%)bound-palmiticacid.Metabolicflexibilityisdenotedbythepercentage
decreaseinPOinthepresenceoffreefattyacidandisexpressedastheratioofCO2
52
productionwithlabeledpyruvateoverCO2productionwithlabeledpyruvateinthe
presenceofpalmitate.OxidativeefficiencyisdenotedbyusingtheratioofCO2/ASM,which
representscompleteandincompleteproductsoffattyacidoxidation.
CitrateSynthase(CS)activitywasassessedbymeasuringthereductionof5,5-
dithio-bis-(2-nitrobenzoicacid)(DTNB)fromtheformationofCoenzymeA(CoASH)over
time.Briefly,tenmicrolitersofa1:5dilutedmusclehomogenatewasadded,induplicate,to
170μlofasolutioncontainingTrisbuffer(0.1M,pH8.3),DNTB(1mM,in0.1MinTris
buffer)andoxaloacetate(0.01M,in0.1MTrisbuffer).Followingatwo-minutebackground
reading,thespectrophotometer(SPECTRAmaxME,MolecularDevicesCorporation,
SunnyvaleCalifornia)wascalibratedand30μlof3mMacetylCoAwasaddedtoinitiatethe
reaction.Absorbancewasmeasuredat405nmat37Cevery12secondsforsevenminutes.
MaximumCSactivitywascalculatedandreportedasμmol/min/mg.
MalateDehydrogense(MDH)activitywasmeasuredspectrophotometricallyat
340nmat37°C.Briefly,tenmicrolitersofsamplewaspipettedintriplicateinwells.Then,
290ulofreactionmedia(0.1Mpotassiumphosphatebuffer,pH7.4plus0.006M
oxaloaceticacid,preparedinpotassiumphosphatebufferplus0.00375MNADH,prepared
inpotassiumphosphatebuffer)wasaddedtothewellsandsampleswerereadforfive
minutesat340nm.TherateofdisappearanceofNADHwasanalyzedandexpressed
relativetoproteincontent.Dataisexpressedasmeans±SEM.
Forthedeterminationofbeta-hydroxyacylcoAdehydrogenase(BHAD),oxidationof
NADHtoNADwasmeasured.Intriplicate,35μlofwholemusclehomogenatewasaddedto
190μlofabuffercontaining0.1Mliquidtriethanolamine,5mMEDTAtetrasodiumsalt
dihydrate,and0.45mMNADH.Thespectrophotometer(SPECTRAmaxPLUS384,Molecular
53
DevicesCorporation,SunnyvaleCalifornia)wascalibratedand15μlof2mMacetoacetyl
CoAaddedtoinitiatethereaction.Absorbancewasmeasuredat340nmevery12seconds
forsixminutesat37C.MaximumBHADactivitywascalculatedandreportedas
μmol/min/mg.
Westernblotanalysis
Frozenmuscletissuesampleswerehomogenizedinice-coldlysisbufferinaBullet
BlenderHomogenizer(NextAdvance,NY)using1.0mmZirconiumOxidebeads(Next
Advance).Sampleswerecentrifugedat14,000gfor15minat4°Ctoremoveinsoluble
components.Supernatantproteinconcentrationsweredeterminedspectrophotometrically
usingthebicinchoninicacidassay(BCA)(ThermoScientific).Lysisbufferwasaddedto
samplesforadjustmenttoequalconcentrationsandcombinedwithequalvolumes2x
Laemellibufferandheatedforfiveminutesat95°C.Equalamountsofproteinwere
separatedonpouredSDS-PAGEgels(TGXFastCastAcrylamideSolutionsKit,Bio-Rad,
Hercules,CA),whichwereactivatedviaultravioletlightexposure(ChemiDocTouch
ImagingSystem,Bio-Rad)priortotransfer.ProteinsweretransferredtoPVDFmembranes
usingaTrans-BlotTurboTransferSystem(Bio-Rad),whichwerethenimaged(Bio-Rad)
forquantificationoftotallaneprotein.PVDFmembraneswereblockedforonehourat
roomtemperaturein5%non-fatdrymilkor5%bovineserumalbuminpriortoovernight
incubationat4°Cwithprimaryantibodies.Membraneswereprobedwithprimary
antibodiesagainstpyruvatedehydrogenasephosphate(PDPc;1:500;SantaCruz
Biotechnology,SantaCruz,CA),pyruvatedehydrogenasekinase4(PDK4;1:500;Santa
Cruz),p38MAPkinase(1:1,000;CellSignalingTechnology,Danvers,MA),phosphorylated
54
p38MAPkinase(1:1,000;CellSignaling).Followingprimaryantibodyincubation,
membraneswereincubatedforonehouratroomtemperaturewithHRP-conjugatedanti-
rabbit,anti-mouse(1:10,000;JacksonImmunoResearchLaboratories,WestGrove,PA),or
anti-goat(1:2,000;SantaCruz)secondaryantibodies.Proteinswerevisualizedvia
chemiluminescence(ClarityWesternECLSubstrate,Bio-Rad,orSuperSignalWestFemto,
ThermoScientific),quantifiedusingImageLabSoftware(v5.2.1,BioRad)andnormalized
tototallaneproteincontent.MolecularweightwasdeterminedbyPrecisionPlusProtein
UnstainedStandards(Bio-Rad).
Statistics
Two-wayrepeatedmeasuresanalysisofvariancewasusedtodeterminedifferences
inmealresponsespreandpost-HFD.MultiplecomparisonswereperformedusingaTukey
post-hocanalysis.Independentt-testswereusedtocomparepercentchangeinprotein
levelsbetweenpreandpost-mealtimepoints,beforeandafteraHFD.Correlationswere
examinedviamultivariateanalysis.Datathatdidnotfollowanormaldistributionwas
loggedbase10,orsquareroottransformed.Alldataisexpressedasmeans±standard
errorofthemean(SEM).Thesignificancelevelissetaprioriatα=.05.
55
RESULTS
Participantcharacteristics
ParticipantcharacteristicsareshowninTable2.Thirteenparticipantscompleted
thestudy.TherewerenodifferencesinweightorBMIaftertheHFDwhencomparedto
baseline(p>0.05).Thisanalysisincludedleanbodymass,fatmassandbodyfat
percentage,noneofwhichweredifferentpretopostHFD.
Table2:ParticipantcharacteristicsVariable(n=13) PreHFD PostHFDAge(yrs) 22.2±0.4 --Height(m) 1.77±0.02 --Weight(kg) 72.09±3.2 71.98±2.9BMI(kg/m2) 23.1±0.9 23.0±0.8BodyFatMass(kg) 16.57±2.1 16.28±2.0BodyFat(%) 22.03±1.7 21.44±1.7LeanMass(kg) 54.15±1.7 54.51±1.9Alldataareexpressedasmean±SEM.
Diet
ThemeanenergyandmacronutrientcontentoftheHFMchallengeandeachdietis
presentedinTable3.Manipulationofthecarbohydrateandfatcontentwasthediffering
factorinthetwodiets(Table3).TheHFMchallengewas~30%ofdailyenergyintakeat
820kcals/meal.
Table3:DietmeanenergyandmacronutrientcontentDietCondition
Energy(kcal/day)
Protein(%) CHO(%) Fat(%) SFA(%kcal)
Habitual 2318±104 16.9 44.3 35.9 13.12-wklead-in(control)
2768±66 15.2 53.9 30.9 9.4
HighFat 2735±73 15.3 30.9 53.9 24.5HFmealchallenge
30%/day820kcal/meal
12%24g/meal
25%52g/meal
63%58g/meal
26%kcal24g/meal
Alldataareexpressedasmean±SEM.
56
Wholebodymeasurements
Fastinginsulinsensitivity,fastingglucoseandfastinginsulindidnotchangein
responsetotheHFD(Table4,p>0.05).Nodifferenceswerefoundinfastingfreefattyacids
betweenpreandpostHFDmeasuresasseeninTable3.Fastingtriglyceridesandfasting
endotoxinswerebothfoundtobesignificantlydifferentaftertheHFD(Table4,p<0.001
andp=0.03respectively).Triglyceridesdecreasedfrom75.4±10.2mg/dLto47.2±6.0
mg/dLandendotoxinsnearlydoubledaftertheHFDfrom1.2±0.1EU/mLto2.3±0.4
EU/mL.Gut(gastroduodenal,intestinal,colonic)permeabilitydidnotchangepretopost
HFD(Table4,p>0.05).
Table4:WholebodyfastingmeasuresFastingMeasures(n=13) PreHFD PostHFDSi([mU/L]/min) 5.6±0.7 4.78±0.6Glucose(mmol/L) 82.1±2.7 81.9±2.7Insulin(uIU/ml) 6.3±2.7 6.5±2.5FreeFattyAcids(uM) 480.9±83.6 462.1±69.1*Triglycerides(mg/dL) 75.4±10.2 47.2±6.0#Endotoxin(EU/mL) 1.2±0.1 2.3±0.4GastroduodenalPermeability(excretionratio)0-5hrs
0.07±0.01
0.08±0.02
IntestinalPermeability0-5hrs(excretionratio)6-24hrs
0.03±0.010.13±0.02
0.04±.010.10±0.01
ColonicPermeability0-5hrs(excretionratio)6-24hrs
0.21±0.070.55±0.09
0.28±0.080.36±0.08
*p<0.001,#p=0.03;Alldataareexpressedasmean±SEM.
Post-HFDserumfreefattyacidsareaunderthecurvewassignificantlyhigherthan
pre-HFDmeasures(Figure2A,p=0.03).Serumtriglyceridesweresignificantlylowerafter
theHFDinresponsetothemeal(Figure2B,p=0.01,pre-HFD=514.7mg/dL/hr,post-HFD
=374.0mg/dL/hr).SerumendotoxinsshowednosignificantdifferencepretopostHFDin
responsetothemeal(Figure2C,p>0.05).
57
Figure2:Mealchallengebloodmeasures
Substratemetabolism
TherewasasignificantHFDxHFMinteractionforskeletalmuscleGO(p=0.002),
FAO(p=0.01),andmetabolicflexibility(p=0.03).Aftercontrolled,lead-infeeding
conditions,postprandialFAO,GO,andmetabolicflexibilityincreased,butaftertheHFD,
thesemeasureswereblunted(Table5).PercentchangeinGO(p=0.003),FAO(p=0.04),PO
(p=0.09)andmetabolicflexibility(p=0.01)ispresentedinFigure3.
TherewasasignificantHFDxHFMinteractionforCSandMDHactivity(p=0.04)as
showninTable5.BothCSandMDHactivityincreasedpostprandiallybeforetheHFD,but
0 1 2 3 40
200
400
600
800
Hours Post Meal Challenge
Seru
m F
ree
Fatty
Aci
ds (u
M)
Pre HFDPost HFD
PreHFD Post HFD0
500
1000
1500
2000
AU
C
* p = 0.03
*
0 1 2 3 40
50
100
150
200
250
Hours Post Meal Challenge
Seru
m T
rigly
cerid
es (m
g/dL
)
Pre HFDPost HFD
PreHFD PostHFD0
200
400
600
800
AU
C
* p = 0.01
*
0 1 2 3 40
1
2
3
4
5
Pre HFDPost HFD
Hours Post Meal Challenge
Ser
um E
ndot
oxin
(EU
/mL)
C
Pre HFD Post HFD0
5
10
15
AUC
A B
58
theiractivitydecreasedfollowingtheHFD.Nointeractionordifferencewasfoundfor
BHAD.
TABLE5:SubstrateMetabolism
PreHFDFasted
PreHFDFed
PostHFDFasted
PostHFDFed
*GlucoseOxidation(nmol/mgprotein/hr)
4.5±0.7 7.3±1.1 6.2±0.7 4.6±0.5
*FattyAcidOxidation(nmol/mgprotein/hr)
7.4±1.0 10.3±1.4 10.7±1.1 8.4±1.1
PyruvateOxidation(nmol/mgprotein/hr)
427.6±33.4 444.1±36.4 386.9±35.5 289.8±21.2
*MetabolicFlexibility(ratioofpyruvateoxidation±FFA)
1.4±0.1 1.8±0.2 1.5±0.1 1.6±0.1
*CS(umol/mgprotein/min)
105.6±14.0 143.3±20.2 104.3±12.9 81.7±13.1
*MDH(umol/mgprotein/min)
1760.9±144.0 2004.1±89.3 1589.9±154.0 1440.1±93.4
BHAD(umol/mgprotein/min)
53.9±6.0 47.8±7.7 52.9±6.7 35.3±4.0
*Significantdifferencefound(p<0.05);alldataareexpressedasmean±SEM.
59
Figure3:Substrateoxidation
*Significantdifferencefound(p<0.05)#(p=0.09)
Pyruvatedehydrogenasecomplex
IncreasedPDK4expressionsuppressesthepyruvatedehydrogenasecomplex,and
conversely,PDPactivates,orincreasestheactivityofthecomplex.Asmeasuredbythe
proteincontentvisualizedinwesternblots,therewasasignificantHFMxHFDinteraction
forPDP(Figure4A,p=0.02).Inresponsetoameal,PDPwasbluntedaftertheHFD
(p=0.02).Inresponsetothemeal,PDK4showsaslightdecreasedexpression,howeveritis
notsignificant(Figure4B,p=0.5).
Pre HFD Post HFD-50
0
50
100
150
Glu
cose
Oxi
datio
n%
Cha
nge
in re
spon
se to
HFM
cha
lleng
e
*
A
Pre HFD Post HFD0
50
100
150
Fatty
Aci
d O
xida
tion
% C
hang
e in
resp
onse
to H
FM c
halle
nge
*
B
Pre HFD Post HFD0
10
20
30
40
50
Met
abol
ic F
lexi
bilit
y%
Cha
nge
in re
spon
se to
HFM
cha
lleng
e*
D
Pre HFD Post HFD-40
-20
0
20
40
60
Pyru
vate
Oxi
datio
n%
Cha
nge
in re
spon
se to
HFM
cha
lleng
e
#
C
60
Figure4:Pyruvatedehydrogenasecomplex
*Significantdifferencefound(p<0.05).
AdaptersandNon-AdaptersinFAOandGO
TobetterunderstandcontributorstoFAOadaptation,amediansplitoffastingFAO
percentchangefrompre-topost-HFDwasexamined(Figure5A,p=0.03).Those
participants’whoincreasedskeletalmuscleFAOabovethemediansplit,inresponseto
HFD,wereclassifiedasadaptersandthosewhofellbelowthemediansplitwereclassified
asnon-adapters.Oxidativeefficiency,whichistheratioofcomplete/incompletefattyacid
oxidation,wassignificantlyhigherinadaptersfollowingaHFDwhencomparedtonon-
adapters(Figure5B,p=0.05).PDK4proteincontentwashigheramongadaptersfollowing
aHFDwhencomparedtonon-adapters(Figure5C,p=0.04),suggestingagreater
pre HFD post HFD0.0
0.5
1.0
1.5
PDP
tota
l pro
tein
arbi
trary
uni
ts
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61
inhibitionofpyruvatedehydrogenasecomplexintheadapters.P38activitytrendedhigher
amongnon-adapters,althoughsignificancewasnotreached(Figure5D,p=0.06).
Figure5:FattyAcidOxidationAdaptation
#Significantdifferencefound(p<0.05).
Adapters Non-Adapters-200
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62
Similarly,amediansplitofGOpercentchangefrompre-topost-HFDwas
conductedtobetterunderstandcontributorstoGOadaptation(Figure6A,p=0.03).Those
participantswhoincreasedskeletalmuscleGOabovethemediansplitinresponsetoHFD
wereclassifiedasadaptersandthosewhofellbelowthemediansplitwereclassifiedas
non-adapters.Endotoxinsweresignificantlyhigheramongnon-adaptersfollowingaHFD
whencomparedtoadapters(Figure6B,p=0.004).Pyruvateoxidation,whichbasedonour
measure,reflectsPDHactivity,wasloweramongnon-adaptersfollowingaHFDwhen
comparedtoadapters(Figure6C,p=0.03).PDK4activitywasloweramongnon-adapters
(Figure6D,p=0.01).
Figure6:GlucoseOxidationAdaptation
#Significantdifferencefound(p<0.05).
Adapters Non-Adapters-200
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63
DISCUSSION
Thepurposeofthisstudywastoinvestigatethepostprandialmetabolicadaptations
thatoccurasaresultofanacuteHFDandtoexaminetheeffectsofaHFDongut
permeabilityandbloodendotoxinsonhealthy,non-obese,sedentaryhumanparticipants.
Inthepresentstudy,fivedaysofHFDinhealthyparticipantsproducesasignificant
postprandialmetabolicadaptationinskeletalmusclewithoutachangeininsulin
sensitivity,bodyweight,orgutpermeability.FAO,GOandmetabolicflexibilitywere
bluntedduringthefastedtofedtransitionaftertheHFD.Thelackofchangeininsulin
sensitivity,bodyweightorgutpermeabilityindicatesthattheseadaptationsarepresenting
attheskeletalmusclelevelbeforetheyarebeingdetectedatthewholebodylevel.
Importanttonoteisthattheparticipantsinthecurrentstudyarehealthy.
Adaptationsobservedmaynotindicateadetrimentalchange,butinstead,anecessary,and
likelynormal,metabolicresponsetotheHFD(andHFmeals).Itisdifficulttodetermineif
thehealthyparticipantswouldreturntobaselineiftheybeganconsumingtheleadindiet
aftertheconclusionoftheHFD;perhapstheymayadjustandadaptfurtherifthey
remainedontheHFD,orpotentially,thefatbalancewouldbeunattainable.Whenexposed
toaHFD,fatbalancecantakeseveraldays,withmanycontributingfactorsassociated27–29.
Perhapsthemajorityoftheparticipantswereintheprocessoffindingthatfatbalance,
whichwouldinturn,affecttheoxidationstatus.Inthepresentstudy,GOandFAOwere
bluntedinresponsetothemealaftertheHFD,butthisobservationismostlikelya
beneficialadaptation.
64
Metabolicflexibility,orswitching,isdefinedasthepreferentialoxidationofthe
substratethatismoreavailable.Adysfunctionintheseprocessesistermedmetabolic
inflexibility,occurringwheneithersubstrateisinefficientlyoxidizedwhileitistheprimary
fuelsource.Verylittleofthemetabolicflexibilityresearchhasbeendoneattheskeletal
musclelevel,mosthavinglookedmorebroadlyatwholebodyflexibility.Metabolic
flexibilityobservedinthisstudymayhavebeenbluntedaccordingtothisdefinition;
however,aswithsubstrateoxidation,thechangesarelikelyabeneficialresponseasthe
participantsadapttobetterutilizeavailablesubstrates.Theirabilitytoswitchbetween
substratesmayhavebecomedifferent,butshouldnotbenecessarilyclassifiedas
inflexibility.Thepresentstudyaddstothebodyofliteraturebyshowingthatpreviousto
wholebodychanges,adaptationsattheskeletalmuscleareoccurring,andthatperhaps
acutemetabolic“inflexibility”observedinahealthypopulationwhenchallengedwitha
HFDorHFmealisnotdetrimental,butanatural,beneficialresponse.
Anumberofmeasureswereanalyzedtounderstandtheunderlyingmechanismsof
thesechangesinskeletalmuscle.CSisoneofthekeyregulatoryenzymesintheenergy
producingmetabolicpathway,formingcitrateneededforthetricarboxylicacidcycle(TCA).
Inthepresentstudy,therewasasignificantHFDxHFMinteraction;beforetheHFD,in
responsetotheHFM,CSactivityincreased,however,aftertheHFD,inresponsetothemeal
CSactivitydecreased.InobeseindividualsandthosewithType2Diabetes,skeletalmuscle
citratesynthaseactivityisattenuated30,31.Thesubjectsinourstudyareleanandhealthy,
thereforethisadaptationmaysuggestamechanismbehindthedecreasedoxidationseen
previoustoweightgainorinsulinresistance.Additionally,MDH,anotherimportant
enzymetotheTCAcycle,catalyzingtheconversiontooxaloacetate,hadasignificantHFDx
65
HFMinteraction,similartoCS.Theseresultsindicateanadaptationpresentinthe
regulatorystepsofoxidationthatiscomparabletotheadaptationobservedinGOandFAO,
andlikelyisoneoftheunderlyingmechanismsforthechangesseen.
FastingendotoxinsnearlydoubledaftertheHFD.Theincreasedendotoxinsseenin
thisstudyaddtoothernotableresearchinanimalmodelsaswellashumanparticipants
whichdemonstrateafteraHFD,endotoxinsweresignificantlyhigherincomparisontothe
control,whichalsoconfirmsourpreviousfindings6–11,26,32.Endotoxincirculationleadsto
dysregulatedsignalsinskeletalmusclethatcontributetoimpairedmetabolicswitching
wherebyregardlessofsubstratesavailable,GOisincreasedandFAOissuppressed.Thisis
detrimentalduetotheHFDyieldingfatasthepredominantsubstrateavailable.Increased
gutpermeabilityhasbeenlinkedtoelevatedcirculatingendotoxins.Thepresentstudydid
notshowachangeingutpermeability,potentiallybecausealongeramountoftimeis
neededforhealthyparticipantstoseeadifferenceingastrointestinalpermeabilityasa
resultofaHFD.Theassayusedtodeterminegutpermeability(sugarprobeurinetest)is
typicallyusedtodetectirritablebowelsyndrome,achroniccondition,andthereforemay
notbesensitiveenoughtotracksmallchangesthatmayhaveoccurredintheacutetime
frameoffivedays23,33–35.Futurestudiesmaywanttoemployameasureofplasmalevelsof
glucagon-likepeptide-2(GLP-2)whichhasbeenshowntodetectgutbarrierfunction36.
ThepostprandialAUCmeasurementsofserumfreefattyacidswereelevatedafter
theHFD.Itisimportanttonotethathighserumfreefattyacidsareassociatedwith
metabolicsyndrome–theelevatedserumfreefattyacidsseeninthecurrentstudycanbe
consideredamarkerofperturbationspriortowholebodydiseasestates.Similarresults
wereobservedinaratmodelpronetoobesitywherefastingserumfreefattyacidswere
66
notdifferent,butinthefedstate,theywereelevated37.Thiseffectmaybeassociatedwitha
compromisedabilityofinsulininthefedstatetoinhibitlipolysisefficiently,which
increasescirculatingfreefattyacids.
Thepyruvatedehydrogenase(PDH)complexisamajorcontrolpointfor
determinationofsubstrateoxidation.ReferringtoFigure4,twoproteins,pyruvate
dehydrogenasekinase4(PDK4)andpyruvatedehydrogenasephosphatase(PDP)were
analyzedtofurtherunderstandtheHFDeffectonthiscomplex.AnincreaseinPDK4
activitysuppressesglycolysisandenhancesFAO,inhibitingtheuseofglucose.Anincrease
inPDPactivityutilizesglucose,promotingGO.Inthepresentstudy,thepostprandialPDP
activitywasbluntedafterthediet,indicatingdisruptionsinactivatingthecycleas
efficientlyasprevioustotheHFD.WewouldexpectPDK4tobeup-regulated,enhancing
FAO,duetothehighvolumeoffatinthedietandmealchallenge.Howeverthisisnot
observedinthepresentstudy,whichindicatesanoveralldecreasedfunctionalityofthe
PDHcomplexaftertheHFD.Whilewedidnotseestatisticalsignificancelikelyduetolow
samplesize,pyruvateoxidationsuppressionaftertheHFDistrending(Figure3C).Our
measureofpyruvateoxidationreflectsPDHactivity.ThePDHcomplexisacontrolpoint
usedtodriveATPsynthesisviaoxidativephosphorylation.Whennotfunctioningproperly,
theinterconnectionofglycolysisorFAOtotheTCAcycleiscompromised,affectingthe
utilizationofsubstrates.
Tofurtherunderstandadaptationsinthepresentstudy,amediansplitofthe
percentchangefrompretopostHFDwascalculatedinbothfastingFAOandfastingGO
measures.Fattyacidsandglucosearetheprimarysubstratesthathavebeenshownto
fluctuatewithchangesindiet.Aswithmosthumanresponsestoanintervention,results
67
arequitevariable.However,theadaptationswerefurtherunderstoodbyanalyzingthe
groupsofparticipantswhofellaboveandbelowthemediansplit.
FastingFAOnon-adaptershaddiminishedoxidativeefficiency(Figure5B)andPDK4
activity(Figure5C).Oxidativeefficiency,asmeasuredbyCO2/ASMratioofFAO,is
indicativeofthebody’scapacitytocompletelyoxidizefattyacidstoCO2.Asexpected,FAO
non-adapters’capacitytodosowassignificantlybluntedaftertheHFD,characterizing
thoseparticipantswithaninabilitytoadapttotheHFD.Incompleteoxidationoffattyacids
leadstoactivationofpro-inflammatorypathways38,39,whichcouldbeacontributortothe
chroniclow-gradeinflammationobservedinmetabolicdiseasestates.
WealsoobservedatrendwhereFAOnon-adaptershadanincreasedp38activity,
whichisinlinewithpreviousresearchfromourlab26.Thelackofsignificance(p=0.06)is
likelyduetothesmallsamplesize.Duetoourotherfindingsofcirculatinginflammatory
markers,adiscussionofp38inthisstudyiswarranted.P38hasthreeisoforms,α,β,andγ,
allofwhichwerecapturedinourassay.Wedonotknowwhichisoformsarechanging,but
tounderstandfurther,p38αisfoundgloballyandoneofitsfunctionsistoregulate
productionofinflammatorymediators40,41;p38β,alsofoundglobally,butmore
concentratedinthebrainandlungs,similarlycontributestoinflammatorymediator
synthesis42;p38γismostsignificantlyfoundinskeletalmuscleandisessentialfor
promotingmitochondrialbiogenesis26,43.Inthecurrentstudy,wecan1)speculatethatthe
increasedp38activityobservedisregulatingproductionofinflammatorymediators
indicatingthatthoserespondingpoorlytoFAOmayhaveanincreasedinflammatory
responseand/orperhapslesslikely2)attributethep38increasetotheγisoform,
indicatingthatp38ispromotingmitochondrialbiogenesis,anadaptationthatmaybe
68
necessarytocompensateforthedecreasedFAOandGOobserved.Low-gradeinflammation
isoftenassociatedwithmetabolicdiseases,thereforethefindingsofthefirstscenario
enhancethebodyofliteraturebyaddingthatFAOnon-adapters,thosewhodonotadaptas
welltotheHFD,haveanincreasedinflammatoryresponseaftertheHFD.Thesecond
scenariomaybeexplainedbytheknowledgethatinobeseandinsulinresistantindividuals,
mitochondrialfunction,sizeandmorphologyareimpaired31,andthatincreased
mitochondrialbiogenesishasbeensuggestedtopreventobesityandglucoseintolerancein
arodentmodel44.ThelatterwouldsuggestadaptationoftheFAOnon-adaptersthatmay
notbenecessaryinthosewhoadaptedandthereforehaveadequatefattyacidoxidation.
FastingGOnon-adaptershadelevatedendotoxinsaftertheHFD(Figure6B),and
decreasedPDHactivity,asmeasuredbypyruvateoxidationandPDK4proteincontent
(Figures6CandDrespectively).Endotoxinsareresponsibleforactivatinganimmune
response,oftenassociatedwithlowlevelsofinflammationandcontributingtometabolic
disordersbywayofmetabolicendotoxemia.Anelevatedlevelofendotoxinsinthenon-
adaptersafterjustfivedaysoftheHFDisindicativeoftheeffectofHFDonthegutandits
contributiontooverallhealth.Inthepresentstudy,theGOnon-adapterssignificantly
decreasedPDHactivityandPDK4proteincontentaftertheHFDwhereasthosewho
respondedwelldidnot.Theseresultsmaybethedrivingfactorbehindthesignificance
foundinFigure4,wherePDPwassignificantlybluntedaftertheHFD,disruptingthePDH
complex.Thisanalysis,andthepracticeofmetabolicallyphenotypingindividualscandrive
furtherquestionsandresearchtobetterdeterminetheeffectsofdietonsubstrate
metabolism.
69
Furtherdirections
Typesoffatswerenotmanipulatedinthepresentstudy.Saturatedfatwasthemost
abundantfatinthediet,astheintentionwastoexamineatypicalhighfatwesterndiet,
whichishighinsaturatedfat.ManipulationofothertypesoffatsintheHFDmayhave
differentoutcomesthanthepresentstudy.Also,welookedattheleveloftheskeletal
musclebutwhatthismeansexactlyforsubstrateoxidationandmetabolicflexibilityatthe
wholebodylevelisyettobedetermined.Finally,metabolicphenotypingshouldbeanarea
offurtherexploration,characterizingparticipantsandtheirmetabolicadaptationsto
differentinterventions.Thisinformationwillcontributetothebodyofliteratureand
informscientistswhoarededicatedtounderstandingmetabolicdiseasestatesandfinding
solutionstoobesity,diabetesandinsulinresistance.
Conclusion
Inconclusion,thepresentstudydemonstratedthatafterfivedaysofaHFD,
adaptationsinbothGOandFAOinskeletalmuscleofhealthyparticipantsareobserved.
Mechanismssuchasincreasedfastingendotoxins,dysregulationofthePDHcomplex,
enzymaticdisruption,andkeyproteinmodulatorshavebeenshowntocontributetothe
adaptationsobserved.Metabolicallyphenotypingbyparticipants’adaptationstosubstrate
oxidationrevealedvaluableinsighttobeusedtofurtherthestudyofindividualchanges
andmetabolicdisease.
70
FIGURELEGENDSFigure2:MealchallengebloodmeasuresBloodwastakenatbaselineandeveryhourforfourhoursafterthemealchallengeandwasanalyzedbyassaykitstodeterminedifferencespreandpostHFD.(A)Serumfreefattyacids(FFA)trendsimilarlyaftertheHFDinresponsetothemeal,althoughsignificantlyhigher(p=0.03).(B)Serumtriglycerides(Tg)aresignificantlyloweraftertheHFDinresponsetoameal(p=0.01).(C)SerumendotoxinsshowsomevariationbeforeandaftertheHFD,althoughnotsignificant.Alldataareexpressedasmean±SEM.Figure3:SubstrateoxidationSubstrateoxidationwasmeasuredusingradiolabeledsubstratesinmusclehomogenates.FivedaysofisocaloricHFDdisruptedpostprandialGOinskeletalmuscle.(A)GOincreasedinresponsetoamealbeforeHFD(+96.9%±36.3)butnotafter(-24.3%±4.5,p=0.003).(B)SkeletalmuscleincreasedFAObeforetheHFDafteramealby106.3%±36.6,butaftertheHFD,thiseffectwasbluntedto15.6%±20.8(p=0.04).(C)POmealresponsebeforetheHFDwas18.3%±20.7andaftertheHFD,itwas-20.42%±6.8(p=0.09)(D)Inresponsetothemeal,skeletalmusclemetabolicflexibilitywassignificantlybluntedfollowingHFD(-24.7%±9.5,p=0.01).Alldataareexpressedasmean±SEM.Figure4:PyruvatedehydrogenasecomplexThepyruvatedehydrogenasecomplexwasanalayzedbydetectingpyruvatedehydrogenasephosphatase(PDP)andpyruvatedehydrogenasekinase4(PDK4)proteinsviawesternblotting.(A)TherewasasignificantHFMxHFDinteractionforPDP(p=0.018).Inresponsetoameal,PDPwasbluntedaftertheHFD(p=0.0241).(B)PDK4showsasimilartrendinresponsetothemeal,althoughnotsignificant.Alldataareexpressedasmean±SEM.Figure5:FattyAcidOxidationAdaptationAmediansplitoffastingFAOpercentchangefrompre-topost-HFDwasdonetodetermineadaptersandnon-adapters(A).(B)Oxidativeefficiencywassignificantlyloweramongnon-adaptersfollowingaHFDwhencomparedtoadapters(p=0.05).(C)PDK4activitywasloweramongnon-adaptersfollowingaHFDwhencomparedtoadapters(p=0.04).(D)p38activitytrendedhigheramongnon-adapters,althoughsignificancewasnotreached(p=0.06).Alldataareexpressedasmean±SEM.Figure6:GlucoseOxidationAdaptationAmediansplitofGOpercentchangefrompre-topost-HFDwasdonetodetermineadaptersandnon-adapters(A).(B)Endotoxinwassignificantlyhigheramongnon-adaptersfollowingaHFDwhencomparedtoadapters(p=0.004).(C)Pyruvateoxidationwasloweramongnon-adaptersfollowingaHFDwhencomparedtoadapters(p=0.03).(D)PDK4activitywasloweramongnon-adapters(p=0.01).Alldataareexpressedasmean±SEM.
71
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CHAPTER6:CONCLUSIONS/FUTUREDIRECTIONS
Skeletalmusclesubstratemetabolismandtheadaptationsthatoccurfollowinga
highfatdietwastheprincipleobjectiveofthisproject.Asecondaryobjectivewasto
determinethechangeingutpermeabilityandcirculatingendotoxinsafteraHFD.
Adaptationswereobservedinsubstrateoxidation,metabolicflexibility,endotoxinsand
manymechanisticstudiesrelatedtometabolicprocesses.Theseadaptationsmaybethe
normalmetabolicresponsewhenhealthyprocessesarechallengedwithunhealthyfood
intake.Furtherresearchisneededtoinvestigatethisidea.Repeatingthisstudywhile
addinganinvivometabolicflexibilitymeasureisneededinordertovalidatetheinvitro
measurementofmetabolicflexibilityusedinthisstudy.Extendingthetimelineofthestudy
andrepeatingcollectionofmeasurementsafterparticipantsreturntothenormaldiet
wouldrevealfurtheradaptations,ormorelikely,areturntobaseline.Otherfuture
directionsmightalsoincludeadietthathasincreasedcaloricintakeduringtheHFD,or
changingthehighfatportionofthediettohighsugarconsumption.Participantswere
sedentary,soaddinganexerciseelementmaychangetheadaptationsobserved.Analyzing
thebacteriallandscapeofthegutpotentiallywouldclarifysomeofthemetabolic
perturbationsobserved.Theobservationoftheadaptersandnon-adapterscouldbe
furtheranalyzedcharacterizingdifferentvariableswithintheresults.Ultimately,future
directionsinsubstrateoxidationandmetabolismshouldincludeanintentionaleffortto
phenotypeandcategorizespecificpopulationsinordertodeterminethedifferencesseenin
subgroups.Investigationofthesedifferencescouldpotentiallyclarifycausationofmany
metabolicperturbations.
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Substrateoxidationattheskeletalmusclelevelisanimportantaspectof
understandingmetabolicdiseasestates.Thisprojectaddsvaluableinsightsabout
adaptationsattheskeletalmusclebeforewholebodydisturbancesoccur.Additionally,this
projectaddsinsighttothediscussionaboutmetabolicendotoxemiaanditspotential
contributiontodisruptedsubstrateoxidation.Continuinginvestigationtodeterminehow
andwhythemetabolicprocessesaredisruptedbydietisnecessary.