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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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).

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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.