RESTING METABOLIC RATE AND DIET-INDUCED THERMOGENESIS

studies in humans on individual differences and on the impact of nutritional and non-nutritional factors

0000 0330 1849 Promotor :dr .J.G.A.J .Hautvast , hoogleraari nd elee rva nd evoedin g end evoedselbereidin g Co-promotor:dr .P.R.M .Deurenberg , universitairhoofddocen t pMö[?-Ol , /^bO

RESTING METABOLIC RATE AND DIET-INDUCED THERMOGENESI S

studiesi n humans on individual differences and on the impact of nutritional and non-nutritional factors

J.A. Weststrate

Proefschrift terverkrijgin gva nd egraa dva n doctor ind e landbouwwetenschappen, opgeza gva nd erecto rmagnificus , dr.H.C .va nde rPlas , inhe topenbaa r teverdedige n opdinsdag 2 me i198 9 desnamiddag st evie ruu ri nd eaul a vand eLandbouwuniversitei t teWageninge n

BIBLIOTHEEK LANDBOUWUNIVERSITEIT WAGEMNGE1V 'S* V-I Uo Aanmij nmoede r

Financial supportb yUnileve r ResearchLaborator y for thepublicatio n of this thesis isgratefull y acknowledged. Stellingen

1.He t ontbreken van gegevens over de reproduceerbaarheid vanmetinge n van het thermogeen effect van voeding betekent niet, dat de reproduceerbaarheid van deze metingen goed is. (dit proefschrift)

2. De hoogte van de ruststofwisselingword t bij obese vrouwen mede bepaald door de lichaamsvetverdeling. (dit proefschrift)

3.D e hoogte van het thermogeen effect van voeding isbi jvrouwe n niet gerelateerd aan het lichaamsvetgehalte end e lichaamsvetverdeling. (dit proefschrift)

4.D e hoogte van het thermogeen effect van voeding hangt mede afva n factoren buiten de voeding. (dit proefschrift)

5.D e bevinding dat bij proefdieren onder normale omstandigheden het grootste deel van koolhydraten na vertering wordt omgezet invet , mag niet naar de mens worden geëxtrapoleerd. (o.a.di t proefschrift) - Björntorp P, Sjöström L.Carbohydrat e storage inman : speculations and some quantitative considerations.Metabolis m 1978;27:1853-65. - Acheson KJ, Flatt JP, Jéquier E.Glycoge n synthesis versus lipogenesis after a 500g carbohydrate meal inman .Metabolis m 1982;31:1234-40.

6. Uit het familiaal voorkomen van bepaalde kenmerken, zoals een verhoogd vetgehalte inhe t lichaam, mag niet geconcludeerd worden dat vooral genetische factoren hiervoor verantwoordelijk zijn. - Garn SM et al.Livin g together as a factor in family-line resemblances. Hum Biol 1979;51:565-87.

7.Observationee l onderzoek naar demat ewaari n variatie in een kenmerk geassocieerd isme t erfelijke - of omgevingsfactoren mag op logische gronden niet causaal geïnterpreteerd worden. - Kempthorne O.Logical ,epistemologica l and statistical aspects of nature-nurture data interpretation. Biometrics 1978;34:1-23.

8.He t dogma ind e voedingswetenschap dat eenvoedingsmidde l nooit als 'gezond'o f 'ongezond' aangemerkt kanworden , staat eenwerkelij k effectieve voedingsvoorlichting ind e weg.

9. Sommige 'light'producte n bevatten nog steeds veel energie.Minde r energierijke varianten van gangbare producten zouden daarom alleen de kwalificatie 'light'moge n krijgen, wanneer zij tenminste 25procen t minder energie bevatten danhu n 'normale'tegenhanger , én inabsolut e zin een energiedichtheid hebben inhe t watervrije product, die niet hoger isda n de energiedichtheid van een gedroogd voedselpakket, zoals samengesteld volgens hetAdvie s Richtlijnen GoedeVoedin g vand e Nederlandse Voedingsraad. 10. Eenverminderin g van hetaandee lva nve t ind e energievoorziening van de gemiddeldeNederlands evoedin g tengunst eva nda tva n koolhydraten zal,naas t eendalin g op populatieniveau van het serumcholesterolgehalte, ook leiden tot eenafnam eva n het gemiddelde lichaamsgewicht.

11. De gewoonte inhe t academische curriculum de eerste jarenva nd e studie vooral te gebruikenvoo r het lerenva n inhe tverlede n geproduceerde kennis en de laatste jarenvoo r deproducti e vannieuw e kennis,leid t totee n laag totaal studierendement.

12. Erval td eeerstkomend e jarenee ngroter ebijdrag e teverwachte n aan de preventie van overgewicht vanhe t opd emark t brengen van een smakelijke, verzadiging-gevende, laag-energetische snackda nva ncongresse n over overgewicht.

13. Bijd e toekenning van onderzoeksgelden aanuniversitair e vakgroepen door derdendien t de originaliteit vand evraagstellin g sterker tewege nda n de afwezigheidva n eenonderzoeklijn .

14.D emogelijkhede n omee nbete rvoedingspatroo n inNederlan d ingang tedoe n vinden,worde n belemmerd door hetverbo d 'gezondheidsclaims' toe te kennen aan producten,di e zijnsamengestel dvolgen she tAdvie s RichtlijnenGoed e Voeding van de Voedingsraad.

15. Hoemee r aandacht ind emedi avoo r gezondheidsproblemen, des te zieker men zichvoelt . zie ook:Barsk yAJ . Theparado x of health.N Engl JMe d 1988;318:414-8.

16.Berichtgevin g ind emedi a overd e aanwezigheid van kankerverwekkende stoffen invoedingsmiddele n dient gepaard tegaa nme t eenevaluati e van het gezondheidsrisico in relatie tothe t gezondheidsrisico vanmee r alledaagse activiteiten, zoalsautorijden ,mati galkoholgebrui k ofd e consumptie van pindakaas. zie ook:Wilso n R, Crouch EAC.Ris k assessment and comparisons:a n introduction.Scienc e1987;236:267-70 .

17. Deveelgehoord e opmerkingva noudere nda t vroeger 'alles'bete rwas ,moe t vooral van toepassingworde n geacht ophe t functionerenva n hun geheugen.

18.D e verstrekking van eenOV-jaarkaar t aan studenten zald e bereikbaarheid van het hoger onderwijsnie tvergroten .

Proefschrift J.A. Weststrate Restingmetaboli c ratean ddiet-induce d thermogenesis: studies inhuman so n individualdifference s and on the impact ofnutritiona l andnon-nutritiona l factors Wageningen, 2me i 1989. Contents

page

Voorwoord 7

Abstract 10

Chapter 1.Genera l Introduction 11

Chapter 2.Energ y exchange and energy balance inhumans :a review 19

Chapter 3.Th e assessment of restingmetaboli c rate, diet-induced thermogenesis and inviv o fuelutilizatio n using a ventilated hood system:methodologica l considerations 59

Chapter 4.Diurna l variation inpostabsorptiv e resting metabolic ratean d diet-induced thermogenesis 105 Am JCli nNut r 1989;i npres s

Chapter 5.Lac k of a systematic sustained effect of prolonged exercise bouts on restingmetaboli c rate in fasting subjects 120 (submitted)

Chapter 6. Surprisingly large impacto fpsychologica l stress on diet-induced thermogenesis butno t on resting metabolic rate 131 (submitted)

-5- Chapter 7.Natur e andmagnitud e of inter-individual differences in restingmetaboli c ratean ddiet-induce d thermogenesis inlea nan dobes e individuals 140 (submitted)

Chapter 8.Restin g energyexpenditur e inwomen :impac to f obesity andbod y fatdistributio n 160 Metabolism 1989;i npres s

Chapter 9.Alcoho l and itsacut e effectso n restingmetaboli c rate anddiet-induce d thermogenesis 179 (submitted)

Chapter 10.Doe spalatabilit y affect thepostprandia l responseo f energyexpenditure ? 197 (submitted)

Chapter 11.Unexpecte d interactionbetwee n theeffect so f short-term carbohydrate overfeeding andprio r exercise on restingmetaboli c ratean d diet-induced thermogenesis 214 (submitted)

Chapter 12.Genera l discussion 234

Summary 249

Samenvatting 252

Curriculumvita e 255 Voorwoord

Vooru ligtee nproefschrif t metee naanta l studiesove rd e energiewisseling bijd emens . Eendee lva nd e onderzoekingenbetref td eenergiewisselin g van mensenme tovergewicht .Dergelij k onderzoek spreektvele naan .Ee ngroo tdee l vand ebevolkin gvoer tee nmoeizaa m gevecht tegenee novervloe d aancalorieën . 'Dikmakers'diene n zicho pvee lplaatse nverleidelij k aan ter lenigingva nd e 'lekkere'trek .Menigee n isnie topgewasse n tegena ldi e calorische verlokkingen.Al s gevolg hiervanworde nnoga lwa tmense n tedik .Vaak ,echter , menenovergewichtig emense nda t zijdoo ree n 'efficiëntere' energiewisseling dik zijn geworden.Dez emense n zouden,bi jwijz eva n spreken,di kworde n alleena lbi jhe t zienva nee nbor deten .Verrassen d genoeg ise r in wetenschappelijke zinno gweini gbeken dove rverschille n tussenmense n ind e efficiëntie vand eenergiewisseling . Eenantwoor d opd evraag ,o fe r personen zijn,di e eenverhoogd e kanshebbe no povergewich tal sgevol gva n een 'efficiëntere'energiewisselin g isva nbelang ;nie tallee n termogelijk e geruststellingva ndiegenen ,di ehe tgevech t tegenovergewich t alsee n verloren zaak beschouwen,maa rvoora lbi jd ebehandelin g enpreventi e van overgewicht.Allee nwetenschappelij k onderzoek kanove rdez e zaak uitsluitsel geven.He t isd ehoo pva nd e onderzoeker datd e onderzoekingen vandi t proefschrift hieraanee nbijdrag e zullenleveren .

Eenproefschrif t dienthe tbewij s te leverenda td epromovendu s zelfstandig origineel onderzoek kanverrichte n enhierove rka n rapporteren.Me tnam e rapportage indaarto e geëigende vaktijdschriften isbelangrijk .Di tbetekent , datproefschrifte nveela lbetaa nui tee nbundelin gva nartikele nme t een inleiding enee nalgemen ediscussi e died ebevindinge n inee npassen d kader plaatsen.Di tproefschrif tvorm thiero pgee nuitzondering .He t beschrijft verschillende onderzoekingenme tuiteenlopend e vraagstellingen. Hetonderzoe k indi tproefschrif t isto t stand gekomendankzi jd e hulpva n velen.Traditiegetrou wwi l ikdi tvoorwoor d gebruikeno md emense n tedanken , diemi jbi jhe tonderzoe k hebbengeholpen . Allereerstwi l ikmij npromoto r Professor Dr.J.G.A.J .Hautvas t hartelijk

-7- dankenvoo rzij nnooi taflatend evertrouwe ni nmij nwerk ,voo rzij n enthousiasmevoo rmij nonderzoe ke nvoo rzij nvermoge nd erod edraa di nhe t onderzoeksteed snaa rvore nt elate nkomen . Ookwi li kmij nco-promoto rDr .P .Deurenber gbedanke nvoo rd euitstekend e collegialeverhoudin ge nd evel ediscussie sdi ewij ,me tnam eo phe tgebie d vand elichaamssamenstelling ,same nvoerden . JaapSeidel lwi li kdanke nvoo rd evel egezellig eure ndi ewi jdoorbrachte n methe tbedenke nva nnieuw ebaanbrekend eonderzoeken ,waarvoo rhelaa she tgel d ontbrak. Ia mindebte dt oProfesso rDr .E .Jéquie ran dDr .Y .Schut zfro mth e "Instituted ePhysiologie "o fth eUniversit yo fLausann ei nSwitserlan dfo r introducingm et oth e'in san douts 'o fth emeasurement so fdiet-induce d thermogenesis. DeDirecti eva nUnileve rResearc hLaboratoriu mi nVlaardinge nbe ni k erkentelijkvoo rd etij ddi ezi jmi jverleende no mdi tproefschrif ta ft e ronden.Mij ncollega' sva nd esecti eFysiologi eva nUnileve rResearc hwi li k dankenvoo rhe tbegri pda tzi jd eafgelope nmaande ntoonde nvoo rmij nnoga l onregelmatigewerktijden . Aanhe tonderzoe k isdoo r1 4doktoraa lstudente nmeegewerkt ,di ei kgraa g persoonlijkwi lbedanke nvoo rd eplezierig esamenwerkin ge nhu nonmisbar e hulp:Gree tVansant ,Cathalin ede nBesten ,Zandri eHofman ,Alid aWapsta ,Jo s Steenbergen,Pete rWeys ,Eri cPoortvliet ,Jacquelin eDekker ,Miria mStoel , LilianeBegheyn ,Kari nva nde rKooy ,Ingri dWunnink ,Leonie kRobroc he nThe o Dopheide. Veeldan kgaa tui tnaa rd emedewerker sva nd eservice-afdelin gva nd eCentral e Dienstva nhe tBiotechnion .Zonde rjulli ehul pwa sdi tproefschrif tnooi t verschenen.Ui tee neindeloz ereek sva nvoddig epapiertje sme teve nzovel e klodderigetekeningetje sva nonderdele nva nd e 'ventilatedhood' ,wiste n jullieee nsystee mvoo renergiewisselingsmetinge nt ebouwen ,dat ,i nstat u nascendi,reed slandelijk ebekendhei dkree gi nee ntelevisieprogramma .Guu s vanMunster ,Arthu rva nMunster ,Ja nTheunissen ,Richar dva nde rVlies ,Ber t Willemsen,Sjaa kViege ne nHan sErmer shartelij kdan kvoo rjulli ehulp .Oo k eenwoor dva ndan kbe ni kverschuldig daa nLe oHerbe nva nd eafdelin g electronicavoo rd econstructi eva nd eelectrisch ecomponente nva nd e 'ventilatedhood' .Zonde rd ehul pva nHen kStomphors te nTo nEren swa si kn u nogbezi gme the tuitrekene nva nd eenergiewisselingsgegeven sva nmij ntweed e experiment.Julli eware nverantwoordelij kvoo rd eautomatiserin gva nhe t 'ventilatedhood 'systeem .

-8- Demedewerker sva nhe tlaboratoriu mva nd eVakgroe pHuman eVoedin gbe ni k erkentelijk voor dehul pbi jd eanalyse sva nmaaltijden ,urine se n bloedmonsters.Ee npaa r personenwi l ikme tnam ebedanken :An s Soffers,Coc k Germing-Nouwen,Jok eBarendse-v.Leeuwe ne nPete rva nd eBovenkamp . Hannieva nOosten-va nde rGoe szorgd eo pkritiek emomente nvoo r voldoende budget opmij n computer-rekeningnummer. Dehul pva nd emedewerker sva nd e tekenkamer vanhe tBiotechnio nwa s onontbeerlijk. Zonder jullieprofessionel e hulphadde nd e verschillende figuren inmij nproefschrif t hoogstwaarschijnlijk eengrot e gelijkenis vertoondme tmij nontwerpe nva nd everschillend e onderdelenva nhe t 'ventilated hood' systeem. Christiaand eVrie smaakt ed e schematische tekeningva nhe t 'ventilated hood' systeem. FransSchoute nbe n ikerkentelij k voord euitvoerin gva n enkele testen ter controleva nhe t 'ventilatedhood 'systeem .D emedewerker sva nd e Bibliotheek vanhe tBiotechnio nmoge noo k nietonvermel d blijven;dan k voor het feitda t julliemij ,n a zovelekere no pd e zwarte lijstgeplaats t tezijn , nog steeds toelatent emidde nva nd ewerke nde rwetenschap .Ee neervoll e vermelding verdienthe t secretariaatva nd eVakgroe pHuman eVoedin g enMarce l van Leuteren.Conny ,Bianca ,Rieki e enMarce lhartelij k dankvoo r de onmisbare hulpbi jhe tvel e type-e nlay-ou twerk . HarryHarsem abe n ikerkentelij k voor hetontwer pva nd e omslag.Meta ,Baukj e enPauline ,bedank tvoo r jullie steun indi edrukk e laatstemaanden . Mijn zussenJane te nNellek ewi l ikgraa gbedanke nvoo rhu nvoortdurend e belangstellingvoo rmij nwerk .Mij nmoede rheef tmi j steedsgestimuleer d om te studerene ndaarbi jmij neige n interessesvooro pt e laten staan;aa nhaa r draag ikdi tproefschrif top . Abstract

RESTINGMETABOLI CRAT EAN DDIET-INDUCE DTHERMOGENESIS : Studies inhuman s on individualdifference san do nth e impact ofnutritiona l and non-nutritional factors

THESIS,DEPARTMEN TO FHUMA NNUTRITION ,WAGENINGE NAGRICULTURA L UNIVERSITY, WAGENINGEN, THENETHERLANDS , 2MA Y 1989

JanA .Weststrat e

This thesis contains studieso n restingmetaboli c rate (RMR)an d diet-induced thermogenesis (DIT)o fhuman susin g indirectcalorimetr y (ventilated hood system)t oasses senerg yexpenditure . A literature surveyo faspect s ofhuma nenerg yexchang e and ofproblem s of energybalanc e is included. At first,methodologica l studieso n themeasuremen to fRMR ,DI Tan d fuel utilization arepresented .Measurement s ofRM Ran d respiratoryquotien t showed good reproducibility, incontras t tothos e ofDI Tan d fuelutilizatio n rates. Secondly,th enatur e andmagnitud e of inter-individual differences inRM Ran d DITwer e assessed.N o significant difference inDI Twa s found between non-obesewome nan d obesewome nwit ha wid e range inbod y fatdistribution . Menha d a significantlyhighe r DITcompare d towomen .Obes ewome nwit h a non-abdominal typeo fbod y fatdistributio n had reduced RMR's in comparison withnon-obes emen ,non-obes ewome nan d obesewome nwit h anabdomina l typeo f body fatdistribution . Thirdly, the impacto fnutritiona l andnon-nutritiona l factorso nRM R and DIT was studied. No significantdiurna lvariatio n inRM Ran d DITwa s found. Psychological stressdi dno taffec tRMR ,bu tpotentiate d DIT.Undul y heavy exercisewa s found tohav ea significant after-effect onRM Ran dDIT ,wherea s moderate toheav yexercis e did nothav ea systematic after-effect onRMR . Ethanol (20g ) induceda significant thermiceffect ,bu tdi d notaffec tDIT . Differences inpalatabilit y amongotherwis e identicalmixe dmeal so r sucrose solutionsdi d notproduc edifference s inDIT .Short-ter m carbohydrate overfeeding increased DIT,bu tno tRMR .Carbohydrat e overfeeding attenuated theafter-effec t onRM Ro fundul yheav yexercise . Thisthesi s showstha t inter-individual differences inRM Ran d DITexist ,eve n after statistically accounting fordifference samon g individuals inage ,bod y weight and body composition.Thi s thesis showsals othat ,i nadditio n to nutritional factors,non-nutritiona l factorsaffec t thepostprandia l rise in energyexpenditure .

-10- Chapter 1. General Introduction

1.1. INTRODUCTION

This thesisdeal swit hth estud yo fhuma n energy exchange. Energy exchange involvesth erelationship s between energy intakean denerg y expenditurean d includes theprocesse s forstorag ean dutilizatio n ofenerg y containing compounds.I nthi s thesisth eenerg y expenditure componento fhuma n energy exchange isstudied ,i nparticular ,th erat eo fenerg y expenditurea tres ti n fasting conditionsan da tres tafte r feeding.Thes e componentso fenerg y expenditure arereferre d toa srestin gmetaboli c ratean ddiet-induce d thermogenesis, respectively. Together theyaccoun t for70-8 5percen t oftota l energy expenditure inth eaverag e adult individual.Becaus eo fthei r quantitative importance fortota l energyexpenditure ,th estud yo frestin g metabolic ratean ddiet-induce d thermogenesis isessentia l forgainin g insight inth edeterminant so finter-individua l variations inoveral l energy requirements.Th equestion sho wmuc h food humans needan dth efactor s that determine their needsar ebasi ct oth escienc eo fnutrition . This thesis investigates methodological aspectso fstudie so nrestin gmetaboli c ratean d diet-induced thermogenesis;i tdescribe sth esiz ean dnatur eo f inter-individual differences inth erat eo frestin g energy expenditure,an d assesses theimpac to fvariou s nutritional andnon-nutritiona l factorso n resting metabolic ratean ddiet-induce d thermogenesis.

1.2.BACKGROUN DO FTHESI S

Inth elas ttw odecade s interest inth estud yo fhuma n energy exchange gained momentum. Thiswas » inparticular ,th eresul to fa nincrease d prevalenceo f problemso fenerg ybalance ,bot h inth edevelope d countriesa si ndevelopin g countries (1-4). Indevelope d countriesth eprevalenc eo foverweigh tan d

-11- obesityattaine d levelstha t caused concernamon gphysician s and nutritionists.Th ewidesprea d occurrence indevelopin g countrieso f energy undernutritionwit h itsclinica l sequelae receivedworldwid e attention. Obesityan d energyundernutritio n are twoside so f thesam ecoin .Bot har e the clinical manifestations ofa prolonge d disturbance ofenerg ybalance .I nth e former,energ y intakeha sexceede d energyexpenditur e and inth e latter the opposite processha soccurred . Initsel f it isno t clearwh y these problems would cause a revival ofth e studyo fhuma nenerg yexchange .Th e firstLa wo f Thermodynamics offersa simple solutiont obot hproblems .Th e obese should decrease their energy intake and theundernourishe d should increase food intake.Obviousl y theproble m isfa rmor e complicated. Interest for studying energyexchang e in relation toobesit y arosea t the suggestion that theobes ewoul d have lower rateso fenerg yexpenditur e and, consequently, lower energy requirements incompariso nwit h the non-obese (5-7). Such lower rateso fenerg yexpenditur ewoul db e of pathogenetic significance. Inaddition ,the ycoul d offer anexplanatio n for the frequently observedweigh t relapse inth eobes eafte rweight loss (8).Th ehypothesi so f lowered energy requirements inth eobes ewa s inagreemen twit h observations on the relevance ofenerg yexpenditur e forgenetic-base d differences inbod y compositionamon gvariou sanima l species (9,10). Interest for studying energyexchang e in relation toundernutritio n stemmed fromobservation s indevelopin g countries that individualswit h surprisingly lowenerg y intakeswer e reported tob e in remarkablygoo dhealt h (11).I twa s suggested that these individualsha d adapted their rateo fenerg y expenditure to the relatively lowenerg y intakes (11).Assessin g thedeterminant s and magnitude of thistyp eo fadaptatio n isconsidere d tob ehighl y relevant for a critical appraisal of the foodavailabilit y situation ina developin g country (11,12). Thus, the studyo fenerg yexchang eha stw o sidesalso .I ndevelope d countries itma yoffe r cluest oth eetiolog yo fproblem so fenerg yovernutritio nan d in developing countries itma yelucidat e thenatur ean dquantif y themagnitud e of a supposed biological adaptationphenomeno nwit h significant food policy implications.

Energyexchang e involvestw ocomponents ,th e first isenerg y intakean d the second isth eus eo f foodenerg yb ymetabolis m fora variet yo fbodil y functionsan d forphysica l activity, ie,energ yexpenditure .Th e current theoryo nenerg y requirements states that it isth e rateo fenerg y expenditure

-12- that fixes the level ofenerg y intake atwhic h energy requirementsar e met (13). This implies thatdifference s inenerg y requirements among individuals with similar degrees ofphysica l activity should be reflected primarily in differences in restingmetaboli c rateo rdiet-induce d thermogenesis.Th e question riseswhethe r inter-individual differences exist inthes e components of energy expenditure.Th e answer tothi squestio n isaffirmativ e with respect to resting metabolic rate (14-18). It iswel l known thatdifference s occur among individuals in restingmetaboli c ratean d that thesedifference s are depended on inter-individual variation inbod yweigh t and body composition (14-18). Personswit h greater amounts ofmetabolicall y active cellswil l have higher restingmetaboli c rates compared toperson swit h smaller amounts of metabolically active cells (14-18).Whethe r inter-individual differences in diet-induced thermogenesis exist and the factors that influence such differences arequestion s that currently canno tb e answered with sufficient certainty. Inth e study ofhuma n energy exchange the following pivotal questions continue to receive much investigative attention:

1.D o individualswit h similar bodyweight , body composition anddegree s of physical activity differ inenerg y expenditure?

2. Inwhic h components ofenerg yexpenditur e do thesedifferences ,whe n they occur,becom e manifest?

3.T owha t extent dopersona l characteristics relate to individual differences inenerg y expenditure?

4.Doe s thegeneti cmake-u po fa n individual affect the efficiency of energy utilization.

5.Doe senerg yexpenditur e adapt in response toenvironmenta l stimuli, in particular, to chronic alterations inth eplan e of nutrition?

6.Di d individuals become obesebecaus e theyha d lowered rateso f energy expenditure?Alternatively , do relatively lowered rateso f energy expenditure occur among theobese ,and , if so,ar e these of pathogenetic significance?

-13- The present thesisdoe sno taddres s all these questions. Itinvestigate s the following questions that are relevant for answers to some of the aforementioned topicso fhuma n energy exchange:

1.Ho w large isth e intra-individualvariatio n in restingmetaboli c rate and diet-induced thermogenesis?D o sex,degre e ofbod y fatnessan d control of theanteceden t diet affect the sizeo f the intra-individual variation in resting energy expenditure?

2.Wha t is thenatur e andmagnitud e of inter-individual differences in resting metabolic ratean ddiet-induce d thermogenesis inlea nan d obese individuals?Doe s the typeo fbod y fatdistributio n inobesit y affect these differences?

3.T owha t extent is restingmetaboli c rateaffecte d bynon-nutritiona l factors, ie,previou s exercise,psychologica l stressan d timeo f the day, and towha tdegre e bynutritiona l factors, ie,ethano l ingestion and short-term carbohydrate overfeeding?

4.T owha t extent isdiet-induce d thermogenesis affected bynon-nutritiona l factors, ie,previou s exercise,psychologica l stress, timeo f theda y and palatability of food, and towha tdegre eb ynutritiona l factors,ie , ethanol ingestion and short-term carbohydrate overfeeding?

In thisthesi s intra-individual variation isdefine d as thevariatio n of an individual abouthi so rhe r truemean . Inter-individual variation is the variation among the truemean s of individualswithi na population . To assess the relative importance of inter-individual variation in restingenergy - expenditure,knowledg e of themagnitud e of the intra-individual variation is important. If intra-individual variation in resting energy expenditure is large, itma y provedifficul t tocharacteriz e individuals fromanother , ie,t o correctly assess thenatur e andmagnitud e of inter-individual variation.

A reviewo f the literature (19,20)show s that itha sbee ndifficul t to distinguish the lean from the obesewit h respect to diet-induced thermogenesis.Ther e isn o satisfactory explanation for thedivergenc e in opinions concerning the importance ofdiet-induce d thermogenesis for the pathogenesis ofhuma n obesity. Thepossibilit y thata relatively large

-14- intra-individualvariatio n indiet-induce d thermogenesisha s contributed to thiscontroversy ,ha sno tbee ninvestigated .Th eproble mo f distinguishing lean subjects fromobes e subjectswit h respectt odiet-induce d thermogenesis couldals ob e related toth eoccurrenc eamon g thelea nan d theobes e of subgroupso f individualswit h relatively lowan dhig h rateso fenerg y expenditure.Thi sthesi saddresse s thesetopic si nsom edepth .I naddition ,t o theestimatio n of the sizeo f intra-an d inter-individual variation in resting energy expenditure,thi s thesiscontain svariou s studieso n the impacto f specific stimuli on restingmetaboli c ratean ddiet-induce d thermogenesis. These stimuli are subdivided intonutritiona l factorsan d non-nutritional factors.Th e factorswer e chosen tostud ythei r impacto n resting energy expenditure becauseo f two reasons.Firstly ,becaus e iti sgenerall y assumed for some of these factors,ie ,fo rpsychologica l stress,previou sexercise , time of theday ,tha t theyaffec tenerg yexpenditure .I n fact,however ,thei r effect onenerg yexpenditur e iscontroversia l orhardl ydocumented .Th e second reason istha t someo f these stimuliwer e expected toaffec t the biochemistry ofpostprandia l energyexpenditur e ina specificwa ya st ocaus ea hypothetized change inth e rateo f restingenerg yexpenditure .Studie swer e carried out totes t thesehypothese s inorde r togai n insight inth enatur e of diet-induced thermogenesis.

1.3.OUTLIN EO FTHESI S

Themajo rpar to f thisthesi scontain sarticle swhic har e inpres so r submitted forpublication .

Chapter 1contain sa genera l introduction.

Chapter 2give sa short reviewo f thecomponents ,regulation ,measuremen t and problemso fenerg yexchang e inhumans .I tprovide sa theoretical framework for the studyo f restingenerg y expenditure inhuman san d includesa formulation of themajo r hypotheses tested inthi sthesis .

Chapter 3consider smethodologica l aspectso f theassessmen to f resting metabolic rate,diet-induce d thermogenesisan d inviv o fuelutilizatio n by indirect calorimetryusin ga ventilate d hood system.Thi schapte r provides data on thenatur ean dmagnitud e of intra-individual variation in resting

-15- metabolic ratean ddiet-induce d thermogenesis.

Chapter 4describe s a studyo nth e impacto fdiurna l variation in resting metabolic ratean ddiet-induce d thermogenesis. Iti sfrequentl yassume d that restingmetaboli c rate showsconsiderabl e diurnalvariation .Thi svariatio n would seriouslyaffec tth eassessmen t ofdiet-induce d thermogenesis,i fno t properly correctedfor .

InChapte r 5th e impacto fpreviou sexercis e on restingmetaboli c rate is discussed.Th eduratio nan dmagnitud e ofth e impacto fpreviou sexercis e on restingmetaboli c rate isa controversia l issue.A prolonged and significant after-effecto fexercis e on restingenerg yexpenditur e may representa significant losso fenerg y for the individual,whic h couldb eusefu l inweigh t control orweight reductiontherapies .

Chapter 6 contains a studyo n theeffec to fmotion-pictur e induced psychological stresso n restingmetaboli c ratean ddiet-induce d thermogenesis. It isgenerall yassume d that "stress"affect senerg yexpenditure .However , littlehar ddat a isavailabl e to support thisassumption .

The nature andmagnitud e of inter-individual differences in restingmetaboli c ratean d diet-induced thermogenesis inlea nan d obese subjectsar ediscusse d inChapte r 7.

Chapter 8describe s the impacto f obesity inwome nan do f the typeo fbod y fat distribution inobesit yo n restingmetaboli c ratean d diet-induced thermogenesis.Th e typeo fbod y fatdistributio n inobesit y isa n important predictor ofmetaboli c complicationso fobesity . In recentyear s obesity researchha s investigated the relationshipsbetwee nbod y fatdistributio n in obesityan dhormona l status,insuli nan dglucos emetabolism , lipidmetabolism , butno t inassocatio nwit h energymetabolism .

The thermic effecto f ethanol and the impacto fethano l ondiet-induce d thermogenesis are reported inChapte r 9. Ethanolprovide so naverag eu p to5 percent ofdail yenerg y intake inadult s inWester ncountries .Alcoho l is reported tob e added on topo f thediet .Surprisingly ,however ,it sus e isno t associatedwit h increased levelso fadiposity .Thi sha s raised the suggestion thatethano l isles sefficientl yuse d asa nenerg y source compared to fato r

-16- carbohydrates.W e studied theefficienc y ofethano l energyutilizatio n by assessing itsthermi c effect infastin gan d fedsubjects . Chapter 10addresse s thequestio nwhethe r palatability affects diet-induced thermogenesis.Ma neat s to satisfyphysiologica l andnon-physiologica l needs. Ingestion of food,bu tpossibl y alsoth emer e sighto r smell of food,elicit s a prompt response inth e secretiono fvariou shormones .Thes e early (pre-absorptive)response sma ymodulat ediet-induce d thermogenesis. Food varying insensor y characteristics isknow n todifferentiall y influence these earlyhormona l responses.I nChapte r 10 itwa s testedwhethe r foodwit h different palatabilitywoul ddifferentiall y affectdiet-induce d thermogenesis.

InChapte r 11w ediscus s the interactionbetwee nexercis e and short-term carbohydrate overfeeding on restingmetaboli c ratean d diet-induced thermogenesis.Prolonge d heavy exercise isknow n todeplet e thebody' s glycogen stores.Carbohydrat e overfeedingma yhav e theopposit eeffect . Diet-induced thermogenesiswoul d be increasedwhe n thebody' sglycoge n levels arehig han d decreasedwit h lowlevel so f liver and skeletal-muscle glycogen. Inthi sChapte r theeffect so f exercise and carbohydrate overfeeding on restingmetaboli c ratean ddiet-induce d thermogenesis are discussed.

Chapter 12contain sa generaldiscussio n of theexperiment s reported inthi s thesis and includes the formulation ofgenera lconclusions .

REFERENCES

1.Bra yGA .Obesity :definition ,diagnose san ddisadvantages .Me d JAus t 1985;142:S2-7. 2.Va n ItallieTB .Healt h implications ofoverweigh t and obesity inth e United States.An n IntMe d1985;103:983-8 . 3.Seidel l JC,d eGroo tCPGM ,va nSonsbee k JLA,Deurenber g P,Hautvas t JGAJ. Associations ofmoderat e and severeoverweigh twit h self-reported illness andmedica l care inDutc hadults .A m JPub lHealt h1986;76:264-9 . 4.Reutlinge r S,Alderma nH .Th eprevalenc e of caloriedeficien t diets in developing countries.Worl d Dev1980;8:239-411 . 5.Mille r DS,Mumfor dP , StockMJ .Glutton y 2.Thermogenesi s inovereatin g man.A m JCli nNut r 1967;20:1223-9. 6. JamesWPT ,Trayhur nP .A n integratedvie wo f themetaboli c and genetic basis forobesity .Lance t1976;2:770-2 . 7.Jéquie r E.Doe sa thermogenic defectpla ya role inth epathogenesi s of humanobesity ? ClinPhysio l1983;3:1-7 . 8.Win gRR ,Jeffer yRW .Outpatient s treatmento fobesity :a comparison of methodology and clinical results.In tJ Obe s1979;3:261-79 .

-17- 9.Thurlb yPL ,Trayhur nP .Th erol eo fthermoregulator ythermogenesi si nth e developmento fobesit yi ngeneticall yobes e (ob/ob)mic epai rfe dwit h leansiblings .B rJ Nut r1979;42:377-85 . 10.Contald oF .Th edevelopmen to fobesit yi ngeneticall yobes erodents .In : CioffieLA ,Jame sWPT ,Va nItalli eTB ,eds .Th ebod yweigh tregulator y system:norma lan ddisturbe dmechanisms .Ne wYork :Rave nPress , 1981;237-42. 11.Sukhatm ePV ,Marge nS .Autoregulator yhomestati cnatur eo fenerg ybalance . AmJ Cli nNut r1982;35:355-65 . 12.Jame sWPT .Researc hrelatin gt oenerg yadaptatio ni nman .In :Schüre nB , ScrimshawNS ,eds .Chroni cenerg ydeficiency :consequence san drelate d issues.Internationa ldietar yenerg yconsultanc ygroup :Lausanne ,1987 . 13.Energ yan dprotei nrequirements .Repor to fa join tFAO/WHO/UN UExper t Consultation,Technica lrepor tserie s724 .Geneva :Worl dHealt h Organization,1985 . 14.Hallida yD ,Hes pR ,Stalle ySF ,Warwic kP ,Altma nDG ,Garro wJS .Restin g metabolicrate ,weight ,surfac eare aan dbod ycompositio ni nobes ewomen . IntJ Obe s1979;3:1-6 . 15.Ravussi nE ,Burnan dB ,Schut zY ,Jéquie rE .Twenty-fou rhou renerg y expenditurean drestin gmetaboli crat ei nobese ,moderatel yobese ,an d controlsubjects .A mJ Cli nNut r1982;35:566-73 . 16.Owe nOE ,Kavl eE ,Owe nRS ,Polansk yM ,Capri oS ,Mozzol iMA ,Kendric kZV , BushmanMC ,Bode nG .A reappraisalo fcalori crequirement si nhealth y women.A mJ Cli nNut r1986;4:1-19 . 17.Owe nDE ,Holu pJC ,D'Alessi oDA ,Crai gES ,Polansk yM ,Smalle yKJ ,Kavl e EC,Bushma nMC ,Owe nLR ,Mozzol iMA ,Kendric kZV ,Bode nGH .A reappraisal ofth ecalori crequirement so fmen .A mJ Cli nNut r1987;46:875-85 . 18.Bogardu sC ,Lillioj aS ,Ravussi nE ,Abbot tW ,Zawadzk iJK ,Youn gA , KnowlerWC ,Jacobowit zR ,Mol lPP .Familia ldependenc eo fth erestin g metabolicrate .N Eng lJ Me d1986;315:96-100 . 19.Wo oR ,Daniels-Kus hR ,Horto nES .Regulatio no fenerg ybalance .An nRe v Nutr1985;5:411-13 . 20.Jéquie rE ,Schut zY .Doe sa defec ti nenerg ymetabolis mcontribut et o humanobesity .I n: Hirsc hJ ,Va nItalli eTB ,eds .Recen tadvance si n obesityresearch :IV .London :Joh nLibbey ,1985;76-81 .

-18- Chapter2 . Energy exchange and energybalanc ei nhumans :a revie w

CONTENTS Page

2.1.INTRODUCTIO N 20

2.2.HISTORICA LPERSPECTIV E 20

2.3.TH ECOMPONENT SO FENERG YEXCHANG E 21

2.3.1.ENERG YEXPENDITUR E 21 2.3.1.1.RESTIN G("BASAL" )METABOLI CRAT E 21 2.3.1.2.DIET-INDUCE DTHERMOGENESI S 25 2.3.1.3.WORK-INDUCE DTHERMOGENESI S 31 2.3.1.4.MEASUREMEN TO FENERG YEXPENDITUR E 32

2.3.2.ENERG YINTAK E 36 2.3.2.1.THEORIE SO NTH EPHYSIOLOGICA LCONTRO LO FFOO D 36 INTAKE 2.3.2.2.CONTRO LO FFOO DINTAK EI NHUMAN S 40

2.3.3.ENERG YBALANC E 42 2.3.3.1.ENERG YBALANC EEQUATIO N 42 2.3.3.2.CONTRO LO FENERG YBALANC E 43

2.3.3.3.ENERG YBALANC EAN DOBESIT Y 47

2.4.CONCLUSION S 49

REFERENCES 50

-19- 2.1. INTRODUCTION

In this review Iwil l brieflydiscus s thecomponent s ofenerg y exchange and their relation toenerg ybalance .

2.2.HISTORICA L PERSPECTIVE

The studyo fenerg yexchang e inbiologica l systems startedmor e thantw o hundred yearsag owit h the inference ofLavoisie r that respiration inanimal s wasbase d on consumption of 'oxygène',a hithert ounknow nelement .I ttoo k more than seventyyear sbefor eanothe r fundamental scientific advancewa smad e inth e study ofenerg yexchang e inbiologica l systems.I n184 2th e German RobertMeye r formulated thePrincipl e ofEnerg yConservation .Thi s Principle isno wknow na s the firstLa wo fThermodynamics .I tstate stha tenerg y canno t be destroyed or created,bu tonl y changed in form.Mor e thanhal f a century later Rubner showed thata nanimal' shea to r energybalanc ewa s consistent with the first Law (1). At the turno f the century similar findingswer e reported byAtwate r andBenedic t forhuman s (2).I nth edecade s following, biochemistsunravelle d themetaboli c pathways involved incellula r heat production and energy transduction (3,4).Durin g thisperio d interest among physiologists andnutritionist s for the studyo fhuma nenerg yexchang e was negligible. Itwa sassume d that the fundamental problems ofenerg y exchange were solved.Energ ybalanc ewoul d be regulated byenerg y intake,whic hwa s under tighthypothalami c control(5) . Interest inth e studyo f the 'fireo f life'revive d inth e 1960san d the 1970s (6-8). Impetus for the revived interest inth e studyo fhuma n energy metabolism came from twodirections .Firstly ,anima l experiments showed that genetic-based differences inbod y compositionwer e related todifference s in energyexpenditur e (9,10).Relate d experiments showed that rats induced toea t a highlypalatabl e diet,increase d their food intake substantially without gaining excessweigh tdu e toa marke d stimulationo fbrow nadipos e tissue thermogenesis (11).Secondly , studies inhuman s indicated that the efficiency of energyutilizatio nand ,consequently , the levelo f energy requirements, varied in relation todegre eo fadiposit y (12,13). Thus, almosta centuryafte r itwa s shown thathuma nenerg y exchange proceeds according to the firstLa wo fThermodynamics ,th e studyo fhuma n energy exchange isa ta poin twher e itneed s the second Lawo fThermodynamic s to

-20- provide theke yt ounderstandin gtw oremainin gproblem so fhuma nenerg y metabolism. Thesear eth epartitionin go fenerg y transformations inth ebod y into those leadingdirectl yt ohea tan dthos e leadingt ointerna lwor kan d subsequenthea t ('efficiencyo funcouple d metabolism'),and ,secondly ,th e efficiencyo fth etransformatio no fchemica l energy infoo d stuffst ointerna l work ('efficiencyo fcouple dmetabolism') .Relate d tothes eproblem sar esom e importantquestion so fhuma nenerg yexchange ,ie ,whethe r individualsma ysho w systematicdifference s inth eefficienc yo fenerg yutilization ,whethe r certainpersona l characteristics covarywit h suchdifferences ,i nwha twa y suchdifference s relatet oenvironmenta l stimuli,and ,finally ,whethe rth e geneticmake-u po fa nindividua l affectsth eefficienc yo fenerg yutilization .

2.3.TH ECOMPONENT SO FENERG Y EXCHANGE

Thehuma nbod y isi nconstan tenerg yexchang ewit h itssurroundings .Chemica l energy isprovide d toth ebod yb yfoo d ingestion.Th eenerg y containedi n macronutrients isliberate db yoxidatio n involvinga continuou suptak eo f oxygenan ddisposa l ofcarbondioxide .Durin goxidatio no fnutrient shea ti s generated tomaintai nbod y temperaturean dfo rth eprovisio no fchemica lwor k (biosynthesis),osmoti cwor k (iongradients )an dmechanica lwor k (muscular contractions).Th etransfe ro fenerg y fromth ebod yt oit ssurrounding si s designated energyexpenditure .Th euptak eo fenerg y from itssurrounding sb y thebod y isknow na senerg yintake .

2.3.1.ENERG Y EXPENDITURE

The transfer ofenerg y fromt obod yt oit ssurrounding s comprisesenerg y expenditure.Tota l energyexpenditur eca nb esubdivide d into threecomponents : restingmetaboli c rate,diet-induce d thermogenesisan dwork-induce d thermogenesis.Thes e elementso fenerg yexpenditur ear ediscusse d inth e following threeparagraphs .

2.3.1.1.RESTIN G ('BASAL')METABOLI C RATE

Resting ('basal')metaboli c rate (RMRo rBMR )i sth erat eo fenerg y

-21- expenditure of an individual,awak ean da tcomplet ephysica l restsom e hours after the lastmea l andphysica l activity (14).Generally , it issimila r to what isknow n as 'basal'metaboli c rate.Basa lmetaboli c rate ismeasure d underhighl y standardized conditions and isdefine d asth e rateo f energy expenditure ofa n individual at completephysica l rest,lyin gdown ,shortl y after being awake,measure d ina thermoneutral state,12-1 4hour safte r the lastmeal ,wit h the individual at sexual reposean d emotionally undisturbed, without disease or fever (14).Th e term 'basal'suggest stha t energy expendituremeasure dunde r these conditionswoul d correspond tominima l energy expenditure.However ,durin g sleep,drowsines so rmeditatio n the rateo f energy expenditure canb e lower compared to 'basal'metaboli c rate (14,15). Most recentauthorativ e reviewso nenerg yexpenditur eus e the term resting metabolic rate (16,17). Throughout thisthesi s thister mwil l beused . Restingmetaboli c rate represents the sumo f theenerg y liberated by the metabolic activitieso fal l the cellso f thebod yunde r the specific conditionso f themeasurement . It includesth ecost so fmaintainin g the integrated systemso f thebod y and thehomeothermi ctemperatur e at rest.Unde r thermoneutral conditions sufficienthea t isgenerate d tomaintai nbod y temperature during oxidationo fnutrient s toprovid eAT P forbiosynthesis , active transport andmuscula r contraction. Inth eaverag eadul t individual restingmetaboli c ratecomprise s 60-75percen to f totaldail yenerg y output (16,17).

Figure 1give sa schematic representationo fvariou s factorsaffectin g resting metabolic rate inhuman sunde r thermoneutral conditionsan d themechanism s involved. Restingmetaboli c rate isdepende d onconstitutiona l factors,ie ,sex ,ag ean d genes,an d environmental factors,ie ,diet ,temperature ,stres san d drugs.Th e constitution of a subjectdetermine shi so rhe rmas so fmetabolicall y 'active' cellsan d the composition ofth emas so factiv e cells.I nthi s respect composition referst oth eproportio no ftissue swit hdifferen tmetaboli c rates (18). Obviously, inth e longter menvironmenta l factors,i nparticula rdiet , alsoaffec t a subject'smas so fmetabolicall y 'active'cell sand ,perhap s also, theproportio no f tissueswit hdifferen tmetaboli c rates.Bot h factors determine primarily theAT Pneed s forbiosynthesis ,activ e transport and muscular contraction,and ,consequently , restingmetaboli c rate. Thus, restingmetaboli c ratediffer samon gpeopl e ofdifferen t sex,bod y weight and age (19-28).However ,whe ndifference s inbod yweigh tan dbod y composition are considered mosto f thesedifference s tend todisappea r

-22- (23-28). In metabolic studies the size of the 'active' mass is usually estimated by a measure of the amount of fat-free or lean mass in the body (23-28). Fat-free mass comprises all 'non-fat' tissues and organs in the body, including bone, skin and extracellular water. The importance of the size of the 'active' mass

CONSTITUTION ENVIRONMENT

DIET /TEMPERATURE SEX/ AGE /GENES "stress"/drugs

SIZE OF COMPOSITION ENDOCRINE FAT-FREE BODY FAT-FREE BODY REGULATORS

EFFICIENCY OXYDATIVE ATP NEEDS FOSFORYLATION -BIOSYNTHESIS EFFICIENCY COUPLED -ACTIVE TRANSPORT. METABOLISM - MUSCULAR CONTRACTION

02 INPUT RMR C02 OUTPUT

Figure1 .'Model 'o fth eimpac to fvariou s factorso nrestin gmetaboli c rate (RMR).Compositio no fth efat-fre emas s representsth eproportio no ftissue s withdifferen tmetaboli c activitiesi nth etota l fat-freemass .Thic k lines indicate established contributions.Interrupte d lines indicate contributions stilli ndoubt .

fordeterminin g restingmetaboli c ratewa sshow ni nnumerou s studies reporting high correlationsbetwee n restingmetaboli c ratean dth esiz eo fth efat-fre e mass (23-28).Averag e restingmetaboli c ratespe runi tweigh to ffat-fre e mass are reportedt ovar ybetwee n11 0an d13 0kJ/d/k gfat-fre emas s (24-28). Since cellso fdifferen t tissuesan dorgan shav ewidel ydifferen t levelso f metabolic activity (18),i tfollow stha tth eaverag emetaboli c ratepe runi t weighto fcel lmas swil l changewhe nth eproportio no ftissue swit h different metabolic rateschanges .Currentl yi ti sver ydifficult ,perhap s impossible, toquantif y exactlyi nviv oth eproportio no fdifferen t tissues (composition) inth efat-fre emas so fth ebod y (29).Thi smean s thatdifference si nrestin g metabolic ratepe rweigh tuni to ffat-fre emas sd ono tnecessaril y indicatea

-23- difference inbasic ,energy-demandin gbiochemica l processesa t the cellular or molecular level.I t isa t thisbasi c level thatenvironmenta l factors,throug h endocrine regulators,ma ymor edirectl yaffec t the rateo fenerg y expenditure and cause inter-individual differences inth e rateo f restingmetaboli c rate. Among theseenvironmenta l factorsdie tan d theenvironmenta l temperature are probably themos t important,bu t stressan d certaindrug sma yals oaffec t restingmetaboli c rate (16,17). Hormones could either affect thedegre eo f coupling ofoxidatio n to phosphorylation, ie,affec tth eefficienc yo foxidativ ephosphorylation ,o r influence theus e ofAT P inmetabolism , ie,affec t thene tefficienc yo f coupledmetabolis m (17).Thyroi dhormone s appear tob e themajo r hormonal determinant ofmammalia n cellular thermogenesis (30).Thyroi dhormone s affect directly the rateo f ionicpumpin g (31,32).A considerable part,perhap s a very large part,o fou rmetaboli c energy is,t ocit eSim san dDanfort h(17) , "...devoted tokeepin g theprimordia l brine outo fou r cells".However , whether theeffec to f thyroid hormoneso n sodiumpumpin g contributes significantly tothyroi d thermogenesis remainst ob eestablishe d (32).I n addition to thyroid hormones,insuli n (33)an dnorepinephrin e (34)ma y also affect ionicpumping .Finally ,norepinephrin e and thyroid hormonesar e reported to increase theus eo fAT P insubstrat e ('futile')cyclin g (35,36). Whether hormonesma yaffec t inma n theefficienc yo fmitochondria l coupling of oxidation toAT Pproductio n isno tknown . Insmal l rodentsi tha sbee n shown thatnorepinephrin e reducesth eefficienc yo foxydativ ephosphorylatio n in brown adipose tissue(35) . Theexten t towhic h subtledifference s inth ehormona l control of cellular thermogenesis areassociate dwit hdifference s inth e rateo f energy expenditure peruni tweight o f fat-freemas s remains tob e investigated. Related to thisquestion ,i sth e impacto fnutritio no nendocrin e regulators of thermogenesis.I nthi s respectnutritionally-induce d changes in sympathetic nervous systemactivit yan d theperiphera lmetabolis m ofthyroi d hormonesar e interesting. Itwa s showntha toverfeedin g increases,o n theon ehand , the activityo f the sympatheticnervou s systeman d thereby catecholamines secretion rates,and ,o n theothe rhand ,th eproductio no f free triiodothyronine,wherea s energy restriction induced opposite effects (37-41). Theobserve d reduction in restingmetaboli c ratedurin g energy restriction (42-59)ha s frequentlybee nascribe d tohormonally-induce d changes inth e efficiency of energyutilization .Recently ,Ravussi ne tal . (54)showe d that thedecreas e in restingmetaboli c rate insubject sdurin gweight losscoul d be

-24- completely accounted forb yth e losso f fat-freemass .Bessar d et al. (52)an d Barrows and Snook (51),however , reporta decreas e afterweigh t loss in restingmetaboli c ratepe runi tweigh t of the fat-freemass . Rozen etal . (55) showed ina short-term study that thedecreas e inmetaboli c rate inobes e euthyroidwome ndurin gweigh t losscoul db eprevente db ya 'physiological' dosis of triiodothyronine.Th e latter study supportsa rolefo r hormonally-induced changes inth eefficienc yo fenerg yutilizatio ndurin g weight loss.However ,mor e research seemsneede d tosettl e thisissue . Two intrigueingobservation so n individual differences in restingmetaboli c rate shouldb ementioned .Recently ,Bogardu s et al. (27)showe d a familial dependency of restingmetaboli c rate inAmerica nPim a Indians.Th e resultso f the study ofBogardu s et al. dono toffe ra nanswe r toth equestio nwhethe r this familial dependencywa s related toa geneti c resembling of familymember s or to similarityamon g familymember s innutritiona l habitsor ,t ogeneralize , inlifestyl e factors.I thas ,however ,bee n reported that restingmetaboli c ratewa smor e alikebetwee nmonozygoti c twins incompariso n todizygoti c twins (56).

2.3.1.2.DIET(AKY)-INDUCE DTHERMOGENESI S

Diet(ary)-inducedthermogenesi s (DIT)i sth e increase inth e rateo f resting energy expenditure in response tofeedin g (14).I nthi s thesisdiet-induce d thermogenesis isdefine d asth eacut eeffec to f food ingestiono n resting metabolic rate,als o referred toa s the thermic (thermogenic)effec to f food or thehea t increment of feeding. Inth eaverag e individual diet-induced thermogenesiswoul d account forabou t1 0percen t of totaldail y energy expenditure (16,17). Therear eman y synonyms inus e todescrib ediet-induce d thermogenesis, these termsar e listed inFigur e 2.I nadditio n toth eplethor a of termsuse d to describe diet-induced thermogenesis, some investigators (57)conside r diet-induced thermogenesisno ta s theacut e impacto f food ingestiono n energy expenditure,bu ta sth echroni c effecto fdie to nth e rateo f energy expenditure. Intha t casedie t issynony mt oth eplan e ofnutrition ,whic h may affect energyexpenditure .Th e impacto fdie to nenerg yexpenditur e would manifest itself inadaptiv e changeso f restingmetaboli c rate,diet-induce d thermogenesis orwork-induce d thermogenesis.T opreven t semantical problems,I propose to refer tosuc hadaptiv e responsesa sdiet-induce d facultative

-25- changes in restingmetaboli c rate,diet-induce d thermogenesiso rwork-induce d thermogenesis, respectively. Thehistor yo fdiet-induce d thermogenesis startswit h thewor k ofRubne r(58) . Rubner observed an increase inoxyge n consumption of fastingdog safte r ingestiono fprotein , fato r sugar.Thi s increase inoxyge n consumptionwas , inparticular, manifes t afterprotei n ingestion.Rubne r called the increase in oxygen consumption after food ingestionb y fastinganimal s the specific dynamic effect (action)o f food.

- Specificdynami c action - Specific dynamic effect - 'Luxusconsumption ' - Heat increment of food,o f feeding - Thermic effect -o f food,o fa meal ,o f glucoseetc . Thermogenic effect Caloric response - tofood ,t oa mea l - Calorigenic effect - Thermogenic-I - Postprandial thermogenesis - Food - Diet induced thermogenesis - Dietary - Glucose (fat, protein)

Figure 2.Term sencountere d inth e literaturedescribin g the increase in energyexpenditur e following the ingestiono f foodo ra mea l(14) .

Inth e first 50-60year so f the 20th centurydiet-induce d thermogenesiswa s nota matte r ofgrea tconcer n tomos tnutritionist s (59-61). Inth e lasttw o decades,however ,th enumbe r ofpublication so nDI Tha s rapidly risen (12,13,52,53,62-85).Th e reasonfo r the revived interest indiet-induce d thermogenesiswa s the suggestiontha t theobes ewoul d have a lower diet-induced thermogenesiswhe n compared toth enon-obes e (12,13). This differencewoul d beo fpathogeneti c significance.I twa s suggested that diet-induced thermogenesiswa sa neffecto r ofdifference s inenerg y requirementsbetwee n leanan d obesesubjects . More recently,diet-induce d thermogenesisha sbee npropose d asa neffecto r of an adaptation ofenerg y expenditure tolowe r energy intakeso r to sustained increasedmetaboli c energyneeds ,ie ,i npregnanc yan d lactation (86-88). Whether diet-induced thermogenesisma yac ta sa neffecto r of inter-individual -26- differences inenerg y requirements isunclear .Controvers y inthi sare ao f the study ofhuma nenerg yexchang e isdisappointingl y high.Seventee n recent studies (12,13,52,53,62-74) reporta blunte d diet-induced thermogenesisi nth e obese,wherea seleve n studies (75-85)di dno tobserv e a systematic difference indiet-induce d thermogenesisbetwee n thelea nan d theobese .Accordin g to D'Alessio et al. (85)th edifference ,i n results isno t simply related toa systematic difference inmethodolog y between the studies supporting a role for diet-induced thermogenesis inth eetiolog yo fobesit yan d studiesdenyin g such a role. Recently, itwa s reported thatdiet-induce d thermogenesiswoul db e reduced, in particular,amon golde rdiabeti c obese subjects (64).Suc h subjectsar e insulin resistant andwoul d have an impaired cellular transport ofglucose , causing lower rateso fglucos e oxidationan d storage.Restorin g the insulin resistance bymean so f clamp studies showed that the thermogenicdefect wa s not related toa defec t inenerg ymetabolis ma tth ecellula r level,bu t to impaired cellular glucose transport(89) . Controversy onth e roleo fdiet-induce d thermogenesis isno tonl ypresen twit h respect to the impacto f thedegre e ofbod y fatconten to ndiet-induce d thermogenesis,bu t foralmos tan yothe r factor thatha sbee n studied for its effecto ndiet-induce d thermogenesis (90).A recentexampl e isth e impacto f thedegre e ofphysica l activityo ndiet-induce d thermogenesis (91-94).Whethe r adaptive changesoccu r indiet-induce d thermogenesisdurin g overfeeding is alsounclea r (5,41). There isn odisagreemen t onth eprimar ydeterminant so f diet-induced thermogenesis.Thes e are the levelo fenerg y intakean d thenutrien t composition of food (80,83,85,93,95).Wit h increasing energyintake , proportion ofprotei no r carbohydrates,diet-induce d thermogenesis increases. According toa recent studyo fD'Alessi o et al. (85)energ y intake islinearl y related todiet-induce d thermogenesis.Th e resultso fth e studyo f D'Alessio et al. are confirmed by findingso fHil le t al.(93) . Flatt (96)ha sprovide d avaluabl e accounto f thebiochemistr y of energy expenditure associatedwit hprotein ,fa to rglucos e ingestion.Diet-induce d thermogenesiswoul d behighes t forproteins ,followe d bycarbohydrate s and, finally,b y fats.Th ebalanc ebetwee n storageo fnutrient s andoxidatio n of nutrientswoul d determinenutrient -o rdiet-induce d thermogenesis.Accordin g toFlatt ,th e energyexpende d indigesting ,absorbin g and transporting nutrients isnegligabl e incompariso nwit h themetaboli c processingo f the ingested nutrients.Evidenc e for thisnotio ncome s froma study that observed

-27- no significant difference indiet-induce d thermogenesisbetwee n enteral and parenteral feeding(97) . Using Flatt'smode l of thebiochemistr y ofenerg y expenditure associated with nutrient ingestion, it canb epredicte d thatmetaboli c states favoring storage of nutrients prior tooxidatio nwil l enhance diet-induced thermogenesis. Flatt'smode l offers apossibilit y tocalculat e from substrate balances the amount of energy expended inpostprandia l metabolism ofnutrients . Various authorshav e shown that theamoun t of energy expended in the postprandial metabolism of the ingested nutrients canno t completely account for the observed thermic response (98,99).Curren t theory on diet-induced thermogenesis states thatdieh-indnre d thermogenesis iscompose d ofiw o distinct components,one ,th e 'obligatory' component, is involved in the postprandial metabolism of thenutrient s (16,17,95).Usin g Flatt'smode l the size of the obligatory component canb e calculated. Inaddditio n to the obligatory component, therewoul d be a 'facultative' component.Thi s component isassume d to contribute about 30-40^percentt o the total thermic response (17,95). The biochemical basis of this facultative component isno t yet elucidated. Food ingestion, inparticula r carbohydrates, can stimulate the activity of the sympathetic nervous system (83,100). It isassume d that this activation leads toa n increase in the turnover of catecholamines affecting, in its turn,th e rate of energy expenditure and consequently diet-induced thermogenesis.

Empirical evidence for the two-component model ofdiet-induce d thermogenesis isgive n by theobserve d reduction indiet-induce d thermogenesis (41)an d glucose-induced thermogenesis (99,101,102) in subjects infused by the beta-blocker propanolol. Ithas ,however ,bee ndifficul t to correlate diet-induced thermogenesiswit h plasma levelso fnorepinephrin e (65,82,83). Recently, itwa sobserved , however,tha t the increment after meal ingestion in plasma appearance rateso fnorepinephrin e correlated significantly with diet-induced thermogenesis (103). It isno t clear which factorsdetermin e the facultative component of diet-induced thermogenesis. Itha sbee n suggested that personal characteristics, ie,th edegre e ofadiposit yma yb e important (104), in particular, thedegre e of insulin resistance that frequently accompanies the obese state. Inaddition , theplan e ofnutritio nma y affect facultative diet-induced thermogenesis.Overfeeding , inparticula r with carbohydrates, would enhance this component (17).A third factor thatmigh t be involved in the thermic response to food are the sensory characteristics of food (105).

-28- The existence ofa facultative component indiet-induce d thermogenesis is inferred from adifferenc e inth e observed thermic response to food ingestion and the thermic response that should have been observed using Flatt'smode l to calculate theenerg y expended inpostprandia l nutrient oxidation and storage pathways.Ther e are someproblem s here.Th e relevance of facultative thermogenesis forenerg y expenditure under normal circumstances after normal mixed meal ingestion hasbee nquestioned , since itspresenc e hasbee n inferred predominantly fromunphysiologica l clamp studies (106). Secondly, nutrient utilization patterns asestimate d from indirect calorimetry dono t reflect dynamic states innutrien t oxidation and storage,bu tonl y net storage and oxidation (107). Thismean s that turnover of anutrien t between its storage andmobilisatio n forms canno tb eassesse dwit h indirect calorimetry. If such turnover would behigh ,thi s could increase thermogenesis. Inthi s respect the quantitation of lipogenesis fromglucos e is interesting. The energy expended intransformin g glucose to fat represents about 25percen t of the energy content of the glucose (96).Thi s indicates that transformation of a large part of the ingested glucose to fatprio r tooxidatio nwil l increase diet-induced thermogenesis when compared toa situationwher emos t of the ingested glucose will be immediately oxydized. The indirect calorimetric method forassessin g nutrientutilizatio n givesestimate s ofne t lipogenesis only. Thismean s that concomitant fat synthesis and fatoxidatio nwil l not be perceived frommeasurement s of gaseous exchange andurinar y nitrogen excretion.Onl y theeffect s of fat synthesis inexces s of fatoxidation ,o r of fatoxidatio n exceeding fat synthesis ongaseou s exchangewil l be measured. This implies that estimating theamoun t ofenerg y expended inth e net transformation of glucose into fatma yunderestimat e the total amount of energy expended inlipogenesis .Thus ,estimatio n of the amount of energy expended inth e socalle d obligatory component ofdiet-induce d thermogenesis may be inerro r too.Consequently , the inference of the existence of a facultative component representing 30-40percen t of total diet-induced thermogenesis could alsob ewrong .

Figure 3present s a 'model'fo r the current theory on thenatur e of diet-induced thermogenesis. Ingestion anddigestio n of food ornutrient s cause the obligatory expenditure of.energy .Afte r absorption nutrients are transported to storage and oxidation sites.Storag e and oxidation ofnutrient s require theobligator y expenditure of energy.Absorbe d foodma ydirectl y stimulate the sympathetic nervous system (SNS)activity , or indirectly through insulin. Increased sympathetic nervous

-29- system activity may lead to facultative thermogenesis. Insuline acts as gatekeeper of the amount of glucose directed to immediate oxidation or storage. Increased storage prior tooxidatio nwil l lead to increased obligatory thermogenesis. Insulinma y alsodirectl yac t to stimulate facultative thermogenesis. Using thismode lw e formulated hypotheses on the impact ofvariou s nutritional and non-nutritional factors on diet-induced thermogenesis. In this thesis we investigated the impact of ethanol,previou s exercise,psychologica l stress, palatability of food and carbohydrate overfeeding on diet-induced

FOOD/NUTRIENTS

INGESTION - DIGESTION ABSORPTION TRANSPORT

INSULIN OXIDATION

STORAGE L SNS ACTIVITY

OBLIGATORY FACULTATIVE OBLIGATORY THERMOGENESIS THERMOGENESIS THERMOGENESIS I _L DIET-INDUCED THERMOGENESIS

Figure 3. 'Model'o f current theoryo n thenatur e of diet-induced thermogenesis (DIT).Thic k lines indicate established contributions, interrupted lines indicate contributions still indoub t (SNS= sympathetic nervous system).

thermogenesis.Ethano lwa s assumed to increase nutrient storage and,hence , diet-induced thermogenesis. In resting conditions,ethano l elimination by -30- oxidationwoul d cause ingestednutrient s tob edirecte d nott ooxidatio n pathways,bu t tostorag epathways .Prolonge d exercise isknow n todeplet e carbohydrate storage inth ebod y (108)an dma yals oaffec t theactivit yo f the sympathetic nervous system (109).W e expected that storage of carbohydrates fromfoo dafte rpreviou sexercis ewoul db e increased inorde r to restore the tissues levelso fglycogen ,henc ediet-induce d thermogenesiswoul d be increased. Psychological stresswa sexpecte d toincreas e diet-induced thermogenesisb yeffect so nth e secretiono f stresshormones ,o r bya stimulationo f theactivit yo f the sympatheticnervou ssystem . Palatability washypothetize d toaffec tth eearl y insulin rise,als oknow na s the cephalic insulin response.Meal swit hhig hpalatabilit ywoul d evokea relativelyhig h cephalic insulin response (110). The levelo fth e cephalic insulin response is positively associatedwit h thetota l insulin réponse,a t least in rats (111). Losso f theearl yphas eo f insulin releaseha sbee n shown inhuman s to impair glucose tolerancean d to reduceth e thermiceffec to f infused glucose (112). We expected that,du e toincrease d insulin secretion,carbohydrat e storage on palatablemeal swoul d be increased and,hence ,diet-induce d themogenesis.I n addition,palatabilit y could increase theactivit yo f the sympathetic nervous systemwit h stimulatoryeffect so n thermogenesis.Carbohydrat e overfeedingwa s expected to increasediet-induce d thermogenesis intw oways . Firstly,b y affecting the stateo f thebody' sglycoge n stores.Secondly ,throug h activationo fth e sympathetic nervoussystem . Itwa s expected thatth e results ofou r studieswoul d enableu s to refineth emode l on thenatur e of diet-induced thermogenesis.

2.3.1.3.WORK-INDUCE D THERMOGENESIS

Work-induced thermogenesis (WIT)i sth eenerg y expendedabov e resting conditionsdu e toth eperformanc e ofphysica lwork . Iti sdetermine d by the type,duratio nan d intensityo fphysica lwork .Physica l activity isa muc h morepoten t stimuluso fenerg yexpenditur e compared todiet .Du e tophysica l activity the rateo f restingenerg yexpenditur e can risewit ha factor 10-20. For the average individual,work-induce d thermogenesiswoul d contribute 15-30 percent totota ldail yenerg yexpenditur e (16,17). It is,however ,difficul t toestimat e accurately theenerg yexpende d inphysica l activity.Thi s applies notonl y toperson s leading theirnorma l lives,bu tals o toperson swhos e energy expenditure isstudie d inwhol ebod ycalorimeters .I nparticula r the

-31- degree of spontaneous,relativel y small,movement s ('fidgeting')ma yb e quantitatively importanta sa neffecto r of inter-individual differences in energy requirements(28) . Dynamic muscularwork , forexampl e cycling,i sgenerall yperforme dwit h an efficiency of 20-25percen t (113). Theoretically,difference s inenerg y requirementsamon g individuals couldb e related toindividua l differences in theamoun t and intensity ofphysica l activityan d inth eefficienc y of the performance ofphysica lwork .A n evaluation ofdifference s inth eamoun tan d intensity ofphysica l activityo f free-living persons iscurrentl yver y difficult.However ,variatio n inwor k efficiency among individuals canb e assessedwit h standard laboratory tests.Ther e islittl e evidence for differences inwor k efficiencybetwee n the leanan d theobes e (114). The delta efficiency ofmuscula rwor k seems similarbetwee n thelea nan d the obese (115). Delta efficiency ispropose d as thebes t indicator of theefficienc y of theperformanc e ofphysica lwor k (116). Thedelt awor k efficiency isth e efficiency ofwor k accomplished calculated bydifferenc e between two steady state levelso fwork ,wherea s thene twor k efficiency isth ewor k doneb y an individual ona nexterna l systempe runi to fenerg yexpende d inth e performance of thatwor k (14).Weigh t losswa s reported toaffec t thene t efficiency ofphysica lwork ,bu tno t thedelt a efficiency (116). Itha sbee n suggested thatexercis ewoul dpotentiat e diet-induced thermogenesis and that theobes ewoul d bedefectiv e inthi s respect (5).Thi s isanothe r areao f controversy on the impacto f thedegre e ofbod y fat content ondiet-induce d thermogenesis.Mor e researchunde r strictly standardized conditions seemsnecessar y tosettl e this issue (114,115). Recently,a n interesting findingwa s reported. Itwa s reported thatAfrica n women expended lessenerg y incarryin g loadstha ndi d their Caucasian counterparts (117). The studyo fMaloi ye tal . (117)doe sno tgiv e ananswe r to thequestio nwhethe r thisdifferenc ewa s the resulto fa highe rwor k efficiency ofphysica lwor k amongth eAfrica nwome n compared toth e Caucasian women, or ofbehaviora l differencesbetwee n thegroup s inth ewa y the loads were carried.

2.3.1.4.MEASUREMEN TO FENERG YEXPENDITUR E

Energy expenditure canb eassesse d bydirec t calorimetry, indirect calorimetry andb ynoncalorimetri c methods.I wil l notdiscus s thesemethod s indept h in

-32- this thesis.Recently ,a fewexcellen t reviews (107,118,119)hav ebee n published on themeasuremen t ofhuma nenerg yexpenditur e and of inviv o fuel utilization.A fewcomment swil l suffice inthi srespect .

Direct calorimetry

Direct calorimetry involvesth emeasuremen to fhea t losses.Thi s isth e oldest of the three techniques.I tha sbee nuse d since the timeo f Lavoisier at the end of the 18th century.Direc tcalorimetr ymeasure s the sumo f radiant heat exchange and ofconvectiv e- , conductive- an d evaporative heat transfer. Ina resting subject the sumo f thesehea tlosse sequals ,i nth e long-run,th ehea t released bymetabolis m inth ebody .Direc t calorimetryha sth edisadvantag e that itca nno tb euse d toasses s short-term effectso f thermogenic stimuli on heatexchang edu e toth e relatively largehea t storage capacityo f thebody . For example,durin g feedingo r short-termexercis e alarg epar to f thehea t that isgenerate d isstore dwithi n thebody .Onl y over aperio d of 24hour so r longer thebod ywil l be inhea tbalanc ewit h itssurroundings .Ove r prolonged periods of timedirec t calorimetryprovide sth emos taccurat e estimate of the rateo fenerg yexpenditure .However ,i t isa lesssuitabl e technique for assessing diet-o rwork-induce d thermogenesis and itdoe sno tprovid e informationo n inviv o fuelutilization .

Indirect calorimetry

Indirect calorimetry isth emetho db ywhic hmetaboli c rate isestimate d from measurements ofoxyge n consumptionan d carbondioxide production.Whe n iti s assumed thatal l theoxyge n consumed isuse d tooxydiz edegradabl e fuelsan d all the carbondioxide thusproduce d is recovered,i t ispossibl e to calculate the total amounto fenerg y 'produced'. If, inadditio n the rateo f nitrogen excretion isknown ,indirec tcalorimetr y canb euse d toquantitat enutrient , ie, fuel or substrate,utilizatio npatterns .Energ y 'production'mean s in this respectconversio no fchemica l energyo fnutrient s intochemica l energyo f 'energy-rich' substances,ie ,ATP ,plu s losso f someenerg ya shea tdurin g the oxidationprocess .Eventually ,however ,i na restingadul t subjectal l energy derived fromth emetabolis mo f food stuffswil l beconverte d intoheat .I ti s essential to realise that inth e long rundirec tan d indirectcalorimet ywil l give similar estimates ofhea t losses fromth ebody . Inth e long runchange s inbod y temperaturewil l cancel outan d the rateo f formationan d degradation

-33- of energy-richbond swil l beequal . Indirect calorimetry canb euse d toasses sacut eeffect so f thermogenic stimuli onmetaboli c heatproductio n inth ebody .Sinc e thebod ydoe sno t possesa noxyge n storage capacityo fquantitativ e importance,oxyge n consumptionwil l immediately rise in response toexposur eo f theorganis m to thermogenic stimuli. There isa variet yo fequation suse d tocalculat emetaboli c rate fromoxyge n consumption,carbondioxid e productionan durinar ynitroge nexcretio n (118). Differencesbetwee n these formulasar equantitativel yunimportan t (118). They are generallydu e toth eus eo f slightlydifferen t constants for theamoun to f oxygenused and carbondioxide produceddurin g combustion inth ebod y of protein, fatsan dcarbohydrates . Two techniquesbase d on indirect calorimetricprinciple s areworthwhil e to mention.Thes ear emeasurement so fenerg y expenditurewit h indirectwhol e body calorimeters,als oknow na s respiration chambers,an dwit hventilate d hood systems.Respiratio n chambersare ,i nparticular ,suitabl e for studying total dailyenerg yexpenditur e of subjectso ranimal s foron eu p tosevera l days. Theventilate d hood system isusefu l formeasurin g the individual components of totaldail yenerg yexpenditure ,ie ,restin gmetaboli c rate,diet -o r work-induced thermogenesis.Th eventilate d hood technique canb e tracedback , at least,t oBenedic t in193 0 (120). However,th edevelopmen t ofmoder n physical gasanalyzers ,eliminatin g the time-consumingvolumetri c analysiso f gaseous exchange,an d ofmoder nphysica l flowmeters,ie ,mas s flowmeters,ha s stimulated the constructionan dus e ofventilate d hood systems inmetaboli c studies.Additiona l impetus for theus eo fventilate d hood systems cameb y the recognition that indirectcalorimetr y canprovid e informationo n the type and rateo f substrateutilizatio n invivo .Thi s typeo f informationma y be important for gaining insight inpattern so fnutrien t assimilation invariou s pathological states,fo rexample ,i ndiabete san d obesity (107).

Noncalorimetricmethod s

There isa variet y ofnoncalorimetri c methods (119)tha t canb euse d to estimate energyexpenditur e inman .Thes emethod sar ebase d on,fo rexample , theus e ofactivit ydiaries ,pulmonar yventilatio no rhear t ratean d mechanical activitymeters .A more recentdevelopmen t isth eus e of isotopic dilutiono fdoubl y labelledwate r (121). Thismetho dwa s introduced in 1955b y Lifsone t al. (122)fo r thestud yo fenerg yexpenditur e insmal lanimals .

-34- Until recently the costo fth e isotopewa s toohig h topermi t itsus e in humans (123). However,i n the1980 sth e firstpublication s appeared inwhic h energy expenditurewa s assessed inhuman sb ymean so f thedoubl y labelled water method (121,124,125).Th eprincipl e of themetho d ist odilut e the body 18 water component ofa perso nwit ha know namoun to fD 90 .Deuteriu mwil l label 18 thewate r pool of thebody ,wherea s the0 ,i naddition ,wil l also label the body'sbicarbonat e pool,du e toth eactio no f carbonic anhydrase.Deuteriu m 18 will leave thebod yb ywate r output onlyan d0 will leave thebod yb ywate r 18 plus carbondioxide output.A sa consequence,th eturnove r inth ebod yo f 0 exceeds thato fdeuterium . Thedifferenc e between the rateso fdeuteriu m and 18 0 turnover inth ebod ywate r isuse d tocalculat e the carbondioxide production rate (126). Toconver t thecarbondioxid e production rate toa n estimate ofenerg yexpenditure ,th eenerg yequivalen to f carbondioxide has to beused .Dependin g on the respiratoryquotien t of themetaboli c mixture oxydized in thebody ,th eenerg yequivalen t of carbondioxidema yvar y between 21.0k J and 27.7kJ . Ifth edietar y intakeo f the subject isestimated ,and , if the subject isi nenerg yequilibriu mdurin g theperio d ofmeasurement ,th e respiratory quotient of thedail ydiet ,als oknow na s the foodquotient ,ca n beuse d toderiv e anestimat e of theenerg yequivalen t of carbondioxide (118). Themajo r advantage of thedoubl y labelledwate r method for theassessmen t of energy expenditure is itsapplicabilit y forus e inperson s living their normal lives (127). Theus e of thedoubl y labelledwate rmetho d in free-living subjects requires theus e ofvariou sassumptions ,fo rexample ,wit h respect to steady state inwate r and energybalanc e of the subjectsdurin g the experimental period, the respiratoryquotien to f thedie tan d the proportion ofwate r that isfractionate d byevaporatio n atepithelia l surfaces (118). Opinions on theapplicabilit y onth edoubl y labelledwate rmetho dvary . Variousauthor s regard themetho d asa nexceptiona l technological advance that permitsaccurat e estimateso f the rateo fenerg y expenditure of free-living adults (127,128).Othe r investigatorsar emor e cautiousan d argue thatmor e research isneede d toestablis h thevalidit yo fvariou sassumption s of the methodwhe napplie d tofree-livin g individualsunde rvariou s conditions of climate, food intake,an dphysica l activity (118). Itshoul d alsob e realised that thedoubl y labelledwate r method givesa nestimat eo f theaverag e energy expenditure during theperio d ofmeasurement ,usuall y 2-3 weeks.I t isno t possible tomeasur e 24-hours energyexpenditure ,day-to-da yvariabilit y in energyexpenditur e or toobtai n information on thequantitativ e importance of thevariou s componentso fenerg y expenditure fortota laverag e energy

-35- expenditure. Inthi s respecta combinationo f theclassica l calorimetric methodsan d thedoubl y labelledwate r methodma yb epromising .

2.3.2.ENERG Y INTAKE

Incontras t toenerg y expenditure,whic h isa constantprocess ,energ y is ingested onlyperiodicall y atmea l timesan dbetwee nmeal sdurin g snacking. It isgenerall y assumed that thebod ycontrol s its food intake,ie ,it senerg y intake, tomee t currentenerg yneed san d tomaintai na nenerg y reserve for timeswhe n food isunavailable .Th e literature on factorsaffectin g the regulation of food intake isenormou s andwil l notb e reviewed here indepth , see for reviewsKissilef fan dVanltalli e (129), Nicholl et al. (130)o r Forbes (131). Inthi s reviewI wil ldircus sbriefl y sometheorie s on the physiological control of food intake and the control of food intake inhumans .

2.3.2.1.THEORIE SO NTH EPHYSIOLOGICA LCONTRO LO F FOOD INTAKE

That food intake isunde r somephysiologica l control isinferre d from observationso n the relative consistency indail yo rweekl y food intake of animalsmaintaine d ona die to fhomogenou s compositionunde r constant ambient temperature (132,133). These observations suggested theexistenc e ofa physiological control system for food intake (134). Itwa shypothetize d that such a control systemwoul d correctdeviation s indail y energybalanc e in order tomaintai n long-term energybalance .Additiona l support for the existence of control of food intake came fromth eobservation s that food intake inanimal svarie d reciprocallywit h caloricdilutio no f food (135,136). Evidence, that itwas ,i nparticular ,th eenerg y contento f the food thatwa s controlled,wa sprovide d byexperiment s that showed compensation in food intakewhe n energyexpenditur ewa svarie d (134,137).Th e existence ofa physiological control of food,ie ,energy , intake inhuman swa s inferred from theobservatio n that,althoug hma nma yea t tonso f foodove r theyears ,bod y weight stays relatively constant (138). Isth eevidenc e fora physiologica l control of food intake sufficient? Two comments canb emad e inthi s respect.Animals ,ie ,rats ,ca nb e easily overfed by offering thema highl ypalatabl e 'cafetaria'die t (139). Secondly, relative weight inma n isno ta sconstan ta s frequently isassumed .Gordo nan d Kannel

-36- showed ina longitudinal study (the 'Framingham'study )tha t inadulthood , bodyweigh t increaseso naverag e 10k gove r aperio d of1 8year s (140). These observations indicate thatcontro l of food intake inanimal sca nb e overruled byenvironmenta l stimuli,ie ,palatabilit yo f food. Inhuman ssom e formo f physiological controlo f food intake isprobabl ymanifest ,otherwis e the increase inweigh tdurin gadulthoo dmigh thav ebee neve nlarge r than the 10k g observed byGordo n andKannel .Th e findingso fGordo nan d Kannel,however , clearly showtha t thecontro lo f food intake inhuman s isprobabl yno t as precisea spreviousl y thought(5) . When it isaccepte d thatbot hhuman san danimal shav e some formo f physiological control of food intake,variou s theoriesma yb euse d toexplai n this control (134,141-144). Thehomeostati c theorieso fth ephysiologica l controlo f food intakehav e receivedmos tattention . Inhomeostati c theories iti spropose d thata variable is regulated and that certaineffector saccomplis h suchregulations . Homeostatic theorieshav e been subdivided into twotypes .Ther ear e those theories thatpropos e that specific chemicals inth ebod yar e regulated ('chemostatted'), ie,fa t ('lipostatted') (134), carbohydrates ('glucostatted') (141), orprotein s ('aminostatted') (142), and those that propose regulationo fenergy ,o r some functiono f it sucha sbod y temperature ('thermostatted') (143), orenerg yexpenditur e bymetabolizin g cells ('energostatted') (144). Nutrient specifichomeostati c theoriesar e nowknow n asth e lipostatic,glucostati c and theaminostati c theories.Th e current opinion istha t these theoriespresen ta toolimite d accounto f the control of sucha complexphenomeno na s food intake (145-148).However ,a fewdecade s ago much researchwa s carried out tovalidat e thevariou s theories.Therefore ,I will brieflymentio n thequintessenc e ofeac h theory.Th e lipostatic theory stated thatdepo t fatwa s the regulatedvariabl e and that rateso f food intake and energy expenditurewer e itseffector s (141). Theglucostati c theory proposed thatutilizatio no fglucos eb yprivilege d cellswa s regulated by initiating feedingwhe nutilizatio nwa s lowan d inhibiting eatingwhe n utilizationwa shig h (134). Theaminostati c theorypropose d thatexcesse san d deficiencies ofplasm a aminoacid swer e responsible for thecontro l of food intake (142).A n integrationo f the lipostatic,glucostati c and aminostatic theorieswa s thethermostati c theorywhic h consideredmetaboli c heat production asth e control of food intake (143). The thermostatic theoryha s been further refined inth eenergostati c theory (144). The latter theory states that theenerg y 'produced'b y themetabolis mo fabsorbe dnutrient s is

-37- monitored to regulate food intake.Specifi c temperature receptors inth e brain would activate and terminate ingestive behavior. The central problem inth evalidatio n of these theories has been to discover the location ofdetector s and effectors for thehomeostati c response. In the pastdecade s four anatomical siteshav e been suggested.Abou t 30year s ago Stellar stated thehypothalamocentri c theory of the control of food intake (149). It states thathunge r isdirectl y proportional to the output of a lateral hypothalamic center,th e 'hunger'centre .Thi s center would elicit feeding. Feedingwoul d be inhibited bya ventromedia l hypothalamic center,th e 'satiety' center,a sa result of specific postfeeding signals. Experimental evidence for thedua l center theory ofhypothalami c control of food intake came from observations that lesions of the lateral hypothalamus induced hypophagia (150)an d stimulation induced eating (151),wherea s lesions of the ventromedial hypothalamus induced hyperphagia (152)an d stimulation inhibited eating (153). Inaddition ,evidenc e wasobtaine d that glucose and free fatty acids affected theneurona l activity inthes e areas, thus relating the dual center theory to the chemostatic, ie,glucostati c and lipostatic theories (134,141). It isno w generally accepted that the concept of thehypothalamu s as having twodistinc t control centres isa gros soversimplificatio n (145). The concept ofdiscret e hunger and satiety centres hasbee n replaced by oneo f diffuse excitatory and inhibitory systems (145). The 'satiety' centre has been associated with twomajo r tracts,ie ,th e serotoninergic pathway, which originates in the raphe nuclei of thepontine-midbrai nare a and passes through theventromedia l hypothalamus,an d theventra l adrenergic bundlewhic h passes through theperiformica lare a inth evicinit y of the ventromedial hypothalamus.Th e hunger centre isassociate d with the dopaminergic nigrostriatal tract. Another suggested major site forhomeostati c control of food intake is the liver (154). According to this theory,hunge r isth e result of adecreas e in the liver glycogen reserves,whic h is signaled to thebrai n bydischarg e from glucoreceptive hepatocytes.A role for the liver inth e control of food intake is supported by the fact that intraportal injection of glucose inhibits feedingwhil e equivalent doses elsewhere are lesseffectiv e (155). The gastro-intestinal systemha sals obee n proposed as amajo r site for regulation of food intake (156-158).Accordin g to this theory,neura l and humoral signalsaris e inth e gastro-intestinal tract that inhibit food intake. Experimental evidence for thismode l came from observations that sham-feeding

-38- was inhibitedb y the infusiono f food inth esmal l intestine (156). In addition,evidenc e fora roleo f thegastro-intestina l tract inth econtro l of food intake came fromobservation s onth e satiatingeffec to f thegu thormon e cholecystokinin (159),whic h isprobabl yneuronall ymediate d bygastri cvaga l efferents (146). Other possiblemediator so f the satiatingeffec to f food in the small intestinear e somatostatinan dpancreati c glucagon (146). Itseem stha tvariou s factorsar e involved inth ephysiologica l control of food intake.Th emoder n theoryo f thecontro l of food intake consistso fa feedback system inwhic hvariou s signalsderive d fromth e gastro-intestinal tract,liver ,th ecirculatin g energy, ie,nutrient ,pool ,depo t fato rmetabo ­ lizing cells,transmi t information toa centra l system, ie,th ebrain ,whic h is responding toan d integrating information fromth evariou speriphera l com­ ponents (148). Thebrai nals o respondst oan d integratesexterna l information. The control of food intakeha sbee ndiagrammaticall ypresente d in figure4 . Inthi ssyste mperiphera l signalsac ta s satiety signals,som e timeafte r food intake these signalsbecom eattenuate d and food intake isterminated . Food intake isinitiate d byputativ ehunge r signals initiating inth ebrain .Ther e hasbee na n intensive search to identify thephysiologica l correlates of putative satietyan d hunger signals.Currently ,a variet yo f signals isknow n that act satiating,ie ,ac t toterminat e feedingo r act tosuppres s appetite (145,146). Thesear e cholecystokinin,bombesin ,calcitonin , corticotropin-releasing factor,thyrotropin-releasin g hormone,neurotensi nan d serotonin.Th e recent search for signalstha twoul d initiatehunge r has shown that, inparticular ,opioi d peptideswoul d acta shunge r signals (145). An interesting hypothesis istha tobesity ,a seffecte db yovereating ,woul d be the resulto fauto-addictio n toendogenou sopioi dpeptide s (145). Concluding Ica n state thatth ephysiologica l controlo f food intake is complex and thatn o factor in isolation seems tooffe r sufficient explanation for the complexbehavio r of foodintake .

-39- COGNITIVE AND EXTEROCEPTIVE INFLUENCES

DEPOT FAT

CIRCULATWG SMALL METABOLIZING INTESTINE ENERGY POOL CELLS "£-

RATE OF TOTAL NCONTROlH L RATEO FTOTA L ENERGY INPUT ENERGY OUTPUT

Figure4 .'Model 'o fcurren thomeostati ctheor yo nth econtro lo ffoo dintak e inhuman s (148). Thecomponent so fth eoveral lsyste mar eshow na sblocks ,whic hcorrespon dt o quantitieso f'fuel 'withi nanatomica lo rphysiologica lentities :th estomach , thesmal lintestine ,th eliver ,th ecirculatin generg ypool ,th eadipos e tissuedepot ,an dth etota lmas so fmetabolicall yactiv ecells .Th eblock sar e connectedt oon eanothe rb ysoli dlines ,whic hrepresen tth eflo wo fsubstrat e betweencomponents .Contro lo fflo wrate sar erepresente db yvalv esymbols . In thisdiagram ,th erat eo fflo wo fenerg yint oth ebod yi sshow na sbein gunde r thecontro lo fth ebrain ,whic hi srespondin gt oan dintegratin ginformatio n (representedb yth edashe dlines )transmitte dfro mvariou scomponent so fth e system.Th easteris ka tth efirs tvalv esymbo lindicate stha tth erat eo f energyintak ei sno tconstan tbu toccur sinstea di ndiscret epacket s(meals ) whoseonse tan dterminatio nar econtrolle db yth ebrai ni nrespons et oa varietyo finput s(adapte dfro mVa nItalli ean dKissilef f (18)).

2.3.2.2.CONTRO LO FFOO DINTAK EI NHUMAN S

Inhuman sfoo dintak ei saffecte dno tonl yb yphysiologica l factors, in particular,energ yneeds ,bu tals ob ynon-physiologica l factors.A variet yo f -40- non-physiological signalsma yac to nth ebrai n toterminat e or to initiate feeding (147). Thesenon-physiologica l factorsma ydiffe r inorigin ,ie , hedonic (palatability, taste,texture ,odor) , social (culture, religion), psychological (learnedpreference san daversions) ,environmenta l (temperature),economi c (cost,availability )o rpharmacologica l (anorectants). Garrowconclude d that 'Inma n control of food intake iscomplex ,an d the primitive hypothalamic reflexesar e soburie dunde r soman y layerso f conditioning, cognitive and social factorstha t theyar ebarel ydiscernible ' (160). Thequestio nma y risewhethe r food intake inhuman s isstil l under physiological control orwhethe r it islargel ydetermine d by exogenous factors. The importance ofphysiologica l controlo f food intake innewborn swa s demonstrated by Fomone t al. (161,162). Innewborn s theeffec to f non-physiological factorso nth e controlo f food intakema yb eassume d tob e ofnegligbl e importancewhe ncompare d toi nadults .Fomo ne t al. (161,162) showed in studieso n the food intake innewborn s thatbabie s fed less energy-dense milk adjusted their intake andat emore .Th e compensationwa s remarkablywell , ie,betwee n8 0percen t and 100percent .I na nelegan t study using normal foods,Poriko se t al. (163)showe d that threeday safte r energy content of thedie tha dbee ncovertl y reduced by substituting the synthetic sweetener aspartame insom e food items for sucrose,lea nadult ssubjects , studied ina metaboli cward ,starte d tocompensat e for the induced energy deficit.Compensatio nwa sno t complete,bu t stabilized after someday sa t 85 percento f theenerg y intakeobserve d inth e controlperiod . Ina subsequent studyo f the same groupwit hadul tobes ean d leansubjects ,n odifferenc e in' caloric compensation between leanan dobes e subjectswa sobserve d (164). These results indicate some formo fcontro l ofenerg y intake inadults .Simila r resultswer e obtained byDurran t et al. (166). Since inth e studieso f Porikos et al. the subjectswer eunawar e of the caloricdilution ,th e response of food intake to the caloricdilutio nma yb econsidere d tob ephysiologicall y in origin. Itca nno tb ediscerne d fromPoriko s etal.' sstudie swhethe r the subjectswer e kepto na weigh tmaintenanc e dieta t the starto f the study. When it isassume d that subjectswer e fed theirenerg y requirementsdurin g the control period,th e results suggest thata covert reduction inth e energy density of thedie t couldb ebeneficia l forweigh t reduction.However ,i n Porikose tal' sstudie sn ocontro l groupwa s investigated toestimat e a spontaneous gradualdeclin e in food intakedu e toadaptatio n toth e experimental conditions.

-41- Little isknow nabou t the relative contribution toth econtro l of food intake ofphysiologica l andnon-physiologica l factors in free-living subjects.Thi s isa subject that should be studiedmor e intensively.

2.3.3.ENERG Y BALANCE

Twomajo r componentsar e involved inth e regulationo fenerg ybalance :energ y intakean d energyoutput .Accordin g toth eLa wo fConservatio n ofEnerg y a completebalanc ebetwee n these twocomponent smus texist .Th e components of energybalance ,it scontro lan d its relation toobesit ywil l bediscussed .

2.3.3.1.ENERG YBALANC E EQUATION

The energybalanc e equation canb eexpresse d inth e following form (166): grossenerg yo f food= energyo f faeces + energyo furin e + energyo fmethan e + metabolic heatproduce d + retrievablewor k + energy retained

Not all food ingested isentirel ydigested , resulting ina losso fenerg y in the faeces.Faece sals ocontain sendogenou smateria l from thealimentar y tract.Th edifferenc e between theenerg y contento f the food and theenerg y contento f the faeces istherefor e referred toa s theapparentl ydigeste d energyo f food.Som e food constituentsar emetabolize d by thebacteria l flora inth e colon.Thi sma y lead toth eproductio no f small amountso f combustible gasses, ie,methane ,representin g a losso fenerg y for the individual.Th e remaining parto fdigestibl e energyar enutrient s thatar eabsorbe d intoth e blood stream tob emetabolized .Th emetabolis m ofprotei n results insom e wasteproduct s thatar e losta senerg y inurine .Th e remainingenerg y isknow n as themetabolisabl eenerg yo f food.Thi smetabolisabl eenerg y (ME)i sused after oxidation for,respectively ,basa lenerg y requirements,indicate d by resting ('basal')metaboli c rate,diet-induce d thermogenesis,work-induce d thermogenesisand ,sometimes ,fo r cold-induced thermogenesis.Unde r thermoneutral conditions,o r in subjectswel l insulated by clothing against

-42- the cold, the latter process isprobabl yo fnegligabl e importance for daily energyexpenditure . Inver y limited circumstances parto f theenerg y expended duringphysica lwor k is retrieved forus e later (retrievable work), ie,i n windingu pa spring.Hea t loss isthe ndelaye dunti l the spring is released. Finally,par to f themetabolisabl e energyma yb e retained inth ebod y for growth,productio n ofmilk , foetus,an d also fora n increase inth ebody' s energy reserves,ie ,fa tstores . The energybalanc e equation isusuall ypresente d ina slightlydifferen t form: metabolisable energy intake (ME)- energyexpenditur e (EE)= gain/loss in body's energy reserves (/\ER),ie ,

ME- EE= /\ER ,whic h equals ME- (RMR+ DI T+ WIT )= ^ER

Inweigh t stableperson s themetabolisabl e energy intakematche s energy expenditure andn one tchang e inth ebody' senerg y stores isobserved . The questionho w thebod ypreserve s itsenerg ybalanc ewithou t significant net storage or losso f energy from thebod y continues tob e of interest to investigators.

2.3.3.2.CONTRO LO FENERGY BALANC E

It follows fromth eenerg ybalanc e equation thata n increase inenerg y intake relative toenerg y expenditure producesenerg y surfeits,increase d fat storage and eventual gain inbod yweight .If ,o nth eothe r hand energy expenditure exceedsenerg y intake,energ y reservesar e lost fromth ebody , fat storesar e mobilized andbod yweigh tdecreases . Which factorscontribut e to thedevelopmen t ofa ne wbalanc e between energy intake and energyoutpu twithou t anyne tchang e infa t stores? Figure 5an d figure 6 showth echange sb ywhic h thebod ywil l gaina new balance between energy intakean d energy output ina situationo f energy imbalance,eithe r causedb ya reduced food intake,ie ,a negativ e energy balance (Figure 5)o rb ya n increase infoo d intake,ie ,a positiv e energy balance (Figure 6), Innegativ e energybalance ,energ y requirementsar e lowered.A decrease in bodyweigh t reduces thecos to fweigh tbearin g activities.Th e losso f fat-freemas swhic h accompaniesweigh t losscause sa lowering of resting

-43- NEGATIVE BALANCE E0UT> EIN

light \ l'metabolic" level of I r- Tefficienc y activity1)1

work i DIT J efficiency'

> t energy cost i WIT | of activity i

RMR {

ENERGY BALANCE E0ur =E,N

Figure 5.'Model 'o fimpac to fa negativ e energybalanc eo nenerg y expenditure.Thic k lines indicate established contributions.Interrupte d lines indicate contributions still indoub t (RMR= restingmetaboli c rate;DI T» diet-induced thermogenesis;WI T= work-induce dthermogenesis) .

Innegativ e energybalance ,energ y requirementsar elowered .A decreas ei n bodyweigh t reducesth ecos to fweigh tbearin g activities.Th elos so f fat-freemas swhic haccompanie sweigh t losscause sa lowerin go frestin g metabolic rate.Th ereductio n infoo d intakedecrease s diet-induced thermogenesis. Individualsma ysho w (facultative)behaviora l changes that reduceth eamoun to fenerg y expended inphysica lactivities . A further reduction inenerg y expenditure couldb ecause db ya nincreas ei n the efficiencyo fenerg yutilization ,ie ,i n'metabolic 'efficiency .Th e latter effectwoul d becomemanifes t in'unexpected ' (notaccounte d forb yth e processes,heade dunde r1 ,2 an d3 )decrease si nrestin gmetaboli c rate, diet-induced thermogenesisan dwork-induce d thermogenesis. -44- Recently, itwa sshown ,however ,tha tth e reduction inenerg yexpenditur e duringweigh t losscoul db e completelyaccounte d forb y the reduction inbod y weight and body composition,decreasin g the costo fphysica l activity ando f restingmetaboli c rate,an db y the reduced energy intake,decreasin g the amounto fenerg yexpende d after food ingestion (54).Ther ewa sn onee d to postulate theexistenc eo fa n increase in 'metabolic'efficiency .However , Leibelan dHirsc hpresente d evidence fora n increase in 'metabolic'efficienc y when they found thatpost-obes eha d significantly lower energy requirements in comparison tolea ncontrol s (167). However,i nthei r studyn omeasurement s wereperforme d of restingmetaboli c rate,diet-induce d thermogenesis orwor k efficiency.Recently ,Geissle r et al. (168)showe d thatpost-obes ewome n had 15percen t lowerdail y energyexpenditur e rateswhe n compared tolea n controls,matche d inbod y compositionan ddegre e ofphysica l activity toth e post-obese.Thes e findingsd o suggestth eexistenc e of somechang e inth e efficiency ofenerg yutilizatio n inth epost-obese .Suc ha changewoul d involve environmentally-induced alterations inth epartitionin g of energy transformations inth ebod y into those leadingdirectl y tohea tan d those leading towor k and subsequenthea tand , secondly, inth eefficienc yo f the transformationo f chemicalenerg y infoo d stuffst ointerna lwork . Iti sclea r thatmor e research isneeded ,no tonl y inpost-obese ,t o investigatewhethe r humans show 'unexpected'metaboli c responsest oa negativ eenerg ybalance . Indevelope d countrieswit h itscalori epollutio na situationwher e energy intake exceedsenerg y expenditure ismor e frequently encountered. Figure6 shows thewa y thebod y reacts toa positiv eenerg ybalance .Ther e isa n increase inbod yweight ,usuall yaccompanie d bya n increase inth e sizeo f the metabolically 'active'mass , ie,i nfat-fre emass .Th eeffect so fenerg y overnutritiono nbod yweigh tan dbod ycompositio n increase theamoun t of energyexpende d inweigh tbearin g activities anda t rest.Sinc emor e energy is ingested,diet-induce d thermogenesis isincreased . Inaddition ,behaviora l adaptation favoringexpenditur e ofenerg y inphysica l activityma yoccur . Similar to the situationo fa nenerg ydeficit ,th equestio n remainswhethe r there are additionalmechanism s favoringexpenditur e ofenergy .Th e existence of suchmechanism sha sbee npostulate d for the first timeb yNeuman n (169) whenh e introduced the 'Luxusconsumption 'phenomenon .Man yyear s later the resultso f theVermon t overfeeding studieso f thegrou po f Simsan d Horton supported theexistenc e of suchmechanism s (170). Inthes e studies itwa s observed thatweigh t gainwa s less thanth eweigh t lossexpecte d tooccu r on thebasi so f theenerg y surplus inth edie t (179). Evidence for the existence

-45- POSITIVE BALANCE EIN > E0UT

weight f j Imetabolic " leveivell ooff i 1" f efficiency acctivityt * ± i worK I D'T j efficiency!

t t energy costi WIT of activity I

RMR f

ENERGY BALANCE E0UT =Em

Figure6 .'Model 'o fimpac to fa positiv eenerg ybalanc eo nenerg y expenditure.Thic k lines indicate established contributions.Interrupte d lines indicate contributions stilli ndoubt . (RMR= restin gmetaboli c rate;DI T= diet-induced thermogenesis;WI T= work-induce dthermogenesis) .

inhuman so fmetaboli c responsest oenerg yovernutritio n favoring energy expenditure is,however ,fa rfro mconclusiv e (17,160). Overfeeding-induced changesi nthyroi d statusan dth eactivit yo fth e sympathetic nervous system coulddecreas eth eefficienc yo funcouple d metabolismo ro fcouple d metablism,ie ,increas eth eus eo fAT Pi nso-calle d substrateo rround-abou t cycles,o rio npumping. Insmal l rodentsi tha sbee n found that overfeeding decreasesth eefficienc yo foxydativ e fosforylationi n brown adipose tissue (171), although thisha sbee ndispute da swel l (172). When thermogenesis increasesdisproportionatel y during overfeeding, this could -46- acta sa protectiv e response toovereating . Thequestio nwhethe r theobes e aredefectiv e inth ecapacit y tosho wa protective response toenerg yovernutritio ncontinue s tob e atopi co f interest both toclinician san d toinvestigator s (17).Anothe r interesting question iswhethe r the change inth ecompositio n of thedie tove r the last centuryha saffecte d the capacity tosho wa protectiv e response toenerg y overnutrition.Th emajo r change incompositio n ofth ediet s ofpeopl e living indevelope d countriesha sbee n the increase inth eproportio n of total energy intakedelivere d by fat (173). Theproportio n ofenerg ydelivere d by fat in thedie tha s increased relative tocalorie sderive d from carbohydrates.Thi s change inth e composition ofth edie tma yb e important foroveral l energy balance fora variet yo f reasons.Firstly ,carbohydrate s generally evokea higher thermic effect incompariso n tofat s (95).Convertin g ingested lipids todepo t fatwil l incura losso fenerg y fromth ebod yo fabou t 4percen t of the ingested fats. Intransformin g carbohydrates todepo t fatabou t 25percen t of the energy ingested,wil l be lost fromth ebod ya shea t (96).I tma yb e hypothetized thatenerg y surfeitswit ha relativelyhig h fatconten twil l lead tohighe r fat storage inth ebod ycompare d tosimila r energy surfeitswit h a higher carbohydrate content.I naddition ,carbohydrat e intakema y increase the activity of the sympathethicnervou s system relative to fat intake (17,100). Empirical evidence for the importance of the fat/carbohydrate ratio inth e diet indeterminin g overall energybalanc e andbod yweigh twa s givenb y Lissner et al. (174). Lissner etal .showe d that subjectso na na d libitum dietwit h a fatconten t of 15-20percen t reduced calorie intake in comparison toa die twit h 30-35percen t or adie twit h 40-45percen t fat.Bod yweigh twa s also reduced. It isno tpossibl e topartitio n thebod yweigh t lossobserve d in the studyo fLissne r et al. ina componentdu e todecrease d energy intake and a componentdu e to increased energyexpenditure .However ,th e results suggest thathabitua lunrestricte d consumptiono f low-fat, ie,o f calorically diluted, dietsma yb ea neffectiv e approach toweigh tcontrol .

2.3.3.3.ENERG YBALANC EAN DOBESIT Y

Obesity isa situationwit ha n increased storageo f fats inth ebody .Obesit y developswhe nenerg y intakeexceed senerg yexpenditur e fora prolonge d period of time.A simple conclusion fromthi sobservatio n is,tha t theobes e are

-47- overindulgerswh od ono tphysiologicall y control food intake asprecisel y as the lean.Experimenta l evidence forthi sconclusio n is,however ,lackin g (164,165). Inabsenc e ofa clea rdefec t inth eobes eo f the (short-term) physiological control of food intake (164,165), itseem s justified toconclud e that theobes e eatmor e thanthei r leancounterparts ,fo rexample ,du e to some particular susceptibility tofoo d intake inducingnon-physiologica l factors. Surprisingly,however ,evidenc e for increased food intakesamon g the obese compared to thelea n isfa r from conclusive (175-179).Thi s seemingly paradoxical finding couldb e related tomethodologica l problemso f accurately quantifying food intake inhumans .I t iswel l knowntha t food intakema y show considerable intra-individualvariation ,implyin g that repeated assessments of food intakema yb e important incomparison so f food intakebetwee n relatively small groupso f leanan d obese subjects (180,181).Anothe r problem in assessing food consumption in free-livingperson s istha t the simple acto f monitoring food intakema yaffec t food intake,possibl ywit h a differential effect on the leanan d theobese .I tha sbee n suggested that theobese ,whe n monitored for their food intake,ten d tounderestimat e their food intake toa greater extentwhe n compared tolea n subjects (127). Inth eabsenc e ofclea r indications that theobes ewoul d overeat,researc hha s shifted itsattentio n toth e studyo fenerg yexpenditur e inobesity . Ifth e obesewoul d havea n increased 'metabolic'efficiency ,thi scoul d explain their obesitywhil e ingesting similar amountso fenerg ya sth elean . Thehypothesi s ofa n increased efficiencyo f foodenerg yus e inth eobes ehas , however,bee ndifficul t toprove .I naddition ,ther e aren o indications for grossdifference s inth edegre e ofphysica l activitybetwee nobes ean d lean subjects (104). Since,accordin g toth e FirstLa wo fThermodynamics ,energ y canno tb e created,ther emus tb e someexplanatio n for theseparadoxica l results.I f obesity isth e resulto fa n increased efficiencyo f foodenerg yuse ,th e absenceo f findinga n increased efficiency inth eobes e state,indicate s that obesity isa nadaptatio n toa metaboli c state favoringenerg y retention.Onl y prospective studies canprovid e adefinit e answer tothi squestion . Alternatively, studyingobes e afterweigh t losscoul db euse d tosolv e this problem,but ,onl yunde r theassumptio n thata chang e inmetaboli c state from obese topost-obes e has similar effectso n thebody' senerg yeconom y compared toa change froma pre-obes e toa nobes e state.Secondly , ifamon g leanan d amongobes e there are individualswit hdifference s in 'metabolic'efficiency , comparingheterogenou s groupso f leanan dobes e individualsma yno t reveal any

-48- significant differences inenerg y expenditure between both groups.A third explanation would be that the obese and the lean are equally efficient inus e of food energy,bu tmethodologica l problems inobtainin g a representative and accurate estimate ofhabitua l food intake,o r of energy expenditure, in particular, due tophysica l activity, among the obese or the lean lead to unexpected results.Finally ,a systematic difference indigestibilit y of food between the leanan d the obese could be important for the etiology of obesity. Ifdigestibilit y would be relatively low inth e leanan d relatively high in theobes e this could favor energy retention inth eobese .However , studies pertaining to this subject have shown that there aren odifference s in digestibility between the leanan d theobes e (182,183). Whatever the cause of obesity is,i t canb epredicte d that any investigator looking for the 'holygrail 'o f the etiology of obesitywil l encounter numerous problems inprovin g his or her hypothesis.Obesit y isprobabl y multifactorial inorigin . Itma ydevelo pdu e toenerg y retention caused by some form of energy overnutrition,aggravate d incertai n groups of the obese by an increased susceptibility to store fata sa result of relatively lowered rateso f energy expenditure. Evidence for thishypothesi s on the etiology of obesity comes from anelegan t studyb yRavussi nan d colleagues (184). Ravussin et al. recently showed ina prospectiv e study that a reduced rate of energy expenditure isa risk factor forbod yweigh t gain.A reduced rate of energy exenditure could,however , inthei r study,accoun tonl y forabou t 35 percent of theenerg y retained inbod yweigh t gain.Th e extent towhic h physiological and environmental factors contribute to the supposed hyperphagia or lowered rates of energy expenditure aswel l asho w these factors interrelate with the genetic make-up ofa pre-obes e individual remain tob e investigated. That the genetic make-up of aperso nma y be important in the response to energy overnutrition of energy expenditure was shownb y Poehlmane t al. (185).

2.4. CONCLUSIONS

The study ofhuma n energy exchange hasexperience d a revival the last 2-3 decades.Durin g thisperio d substantial insightwa sgaine d in factors affecting energy output.Salien t findings have been that individuals can differ in their efficiency of energyutilizatio n and that a reduced rate of energy expenditure can contribute tobod yweigh t gain in free-living

-49- individuals.However ,th enatur ean dmagnitud eo finter-individua ldifference s inth eefficienc yo fenerg yutilizatio na swel la sthei rrelatio nt o'nature ' and 'nurture'ar elargel yunknown .

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-58- Chapter3 . Theassessmen t ofrestin gmetaboli crate ,diet-induce d thermogenesis and in vivofue l utilization usinga ventilate d hood system: methodological considerations

JANA WESTSTRAT EMSC ,PAU LDEURENBER GPHD ,JOSEP HGA JHAUTVAS TH D

ABSTRACT

Differentaspect so fth emethodolog yo fassessin gpostabsorptiv erestin g metabolicrat e (RMR),diet-induce dthermogenesi s (DIT)an di nviv ofue l utilizationrate s(FUR's )wer einvestigate di nhuman susin ga nindirec t calorimetricventilate dhoo dsystem .Th ereproducibilit yan dbetween-perso n variabilityi nRMR ,DIT ,postabsorptiv ean dpostprandia l respiratoryquotient s (RQ's)an dFUR' swer eassesse di na grou po f3 7non-obes eme nan di n2 2 non-obesewome nan d3 2obes ewomen .RM Rreproducibilit ywa sassesse di na groupo f4 9men .Th eimpac to fstric tcontro lo fth eanteceden tdie to nth e reproducibilityo fRMR ,DIT ,RQ' san dFUR' swa smeasure di na separat estudy . Diurnalvariatio ni nRMR ,th eeffec to fth ephas eo fth emenstrua lcycl eo n RMRan dDIT ,th eeffec to fth eenerg yconten to fth etes tmea lan do fth e durationo fth epostprandia lperio do nth ecalculatio no fDI Twer eals o investigated.Th eaverag emethod-fre ewithin-perso ncoefficient so fvariatio n ofRM Ran dRQ' swer e5- 6percent .Th ewithin-perso nvariatio ni nDI Tan dFUR' s were,respectively ,o naverag e2 8percen tfo rDI Tan d2 0t o5 0percen tfo r FUR's,dependin go nth etyp eo fnutrien tan dth eprandia lstate .RM Rdi dno t showa significan tchang efro mearl ymornin gt oearl yafternoon .N osystemati c effecto fth ephas eo fth emenstrua lcycl eo nRM Ran dDI Twa sobserved .I n bothsexe sDI Twa spositivel yassociate dwit hth eenerg yconten to fth etes t meal.Wit henergy'intake sbelo w260 0k Ji nme nan dbelo w210 0k Ji nwome n around9 0percen to fth etota lDI Tresponse ,observe di nfou rhours ,coul db e assessedwithi nthre ehours .DI Twa sno tsignificantl yassociate dwit hbod y weight,th esiz eo fth efa tmas so rth efat-fre emass .

-59- It isconclude d thatth e low reproducibilityo fDI Tmeasurement sha s important considerations forth edesig nan d evaluation of studieso nDIT .

Keywords: indirectcalorimetry , restingmetaboli c rate,diet-induce d thermogenesis, respiratoryquotient ,fue lutilization ,methodology , reproducibility,obese ,non-obese ,anteceden tdiet ,diurna lvariation , menstrual cycle,huma n

INTRODUCTION

At the turno f thecentur yRubne r observed that the increase inhea t production, ie,i nenerg yexpenditure ,wa s greaterwit hprotei n thanwit h fat or sugar ingestion (1).Rubne r called the increase inhea tproductio n after food ingestion the 'specificdynami c effecto f food'.A t the same time that Rubner developed hisconcep to n the specificdynami c effect of food,Neuman n described the 'Luxusconsumption 'phenomeno n (2).Luxu sconsumptio n referst o a process ofmetaboli cwastag e of ingested excessenergy .Durin g Luxus consumption excess energy isno tuse d forproportiona l increases ingrowt ho r inbod y energy reserves,bu t for increased thermogenesis. BothRubne r andNeuman n couldno tpossibl yhav e foreseen thatmor e than6 0 years later interest inth especifi cdynami c effecto f foodan d inLuxu s consumptionwoul d increase dramatically. Until the 1960sth e specificdynami c effecto f foodan dLuxu s consumptionwer e matterso f little concern tomos tnutritionist s orphysiologist s (3,4). Inth e last threedecade s interest inbot htopic shas ,however ,bee n reawakened (5,6). During this timea plethor a ofterm shav ebee nuse d todescrib e the specific dynamic effecto f foodan d theLuxu sconsumptio nphenomenon .Th e termsmos tuse dnowaday sar e thethermi c effecto f foodan d diet-induced thermogenesis (DIT) (5,6). Both termsar e considered here to refer toth e immediate responseo fenerg y intake tofoo d ingestion,irrespectiv e of the nutritional statuso f thesubject . The reasons forth e revived interest inDI Twa s theemergin gvie w that thermogenesiswoul dpla ya n important role inth e regulationo fbod yweigh t andbod y energy stores (7,8). Previously,th ecentra l problem ofweigh t or energybalanc e regulationwa s considered tob e the regulationo fenerg y intake (9,10).Anima l experiments,however ,showe d that,i nadditio n tothi sfactor , the regulation ofenerg youtpu twa s important for thecontro l ofbod yweigh t

-60- (7,11,12). These findings suggested that inhuman sa defectiv e regulationo f energyoutpu t could alsob e responsible forth edevelopmen t ormaintenanc e of obesity.Thi shypothesi s led toa proliferatio n of studiescomparin g the obese with thenon-obes e orpost-obes e on totaldail yenerg y expenditure or on some component ofdail yenerg youtput ,usuall y the resting ('basal')metaboli c rate (RMR)an dDIT .Wit h respectt oDI Tth emos t significant outcome ofal l these studieswas ,tha tDI Twoul d beblunte d inth eobese ,an d inth epost-obese , and therefore of significance inth epathogenesi so rmaintenanc e of obesity (13-29). Theevidenc e for thisvie w is,however ,fa r fromconclusiv e (30-42). Since 1924a tleas t 17 reports (13-29)hav ebee npublishe d inwhic h itwa s shown thatth eobes eha d adefec t inDIT ;i na tleas t1 3article s (30-42)n o suchdefec twa s reported.Conflictin g resultsar e reported foralmos t any other factor affectingDI Ttha tha sbee n investigated (43-58). The reasons for these conflicting resultsar eobscur e (42).I t isclea r that, progress ingainin g insight inth eetiolog yo fproblem so fenerg ybalanc e is hampered considerably by thesecontroversies .

Thediscrepanc y in resultsi nthi s fieldo f studyo fpostprandia l metabolism may, inpart ,b e related tosom e specificmethodologica l problems of the measurement ofDIT .Fo rexample ,non eo f the studies reporting onDIT ,presen t data on thewithin-perso nvariability , ie,th e reproducibility,o fDI T measurements. Inth e reportpresente d here,th emethodolog y forassessin gDI T inhuman s is systematically reappraised.A description isgive no fa computerized open-circuit ventilated hood system,constructe d forassessin g RMRan dDIT . Resultsar epresente d of repeated DITan dRM Rmeasurement s obtained in9 1 subjects,male san d females,th e latter groupwit ha wid e range inbod y fat content. Special attention isgive n toth e reproducibility ofRM R and DIT measurements,th equantitatio n ofth ethermi c response,th e relation between DITan d theenerg y intake of the ingestedmea l and theduratio no fDI T measurements.I naddition ,th e impacto fth ephas eo f themenstrua l cycleo n RMR and DIT isinvestigated . The resultso f thisstud yprovid e important information onmethodologica l aspectso fDI Tmeasurements .

-61- SUBJECTSAN DMETHOD S

Theventilate dhoo d system

An open-circuit indirectcalorimetri cventilate d hood systemwa sbuil t to measure continuously the rateo fenerg yexpenditur e fora prolonge d period of time (upt o6 hours )wit hminima ldiscomfor t for the subject.Th eprincipl e of a ventilated flow-through system is,tha ta streamo fai r isforce d topas s across the faceo fa subjectan dmixe swit han d collects anyexpire d aira s it does. The expired air iscollecte d bya transparen t perspexhoo d placed over the subject'shead ,whic h ismad e airtight around thenec kwit ha cord.Th e rateo fenerg yexpenditur e canb e calculated bydeterminin g theamoun t ofai r flowing through thehoo d andb ymeasurin g theoxyge n (0,)an d the carbondioxide (CO,)concentration s inth e incomingan doutflowin gair . Figure 1give sa schematic representation of thesyste muse d inWageningen .

Figure 1.Schemati c outlineo f theWageninge nVentilate d Hood System. VH indicatesventilate d hood, FTi sflowmete r (flowtransmitter)an d FC is flowcontroller.No t shown inthi soutlin ear e themulti-pe n chart recorder and the loadcells .

-62- Througha perspe xhoo d(volum e3 01 )wit ha nai rinle to nto pan da nai r outleta tth erigh tsid eo fth ehoo dfres hfiltere datmospheri cai ri sdraw n bynegativ epressur ecreate db ya pum pdownstrea m (OceanSCL210 ,Dieren ,th e Netherlands).Airflo wthroug hth ehoo di smeasure di nth eoutle tairstrea mb y a thermalmas sflowmete r (Brooks5812N ,Veenendaal ,th eNetherlands) .Thi s devicemeasure sth emas so fga san di sunaffected ,withi ncertai nlimits ,b y temperaturean dpressur evariations .I tis ,however ,affecte db yhumidit yan d atth epoin twher eliqueficatio nstarts ,i ti simpossibl et omeasur eth emas s flowo fa ga saccurately .I nrestin gsubject scondensatio no fwate rvapou ri n theoutle tairstrea mo fa ventilate dflow-throug hsyste mdoe sno toccu ran d theimpac to fsmal lvariation si nhumidit yo nth emeasuremen to fairflo wi s negligible.Th emas sflowmete ri scalibrate da tleas tonc ea yea rt owithi n 0.2percen to findicate dflowvalue si nth erang ebetwee n2 0an d5 01/mi na t thesupplier' sfactor yusin ga ver yprecis egasvolum emete rwit hbuilt-i n temperaturean dpressur esensors .Flo wreading so fth emas sflowmete rar e directlyconverte dt oSTP Dconditions .Prio rt oth eflo wmeasuremen ta smal l andconstan tquantit y (0.41/mi nSTPD )o fai ri scontinuousl ywithdraw nb ya n airtightpum pfo rga sanalysis .Sampl ega si sthoroughl ymixe di na smal l(0. 4 1/min)perspe xmixin gchambe rcontainin ga smal lcirculatin gfa n(Microne lA G V244L,Zürich ,Switzerland) .Befor egasanalysi sth esampl ei spasse dthroug h twoseriall yplace dperspe xtubes ,eac h2 2m mi ndiamete ran d25 0m mi n length,containin gth edryin gagen tCaCl 2(Merc k2387 ,Darmstadt , West-Germany).Throug ha syste mo fcomputer-drive nsolenoi dvalve s(Bürker t 211A,Ingelfingen ,Germany )sampl ega si sobtaine dfro meithe rth eventilate d hood,standar dga smixture so rfres hfiltere datmospheri cair .Th eoxyge n analyzer (Servomex1100A ,Zoetermeer ,th eNetherlands )i splace da tth eoutle t ofth ecarbondioxid eanalyze r (AnalyticalDevelopmen tCompan ySS100 , Hoddesdon,UK) .A bypas ssyste mo ftw ovisua lflowmeter s(Brook s1355 , Veenendaal,th eNetherlands )wit hneedl evalve smeasure sth etota lsampl eflo w andi suse dt oregulat eth eflo wthroug hth eoxyge nanalyze r (0.10-0.15 1/min).Ga sanalysi si sperforme dwith ,respectively ,a ninfrare d carbondioxideanalyze rwit ha ful lscal erang eo f0- 1percent ,an da paramagneticoxyge nanalyze rse tt oa full-scal erang eo f20-2 1percent .Bot h analyzershav ea resolutio no f0.0 1percen tan dar eaccurat eto ,respectively , 2percen to fful lscal e(carbondioxide )an d1 percen t (oxygen).Bot hanalyzer s werefoun dt ob ever ystabl ei nzer oan dsensitivit ysettin gove rth ecours e ofmultipl e (n= 10 )5- 6hour smeasuremen tsessions .Zer odrif twa s-0.00 2+ 0.002percen t (offul lscale )fo rth eCO ,analyze ran d0.00 3+ 0.00 2percen t

-63- (offul lscale )fo rth e0 ,analyzer .Span ,ie ,sensitivity ,drift swere , respectively-0.00 3+ 0.00 3percen t (offul lscale )fo rth eCO ~analyze ran d 0.003+ 0.00 5percen t (offul lscale )fo rth e0 ,analyzer .Par to fth espa n drifti nth e0 ,analyze ri sno tcause db yrando mdrif to fth eanalyzer ,bu tb y shiftsi nbarometri cpressur eo fth eai ruse dt ocalibrat eth eanalyzer . Analyzersar ecalibrate dusin gdrie dstandar dga smixture san ddrie dfiltere d freshatmospheri cair .Zer osetting sar eperforme db ypassin g10 0percen t nitrogenthroug hth eanalyzer s (0.41/min ,a ta pressur eo f0. 4bar) .Th espa n ofth ecarbondioxid eanalyze ri sse tb ypassin ga specia lga smixtur e(0.6 0 percentcarbondioxide ,20.5 0percen toxygen ,78.9 0percen tnitrogen )throug h theanalyze r (0.41/min ,a ta pressur eo f0. 4bar) .Th espa no fth eoxyge n analyzeri sse ta t20.9 5percen tusin gfres hdrie dfiltere datmospheri cair . Duringth ecours eo fmeasurement so fenerg yexpenditur eth espa no fth eoxyge n analyzeri scontrolle donc eever y30-6 0minutes .Th eflo wthroug hth ehoo di s adjustedt okee pcarbondioxid ereading si nth erang eo f0.5 0t o0.8 0percen t andoxyge ni nth erang eo f20.1 0t o20.4 0percent .Flo wadjustment sar emad e bya flo wmeasur ean da flo wcontrol-uni t (Brooks5871 ,Veenendaal ,th e Netherlands)whos eoutpu ti sconnecte dt oa motor-drive nvalv e (Brooks5837 , Veenendaal,th eNetherlands )place ddownstrea mth etherma lmas sflo wmeter .I n adults,flo wrate sbetwee n2 5an d5 01/min ,dependin go nbod ysiz ean dbod y composition,ar eused .Subject' smovement sar erecorde db ya loa dcel l(Toky o SokkiKenkuyj oTKA-200A ,Tokyo ,Japan )place dunde ra le go fth ehospita lbed , thati suse db yth esubjec tdurin gth ega sexchang emeasurement .Th eprincipl e ofa loa dcel li sth eregistratio no fchange si na vertica lload ,ie , pressure.Pressur ei sexerte do nstrai ngauge si nth ecentr eo fth eloa dcell . Achang ei npressur ecause sa nelectrica lsigna lwhic hi samplifie dan d digitizedb ya nanalogue-digita lconverte rfo rcompute rprocessing . Outputvoltag esignal so fth ecarbondioxid ean doxyge nanalyzer ,th emas sflo w meteran dloa dcel lar emeasure dan ddigitize db ya data-acquisitio nsyste m (HewlettPackar d3497A ,Pal oAlto ,USA )interface dt oa deskto pcompute r (HewlettPackar d86 Bo r85 ,Pal oAlto ,USA) ,whic hi sconnecte dto , respectively,a diskdriv e (HewlettPackar d9121 ,Pal oAlto ,USA) ,containin g theprogra mfo rth ecalculatio no fenerg yexpenditure ,an da printer .Flo wan d gasconcentratio nmeasurement swer etake never y1 0seconds ,th eoutpu to fth e loadcel lwa smeasure dwit ha frequenc yo f5 times/sec .Th ecompute r calculatedan dprinte dth evalue so foxyge nconsume d (VO,a tSTPD) , carbondioxideproduce d (VCO,a tSTPD) ,th erespirator yquotien t (VCOj/vO,)an d therat eo fenerg yexpenditur e (inkJ/min )integrate dove rperiod so feithe r

-64- 1,2 o r5 minute(s) .Outpu tsignal so fth ega sanalyzers ,mas sflowmete ran d loadcel lar eals ovisualize do na multi-pe nchar trecorde r (Kipp&ZonenBD101 , Delft,th eNetherlands) .Energ yexpenditur emeasurement sar eperforme d ina noise-an dtemperatur econtrolle droo m(23-2 6 °C).Roo mtemperatur ei s measuredb ya ceramic-platin aelectrod e (HeraeusPTlOO ,Wij kbi jDuurstede , theNetherlands) ,calibrate dt o0.0 2 °C)an dals oprinte da t1 ,2 o r5 minute(s)intervals . A firstventilate dhoo dsyste mwa sbuil ti n1985 ,a secon dsyste mwa s constructedi n198 6an di n198 8tw omor esystem swer ebuilt .

Calculationo fenerg yexpenditur ean dfue lutilizatio n

Therat eo foxyge nconsumptio nan dcarbondioxid eproductio n (atSTPD )ar e calculatedusin gequation spublishe db yJéquie re tal . (59).

*> 2= l *[v OTtMAro2-AF«>2*Fino2)]. 1 -F in°2

<2>*» 2 -*ou t* F002- where:

F.0 ,= fractio no foxyge na tth einlet . V .= rat eo fai rflo wa toutle t(1/mi na tSTPD) .

AF02 =F in02- F out02.

AFC02 =F outC02- F. nCo2.

F .C02= fractio no fC0 2a tth eoutlet . F.Co 2= fractio no fCCX ,a tth einlet , in 2

Whenth erespirator yquotien ti sdifferen tfro m1.0 ,th einspire dvolum ei s notequa lt oth eexpire dvolum ean dtherefor eth einflo wo fai r (V. )i s differentfro mth emeasure doutflo w (V.). Measurementso fga sconcentration s atth einle tar ethu smad ei na differen tvolum eo fga swhe ncompare dt o measurementso f0 2an dC0 2a tth eoutlet .Th eHaldan ecorrectio n (59)i suse d toadjus tmeasurement so finle tga sconcentration sfo ra differenc ebetwee n theinle tan doutle tga svolumes .Th eHaldan ecorrectio no finle tga s concentrationsi sparticularl yimportan tfo r0, ,sinc e0 2ha sa relativel y largeconcentratio ni nth einspire dair .

-65- Therat eo fenerg yexpenditur eo rmetaboli crat ei scalculate dusin gth e formula(59) :

(3)M R= 4.184*[((4.686+1.096*(R O -0.707))*VO2+4.60*VO2p)]. where:

MR= metaboli crate ,ie ,rat eo fenerg yexpenditure ,i nkJ/min .

RQ =th enonprotei nrespirator yquotient ,ie ,VC0 2 /V02 . VCO, =th evolum eo fcarbondioxid eproduce di nnonprotein ,ie ,carbohydrat e

andfat ,oxidatio n= VCO, -VC0 2 ,i n1/mi n(STPD) .

VO? =th evolum eo foxyge nuse dfo rnonprotei noxidatio n=

vt>2- V0 2 ,i n1/mi n (STPD). VCO, =th evolum eo fcarbondioxid eproduce di nprotei noxidatio n= N * 6.2 5* 0.7739,i n1/mi n (STPD).

VD2 =th evolum eo foxyge nuse di nprotei noxidatio n= N * 6.2 5 *0.9936 ,i n 1/min (STPD). N= tota lurinar ynitroge nexcretion ,i ng/min . P= protei n= 6.2 5 *N ,i ng/min .

Ata nR Q of0.707 ,i nadditio nt oprotein ,fa ti soxydize di nth ebod yan d theenergeti cequivalen to f1 1 o foxyge nuse dfo roxidatio ni sequa lt otha t offat ,ie ,19.6 1kJ .A ta nRQ_ _o f1.00 ,i nadditio nt oprotein , carbohydratesar eoxydize dan dth eenergeti cequivalen to f1 1 o foxyge nuse d foroxidatio ni sequa lt otha tobtaine da tglucos eoxidation ,ie ,20.9 5kJ . Duringrelativel yshor tenerg yexpenditur emeasurements ,fo rexample ,fo r assessingrestin gmetaboli crate ,tota lurinar ynitroge nexcretio nca nno tb e assessed.Unde rthes ecircumstance smetaboli crat ei scalculate dwit hth e followingformula :

(4)M R= 4.184*t(4.686+1.096*(RQ-0.707))*VO 2]

where:

RQ= "online "measure d respiratoryquotient ,ie ,respirator yexchang eratio ,

V02 ="o nline "measure doxyge nconsumption ,i n1/mi n (STPD).

v"C02= "o nline "measure dcarbondioxid eproduction ,i n1/mi n (STPD).

-66- Wehav eestablishe dtha twit hthi sformul aenerg yexpenditur ei sdetermine d witha ver ysmal lerro r (<1% )compare dt ometaboli crat edetermine dwit h formula no3 .

Inviv one tfue lutilizatio nrate sar ecalculate dusin gth eLusk' sconstant s forfat ,carbohydrat ean dprotei noxidatio n(60) : 6.25g o fprotei nar eoxidize di nth ebod yt oproduc e1 g o fure anitrogen , requiring966. 3m lo f0 ,an dproducin g773. 9g o fCO, .Th eR Qfo rlipi d oxidationi s0.70 7an dfo rcarbohydrate s1.00 .Oxyge nconsume di s2019. 3ml/ g offat ,828. 8ml/ go fglycoge nan d745. 8g/m lo fglucose .Th econstant sfo r glycogenwer euse dt ocalculat ecarbohydrat eutilizatio nrat ei nth efastin g state.

Thus,th efollowin gmetaboli cmixtur eequation sar ederived : inth efastin gstate :

(5)vD 2= 0.8288*Gl y+ 2.019* F+ 6.25*N*0.9663 ,

(6)VC0 2= 0.8288*Gl y+ 1.4276* F+ 6.25*N*0.7739 , inth epostprandia lstate :

(7)V0 2= 0.7456*Gl u+ 2.019* F+ 6.25*N*0.9663 ,

(8)VC0 2= 0.7456*Gl u+ 1.4276* F+ 6.25*N*0.7739 , whereVC uan dVCO ,ar eexpresse di n1/mi nan dGly,Glu ,F an dN i ng/min .

Gly= amoun to fglycoge noxidize d Glu= amoun to fglucos eoxidize d F =amoun to ffa toxidize d N =amoun to ftotal -o rurea-nitroge nexcreted . SinceN an dVO ,an dVCO ,ar emeasured ,thi ssyste mwit hmetaboli cmixture s equationsca nb esolve dan dth eamount so ffat ,glucose ,glycoge no rprotei n thatar eoxydized ,ca nb ecalculate dusin gth efollowin gformula's :

-67- carbohydrates: in the fasting state:

(9)Gl y= 4.118*vC02- 2.911*V02- 2.325*N. inth epostprandia l state:

(10)Gl u= 4.5768*VD02- 4.5768*v02- 2.595*N. fat:

(11)F = 1.69*V02- 1.69*VC02- 2.032*N. protein: (12)P = 6.25*N .

A negative fatoxidatio n rateoccur swhe n theR Q exceeds 1.00.Thi s is ^ np indicative ofne tlipogenesi s fromglucose ,ie ,lipogenesi s inexces so f concomitant fatoxidation .Th e rateo fne tnewl y synthesized fat isequa l to thenegativ e fatoxidatio n rate.I t istake n that2.82 5g o fglucos e areuse d to synthesize 1g o fa representative fat,ie ,o f palmitoyl-stearoyl-oleoyl-glycerol (61).Th e amounto fglucos e stored (non-oxidativestorage )a slipi d ismeasure d in indirect calorimetrya s glucose oxidation. Ifne t lipogenesis occurs,observe d glucose oxidation rates are corrected for theamoun to fglucos e converted intolipid .

SUBJECTS

In total 103 subjects,4 9non-obes emen ,2 2non-obes ewome nan d 32obes e women,participate d inth e studiespresente d here.Al l studieswer e conducted between January 1986an d august 1988.I n9 1o f these subjects,3 7me nan d 54 women, repeatedmeasurement s ofRM Ran dDI Twer eobtained . For 12me n repeated measurements ofRM Ronl ywer e available.Fifty-fou r of the subjects participated instudie s carried out specifically for investigating methodological aspectso fRM Ran dDI Tmeasurements ;i nth eothe r 49persons , investigating aspectso fRM Ran dDI Tmethodolog ywa sno t themajo r objective

-68- of study.Subjec t characteristicsar eshow ni nTabl e1 fo rth eme nan di n Table2 fo rwomen . Inadditio nt ostudie si nthes egroups ,a grou po f6 me n and 5wome nparticipate d ina stud yt oasses sdiurna l variationi nRMR . Subjectswer e recruitedfo rparticipatio nb yadvertisemen t inregiona l newspapers,th eUniversit yweekl yperiodical ,b yposte ro rthroug h informal contacts.Afte r applying forparticipation , subjects receiveda brochur ewit h detailed informationo nth enatur ean dpurpos eo fth eexperiment(s) .I n addition,subject s receiveda questionnair e forobtainin g informationo npas t andpresen thealt hstatus ,nutritiona lhabits ,smokin gan d(alcoholi c beverages)drinkin gbehaviour ,habitua l activitypatter nan dsportin g activities. Subjectseligibl efo rparticipatio nwer e invitedfo ra firs tvisi t toth elaborator yt ofamiliariz e themwit hth eventilate d hood system.Afte r receiving their informed consent subjectswer e allowedt oparticipat e inth e studies.Th eprotoco lo feac h studywa ssubmitte dt oan dapprove do fb yth e Medical-Ethical Committeeo fth eWageninge nAgricultura l University.

Table1 .Characteristic so fme nparticipatin g inreproducibilit y studieso f BMR,DI Tan dfue lutilizatio n rates.

Stud/ 1 Study 2 Study 3 Study 4 Study 5 StucV 6 Total < Range >

Sample size (n) 12 5 4 10 6 12 8.2±1.5 4-12 FMI replicates (k) 4 4 4 6 6 4 4.7+0.4 4-6 nrr replicates

Value* s are given as •ram + sen Study 6 not included in calculatim

Entry criteria couldvar yamon gth edifferen t studies,fo rexample ,wit h respectt obod yfa tcontent .I ngeneral ,however ,a subjec twa seligibl efo r participationwhe nh eo rsh ewa sapparentl yhealth y (noevidenc eo fpas to r present thyroiddisorder san ddiabete smellitus) ,di dno tus edrug sknow nt o affectenerg ymetabolism ,ha dn oproteinuri ao rglycosuria ,wa seatin ga normal balanced diet,smoke d lesstha n1 0cigarettes ,consume dn omor e than5 alcoholicdrinks/d ,di dno texercis emor e than7 hours/wee kan dha dha da -69- stablebod yweigh t (within2. 5k go fbod yweigh ta tentry )fo ra perio do fa t least6 month sprio r toth ebeginnin go fth estudy .Wome nha dt ob ei nth e premenopausal state.Obesit ywa sdefine da sa bod yfa tcontent ,assesse d with theunderwate rweighin g technique,exceedin g2 5percen t inmen ,o rexceedin g 35percen t inwomen .I nth eobes ewome n fastingbloo d glucose levelswer eal l within thenorma l range.

Table2 .Characteristic so fwome nparticipatin g inreproducibilit y studieso f RMR, DITan d substrateutilizatio n rates.

stucfy 1 Stuty 2 Stucty 3 Stuty 4 StlKfy 5 StucV 6 Total < Bange >

Sample size (n) 6 5 5 6 17 15 9.0+2 2 5-17

RMR replicates (k) 4 4 12 6 4 2 5.3+1.4 2-12 DIT replicates 28.8+1.2 27.7±2.3 28.4+1.1 28.1+2.0 47.8+1.3 44.8+0.9 39.0+1.4 19.9-55.9 Fat-free mass (kg) 43.3+1.1 44.4+1.2 47.2+2.0 43.8+2.2 47.6+1.4 49.0±1.2 46.8+0.7 36.6-60.6 Fat mass (kg) 17.6+1.2 17.4+2.1 18.8+1.1 17.5+2.3 45.0±3.2 40.3±2.0 32.6±2.1 10.0-70.4

Values are given as i

METHODS

Variabilityan daccurac yo fth emetho d

Theprecisio no fth ega sanalyzer san dth ecalibratio nprocedur ewa smeasure d overa perio do f2. 5year sb y6 0ethano l (Merck 983,Darmstadt ,West-Germany ) combustion testso fo naverag e2 hour sduratio n (range1- 4hours) . Twentyo f these testswer euse dt ochec k theprecisio nan dvalidit yo fth eentir e ventilated hood system.T ooxydiz e1 g o fethano l1.46 01 o foxyge ni sneede d and 0.9721 o fcarbondioxid e isproduce d (RQ= 0.666) .Whe na know n quantity of ethanol iscombusted ,th evolume so foxyge n consumedan dcarbondioxid e produced, involved inth eoxidatio nproces sca nb ecalculated . These calculated valuesca nb ecompare dt oth eobserve d values.T ocalculat eth e variabilityo fth emetho d ina comprehensibl e form,w eextrapolate dth e ethanol combustionst oa nethano l combustion rateneede d forth eaverag e male sedentary individualwit ha restingmetaboli c rateo f700 0kJ/d .Thi swoul d -70- correspondt oa nethano lcombustio no f23 6g assumin ga nenergeti cequivalen t ofethano loxidatio no f29. 7kJ .Fo rth eoxidatio no f23 6g o fethano l34 51 0*ar eneede dan d23 01 o fC0 ~ar eproduced .

Within-person variability in RMR, DIT, FQan d fuel utilization rates

RMRvariabilit ywa smeasure di n4 9me nparticipatin gi n6 differen tstudies , DIT, RQ and fuel utilization rate variability was evaluated in 5 different studiesi n3 7men .I n5 4women ,6 studie swer euse dt oestimat eth e within-person variability in variables of energy metabolism. All studies were conducted within a period of 6 weeks, except for study no 3 and 4 in women, which were carried out over a period of 3 months. A brief description of the differentstudie si sgive nbelow :

- Men:

Study1 ;n=1 2 thesesubject sparticipate di na stud yt omeasur eth ethermi ceffec to fethano lan dth eimpac to fethano lo nEŒT .FM Rva s measuredfou rtime sunde rth esam econdition san dDI T (4hours )va smeasure dtw otime sunde rth esam econditions .Betwee n repeatedmeasurement sva sa tim einterva lo fa tleas ttw odays . Study2 :r*= 5 Thesesubject sparticipate di na stud yt oasses sth eimpac to fpalatabilit yo nDIT .Th enumbe ro freplicate san d time-intervalbetwee nreplicate so fRM Ran dDI Tmeasurement swer esimila rt othos ei nstud y1 . Study 3:n= 4 Thesesubject sparticipate di na stud yt omeasur eth eeffec to fa two-week scontrolle ddietar ytria lo nth ewithin-perso n variabilityi nRMR ,EXIT ,R Qan dfue lutilizatio n rates.RM Ran dDI Twer emeasure dfou rtime sunde rth esam econdition s witha time-interva lo f3- 4day sbetwee n replicatemeasurements . Study4 :n=1 0 This study vas performed to measure the impact of diurnal variation (nwming versus afternoon) on PMRan d DIT (62). The time-interval between replicate RMRan d DIT measurements was at least two days. Study 5: n=6 This studywa sspecificall ydesire dt oestimat eth ewithin-perso nvariabilit yi nRMR ,DIT ,R Qan dfue lutilizatio n rates.RM Ran dDI Twer emeasure do nthre econsecutiv emorning si ntw operiods ,separate db ya fiv eweek speriod .Thi s studywa sth efirs tstud yw eperformed .Th eresult so fthi sstud yshowe da significan t timeo fmeasuremen t effecto nRMR , butno to nDIT .RM Rmeasure do nth emornin go fth efirs tda yo feac hperio dwa sslightl yhighe rtha no nth emorning so f thesecon dan dthir dday ,respectively ,3 an d7percent .Thi swa sprobabl ydu et oa smal lcarry-ove r effecto fth efirs t measurementda yo nth esecon dday ,an do fth esecon dda yo nth ethir dday .C na da ytha tenerg yexchang ei smeasure d food intakean dactivit ypatter nar egenerall yquit edifferen tfro mfoo dintak ean dactivit ya sobserve do na 'normal'day .A s a resulto fthi sstud ya time-interva lbetwee nreplicate so fRM Ran dEU To fa tleas ttw oday swa schosen . Study6 :n*1 2 Inthi sstud yth eimpac to nRM Ran dDI To ftyp eo fmotio npictur ewa sassessed .Th etyp eo fmotio npictur edi dno t significantlyaffec tth eRMR .O feac hsubjec tfou rreplicate so fRM Rwer eavailable .

-71- - Women:

Study Ir n=6 Thisstud ywa sdesigne dt otes tth eimpac to fpalatabilit yo nDIT .I neac hwome nfou r replicateso fth eBf Ban dtw oo fDI T weremeasured .I nfou rwome nth eeffec to nFM Ran dDI To fphase ,ie ,pre-ovula rversu spost-ovular ,o fth emenstrua lcycl e couldb eassessed .Ovulatio nwa sestimate dt ooccu rtw oweek sbefor eth eonse to fmanses .Replicat emeasurement so fFM R andDI Twer eperforme dwit ha time-interva lo fa tleas ttw oday sbetwee n replicates.Th ewome nwer emeasure da tleas t threeday safte rth estar to fth emenstruation . Study2 :n= 5 Inthi sstud yth eeffec to fa two-week scontrolle ddietar ytria lo nth ewithin-perso nvariabilit yo nFMR ,DIT ,E Qan dfue l utilization rateswa sassessed .Fou r replicatemeasurement so fFM Ran dDT Twer eavailabl eo feac hwoman .Th etim einterva l between replicatemeasurement swa s3- 4days .Woma nwer emeasure da tleas tthre eday safte rth eonse to fmenstruation .I n twowome nth eimpac to fth ephas eo fth emenstrua lcycl eo nFM Ran dDI Tcoul db edetermined .Th eothe rwome nuse dora l contraceptives. Study3 :n= 5 This studywa sspecificall yperforme dt omeasur eth eeffec to fth estag eo fth emenstrua lcycl eo nFM Ran dDTT .Th ewome n weremeasure dove ra perio do fthre ecycli .FM Rwa smeasure d6 time si nth epre-ovula ran d6 time si nth epostovula rphas e ofth ecycle .DT Twa smeasured ,respectively ,3 time spre-ovularl yan d3 time spost-ovularly .Ga sexchang emeasurement s in thepre-ovula rphas ewer ealway sperforme da tleas tthre eday safte rth eonse to fmenstruation .Th etime-interva lbetwee n replicates ineac hstag eo fth ecycl ewa si ngenera lthre edays .Ovulatio nwa sassesse dwit ha nI Hovulatio ntes t (NourypharmaI HColor ,Oss ,th eNetherlands) . Study4 :n= 6 This studywa scarrie dout t odetermin eth ewithin-perso nvariabilit yi nwome nusin gora lcontraceptives .FM Ran dDT Twer e measured for6 an d3 times ,respectively ,ove ra perio do fthre emonths .FM Rmeasurement swer eperforme di ngenera lwit ha two-weeksinterva lbetwee nth e replicatemeasurements ,DT Twa sassesse donc eever ymont hwit ho naverag ea fourweek s time-intervalbetwee nth esuccessiv emeasurements . Gasexchang emeasurement swer ealway sperforme da t leastthre eday safte rth eonse to fmenstrua lbleeding . Study 5:n=1 7 Inthi sstud yonl yobes ewome nparticipated .Th epurpos eo fth estud ywa st omeasur eth ewithin-perso nvariabilit yo fFMR , DTT,R Qan dfue lutilizatio n ratesi nobes ewomen .O feac hwome nfou r replicateFM Ran dDI Tmeasurement swer eavailable . Thetime-interva lbetwee nth e replicatemeasurement swa sa tleas ttw odays .Ga sexchang emeasurement swer eperforme da t leastthre eday safte rth estar to fth emenstruation ,m 12wome n2 FM Ran dDT Tmeasurement swer eobtaine di nth e pre-ovularan di nth epost-ovula rphas eo fth ecycle . Study6 :n=1 5 Similart ostud y5 ,i nstud y6 onl yobes ewoma nparticipated .Th epurpos eo fthi sstud ywa st oasses sth eimpac to fbod y fatdistributio nan dweigh tlos so nFM Ran dEXIT .I n1 5o fthes ewome ntw oinitia lFM Ran dDT Tmeasurement swer eobtained . Measurementswer eperforme da tleas tthre eday safte rth estar to fth emenstruation ,but n oattemp twa smad et omeasur e thewome ni na specifi cphas eo fth ecycle .Par to fthi s studyha sbee npublishe d(63) .

Impact of control of the antecedent diet on the within-person variability in RMR, DIT, RQ's and fuel utilization rates

Nine subjects, 4 men and 5 women, ie, study 3 in men (Table 1) and study 2 in women (Table 2), participated in a two-weeks controlled dietary trial. Subjects received their total daily food intake, adjusted to individual energy needs, from the laboratory. They were instructed to eat all the food we providedt othe man dno tt oea tothe rfoo ditem sdurin gth ecours eo fth e study and to keep to the same activity pattern. Energy needs were assessed using the mean of two RMRmeasurements , performed on the same day, ie, in the morning and in the afternoon. This value was extrapolated to 24 hours. Energy needs were calculated assuming that the RMR contributed, respectively, 70 -72- percentt otota ldail yenerg youtpu ti nsedentar y subjectswit ho naverag e1- 2 hours/week sporting activitiesan d6 7percen t insubject shavin ga naverag eo f 3-5hours/wee k sporting activities.Eac hda yon esupplementar y consumptiono f an alcoholicbeverag eo ro forang e juicewa spermitted . Food itemswer e provided ina 4-day srotatin gmen uwit hmino r differencesi nenerg y content (coefficiento fvariatio n (CV)< 5 percent) )an dnutrien t composition (CV-protein,fat< 1 0percent ,CV-carbohydrate s <1 5percent )betwee nth efou r menus.Energ y intakean dnutrien t intakewer edetermine d fromchemica l analysiso fthre eduplicat e portionso fa tota l 4-daysmen ufo ra hypothetica l individualwit ha nestimate d energy intakeo f8-8. 5MJ/d ,8.5- 9MJ/ do r9-9. 5 MJ/d.Protei n (64),fa t(65 )an dcarbohydrate s (66) weredetermine d after assessingmoistur ean das hconten t (67).Mea n energy intakewa s9. 3+ 0. 5MJ/ d (range:7. 3- 12. 4MJ/d) ,wit ho naverag e1 5percen to fth eenerg y derived fromprotein ,3 5percen t fromfa tan d5 0percen t fromcarbohydrates .

Constancyo fth eRM R

Iti ssometime sadvocate d tous ea non-calori c sham feeding treatmenti n studieso nDI Ta sa contro lo nth econstanc yo fth ebaseline ,ie ,th eRM R (68).Th econstanc yo fth ebaselin e isdisputed , sinceth eRM Rwoul d show

pre-and postprandial metabolic rote (kj/min) 70-

___RMR|10V.

constant RMR

RMR |»V.

12 3 4 postprandial time (tpin h)

Figure2 .Postprandia l energyexpenditur e (PEE)wit ha nextrapolate d changing or constant restingmetaboli c rate (RMR).

considerable diurnalvariatio n (68).Variou s investigators reportth eus eo f

-73- suchnon-calori csha mfeedin gtest s (34-36,38).Others ,however ,d ono trepor t theus eo fsuc ha contro ltreatment ,assumin gn osignifican tdiurna lvariatio n inRM R (15,17,20).Whe nth eRM Rshow sa nincreasin go rdecreasin gtren ddurin g theday ,thi sma yseriousl yaffec tth ecalculatio no fth eDI Tresponse .Thi s isillustrate di nFigur e2 . Witha constan tRM Rth etota lthermi ceffec twoul dbe : 240 (durationo fpostprandia ltim e (Tp))*[ PE E- RM R ],eg ,240*[4.6 5- 4.10 ] =15 6kJ ,assumin ga naverag epostprandia lenerg yexpenditur e (PEE)o f4.6 5 kJ/minan da nRM Ro f4.1 0kJ/min . Whenth eRM Rwoul dincreas e1 0percent ,DI Twoul db ereduce db y3 8percen tt o 96KJ .O nth eothe rhan dwhe nth eRM Rwoul ddecreas e1 0percent ,DI Twoul d increase3 8percen tt o21 6kJ .Thes ear esubstantia leffect san di ti s importantt ochec kth econstanc yo fth ebaseline ,ie ,th esuitabilit yo f extrapolatingth eRM Rove rth epostprandia lperio dfo rcalculatin gth ethermi c response. Weassesse dth econstanc yo fth eRM Ri nthre edifferen tways .I na firs tstud y inme n (Table1 ,stud y 4), thedifferenc ewa sdetermine dbetwee nmornin gan d afternoonRM Ran dDI Tmeasurements ,assesse do ndifferen tdays .W ehav e reportedpreviousl yo nth eresult so fthi sstud y (62).I na secon dstudy ,w e measuredi n2 0subjects ,1 1me nan d9 women ,th edifferenc ebetwee ntw o1-hou r RMRmeasurements ,performe do nth esam eday .Th efirs tRM Rwa smeasure d inth e morningbetwee n7.3 0an d9.0 0hour safte ra novernigh tfas tan dth esecon don e wasperforme d inth eafternoo nbetwee n13.3 0an d15.0 0hour swit hth esubject s ina 4. 5hour spostabsorptiv estate .Fo rthi sstudy ,subject sreceive da smal l (2MJ )standardize dbreakfast .Unti lth esecon dRM Rmeasuremen tsubject swer e notallowe dt oengag ei nheav yprolonge dexercise ,sleepin go rt odrin kcoffe e ortea .Minera lwate ran dsugar-fre enon-alcoholi cdrink swer eallowe da d libitum.I na thir dstudy ,w emeasure dRM Ri n1 2subjects ,al lmen ,fo ra periodo f2. 5hour sdurin ga morning .

Impacto fenerg yconten to fa tes tmea lo nDI Tan dduratio no fmeasuremen to n calculationo fDIT .

In3 4wome nDI Twa smeasure dfo ro naverag e3. 9hour s(3- 4 hours)t oa mea l ofo naverag e188 4k J (varyingo naverag ebetwee n181 0an d200 0kJ) . In2 0 women,w emeasure dDI Tfo ro naverag e3. 1hour s( 3- 3. 5hours )t oa mea lo f onaverag e132 1k J (varyingo naverag ebetwee n131 5an d134 0kJ) . In1 2me n DITwa smeasure d repeatedlyfo ra perio do f4 hour st oa mea lwit ha nenerg y

-74- contento fo naverag e255 0kJ .I n2 1me nDI Twa sassesse dfo ro naverag e3. 9 hours(3- 4 hours)t oa mea lo fo naverag e195 2k J (varyingo naverag e between190 0an d200 0kJ )an di n4 me nth ethermi ceffec to ffoo dwa sassesse d for3. 5hour sexactly ,t oa mea lo fo naverag e134 0kJ .Th eaverag ehourl y increasei nmetaboli crat eafte ringestio no fthes edifferen ttes tmeal swa s comparedamon gth evariou sgroups ,subdivide daccordin gt osex .I naddition , thedistributio n(i npercen to ftota lresponse )o fth eentir eDI Trespons e overth efirst ,second ,third ,and ,i fpossible ,ove rth efourt hhou ro fth e postprandialperio dwa scalculate dan dcompare damon gth evariou sgroups .Th e completenesso fth eDI Trespons ewa sevaluate db ycalculatin gth edifferenc e (expressed ina percentag eo fth eRMR )betwee nth eaverag erat eo fenerg y expenditureove rth elas thou ro fth epostprandia lperio dan dth eRMR .

Gasexchang emeasurement s

Allga sexchang emeasurement swer eperforme dwit ha ventilate dhoo dsystem ,a s describedi ndetai lbefore .

Restingmetaboli crat e

Restingmetaboli crat e(RMR )wa sgenerall ymeasure di nth emornin gbetwee n 7.30an d9.0 0hour safte ra novernigh tfast .I nsom estudies ,ie ,i nstudie s 1,3 an d4 i nme nan di nstud y2 i nwomen ,RM Rwa sassesse di nth eafternoo n between12.3 0an d14.0 0hour so rbetwee n13.3 0an d15.0 0hours ,a tleas t4. 5 hoursafte ra smal l (<2 MJ )standardize dbreakfast .Subject swer ealway s instructedno tt operfor mheav yphysica lactivit yth eevenin gbefor eRM R measurements.Fo reac hga sexchang emeasuremen tsubject swer ebrough tb yca r toth elaboratory .Afte rvoidin gthe yla yi na supin e (semi-recumbent) position,bu tawak ean dmotionles sunde rth eventilate dhoo do na hospita lbe d for3 0minute sbefor eth eactua lstar to fth eRM Rmeasurement .RM Rwa sthe n continuouslymeasure dfo ron ehour .Metaboli crate ,oxyge nconsumption , carbondioxideproductio nan drespirator yquotien treading swer eintegrate db y thecompute rove rperiod so ftw oo rfiv eminutes .Thes ereading swer eaverage d overa perio do f1 hou rt oobtai nth eRMR .Occasionall ysubject shyper -o r hypoventilated forsom eperio ddurin gth eRM Rmeasurements .Thes ereading s wereno tinclude di nth ecalculatio no fth eRMR .Metaboli crat ewa sals o correctedfo rth eimpac to fspontaneou sphysica lactivity ,a sassesse db yloa d cells,o noxyge nconsumptio nan dcarbondioxid eproductio nrates .I naddition ,

-75- valueswer e adjustedfo ran ydrif ti nth espa n readingo fth eoxyge nanalyzer , forexample ,du et ochange si nbarometri c pressuredurin gth ecours eo fa on e hour measurement session.Measurement swer e performed ina nois ean d temperature controlled room.Durin gth eRM Rmeasurement s subjectswer e kept awakeb yshowin g themmotio npictures .

Diet-induced thermogenesis

Diet-induced thermogenesis (DIT)wa sassesse d aftera ninitia lRM R measurement,whic hwa suse da sa baselin e inth ecalculatio no fth eDI T response.Th emeal suse dt oevok ea thermi c responsewere ,i ngeneral ,liqui d mixedmeals ,containin gyoghurt ,sucrose ,orang e juice,a protei n supplement andvegetabl e oil.Meal svarie d inenerg y contentamon gth evariou s studies from130 0t o260 0kJ .Nutrien t compositionwa sver y similar amongth evariou s meals. Table 3show saverag eenerg y contentan dnutrien t compositiono fth e various testmeals .I non estudy ,ie ,stud y6 i nwomen ,a testmea l composedo f normal solid foods,ie ,bread ,margarine ,appl e syrup,chees ean dorang e juice,wa sused .N osignifican tdifferenc e inthermi c effectbetwee n thismea l anda liqui d testmea lo fsimila r energy contentwa sobserved .

Table 3.Energ yconten tan dnutrien t compositiono fmeal si nreproducibilit y studieso fDIT .

MEN WOMEN Energy Protein Fat CBD Energy Protein Fat an kJ 9 % 9 % 9 % kJ 9 % 9 % g %

Stuty1 2550 19.8 13.0 18.6 27.0 90.8 60.0 2000 14.4 12.0 17.6 33.1 67.5 54.9 2 2000 14.4 12.0 17.6 33.1 67.5 54.9 1340 10.4 13.0 12.0 33.6 43.9 53.4 3 1340 10.4 13.0 12.0 33.6 43.9 53.4 1950 13.0 11.1 18.5 35.7 63.6 53.2 4 1900 11.6 10.2 16.7 33.1 66.1 56.7 1920 12.8 11.0 18.1 35.4 62.7 53.4 5 2000 14.4 12.0 18.6 35.0 65.0 53.0 1810 12.2 11.3 16.4 34.1 60.7 54.6 6 - 1315 9.8 12.4 11.6 33.2 43.9 54.4 Mean+ 1960 14.1 12.0 16.7 32.4 66.7 55.6 1723 12.1 11.8 15.7 34.2 57.1 54.0 son 194 1.6 0.5 1.2 1.4 7.4 1.3 1218 0.7 0.3 1.3 0.5 4.3 0.3

Liquid testmeal shav evariou s advantagesove r solidmeals .Liqui d testmeal s canb eprepare d inbulk ,whic hwil lminimiz edifference s in.compositio n among testmeals .Secondly , liquidmeal sca nb eingeste dwit hminima l disturbanceo f

-76- theenerg yexchang emeasurements ,fo rexample ,b ypassin g a strawthroug h an opening inth eventilate dhoo dan daskin gth e subjectst odrin k themeal . To ingesta solidmeal , it isnecessar y to remove theventilate d hood fromth e subjectshead ,whic hwil l causea relativelygreate rdisturbanc e of theenerg y exchangemeasurements .Thirdly ,th e compositiono f liquidmeal sca nb e manipulatedmor eeasil y thanth e compositiono fa solidmeal . Subjects ingested the liquidmeal salway swithi n 5minutes ,th e solidmea l was eaten ina perio d of 10-15minutes .Afte r ingestiono f the testmea l the rate ofenerg yexpenditur ewa s continuously assessed foreithe r 180,21 0o r 240 minutes. Once everyhou r the spano f theoxyge nanalyze rwa s checked against fresh dried filtered atmospheric airdurin g fiveminutes .Durin g thisperio d subjectswer e allowed tomov ebriefly .Subject swer e asked to remainawak e and motionlessdurin g the remaining timeo f themeasurements .Durin g the measurements subjectswatche dmotio npictures .Occasionally , subjectsmove d a leg,ar mo r another parto fthei rbody .Whe nnecessary ,metaboli c rate readingswer e adjusted for thistyp eo fdisturbance . Oxygen consumption,carbondioxid e production, respiratoryquotien t and metabolic ratewer e integrated overperiod so f fiveminutes .Thes e periods were averaged toobtai naverag ehourl ypostprandia l energyexpenditur e rates (PEE). The totalDI Twa sdefine da sth e integrated (over the total postprandial period (Tp))differenc e between thepreprandia l (RMR)baselin e energyexpenditur e ratean d theaverag e postprandialenerg yexpenditur e rate, ie,DIT =T p * (PEE-RMR).Occasionall y energy expenditure at theen d of the postprandial periodwa s lowerwhe ncompare d toth eRMR . Forthes e subjectsRM R was redefined asth emea no f theactua lRM Rmeasuremen t and the lower postprandial rateo fenerg yexpenditure .Afte r theassessmen to fth e thermic effect of food, subjectsvoide d andurin ewa s collected fordeterminin g the totalamount o f totalurinar ynitrogen .Tota lnitroge nexcretio nwa sassesse d by the Kjeldahlmethod .

The energy contentan dmacronutrien tcompositio no f the testmeal swer e chemically measured ina random 5percen t sampleo feac hbatc ho f testmeals . Protein (64),fa t (65)an d carbohydratesan d lacticaci d (66)wer e determined after assessingmoistur e andas h content(66) . Generally, subjectswer e familiarizedwit h theequipmen t forassessin g gas exchange bymeasurin g restingenerg yexpenditur e ona seperateda y for between 30an d 45minute sbefor e the starto fth eactua lexperiment .

-77- Body composition

Bodyweigh twa smeasure d toth eneares t 0.05 kg (Berkel ED-60T,Rotterdam , the Netherlands) insubject swearin gunderwea r or swimming clothesonly ,afte r an overnight fast,o ra t least 4.5 hoursafte r the lastmeal ,an d aftervoiding . Theweigh t ofth e fatan d the fat-freebod ymas swa sdetermine d by hydrodensitometry. Subjectswer eweighe d underwaterwit h simultanuous assessment of the residual lungvolum eb ya heliu mdilutio n technique.Tota l bodydensit ywa s calculated and body fatpercentage ,fat-fre ebod ymas san d body fatmas swer e estimatedusin gSiri' sformula .Fat-fre ebod ymas swa suse d asa nestimat e of themetabolicall y activemas so f thebod y(69) .

Statistics

Variability of themetho d andwithin-perso n variability

Reproducibility trialswer e analyzed using the repeatedmeasure smode l as described byWine r (70).Tim e ofmeasuremen twa s treated asth ewithin-subjec t factor.Th ewithin-subjec t factorwa s treated inth eanalysi so fvarianc e asa fixed effect factor,a factor for cases (subjects)wa suse d asth eonl y random effect factor.Th e subject factorwa scrosse dwit h thewithi n factor,ie ,eac h casewa sobserve d atal l combinationso f thewithi n factor.Th emea n squareo f theerro r termo f thewithin-subjec t factorwa s takena sth ebes tunbiase d estimator of thewithin-subjec t variance of thevariabl eunde r study. Instud y 5 inme na significant timeo fmeasurement ,ie ,sequence ,effec to nRM Rwa s observed. The estimator ofth ewithin-subjec t variance inRM Rwa s corrected, according toWine r (70)fo r thiseffect .Assumption s required for the validity of F tests in the fixed-effects analysiso fvarianc emode l are that the observations aremutuall y independent,normall ydistributed , andhav e equal variances. Inth e repeated-measures model observations obtained inth e same subjects are generally correlated.A sa consequence Ftes twer emad e ina wa y thatallow s some relaxationo f theassumption s ofcomplet e independency,a s described byWine r (70)an d inDixo n(71) . For each subjectw e alsocalculate d thecoefficien t ofvariatio n (CV)o f repeated RMR andDI Tmeasurements . Reproducibility ofbod ycompositio nmeasurement sha sbee n reported before (72).

-78- Between-personvariabilit y

For eachperso nRMR ,DIT ,R Qan d fuelutilizatio n rateswer eaverage d over the repeatedmeasures .Analyse so f thebetween-perso nvarianc e ofRMR ,DIT ,R Qan d fuelutilizatio n rateswer ebase d onthes eaverag evalues .Sinc epar to f the total between-personvarianc e inRMR ,DIT ,R Qan d fuelutilizatio n ratescoul d be related todifference samon g individuals inbod yweight ,bod y composition, age (RMR)o rmacronutrien tan d energy intake (RQ,DIT ,fue lutilizatio n rates),multipl e regressionanalyse swer euse d toestimat e thevarianc e among individuals inthes evariable saccounte d fordifference s inbod yweight ,bod y composition,ag e ormacronutrien tan denerg y intake.Th evarianc e not accounted forb y the lineareffect so f thevariou spredicto r variables (covariates)equal sth e residualmea n square of theerro r termo f the analysis ofvarianc e of the regressionmodels .Thi s residualvarianc e is referred toa s the 'adjusted' (forth e lineareffect so f covariates)between-perso nvariance . RMR, fastingR Qan d fuelutiliatio n rateswer eadjuste d for fat-freemass , fat mass and age.DI Tan d postprandial RQan d fuelutilizatio n rateswer e adjusted for the samevariable s including theenerg yo rmacronutrien t intakewit h the testmeal .Norma l probabilityplot so f the residualswer euse d toevaluat e differences from the normalityassumption s of thevariou s regressionmodels . Ifn odeviatio n fromnormalit yoccurre d theadjuste d between-person variance wasassume d tob edistribute d asa Chi-squar edistribution .

- Between-groupdifference s invariable so fenerg ymetabolis mwer e tested using normal analysiso fvarianc eprocedures .I naddition ,analysi so f variance statisticsno tassumin gequalit yo fgrou pvariance s ineac hgroup , ie, theWelc han dBrown-Forsyth e statistic (70,71),wer euse d toevaluat e the significance ofgrou pdifferences .

- Differences invariable sbetwee n twotreatments ,ie ,pre-ovula r versus post-ovular andmornin gversu safternoo nenerg yexpenditur e rates,wer e evaluatedusin g thepaire d (within-person)compariso nt-tes twit h a two-sided rejection regionan d a confidence levelo f 95percent .

- Correlation coefficientswer e computed asPearson' s product-moment correlation coefficients.Adjustin g forlinea reffect so fa variabl e ona correlationwa sperforme d by thepartia l correlationprocedure .

-79- -Al ldescriptiv estatistic sar ecompute da smea n+ sem .

RESULTS

Accuracyo fth emetho d

Therespirator yquotien to f6 0ethano lcombustion stes twa sfoun dt ob e0.66 6 + 0.001 (range:0.63 5- 0.695 )wit ha coefficien to fvariatio no f1. 5percent .

In2 0whol esyste mcheck sth eobserve dVD ?an dVCO ,wer e34 0+ 1 01/ dan d22 7 +8 1/d , respectively,wit ha R Qo f0.668 .Thi swoul dresul ti na calculate d energyexpenditur eo f692 9+ 19 2kJ/d ,wit ha coefficien to fvariatio no fth e methodo f2. 8percent .Oxyge nwa sunderestimate dwit h1. 5percen tan d carbondioxidewit h1. 3percent .

Variabilityo fFHR ,DIT ,R Qan dfue lutilizatio nrate si nperson s

Table4 show sth ewithin-perso nvariabilit yo fRM Ran dDI Ti nmen .Th e 2 within-personvariance swere ,respectively ,i nRM R0.09 5+ 0.01 2 (kj/min), i n

DIT217 2+ 52 6(kJ) 2o r5.7 8± 1.2 8 (percent)2o fth eenerg yintake .Th e

Table4 .Result so freproducibilit ystudie so fRM Ran dDI Ti nmen .

StuaV 1 Stucty 2 Stuay 3 Stuay 4 Stuay 5 Stuay 6 Total < Range > (n = 12) (n = 5) • (n = 4) (n = 10) (n =6) (n = 12) (n = 49)

FMR (kj/hrin) 5.34+0.27 5.46+0.25 5.24+0.29 4.76+0.14 4.92+0.16 4.69+0.10 5.02+0.09 4.16-7.29 Dn (kJ) 185±20 188+11 123+11 134+13 178±15 - 164+9 76-329 Drr-% ME (%) 7.17±0.76 9.32+0.53 9.08+0.83 7.13+0.71 8.91+0.76 - 7.94+0.37 4.02-12.76

Within-person variance: RMR (kj/iirin) 0.112 0.117 0.108 0.115 0.044 0.072 0.095+0.012 0.044-O.115 nrr (kj| 3146 1311 1166 3722 1513 - 2172+526 1166-3722 nrr-% ME (%) 4.90 3.28 6.58 10.38 3.77 - 5.78±1.28 3.28-10.38

Within-person CV's: RMR 6.0+1.5 5.4+2.1 5.9+1.2 6.9+0.9 5.2+0.2 5.4+0.5 6.O1O.6 0.1-16.5 DTT 27.7+6.7 14.1+4.5 22.6+3.2 39.4+5.4 22.0+5.4 - 27.5±3.0 0.9-68.7 nrr-% ME 27.9+6.7 14.2+4.7 22.8+3.1 39.4+5.4 21.7+3.3 - 27.6±3.0 1.0-69.1

Vtelue* s are given as WBOD ± sea SbicV 6 not included in calcul^ticn Ml = resting •etabolic rate; EOT = diet-indiced therBogeneeis; %-*K= percentage of energy contait of test Maal; CV = coefficient of variation

-80- calculatedcoefficient so fwithin-perso nvariatio nwere ,respectively ,o n average6. 0+ 0. 6percen tfo rth eRM Ran d27. 5+ 3. 0percen tfo rth eDIT .Th e within-subjectvariabilit yi nRM Rwas , expresseda sth eroo tmea nsquar eo f 2 thepoole dwithi nsubjec tvariance s(S D ),0.30 8kJ/min .I fi ti sassume d thatth eintra-individua lvariabilit yi nRM Ro fa subjec ti snormall y 2 distributedan duncorrelate dwit hth erando mmeasuremen terro r (SD ), then 2 2 thecontributio no fth esubject' svarianc e (SD )t oth etota lobserve dS D

isgive nby :

2 2 SDs =V(SDw -SDm ).

Thisresult sfo rme ni na nS D of0.27 7kJ/mi ncorrespondin gt oa method-fre e coefficiento fwithin-subjec tvariatio no f5. 5percent . Thecorrelatio nbetwee nth ecalculate dcoefficient so fvariatio nfo rth eRM R andfo rth eDI Twa sno tsignifican t( r= 0.01) .Th eaverag eintercorrelatj.on s inme namon gth erepeate dRM Ran dDI Tmeasurement swer e0.7 2an d0.22 , respectively. Table5 show sth ewithin-perso nvariabilit yo fRM Ran dDI Ti nwomen .Th e 2 within-personvariance swere ,respectively ,i nRM R0.07 0± 0.00 9(kj/min ), ? 2 andi nDI T124 7+ 14 3(kJ ) or4.6 6+ 0.7 0(percent ) ofenerg yintake .Th e calculatedcoefficient sfo rth ewithin-perso nvariatio nwer e6. 0+ 0. 4fo rth e RMRan d28. 5+ 2. 6percen tfo rth eDIT .Th eS D ofth eRM Ri nwome nwa s0.26 5 kJ/min,th emethod-fre eS D was0.24 2kJ/mi ncorrespondin gt oa method-fre e coefficiento fwithin-subjec tvariatio no f6. 1percent . Similart omen ,i nwome nth ecalculate dcoefficient so fvariatio nfo rth eRM R showeda non-significan tcorrelatio nwit hthos eo fth eDI T(r«0.06) .Th e averageintercorrelation si nwome namon gth ereplicat emeasurement so fth eRM R andDI Twere ,respectively ,0.8 0an d0.41 . Exceptfo rstud y5 i nmen ,non eo fth estudie sshowe da significan ttim eo f measurementeffec to nRM Ran dDIT . Table6 an d7 sho wwithin-perso nvariance si nfastin gan dpostprandia lRQ' s andfue lutilizatio nrate si nme nan di nwomen ,respectively .Bot hfastin gan d postprandialRQ' san dnonprotei nRQ' sshowe drelativel ylo wwithin-perso n variance.Averag eCV' sfo rth eintra-individua lvariatio ni nthes evariable s werebetwee n4 an d6 percent .Intra-individua lvariatio ni nfue lutilizatio n rateswa sgenerall yhighe rwhe ncompare dt ovariatio ni nRQ's .Th elowes tCV' s wereobserve dfo rprotei noxidatio nrates ,ie ,o naverag e2 0percen ti nme n -81- Table 5. Results of reproducibility studies of RMRan d DIT in women.

Study1 Study2 Stuoy3 Study4 Stuoy5 Stuoy6 Total (n=6) (n=5 ) (n=5 ) In»6 ) (n= 17 ) (n= 15) (n= 54)

EMR (kj/tnin) 3.88+0.io 3.98+0.14 3.91+0.21 3.59+0.18 4.18+0.16 3.97+0.13 3.98+0.07 3.10-5.70 DIT (kJ) 127+17 66+10 114+17 128+14 128+7 89+8 110±5 25-205 DIT-%M E (%) 6.30+0.84 4.89+0.77 5.78+0.86 6.60±0.7 7.03+0.3 6.69+0.57 6.48+0.26 1.84-11.23

WLthin-person variance: FJ1R (kJ/tnin) 0.088 0.081 0.058 0.051 0.096 0.043 0.070+0.009 0.043-0.096 DIT (kj>2 868 1008 1627 1089 1736 1155 1247+143 868-1736 2 Drr-%H E (%) 2.72 5.80 4.24 2.68 5.82 6.69 4.66+0.70 2.72-6.69

Within-personCV's :

SMR 6.8+1.5 6.2+1.6 6.2+0.8 5.7+0.8 7.0+0.4 4.8+0.6 6.0+0.4 0.5-13.2 DIT 24.0+13.8 41.2+9.1 32.5+6.5 22.7+3.1 28.9+2.6 35.2+6.4 28.5+2.6 0.1-72.0 DTT-%M E 24.1+13.8 41.4+9.1 32.7+6.5 21.5±3.1 29.3+2.7 35.6+6.4 28.6+2.6 0.1-72.2

Values are given as aeon ± seal RR = resting metabolic rate; EOT = (net-induced themogenasis; %-M3= percentage of energy content of test MealOT; = coefficient of variation

Table 6. Results of reproducibility studies of fuel utilization rates in men.

Variables studied Stuoy1 Study2 Stuoy3 Study4 Study5 Total (n= 12 ) (n= 5 ) (n= 4 ) (n= 10) (n= 6 ) (n= 37)

- Pastabsorptive:

1. BQ 0.81+0.01 0.83+0.01 0.80+0.01 0.84+0.01 0.85+0.01 0.83+0.01 0.75-0.87 2. npBQ 0.81+0.01 0.84+0.02 0.80+0.01 0.85+0.01 0.86+0.01 0.84+0.01 0.73-0.90 3. Fatoxidatio n (nq/nàn) 62+6 61+9 68+5 48±5 44+5 56+3 26-92 4. CHO oxidation (nq/Wn) 85+11 111±9 82+6 115±8 120+4 102+5 17-139 5. Protein oxidation{nq/todn) 78+5 57±4 58±12 56±3 57±3 64±3 40-112

- Postprandial:

6. FQ 0.88+0.01 0.88+0.01 0.83+0.01 0.88+0.01 0.89+0.01 0.88+0.01 0.79-0.94 7. npBQ 0.90+0.02 0.90+0.02 0.83+0.01 0.90+0.02 0.92+0.01 0.90+0.01 0.79-0.99 8.Fa toxidatio n (mg/hon) 39+6 44+9 64±5 37±8 30+3 41+3 4-96 9. CHD oxidation (mj/hrin) 198+13 215±9 134+10 178+11 215±11 190+7 112-268

Within-person variance: 1. RQ.10 1.63 2.16 1.07 1.37 0.89 1.42+0.22 0.89-1.63 2. npRQ.lo" 2.84 3.81 1.78 1.51 1.61 2.31+0.44 1.51-3.81 3. Fat oxidation (mj/inin) 235 403 263 360 200 292+131 200-403 4. CBD oxidation (nq/iivin) 2068 2592 989 1370 791 1562+337 791-2592 5. Protein oxid (mg/inin) 137 195 322 52 83 158+48 52-322 6. RQ.10"3 3.70 1.33 0.70 1.00 0.94 1.53+0.55 0.70-3.70 7. npEQ.10- 6.06 1.86 1.66 1.82 1.37 2.55+0.88 1.37-6.06 8. Fat oxidation (nig/tain) 1127 174 214 241 204 392+184 174-1127 9. OD oxioation (ing/tairi) 7028 2961 1095 1252 1406 2749+1121 1095-7028

Values are given as nan ± sat BQ= respiratory guotioit; np =ncn protein; CH>= carixhydrate (glycogen or gluoose); oxid = oxidation

-82- and1 9percen t inwomen .Fastin g fatoxidatio n rates showedCV' s of, respectively, 31percen ti nme nan d3 0percen t inwomen . Fasting carbohydrate,ie ,glycogen ,oxidatio n rates showed higher CV's, ie, of4 0percen t inme nan d3 0percen t inwomen .Within-perso nCV' sfo r postprandial fatoxidatio nwer e4 9percen ti nme nan d5 0percen ti n women,wherea sth eCV' sfo rpostprandia l glucoseoxidatio nwer e2 7 and2 0percent , respectively.Averag e fastingRQ' swer e0.8 3+ 0.0 1 witha rang eo f0.7 5t o0.90 .Postprandia lRQ' swer eo naverag e0.8 8 +0.01 witha rang eo f0.7 9- 0.94 . Innon eo fth esubject sw e observed average nonproteinpostprandia lRQ' sexceedin g1.00 .

Table7 .Result so freproducibilit y studieso ffue lutilizatio n ratesi nwomen .

Variablesstudie d Study1 Study2 Study3 Study4 Study5 Study6 Total (n=6 ) (n= 5 ) (n=5 | (n= 6 ) (n= 17 ) (n= 15 ) (n= 54)

- Postabsorptive:

1. »0 0.83+0.01 0.82+0.01 0.86+0.01 0.87+0.01 0.83+0.01 0.82+0.01 0.83+0.00 0.75-0.90 2. npHC 0.84+0.01 0.82+0.01 0.88+0.02 0.89+0.01 0.83+0.0 0.83+0.01 0.84+0.01 0.73-0.93 3.Fat oxi d (nvj/hdn) 43±3 49+3 30+6 25+3 44+3 40+4 40+2 13-76 4.O O oxid (mg/hin) 83+7 73+3 103+5 103+4 81+4 69+7 81+3 13-123 5.Protei noxi d (mg/hrin) 38+4 42+2 47+2 42+4 54±2 62+4 51+2 21-88

- Postprandial: 6. HB 0.89+0.01 0.87+0.01 0.88+0.01 0.90+0.01 0.88+0.01 0.88+0.01 0.88+0.00 0.80-0.93 7. npBQ 0.90+0.01 0.89+0.01 0.91+0.02 0.92+0.01 0.90+0.01 0.90+0.01 0.90+0.01 0.80-0.99 8. rat oxid (nq/tain) 30±2 32±2 27+7 21+2 30+2 27+4 28+2 2-52 9. OD oxid (ng/tam) 162+6 140+10 153+7 159±8 160+9 141+9 152+4 71-220

Within-perso n variance: 1. RQ.10 0.97 0.48 1.33 1.11 1.35 0.38 0.94+0.17 0.38-1.33 2. npRQ.10"3 1.50 0.76 1.33 2.21 2.51 0.71 1.50+0.30 0.71-2.51 3. Fatoxi d (nq.min) 137 82 193 150 235 57 142+27 57-235 4. CK>oxi d (mo/tain) 640 420 808 329 959 381 590+104 329-808 5. Proteinoxid(mg/hin ) 15 102 85 58 118 174 92+22 15-174 6. BQ.10 0.99 1.10 1.10 1.71 1.53 1.23 1.28+0.12 0.99-1.71 7. npPQ.10"3 1.52 1.56 1.07 3.08 2.68 2.56 2.08+0.33 1.07-3.08 8. Fatoxi d (irç/hdn) 174 181 180 255 260 155 201+18 155-260 9. OO oxid (mcfftdn) 585 1296 998 406 1184 1126 933+145 406-1296

Values aie given as Been + sea BQ= respiratory quotient; np = nonprotein; OD = carbohydrate (glycDgen/glucose); oxid = oxitfetion

-83- Impact ofcontro lo fth eanteceden tdie to nwithin-perso nvariabilit yo fPMR , DIT, RQ,an dfue lutilizatio n rates

The results,presente d forme ni nTable s4 an d6 (study3 )an dfo rwome ni n Tables4 an d7 (study 2), indicaten oclea r impacto fcontro lo fth e antecedentdie to nth ewithin-perso nvariabilit yo fRMR ,DIT ,R Qan dfue l utilization rates,whe n comparingth evariou sestimator so fwithin-perso n variances invariable so fenerg ymetabolis mamon gth edifferen tstudies .

Variabilityo fRMR ,DIT ,R Qan dfue lutilizatio n ratesbetwee nperson s

Figure 3an dTabl e8 sho wvariabilit yo fth emethod ,within-perso n variability and between-personvariabilit y inRM Ran dDIT .

resting metabolic rate (kJ/min) feg WOMEN f=JMEN

0.20-i nil -E7HG0 WITHIN A3J.3ETW. BETWEEI N cierXi-.S'jcsd fr.armoasnesis loglkJ] dier-induced fhermogenesis (%)' fy/A WOMEN f \ WOMEN MO- S MEN i U)0- 2.C0- • 2J00

i 0 1 METHOO WITHIN ADJ.BETW BETWEEN '£\MÎ7H00 WITHIN A0J.9ETW. 5ETWEEN 1

Figure 3.Within -an dbetween-perso nvariance si nme nan dwome no frestin g metabolic ratean ddiet-induce d thennogenesis.

Thebetween-perso nvariabilit yi nRM Rwa ssignificantl yhigher ,eve nafte r adjusting forcovariates ,compare dt oth ewithin-perso nvariability .Th erati o of (adjusted)between-perso n towithin-perso nvarianc ewa s2. 2i nme nan d2. 0

-84- inwomen .I ncontrast ,n osignifican tdifferenc e between (adjusted) inter-individualan dintra-individua lvariatio ni nDI Twa sfoun di nmen ,wit h ratioso fbetwee nan dwithin-perso nvariabilit y around1 .I nwome nth e adjusted beteen-personvarianc ei nDI Twa seve n slightly lower thanth e within-personvariatio ni nDIT .Th eratio' so f(adjusted )between-perso nan d within-personvariatio ni nRQ' san dfue lutilizatio n rateswer eal lwithi nth e rangeo f0. 5t o1.7 .Excep tfo rpostprandia lRQ' san dfa toxidatio n rates,n o significantdifference sbetwee nth e(adjusted )inter-individua lan d intra-individual variationswer e found.

Table8 .Between-perso nvarianc eo fRMR ,DIT ,R Qan dfue lutilizatio n ratesi n menan dwomen .

Men Women Between Mj. between* Between A3j. Between*

ra« {KJ/ndn)2 0.415 0.212 0.278 0.142 DTP (kJ)2 4643 2626 1385 892 2 nrr-% >E (%) 5.16 5.31 3.65 3.27

Postabsorptive na . icf3 1.02 1.80 0.961 0.80 nBQ. l

Postprandial : BQ . l

* = variance adjusted for fat-free i nss, fat nass. age (pœtabsoiptive ) ni energy or aacranutrient intake

BMR= restingMetaboli czate ;DI T= diet-induce dtheEaogenesis ;% f E= percentag eo fenerg yconten to ftes taBal ;B Q= respiratoryquotient ;n p= nonprotein ;(3 D= carbohydrat e (glycogeno rglucose )

Constancyo fth eRM R

The resultso fth ethre e studieso nth econstanc yo fth eRM Rar epresente di n Table9 .Non eo fth ethre e studies showeda significan tdiurna lvariatio ni n theRMR .I nth efirs t studyth eaverag edifferenc e betweenmornin gRM Ran d afternoonRM Rwa s0.1 4+ 0.0 9kJ/min ,ie ,2. 9+ 1. 9percen to fmornin gRMR .

-85- Inth esecon dstud yth edifferenc ebetwee nmornin gan dafternoo nRM Rwa s-0.0 3 +0.0 6kJ/min ,ie ,-0. 7+ 1. 4percen to fmornin gRMR .I nth ethir dstud yn o significantchang ei nRM Rove ra 2. 5hour smeasuremen tperio dwa sfound .Th e differencebetwee nth eRM Rassesse dove rth efirs t3 0minute so fthi sperio d andth eRM Rmeasure dove rth elas t3 0minute swa s0.0 9+ 0.17 ,ie ,1. 9+ 3. 6 percento fth einitia lRM Rvalue .

Table9 .Result so fstudie so nth econstanc yo fth eRMR .

Variables Stuty 1 (n = 10) Study 2 (n = 20) Study 3 (n = 12)

Age yrs 22.2±0.5 23.1+0.5 24.8+0.6 Body weight kg 75.5+2.2 65.0+2.1 70.2+1.2 Body fat % 15.1+1.2 19.4+1.5 15.0+1.4 Morning WR kJ/lnin 4.81+0.18 4.58+0.14 - Afternoon WIR kJ/inin 4.67+0.11 4.61±0.14 - Morning W© 0-30 min kJ/min - - 4.67+0.11 30 - 60 kJ/min - - 4.78+0.11 60 - 90 kJ/min - - 4.71+p.ll 90 -120 kJ/min - - 4.68+0.10 120 -150 kJ/min 4.76+0.10

indue* s axe given as aean ± seal W/r ratio UJ9, age in van: 32.5 + 0.9 yrs. weight: 71.4 ± 2.3 kg, body fat content: 14.9 + 1.2 percent; age in i 22.6 + 0.4 yrs, wight: 65.0 + 2.0 kg, body fat content: 23.8 + 1.4 percent EKl = resting Metabolic rate

Impacto fphas eo fth emenstrua lcycl eo nRM Ran dDI T

Table1 0show sth eresult so fth eimpac to fth ephas e (pre-ovularversu s post-ovular)o fth emenstrua lcycl eo nRM Ran dDIT .N osignifican tdifferenc e inRM Ran dDI Tbetwee nth etw ophase so fth ecycl ewa sfound .Th eaverag e numbero freplicate si neac hphas eo fth ecycl ewa s2. 9 forth eRM Ran d2. 0 forth eDIT .Th ewithin-perso ncoefficient so fvariatio ni nRM Ran dDI To f thesewome nwer ecompare dwit hthos eo fth ewome nusin gora lcontraceptives . CV'so fRM Rwere ,respectively ,5. 7+ 0. 7percen tfo rth ewome nwit ha norma l menstrualcycl ean d7. 5+ 0. 7fo rth ewome nusin gora lcontraceptives .CV' s forDI Twere ,respectively ,31. 5+ 3. 4fo rwome nwit hnorma lmenstrua lcycle s and26. 1+ 4. 0 forwome nusin gora lcontraceptives .A significantdifferenc e (p<0.05)i nCV' sfo rRM Rbetwee nbot hgroup swa sfound ;CV' sfo rDI Tdi dno t differsignificantl ybetwee nbot hgroups . -86- Table10 .RM Ran dDI To fwome ni nth epre-ovula ran dpost-ovula rphas eo fth e menstrual cycle.

Subject no Age Weight FFM PMÏpr e FMIpos t DITpr e DITpos t DTTpr e DIT post (yrs) (kg) (kg) (kJ/tnin) (kj/hûiï) (kJ) (U) (%)* (%)

1. 20 74.6 54.5 3.78 4.00 100 62 5.09 3.18 2. 23 59.3 43.0 3.32 3.41 127 123 6.44 6.22 3. 22 62.1 43.9 3.87 3.76 104 111 5.29 5.62 4. 25 68.7 46.5 3.76 3.84 114 81 5.82 4.12 5. 24 65.0 47.9 4.70 4.65 164 126 8.39 6.41 6. 21 64.3 45.8 4.08 4.30 147 155 7.35 7.75 7. 21 61.1 43.5 3.90 4.21 169 152 8.45 7.60 8. 20 65.5 45.2 3.86 3.82 151 127 7.55 6.35 9. 25 51.8 39.1 3.62 3.83 157 171 7.85 8.55 10. 22 67.1 44.3 4.13 3.67 72 95 5.44 7.12 11. 22 50.0 40.3 3.93 3.97 49 45 3.71 3.35 12. 42 81.2 43.9 3.72 4.30 116 100 6.41 5.52 13. 32 101.7 50.4 3.72 3.67 79 158 4.47 8.73 14. 43 74.2 50.1 3.69 3.47 94 112 5.20 6.27 15. 41 74.2 42.6 3.54 3.25 98 112 5.43 6.29 16. 44 65.5 40.3 3.84 3.43 152 198 8.41 10.95 17. 39 72.2 43.1 3.75 3.81 158 94 8.75 5.26 IS. 44 92.7 48.4 5.54 5.87 147 65 8.12 3.60 19. 40 103.4 47.5 5.62 5.54 123 148 6.99 8.18 20. 45 91.2 50.7 4.00 4.13 72 76 3.97 4.36 21. 40 101.3 46.6 3.94 4.03 138 112 7.63 6.21 22. 36 95.9 50.7 3.86 4.04 122 196 6.81 10.91 23. 40 83.6 41.5 3.90 4.15 93 111 5.32 6.37 ffean± sem 31.7±2.0 75.1±3.3 45.6+0.8 4.00+0.12 4.05+0.13 119+7 119+8 6.47+0.32 6.47+0.44

Bqvessed in percentage of energy UUMJMHL of test Beal Pbx cases 1-5 nailer of replicate aeasureaents for each these of cycle are 6 for HH and 3 for DIT; for cases 6-9 2 replicates for Mt and none for DTTan d for cases 10-23 2 replicates for RH and 2 for DIT FM = fat-free BBSS; RK = resting aBtabolic rate; DTT= diet-induced theraogEnesis; Pre = pre-ovular; ftjst^ post-ovular

Impacto fenerg y intakean dduratio no fpostprandia lmeasuremen to nth e calculationo fDI T

Figure4 show spostprandia l energyexpenditur e ratesi nthre e groupso fmen , who ingesteddifferen t amountso fenerg yi nth emeal suse d toevok eth e thermic response.N osignifican tdifferenc e amongth ethre e groupsi nth e postprandial riseo fenerg yexpenditur e duringth efirs ttw ohour so fth e postprandial periodwa sfound .I nth ethir dhou ro fth epostprandia l period, thepostprandia l rateo fenerg y expenditurewa ssignificantl y lower inth e groupwit hth elo wenerg y intake comparedt oth egroup swit henerg y intakeso f

-87- diet induced thermogenesis (kJ/min •. nn= = 2 21 EEii nr ~ 1950 kJ 1.20- T T à n= 12 Ein = 2550 kJ ? Tv • n= 4 Ein = 1340 kJ

0 12 3 4 time after meal ingestion (h)

Figure4 .Diet-induce dthermogenesi s (mean+ sent )i nme nafte ringestio no f mealswit hdifferen tenerg ycontent .

onaverag e195 2k Jan d255 0kJ .I nsubject swit hth elo wenerg yintak e8 6 percento fth etota lobserve dthermi ceffec t(ove r3. 5hours )wa sobserve di n thefirs ttw ohour so fth epostprandia lperiod ,compare dt o7 5percen ti n subjectswit hhighe renerg yintakes .A tth een do fth epostprandia l measurementperio denerg yexpenditur ewa selevate d4 percen tabov eth eRH Ri n subjectso fth egroup swit hhighe renerg yintake san d0 percen ti nsubject s withlo wenerg yintake .I nseve nsubject senerg yexpenditur ea tth een do fth e postprandialperio dwa slowe rcompare dt oRMR ,wit ha minimu mo f9 8percen to f theRMR .Tota lDI Tvalue sincrease dsignificantl yo nth emeal swit hhighe r energycontent .Th ecorrelatio ncoefficien t (r)betwee ntota lDI Tan denerg y intakei nth etestmea lwa s0.3 9 (p=0.05). Nosignifican tcorrelatio nwa sfoun di nme nbetwee nDI Tand ,respectively , bodyweigh t (r= 0.11) ,th efat-fre emas s( r= 0.07) ,fa tmas s( r= 0.16 )o r age (r= -0.02) ,adjuste dfo rlinea reffect so nth ecorrelation so f differencesi nenerg yintak ei nth etes tmeal .N odifferenc ei nDI Tamon gth e threegroup swa sobserve di nDI Texpresse da sa percentag eo fth eenerg y contento fth etes tmeal ,ie ,8.1 6± 0.4 6percen t (1952kJ) ,7.1 7± 0.7 6

-88- percent (2550kJ )an d9.0 8+ 0.8 3percen t (1340kJ) . Figure5 show s increases inpostprandia l energyexpenditur e forfemal e subjects receiving either mealso fo naverag e188 4kJ ,o ro fo naverag e132 1 kJ.

induced thermogenesis (kJ/min

. n= 2 0 Ein»132 0 kJ i n= 3 4 Ein=1880 kJ

0 12 3 4 time after meal ingestion (h)

Figure 5.Diet-induce d thermogenesis (mean+ sem )i nwome nafte r ingestiono f mealswit hdifferen t energycontent .

Therewa sa significan tdifferenc e inth eris eo fpostprandia l energy expenditure duringth esecon dan dthir dhou ro fth epostprandia l period betweenbot h groups.I nsubject s receivingth elo wenerg ymeals ,o naverag e8 3 percento fth etota l thermic responsewa smeasure d inth efirs ttw ohour so f thepostprandia l period.I nsubject s receivingth ehighe r energymeals ,o n average6 6percen to fth etota l responsewa sobserve d inth efirs t12 0 minutes.A tth een do fth epostprandia lmeasuremen tperiod ,energ y expenditure was stillelevate db y7. 2+ 0. 8percen ti nth egrou p receivingth ehighe r energymeal san db y6. 5+ 1. 3percen ti nth egrou p receivingth elowe r energy meals. Infou rwome n lower rateso fenerg yexpenditur ewer e observeda tth e endo fth epostprandia l measurement periodwhe n comparedt oth eRMR ,wit ha minimumo f9 8percen to fth eRMR .Tota lDI Tvalue swer e significantly higher (p<0.05)i nwome nwit hhighe r energy intakesi nth etes tmeals .Th e correlation coefficient betweenDI Tan dth eenerg y contento fth etes tmea l

-89- was0.5 3 (p<0.001)i nwomen . Nosignifican tcorrelation swer efoun di nthes ewome nbetwee nDI Tan dth esiz e ofth efat-fre emas s( r= 0.02) .DI Twa ssignificantl ycorrelate dwit hbod y weight (r= 0.28 ,p<0.05)) ,bod yfa tmas s (r= 0.33 ,p<0.05 )an dag e( r= 0.35 ,p<0.05) ,adjuste dfo rlinea reffect so n thecorrelation so fdifference si nenerg yconten to fth etes tmeal .N o significantdifference swer efoun dbetwee nbot hgroup si nDI Texpresse d ina percentageo fth eenerg yconten to fth etes tmeal ,respectively ,6.6 4+ 0.2 9 percenti nth egrou pwit hhig henerg yintak ean d6.2 4+ 0.5 0i ngrou pwit hlo w energyintake .

DISCUSSION

Thisstud ywa sperforme dt oevaluat evariou smethodologica laspect so fth e assessmento frestin gmetaboli crat e (RMR),diet-induce dthermogenesi s(DIT ) andi nviv ofue lutilizatio nrates .I ti sth efirs tstud ytha treport sdat ao n thereproducibilit yo fmeasurement so fDI Ti nme nan dwome nusin ga ventilate d hoodsystem .A tth estar to fth estud ya ventilate dhoo dsyste mwa sbuil ti n ordert ob eabl et omeasur eth erat eo fenerg yexpenditur econtinuousl yfo r prolongedperiod so ftim ewit hminima ldiscomfor tfo rth esubject .Th e accuracyo fthi smetho di nassessin generg yexpenditur ewa steste dan dstudie s wereperforme dt oasses sth ewithin-subjec tvariabilit yi nRM Ran dDIT .Th e impacto fth eenerg yintak eo fth eingeste dmeal ,o fth eduratio no fth e postprandialmeasuremen to nDIT ,o fth ephas eo fth emenstrua lcycl eo nRM R andDIT ,o fcontro lo fth eanteceden tdie to nth ewithin-perso nvariabilit yo f RMRan dDI Tan do fdiurna lvariatio no nRM Rwer eals oinvestigated .Al l studies,wit hth eexceptio no ftw ostudie si nwome n(Tabl e2 ,stud yn o3 an d 4),wer ecarrie dou ti na perio do fa tmos t6 week sduratio nt opreven t seasonaleffect so nenerg yexpenditure . Theresult so fthi sstud yhav eimportan tconsequence sfo rth estud yo f postprandialhuma nenerg ymetabolism .A findin go fgrea timportanc ei sth e relativelylarg ewithin-subjec tvariatio ni nDIT ,whic hcoul dno tb e contributedt ovariatio ni nth emetho dan dwhic hseeme dno tsubjec tt o reductionwhe nth eanteceden tdie twa scontrolled .

-90- Accuracy of themetho d andwithin-subjec tvariatio n inRMR ,DIT ,R Qan d fuel utilization

The randoman d systematic error ofassessin g energyexpenditur ewit h indirect calorimetryusin ga ventilate dhoo d systemwa s inaccordanc ewit h thedat ao n theaccurac yo f the indirect calorimetrymetho duse d in respiration chambers (73,74). The randomerro rwa s 2.7 percentan d the systematic errorwa s 1.5 percent.Th evariabilit y ofth emetho dwa smuc h smallerwhe n compared toth e within-subject variability inth eRM Ran d inDIT .Th evariabilit y inth e method contributed lesstha n1 0percen t toth etota lobserve d within-person variability inRMR .Th e totalC V for repeatedwithin-perso nmeasurement so f RMRwer e alsocalculate d asth eaverag e of the individual CV's.Ou r results agreewel lwit h other reportso n thewithin-perso nvariabilit y inRMR ,whic h reportaverag ewithin-subjec t CV's forth eRM R rangingbetwee n2 an d 7percen t (75-78). Iti sno tpossibl e topartitio n themethod-fre e within-subject variation into specific sources.RM Rmeasurement swer eperforme d under strictly standardized conditions,bu t therewa s somevariatio n in conditions underwhic h theRM Rwa sassessed .A t eachRM Rmeasuremen t subjectswatche d differentmotio npictures ,whic hma yhav e causedvariatio n inRM R measurements. Inaddition ,ther ema yhav ebee nvariatio n among the repeated measurementswithi na subject in subject'sbehavior , ie,on eda ya subject liescalmly ,awake ,motionles s inth e same semi-recumbentpostur eunde r the hood,breathin g regularly,o nanothe r daya subjectdoze sof f frequently, is restless,ha sman y spontaneous smallmovement s and changeso fpostur e and is breathing irregularlywit h alternating periodso fhyper-an d hypoventilation. Finally,ther ema yb ebiologica lwithin-perso nvariatio n inRM R fromda y to day.Thi svariatio n couldb e related tovariation s in feeding,drinkin g or activitypatter n inth eday sbefor e theRM Rmeasurements .I nmos to f the studiesher e reported, subjectswer emeasure d after anovernigh t fast. Subjectswer e eating normalbalance ddiets ,bu t theirdiet swer eno tunde r strictcontrol .

There isconsiderabl ewithin-perso nvariatio n inenerg yan dnutrien t intake fromda y toda y (78).Thi svariatio nma yb ea sourceo fadditiona l variation inth eRMR , irrespective of the fact that theacut eeffect s of food intake on RMRwer eminimize d bymeasurin g inth epostabsorptiv e state.W e tested this hypothesisb ymeasurin g thewithin-subjec tvariatio n inRM R insubject swh o were put ona controlled dietary intake regime fortw oweek s andwhos e activitypatter n remained asconstan t aspossibl e throughout the experimental

-91- period.Contro l of theanteceden t dietdi d not seem tohav emarke d effects on thewithin-perso nvariability , suggesting thatvariatio n in subject's behavior and inmeasuremen t circumstancesma yb egreate r sourceso fwithin-subjec t variation thanth eabsenc eo f strictcontro l of theanteceden tdiet . Incontras t toth e relatively smallwithin-perso nvariatio n inRMR ,th e intra-individualvariatio n inDI Twa smuc h larger.I nthi s studyDI T reproducibilitywa s found tob eo f similarmagnitud e inobes ewhe n compared to non-obesewomen .Th ewithin-subjec tvariatio n seemed not subject to systematic reductionwhe n theanteceden tdie twa s controlled,bu t it should benote d that for these subjectsn oestimate swer eavailabl e of thewithin-perso n variability ofDI Tunde r normalmeasuremen tconditions . Comparisono f thewithin-perso nvariabilit y ofDI Tmeasurement s asobserve d in this studywit hdat a ofothe r studies isdifficult .T o thebes to fou r knowledge noothe rgroup shav epublishe d datao nth e reproducibility of DIT measurements obtained bya ventilate d hood system,hav ebee npublished . Ravussine t al. (74)repor ta hig hwithin-perso nvariabilit y inDI T calculated from thedifferenc e between 24-hoursenerg yexpenditure ,correcte d for work-induced thermogenesis,an d thebasa lmetaboli c rateextrapolate d toa 2 4 hoursbasis .Th e lack ofdat ao n the reproducibility ofDI Tmeasurement s is surprising.Difference s inDI Thav ebecom e a subjecto fparamoun t interest for many groups involved in research on,fo rexample ,th eetiolog y of obesity (13-40). The sourceso f thewithin-perso nvariatio n inDI Tma yb e similar tothos eo f theRMR , ie,variatio n among trials inmeasuremen t circumstances (motion pictures),an d insubject' sbehavior .Sinc eDI T isa response of relatively smallmagnitude ,assesse d over aprolonge d period,thes e factorsma yhav e a relativelygreate r impacto n the reproducibility ofDI Tmeasurement s compared tothei r impacto n the reproducibilityo f theRM Rmeasurements .Subject swit h a relatively low reproducibility ofRM Rmeasurement sha d notnecessaril y the highestwithin-perso nvariatio n inDIT .Thi swa s illustrated by the non-significant correlationbetwee nwithin-perso nvariatio n inth eRM Ran d in DIT.A n additional sourceo f intra-individual variation inDI Tma yb e biological day-to-dayvariatio n inth e postprandial processing ofnutrients . Acheson et al. (79)showe d that the stateo f thebody' sglycoge n stores influenced DIT.Th e amounto fglycoge n stored in thebod y isaffecte d bydie t and physical activity (80).Therefor e inthi s studyonl y subjectseatin g normalmixe dmeal swer e allowed toparticipate .Subject swer e also instructed not toperfor mheav y exercise on theda yprecedin g theDI Tassessmen t to

-92- prevent serious depletion of thebody' s glycogen stores. We found thatmos t subjects considered theDI Tmeasurement s as tedious measurements,becaus e of their longduratio n and the requirement to perform themeasuremen t under RMR conditions.Divertin g their attention by showing motionpicture swa sessentia l topreven t the subjects from falling asleep or becoming restless and bored.

Within-subjectvariatio n inR Qan d fuelutilizatio n rates

Postabsorptive and postprandial respiratory quotients showed good reproducibility.Within-perso nCV' s for repeated measurements were, in general,belo w6 percent . Fuelutilizatio n rates,bot h postabsorptively as postprandially, showed greater within-subject variances.Thi s indicates that the type ofmetaboli c mixture utilised inth epostabsorptiv e or postprandial phase is relatively constant ina n individual.Th e amount of nutrients oxydized to obtain agive n type ofmetaboli c mixture may showmuc h larger within-subject variation. Control of theanteceden t dietdi d not seem to reduce thewithin-subjec t variation inRQ' s and fuelutilizatio n rates.

Between-subjectvariatio n inRMR , DIT,R Qan d fuelutilizatio n rates

In this study itwa s found that thebetween-perso n variation inRMR ,wit ho r without adjusting for the covariates age,fat-fre e mass and fatmas s was significantly greater when compared toth ewithin-subjec t variation. This finding confirms results obtained ina previou s study (74).Th e reasonswh y individuals of agive n age,fa tmas s and fat-freemas sma y differ inRM R are now subject ofmuc h research (6).Par to f the (adjusted) inter-individual differences inRM Rma y be related todifference s inhormonal ,ie ,thyroid , status or activity of the sympathetic nervous system(6) . The inter-individual variations inDIT ,R Qan d fuelutilizatio n rateswere , after adjusting for covariates,generall y of the samemagnitud e or smaller in comparison to the intra-individualvariation .

-93- Importanceo fwithin -an dbetween-person-variatio ni nRMR ,DIT ,fo r study designan d evaluation Knowledge of thewithin-perso n andbetween-perso nvariatio n invariable so f energymetabolis m iso fparamoun t importance for thedesig nan d evaluation of studiesassessin g changes inRM R orDI Twithi n the same individualdurin ga treatment,o r inth e comparisono fRM R orDI Tbetwee n independent sampleso f individuals classified ashig ho r lowwit h respect to,fo rexample ,bod y fat content. Inth elatte r case,averag e RMRan dDI T for individuals classified into these twolevel sar e generally compared to see ifthe yar e significantly different. If intra-individualvariabilit y islarg ean d thenumbe r of measurements foreac h subject issmall ,th epowe ro f thewithin-subjec t or between-group comparisonsma yb e low.Thi smean s that theprobabilit y of rejecting thenul lhypothesi so fn oeffec to f treatment (paired comparison)o r ofn odifferenc e invariable so fenerg ymetabolis m (betweengrou p comparison) mayb e small,eve n ifth enul lhypothese sar e falsean d the true treatment effects or truebetween-grou pdifference sar equit e large.A well-designed studyaimin g atth eassessmen t of treatment-induced within-person changes ina variableunde r studyo r atth e comparisonbetwee n interestingvariable so f independent samplemean s should,o nth eon ehand ,hav ea grea tchance ,ie , power,o fdetectin g a trueeffec to f treatmento rdifferenc e and,o n the other hand,a small chance,ie ,confidence ,o n falselydetectin g aneffec t or difference. Inothe rword s thetyp e 1erro r (falsepositive )an d the type 2 error (falsenegative ) should be sufficiently small.Th eprobabilit y of the type 1error ,generall y called(X ,i sspecifie db y the investigator.However , theprobabilit y thata significant resultwil l beobtaine d ifa realeffec to r difference exists,depend s largelyo nth e sample size.A s thetota l sample size increases,th etyp e 2error ,generall y called ß,decreases .Thi smean s that ifa statistical test fails to reach significance,th epowe r of the test becomesa critical factor in reachinga n inference.Th e failure toachiev e statistical significance mayb e relatedmor e toth e lowpowe r ofa n experiment than toth eactua l lack ofeffec to fa treatmento rdifferenc e betweengroups . Thismean s thata nexperimen twit h inadequate sample size isdoome d to failure before itbegins .Thi sma y serve only toconfus e the issueo fdeterminin g the effecto fa treatment oro fassessin g differencesbetwee ngroups .Therefor e each investigator should take stepst oensur e that thepowe r ofa n experiement is sufficient to justify theeffor t involved. Ifa nexperimen tha sgrea t power, say 0.95, the failure toachiev e significance may be properly interpreted asprobabl y indicating anegligibl e treatmenteffec to r negligible

-94- relevantbetween-grou pdifferences .Man y statisticians,bu tno tall ,argu e that theprope r interpretation ofa negativ e result shouldb ebase dupo n a consideration of thepowe r ofa nexperimen t (81-83). Inappendice s 1an d2 sample size calculations aregive n atspecifie doc -an dß-level san d specified true treatmenteffect so rbetween-grou pdifference s inDIT . From these calculations itappear s that studieswit h sample sizes smaller than 10person san dperformin gon emeasuremen tpe r subjectma yhav epowe r levels below8 0percen t ofassessin g true,relativel y large,ie ,25-5 0percent , treatment effectso rbetween-grou pdifference s inDIT . Thequestio n riseswhethe r these findingshav e consequences for the interpretationo f studieso ndifference s inDI Tbetwee n theobese -an d non-obese.A literature survey shows thateigh t (30,31,34,36,37,39,40,42) of the thirteen studies reporting nodifferenc e inDI Tbetwee nobes e and non-obese,probabl yha d apowe r lower than8 0percen t of findinga true between-groupdifferenc e inDI To f2 5percent .

Constancy of the RMR

The results ofTabl e 9 showtha tRM Rdi dno t showsystemati c diurnal variation frommornin guntil l earlyafternoon .Thi s indicates that theRM R remains constantdurin g theassessmen t ofpostprandia l energyexpenditure ,a t least duringmeasurement s ofenerg yexpenditur e overa morning .Thi s implicates that inth e assessment of the thermic effecto f foodn o sham feeding control experiment isneede d tocorrec t forchange s inth ebasa l energyexpenditure . Our findingsar e inaccordanc ewit hmos tothe r studiesassessin g changes during theda y inRM R (34-36),wit h theexceptio no f some older studies (84,85). Iti swel l knowntha tRM R showsdiurna lvariatio ndu e toth e thermic after-effects ofexercis e and food ingestiono rdu e toslee p (20).Thi s study shows that inth eabsenc eo f thermogenic stimuli thepostabsorptiv e RMR shows very little change in themornin g over the courseo fa 2.5 hoursmeasuremen t period orbetwee nmornin g andafternoo nassessments ,comparin g singleRM R measurements performed on the sameday ,o r comparing RMR'sassesse d repeatedly onmornings orafternoons .

-95- Impact of thephas eo f themenstrua l cycleo nRM R and DIT

Table 10show s that thephas e of themenstrua l cycledi d not significantly influence RMRo rDIT .RM Rwa s slightly, ie,1. 2 percent,highe r during the post-ovularphas e of thecycl e compared toth epre-ovula rphase .O n average pre-ovularan d post-ovular RMR andDI Twere ,respectively ,assesse d for about three and two times in2 3subjects ,althoug h thevariatio n innumbe r of replicateswa squit e large.Thi smean s thatwit h this sample size,tru e differences between the twophase so f thecycl eo fa t least 2percen t inth e RMR or at least2 0percen t inDI Tcoul d havebee ndetecte dwit ha powe r of 90 percentan d a confidence levelo f 95percen t (two-sided),assumin g a correlationbetwee n the twophase so f thecycl eo f 0.80 inRM Ran d of 0.40 in DIT. Smaller differences inRM Ro rDI Tbetwee n thepre -an d post-ovular stage of the cyclema yb ephysiologicall y irrelevant.Wit h respect toth eRM R the results of thisstud y support the findingso f several authors (86-90),bu tar e incontras twit h resultso fothe r studies (91-94). The reasons for these differences areno tquit e clear,bu tma yb e related todifference s in experimental protocol,technique suse d formeasurin g energy expenditure,th e number of subjects studied,th e frequence ofmeasurement sdurin g themenstrua l cycle and themetho duse d toasses sovulation . Inth epresen t studymeasurement swer ealway sperforme d at least three days after theonse to fmenses .Th e rationalebehin d thisprotoco l demand isth e fact thatman ywome nhav ehealt h complaints shortlybefor e or during menstruation.Thi sma y affecta subject'sbehavio r during energy expenditure measurements,whic hma y causeunwante d effects onmeasurements .Fo rexample , already in the 1920s itha sbee n reported thatbasa lmetaboli c rate is lowered duringmenstruatio n incompariso nwit hothe r phaseso f themenstrua l cycle (95). Inthi s studymos to f thepre-ovula r energyexpenditur e measurementswer emad e ina perio d between 4an d 10day safte r theonse to fmenstruation . For most subjects ovulationwa s assumed tooccu r 14day sbefor e the starto fmenstrua l flow. In subjects 1t o 5ovulatio nwa sconclusivel y identified using an ovulation test.Also ,i nthes e subjectsn o significant effecto f the stage of theovula r cycleo nRM R orDI Twa s observed. No studieshav e reported on the impacto f the stageo f themenstrua l cycleo n DIT. Frequently iti sassume d that thephas eo f themenstrua l cycle affects theRM R (96).I nfact ,th enumbe r of studiesno t showingan yeffec t of the stage of the cycleo n theRM Rar e inbalanc ewit h thenumbe r of studies that

-96- do reporta neffect .I ngeneral ,th epost-ovula r rise inRM R isattribute d to the increased progesterone secretion intha tphas e of the cycle (91-94). The thermogenic effects ofprogesteron e are,however ,no tclea r (97).I na recent study,progesteron e administrationwa s found todecreas e RMR inwome n instead of increasingRM R(97) . Variability inRM R andDI Twa sals ocompare d betweenwome nusin g oral contraceptives andwome nhavin gnorma lmenstrua l cycles.Variatio n inRM Rwa s significantlyhighe r inwome nusin g oral contraceptives incompariso n towome n having normal cycles.Thi swa sdu e toth e fact that 5wome nusin g oral contraceptivesha dparticularl yhig hCV' s for theirRMR ,exceedin g 10percent . We expected that thewithin-perso n variability ofRM R inwome nusin g oral contraceptiveswoul d be smallerwhe n compared toth evariabilit y observed in womenhavin g normalmenstrua l cycles.I nwome nusin gora l contraceptives hormonal profileswil l be relativelymor e constantove r thecours e ofa cycle . As a consequence thebiologica lwithin-perso nvariatio n inRM Rwa sexpecte d to be smaller inwome nusin g oral contraceptiveswhe n compared towome nwit h normal ovular cycles.Thi shypothesi swa sno t supportedb y our findings.N o systematicdifferenc e betweenbot hgroup s invariatio n inDI Twa s observed.

Impact ofenerg y intakean dduratio n ofpostprandia l measurement on the calculationo fDI T

There isa grea tvariet yamon g thevariou s studieso nDI T inth e energy content of themea l givenan d induratio no f thepostprandia l measurement period (13-42). Thus,meal s ornutrien t solutionsar ebein ggive nvaryin g in energy contentbetwee n 800k J (14)an d800 0 (42)kJ ,wherea s the postprandial measurement periodvarie sbetwee n 1.5 (27)an d 8hour s (42).I tha sbee n pointed outb yvariou sauthor s (20,42)tha t theduratio no f the postprandial measurement period shouldb eo f sufficient length inorde r tomeasur e the largestpar to f the thermic response tofood .However ,i tha sno t been reportedwhic h proportion of the thermiceffec to f food,usin g different energy loads,i smeasure dwithi n fixed time intervalso f the postingestive period. In thisstudy , itwa s found thatDI T increaseswit h greater energyintakes , confirmingpreviou s findings (42).I nmen ,DI Twa spositivel yassociate d with energy intake,bu tn o significant correlation betweenDI Tan dbod yweight ,th e size of the fatmas s or fat-freemas swa s found.I nwomen ,DI Twa s also significantly positively correlated toth eenerg y content of the testmeals ,

-97- butno t to the sizeo f the fat-freemass . Low,bu tsignifican t correlations were found betweenDI Tan dbod yweight ,fa tmas san dage .Thi s indicates that some of theolde r obese subjectsha d relativelyhighe r DIT'swhe n compared to theyounge r lean subjects.Th epostprandia l responsepattern s indicate that in menan dwome na DI T responset o ingestiono fa mixe d liquidmea lwit h an energy contentbelo w150 0k J canb enearl y completely assessedwithi n three hours.Th eDI T response tomeal swit h larger energy content,ie ,betwee n 1500 kJ and 2600k J inme nan dbetwee n 1500an d 2000k J inwomen ,ca nals ob e assessedwit h sufficient accuracywithi n threehours .Th edat a show that adding a fourthhou r toth epostprandia l period increased theobserve d thermic effect by 10-15percen t only.W e have experienced thatafte r threehour sman y subjectsbecom e restlessan dbore d by themeasurement .Reducin g the postprandial measurementperio d tothre ehour sdoe sno thav e substantial effectso n thecalculatio n of theDI T response tomeal so fa nenerg y content of 2000kJ ,bu twil lpreven t someo f thebehavioura l problems occurringwhe n subjectsar emeasure d fora mor eprolonge d period oftime . Sometimes theenerg y contento fth emea l ornutrien t solution isgive na sa dosispe r kg (ideal)weigh t (13,34,41)o r fat-freebod ymas s (20,42).Whe n such aprocedur e isused , it isimplicitl y assumed that the thermic response iscorrelate d to (ideal)bod yweigh t or toth e sizeo f the fat-freemass ,an d thatb y adjusting for inter-individual differences inthes e variables inter-individual comparison ofDI T responseswil lb emor evalid .However ,dat a to support thisvie whav eno tbee n reported.Th epresen t study showed a low, and for themost ,excep t inwomen ,par tnon-significan t correlation between DITan d bodyweigh t and the sizeo f the fat-free mass.Thi s isi n accordance with the findings of a recent report (42)an d supportsa notio npu t forward by Garrow,quote d in (96),ie ,tha t "a logo fa give n sizegive so f the same amount ofhea t ina smallo r large oven".Ou r findingssugges t that comparing DIT responsesamon g individuals toequa l caloric loads shouldb e advocated. This supports thevie wo f Sjöström(98) .

Inconclusion ,thi s study shows thatRM Ran dpostabsorptiv e and postprandial RQ's canb emeasure dwit h sufficientlyhig h reproducibility inhuman swit h a ventilated hood system.DI Tan d fuelutilizatio n rates show considerably largerwithin-perso nvariation ,comparabl e inmagnitud e toth e between-person variation.Th ewithin-perso nvariatio n inDI T seemed not subject to reduction when the antecedent dietwa scontrolled .Postabsorptiv e RMRdi dno t show substantial variation over amorning ,eliminatin g thenee d for sham-feeding

-98- control experiments inth eassesssmen to fDIT .Th ephas e of themenstrua l cycledi dno tmarkedl y affectRM Ro rDIT ,suggestin g that inmeasurement s of RMRan dDI T inwome n the stage ofth ecycl e isa factor of negligible relevance inth e studydesign .Th e thermic effect tomeal swit h an energy content betweenabou t1.5-2. 5M J canb enearl y completely assessed within threehours .Ther e isn onee d to standardize theenerg y content of the test meal tobod yweigh to r toth e sizeo f thefat-fre e mass.

ACKNOWLEDGEMENTS

Theauthor sar e indebted toZandri eHofman ,Jo sSteenbergen ,Pete rWeys , Eric Poortvliet,The oDopheide ,Gree tVansant ,Cathalin e denBesten ,Alid aWapstra , Jacqueline Dekker,Miria m Stoel,Lilian eBegheyn ,Kari nva nde r Kooy,Leonie k Robrochan d IngridWunnin k forhel p inconductin g theexperiments .W e are also indebted to the staff of the laboratory of theDepartmen t ofHuma n Nutrition for their help inblood ,urin e and foodanalyses ,i nparticula r toMis sAn s Soffers.W ewoul d also like to thank MissSaski aMeyboo m forhe r help in preparing the food items for the subjects receiving controlled diets.Finally , wewoul d like tothan k all subjects forthei r enthousiastic participation in thevariou sstudies .

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-103- Appendix1 . Numbero fsubjects ,accordin gt osex ,require dt otes ta chang e withinsubject so f10 ,2 5o r5 0percen ti ndiet-induce d thermogenesiswit ha significanc eleve lo f

Ejectedchang e (%)*i nBI T 10 25 50 ß- level ß- level ß- level 0.2 0.1 0.2 0.1 0.2 0.1

Noo fmeasurement s Sex Correlation 1 M 0 129 172 22 29 8 9 0.2 103 138 18 24 4 6 0 164 219 28 37 9 11 0.4 99 132 18 23 6 8

0 66 87 13 16 5 6 0.2 48 63 10 12 4 5 0 83 111 15 20 6 7 0.4 38 49 8 10 4 4

rtoo fmeasurement s Sex 1 M 308 412 50 67 14 18 F 233 311 38 51 11 14

2 M 154 206 25 34 7 9 F 117 156 19 26 6 7

& = 0.05, two-sided Rater of subjects calculated according to lachin (83), using the adjusted between-person variation, according to ! found in this study * Expected change expressed as percentage of •aonOTT value s (in kJ) observed, in wen and vanen EBT = diet—induced thenaogenesis

-104- Chapter 4. Diurnal variation inpostabsorptiv e restingmetaboli crat e and diet- induced thermogenesis

JAN A WESTSTRATE MSC, PETER JM WEYS BSC, ERIC J POORTVLIET BSC, PAUL

DEURENBERG PHD, JOSEPH GAJ HAUTVAST MD

ABSTRACT

Postabsorptive restingmetaboli c rate (RMR)an ddiet-induce d thermogenesis (DIT)wer e repeatedlyassesse do ndifferen tday si nte nyoun gnorma l weight meno nmorning san dafternoons .N osignifican tdiurna lvariatio nwa sfoun di n RMR,DIT ,tota lpostprandia l substrateoxidatio nan doveral l postprandial nutrientbalances .Th epatter no fpostprandia l substrateoxidatio n indicated an increased glucoseoxidatio ni nth efirs thou r after ingestiono fa mea lo n morning testscompare dt oafternoo n tests.Thi swa sprobabl y relatedt o differences inth edegre eo fth epostabsorptiv e state,ie ,12-1 4hour so n morning testsan d6- 7hour so nafternoo ntests .

Keywords: indirect calorimetry, restingmetaboli c rate,diet-induce d thermogenesis,diurna lvariation ,substrat e oxidation,huma n

INTRODUCTION

Rhythmicvariation s inenerg ymetabolis mar ewel ldocumente d foranimal s (1). Cyclical changes inenerg ymetabolis mhav eals obee npostulate d forhuman s (1).Amon g them,change si nenerg yexpenditur edurin gth emenstrua l cyclehav e been studied relatively frequent (2-5). Less informationi savailabl efo r humanso nvariatio n inenerg ymetabolis mdurin gth eday ,ie ,o ndiurna l variation inenerg yexpenditur e (6-8). Iti swel l known thatenerg y expenditurevarie sdurin gth eda ydu et oth ethermi ceffect so ffoo dan d physical activity (9). Veryfe winvestigators ,however ,hav e studied changes

-105- during theda y inenerg yexpenditur e inth eabsenc eo f thermogenic stimuli (6-8). Inaddition ,n o studies reporto ndiurna lvariatio no f energy metabolism in response tothermogeni cstimuli . Inpreviou s studieso nhumans ,evidenc ewa s obtained fora gradua l increase during theda y inpostabsorptiv e restingenerg yexpenditure ,ie ,i n postabsorptive restingmetaboli c rate (6-8).Mor e recent studiesd ono t support these findings (10-13). Inth elatte r studies,however ,diurna l variation inpostabsorptiv e restingmetaboli c ratewa sno t themai n objective of study,an d energyexpenditur ewa susuall ymeasure ddurin g themornin g only. It isimportan t toclarif y the issueo fdiurna lvariatio n inenerg y expenditure,sinc e thisma yaffec tth e interpretationo f studieso ndiet - induced thermogenesis.Diet-induce d thermogenesisca nb edefine d as the increase inenerg yexpenditur edu e toingestio no f food.Frequently , in studieso ndiet-induce d thermogenesisa non-calori c controlo r sham feeding test isused toadjus t forpossibl ediurna lvariatio n inpostabsorptiv e restingenerg yexpenditur e (10-13). However,othe r investigatorsassum e no significant changes inpostabsorptiv e restingmetaboli c ratedurin g theda y andd ono t report such sham feeding tests (14-17). Besidesdiurna lvariatio n in restingenerg yexpenditur e inth eabsenc e of food,change sdurin g theda y inth e responseo fenerg ymetabolis m to foodma y be important.Diurna lvariatio n inth e responseo fenerg ymetabolis m to food mayaffec tdail ynutrien t andenerg ybalance . The present studywa sdesigne d toinvestigat ewhethe r repeatedmeasurement s of restingmetaboli c ratean ddiet-induce d thermogenesisvarie d systematically with time of theday , ie,betwee nmorning san d afternoons.N o significant diurnalvariatio n in restingmetaboli c ratean d indiet-induce d thermogenesis was found,bu tpostprandia l substrateoxidatio n showed adifferen t pattern in themornin g incompariso n toth eafternoon .

SUBJECTSAN D METHODS

SUBJECTS

Tenyoun gmen ,al l students,participate d inth e study.Volunteer swer e invited forparticipatio n inth e studyb yadvertisemen t inth eUniversit y weeklyperiodical .Dat a concerning somephysica l characteristicsan d age are presented inTabl e 1.Eac h individualwa s fullyinforme d on thenatur ean d

-106- purposeo fth eexperiment ,befor egivin ghi swritte nconsen tt oparticipate . Eachsubjec treceive da questionnair et oasses spas tan dpresen thealt hstatu s andnutritiona lhabits .Non eo fth esubject swa so na specia ldiet ,al l subjectswer eeatin gnorma lbalance ddiet san dnon eo fth esubject swa susin g anymedicatio na tth etim eo fth estud yo rha dexperience dgreate rbod yweigh t fluctuationtha n2. 5k gi nth ehal fyea rprecedin gth estudy .Al lsubject s werenon-smoker san dapparentl yhealth ya sassesse db ymedica lquestionnaire . Theprotoco lwa ssubmitte dt oan dapprove do fb yth eMedical-Ethica lCommitte e ofth eWageninge nAgricultura lUniversity .

METHODS

Experimentaldesig n

Restingmetaboli crat e(RMR )wa smeasure dsi xtime so ndifferen tday s ineac h subject,thre etime si nth emornin gand ,similarly ,thre etime si nth e afternoon.Betwee neac hsessio nther ewa sa tim espa no fa tleas ttw odays . Allsubject scomplete dth eexperimen ti nfou rweeks .Mornin gRMR' swer e measuredwit hth esubjec ti na 12-1 4hour spostabsorptiv estate .Afternoo n RMR'swer emeasure dwit hth esubjec ti na 6- 7hour spostabsorptiv e state.A t theda yo fth eafternoo nsession sth esubject swer eallowe dt ohav ea smal l normalDutc hbreakfas to fabou t200 0k J(1 0percen tprotein ,3 5percen tfat , 55percen tcarbohydrates )betwee n7.4 5an d8.0 0hours .Th esubject swer e instructedt orefrai nfro msleeping ,coffe edrinkin gan dmoderat et oheav y physicalactivit yo nth emorning sbefor eth eafternoo ntests .The ywer eals o instructedno tt operfor mheav yphysica lexercis eo nth eevenin gbefor eth e morningtests .Th eorde ro fRM Rmeasurement swa srandomize dbetwee nth e morningan dafternoo ntests . Ontw omorning san do ntw oafternoon safte rth eRM Rmeasurement ,energ y expenditurei nrespons et oa mea lwa smeasure di neac hsubjec tt oasses s diet-inducedthermogenesis .Th eincreas ei nrestin gmetaboli crat edu et oth e ingestiono fth emea lwa sdefine da sdiet-induce dthermogenesi s (DIT).Th e mealwa sa yoghurt-base dliqui dformul acontainin go naverag e190 0k J(1 0 percentprotein ,3 3percen tfat ,5 7percen tcarbohydrates) .Th eorde ro fth e DITmeasurement swa srandomize dbetwee nth emornin gan dafternoo ntests . Tofamiliariz eth esubject swit hth eequipmen tfo rmeasurin gRM Ran dDIT ,

-107- resting energyexpenditur ewa smeasure d ineac h subjecto na separate day before the starto f theexperimen t forabou t 30-45minutes .

Gasexchang e measurements

Restingmetaboli c rate

On theda yo feac hRM Rmeasurement ,th e subjectswer e taken toth e laboratory by car.Afte rvoidin g theyla y supinebu tawak e ona hospita lbed .Afte r a 30 minutes restperiod ,RM Rwa s continuouslymeasure d between 08.00an d 09.00 hours inth emornin g or 14.00an d 15.00hour s inth eafternoo n ata n environmental temperature of23-25°C .Durin g theRM Rmeasurement s the subjects watchedvide ofilms .

Postprandial energy expenditure

After theRM Rmeasuremen t the subjectswer e given the testmeal , five minutes after theyha d finished eating thetes tmeal ,energ yexpenditur ewa smeasure d continuously for fourhours .Durin g thepostprandia l energy expenditure measurements the subjectswatche dvide o films.Roo mtemperatur ewa s always kept at 23-25°C. The subjectswer e instructed tob ea s relaxed aspossibl e and tominimiz e movements.Afte r thega sexchang emeasurements ,urin ewa s collected for determining urea-nitrogen and totalurinar ynitrogen . All gasexchang emeasurement swer e performed usinga ventilate d hood system. A transparent perspexventilate d hoodwa splace d over the subjectshea d andmad e airtight around theneck .A pump (OceanSCL210 ,Dieren ,Th e Netherlands) drawed freshoutsid e air through thehood .Th e flow rate through thehoo dwa s measured bya thermalmas s flowmeter (Brooks,5812N ,Veenendaal ,Th e Netherlands). The flow ratewa skep tbetwee n 30an d 401/mi nb ya nelectri ­ callyoperate d controlvalv e (Brooks,mode l 5837,Veenendaal ,Th eNether ­ lands).A small sampleo fabou t 0.4 1/min ofth eai r leaving thehoo dwa s analysed foroxyge n (0,)an d carbondioxid e (CO,)conten tby , respectively, a paramagnetic 0,-analyzer,adjuste d toa full scale rangeo f 20-21 percent (Servomex,1100A ,Zoetermeer ,Th eNetherlands )an da n infraredCXU-analyzer , full scale range 0-1 percent (Analytical Development Company,SS100 , Hoddesdon,UK) .Th eanalyzer swer e calibrated eachmornin g or afternoon sessionusin gdrie d gasmixture swit h known0 2 andCO ,content .O ,an d CO,

-108- concentrations indrie d freshoutsid eai rwer emeasure d for fiveminute s every 30minute sdurin g theRM Rmeasuremen t and everyhou rdurin g the postprandial energyexpenditur emeasurements .Ai r flow rate,0 ,an d C0? concentration of the inflowingan d outflowing airwer e computed on lineb ya n automaticdat a acquisitionuni t (HewlettPackar d 3497A,Pal oAlto ,USA ) interfaced toa microcompute r (HewlettPackar d 85,Pal oAlto ,USA) .Th e outputs of thegasanalyzer s and the flowmeterwer e alsocontinuousl y recorded bya multi-pe n recorder (Kipp& Zone nBDlOl ,Delft ,Th eNetherlands) .0 ? and CO,concentratio n ofth eoutflowin g air andai r flow rate through thehoo d weremeasure d every 30second san d integrated over fiveminute s interval periods toobtai n0 ,consumption ,CO ,production ,respirator yquotien t (RQ) andmetaboli c rateaccordin g toequation sdescribe d byJéquie r(18) .

Calculation ofdiet-induce d thermogenesisan d substrate oxidation

The0 ,consumptio nan d CO,productio nwer e corrected formovement so f the subjectswhe nnecessary .T oobtai n themea n restingenerg yexpenditur e for eachhou r after ingestiono f the testmeal , the0 ,consumptio nan d CO, productionwer e averaged over eachhou rusin g the fiveminute s intervaldata . Thesevalue swer euse d tocalculat e theaverag eDI T for the individual hours after the testmea l and for thetota lmea nDI Tove r fourhours .DI Twa s calculated by subtracting theRM Rvalu e fromth e respectiveaverag ehourl yo r totalmea npostprandia l energy expenditure.Th eDI T isexpresse d asa percentage of theenerg y contento f the testmeal . Proteinoxidatio nwa sestimate d bydeterminatio n ofurinar y urea-nitrogen excretion,wit ha testkit (Boehringer MannheimB Vki t 396346,Almere , the Netherlands)accordin g toGutma nan dBergmeye r (19).Carbohydrat e and fat utilization during RMRan ddurin g the fourhour safte r ingestiono f the test mealwer e calculated using thenon-protei n respiratoryquotien t (18).Oxyge n and carbondioxid eequivalent s for substrate oxidationbefor e andafte r the ingestiono f thetes tmea lwer e obtained fromLillioj a et al.(20) . Carbohydrate oxidationwa s corrected forne td enov olipogenesi s from glucose aspreviousl y described (17).A non-protein respiratoryquotien t exceeding 1.00 indicated netd enov olipogenesis ,ie ,lipogenesi s exceeding concomitant fatoxidation .

-109- Body composition

Fat-freemas swa sestimate d bydensitometry .Tota lbod ydensit yo f each subjectwa smeasure d oncedurin g theexperimen t induplicat e by theunde r waterweighin gmetho dwit h simultaneousmeasuremen t ofth e residual lung volume bya heliu mdilutio n technique.Fat-fre emas swa s calculated from total bodydensit yusin g Siri'sequatio n(21) .

Statistics

The resultsar eexpresse d asmea nvalue swit h standard errorso f themea n (sem).Analysi so fvarianc e using themode l fora single factor experiment with repeatedmeasurement swa suse d totes twhethe r RMRan dDI Tvarie d systematicallybetwee nmorning san d afternoons.Paire d two-tailed Student's t-testswer e performed toevaluat edifference s inmea nRMR ,DI Tan d substrate oxidation ratesbetwee nmornin g and afternoon sessions.Confidenc e intervals areuse d toexpres smea ndifference s inRMR ,DI Tan d substrateoxidatio n rates betweenmornin g and afternoon sessions.Pearson' sproduct-momen t correlation coefficientswer euse d incorrelatio nanalyses .

RESULTS

Table 1show stha tal l subjectsha dbod yweight san dbod y fatpercentage s within thenorma l range.

Table 2give s restingmetaboli c rates insubjects ,separatel y for themornin g and afternoon sessions.Analysi so fvarianc e showed no significantda y and time ofda yeffec t onRMR .Th e 95percen t confidence interval for the average difference between themea nmornin g andafternoo nvalue s inRM R is-0.0 7 to +0.35kJ/min .Therefore ,fo reac hperso nmea nmornin gan d afternoon RMRvalue s were used insubsequen t analyses.Mornin g RMR'swer e significantly correlated with afternoonRMR's , (r= 0.95,p < 0.01).

-110- Table1 .Ag ean dsom ephysica lcharacteristic so fth esubjects .

subjects age weight bodymas s fat-free bodyfa t index mass percentage yrs kg kg/m kg %

1 22 65.5 21.7 53.9 17.6 2 20 86.1 25.9 71.3 17.1 3 23 74.3 21.8 59.9 19.3 4 20 78.9 21.3 68.0 13.7 5 21 75.2 21.4 61.7 17.9 6 24 74.9 22.8 61.9 17.3 7 25 79.4 22.0 69.5 12.5 8 22 65.2 19.5 59.9 7.8 9 23 71.9 22.2 64.5 10.3 10 22 83.5 24.4 69.1 17.2 mean+ se m 22± 0.5 75.4+ 2. 2 22.3+ 0. 6 64.0 ± 1.7 15.1± 1. 2

Table2 .Restin gmetaboli crat ei nsubject so ndifferen ttime so fth eday . subjects Restingmetaboli crat e(kJ/min )

morning afternoon total mean+ sem * mean+ sem* mean +sem T

1 4.45 + 0.11 4.54+ 0.07 4.50 + 0.06 2 5.87 + 0.24 5.45+ 0.07 5.66 + 0.15 3 4.61 + 0.27 4.74+ 0.35 4.68 + 0.20 4 5.43 + 0.35 4.82+ 0.33 5.12 + 0.62 5 4.56 + 0.06 4.31+ 0.20 4.43 + 0.11 6 4.52 + 0.24 4.66+ 0.26 4.58 + 0.16 7 5.24 + 0.11 4.69+ 0.11 4.96 + 0.14 8 4.37 + 0.28 4.47+ 0.04 4.42 + 0.13 9 4.09 + 0.21 4.19+ 0.05 4.14 + 0.10 10 5.02 + 0.04 4.87+ 0.04 4.94 ± 0.04 mean+ se m 4.81+ 0.1 8 4.67+ 0.11 4.74 + 0.15

*mea no fthre emeasurement s fmea no fsi xmeasurement s

Datao ndiet-induce dthermogenesi si nsubject sove ra fou rhour sperio dafte r ingestiono fa mea lo ndifferen ttime so fth eda yar epresente di nTabl e3 , separatelyfo rmorning san dafternoons .Analysi so fvarianc eshowe dn o significantda yan dtim eo fda yeffec to ntota lDIT .Therefore ,fo reac h personth emea nmornin gan dafternoo nDI Tvalue swer euse di nsubsequen t

-111- analyses.Th e9 5percen tconfidenc einterva lfo rth emea ndifferenc ebetwee n themornin gan dafternoo nDI Tvalue swa s-1. 8t o+ 2. 6percen to fth eenerg y contento fth etes tmea l(-3 4 to+ 5 0kj) .Averag emornin gDI Tvalue swer e significantlycorrelate dwit haverag eafternoo nDI Tvalue s( r= 0.89 ,p < 0.01).

Table 3.Diet-induce d thermogenesisi nsubject safte r ingestiono fa mea lo n differenttime so fth eday .

subjects Diet-inducedthermogenesi s (%o fenerg yconten to ftes tmeal) *

morning afternoon total , mean+ se m mean+ sem ' mean+ sem '

1 7.5 0.6 5 2 + 1 5 6 3 + 1 0 2 6.9 1.3 13 6 + 6 3 10 2 + 1 6 3 7.8 0.7 4 8 + 1 3 6 3 + 1 0 4 9.3 4.5 9 8 + 1 2 9 5 + 1 9 5 6.1 0.5 6 4 + 2 8 6 2 + 1 2 6 6.3 1.4 4 7 + 1 0 5 5 + 0 9 7 5.2 0.9 7 6 + 3 4 6 4 + 1 6 8 6.3 0.3 5 4 + 0 5 5 8 + 0 4 9 7.0 0.8 4 6 + 1 2 5 8 + 0 .9 10 10.2 0.0 6 8 + 1 6 8 5 + 1 .2 mean+ se m 7.4+ 0. 5 6 9 + 0 9 7 1 + 0 .5

* testmea l contained onaverag e 1906 kj.Th ecoefficien t ofvariatio nfo r energyintak ewa s0. 5percent . f meano ftw omeasurement s 4: meano ffou rmeasurement s

Figure1 show sdiet-induce dthermogenesi sseparatel yfo reac hhou rafte r ingestiono fth emea li nsubject so nmornin go rafternoo ntests .N o significanttim eo fda yeffec to nth emea nhourl yDI Tvalue swa sobserved .O n averageDI Twa sslightl yhighe ro nmornin gsession si nth efirs thou rafte r ingestiono fth emea li ncompariso nwit hafternoo nsessions ,wherea sfo rth e thirdan dfourt hhou rth eopposit ecas ewa strue .Th eaverag eDI Ti nth e fourthhou rafte rth eingestio no fth etes tmea lwa sabou t0. 1kJ/mi n (2-3pe r cent)abov eth ecorrespondin gpostabsorptiv erestin gmetaboli crat eo nmornin g sessionsan dabou t0. 2kJ/mi n (4-5pe rcent )abov eth ecorrespondin g postabsorptive restingmetaboli crat eo nafternoo nsessions . Figure2 represent srespirator yquotient si nsubject sbefor ean dafte r ingestiono fa tes tmea lo ndifferen ttime so fth eday .RQ' swer e

-112- diet-induced thérmogenesis [kJ/min] D morning afternoon

2 3 4 1-4 time after meal ingestion (h)

Figure1 :Diet-induce dthermogenesi s(mea n+ sem )i nsubject safte ringestio n ofa mea lo ndifferen ttime so fth eday .

respiratory quotient 1.94- • morning 0.921 • afternoon 0.90

I

RMRMEAL 12 3 4 1-4 before/after meal ingestion (h)

Figure 2: Respiratory quotients (mean + sem) in subjects before and after ingestion of a meal on different times of the day.

-113- significantly higher on mornings in the first hour after ingestion of the meal in comparison with afternoon RQ's. The reverse was true for the third and fourth hour after ingestion of the test meal. Table 4 gives carbohydrate and fat oxidation in subjects before and after ingestion of a meal, separately for the morning and afternoon sessions. The results are expressed in absolute amounts and as a percentage of total energy expenditure. Before ingestion of the test meal, during the RMR measurement, no significant differences were obeserved in relative or absolute amount of oxidation of these nutrients between morning and afternoon sessions.

Table 4. Mean carbohydrate and fat oxidation in absolute amounts and as a percentage of total energy expenditure in subjects before and after ingestion of a meal on different times of the day. oxidation carbohydrates* fats during : morning afternoon morning afternoon mean mean mean mean

RMRt %kJ/min 39 39 40 40 mg/min 117 115 50 47S PEElt %kJ/min 64 57§ 16 24§ mg/min 234 196§ 24 33 PEE2 %kJ/min 66 66 14 14 mg/min 241 226 21 20 PEE3 %kJ/min 46 54§ 33 25§ mg/min 152 177 46 34§ PEE4 %kj/min 27 37§ 51 42§ mg/min 84 133§ 65 53 PEEt %kJ/min 52 54 28 26 mg/min 178 178 39 35

* expressed on a glucose basis -f-RMR : postabsorptive resting metabolic rate •£PEE : postprandial energy expenditure; PEEl, during first hour; PEE2, during second hour; PEE3, during third hour; PEE4, during fourth hour; PEEt, during total postprandial period S significant difference between morning and afternoon values at p < 0.05

Carbohydrate oxidation was significantly higher and fat oxidation significantly lower on mornings in the first hour after ingestion of the test meal than on afternoons. During the third and fourth hour of the postprandial period, carbohydrate oxidation was lower in the morning in comparison with the afternoon, in contrast to fat oxidation. Protein oxidation was estimated for each session by the average (5.5 hours) urinary urea-nitrogen excretion. Protein oxidation was not significantly different between morning tests and -114- afternoon tests,57+ 3 mg/minversu s 53+3 mg/min. Table5 show senerg yan dnutrien t balancesi nsubject s after ingestiono f mealso ndifferen t timeso fth eday .N osignifican tdifference s between morningan dafternoo n testsi ntota loxidatio nan dstorag eo rbalanc eo fth e variousnutrient swer e observed.O naverage ,energ ybalance ,ie ,energ y retention,wa sslightl yhighe ro nafternoo n thano nmornin g sessions.Th e9 5 percent confidence interval forth emea ndifferenc e inenerg y retention betweenmornin gan dafternoo n testsi s- 1t o+13 3kJ .Ne t"d enov o lipogenesis" fromcarbohydrate sdi dno tdiffe r betweenmorning san d afternoons.O naverag e less than3 percen to fth eingeste d carbohydrateswa s converted tofat .

Table5 .Nutrien tan denerg ybalanc e insubject safte r ingestiono fa mea lo n different timeso fth eday .

energy carbohydrates fat protein iroming afternoon noming afternoon morning afternoon iroming afternoon kj kj gg gggg

intake 1902± 3 1911+ 4 * 69.2+ 0. 1 69.5+ 0. 2 16.6+ 0. 016. 7+ 0. 0 11.7+ 0. 0 11.7+ 0. 0 oxidation 41.7±3. 1 41.8± 2. 2 9.0 ±1. 9 8.0+ 1. 6 13.8+ 0. 8 12.8± 0. 7 expenditure 1306 ±53 1248 ±43 net lipogenesis 0.7 ± 0.3 0.4 ± 0.2 balance 597+54 664+421" 27.5 ± 2.3 27.7 ± 2.3 8.4 ± 2.1 9.2 ± 1.7 -2.1 ± 0.8 -1.1 ± 0.7

* insults expressed as aeen ± Bern + significant difference Letwuen aDming and afternoon values «tp^ 0.05 (borderline significance)

DISCUSSION

This studywa sdesigne d todetermin e theeffec to ftim eo fth eday ,mornin g versusafternoon ,o nrestin gmetaboli c ratean ddiet-induce d thermogenesis. The results shown osignifican tdiurna l variationi nrestin gmetaboli c rate and diet-induced thermogenesis.Postprandia l substrate oxidationwa saffecte d by timeo fth eday ,bu tthi seffec twa stransien tan doveral l postprandial nutrientbalance sdi dno tsho wsignifican tdiurna lvariation .Mornin gan d afternoonmeasurement so fenerg yexpenditur ewer eperforme do ndifferen t days. Inthi sway ,i twa sexclude d thatmorning«measurement swoul d havea residual effecto nafternoo nassessment so fenerg yexpenditure .Th edesig no fth e

-115- study,however ,allowe d fornorma l intra-individualbetween-da yvariatio n in morning and afternoonRM Ran dDIT .Thi sma yhav e obscured the finding of diurnalvariatio n inRM Ran dDI To fa relatively smallmagnitude .T ominimiz e thispossibilit y RMRan dDI Twer e assessed repeatedlybot ho nmorning s and on afternoons.

Diurnalvariatio n inRM R

Several investigatorshav e studied inma n temporalorde r ina numbe r of physiological variables.Fo rman yvariables ,ie ,hormon ebloo d levelsan d urinaryhormon e excretions,diurna l rhythmshav ebee nwel l established(22) . Information oncyclica l changesdurin g theda y inhuma nenerg ymetabolis m is scarce.Th e firstpar to f this study isabou tdiurna lvariatio n inpost - absorptive restingmetaboli c rate.Earl y reports (6-8)sho wtha t inma n postabsorptive restingmetaboli c rate increasesdurin g theday .However ,th e resultso f these studiesma yhav ebee n influenced byeffect so fvariatio n during themeasuremen t period inth eduratio no fth epostabsorptiv e state. Thus,metaboli c ratemeasurement s later on theda ywer eusuall yperforme d in subjectsunde r greater postabsorptive stress.Thi sma y initsel f have caused the reported risedurin g theda y inpostabsorptiv e restingmetaboli c rate.I n spite of theabsenc e ofclea rdat ao ndiurna lvariatio n inpostabsorptiv e restingmetaboli c rate inman ,i ti sgenerall ybelieve d that suchvariatio n exists (10-13,23).Obviously , restingenerg yexpenditur evarie sdurin g theda y due to the thermic effectso f feedingan dphysica l activity (9).I naddition , nocturnal restingmetaboli c ratema yb e slightlydifferen t fromdiurna l restingmetaboli c rate (24).Th equestio n is,whethe r there isa diurnal rhythm inma n in restingmetaboli c ratea ta give n fasting state.Whe n sucha rhythmexists ,it sphysiologica l andbiochemica l basiscoul d be related to hormonally-mediated changesdurin g theda y inenergy-demandin g processes,ie , insodiu mpumpin g orprotei n synthesis.A tpresent ,however ,n o diurnal variation in turnover rateso f thermogenichormone sha sbee n reported (25)an d a mechanism forpossibl e diurnalvariatio n in restingenerg yexpenditur e is unclear.

To studydiurna lvariatio n inenerg y expenditure,on e should ideallymeasur e restingmetaboli c rate in subjectsunde r the samecondition s atdifferen t timeso f theday .Usuall y inpreviou s studieso nthi s subject,variatio nwa s introduced inth eduratio no f the fasting statebetwee ndifferen t timeso f the day (6-13). Inth epresen t studymornin g and afternoon testsals ovarie d in

-116- durationo f thepostabsorptiv e state.Bu t incontras t topreviou s studies,th e subjectsha d a shorter durationo f the fasto n theafternoo n tests thano n the morning tests.Thi sma yhav e confounded theafternoo nmeasurement s of resting metabolic ratewit h residualdiet(breakfast)-induce d thermogenesis.However , the results showtha t residualdiet-induce d thermogenesisdi dno t significantly increaseafternoo nRMR' si ncompariso nwit hmornin g postabsorptive RMR's. Incontrast ,averag e restingmetaboli c ratewa s slightly,bu tno t significantly lowero nafternoo n thano nmornin g tests.Th e absence of significantdiurna l variation inpostabsorptiv e resting metabolic ratewa sno tdu e to large inter-individual differences in response totim eo f theda yo fenerg ymetabolism , sincemornin gRM Rvalue swer ehighl y correlated with afternoonRM Rvalues .I naddition ,th e resulto f this studyagree swit h other reports inwhich ,fo r severalhour sdurin g themorning ,n o significant effect of sham feedingwa sobserve d on restingmetaboli c rate (10-13).

Diurnalvariation inDI T

The secondpar to f the studydeal swit h thequestio no f systematicvariatio n with time of theda y inth e response ofenerg ymetabolis m to food. This questionha sno tbee naddresse d before.Th e resultssho wtha t timeo f theda y didno t significantly influencediet-induce d thermogenesis (DIT). Similar to the findings forth eRMR ,thi swa sno tdu e togrea t inter-individualvaria ­ bility indifferenc e inDI Tbetwee nmornin g andafternoo n tests.I naddition , it isunlikel y thatth eabsenc eo fdiurna lvariatio n inDI Twa s causedb ya too shortmeasuremen tperiod ,sinc emea nDI Tha d reduced almost tozer o inth e fourthhou r after ingestiono f themea lbot h inth emornin g as inth eafter ­ noon.DI Twa sassesse dwithou tusin ga noncalori c control test.I tha sbee n argued (23)tha t sucha test should beuse d toasses sdiurna lvariatio n in postabsorptive restingmetaboli c rate.However ,severa l investigators calculate DIT inth ewa ya sha sbee ndon e inthi s study (14-17). Furthermore, whenmetaboli c ratemeasurement so n such testsar e confounded byvariatio n in theduratio no f the fasting statedurin g thetest ,measure d changes in resting metabolic ratema yb e inducedb y anadaptatio n toa n increasingly prolonged fasting state andma yno t reflectdiurna lvariatio ni npostabsorptiv e resting energyexpenditure . Inaddition ,i ti sa matte r of conjecture toinfe r that changes in restingenerg yexpenditur e inth eabsenc e of foodwil l also occur after ingestiono f food. Totalpostprandia l substrateoxidatio nan dnutrien tbalance swer eno tsig -

-117- nificantlydifferen tbetwee nmornin g andafternoo n tests.Ther ewas ,howeve r a transient effect of timeo f theda yo n substrateoxidatio n inth e first,thir d and fourthhou r after ingestiono fth emeal .Th eeffec to f time of theda yo n postprandial substrate oxidation ismos t likely related todifference s between morning and afternoon sessions inth eduratio no f thepostabsorptiv e state.I t canb e expected thatafte r a 12-14hour s fasttota lbod yglycoge n storesar e moredeplete d thanafte r a 6-7 hours fast.Previou s studies inma nhav e shown thatglucos e givenorall yo r intravenously istake nu ppredominantl y bymuscl e for glycogen synthesis (26,27). Ina mor e glycogendeplete d state,th e activity ofglycoge n synthetase seems increased (28),whic hma yb e associated with anenhance d insulin response or insulinactio na t thecellula r level (29). Thismay , inturn ,b e related toa n increased glucoseoxidation , since inth e resting state,glucos eoxidatio nu p tolevel so fabou t 4mg/min.k g is associatedwit h increased glucose storage,ie , glycogen synthesis (30).Suc h high rateso fglucos eoxidatio ndi d notoccu r inthi s study.Du e toth e relatively increased glucose oxidation inth e firsthou r after ingestiono f themeal , lessglucos ema yb eavailabl e inth ecirculatio n foroxidatio n in the third and fourthhou r of thepostprandia l period.Thi sma yenhanc e fat oxidation tomee t the tissuedemand s forenergy .Thi smechanis m is supported by the findingso f thisstudy . Inconclusio nw e can state thattim e ofth eda yha dn osignifican t effect on restingmetaboli c ratean ddiet-induce d thermogenesis.I naddition ,n o systematicdiurna lvariatio nwa sobserve d intota lpr e and postprandial substrate oxidation.However ,hourl ypostprandia l substrate oxidation indicated an increased glucose oxidation in subjects ina mor e prolonged fasting state.

ACKNOWLEDGEMENTS

The authorswoul d like tothan kM rMarce lva n Leuteren for typing themanu ­ scriptan dM sAn s Soffersfo rhelpin gwit h theurinar yurea-nitroge nanalyses . We alsowoul d like tothan k thevolunteer s for their kind and enthousiastic participation inth eexperiment .

-118- REFERENCES

1.Aschof fJ ,Poh lH .Rhythmi cvariation si nenerg ymetabolism .Fe dPro c 1970;29:1541-52. 2.Wakeha mG .Basa lmetabolis man dth emenstrua lcycle .J Bio lChe m1923 ; 23:555-67. 3.William sWW .Th ebasa lmetaboli crate ,basa lbod ytemperatures ,an dth e ovariancycle .A mJ Obste tGyneco l1943;46:662-7 . 4.Solomo nSJ ,Kurze rMS ,Callowa yDH .Menstrua lcycl ean dbasa lmetaboli c ratei nwomen .A mJ Cli nNut r1982;36:611-6 . 5.Web bP .24-hou renerg yexpenditur ean dth emenstrua lcycle .A mJ Cli nNut r 1986;44:614-9. 6.Benedic tFG .Factor saffectin gbasa lmetabolism .J Bio lChe m1915;20 : 263-99. 7.Buskir kER ,Iampietr oPF ,Welc hBE .Varation si nrestin gmetabolis mwit h changesi nfood ,excercis ean dclimate .Metabolis m1957;6:144-53 . 8.Baile yD ,Harr yD ,Johnso nRE ,Kuppra tI .Oscillation si noxyge n consumptiono fma na trest .J App lPhysio l1973;34:467-70 . 9.Wo oR ,Daniels-Kus hR ,Horto nES .Regulatio no fenerg ybalance .An nRe v Nutr1985;5:411-33 . 10.Sharie fNN ,Macdonal dI .Difference si ndietary-induce dthermogensi swit h variouscarbohydrate si nnorma lan doverweigh tman .A mJ Cli nNut r1982 ; 35:267-72. 11.Well eS ,Campbel lRG .Norma lthermi ceffec to fglucos ei nobes ewomen .A m JCli nNut r1983;37:87-92 . 12.Nai rKS ,Hallida yD ,Garro wJS .Thermi crespons et oisoenergeti cprotein , carbohydrateo rfa tmeal si nlea nan dobes esubjects .Cli nSe i1983;65 : 307-12. 13.Feli gP ,Cunningha mJ ,Levit tM ,Hendle rR ,Nade lE .Energ yexpenditur ei n obesityi nfastin gan dpostprandia lstate .A mJ Physio l1983;244 :E45-51 . 14.Schut zY ,Gola yA ,Felbe rJP ,Jéquie rE .Decrease dglucose-induce d thermogenesisafte rweigh tlos si nobes esubjects :a predisposin gfacto r forrelaps eo fobesity ?A mJ Cli nNut r1984;39:380-7 . 15.Tapp yL ,Randi nJP ,Felbe rJP ,Chioler oR ,Simonso nDC ,Jéquie rE , DeFronzoRA .Compariso no fthermi ceffec to ffructos ean dglucos ei n normalhumans .A mJ Physio l1986;250:E718-24 . 16.Illingwot hPJ ,Jun gRT ,Howi ePW ,Isle sTE .Reductio ni npostprandia l energyexpenditur edurin gpregnancy .B rMe dJ 1987;294:1573-6 . 17.De nBeste nC ,Vansan tC ,Weststrat eJA ,Deurenber gP .Restin gmetaboli c ratean ddiet-induce dthermogenesi si nabdomina lan dgluteal-femora lobes e womenbefor ean dafte rweigh treduction .A mJ Cli nNut r1988:i npress . 18.Jéquie rE .Direc tan dindirec tcalorimetry .In :Garro wJS ,Hallida yD , eds.Substrat ean denerg ymetabolism .London :Joh nLibbey ,1985:82-92 . 19 GutmanI ,Bergmeye rHU .Urea .In :Bergmeye rHU ,ed .Method so fenzymati c analysis,2n ded .Ne wYork :Verla gChemi eWeinhei man dAcademi cPres sInc , 1974:1791-8. 20.Lillioj aS ,Bogardu sC ,Mot tDM ,Kenned yAL ,Knowle rWC ,Howar dBV . Relationshipbetwee ninsulin-mediate dglucos edisposa lan dlipi d metabolismi nman .J Cli nInves t1985;75:1106-15 . 21.Sir iWE .Th egros scompositio no fth ebody .Ad vBio lMe dPhy s 1956;4:239-280. 22.Halber gF .Chronobiolgy .An nRe vPhysio l1969;31:675-725 . 23.Garro wJS .Energ ybalanc ean dobesit yi nman .2n ded .Amsterdam : Elsevier/Northhollan dBiomedica lPress ,1978:96-7 . 24.Schut zY ,Bessar dT ,Jéquie rE .Diet-induce dthermogensi smeasure dove ra wholeda yi nobes ean dnonobes ewomen .A mJ Cli nNut r1984;40:542-52 .

-119- 25.Greenspa nFS ,Forsha mPH .Basi c& Clinica lEndocrinolgy .2n ded .Norwalk : Appleton-Century-Crofts,1986 . 26 DeFronzoRA ,Jaco tE ,Jéquie rE ,Maede rJ ,Wahre nJ ,Felbe rJP .Th eeffec t ofinsuli no nth edisposa lo fintravenou sglucose .Result sfro mindirec t calorimetryan dhepati can dfemora lvenou scatherization .Diabete s 1981;30:1000-7. 27.Kat zLD ,Glickma nMG ,Rapopor tS ,Ferrannin iE ,DeFronz oRA .Splanchni c andperiphera ldisposa lo fora lglucos ei nman .Diabete s1983;32:675-9 . 28.Bogardu sC ,Thuille zP ,Ravussi nE ,Vasque zB ,Narimig aM ,Azha rS .Effec t ofmuscl eglycoge ndepletio no ni nviv oinsuli nactio ni nman .J Cli n Invest1983;72:1605-10 . 29.Bogardu sC ,Lillioj aS ,Ston eK ,Mot tD .Correlatio nbetwee nmuscl e glycogensynthas eactivit yan di nviv oinsuli nactio ni nman .J Cli n Invest1984;73:1185-90 . 30 ThiebaudD ,Jaco tE ,DeFronz oRA ,Maede rE ,Jéquie rE ,Felbe rJP .Th e effecto fgrade ddose so finsuli no ntota lglucos euptake ,glucos e oxidation,an dglucos estorag ei nman .Diabete s1982;31:957-63 . Chapter 5. Lack of asystemati csustaine d effect ofprolonge d exercisebout so n resting metabolicrat e infastin g subjects

JANA WESTSTRAT EMSC ,PETE RWEY SBSC ,ERI CPOORTVLIE TBSC ,PAU LDEURENBER G PHD,JOSEP HGA JHAUTVAS TM D

ABSTRACT

Restingmetaboli crat e(RMR )an drespirator yquotien t (RQ)wer emeasure di n nineyoun gnorma lweigh tme nafte ra novernigh tfas to nthre eoccasions ,twic e withan donc ewithou texercis eo nth epreviou sevening .Exercis ewa scyclin g for9 0mi na t10 0W o r17 5W .A Lati nsquar eexperimenta ldesig nwa suse dt o balanceth eorde ro ftreatmen twit hth etyp eo ftreatment .Energ yexpenditur e wasmeasure db yindirec tcalorimetr yusin ga ventilate dhoo dsystem . Nosignifican t10-hour safter-effec to fexercis eo nRM Rwa sfound .Th e differencesi nRM Rbetwee nth econtro lan dth e10 0W o r17 5W treatment swere , respectively,0. 1+ 1. 3 (mean+ sem )percen tan d2. 4+ 1. 6percen to fth e controlvalu e (4.68+ 0.1 3 kJ/min).O nth e17 5W exercis etreatmen tRQ' s decreasedsignificantl yfro m0.8 5+ 0.0 1 (control)t o0.8 2+ 0.0 1 (p<0.05). Thisindicate stha tsingl ebout so fprolonge dexercis eca nhav ea persistin g effecto nth etyp eo fsubstrat eoxydize di nth efastin gstate .

Keywords :indirec tcalorimetry ,exercise ,fasting ,restin gmetaboli crate , respiratoryquotients ,huma n

INTRODUCTION

Thequestio nwhethe rexercis eha sa sustaine deffec to nrestin gmetaboli crat e remainscontroversia l (1-10).Ther ei sn odisagreemen to nth eimmediat eeffec t ofexercis eo nenerg yexpenditur eo roxyge nconsumption .I ti sals ogenerall y acceptedtha tdurin gth epostexercis erecover yperiod ,oxyge nconsumptio n

-120- declines gradually to itspre-exercis e level (11).Thi s increased oxygen consumption after exercise hasbee nknow na s the lacticoxyge ndebt , indicating that itwoul d correspond toth eamoun t ofoxyge nneede d to eliminate the lactateproduce ddurin gexercis e (11).Mor e recently,thi s conceptha sbee nheavil ycriticize d and itwa spropose d to rename the lactic oxygendeb t toexces spostexercis e oxygen consumption (EPOC) (12).Th emajo r parto fEPO Cwoul db edu e toa nelevatio n intissu e temperature and increased levels of circulating catecholamines (12).However ,th ebiochemica l andphy ­ siologicalbasi so fEPO Car eno tye tcompletel y elucidated. Controversy arisesparticularl y inestimatin g themagnitud e andduratio n of EPOC. Estimates of theduratio no f EPOCvar y from fortyminute s tomor e than twenty-four hours (1-10). It isimportan t toclarif y this issue,sinc e a persisting EPOCma y represent a significant losso fenerg y for theindividual . Thisma yb e important forweight-consciou s individuals.Th e reported differences inestimate so f theduratio no fEPO Cma y inpar tb edu e to variation among the studies inmethodolog yuse d toasses sEPOC .Studie s reporting on EPOCdiffe r considerably in,fo rexample ,sampl e size,techniqu e formeasurin g oxygenconsumption ,intensity ,duratio nan d typeo f exercise used,and , incontro l of food intakedurin g thepostexercis e period (1-10). In most studies thenumbe r of subjectswa s less thansi xperson s (1-3,6,9). Such studiesma yno thav e had enoughpowe r todetec t a smallpersistin g EPOC. However,studie s thatha d relatively larger sample sizes (5,7,8,10)als ovar y considerably inmethodolog yo fth eassessmen to fEPOC .Consequently , estimates in these studieso f theduratio nan dmagnitud e ofEPO Car ever ydissimilar . Thepresen t studywa sundertake n todetermin ewhethe r prolonged exercise bouts have a sustained effect onoxyge n consumption,ie ,o n restingmetaboli c rate, measured 10hour safte r theexercise .Tw o levelso fexercis e intensitywer e used todetermin ewhethe r there isa threshol d levelo fexercis e intensity before EPOCwoul d be significantly increased.Sinc eexercis ema y potentiate the thermic effect of food (13),subject swer e fasteddurin gan d after exercise.Th e resultso f this study showtha tmoderat e degreeso fexercise , performed theevenin gbefor eassessmen to f restingmetaboli c rate,d o not systematically affect restingmetaboli c rate.

-121- SUBJECTSAN DMETHOD S

SUBJECTS

Nineyoun gme nparticipate di nth estudy .Volunteer swer einvite dt o participatei nth estud yb yadvertisemen ti nth eUniversit yweekl yperiodical . Eachindividua lwa sfull yinforme do nth enatur ean dpurpos eo fth e experiment,befor egivin ghi sconsen tt oparticipate .Non eo fth esubject swa s ona specia ldie tan dnon ewa susin gan ymedication .Al lsubject swer e non-smokersan dwer epracticin gsport sfo rbetwee n1 an d4 hour sweekly .A questionnairewa suse dt oobtai ninformatio no nth epas tan dpresen thealt h statusan do nth enutritiona lhabit so fth eparticipants .Al lsubject swer e consideredb ya physicia n (JGAJH)t ob ei ngoo dhealth .I naddition ,onl y subjectswer eselecte dwh oha dno texperience dgreate rweigh tchange stha n2. 5 kgi nth ehal fyea rprecedin gth estar to fth estudy .Al lsubject swer eeatin g normalbalance ddiet san dwer einstructe dt okee pt oth esam eeatin gan d drinkingpatter ndurin gth ecours eo fth estudy .Th eprotoco lo fth estud ywa s submittedt oan dapprove do fb yth eMedical-Ethica lCommitte eo fth e WageningenAgricultura lUniversity .

METHODS

Experimentaldesig n

Restingmetaboli crat e(RMR )wa smeasure dthre etime si neac hsubject .RM R measurementswer ealway sperforme dbetwee n08.0 0an d09.0 0hour swit hth e subjecti na 12-1 4hour spostabsorptiv estate .Betwee neac hsessio nther ewa s atim einterva lo fa tleas t3 days .Eac hsubjec tcomplete dth eexperimen t within2 weeks .RM Rwa smeasure do ntw omornings ,afte rth esubject sha d exercisedo na cycl eergomete rth eevenin gbefor eth etest .A RM Rmeasuremen t withoutexercis eo nth epreviou sevenin gserve da sa contro lmeasure .Cyclin g wasalway sperforme dbetwee n20.0 0an d22.0 0hours ,afte rth esubject sha dha d theirevenin gmeal .Th eevenin gmea lwa scompose do fordinar yfood san d provideda nestimate denerg yintak eo f3- 5MJ .Durin gan dafte rth eexercis e period,th esubject swer eallowe dt odrin kwate ronly .Th eexercis eperio d consistedo fcyclin gfo r9 0minute sa trespectivel y10 0W ,ie ,a tabou t25-3 5 percento fmaxima laerobi cpowe r (6,8,10,11),an da t17 5W ,ie ,a tabou t60-7 0 percento fmaxima laerobi ccapacit y (6,8,10,11).Th eexercis ewa sperforme di n

-122- the laboratory and always supervised byon eo f theinvestigators . Sincea systematic additive effecto nRM Rassociate dwit h theorde r of treatment couldno tb e excluded,a Latin-squar e experimental designwa suse d tobalanc e theorde r of treatmentwit h thetyp e oftreatment .T o familiarize the subjectswit h theequipmen t formeasurin g RMR, restingenerg y expenditure wasmeasure d ona separateda ybefor e starting the study inth eafternoo n for about 30-45minutes .

Gasexchang e measurements

Restingmetaboli c rate

On themornin g ofeac hRM Rmeasurement ,th e subjectwa s takent oth e laboratoryb y car atabou t 07.30hours .Afte r a 30minute s restperiod ,RM R wasmeasure d continuously foron ehou rbetwee n 08.00an d 09.00hour s ata n environmental temperature of 24°Cwit h the subject lying ina supine position ona hospita l bed.Durin g theRM Rmeasurements ,th e subjectswatche d video tapes.A ventilated hood systemwa suse d toasses senerg yexpenditure .A transparent plasticventilate d hoodwa splace d over the subjectshea d and made airtight around theneck .A pump (OceanSCL210 ,Dieren ,Th e Netherlands) drawed freshoutsid e air through thehood .Th e flow rate through thehoo dwa s measured by a thermalmas s flowmeter (Brooks,5812N ,Veenendaal ,Th e Netherlands). The flow ratewa skep tbetwee n 30an d 401/mi nb ya nelectri ­ callyoperate d controlvalv e (Brooks,5837 ,Veenendaal ,Th eNetherlands) . A small sample (0.41/min )o f theai r leaving thehoo dwa s analysed for oxygen

(0?)an d carbondioxide (CO.,)conten tby , respectively,a paramagnetic Oj-analyzer,adjuste d toa full scale rangeo f 20-21percen t (Servomex 1100A, Zoetermeer,Th eNetherlands )an d an infrared COp-analyzer, full scale range 0-1 percent (Analytical Development Company,SS100 ,Hoddesdon ,UK) .Th eana ­ lyzerswer e calibrated eachmornin gusin gdrie d gasmixture swit h known0 ,an d CO,content .0 ~an d CO,concentration s indrie d freshoutsid e airwer e measured thricedurin g theRM Rmeasurement .Ai r flow rate,0 ,an d CO, concentration ofth e inflowing and outflowing airwer e computed on line through anautomati c data acquisition system (HewlettPackar d 3497A,Pal o Alto,USA ) interfaced toa microcompute r (HewlettPackar d 85,Pal oAlto ,USA) .

0? and CO,concentratio n of theoutflowin g airan d air flow rate through the hoodwer emeasure d every 30second s and integrated over 5minute s interval periods toobtai nO ,consumptio n (VO_), CO? production (VCO?), respiratory

-123- quotient (RQ)an dRM Raccordin gt oa modificatio no fa nequatio ndescribe db y Jéquier (14), ie: VCO, RMR (kJ/tain)= 4.18 4 .[(4.68 6+ 1.09 6 .(—;— 20.707) ) .VO, ]an d V02 2

RQ= -— 2 .

Bodycompositio n

Fat-freemas san dfa tmas swer eestimate db ydensitometry .Tota lbod ydensit y ofeac hsubjec twa smeasure donc edurin gth eexperimen ti nduplicat eb yth e underwate rweighin gmetho dwit hsimultaneou smeasuremen to fresidua llun g volumeb ya heliu mdilutio ntechnique .Fat-fre emas san dfa tmas swer e calculatedfro mtota lbod ydensit yusin gSiri' sequatio n(15) .

Statisticalanalysi s

Theresult sar eexpresse da smea nvalue swit hstandar derror so fth emea n (sem).Analysi so fvarianc ewa suse dt oevaluat eth edat afro mth eLati n squaredesig n (16).Difference si noxyge nconsumption ,restin gmetaboli crat e andrespirator yquotient sbetwee npair so fdifferen ttreatment swer eevaluate d bypaire dt-test susin ga two-side dsignificanc eleve lo f5 percent .

RESULTS

Table1 show sag ean dsom ephysica lcharacteristic so fth esubject spar ­ ticipatingi nthi sstudy .Th esubject sforme da homogeneou sgrou pwit hrespec t toage ,weight ,bod ymas sindex ,fat-fre emas san dbod yfa tpercentage .Non e ofth esubject sha da bod yfa tconten tlowe rtha n1 2o rhighe rtha n2 0percen t bodyweight . Table2 give sdat ao npostabsorptiv eVO, ,VCO, ,R Qan dRMR ,separatel yfo rth e controlan dexercis etreatments .N osignifican tdifference si nVO- ,VCO ,an d RMRwer efoun damon gth edifferen ttreatments .RMR' so nth e10 0W exercis e treatmentdiffere do naverag e0.0 4+ 0.0 6kJ/mi nfro mcontro lRMR's .Th emea n differencebetwee nRMR' so nth e17 5W exercis etreatmen tan dth econtro lRMR' s was-0.1 1+ 0.0 8kJ/min .Generally ,th eRQ' swer elowe ro nth eexercis e treatmentwhe ncompare dt oth econtro lRQ's .However ,onl yth edifference ,ie ,

-124- 0.034+ 0.013,i nRQ' sbetwee nth e17 5W exercise treatmentan dth econtro l treatment reached statistical significance (p= 0.03).

Table1 .Ag ean dsam ephysica l characteristicso fth esubjects . subjects age ight body mass fat-free body fat index mass* percentage yr kg kg/m kg %

18 81.2 24.4 68.5 15.6 2 24 68.3 21.6 58.4 14.5 3 22 66.6 22.2 54.8 17.6 4 20 83.1 25.4 68.9 17.1 5 21 76.4 21.6 66.4 13.0 6 24 74.4 22.6 61.5 17.3 7 23 74.0 21.7 59.7 19.3 8 28 77.8 21.4 68.2 12.4 9 23 71.3 22.0 57.6 19.2 mean + sem 22.6 + 0.9 74 .8 + 1.8 22 .5 + 0.5 62 .7 + 1.8 16 .2 + 0.8

*assesse d fromtota lbod ydensity .

Table 2: Postabsorptive oxygen consumption, carbondioxide production, respiratory quotient and energy expenditure in subjects with or without previousexercise . subjects control exercise loo w exercise 175 W VD ? voo2 PQ BPE* ™2 m BQ RMR VD2 *3> BQ Em ml/min ml/inin kj/min ml/min ml/min kj/min ml/min ml/min kj/min

1 231 208 0.90 4.73 240 204 0.85 4.86 244 206 0.84 4.94 2 216 173 0.80 4.33 214 181 0.85 4.33 224 178 0.80 4.48 3 202 170 0.84 4.09 209 179 0.86 4.24 222 180 0.81 4.46 4 262 240 0.92 5.39 274 226 0.83 5.52 255 205 0.80 5.11 5 251 212 0.85 5.08 239 195 0.82 4.81 265 211 0.80 5.30 6 229 189 0.83 4.61 218 175 0.80 4.37 221 186 0.84 4.47 7 233 197 0.85 4.72 225 176 0.78 4.49 254 208 0.82 5.11 8 234 195 0.83 4.72 227 190 0.84 4.59 231 195 0.84 4.67 9 218 185 0.85 4.41 225 190 0.84 4.55 227 181 0.80 4.55 mean + 231 197 0.85 4.68 230 191 0.83 4.64 238 194 0.82 4.79 sem 6.0 7.2 0.01 0.13 6.5 5.4 0.00 0.13 5.6 4.5 0.01 0.11 fc> = ootygen ccnsuaption; fa>2 - cadxndioatide production HQ = respiratory quotient; Hfl = resting metabolic rate * RR calculated as HV = 4.184. < (4.686 + 1.096. (RB-0.707) ).*>,) according to a accUfication of an equation published by Jigjuier (14)

-125- tothei r controlvalue .I ntw osubjects ,n o3 an d7 ,th edifferenc e between control RMRan dRM Ro nth e17 5W exercise treatmentwa srelativel y large,ie , between 5an d1 0percent .

resting metabolic rate (kJ/min) 5.80- control 100 W exercise 175W exercis e

U •- 5.20 5

4.60

4.00

I

Figure1 :Postabsorptiv e restingmetaboli c ratei nsubject swit ho rwithou t previousexercise .

DISCUSSION

Thepresen t studywa sdesigne dt odetermin ewhethe r exercise causesa nexces s postexercise oxygen consumption (EPOC)unde r fasting conditions after1 0 hours.Th eresult s showtha tpostabsorptiv e restingmetaboli c rateswer eno t significantly increased1 0hour safte r exercise incompariso nwit h control values.However ,th epostabsorptiv e respiratoryquotient so nth emornin g after the17 5W exercisewer e lowerwhe n comparedt oth econtro lmorning . Thus, exercise,dependin go nit sintensity ,ca nhav ea nafter-effec to nsubstrat e oxidation. Most recent reportso nth eduratio nan dmagnitud eo fEPO Cagre ewit hou r findings (4-6,9), withth eexceptio no ftw ostudie s (7,8).On eo fthes estu -

-126- diesshowe da variabl eeffec to fpreviou sexercis eo nrestin gmetaboli crat e after1 8hours .I naddition ,n osignifican teffec to fexercis eo nsleepin g metabolicrat ewa sfoun d (7).Maehlu me tal . (8)foun dtha toxyge nconsumptio n waselevate dwit habou t1 5pe rcen tfo rabou t1 2hour safte rexercis ei n comparisonwit hcontro lvalues .However ,a clos einspectio no fth edat ao f thisstud yreveal stha toxyge nconsumptio nwa ssignificantl yelevate dfo r about5 hour safte rexercis ean dno tfo rabou t1 2hour safte rexercise .Thi s meanstha tth eresult so fou rstud yd ono tcontras twit hth edat ao fth e latterstudy .Recently ,Bah re tal . (10)reporte dtha t1 2hour sEPO Cwa s proportionalt oexercis eduratio na ta wor kintensit yo f7 0percen to fmaxima l oxygenconsumption .Beside sdifference si nth eintensit yo fth eapplie d exercisebetwee nBah re tal.' sstud yan dth epresen tstudy ,ther ear e differencesbetwee nbot hstudie si nth econtro lo ffoo dintake .I nBah re t al.'sstud ysubject sreceive dmeal sdurin gth eassessmen to fEPOC ,wherea si n thisstud ysubject swer efaste ddurin gan dafte rth eexercise .Par to fth e EPOCobserve db yBah re tal .ma yb erelate dt oa synergisti ceffec to f previousexercis eo ndiet-induce dthermogenesi s(13) . Thisstud ydi dno tsho wa significan tpersistin g1 0hour sEPO Cunde rfastin g conditions.Usin gdat afro mtabl e2 w ecalculate dtha tth eprobabilit yt o detecta nincreas ei nRM Ri nrespons et opreviou sexercis eo fabou t0. 2kJ/mi n (4-5percent )wa s9 5percen tfo rth e10 0W an d9 0percen tfo rth e17 5W exercisetreatment s ((* = 0.05 ,one-sided) .Thi smean stha twit hth epresen t design,increase si npostabsorptiv eRMR' sles stha n4 percen tcoul dno thav e beendetecte dwit hsufficien tpower .Whethe rsmalle rincrease si nRM Rar e physiologicallyimportan ti sa matte ro fconjecture . Onlytw operson sshowe dconsistentl yhighe rRMR' safte rexercis ecompare dt o controlvalues ,an donl ytw operson sshowe drelativel ylarg eincrease si nRM R afterexercise ,ie ,betwee n5 an d1 0percen to fcontro lvalues .I nthes e personsth eincreas ei nRM Rafte rexercis ecoul db erelate dt oa persisten t EPOC.O nth eothe rhand ,eve ni nthes eindividual sdifference si nRMR' samon g thethre etreatment sma yb esimpl ya reflectio no fordinar yintra-individua l variationi nRMR . Usingdat afro mth eliteratur e (6,8,10,11)w eestimate dtha ti nou rsubject s the10 0W exercis etreatmen trepresente da naverag eexercis eintensit yo f 25-35percen to fth emaxima laerobi cpower ,wherea sth e17 5W exercis e treatmentwa sconducte da t60-7 0percen to fth eaerobi ccapacity .Th elatte r treatmentseem scomparabl ei nintensit ywit hth etreatment suse db yBielinsk i etal . (7),Maehlu me tal . (8)an dBah re ta l(10) .Th e10 0W exercis e

-127- treatment iscomparabl e or slightly lower in intensity compared to the exercise leveluse d byPac ye t al. (6).I tha sbee n suggested that variation inestimat e of themagnitud e andduratio n of EPOC is related to differences among thevariou s studies inexercis e intensity (17).Breh m andGuti n (17) suggested the existence of a threshold value ofabou t 50percen t of the maximal oxygen consumption before EPOCwoul d be significantly increased. The 175W exercise treatment employed inthi s study iscertainl y above this threshold value, since our subjectswer e all normal males notengage d in athletics. We calculated that the exercise treatments used inthi s study corresponded to anaverag e energy expenditure of about 2.5 MJ (100W , 90min )an d of about 4 MJ (175W , 90min) .I tcanno t be excluded thatmor e prolonged heavier exercise can cause a sustained increase inenerg y expenditure. Extreme prolonged intense exercise may cause long-lasting metabolic disturbances andma y also result indamag e of the tissues.Restorin g themetaboli c disturbances and tissue damage may, inthi s case,elevat e energy expenditure during recovery for longer periods of time thanafte r less extreme typeso f exercise.O n the other hand, ifEPO C isonl y significantly increased after extreme prolonged heavy exercise, this finding mayb eo fn o practical importance for the average individual engaged in recreational physical activity. The effect of exercise on substrate oxidation has alsobee n reported by other investigators (7-10). Sustained moderate toheav y exercise decreases the body glycogen stores (18)an dma y reduce postabsorptive hepatic glucose production. As a corollary, glucose oxidation byperiphera l tissueswil l be reduced. Instead of glucose, free fattyacid sar euse d as substrates for energy metabolism. Ingeneralizin g the results of this study toothe r situations inwhic h exercise-training isemployed , forexample ,a s anadjuvan t inweigh t reduction programmes,a caveat iswarranted . The results of this study canno t be used toevaluat e thepossibl e use of exercise-training programmes on long-term energy expenditure (19-21). Exercise-training programmesma y induce an increase in the fat-free bodymass ,whic h in itself canelevat e energy expen­ diture (22).Furthermore , exercise programmes mayhav e different biochemical and physiological consequences compared to single sustained bouts of exercise. In conclusionw e can state that single sustained bouts ofmoderat e to heavy exercise did not have a systematic prolonged effect onpostabsorptiv e resting metabolic rate.The ymay ,however , increase theamoun t of fatoxydize d in the postabsorptive state.

-128- ACKNOWLEDGEMENTS

Theauthor swoul dlik et othan kM rM .va nLeutere nfo rhi shel pi nth e conductanceo fth estudy .W ear eals oindebte dt oth evolunteer sfo rthei r participationi nthi sstudy .

REFERENCES

1.Edward sHT ,Thorndik eA ,Dil lDB .Th eenerg yrequiremen ti nstrenuou s muscularexercise .Ne wEng lJ Me d1935;213:532-5 . 2.Passmor eR ,Johnso nRE .Som emetaboli cchange sfollowin gprolonge d moderateexercise .Metabolis m1960;9:452-5 . 3.Vrie sH Ade ,Gra yDE .Aftereffect so fexercis eupo nrestin gmetaboli c rate.Re sQuarterl y1963;34:314-21 . 4.Dalloss oH ,Jame sWPT .Dietar ythermogenesi san dexercise .Pro cNut rSo c 1981;41:35A(abstract) . 5.Freedman-Akaba sS ,Col tE ,Kissilef fH ,Pi-Sunye rFX .Lac ko fsustaine d increasei nVO -followin gexercis ei nfi tan dunfi tsubjects .A mJ Cli n Nutr1985;41:535-9 . 6.Pac yPJ ,Barto nN ,Webste rJD ,Garro wJS .Th eenerg ycos to faerobi c exercisei nfe dan dfaste dnorma lsubjects .A mJ Cli nNut r1985;42 :764-8 . 7.Bielinsk iR ,Schut zY ,Jéquie rE .Energ ymetabolis mdurin gth epost ­ exercise recoveryi nman .A mJ Cli nNut r1985;42:69-82 . 8.Maehlu mS ,Grandmontagn eM ,Newsholm eEA ,Sejerste dOM .Magnitud ean d durationo fexces spostexercis eoxyge nconsumptio ni nhealth yyoun gmales . Metabolism1986;35:425-9 . 9.Devli nJT ,Horto nES .Potentiatio no fth ethermi ceffec to finsuli nb y exercise:difference sbetwee nlean ,obes ean dnon-insulin-dependen t diabeticmen .A mJ Cli nNut r1986;43:884-90 . 10.Bah rR ,Ingne sI ,Vaag e0 ,Sejerste dOM ,Newsholm eEA .Effec to fduratio n ofexercis eo nexces spostexercis e0 7consumption .J App lPhysio l1987;62 : 485-90. 11.Âstran dPO ,Rodah lK .Textboo ko fwor kphysiology .3r ded .Ne wYork : McGraw-HillBoo kCompany ,1986;300 . 12.Gaesse rGA ,Brook sG .Metaboli cbase so fexces spost-exercis eoxyge n consumption:a review .Me dSe iSport sExce r1984;16:29-43 . 13.Youn gJC ,Treadwa yJL ,Balo nTW ,Garra sHP ,Ruderma nNB .Prio rexercis e potentiatesth ethermi ceffec to fa carbohydrat eload .Metabolis m 1986;35:1048-53. 14.Jéquie rE .Direc tan dindirec tcalorimetry .In :Garro wJS ,Hallida yD , eds.Substrat ean denerg ymetabolism .London :Joh nLibbey ,1985:82-92 . 15.Sir iWE .Th egros scompositio no fth ebody .Ad vBio lMe dPhy s 1956;4:239-80. 16.Wine rBJ .Statistica lprinciple si nexperimenta ldesign .2n ded .Ne wYork : McGraw-HillBoo kCompany ,1971 . 17.Breh mB ,Guti nB .Recover yenerg yexpenditur efo rstead ystat eexercis ei n runnersan dnonexercisers .Me dSe iSport sExercis e1986;18:205-10 . 18.Ahlbor gG ,Feli gP ,Hagenfeld tL ,Hendle rR ,Wahre nJ .Substrat eturnove r duringprolonge dexercis ei nman .Splanchni can dle gmetabolis mo fglu ­ cose,fre efatt yacid san damin oacids .J CLi nInves t1974;53:1080-90 . 19.Lenno nD ,Nagl eF ,Stratma nF ,Shrag oE ,Denni sS .Die tan dexercis e traininaeffect so nrestin gmetaboli crate .In tJ Obe s1985;9:39-47 .

-129- 20.Trembla yA ,Fontain eE ,Poehlma nET ,Mitchel lD ,Perro nL ,Bouchar dC .Th e effecto fexercise-trainin go nrestin gmetaboli crat ei nlea nan dmoderat e obeseindividuals .In tJ Obe s1986;10:511-7 . 21.Dal eD van ,Sari sWHM ,Schoffele nPFM ,te nHoo rF .Doe sexercis egiv ea n additionaleffec ti nweigh treductio nregimes ?In tJ Obe s1987;11:367-75 . 22.Web bP .Energ yexpenditur ean dfat-fre emas si nme nan dwomen .A mJ Cli n Nutr1981;34:1816-26 .

-130- Chapter 6. Surprisingly largeimpac t ofpsychologica l stresso n diet-induced thermogenesisbu t not onrestin g metabolic rate

JANA WESTSTRAT EMSC ,KARI NVA NDE RKOO YMSC ,PAU LDEURENBER GPHD , JOSEPHGA JHAUTVAS TM D

ABSTRACT

The effecto fpsychologica l stresso nrestin gmetaboli c ratean ddiet-induce d thermogenesiswa sassesse d in1 2health yyoun gnon-obes emen ,bod yweigh t70. 2 + 1.2k g (mean+ sem),ag e2 5± 0. 6years .Tw otype so fmotio npicture s (video films)wer e shownt oth esubject sdurin gth emeasurements ,ie ,stress-inducin g horror filmsan dromanti c family films (control). Resting metabolic ratean drespirator y quotientswer eno tsignificantl y influencedb yth etyp eo ffil m shownt oth esubjects .Diet-induce d thermogenesiswa ssignicantl y increasedo nth estress-inducin g treatment,0.9 5 + 0.05kJ/mi n (mean+ sem )versu s0.7 6+ 0.0 6kJ/mi n (control). No significant effecto fpsychologica l stresso npostprandia l substrate oxidation rates,nutrien tbalance san durinar y catecholamines excretionwa s observed.

Keywords: indirect calorimetry, restingmetaboli c rate,diet-induce d thermogenesis,stress ,anxiety ,human .

INTRODUCTION

Dailyhuma nenerg y expenditure consistso fthre emajo r components (1,2).Th e largestamon g these isth ebasa l (BMR)o rrestin gmetaboli c rate (RMR).Th e second componenti sth eenerg yexpenditur edu et oth eperformanc eo fexterna l work.Th ethir dmajo r componenti sth eincreas e inrestin gmetaboli c ratedu e

-131- toth ethermi ceffec to ffood ,als oknow na sdiet-induce dthermogenesi s(DIT) . RMRan dDI Tcontribut eabou t70-8 5percen tt odail yenerg yexpenditur ei nth e averageindividual . Inrecen tyear sther eha sbee nconsiderabl einteres ti ninter-individua l differencesi nRM Ran dDI T (3-9).Inter-individua lvariatio ni nRM Rprimaril y reflectsdifference sbetwee nindividual si nbod yweigh tan dbod ycompositio n (5,9).Controvers yexist so nth enatur ean ddeterminant so finter-individua l variationi nDI T (3,6,8). Conflictingresult si nthi sare ao fresearc hmigh tb erelate dt oth e methodologyuse dfo rmeasurin gth ethermogeni crespons et omea lingestio n (10).Variou sinvestigator s(5,7,11 )repor ttha tsubject swer eentertaine db y musico rmotio npicture sdurin gth eassessmen to fenerg yexpenditure .Thi sma y haveha da nimpac to nth ementa lstat eo fth esubjec tan dlikewis eo nenerg y expenditure.Unfortunatel yther ear ever ylittl edat ao nth eimpac to f disturbanceo fth ementa lstat eo nRM Ran dDIT . Thepurpos eo fthi sstud ywa st odetermin ewhethe remotionall yupsettin gan d frighteningmotio npicture saffecte dRM Ran dDI Ti ncompariso nt oromanti c familyfilms .W ehypothetize dtha ta nemotionall yupsettin gfil mwoul dlea dt o anincreas ei nenerg yexpenditur emediate db ya stimulatio no fth esympatheti c nervoussystem .Urinar ycatecholamine sexcretio nwa suse da sa ninde xo f sympatheticactivit y(12) .

SUBJECTSAN DMETHOD S

SUBJECTS

Twelveyoun ghealth ynorma lweigh tme nparticipate di nthi sstudy .Subject s wereeithe rgraduate so rundergraduate so fth eUniversity .Al lsubject swer e agedbetwee n2 2an d2 9year s(2 5year s+0. 6mea n+ sem) ,ha da bod ymas s 2 2 (Quetelet)inde xbetwee n18. 9an d22. 9kg/ m (21.0± 0. 3kg/ m )an da bod yfa t percentagebetwee n7 an d2 2percen t (15.0+ 1. 4percent) .Bod yfa tpercentag e wasassesse db ydensitometr ya spreviousl ydescribe d (11).Al lsubject swer e apparentlyhealth ya sevaluate db ya medica lquestionnaire .Non eo fth e subjectswa so na die ta tth etim eo fth estudy ,use dan ymedicatio no rha d experienceda bod yweigh treductio no fmor etha n2. 5k gi nth ehal fyea r precedingth estud yo rha dglycosuria .Al lsubject swer eeatin gnorma l balancedmeals .Th eprotoco lwa sfull yexplaine dt oth esubjects ,bu tnon eo f -132- the subjectskne wth epurpos eo f thestudy .Subject swer e informed thatth e studywa s intended to investigate theeffec to fmea l ingestionan dbod y temperature on totalbod y impedance,a measur e ofbod y composition (13).Th e protocol of the studywa s submitted toan dapprove d ofb y the Medical-Ethical Committee of theWageninge nAgricultura l University.

METHODS

Experimental design

Ineac h subject energyexpenditur ewa smeasure d on fourmorning sbetwee n 08.00 and 13.00hour swithi na perio d of sixweeks .Betwee neac h testwa s atime - interval ofa tleas ttw odays . In the firstexperimen t (A),th eeffec to fpsychologica l stresso n resting metabolic rate (RMR)wa smeasure d induplicat e ineac h subjectafte r a 12-14 hoursovernigh t fast.Hal f ofth e subjects,randoml y selected, started the firstRM Rmeasuremen twit h the stress-inducing treatment (horror films)an d switched to thecontro l treatment (romantic-family type films)afte r 1.5 hours.Th eothe rhal f of the subjects received thetreatment s inth e reverse order.A t the secondRM Rmeasuremen t the sameprocedur ewa s followed,bu t subjectswh oha d started the firstRM Rmeasuremen twit h the stress-inducing treatmentno w startedwit h thecontro l treatment andvic eversa . In the second experiment (B),diet-induce d thermogenesis (DIT)wa smeasure d twice ineac h subjectafte r ingestiono fa standardizedmixe d liquid testmea l witha nenerg yconten to fo naverag e 1900k J (10.8percen tprotein ,32. 9 percent fat,56. 3percen t carbohydrates).Subject swer e randomlyallocate d to the stress-inducing or control treatment after ingestion of themeal . Energy expenditurewa sassesse dwit ha ventilate d hood systema spreviousl y described (11).Fo ra DI Tassessment ,RMR , ie,premea l "baseline" resting energyexpenditure ,wa sbase d ona non ehou r continuous registration of oxygen consumption and carbondioxide production,whil e the subjectswatche d 'normal', ie,control ,motio npictures .Afte rmea l ingestionpostprandia l energy expenditure (PEE)wa smeasure d continuously for fourhours .Th e difference between themea nPE Ean d theRM Rwa suse d tocalculat eDI T inkJ/mi nand , integrated over the totalmeasuremen tperiod , inkJ . After theDI Tassessment ,urin ewa s collected fordeterminatio n of catecholamines excretion rates.Catecholamine s excretion rateswer e also

-133- assessed inurin e collected over the night preceding the DITmeasurements . These valueswer euse d asbaselin e excretion rates.Catecholamine s were analyzed according toSmede s et al. (14)an dWesterin k et al.(15) . Substrate oxidation rates and nutrient balanceswer e calculated using the nonprotein respiratory quotients and urinaryurea-nitroge n excretion rates for estimating'proteinoxidatio n asdescribe d by den Besten et al.(11) .

Statistical analysis

Significance ofdifference s in several variables between the experimental conditionswa sevaluate dwit h two-sided paired t-tests.Al lvalue s are given asmea n + sem.

RESULTS

resting metabolic rate (kJ/min)

5.00

4.90

4.80

4.701

46 0

4.50 a stress • control

30 60 90 1ST 150 mm

Figure 1.Restin g metabolic rate (mean+ sem)measure d for 2.5 hours in the same subjects on two separatemorning swit h different types of motion pictures (• = control, D= stress-inducing) shown to the subjects during each measurement. -134- Figure 1show sRMR' smeasure d inexperimen tA .Ther ewa sn o significant difference inRM Rbetwee nth etw otreatments .O naverag eRM Rincrease d0.0 4 kJ/min, less than1 percent ,whe n filmswer e changed from stress-inducingt o control type. Figure2 show spre -an dpostprandia l energy expenditure measured inexperimen t B. RMR'swer eno tsignificantl y different between thetw otreatments , ie,4.6 9 + 0.10kJ/mi n (control)versu s4.6 7+ 0.0 8kJ/mi n (stress). Diet-induced thermogenesis,wa ssignificantl y highero nth estress-inducin g treatment,ie , 0.95 + 0.05kJ/mi n compared to0.7 6+ 0.0 6kJ/mi n forth econtro l treatment (p=0.021).Th edifferenc e intota l integratedDI Tbetwee nth etreatment swa s about 45+ 1 7k jo r2. 5+ 0. 9percen to fth eenerg y contento fth etes t meal. Average postprandial respiratory quotientsdi dno tdiffe r significantly between both treatments.The ywere r respectively, 0.91+ 0.006an d0.9 1± 0.009.

pre-and postprandial metabolic rate (kJ/min)

6.00

5.50

5.00

Figure 2.Postprandia l energy expenditure (mean+ sem )measure d for4 hour si n the same subjectswit h (a)o rwithou t (•) stress-inducing motion pictures.

Table 1give s urinary catecholamines excretion forth etw otreatments . Baseline levelsfo rth eexcretio no fcatecholamine swer eno tsignificantl y

-135- differentbetwee n the treatments,neithe rwer e the excretion rates measured over the treatment period or ratioso fbaselin e and treatment period excretions.Tota l or individual catecholamines excretion rateso r baseline treatment period ratioswer e not significantly correlated withDIT .

Table 1.Urinar y catecholamines excretion inme nbefor e anddurin g energy expenditure measurements underdifferen tattentio n diverting conditions.

Catecholamines Control (n=12) Stress (n=12) before* during"!"during/befor e before during during/before /yg/h fjg/h /jg/h //g/h adrenaline 0.43+0.09 0.81+0.10 2.72+0.46 0.34+0.08 0.81+0.09 3.18±0.50 noradrenaline 1.53±0.14 2.31±0.15 1.66+0.19 1.35±0.08 2.09+0.18 1.61±0.20 dopamine 17.3+1.7 22.9±2.5 1.41+0.14 16.9+0.9 21.0±1.7 1.23±0.06

Allvalue sar e givena smea n+ sem *measure d overa n 11hour sperio d immediately before the starto f the energy expenditure measurements + measured over a 5.5 hoursperio d during energyexpenditur e measurements

Postprandial substrate oxidation ratesan d nutrient balances didno t differ either between both treatments (datano t shown).

DISCUSSION

This study showsa surprisingly large impact ofpsychologica l stress on diet-induced thermogenesis,bu tno t on restingmetaboli c rate. Most reviews on factors affecting energy output inhuman smentio n that anxiety or stressma y influence energy expenditure (16,17,18).Relativel y fewstudies , however,giv e quantitative data on the impacto f some sort ofemotiona l upset onhuma n energy expenditure (19-22). The resultso f the latter studies do not permit anaccurat e estimation of the impact of stresso nenerg y expenditure. Only one recent study reportso n the impact ofa mor eusua l type of stress, ie,o f arduous clericalwork ,o ndail y energy expenditure(23) . In the studyher e reported, stresswa s induced bymotio npictures .Th eus e of a motion picture't o induce stressunde r laboratory conditions is common practice in the field ofexperimenta l psychology (24).I twa sexpecte d that an emotionally upsetting and frightening filmwoul d lead toactivatio n of the sympathetic nervous system, causing a rise inenerg y expenditure.T o our surprise no significant effect of stresso nRM Rwa s found,wherea s DITwa s -136- significantly increased byabou t 0.20 kJ/min.O naverag eonl y 0.04 kJ/mino f this increasema yhav ebee ndu e toth e impacto f stresso nRMR .Th e absence of aneffec to f stresso nRM R indicatestha tunde r fastingcondition sa n emotionallyupsettin g film isno ta sufficient stimuluso f the sympathetic nervous system.Th ecombinatio no fexposur e topsychologica l stressan d ingestion of food resulted inincrease d rateso fenerg yexpenditure , suggesting a synergistic actiono f stressan d food ingestiono n the activity of the sympathetic nervous system. Ingestion of food,especiall yo fcarbohydrates ,affect sth eactivit yo f the sympathetic nervous system (25).A relatively largepart , 30-40percent ,o f the totalDI T response isthough tt ob emediate d bya n increase inth e activityo f the sympatheticnervou s system (2). An increased activityo f the sympathetic nervous system isusuall y reflected in increased urinary catecholaminesexcretio n rates (12).I nthi s study,however ,n o significant correlationwa s foundbetwee n totalDI Tan durinar ycatecholamine s excretion rates,suggestin g thaturinar y catecholaminesexcretio n ratesar eno ta sensitive indicator of stimulationo f the sympatheticnervou s system. Postprandial substrateoxidatio n ratesan dnutrien tbalance s indicated no significantdifference sbetwee n the treatments inth emetabolis mo f the ingestednutrients .Thi s indicates that theeffec to f stresso nDI Twa sdu e to other factors increasing thermogenesis,fo rexampl eb y a stimulationo f ionic pumping (26)o r substrate cycling in skeletalmuscle s (27).I naddition ,i ti s possible that skeletalmuscl e tonewa shighe r in subjectsexpose d toth e stresstreatmen t incompariso nwit h thecontro l treatment.Th eenerg y expended by such isometricmuscl e contractionma yhav e elevated total energy expenditure.Finally ,tota lpostprandia l energyexpenditur ema yhav e been elevated bymino r movements.However ,a n increase inmuscl e tonean d inmino r movementsdu e tostres swoul d alsohav e elevated RMR,whic hwa sno t found. Whatever the cause of the increased DIT,thi s studyclearl y shows that stress-inducingmotio npicture s can significantly influence diet-induced thermogenesis.Th e findingso f this study implicate that investigators ofRM R and DIT should explicitly stateunde rwha t conditions energy expenditurewa s assessed.

-137- ACKNOWLEDGEMENTS

Theauthor swoul dlik eth othan kM rRoe lBruntin ko fth eInstitut eo fAnima l Physiologyo fth eUniversit yo fGroninge n(th eNetherlands )fo rth eanalyse s ofth eurinar ycatecholamines ,M rMarce lva nLeutere nfo rpreparin gth e manuscriptan dlast ,bu tno tleast ,th esubject sfo rthei renthousiasti c participationi na sometime sstressfu lexperiment .

REFERENCES

1.Danfort hE .Die tan dobesity .A mJ Cli nNut r1985;41:1132-45 . 2.Sim sEAH ,Danfort hE .Expenditur ean dstorag eo fenerg yi nman .J Cli n Invest1987;79:1019-25 . 3.Pitte tP ,Chappui sP ,Acheso nK ,d eTechterman nF ,Jéquie rE .Thermi c effecto fglucos ei nobes esubject sstudie db ydirec tan dindirec t calorimetry.B rJ Nut r1976;35:281-92 . 4.Jame sWPT ,Davie sHL ,Baile sJ ,Daunce yHJ .Elevate dmetaboli crate si n obesity.Lance t1978;1:1122-5 . 5.Ravussi nE ,Burnan dB ,Schut zY ,Jéquie rE .Twenty-four-hou renerg y expenditurean drestin gmetaboli crat ei nobese ,moderat eobese ,an d controlsubjects .A mJ Cli nNut r1982;35:566-73 . 6.Feli gP ,Cunningha mJ ,Levit tM ,Hendle rR ,Nade lE .Energ yexpenditur ei n obesityi nfastin gan dpostprandia lstate .A mJ Physio l1983;244:E45-51 . 7.Schut zY ,Bessar dT ,Jéquie rE .Diet-induce dthermogenesi smeasure dove ra wholeda yi nobes ean dnonobes ewomen .A mJ Cli nNut r1984;40:542-52 . 8.Sega lKR ,Guti nB ,Nyma nAM ,Pi-Sunye rFX .Thermi ceffec to ffoo da trest , duringexercise ,an dafte rexercis ei nlea nan dobes eme no fsimila rbod y weight.J Cli nInves t1985;76:1107-12 . 9.Bogardu sC ,Lillioj aS ,Ravussi nE ,Abbot tW ,Zawadzk iJK ,Youn gA , KnowlerWC ,Jacobowit zR ,Mol lPP .Familia ldependenc eo fth erestin g metabolicrate .N Eng lJ Me d1986;315:96-100 . 10.Jéquie rE .Energ yexpenditur ei nobesity .Cli nEndocrino lMeta b 1984;13:563-80. 11.De nBeste nC ,Vansan tG ,Weststrat eJA ,Deurenber gP .Restin gmetaboli c ratean ddiet-induce dthermogenesi si nabdomina lan dgluteal-femora lobes e womenbefor ean dafte rweigh treduction .A mJ Cli nNut r1988;47:840-7 . 12.Fitzgeral dGA ,Doller yCT .Biochemica lindice so fsympathi efunctio ni n man.TIP S1979:84-7 . 13.Deurenber gP ,Weststrat eJA ,Hautvas tJGAJ .Durin gweigh tlos schange si n fatfre emas smeasure db ybioelectrica limpedanc ediffe rfro mchange si n fatfre emas smeasure db ydensitometry .A mJ Cli nNut r1989 :i npress . 14.Smede sF ,Kraa kJC ,Popp eH .Simpl ean dfas tsolven textractio nsyste mfo r selectivean dquantitativ eisolatio no fadrenaline ,noradrenalin ean d dopaminefro mplasm aan durine .J Chro m1982;231:25-9 . 15.Westerin kHC ,Boske rFJ ,O'Hanlo nJF .Us eo falumina ,Sephade xG—1 0an d ion-exchangecolumn st opurif ysample sfo rdeterminatio no fepinephrine , norepinephrine,dopamine ,homovanilli caci dan d5-hydroxyindoleaceti caci d inurine .Cli nChe m1982;28:1745-8 . 16.Garro wJS .Energ ybalanc ean dobesit yi nman .2n ded .Amsterdam :Elsevie r /NorthHollan dBiomedica lPress ,1978 .

-138- 17.Wo oR ,Daniels-Kus hR ,Horto nES .Regulatio no fenerg ybalance .An nRe v Nutr1985;5:411-33 . 18.Sim sEAH .Energ ybalanc ei nhuma nbeings :th eproblem so fplenitude .Vi t Horm1986;43:1-101 . 19.Landi sC .Studie so femotiona lreactions .IV .Metaboli crate .A mJ Physio l 1925;74:188-203. 20.Blaz aSE ,Garro wJS .Th eeffec to fanxiet yo nmetaboli crate .Pro cNut r Soc1980;39:13 A(abstract) . 21.Damask ,MC ,Askanaz iJ ,Weissma nC ,Elwy nDH ,Kinne yJM .Artifact si n measurementso frestin generg yexpenditure .Cri tCar eMe d1983;11:750-2 . 22.Udalo vJu ,Sibunee vAG .Effec to fnervou sstrai no nsom easpect so f metabolism.Nut rAbst rRe v1964;34:524-5 . 23.Webste rJS ,Wels hG ,Pac yP ,Garro wJS .Descriptio no fa huma ndirec t calorimeter,wit ha not eo nth eenerg ycos to fclerica lwork .B rJ Nut r 1986;55:1-6. 24.Lazaru sRS ,Speisma nJC ,Mordkof fAM ,Daviso nLA .A laborator ystud yo f phychologicalstres sproduce db ya motio npictur efilm .Psycho lMonograp h 1962;76:43-51. 25.Well eS ,Lilaviva tU ,Campbel lRG .Thermi ceffec to ffeedin gi nman : increasedplasm anorepinephrin elevel sfollowin gglucos ebu tno tprotei n orfa tconsumption .Metabolis m1981;30:953-8 . 26.Himms-Hage nJ .Brow nadipos etissu emetabolis man dthermogenesis .An nRe v Nutr1985;5:69-94 . 27.Faghe rB ,Liebhol dH ,Montr iM ,Morrit zU .Thermogenesi si nhuma nskeleta l musclea smeasure db ydirec tmicrocalorimetr yan dmuscl econtractil e performancedurin gß-adrenocepto rblockade .Cli nSe i1986;70:435-41 .

-139- Chapter 7. Nature and magnitude of inter-individueldifference s inrestin g metabolicrat ean d diet-induced thermogenesisi nlea nan dobes e individuals

JANA WESTSTRAT EMSC ,PAU LDEURENBER GPHD ,JOSEP HGA JHAUTVAS TM D

ABSTRACT

Restingmetaboli crat e(RMR )wa smeasure di n15 4subjects ,7 6non-obes emen , 44non-obes ewome nan d3 4obes ewomen .Diet-induce dthermogenesi s (DIT)an di n vivofue lutilizatio nrate swer emeasure di na subgrou po f12 6individuals ,5 5 men,3 7non-obes ean d3 4obes ewomen .RM Rwa spositivel yassociate d (r= 0.80 ) withth esiz eo fth efat-fre emas s (FFM),ie ,RM R(kj/d )= 181 3+ 86*FF M(kg) . The5t han dth e95t hpercentil evalue sfo rth edistributio no fth eRM Rpe rk g fat-freemas s(FFM )wer e10 0an d15 0kJ/d ,respectively .RM Rpe rk gFF Mwa s significantlylowe ri nme ncompare dt owomen ,11 5+ 1 (mea n+ sem )kJ/ dversu s 126+ 2 kJ/ d (p<0.001).RM Radjuste dfo rFFM ,ag ean dfa tmas swa s significantly (p<0.05)lowe ramon gobes ewome nwit ha non-abdomina ltyp eo f bodyfa tdistributio ncompare dt onon-obes emen ,non-obes ewome nan dobes e womenwit ha nabdomina ltyp eo fbod yfa tdistribution .Inter-individua l variationi nRMR ,adjuste dfo rFFM ,ag ean dfa tmass ,wa ssimila ramon gth e variousgroup san daverage d1 0percent . DITt oa mixe dtes tmea lo f177 5+ 3 4k Jwa s7. 5+ 0. 2percen to fth eingeste d energy.DI Twa spositivel y (r= 0.52 )relate dt oth eenerg yconten to fth e testmeal .Th e5t han d95t hpercentil epoint sfo rth edistributio no fDI Twer e 4.0an d12. 0percen t (ofenerg yconten to ftes tmeal) ,respectively .DI Twa s significantlyhighe ri nme ncompare dt owomen ,8. 4+ 0. 3versu s6. 8+ 0. 3 percent (p<0.001).I nmen ,o naverag e7 7percen to fDI Twa scalculate dt ob e relatedt oenerg ycost sfo rprocessin gth eingeste dnutrients ,i nwome nthi s waso naverag e9 9percent .I neac hsex ,DI Twa sno tsignificantl yassociate d withFFM ,bod yweigh to rbod yfa tcontent .Inter-individua lvariatio ni nDI T amongth evariou sgroup saverage d3 6percen tan dwas ,respectively ,3 0percen t

-140- inmen ,4 3percen t innon-obes ewome nan d 26percen tamon gobes ewomen . Postabsorptive andpostprandia l inviv o fuelutilizatio npattern swer e similar inme nan dwomen .Afte rmea l ingestioncarbohydrat e oxidation increased from onaverag e 37t o5 1percen to f total energyexpenditur e and fatoxidatio n decreased fromo naverag e 40t o2 6percent .O naverag e lesstha n 5percen to f the ingested glucosewa s converted tofat . It isconclude d thata relatively lowRM Ro rDI T isno ta n exclusive characteristic of theobese . keywords: indirectcalorimetry , restingmetaboli c rate,diet-induce d thermogenesis, fuelutilization ,men ,women ,obesity , inter-individual variation

INTRODUCTION

Energy requirements foradult sar e assumed tob eprimaril ydependen t onage , sex, bodyweigh t andphysica l activity (1).Th e current theoryo nhuma n energy requirementsattribute svariatio n inenerg yneed samon g individualso f given age, sexan dbod yweigh t todifference s inth edegre eo fphysica l activity.I t isassume d thatage ,se xan dbod yweigh tdetermin emaintenanc e requirements to which incrementsar e added,dependin g onphysica l activity, toderiv e total energyneeds .Maintenanc e requirementswoul d be indicated by the level of the resting ("basal")metaboli c rate (RMR). TheRM R canb edirectl yassesse dusin g (in)direct calorimetryo r itca nb e estimated froma variet y of simple prediction equations (2-7). Inadditio n toth eenerg yexpende d for maintenance and physical activity,energ y isdissipate d by thebod y inth eprocessin g of ingestednutrients ,ie ,i ndiet-induce d thermogenesis (DIT)(8) . Thevalidit yo f thecurren t theoryo nhuma nenerg y requirementsmay ,however , be questioned.A t first,Sukhatm ean dMarge n (9)hav epointe d out,tha t individuals canadap t tovaryin generg y intakesno tb yalteration s inth e level ofphysica l activity,bu ta sa resulto f changes inth eefficienc y of energyutilization .Thes e changeswoul dmanifes t themselves inadaptiv e fluctuations ofth e level ofenerg yexpenditur e inon eo rmor e componentso f dailyenerg y output.Whethe r individualsma y showth e relatively large changes inefficienc yo fenerg yutilizatio na spropose d by Sukhatme andMargen , isa t present amatte r of conjecture.Secondly ,evidenc e isaccumulatin g from metabolic studies for inter-individual differences in resting energy

-141- expenditure among individualso fgive n sex,age ,bod yweigh t and body composition (10,11).However ,th enatur ean dmagnitud e of these differences remain tob edefinitel yassessed .Resolvin g this issuema yb e important for various reasons,ie ,fo rth ediagnosi s ofmetaboli c disordersa shypo -an d hyperthyroidism, for the refinemento festimate so fhuma nenerg y requirements, and, for the identification,amon g the lean,o f individuals,tha twoul d be at increased risk forbod yweigh tgain ,and ,amon g theobese ,o f individuals that would beparticularl y resistant tobod yweigh t reduction. Inth epresen t study restingmetaboli c ratean ddiet-induce d thermogenesis weremeasure d ina groupo f 154me nan dwomen ,an d ina subgroupo f 126 persons, respectively.Th eaim so fth e studywer e toasses sth emagnitud e and determinants of inter-individual differences inRM Ran d DIT.Informatio nwa s alsoobtaine d ina subsampleo f 91 subjectso nth e intra-individualvariatio n inRM Ran dDIT .

SUBJECTSAN DMETHOD S

SUBJECTS

Between January198 6an d august 1988 restingmetaboli c rate (RMR)wa smeasure d ina grou po f 154subjects ;i n12 6o fthes e individuals diet-induced thermogenesis (DIT)wa sals oassessed . The subjectsparticipate d inongoin g studieso nmethodologica l aspectso f the measurement ofpostprandia l energyexpenditure ,o r instudie so n (non)-nutritional influenceso nRM Ran dDIT .Durin g thisperio d a totalo f 570 RMRmeasurement s and a total of 360DI Tmeasurement swer eobtaine d inthes e subjects.Volunteer swer e invited toparticipat e inth e studiesb y advertisements in regionalnewspapers ,th eUniversit yweekl yperiodical ,b y poster or through informal contacts.Afte r applying forparticipation , subjects received a letter informing themo n thenatur ean d purpose of the experiment.Subject s also received oneo r twoquestionnaire(s ) for obtaining informationo npas tan d presenthealt h status,nutritiona l habits,smokin gan d (alcoholicbeverages )drinkin gbehaviour ,habitua l activitypatter nan d sporting activities.Subject seligibl e forparticipatio nwer e invited fora firstvisi t toth e laboratory to familiarize themwit h theapparatu s for measuring energy expenditure.Afte r giving their informed consent subjects were allowed toparticipate .Th eprotoco l ofeac h studywa s submitted toan d

-142- approved ofb y theMedical-Ethica l Committeeo f theWageninge nAgricultura l University. Subjectsparticipatin g inth e studieswer e all inapparen t good healthha dn oevidenc eo fpas to rpresen t thyroiddisorder s ordiabete s mellitus,n oglycosuri a orproteinuria ,wer eno tusin gdrug sknow n toaffec t energymetabolism ,wer e alleatin gnorma lbalance ddiets ,smoke d less than1 0 cigarettes/d,drun k nomor e than 5alcoholi cdrinks/d ,di d notexercis e more than 7hours/Week ,an dha dha d stable (within2. 5 kgo fbod yweight at entry) bodyweight s atleas t 6month sprio r toth ebeginnin g of the study.Wome nwer e inth epremenopausa l state.I nth eobes e fastingbloo d glucosevalues wer e all within thenorma l range,ie ,betwee n 3.9 and 5.6 mmol/1(12) .

METHODS

Gasexchang e measurements

Gasexchang e measurementswer eperforme dwit h aventilate d hood system,whic h hasbee ndescribe d indetai lbefor e (13,14).Briefly ,a subject'shea d is coveredwit h a transparentperspe xhoo dwit ha nai r inleto nto pan d anai r outlet on the side.Th ehoo d ismad e airtightaroun d thenec kwit ha cord. Freshai r isdraw n throughth ehoo d bynegativ epressur e created by apum p placed downstream. The freshai rmixe swit h the subjectsexpire d air,whic h causesa decreas e inoxyge n contentan d an increase incarbondioxid e content of theai r leaving thehood , relative toth ega s concentrationso f theai r entering thehood .Accurat e measurement ofth eoxyge n (Servomex 1100A, Zoetermeer,th eNetherlands )an d carbondioxide (Analytical Development Company SS100,Hoddesdon ,UK )conten to f the inflowing andoutflowin g air combined withmeasuremen to f thevolum eo fai r (Brooksmassflowmete r 5812N,Veenendaal , theNetherlands ) flowing through thehoo dpermit sa calculatio no f the amount of oxygenuse db y the subjectan d theamoun to f carbondioxide produced. The analyzersan d flowmetervoltag e outputswer edigitize d (HewlettPackar d 3497A, PaloAlto ,US )an d connected toa deskto pcompute r (HewlettPackar d 86Bo r85 , PaloAlto ,US) .Continuou s integrated calorimetricmeasurement swer e recorded by the computer andprinte d over 2o r 5minute s intervals.Th e rateo f oxygen consumption and carbondioxide production serve asa basi s for the calculation ofmetaboli c rate.Metaboli c ratewa s calculated bya compute r using an equationpublishe d byJéquie r et al. (15).Fro m repeated ethanol combustions

-143- we calculated a randomerro ro f 2.8 percento fenerg yexpenditur e measurements with theventilate d hood system.

Restingmetaboli c rate

Restingmetaboli c rate (RMR)wa sgenerall ymeasure d inth emornin g between 7.30 hoursan d 9.00 hours,afte ra novernigh t fast,fo ra perio do fon ehou r continuously after a 30minute sacclimatizatio nperiod .Subject swer e instructed to refrain fromheav yphysica l exercise theevenin gbefor e theRM R measurements.Durin g theacclimatizatio nperio d thehoo dwa salread yplaced , but subjectswer e stillallowe d tomov e inorde r tofin da mos t comfortable position for theRM Rmeasurement .Subject swer emeasure d ina supine (semi-recumbent)positio n ina nois ean d temperature controlled (23-26°C)roo m during theRM Rmeasurement .Subject swer e asked to remainmotionless ,bu t awake. Subjectswatche dmotio npicture sdurin g themeasurement . Insom e subjectsRM Rwa smeasure d inth eafternoo nbetwee n 13.30an d 15.00hour swit h the subjects ina 4.5 hourspostabsorptiv e stateafte ra standardized breakfasto f about 2MJ .W e havepreviousl y showntha tRMR' smeasure d under these conditions inth emornin gd ono tdiffe r systematically fromRMR' s measured inth eafternoo n (13).RM Rwa s calculated omittingmetaboli c rate readingsdisturbe d by spontaneousmovement so rhypo -o rhyperventilation . For 103 subjects repeated RMRmeasurement swer e available toasses s intra-individualvariabilit y inenerg yexpenditure .

Diet-induced thermogenesis

In12 6 individualsdiet-induce d thermogenesis (DIT)wa smeasured .Afte r an initial RMRmeasuremen t subjects received amixe d liquidmea l composed of yoghurt,sucrose ,orang e juice,vegetabl e fatan d aprotei n supplementwit h an averagenutrien t compositiono f 12percen tprotein ,5 5percen t carbohydrates and 33percen t fat.Th eenerg y contento f the testmea lvaried ,dependin g on the type of study,betwee nabou t 1300k Jan d 2600kJ .Mea nenerg y contentwa s 1775+ 34kJ .Th e liquidmea lwa seate nwithi n 5minutes .Th emea lwa s sipped througha plasti c tubewhic hwa spasse d througha nopenin g inth ehood . Fifteen subjects received anorma l solidmixe dmea lwhic hwa s eatenwithi n 15 minutes.N o systematicdifferenc e inDI Tbetwee na liquid or solidmea lwa s observed (14). After the subjectsha d eaten theirmeal ,energ yexpenditur ewa s measured continuously fora perio d of 3-4 hours,dependin g on the typeo f

-144- study.Th eduratio no f thepostprandia lmeasurement sperio dwa s onaverag e 224 + 2minutes .DI Twa sdefine d asth e cumulative increase inenerg y expenditure aftermea l ingestionabov e thepremea l energyexpenditure , ie,th eRMR . In some subjectsenerg yexpenditur e at theen do f thepostprandia l period had decreased toa levelbelo wth ecorrespondin gRMR .Fo r these subjectsa new baseline for thecalculatio no fDI Twa sdefined , ie,th emea no f theRM Ran d theenerg yexpenditur ea tth een do fth epostprandia lperiod .Th e energy expended for theprocessin g ofth e ingestednutrient s (inkJ )wa sdivide d by the energy contento f thetes tmea l toexpres sDI T (16).Thi sallow sa comparison of thermic responses tomeal swit hdifferin g energy contents(17) . In9 1o f the subjects repeated DITmeasurement swer e obtained toasses s intra-individualvariability . The energy contento f thetes tmeal swa sdetermine d by chemical analyseso f duplicate portionso f themeals .

Substrateutilizatio n rates

Pre-an dpostprandia l substrateoxidatio n ratesan dnutrien t balanceswer e calculated aspreviousl y described (13,14),usin g thenonprotei n respiratory quotient.Th enonprotei n respiratoryquotien twa scalculate d from oxygen consumption,carbondioxid e productionan d totalurinar ynitroge nexcretion . After the gasexchang emeasurement s subjectsvoide d fordeterminatio n of total nitrogenexcretion .Tota lnitroge nexcretio nwa sdetermine dwit h the Kjeldahl method.Th e energy costs forprocessin g the ingested nutrientswer e calculated using factorsgive nb y Flatt for theenerg y costso fprocessin g the ingested nutrients through oxidationo r storagepathway s(18) .

Body composition

Bodyweight wa smeasure d toth eneares t 0.05 kg (Berkel ED-60T,Rotterdam , the Netherlands)wit h the subjects ina novernigh t fasting,o r 4.5 hours postabsorptive state,afte rvoidin g andwearin gunderwea r or swimming clothes only.Bod y compositionwa sdetermine dusin gdensitometry . Subjectswer e measured underwaterwit h a simultaneous assessment of residual lungvolum e according toa heliu mdilutio nmethod .Tota lbod ydensit ywa s calculated and the sizeo f the fat-freemas san d the fatmas swer e estimated aspreviousl y described(13) .

-145- Statistics

Differences incontinuou s variables among groupswer e evaluated using analysis ofvarianc eo runpaire d Student's t-tests.Adjustin g groupmean sfo r covariateswa sperforme d byanalysi so fcovarianc e (19).Linea r relationships between resting metabolic ratean dbod y composition variableswer e assessed using univariate ormultipl e regression analyses (20).Correlatio n coefficients arePearson' s product-moment orpartia l correlation coefficients. Inter-individual variation inRM Ran dDI Twa scalculate d after assessing linear effectso nRM Ro rDI To fbod y composition (RMR)variable san denerg y intakeo ftes tmeal s (DIT)i nmultipl e regression models.Th eresidua l mean squareo fthes emodel swa suse da sa nestimato r ofth e 'adjusted' between-personvariation .

RESULTS

Table 1show sphysica l characteristics, restingmetaboli c ratean d respiratory quotientso fsubject s participating inth etw ostudies .Beside s systematic

Table 1.Physica l characteristics andcharacteristic s ofpostabsorptiv e resting energyexpenditur e ofsubject s intw ostudies .

Study 1 Study 2

Inter-individual variation in resting Inter-individual variation in diet-induced metabolic rate thernogenesis and in vivo fuel utilization

Women (n=78) Man (n=55) Honen (n=71| variables studied age (yrs) 29.3 ± 0.4 (18-J1) 31.7 + 1.1 (18-49) 24.3 ± 0.5 (20-41) 32.3 ± 1.1 (20-49) wight (kg) 73.9 ± 0.9 (58.4-102.0) 74.1 ± 2.0 (50.3-125.9) 73.4 + 1.0 (58.4-101.8) 75.3 + 2.2(50.3-125.9) fat-free mass (kg) 62.7 ± 0.8 (48.8-91.4) 46.1 ± 0.5 (36.6-60.6) 62.4 ± 0.9 (48.8-91.4) 46.3 ± 0.6 (36.6-60.6) fat mass (kg) 11.2 ± 0.4 (3.4-25.2) 28.0 ± 1.7 (9.1-70.4) 11.0 + 0.5 (3.4-19.1) 29.0 ± 1.8 (9.1-70.4) bodyfa t content (%) 15.0 ± 0.5 (5.5-24.7) 35.5 + 1.2 (14.6-55.9) 14.9 + 0.5 (5.5-22.3) 36.3 ± 1.2 (14.6-55.9) WR (kj/min) 4.99 ± 0.07 (3.93-7.29) 4.01 + 0.06 (3.10-5.70) 5.00 + 0.09 (3.93-7.29) 4.01 ± 0.06 (3.10-5.70) WR (kJ/kgFTM.d) 114.9 ± 1.3 (92.8-149.6) 125.8 + 1.6(100.8-155.6)* 115.7 + 1.6 (92.8-148.8) 125.1 + 1.7(100.8-155.6)* BQ - IWR 0.838 ± 0.005(0.746-0.940) 0.831 + 0.004 (0.754-0.929) 0.836 ± 0.005(0.746-0.940) 0.833 ± 0.004(0.754-0.929)

AH values are given as aean ± sea (range) HR = postabBorptive resting •etabolijc rate; kg FHf = fat-free i s; BQ = respiratory quotient * significant difference betuBen sen and wmea at p < 0.001

-146- differences betweenme nan dwome ni nbod y composition variables,ie ,i n fat-free mass,fa tmas san dbod yfa tcontent ,RM Rexpresse do na fat-fre e mass basiswa ssignificantl y higher inwome n compared tomen .Wome nha do naverag e 9 percent higher RMR'spe rk gfat-fre emas s thanme ndid . Fivepercen to fth e total populationha dRMR' spe rk gfat-fre emas sbelo w10 0kJ/ dan dfiv e percent above15 0kJ/d . Inter-individual coefficientso fvariatio ni nrestin g metabolic rateafte r adjusting forfat-fre e mass,ag ean dfa tmas swere , respectively,9. 6percen t inmen ,11. 3percen t innon-obes ewome nan d10. 7 percent inobes ewomen , intra-individualvariatio n inRM Rwa s5 percen t inme n and6 percen t inth enon-obes ean dth eobes ewomen .

resting metabolicrat e(kJ/d )

N =154 10400 r =0.80 RMR=1813*86t FFM

8800

•• • • •• •• •• • • •• •• • • • 7200- • •• • •• • • • • • • • ••• • • •• • • • •#•• •• •• • • • •« • • •• • •• • • • 5600 •••••M •• •• • * •• • ••• • M • • •• M* • •

r- 1 1 1 1 1 -r w35 49 65 77 91 fat-freemas s(kg )

Figure1 :Relatio n between fat-freemas s (FFM)an drestin gmetaboli c rate (RMR)i n15 4subjects .

Figure 1sho wth erelationshi p between fat-freemas san dRMR .N osignifican t differences amongmen ,non-obes e womenan dobes ewome n inslope so r intercepts ofth eregressio n equationswer e found.I nth eobes ewomen ,i nadditio nt oth e sizeo fth efat-fre e mass,th esiz eo fth efa tmas swa ssignificantl y positively related toRM R(r=0.68 ,p<0.001) , evenafte r adjusting forth e

-147- lineareffect so fth efat-fre emas so nfatmas s(r=0.44 ,p<0.01) .Comparin g RMR's,adjuste dfo rage ,fat-fre emas san dfa tmass ,amon gmen ,lea nwome nan d obesewome nreveale dsignificantl ylowe rRMR' samon gth eobes ewhe ncompare d toth enon-obes ewome nan dmen .Adjuste dRMR' swere ,respectively ,4.5 9+ 0.1 1 kJ/min (men),4.5 9+ 0.1 0kJ/mi n (leanwomen )an d4.1 8+ 0.1 8kJ/mi n(obes e women).A furtheranalysi sshowe dtha tth elowe radjuste dRMR' swere ,i n particular,characteristi cfo rth enon-abdomina lobes ewomen ,ie ,fo rth e womenwit ha waist-to-hip sgirt hrati obelo w0.85 .I nthes ewome nadjuste d RMR'swer e4.1 3+ 0.1 8kJ/min ,wherea si nwome nwit ha nabdomina ltyp eo fbod y fatdistributio nadjuste dRMR' swer e4.5 2+ 0.2 3kJ/min .

Table2 .Restin gmetaboli crate ,diet-induce dthermogenesi san di nviv ofue l utilizationi nme nan dwomen .

Men(n=55 ) Women (n=71)

Rtt (kJ/min) 5.00 + 0.09 (3.93-7.29) 4.01 ± 0.06 (3.10-5.70)* DIT (kJ) 164 + 7 (68-329) 109 ± 5 (-5-209)* DIT - % energy content meal (%) 8.37 ± 0.33 (4.02-14.39) 6.78 ± 0.31 (-0.35-15.49)* DTT- % increase WR (%) 13.9 ± 0.7 (6.9-32.4) 12.3 ± 0.5 (-1.0-22.5) basal fat oxidation (10 .mg/fciin.kgFFM) 799 ± 40 (50-1623) 876 ± 37 (61-1623) basal CH3 oxidation (10 .ng/fain.kgFTM) 1748 + 78 (262-3288) 1825 + 67 (247-3300) postprandial fat oxidation (10 .mjAiin.kgFFM) 594 + 44 (-47-1466) 639 + 35 (-254-1346) postprandial d£) oxidation (10 .mg/hdn.kgFTM) 3106 + 89 (1569-4388) 3273 + 87 (1326-4975) protein oxidation (10 .ncytoin.kgFTM) 963 + 24 (649-1364) 1069 ± 29 (538-1758)* basal fat oxidation (% JWR) 39.6 ± 1.8 (2.5-«9.6) 39.9 ± 1.5 (2.9-71.2) basal OÜ oxidation (% Em) 37.6 ± 1.6 (5.4-67.9) 36.3 + 1.2 (4.8-60.4) basal protein oxidation (% tm.) 22.5 ± 0.5 (14.3-34.5) 23.3 ± 0.7 (10.9-40.6) postprandial fat oxidation (% PEEI 26.3 ± 1.7 (-0.4-59.2) 26.3 ± 1.3 (-10.7-57.2) postprandial CK> oxidation (% PEE) 54.0 ± 1.5 (26.9-73.8) 53.3 ± 1.2 (23.3-70.8) postprandial protein oxidation (% PEE) 19.8 ± 0.5 (13.3-26.7) 20.8 + 0.6 (9.1-35.7)

Values axe given as nan ± seal (range) IHt = postabsorptiue resting •Btabolic rate; DTT = diet-induced therMigenesis; kgFTH = kg fat free »ass; OD = carbohydrate (glycogen or glucose); FEE= postprandial energy expenditure * significant difference betjuean BBnan d women at p < 0.001

Table2 show sRMR ,DI Tan di nviv ofue lutilizatio nrate si nme nan dwomen . DITwa ssignificantl y (p<0.001)associate dwit hth eenerg yintak eo fth etes t meal (r= 0.52) .Th e5t han d95t hpercentil evalue so fth eDI Tdistributio ni n thetota lsampl ewer e4 an d1 2percen t (ofenerg yconten to fth etes tmeal) , respectively.A significan tdifferenc eo f2 4percen twa sobserve di nDI T betweenme nan dwomen .N osignifican tdifferenc ewa sobserve dbetwee nlea n

-148- women and obesewome n inDIT .DI Twas ,respectively , 6.65 + 0.56 percent in the leanan d 5.71 + 0.99 percent inth eobese .DI Twa sno t significantly affected inth eobes ewome nb y type ofbod y fatdistribution .Th e differences inDI Tbetwee n the sexesoccurre d during theentir e first threehour s of the postprandial period andparallelle d differences incarbohydrat e oxidation,bu t not in fatoxidation .Oxidatio n ratesdurin g the first threehour so f the postprandial periodwere ,respectively , forcarbohydrate s 217+ 8mg/mi n in menversu s 163+ 5mg/mi n inwome n (p<0.01),an d for fat 31+3 mg/min inme n versus 26+2 mg/min inwomen .Protei noxidatio ndiffere d also significantly between the sexes,ie ,60+ 2 mg/min inme nan d 50+2 mg/min inwomen .N o differences inbasa l orpostprandia l fuelutilizatio n ratesexpresse d ona fat-freemas sbasi sbetwee nth e sexeswa sobserved ,excep t for protein oxidation,whic hwa so naverag e 11percen thighe r inwome ncompare d tomen . Inbot h sexes,fa toxidatio n contributed inth ebasa l stateo naverag e 40 percent to totalbasa l energy expenditure.Carbohydrat e oxidation contributed 37percen t to totalbasa l energyexpenditure . Inth epostprandia l state,fa t oxidationwa s reduced to2 6percen to f totalenerg yexpenditure .A reciprocal changewa s observed inpostprandia l carbohydrate oxidation. In2 3person s thereoccurre d during thepostprandia l period occasionallyne t transformation ofglucos e intofat .I nth eothe r 103subject sn one tlipogenesi soccurred . On average, inmen ,4 percen t of the ingested glucosewa s converted to fat,3 3 percentwa s stored asglycoge nan d 63percen twa s oxidized. Inwome n these figureswere ,respectively , 5,3 9an d 56percent .O naverage ,5 0percen t of theamoun t of ingested fatwa soxydize d inth epostprandia l period inbot h sexes.

The coefficients of inter-individual variation inDI Twer e 29percen t inmen , 43percen t innon-obes ewome nan d 26percen t inobes ewomen . Inter-individual variation inDI Twa s similar tointra-individua lvariatio n inDIT .Th e coefficients of inter-individual variation,adjuste d for fat-free mass, of in vivo fuelutilizatio n rates inth epostabsorptiv e statewere ,respectively , 36 percent for fat,3 0percen t for carbohydrates and 21percen t forprotein . Inter-individual variation of fuelutilizatio n rates in the postprandial state,adjuste d for fat-freemas san dnutrien t intakewit h the testmeal ,wa s respectively, 50percen t for fat,2 1percen t for carbohydrates and 20percen t forprotein . Inter-individualvariatio n infue lutilizatio n rateswa s similar amongmen ,non-obes ewome nan dobes ewomen . Table 3show sdifference s inbod y composition andenerg ymetabolis m between subjects characterized by relatively low (<10t hpercentil e of sex-specific

-149- frequencydistribution )o rhig h( >90t hpercentile )RM Rpe rk gfat-fre e mass. Significant differencesbetwee nbot hgroups ,separatel yfo rme nan dwomen , were observed inth esiz eo fth efat-fre emas san di nbasa l fatoxidatio n rates.I naddition ,i nwome na significan tdifferenc e betweenbot h groupsi n DITwa sobserved .Wome nwit h relativelylo wRMR' swer e forth emos t part (n= 6)obes ewome n characterizedb ya non-abdomina l typeo fbod yfa tdistributio n withwaist-to-hip sgirt h ratioo f0.7 5+0.01 .

Table3 .Bod ycompositio nan denerg yexpenditur e insubject s characterizedb y relativelylo wo rhig h restingmetaboli c ratespe rk gfat-fre emass .

Men «—

low (n=7) high (n=8) low (n=8) high (n=8)

weight (kg) 78.2+ 3. 9 68.8+ 2.9 85.1± 4. 1 61.7± 3.4** * bodyfa tconten t (%) 14.9+ 1.6 14.5 ± 1.4 40.4+ 2.4 28.6+ 2.7** fat-freemas s (kg) 66.2± 2. 1 58.7± 2.5 * 50.3± 1.9 43.5+ 1.3** restingmetaboli crat e (kJ/d.kgFFM) 98.2± 0. 9 137.8± 2.8** * 103.1± 0. 8 152.4± 0.7** * diet-inckicedthernogenesi s (%Ein ) 7.65 ± 1.90 7.99+ 0.78 5.64± 0.59 10.15± 1.54 * fatoxidatio n (10 .mj/min.kgrTM) 750± 7 1 1087 ±105 * 594± 8 3 1200+ 97*** CH3oxidatio n (10 .mg/min.kgFFM) 1298± 19 6 1784± 172 * 1534± 15 0 2186± 27 1 proteinoxidatio n (10 -mg/min.kgFYM) 839+ 54 1123± 71 * 1113± 8 8 1047± 11 2

Values axe given as BMI + see hgpm = kg fat-free eass; Bin = energy UJILHUL of test eaBl; OD = carbulaJüLale (glycogen or glucose) * significant difference between low and high groups at p < 0.05 ** irddaa at p < 0.01 i at p < 0.001

Table4 show s some comparisonso nvariable sbetwee n subjects characterizedb y a relatively highDI Tan dsubject swit ha relativel ylo wDIT .Fo rmen ,n o significant differences betweenbot hgroup si nan yo fth evariable swa s observed.Wome nwit h relatively highDIT' sha dhighe r postprandial carbohydrate oxidation ratesan dRMR' s comparedt owome nwit h relativelylo w DIT'S. Inaddition ,w ecompare dth emeasure d RMR'si nthi s studywit hRMR' s predicted accordingt oformula s recentlypublishe db yOwe ne tal . (6,7). Inmen , the measured RMR'swer e3. 9+ 1. 1percen thighe r (p<0.001)compare dt opredicte d RMR's.Als oi nwomen ,measure dRMR' swer e systematically higher (p<0.001) comparedt opredicte d RMR's,o naverag eb y2. 9+ 1. 3percent .

-150- Table4 .Bod y compositionan denerg yexpenditur e insubject scharacterize db y relativelylo wo rhig h thermic responset ofood .

Man Women

low (n=6) high (n=6) low (n=7) high (n=8)

weight (kg) 67.8 ± 2.2 73.0 ± 2.2 72.2 + 7.6 75.0 ± 9.4 body fat content (%) 12.0 + 1.6 15.0 + 1.6 33.1 ± 4.0 33.3 ± 5.0 fat-free mass (kg) 59.8 + 2.6 61.7 ± 1.9 46.6 + 2.2 46.8 ± 2.0 resting metabolic rate (kJ/d.kgFFM) 107.9 ± 5.9 110.7 ± 3.1 125.3 ± 5.5 142.6 ± 4.7* diet-induced thenrogenesis (% Ein) 4.51 + 0.20 12.21 ± 0.46 2.54 ± 0.55 12.06 + 0.76 fat oxidation (10 .irg/hiin.kgïTM) 568 + 92 608 ± 186 762 ± 111 877 + 81 CH3 oxidation (10 .mg/lran.kglTM) 2690 ± 131 3080 ± 358 1433 ± 237 1974 ± 222* protein oxidation (10 .mg/min.kgrTTI) 886 ± 74 942 ± 80 1000 ± 127 1105 ± 90

Values aie given as aeon + sen kgFFM = kg fat-free BOSS; Ein = energy caitmt of test real; Of) = carbohydrate (glycogen or glucose) * significant difference between low and high groups at p < 0.05

DISCUSSION

Inthi sstud yw eassesse dth enatur ean dmagnitud eo finter-individua l differences inrestin generg yexpenditur ean di nviv o fuelutilizatio nrates . Itwa sfoun d thatsex ,th edegre eo fbod y fatnessan dtyp eo fbod yfa t distributionar eimportan tdeterminant so fth eamoun to fenerg yexpende di n thepostabsorptiv e resting state,ie ,i nRMR .Bod y fatnesso rbod yfa t distributiondi dno tsystematicall y affectDIT ,i ncontras tt osex . Inter-individualvariatio ni nRM Rwa ssimila ramon gnon-obes emen ,non-obes e womenan dobes ewomen .Between-perso nvariatio ni nDI Twa ssimila rt oth e intra-individualvariation ,wit h slightdifference samon gmen ,non-obes ewome n and obesewomen . The totalamoun to fenerg yexpende d inRM Ran dDI Tcomprise si nth eaverag e individual 70-85percen to ftota ldail yenerg youtpu t (8).Th emajo r determinantso frestin gmetaboli c ratear ebod yweigh tan dbod y composition (10),an dfo rdiet-induce d thermogenesis,energ y intakean dnutrien t composition (17,21). Importantquestion st ob eanswere d instudie so nproblem so fenerg ybalanc ei n humans are,whethe r obese individualswoul dhav e relatively lower rateso f energy expenditureand ,secondly ,whethe r subjects susceptiblet obod yweigh t

-151- gainar e also characterized by relatively low rateso fenerg yexpenditure . Sucha characteristicwoul db e indicative ofa n increased efficiency of energy utilization, sometimes referred toa sa n increased "metabolic"efficienc y (22).A n increased metabolic efficiency hasbee n reported invariou s animal strainsproduce d by selective genetic breeding (23-25).A n increased efficiency of energyutilizatio n among theobes e could explain the seemingly anomalous finding of similar energy intakesamon g theobes e and the lean (26-28). However,evidenc e supporting thehypothesi so f increased metabolic efficiency among theobes e isfa r fromconclusiv e (8).Thi sma yb e causedb ya variety of reasons.Firs to fall ,i t ispossibl e thatamon g lean subjects differences inth eefficienc y ofenerg yutilizatio n exist.Comparin g obese with lean control subjectsma y thenno t revealan ydifference s inth e rateo f energyexpenditure .Secondly ,amon g theobes e therema yb evariatio n in metabolic efficiency, andonl y specific groupso fobes e subjectsma yb e characterized bya nenhance d metabolicefficiency .Thirdly ,th eobes e state mayb e anadaptatio n toa pre-obes e state characterized bya relatively low rateo fenerg yexpenditure .Onc eobesit y isestablished , the relatively low rateo f energy expenditure isno t longermanifest .Th evalidit y of the latter proposition canonl yb e tested inprospectiv e studieso f lean individualswit h relatively low rateso fenerg yexpenditure .Cross-sectiona l studies among lean andobes e subjects,a sth estud yher e reported,ma yb euse d toasses s the nature andmagnitud e of inter-individual variations in rateso f energy expenditure.Suc hstudie sma yanswe r thequestio nwhethe r there isvariatio n among the leanan d theobes e inmetaboli c efficiency. Such studiesma y alsob e ofgrea tvalu e for formulatinghypothese sabou t longitudinal outcomes in individualswit hdifference s inmetaboli c efficiency interm so fbod yweigh t gain.

This study shows thatvariatio n in indicators (RMR,DIT )o f metabolic efficiency isa slarg eamon gnon-obes ea samon g theobese .Thi smean s that relatively low rateso fenerg yexpenditur e areno ta nexclusiv e characteristic ofobes e individuals.Thi s findingconfirm s recent reports (6,7,10,11). The variation inRMR' spe r kg fat-freemas swa s 12percen twit h 5percen t of the populationhavin gRMR' sbelo w 100kJ/d.k g fat-freemas s and fivepercen to f thepopulatio nwit hRMR' sabov e 150kJ/d.k g fat-freemass . These subjectsma y tentatively be characterized, respectively,a shypo -an dhypemetaboli c with respect to theRMR . Itseem sunlikel y thatdifference s inth ecompositio n of the fat-freemass , intra-individualvariatio n inRM Ro rmeasuremen t error of the assessmento fbod y composition could account forth edifference s inRM R

-152- perk gfat-fre emas sbetwee nthes esubjects . Itwa sfoun dtha tRM Rpe rk gfat-fre emas si ssignificantl ylowe ri nme nwhe n comparedt owomen .T oth ebes to fou rknowledg ethi sfindin gha sno tbee n reportedbefore .Thi sresul tcoul db ecause db ya highe rproportio no f metabolicallyles s 'active'tissue si nth etota lamoun to ffat-fre emas si n menwhe ncompare dt owomen .RM Ri sdetermine dfo ro naverag e6 5percen t(29 ) byorga nactivity ,wherea sth emetaboli cactivit yo fskeleta lmuscle s contributeso naverag e2 3percen tt oRM R (29).A greate rproportio no f skeletalmuscle si nth etota lfat-fre emas si nme ncompare dt owome nma y explainth erelativel ylowe rRMR' samon gmen .However ,n oaccurat edat aar e availableo nth eproportio no fskeleta lmuscl ei nth etota lfat-fre emas si n menan dwome nt otes tthi shypothesis .I twoul db einterestin gt orelat eRM R tofat-fre emas si nme no rwome nwit ha relativ ehypertroph yo fskeleta l muscles,ie ,i nbod ybuilders .I naddition ,t odifference si nth ecompositio n ofth efat-fre emas sbetwee nth esexes ,i ti spossibl ethat ,a tsimila r compositiono fth efat-fre emass ,me nhav elowe rrate so fenerg yexpenditur e thanwome ndo .Th epresen tstud ygive sn odat at otes tthi shypothesis . Inaccordanc ewit hothe rfinding s (5-7,10,11),thi sstud yals oshow sa significantpositiv erelatio no fth eRM Rt ofat-fre emass .O naverag e6 4 percento fth etota lvariatio ni nRM Rwa srelate dt ovariatio ni nth efat-fre e mass.Th epredictio nequatio no fRM Rfro mfat-fre emas si sver ysimila rt o equationspublishe db yothe rinvestigator s (5-7,10,11).RMR' sobserve di nthi s studywer eo naverag e3 percen thighe ri ncompariso nt oRMR's ,predicte d accordingt orecentl ypublishe dequation so fOwe ne tal . (6,7).Owe ne tal . showtha tmos tpredictio nequation soverestimat eRM Rb y7-1 4percent .Ou r studysupport sOwe ne tal.' sconclusions ,althoug hth eoverestimatio no fRM R appearst ob eslightl ylowe ri nou rstud yi ncompariso nwit hOwe ne tal's . study.

Itwa sobserve dtha ti nth eobes ewomen ,i nadditio nt ofat-fre emass ,th e sizeo fth efa tmas swa spositivel yrelate dt oRMR .Thi si si naccordanc ewit h findingso fHofman se tal . (30).A positiv eassociatio nbetwee nth esiz eo f thefa tmas san dth eRM Ra ta give nfat-fre emass ,indicate sthat ,a t increasedbod yfa tmasses ,th eproportio no fmetabolicall y 'active'tisue si n thetota lfat-fre emas si srelativel yenlarged ,o rtha tth erat eo fenerg y expenditurei ntissue scomprisin gth efat-fre emas si srelativel yenhanced .I t ispossibl etha ti nobes eindividual swit ha give nfat-fre emass ,increasin g amountso ftota lbod yfa tcaus ea highe rwor kloa dt ovariou sorgan ssuc ha s

-153- theheart,lung so r kidneys,o r thatthe ycaus ea relative enlargement of these organs. We alsocompare d individualswit h relatively lowRMR' swit h individualswit h relativelyhig hRMR's . Itwa s found, inme nan dwomen ,tha t individualswit h reduced RMR'swer e characterized by a relatively enlarged sizeo f the fat-free mass.Again ,thi sma y indicate that inthes e individuals theproportio n inth e total fat-freebod ymas so f skeletalmuscle san d other relatively less metabolically 'active'tissues ,ie ,bon eo r skin,i s relatively increased.W e observed thathighe r RMR'swer e inparticula r related tohighe r rateso f fat oxidationpe r kg fat-free mass.Th edegre e of fatoxidatio n isunde r hormonal control (12).Catecholamine s increase lipolysisan d enhance fatoxidation . Catecholamines mayals o increase thermogenesis through stimulatory effectso n ionpumpin g or substrate cycling (8).I ti spossibl e that theenhance d basal fatoxidatio n rates insubject swit h relatively lowRMR' sar ea reflection of a higher sympathetic activity inthes e individuals.I nwomen ,i twa s found that the typeo fbod y fatdistributio n significantly affected theRMR .Obes e womenwit h anon-abdomina l typeo fbod y fatdistributio nwer e characterized by reduced RMR'scompare d toabdomina l obesewomen ,lea nme no r leanwomen .W e have recently reported indetai l on the impacto fbod y fatdistributio n in obesewome no n restingmetaboli c rate (14).I nadditio nt othi sstudy ,th e present study shows thatRMR' s inwome nwit hnon-abdomina l obesitywer e reduced,no tonl y incompariso nwit hage-matche d non-obesewomen ,bu tals o in comparisonwit hyounge r non-obeseme nan dwomen .Th epresen t studydoe sno t allow to infero n themechanis mbehin d this finding.Mos t likely the differences inadjuste dRMR' samon g thevariou s groupsar e related toa difference inth eendocrinologica l state.Suc ha differenc e could, in its turn,affec tdirectl yo r indirectly the rateo f thermogenic processes in tissues. It isals opossibl e thatdifference s inth e compositiono f the fat-freemas s cause thedifference s inadjuste d RMR's.Th e findingo f relatively reducedRMR' samon g theobes e in relation totyp eo fbod y fat distribution isunique .Relativel y reduced rateso fenerg yexpenditur e have been reported forpost-obes ewome n (32).I tha sals obee n reported that in wrestlers repeated cycleso fweight lossan d regainar eassociate d with changes inth eRM Rpe r kg fat-freemas s (33).Wrestlers ,wh oha d experienced repeated cyclesq fweight gainan d loss,ha d 14percen t lowerRMR' spe r kg fat-freemas s incompariso n towrestler swit h relativelymino rweigh t fluctuations.Thi ssuggest s thepossibilit y thatperiodi c reductions inbod y weight combinedwit h subsequentweight relapse can reduceRMR's . Inthi s study

-154- nodifferenc ewa sobserve d betweenabdomina l andnon-abdomina l obesewome n in self-reportedweigh thistory .Th emajorit y ofwome n reported tohav e had severalunsuccesfu l attempts to reducebod yweight .W e found (14)tha t non-abdominal obesity is (pre)pubertal inonset ,wherea sabdomina l obese obesity iso fth eadult-onse t type.Thi ssuggest stha tth e findingo f relatively reducedRMR' s inth enon-abdomina l obesema yb eo f etiological significance for theobesity . The second component of restingenerg yexpenditur e thatwa s investigated in this study isDIT .Severa l investigators (34,35)hav e reported that inobes e individualsDI Twoul d beblunte d incompariso nwit h thenon-obese .Thes e findingshave ,however ,no tbee n corroborated byothe r investigators (5,6,17,22). Inth epresen t studyn o significantdifferenc e inDI Twa s found betweennon-obes ewome nan d obesewomen . Variation inDI T for thetota lpopulatio nwa s 36percent ,wit h fivepercen t of thepopulatio n having,a DI Tbelo w 4percen tan d fivepercen thavin g a DIT greater than 12percen t (energyconten to f testmeal) . These subjectsma y tentatively be characterized as subjectswit ha hyper-efficienc y ora hypo-efficiency ofpostprandia l energyutilization , respectively. Itseem s unlikely that intra-individualvariatio n inDI Tca naccoun t for the differences inDI Tbetwee n thesesubjects . A further analysis ofDI Tamon g theobes e in relation totyp e ofbod y fat distribution revealed thatbod y fatdistributio nwa sno tassociate d with differences inDIT .A nunexpecte d findingwa s the significantdifferenc e in DITbetwee n the sexes.Me nha d a significant higher DITtha nwome ndid . This indicates thatme nhav ea lower efficiency ofpostprandia l energy utilization compared towomen .Thi s findingma yb eexplaine d byassumin gdifference s in energy intakebetwee n the sexes.I tha sbee n customary tostandardiz e the energy content of the testmeal swit h respect toth e sizeo f the fat-freemas s (17,34)o rwit h respect to restingmetaboli c rate (36).I nthi s study a higher DITwa sobserve d amongme n compared towomen ,despit e the fact,tha tme nha d onaverag e 10percen t lower energy intakes from testmeal s thanwome ndid , either per kg fat-freemas so r inpercentag e of restingmetaboli c rate.I nme n the average energy costso fabsorbin g andprocessin g of the ingested nutrients were calculated at 126 kJ,whic h is7 7percen to f thetota l thermic response. This figure isi ngoo d agreementwit hdat a on the relative contribution of the socalle d 'obligatory' component of thetota lDI T response (8,15). Forwome n we calculated that the costso fprocessin g the ingested nutrients averaged 108 kJ,whic h is9 9percen to f the total DIT response.Thi s suggests that the

-155- difference between the sexes inDI T iscause d by thepresenc eo fa n additional thermic response inme nt ofood ,tha ti sno tdirectl y relatedt oth e postprandial processing ofth enutrients .Thi s componentha sbee n referred to asth e 'facultative'componen to f theDI T (8,15). The facultative componento f theDI T isthough t tob emediate db y theactivit yo f the sympathetic nervous system. Food ingestionwoul d increase theactivit yo f the sympathetic nervous system causinga n increased turnover ofcatecholamines .Thi s increase in catecholamines turnoverwoul d enhance the rateo fenerg yexpenditur e of variousmetaboli c processes(8) . These processesmay , inparticular ,b e stimulated inskeleta lmuscles . Recently, itwa s reported that inme nabou t 50percen to f the increase in thermogenesis inducedb y the sympathomimeticumephedrin e took place in skeletalmuscle s (37).'Sinc e inme n theproportio no f skeletalmuscle s in totalbod yweigh t is relativelyhighe r than inwomen ,thi sma yoffe r some explanation for themechanis mbehin d theobserve d difference inDI Tbetwee n the sexes.Th edifferenc e inDI Tbetwee nme nan dwome n suggeststha t in comparisonso fDI T inlea nan d obese,analyse s shouldb eperformed , stratified according tosex ,o r thatgroup s shouldb ematche d inse xdistribution . We alsocompare d individualswit h relatively lowDIT' swit h individualswit h relativelyhig hDlT's . Inme nn o significantdifference s inbod y composition orvariable so fenerg ymetabolis mbetwee nbot hgroup swer e found,suggestin g thatothe r factorswer e involved thatcause d thedifferenc e inDI Tbetee nbot h groups, forexample ,th edegre eo f sympatheticnervou s systemactivity . In women the rateo fpostprandia l glucoseoxidatio npe rk g fat-freemas swa s significantlydifferen tbetwee nbot hgroups ,suggestin g thatpar to f the difference inDI Twa s related todifference s inth eprocessin g of ingested carbohydrates. The patterno f fuelutilizatio n inth epostabsorptiv e statean d inth e postprandial statewa s similar inme nan dwomen .I nth ebasa l state,fa t oxidation contributed thegreates tpar tt otota lenerg ydissipation .Thi s is inaccordanc ewit hgenera l textboo k knowledge (12). Aftermea l ingestion, therewa sa relative increase inglucos eoxidatio nparallele d bya relative decrease in fatoxidation .Afte rmea l ingestion, insulin secretion rises rapidly causinga decreas e inlipolysi san d an increase in carbohydrate oxidation.Th epostprandia l rise ininsuli n secretionan d incarbohydrat e oxidationgenerall y lasts fora fewhours .Durin g thisperio d thepostprandia l substrateutilizatio npatter ngraduall ybecome smor e similar toth e postabsorptive substrateutilizatio npattern .I naccordanc ewit h recent

-156- findings (38,39),th epresen tstud yshew stha tpostprandia lne tlipogenesi s fromglucose ,ingeste di nmoderat eamount sfro ma mixe dmeal ,i so fnegligibl e importancefo rth estorin go fingeste dcarbohydrates . Inconclusion ,th epresen tstud yshow stha tsex ,bod yfa tconten tan dtyp eo f bodyfa tdistributio nar eimportan tdeterminant so fRHR .Se xals oaffecte d DIT.Inter-individua lvariatio ni nRM Ran dDI Twa scomparabl eamon gth elea n andth eobese .Thi ssuggest stha tamon gth elea nan damon gth eobes ether ema y beindividual swit hdifferen tefficiencie si nth eutilizatio no fenergy .

ACKNOWLEDGEMENTS

Theauthor swoul dlik et othan kth esubject sfo rthei renthousiasti c participationi nth estudy .W ear eals oindebte dt ostaf fan dstudent so fth e Departmento fHuma nNutritio nfo rthei rhel pi nconductin gth estudy .

REFERENCES

1.Energ yan dprotei nrequirements .Repor to fa join tFAO/WHO/UN UExper t consultation.Technica lrepor tserie s724 .Worl dHealt hOrganization : Geneva,1985 . 2.Harri sJA ,Benedic tFG .A biométri estud yo fbasa lmetabolis mi nman . WashingtonDC :Th eCarnegi eInstitute ,1919:1-266 . 3.Boothb yWM ,Berkso nJ ,Dun nHL .Studie so fth eenerg yo fmetabolis mo f normalindividuals :a standar dfo rbasa lmetabolism ,wit ha nomogra mfo r clinicalapplication .A m J Physiol1936;116:468-84 . 4.Robertso nJD ,Rei dDD .Standard sfo rth ebasa lmetabolis mo fnorma lpeopl e inBritain .Lance t1952;1:940-3 . 5.Cunningha mJJ .A reanalysiso fth efactor sinfluencin gbasa lmetaboli c ratei nnorma ladults .A mJ Cli nNut r1980;33:2372-4 . 6.Owe nOE ,Holu pJL ,D'Alessi oDA ,Crai gES ,Polansk yM ,Smalle yKJ ,Kavl e EC,Bushman nMC ,Owe nLR ,Mozzol iMA ,Kendric kZV ,Bode nGH .A reappraisa l ofth ecalori crequirement so fmen .A mJ Cli nNut r1987 ;46:875-85 . 7.Owe nDE ,Kavl eE ,Owe nRS ,Polansk yM ,Capri oS ,Mozzol iMA ,Kendric kZV , BushmannMC ,Bode nG .A reappraisalo fcalori erequirement si nhealth y women.A mJ Cli nNut r1986;44:1-9 . 8.Sim sEAH ,Danfort hE .Expenditur ean dstorag eo fenerg yi nmen .J Cli n Invest1987;79:1019-25 . 9.Sukhatm ePV ,Marge nS .Antoregulator yhomestati cnatur eo fenerg ybalance . AmJ Cli nNut r1982;35:355-65 . 10.Bogardu sC ,Lillioj aS ,Ravussi nE ,Abbot tW ,Zawadzk iJK ,Youn gA , KnowlerWC ,Jacobowit zR ,Mol lPP .Familia ldependenc eo fth erestin g metabolicrate .N Eng lJ Me d1986;315:96-100 . 11.Ravussi nE ,Lillioj aS ,Knowle rWC ,Christi nL ,Freymón dD ,Abbot tWGH , BoyceV ,Howar dBV ,Bogardu sC .Reduce drat eo fenerg yexpenditur ea sa riskfacto rfo rbody-weigh tgain .N Eng lJ Me d1988;318:467-72 .

-157- 12.Ganon gWF .Revie wo fmedica lphysiology .9t hed .California :Lang emedica l publications,1979 . 13.Weststrat eJA ,Wey sJPM ,Poortvlie tEJ ,Deurenber gP ,Hautvas tJGAJ . Diurnalvariatio ni npostabsorptiv erestin gmetaboli crat ean d diet-induced thermogenesis.A mJ Cli nNut r1989 ;i npress . 14.Weststrat eJA ,Dekke rJ ,Stoe lM ,Beghey nL ,Deurenber gP ,Hautvas tJGAJ . Restingenerg yexpenditur ei nwomen :impac to fobesit yan dbod yfa t distribution.Metabolis m1989 ;i npress . 15.Jéquie rE ,Acheso nK ,Schut zY .Assessmen to fenerg yexpenditur ean dfue l utilizationi nman .An nRe vNut r1987;7:187-208 . 16.Schutz ,Terminology ,factor san dconstant si nstudie so nenerg ymetabolis m ofhumans .In :va nE sAJH ,ed .Huma nenerg ymetabolism :physica lactivit y andenerg yexpenditur emeasurement si nepidemiologica l researchbase dupo n directan dindirec tcalorimetry .Euronu trepor tn o5 .Wageningen : StichtingNederland sInstituu tvoo rd eVoeding ,1985 :153-68 . 17.D'Alessi oDA ,Kavl eEC ,Mozzol iMA ,Smalle yKJ ,Polansk yM ,Kendric kZV , OwenLR ,Bushma nMV ,Bode nG ,Owe nOE .Thermi eeffec to ffoo di nlea nan d obesemen .J Cli nInves t1988;81:1781-9 . 18.Flat tJP .Biochemistr yo fenerg yexpenditure .In :Bra yG ,ed .Recen t advancesi nobesit yresearch :II .London :Joh nLibbey ,1978:211-28 . 19.Wine rBJ .Statistica lprinciple si nexperimenta ldesign .2n ded .Ne wYork : McGraw-HillBoo kCompany ,1971 . 20.Pec kEA ,Montgomer yDC .Introductio nt olinea rregressio nanalysis .Ne w York:Joh nWile y& Sons ,1982 . 21.Jéquie rE .Thermogenesi sinduce db ynutrien tadministratio ni nmen . Infusionstherapie1984;11:184-8 . 22.Feli gP ,Cunningha mJ ,Levit tM ,Hendle rR ,Nade lE .Energ yexpenditur ei n obesityi nfastin gan dpostprandia lstate .A mJ Physio l1983;244:E45-51 . 23.Canolt yNL ,Koon gLF .Utilizatio no fenerg yfo rmaintenanc ean dfo rfa t andlea ngain sb ymic eselecte dfo rrapi dpostweanin ggrowt hrate .J Nut r 1976;106:1202-8. 24.Ferrel lCL ,Jenkin sTG .Energ yutilizatio nb ymature ,nonpregnant , nonlactatingcow so fdifferen ttypes .J Ani mSe i1984;58:234-43 . 25.Koon gLJ ,Nienabe rJA ,Mersman nHJ .Effec to fplan eo fnutritio no norga n sizean dfastin gmea tproductio ni ngeneticall yobes ean dlea npigs .J Nutr1983;113:1626-31 . 26.Stefani kPA ,Heal dF Pjr ,Maye rJ .Calori cintak ei nrelatio nt oenerg y outputo fobes ean dnon-obes eadolescen tboys .A mJ Cli nNut r 1959;7:55-62. 27.Maxfiel dE ,Konish iF .Pattern so ffoo dintak ean dphysica lactivit yi n obesity.J A mDie tAsso c1966;49:406-8 . 28.Kromhou tD .Energ yan dmacronutrien tintak ei nlea nan dobes emiddle-age d men(th eZutphe nstudy) .A mJ Cli nNut r1983;37:295-9 . 29.Grand eF .Energ yexpenditur eo forgan san dtissues .In :Kinne yJM ,ed . Assessmento fenerg ymetabolis mi nhealt han ddisease .Columbus :Ros s Laboratories,1980:88-92 . 30.Hoffman sM ,Pfeiffe rWA ,Gundlac hBL ,Nijkrak eHGM ,Oud eOphui sAJM , HautvastJGAJ .Restin gmetaboli crat ei nobes ean dnorma lweigh twomen , intJ Obe s1979;3:111-8 . 31.Horwit zBA .Metaboli caspect so fthermogenesis :neurona lan dhormona l control.Fe dPro c1979;38:2147-9 . 32.Geissle rCA, 'Mille rDS ,Sha hM .Th edail ymetaboli crat eo fth epost-obes e andlean .A mJ Cli nNut r1987;45:914-20 . 33.Stee nSN ,Opplige rRA ,Brownel lKD .Metaboli ceffect so frepeate dweigh t lossan dregai ni nadolescen twrestlers .JAM A1988;260:47-50 . 34.Schut zY ,Bessar dT ,Jéquie rE .Diet-induce dthermogenesi smeasure dove ra wholeda yi nobes ean dnon-obes ewomen .A mJ Cli nNut r1984:542-52 .

-158- 35. SwaminathanR ,Kin gRFGJ ,Holmfiel dJ , SiwekRA ,Bake rM ,Wale sJK . Thermic effect of feedingcarbohydrate ,fat ,protei nan dmixe dmea l in leanan d obese subjects.A m JCli nNut r1985;43:177-81 . 36.Bessar d T,Schut zY ,Jéquie r E.Energ yexpenditur e and postprandial thermogenesisi nobes ewome nbefor ean dafte rweigh t loss.A m JCli nNut r 1983;63:680-93. 37.Astru pA , BiilowJ ,Madse nJ , ChristensenNJ .Contributio n ofBA Tan d skeletalmuscl e tothermogenesi s induced byephedrin e inmen .A m J Physiol 1985;248:E507-15. 38.Acheso n KJ,Flat tJP ,Jéquie r E.Glycoge n synthesisversu s lipogenesis after a 500gra mcarbohydrat e meal inmen .Metabolis m1982;31:1234-40 . 39.Acheso n KJ,Schut zY ,Ravussi nE ,Jéquie r E,Flat tJP .Nutritiona l influenceso n lipogenesisan d thermogenesis after a carbohydrate meal.A m J Physiol 1984;246:E62-70.

-159- Chapter 8. Resting energy expenditure in women:impac t of obesity and body fat distribution

JANA WESTSTRAT EMSC ,JACQUELIN EDEKKE RBSC ,MIRIA MSTOE LBSC ,LILIAN E BEGHEIJNBSC ,PAU LDEURENBER GPHD ,JOSEP HGA JHAUTVAS TM D

ABSTRACT

Restingmetaboli crat e(RMR )an ddiet-induce dthermogenesi s (DIT)wer e repeatedlyassesse dwit ha nindirec tcalorimetri cventilate dhoo dsyste mi na groupo f3 2health ypremenopausa lobes ewomen ,bod yfa tpercentag e46. 4+ 0. 9 (mean+ sem),ag e38. 5+ 0. 9years .RM Ran dDI Twer eals omeasure d ina grou p of1 0health ypremenopausa lnon-obes ewomen ,bod yfa tpercentag e31. 3+ 1.7 , age37. 7+ 2. 4years . Theobes ewome nwer esubdivide daccordin gt oth ewaist-to-hip sgirt hrati o (WHR)int othre egroup swit ha differen ttyp eo fbod yfa tdistribution .A gluteal-femoralobes egrou p(n=10) ,WH R< 0.79 ,a nintermediat eobes egrou p (n=10),0,7 9 0.85 .N o significantdifference swer eobserve damon gth eobes egroup si nage ,bod y weight,bod yfa tmas san dfat-fre ebod ymass .Bod yfa tdistributio nwa sno t associatedwit hdifference si nDIT ,pre -an dpostprandia l respiratory quotientsan dsubstrat eoxidatio nrates .Th eabdomina lobes ewome nha d significantlyhighe rRMR' sadjuste dfo rage ,fa tmas san dfat-fre ebod ymas s (6075+ 20 0kJ/d )i ncompariso nt oth egluteal-femora lobes ewome n (5502+ 205kJ/d) ,an di ncompariso nt oobes ewome nwit ha nintermediat ebod yfa t distribution (5517+ 19 3kJ/d) ,bu tno ti ncompariso nt oa non-obes econtro l group,679 0+ 26 1kJ/d .I ti sconclude dtha twithi nth etota lgrou po fobes e women,th enon-abdomina lobes eca nb echaracterize db yrelativel yreduce d restingmetaboli crate si ncompariso nwit heithe rabdomina lobes ewome no r withnon-obes ewomen .

-160- Keywords: indirect calorimetry, restingmetaboli c rate,diet-induce d thermogenesis,obesity ,women ,bod y fatdistribution .

INTRODUCTION

Obesity isth econsequenc e ofa prolonge d imbalancebetwee nenerg y intakean d energyexpenditure ,cause db y eitherpersisten thyperphagi a or a reduced rate ofenerg y expenditure.I nspit eo fmuc h research itha sno tbee npossibl e to define fora givenobes e individual the relative etiological importance of these factors (1-3).T odetermin e the contributiono fhyperphagi a or a reduced rateo fenerg yexpenditur et oth eetiolog yo fobesity ,prospectiv e studiesar eneede d inwhic hdail y food intakean denerg yexpenditur e are accuratelymeasure d forprolonge d periodso f time inperson sleadin g their normal lives.Suc h studiesma yanswe r thequestio nwhethe r individualswit h similar bodyweights ,bod ycompositio nan ddegree so fphysica l activityhav e similar ordifferen t energy requirements, ie,rate so fenerg yexpenditure . Thereare ,however ,ver y fewprospectiv edat a onenerg yexpenditur e (4).Th e majorityo f studiesar eo fa cross-sectiona l nature (5-11),usuall y involving a comparisono fobes e individualswit hnon-obes e subjectso non e ormor e componentso fdail yenerg yexpenditure .Generall ythes e studies showtha tth e obese have anenlarge dbod y fat-freemas sand ,consequently ,elevate d resting metabolic rates.Restin gmetaboli c rateadjuste d for fat-freebod ymas s would,however ,b e similar among theobes e incompariso nwit h thenon-obese . Controversy existso n thequestio nwhethe r theobes ear e characterized bya defectivediet-induce d thermogenesis.Som e investigators (5,6,8,11)repor ta diminution indiet-induce d thermogenesis inobes e individuals in comparison withlea n controls,wherea s other investigators couldno t confirm these findings (7,9,10). The reason for thediscrepanc y in results isno t clear.I t could be that theobes e canb e subdivided into subgroupswit han dwithou t reduceddiet-induce d thermogenesis ordecrease d restingmetaboli c rate.I n this respect subdividingobesit yaccordin g totyp eo fbod y fatdistributio n mayb e important (12-14). Itwa s showntha ti nobes e individualswit h a relativepredominanc e of fat inth egluteal-femora l region theobesit ywa s of earlier, ie,childhood ,onset ,wherea s inindividual swit h a relatively greater storage of fat inth eabdomina l region,th eobesit ywa so f adult-onset (12-14). Itmigh tb ehypothetize d thatchildhoo d obesity,ie , gluteal-femoral obesity, ismor e closelyassociate dwit hdecrease d energy

-161- expenditure thanadult-onse t obesity,whic hmigh t bemor e environmentally induced. This studywa sdesigne d to test thishypothesi s by assessing the impact of body fatdistributio n on restingmetaboli c rate and diet-induced thermogenesis. Inaddition , restingmetaboli c ratean d diet-induced thermogenesis weremeasure d inage-matche d non-obese subjects. Resting metabolic rate and diet-induced thermogenesis were repeatedly assessed in each obese individual toallo w forday-to-da y within-person variation in diet-induced thermogenesis (13,15). The results showa significant impact ofbod y fatdistributio n on resting metabolic rate,bu tno t ondiet-induce d thermogenesis.

SUBJECTSAN D METHODS

SUBJECTS

The obese subjects for this studywer e invited for participation inth e study by advertisements in regional newspapers.Thirty-tw o of the forty-five obese women,wh o responded were selected forparticipatio n inth e study on the basis of their body fatpercentag e (>35percen t asassesse d by densitometry), age (between 20-45 years), premenopausal state,smokin g behavior (smoking less than 10 cigarettes/d),healt h status (noevidenc e of past or present hypothyroidism, hyperthyroidism, diabetesmellitus ) and druguse . Subjects taking medicines known toaffec t energymetabolis m were excluded from the study.Healt h statuswa s evaluated using amedica l questionnaire by a physician (JGAJH). Only those subjectswer e selected who had not experienced greater weight changes than 2,5 kg in thehal fyea r preceding the start of the study.B yquestionnair e informationwa sobtaine d inth e obesewome n on nutritional habits,weigh t history,onse t ofobesit y and family history of obesity. The tennon-obes e subjectswer emember s of theUniversit y staff,wh o met the same entry criteria as the obese subjects except for body fat percentage,whic h had tob eunde r 35procent .Eac h individual was fully informed on thenatur e and purpose of the study and allwome n gave their written informed consent. The protocol of the studywa s submitted toan d accepted by the Medical Ethical Committee of theDepartmen t ofHuma nNutrition ,o f the Wageningen Agricultural University.

-162- METHODS

Experimental design

Restingmetaboli c rate (RMR)wa smeasure d inth emornin g after an overnight fast,o naverag e three times inth eobes e individuals (infiftee n obese subjectsRM Rwa smeasure d induplicate , inseventee nobes e subjectsRM R four times)an d once in thenon-obes e subjects.I tshoul db enote d that the control groupwa sadde d toth e studyafte r the studyo n theobes e subjects was finished.Ga sexchang emeasurement swer e performed inth eobes e during springan d inth enon-obes e during summer.Th emethodolog y andprocedure s of the gasexchang emeasurement swere ,however ,simila r for theobes e and the non-obese group.Betwee neac hmeasuremen twer ea t least twodays ,al l obese subjects completed the study inthre eweeks .N oattemp twa smad e tomeasur e RMR and DIT ina specificphas e of themenstrua l cycle.Al l gas exchange measurementswere ,however ,mad e at least 3day safte r theonse to f menstruation. For twelve obese subjectsa balance d number (n=2)o f gas exchangemeasurement swa savailabl e foreac hperio d of themenstrua l cycle (pre-an d post-ovulary).Ovulatio nwa sestimate d tooccu r 14day sbefor e the onset ofmenstruation .N o significantdifference s inRM Ran d DITbetwee n the twophase s of themenstrua l cyclewer e found.Therefore ,al lRM Ran d DIT measurementswer epoole d foreac h individual and themea nvalue swer euse d in theanalysis .Th ewome nwer e instructed tokee p tothei rnorma l eating and drinkingpatter n three tofou rday sprecedin g the studyan d toabstai n from strenuousphysica l activity theda y immediately before the tests.A dietary historywa sobtaine d fromeac hobes ewoma n toasses shabitua l food consumption.Subject swh o ate lesstha n20 0g/ d of carbohydrateswer e instructed toincreas e theconsumptio n of carbohydrate-rich products,sinc e a relatively lowcarbohydrat e consumptionma ydecreas e diet-induced thermogenesis (16).I n seventeen (4gluteal-femoral , 5intermediate ,8 abdominal)obes e subjectsdiet-induce d thermogenesis (DIT)wa smeasure d four times for fourhour s after ingestiono fa mixe d liquidmea l (mealA )o n the morning after acorrespondin g preprandial restingmetaboli c ratemeasurement . In fifteen (6gluteal-femoral , 5intermediate ,4 abdominal )obes e subjects DITwa smeasure d induplicat e for threehour safte r ingestiono fa solid mixedmea l (meal B). Inth enon-obes e subjectsDI Twa smeasure d for three hours after ingestiono fa liqui dmixe dmea l (meal C). The cumulative postprandial increase of thepremea lRM Rwa sdefine d asdiet-induce d

-163- thermogenesis.Durin g theexperimen t fifteen testmeals of typeA , 6o f type B and 5o f typeC wer e chemically analyzed forenerg yan dnutrien t content. MealA was composed ofa yoghurt-base d liquidmea l containing onaverag e 1810 kJ ofmetabolisabl e energy (11.3percen tprotein ,34. 1percen t fat,54. 6 percent carbohydrates).Mea lB wa scompose d ofa norma lDutc h breakfast (bread,margarine ,orang e juice,cheese ,appl e syrup)containin g on average 1315k J (12.4percen tprotein ,33. 2percen t fat,54. 4percen tcarbohydrates) . Meal C contained onaverag e 1310k J (12.2percen tprotein, 3 0percen t fat, 57.8percen t carbohydrates).Beside sdifference s inth emetabolisabl e energy content,mea lA ,B an d Cwer ealmos t identical incomposition .Mea lA ,mea l B andmea l C represented similar caloric loads for thepostprandia l period,ie , about 450kJ/ h formea lA and about 440kJ/ h formea lB andmea l C.Th e results showed nodifferenc e inDIT ,expresse d asa percentag e of the energy content of the testmea l between the testmeal sA andB , ie, 7.03+0.30 (mean+ sem)percen t formea lA and 6.69 + 0.57 percent formea lB . Therefore inth eanalyse s theDI T resultso f theobes e subjectswer e pooled. Inth eobes e subjects receivingmea lA , everymornin g before the start of the gasexchang emeasurement s anovernight-fastin g venousbloo d sampleo f 10m l was taken fordetermin gserumlipid san dbloo d glucose levels.I nth e obese subjects receivingmea lB every first testmornin go f theDI Tmeasuremen t an overnight-fasting venousbloo d sample of 10m lwa s taken.N o bloodsamples were taken inth enon-obes e subjects.

Gasexchang e measurements

Restingmetaboli c rate

On theda yo feac hRM Rmeasuremen t the subjectwa s taken to thelaborator y by car.Afte r voiding they laysupin e butawak ean dmotionles s ona hospita l bed.Th e subjectswor e light indoor clothing.Afte r a 30minute s restperio d with thehoo dplace d over the subjectshea d RMRwa s continuously measured between 08.00an d 09.00hour sa ta nenvironmenta l temperature of 23-25°C. During theRM Rmeasuremen t subjectswatche d romantic family typemotio n picture filmso nvideo .

Postprandial energy expenditure

After theRM Rmeasuremen t the subjectswer e given the testmeal .Mea lA and

-164- meal Cwer e given througha strawove r 3-5 minutesafte rwhic h postprandial energy expenditurewa smeasure d continuously for,respectively , 3o r 4hours . MealB wa s eatenwithi n 10-15minute safte r removement of thehood . Fiveo r threeminute s after themea lha dbee neate nan d thehoo d hadbee n replaced postprandial energyexpenditur ewa smeasure d continuously for threehours . During thepostprandia l energyexpenditur emeasuremen t the subjectswer e awake, remainedmotionles san dwatche d video tapes.Afte r thega s exchange measurements,urin ewa s collected fordeterminin g totalurinar ynitrogen . All gasexchang emeasurement swer eperforme dwit ha ventilate d hood system.A transparent perspexventilate d hoodwa splace d over thehea d of the subject andmad e airtight around theneck .A pump (Ocean SCL210,Dieren ,Th e Netherlands)drawe d fresh outsideai r through thehood .Th e flow rate through thehoo dwa smeasure d by atherma lmas s flowmeter (Brooks,model 5812N , Veenendaal,Th eNetherlands) .Th e flow ratewa skep tbetwee n 30an d 401/mi n by anelectricall y operated controlvalv e (Brooks,mode l 5837,Veenendaal , TheNetherlands) .A small sampleo f 0.4 1/min of theai r leaving thehoo dwa s analyzed foroxyge n (0,)an d carbondioxide (CO~)conten tby , respectively,a paramagnetic 0,-analyzer,adjuste d toa full scale rangeo f 20-21 percent (Servomex 1100A,Zoetermeer ,Th eNetherlands )an da n infrared CCU-analyzer, full scale range 0-1 percent (Analytical Development Company SS100, Hoddesdon,UK) .Th eanalyzer swer e calibrated eachmornin gusin gdrie d gas

mixtureswit h known0 ? andCO ,content .C u and CO? concentrations indrie d freshoutsid e airwer emeasure d for fiveminute s every 30minute sdurin g the RMRmeasurement s and everyhou rdurin g thepostprandia l energy expenditure measurements.Ai r flowrate ,O ,an dCO ,concentratio n of the inflowing and outflowingai rwer e computed onlin eb ya nautomati cdat aacquisitio n unit (HewlettPackar d 3497A,Pal oAlto ,USA )interface d toa microcompute r (HewlettPackar d 86Bo r 85,Pal oAlto ,USA) .Th eoutput so f thega s analyzers and the flowmeterwer e alsocontinuousl y recorded bya multi-pe n recorder (Kipp&Zonen BD101,Delft ,Th eNetherlands) .0 ,an d CO,concentratio n of the outflowing air and air flowrat e through thehoo dwer emeasure d every 30 secondsO and integrated over fiveminute s intervalperiod s toobtai n0 ~

consumption,CO ? production,

respiratoryquotient san dmetaboli c rateaccordin g toequation sdescribe d by jéquier et al.(17) .

-165- Calculation ofDI Tan d substrateoxidatio n rates

To obtain themea n energyexpenditur e foreac hhou r after ingestiono f the meal, theC U consumption and C0? productionwer e averaged overeac hhou r using the fiveminute s intervaldata .Thes evalue swer euse d tocalculat e the average DIT for the individualhour safte r the testmea l and for the total meanDI Tove r four or threehours .DI Twa s calculated by subtracting theRM R value fromth e respectiveaverag ehourl yo r totalmea npostprandia l energy expenditure (PEE), ie,DIT=PEE-RMR .Th eDI T isexpresse d inkJ , ina percentage of the increase inth epremea lRM Ran d ina percentag e of the energy content of the testmeal . Proteinoxidatio nwa s calculated from totalurinar ynitroge nexcretion . Carbohydratean d fatoxidatio ndurin gRM Ran d thepostprandia l energy expenditurewer e calculated using thenonprotei n respiratoryquotients . Oxygenan d carbondioxid e equivalents for substrate oxidationbefor e and after the ingestiono f the testmea lwer e obtained fromLillioj ae t al.(18) . A nonprotein respiratoryquotien t exceeding 1.00 indicatedne td e novo lipogenesis,ie ,lipogenesi sexceedin g concomitant fatoxidation . Carbohydrate oxidationwa s corrected forne td enov olipogenesi s from glucose aspreviousl y described(13) .

Bodycompositio n andbod y fatdistributio n

Fat-freemas swa sestimate d bydensitometry .Tota lbod ydensit yo f each subjectwa smeasure d by theunde rwate rweighin gmetho dwit h simultaneous measuremento f residual lungvolum eb ya heliu mdilutio n technique.Fat-fre e bodymas swa s calculated fromtota lbod ydensit ya spreviousl y described (13). Body fatdistributio nwa sassesse d inth eobes ewome nusin g thewaist-to-hip s girth ratio (19).Thre e categories ofbod y fatdistributio nwer edefined ,ie , a gluteal-femoral typeo fbod y fatdistributio nwhe nth eWH R <0.79 , an intermediate typeo fbod y fatdistributio nwit h theWH Rbetwee n 0.79 and 0.85 and anabdomina l typeo fbod y fatdistributio nwit h theWH R > 0.85.

Blood analysis

Bloodvariable swer emeasure d inpostabsorptiv evenou sbloo d samples.Bloo d glucosewa sdetermine d by theglucos e 4-amino-antipyrene (GOD-PAP)metho d

-166- witha ki tsupplie db yBoehringe rMannhei mQmB H(Almere ,th eNetherlands) . Totalseru mcholestero lwa sobtaine db yth echolesterol-oxidas e 4-aminoantipyrene (CHOD-PAP)hig hperformanc emetho dwit ha ki tals osupplie d byBoehringe rMannheim .Seru mhig hdensit ylipoprotei n (HDL)cholestero lwa s determinedb ya precipitatio ntechniqu e (20).Seru mtriglyceride swer e assayeda sdescribe db ySulliva ne tal .(21) .

Statistics

Thegroup so fobes eindividual ssubdivide daccordin gt otyp eo fbod yfa t distributionan dth econtro lgrou po fth enon-obes esubject swer ecompare do n continuousvariable susin ganalysi so fvariance .Analysi so fcovarianc ewa s usedt ocompar eamon ggroup sRM Ran dDI Tadjuste dfo rfat-fre ebod ymass ,ag e andfa tmass .Two-side dStudent' st-test swer euse dt oevaluat edifference s insevera lvariable sbetwee nobes esubject sfro mth ehighes tan dlowes t quintileo fth edistributio no fDI T(percentag eo fenerg yconten to ftes t meal)an dRM R (kJ/d.kgfat-fre emass) .Al lresult sar eexpresse da smea n+ semvalues .

RESULTS

Table1 show ssom echaracteristic so fth eobes ewome naccordin gt otyp eo f bodyfa tdistributio nan dfo rth enon-obes ewomen .N osignifican t differences,excep ti nth eWHR ,wer eobserve damon gth eobes esubgroups .O n averageth eabdomina lobes eha dhighe rbod yfa tan dfat-fre ebod ymasse si n comparisont osubject sfro mth eothe rgroups ,bu tth edifference swer eno t significant.Th enon-obes esubject sha dlowe rvalue sfo ral lvariables , exceptfo rage ,i ncompariso nwit hth eobese . Table2 show sseru mlipid san dbloo dglucos elevel si nth eobes ewome n accordingt otyp eo fbod yfa tdistribution .Wit hincreasin gWH Rtota lseru m cholesterol,seru mtriglyceride san dbloo dglucos elevel sincreased ,wherea s therevers ewa sfoun dfo rth eleve lo fseru mHDL-cholesterol .Th edifferenc e inbloo dglucos elevel samon gth egroup swa sth emos tmarked .

-167- Table1 .Som echaracteristic so fparticipatin g obesean dnon-obes ewomen .

Cfoese IMralue Body fat distribution F-ratio Gluteal-femoral Intermediate Ptxkmrnal V«R < 0.79 0.79 0.85 n=10 n=10 n=12

Age (yr) 35.3± 2.2 40.7+ 0.7 39.4± 1.4 37.7± 2.4 0.19 Bodyueigh t (kg) 89.6± 4.6 84.6+ 4.6 97.7± 4.0 64.4± 2.5 <0.000 1 Bodymas sindex T (kg/tn2) 32.2± 1.4 31.6+ 1.1 35.4± 1.4 21.9± 0.7 <0.000 1 Fat-freemas s (kg) 41.6+ 1.2 46.6± 1.9 50.2± 1.5 44.1+ 1.4 0.05 Fat-mass (kg) 42.0± 3. 7 38.0± 2.9 47.5± 3.1 20.4± 1.6 <0.000 1 Fatpercentag e (%) 46.2± 1.6 44.5± 1.3 48.1+ 1.4 31.3± 1.7 <0.000 1 TOR 0.744+ 0.011 0.821 ±0.00 5 0.902+ 0.012 notassesse d <0.000 1

Value* s are given as mm + sea waist-to-hips girth ratio t tody weiojit (kg)/body height2(H)

Table2 .Serumlipid san dbloo d glucosei nobes ewome n subdivided accordingt obod yfa tdistribution .

body fat distribution Gluteal-femoral Intermediate Abdominal P-value n= 9 n= 10 n = 12 F ratio

Cholesterol (imol/1) 5.04 ± 0.24 5.34± 0.23 5.45± 0.23 0.47 HDL-cholesterol (rnnol/l) 1.31 + 0.11 1.16± 0.0 7 1.07+ 0.06 0.13 HCD-c/total-c 0.26 + 0.02 0.22+ 0.01 0.20± 0.01 0.03 Triglycerides (mrol/l) 0.91 + 0.08 1.59± 0.18 1.71+ 0.26 0.03 Blood glucose (mrol/l) 4.46 + 0.19 4.82+ 0.12 5.22+ 0.16 0.009

Value* s are given as Bean + SCB HXi-cholesteroL-total ctalesterol ratio

Nodifference s inself-reporte dweigh thistor yo rfamil yhistor yo fobesit y were found amongth evariou s groupso fth eobese .However ,8 0percen to fth e gluteal-femoral obese reporteda nonse to fobesit ybefor e theag eo f2 0year s incompariso nwit h 50percen to fth esubject swit ha nintermediat e typeo f bodyfa tdistributio nan d2 5percen to fth eabdomina l obesewomen . Table 3show s restingmetaboli c rate (RMR)diet-induce d thermogenesis(DIT ) and respiratory quotients (RQ)fo rth edifferen t obesegroup san dth e non-obese group.RM Ran dpostprandia l energyexpenditur e (PEE)(dat ano t shown)differe d significantly amongth egroups ,th eabdomina l obesean dth e

-168- non-obese subjectsha d onaverag ehighe rRMR' san d PEE'stha n subjectswit h a non-abdominal type ofbod y fatdistribution .Th eRM Rexpresse d inkj/d.k g fat-freemass ,wa sals o significantly different among thegroups ,bu t thiswa s not foundwhe n thenon-obes e subjectswer eexclude d from theanalysis . Variation among thenon-obes e subjects inRM Rpe r kg fat-freemas swa s relatively larger compared toth evariatio namon g theothe r groups.Thi swa s due to the fact that threenon-obes ewome nha d relatively largeRMR' spe r kg fat-free mass (FFM), ie,betwee n 150an d 155kj/d.k g FFM. Inth eothe r non-obesewome nRMR' spe r kg fat-freemas swer e allbetwee n 115an d 140 kj/d.kg FFM.DI Tan d RQ's, including thenonprotei n RQ's (datano t shown),di d notdiffe r either among thegroups .Pre -an dhourl ypostprandia l RQ'swer e not significantly differentamon g the four groups.A t theen d of the postprandial measurement period energyexpenditur ewa s onaverag e stillelevate dwit h about 7-8 percent above thepremea l RMR.Thi swa sno tdifferen t among the four groups. Themea n coefficient ofvariatio n (CV)fo r theRM Rmeasurement s inth e obese was 6.0 + 0.5 percent (range,0. 7 - 12.7percent) . The intra-individual day-to-dayvariatio n inth eobes e inDI To f 31.9+ 2.7 percent (range,0. 1 to 60.7percent )wa smuc h larger than thewithin-perso nvariatio n inRMR . No significant sequence effect,ie ,tim eo fmeasuremen t effect,o nRM R or DITwa s observed inth e obese subjects forwho m repeatedmeasurement swer eavailable . The average difference between themea n firstRMR ,o rDIT ,an d themea n last RMR, or DIT,measurement swere ,respectively , 2,ie ,4.0 5 (firstRMR )versu s 4.13 (lastRMR )kJ/min ,an d 4percent ,ie ,10 5 (firstDIT )versu s 109 (last DIT)kJ . Figure 1show s the increase in resting energyexpenditur e inth e obese subjects after ingestiono f the testmea l incompariso nwit h thepremea l RMR. Themajo r part of theDI Toccurre d inth e first twohour so f the postprandial period, ie,abou t 70percen to f thetota l calculated response.N o significant differenceswer e observed in responsepatter n among thevariou sgroups . Table 4show spre -an dpostprandia l substrate oxidation ratesan d nutrient balances for thevariou s groups.Non e of thesevariables ,excep t the glucose balance differed significantly among thegroups . Obese individualswh ower e either inth ehighes tquartil e of the distribution ofRM R (kJ/d.kg fat-freemass )o r inth ehighes tquartil e of theDI T (percentage of theenerg y content of the testmeal )distributio nwer e compared onvariou svariable swit h subjectswh ower e inth elowes tquartile so f the corresponding distributions.Tabl e 5list s the resultso f thesecomparisons .

-169- Individuals witha relativel y highRM Rwer e characterizedb yamor e abdominal typeo fbod yfa tdistributio n (p=0.05).

Table3 .Restin gmetaboli c rate,diet-induce d thermogenesisan dpre-an d postprandial respiratory quotients inobes ean dnon-obes ewomen .

Obese Iton-obese IMralue Body fat distribution F ratio Gluteal-fenoral Intermediate Abdominal n=10 n=10 n=12 n=10

EM! {kJ/hiin) 3.95 + 0.14 3.78 + 0.13 4.46 ± 0.19 4.35 ± 0.14 0.01 KMR (kJ/d.kgfFM) 119.5 ± 3.7 117.8 ± 3.7 128.4 + 3.8 140.2 + 5.5 0.0005 RQ - preprandial 0.83 + 0.01 0.82 + 0.01 0.82 + 0.01 0.84 ± 0.01 0.62 EQ - postprandial 0.86 ± 0.01 0.87 + 0.01 0.88 ± 0.01 0.87 + 0.02 0.86 Drr

Values aregive n as aBan ±se a BMt= resting

diet-induced thermogenesis ( kJ/min) 0.80T | | glu teal-femoral

lll'J intermediate

HI abdominal 0.60

0A0-

0.20

time after meal ingestion (h)

Figure1 .Diet-induce d thermogenesis (mean+ sem)i nobes ewome n subdivided according tobod yfa tdistribution . -170- Table4 .Pre -an dpostprandia l substrate oxidation ratesan dpostprandia l nutrient balancesi nobes ean dnon-obes ewomen .

Obese Non-obese P value Body fat distribution F ratio Gluteal-femoral Intenrediate abdominal n = 9 n= 10 n= 11 n = 10

- Preprandial oxidation: Protein (ïiq/ïnin.kgFFM ) 1.17 ± 0.09 1.19 ± 0.03 1.26 ± 0.06 1.18 ± 0.13 0.85 Fat (mj/inin.kgFFM) 0.74 + 0.10 0.84 ± 0.08 0.97 + 0.09 0.97 + 0.17 0.44 Glucose (jrei/hiin.kgFFM) 1.76 ± 0.19 1.46 + 0.15 1.57 ± 0.15 2.09 ± 0.26 0.11

- Postprandial, oxidation: Fat (nq/hiin.kgFFM) 0.49 ± 0.09 0.60 + 0.07 0.58 + 0.09 0.80 ± 0.16 0.24 Glucose (nq/ïiün.kgFFM) 3.26 ± 0.20 2.95 ± 0.21 3.33 + 0.26 3.46 ± 0.40 0.61

- Nutrient balance': Protein (g) - 1.0 + 0.8 - 1.2 ± 0.6 - 2.9 + 0.9 - 0.6 + 1.3 0.27 Fat (g) 8.1 ± 0.7 7.3 + 1.0 8.0 + 1.2 5.4 ± 1.3 0.31 Glucose (g) 22.4 ± 2.5 25.1 ± 2.2 23.8 ± 2.4 13.9 ± 3-5 0.03

Values are gùm as aeon ± sea

T inbakB (g) - cuwtative oxidation (g)

Inaddition ,RMR' san dDIT' sadjuste d forfat-fre ebod ymass ,ag ean dfatmass , were compared among groupsdifferin g inbod yfa tdistribution .Th eRMR , extrapolated to24-hou ran dadjuste d forthes e covariateswa ssignificantl y higher inth eabdomina l obesewhe n comparedt oth egluteal-femora l obese, respectively 6075+ 20 0versu s 5502+ 20 5kj/ d (p=0.04).Als oi ncompariso n with subjectswit ha nintermediat e typeo fbod yfa tdistribution ,th e abdominal obeseha dhighe r adjusted RMR's,ie ,607 5+ 20 0versu s 5517+ 19 3 kJ/d (p=0.05).Th eadjuste dRM Ri nth enon-obes e groupwa s679 0+ 26 1 kJ/d, whichwa ssignificantl y (p<0.01)highe r thanth eadjuste d RMR'si nth eobes e with eithera gluteal-femora l orintermediat e typeo fbod yfa tdistribution . No significant difference inadjuste dRM Rwa sfoun dbetwee nth enon-obes ean d the abdominal obese.Excludin gth ethre e control subjectswit h relatively high RMR'spe rk gfat-fre emas s fromth eanalysi sdi dno tproduc ea significant changei nresults .Afte rth eexclusio no fthes e subjects fromth eanalysis , adjusted RMR'swer e respectively, 5399+ 20 1kJ/ di nth egluteal-femora l obese, 5455+ 19 0kJ/ di nth eobes ewit ha nintermediat e typeo fbod yfa t distribution, 5995+ 18 0kJ/ di nth eabdomina l obesean d632 9± 31 1kJ/ di n

-171- the control women (n=7).Th edifference s between,o nth eon ehand ,th e abdominal obesean dnon-obes ewomen ,and ,o nth eothe r hand,th eothe rtw o obese groups,remaine d significanta tth ep < 0.0 5level .N odifference s were found amongth egroup si nDIT ,adjuste d forthes e covariates.

Table5 .Restin gmetaboli c rate,diet-induce d thermogenesisan dvariou s other variables inobes ewome n categorized inth ehighes tan dlowes tquartile so f thedistributio no fparameter s indicativeo fefficienc yo fenerg y metabolism.

FM! - kgFFM DET-% ME-mr&KE t Lowest Highest bauest Highest n=8 n=9 n=8 n=8

EMR (kJ/d/kgFfM) 106.6 1.4 138.4 2.1* 113.2 2.8 125.2 5.5 DTT (% ME intake) (%) 6.57 0.S9 1.58 0.57 4.50 0.38 9.49 0.34 t Fat-free mass (kg) 50.0 1.5 47.0 2.1 51.5 1.5 47.6 1.7 Fat mass (kg) 41.3 2.9 43.5 4.4 44.1 3.1 46.5 4.6 *HR 5 0.81 0.01 0.86 0.0$ 0.84 O.03 0.81 0.03 - Preprandial oxidation: Fat (mg/min.kgFFM) 0.63 0.09 1.04 0.0?f 0.76 0.11 II 0.87 0.14 - Pastprandial oxidation: Glucose (ng/lnin.kgFFM) 2.81 0.20 3.79 0.1?t 2.62 0.30 II 3.34 0.24

Value* s are given as nan + sea resting Metabolic rate expressed per kg fat-free Mess I diet—juli ml themogenesis expressed dn percentage of «atjihniî««M a energy content of test •eal î significant (p<0.05) difference aacng groups S wrist-to-hips girth ratio II fat and glucose oxidation rates assessed in 7 subjects free the lowest guartiXe.

DISCUSSION

Obesity isa comple x disorder,whos e etiology isincompletel y understoodi n the absenceo fclea r hormonalo rgeneti c disorders.I ti sdifficul tt o characterize theobes ea sindividual swit h reduced energy requirementso r energy expenditure. Some investigators reporta defectiv e diet-induced thermogenesis inobes e individuals,wherea s others couldno tobserv ea reduced diet-induced thermogenesis inobes e individuals incompariso nwit h lean controls (5-11).Th econtrovers yo ndefectiv e thermogenesis inth eobes e might bedu et ovariatio n amongth eobes e inenerg y expenditure.Th estud y here reportedha sinvestigate d this issue.I ti suniqu e intw orespects .I nth e firstplace ,i tstudie sth eimpac to fbod yfa tdistributio no nvariatio ni n energy expenditure ina relatively large groupo fobes ewomen ,and ,secondly ,

-172- ineac hobes e individual,energ yexpenditur ewa s repeatedlyassesse d toobtai n a moreprecis eestimat e of the individuals 'true'DI Tan dRMR . Inaddition , energy expenditurewa smeasure d inage-matche d non-obesewomen . The results shown o cleardifference s among theobes e andnon-obes ewome n in diet-induced thermogenesis.Restin gmetaboli c rate,adjuste d for fat-free body mass,ag e and fatmasswas ,however ,significantl y lower inobes ewome nwit ha gluteal-femoral or intermediate typeo fbod y fatdistributio n incompariso n with obesewome nwit ha nabdomina l typeo fbod y fatdistributio n and the non-obesewomen .Th e resultso f this study support thehypothesi s thatbod y fatdistributio n affectsenerg yexpenditur e inobesity . Currently,onl yon e studyha s investigated the relationbetwee nbod y fat distribution and energyexpenditur e inobesit y (13).Th e studyo fDe nBeste n et al. (13)showe d ina limite d number of subjectstha tbod y fatdistributio n did not significantly influencediet-induce d thermogenesisan d postabsorptive restingmetaboli c rate.However ,a trendwa sobserve d toward higher RMR's (kJ/d.kg fat-freemass )i nth eabdomina l obese incompariso nwit h the non-abdominal obese.Th epresen t studyuse sa considerabl e larger groupo f subjectsan dmor emeasurement s ofenerg yexpenditur e ineac h subject thandi d the studyo fDe nBeste ne ta l (13).I naddition ,value s forRM Ran d DITwer e obtained ina non-obes e controlgroup . The averagewithin-perso nday-to-da yvariatio n inRM R inth eobes ewa s relatively small incompariso n toth evariatio n inDIT .Th ewithin-perso n variation inRM Robserve d inthi s studywa s comparable tovalue s found by investigatorswh o studiedRM R in respiration chambers inindividual so n controlled diets (14).Suprisingl y fewstudie s reporto n reproducibility of DITmeasurement s (13,15). Both studies reporthig hwithin-perso n day-to-day variation inDIT ,ie ,o fabou t 30-60percent .A highwithin-perso n variation inDI Tmean s that studiescomparin g relatively smallgroup so f randomly selected obese individualswit h randomly selected leancontrol smigh thav eha d insufficient statistical power todetec tmeaningfu l physiological differences inDI Tbetwee nobes e and leansubjects . Pre-, and totalpostprandia l energyexpenditur ewer eo naverag e significantly lower inth enon-abdomina l obese incompariso nwit h theabdomina l obese or the non-obese subjects.Als owhe n theRM R (kJ/d.kg fat-freemass )wa s compared among thegroup s itwa s found thatth eabdomina l obese and thenon-obes e had stillhighe r pre-an d postprandial rateso fenerg yexpenditure . Respiratory quotients showed a characteristic pattern.Fastin gRQ' swer e between 0.80 and 0.85,whic h isi ngoo dagreemen twit h findings fromothe r

-173- studies (5,6,13). Inth e fasting stateabou t 40percen t of the energy dissipatedwa sderive d fromfa toxidation ,an d about2 5percen t fromprotei n oxidation. Inth epostprandia l state about6 0percen t of theenerg y expended wasderive d fromglucos eoxidation .Thi s isi nagreemen twit h findingso f Flatte t al. (22)wh o showed that carbohydrate intake elicitsa promp t increase inth e rateo fglucos e oxidation. Nutrient balanceswer eo naverag eals o similar among thedifferen t obese groups, indicating nodifference s among thegroup s inoxidativ e and non-oxidative storage of ingested carbohydrates andfat . A comparison ofobes e subjects characterized by cleardifference s inDI Tan d RMR's revealed that itwa sdifficul t tocharacteriz e subjectswit h a relativelyhig hDI Tfro m subjectswit ha relativelylo wDIT .Age ,bod y composition and body fatdistributio ndi d notdiffe r significantly among these subjects.Obes e individualswit h ahighe r DITha d onaverag e slightly,bu t not significantly,greate r postprandial glucoseoxidatio n rates thanobes e subjectswit h a lower DIT.Increase d cellular oxidationo fglucos emigh t be associatedwit h increased transport ofglucos e intoth e cells for nonoxidative storage, ie,formatio no fglycoge no r fat.Thes e latter twoprocesse s require more energy thanoxidatio no fglucos ean d could, thereby,contribut e toa n increased DIT (23).I naddition ,i tha sbee n reported thatDI Ti sonl y partly determined by themetabolis mo f the ingestedmacronutrients .Activatio n of the sympatheticnervou s systemb y ingestiono fnutrient smigh tals ob e important indeterminin g theleve l ofa subject'sDI T (2).N o informationwa s obtained inth epresen t study onactivatio n of the sympathetic nervous systemb y food ingestion. Obese individualswit h relativelyhig hRMR' swer e characterized, in particular,b y relativelyhig hpreprandia l fatoxidatio n rates,hig h postprandial glucoseoxidatio n ratesan da mor e abdominal typeo fbod y fat distribution.Whe nRMR' sadjuste d forage ,fa tmas san d fat-freemas s (4)wer e compared among thedifferen t groupso fbod y fatdistribution ,i twa s found thatwit h an increasingwaist-to-hip s girth ratio these adjusted RMR's also increased. Thus,RMR' sadjuste d forage ,fa tmas san d fat-freemas swer e on average 11percen t (about 0.6 MJ/d)lowe r inth egluteal-femora lobes e and the obesewit h an intermediate typeo fbod y fatdistributio nwhe n compared toth e abdominal obese.Th eDI Tadjuste d for these covariatesdi d notdiffe r significantly among the three groupso fobes e individuals.N odifference s in adjusted DIT'swer e foundbetwee n theobes ean d thenon-obes esubjects , whereas adjusted RMR'swer ehighes t inth enon-obes e subjects.Th e relatively

-174- high adjusted RMR'samon g thenon-obes e canb epartl yattribute d toth e fact thatthre eo f thenon-obes eha d relativelyhig hRMR' spe r kg fat-freemass . It ispossibl e thatRM R inthes ewome nwa selevated , forexample ,du e toanxiet y caused by theRM Rmeasuremen t ordu e tosom eundetecte d minor spontaneous movementsdurin g theRM Rmeasurement .However ,excludin g thesewome n from the analysis,di d notproduc e a significantlydifferen t result. The reasons for thedifferenc e inRM Radjuste d forage ,fa tmas san d fat-free massamon g thevariou s groupsar eno tdirectl y clear.Fastin g energy expenditure at rest isdetermine d largelyb y themetaboli c activityo f the organs and toa smaller extentb y themetaboli c activity of skeletal muscles (22). Itcoul d be that theproportio no forgan s inth etota l fat-freemas s is higher inth enon-obes e incompariso nwit h theobese ,an dtha tamon g theobes e thisproportio n ishighe r inth eabdomina l obese than inth eobes ewit h a non-abdominal typeo fbod y fatdistribution . Iti sals opossibl e that the metabolic activity of theorgan sdiffer samon g thevariou sgroups ,fo r example,b ydifference s in rateso f ionpumping ,protei n turnover or various substrate cycles,whic h could be related todifference s inhormona l statuso r activityo f the sympathetic nervous system.However ,informatio n on the metabolic activityo f theseprocesse s in relation tobod y fatpercentag e and body fatdistributio n islackin g orscarce .

It iswel l known from invitr o studies (25)tha t themetaboli c activity of abdominal fat ishighe r thanth eactivit yo fgluteal-femora l fat,bu twhethe r thisdifferenc e isassociate d inviv owit hhighe r rateso fenerg y expenditure inabdomina l fat than ingluteal-femora l fat isunknown .Eve nwhe n the activityo f the triglyceride-free fattyaci d substrate cycle is relatively elevated inabdomina l fat,i t seemsunlikel y thatthi scoul d explain the total difference ofmor e than1 0percen t inadjuste d RMR'samon g theobes e subgroups. Itha sals obee n found that skeletalmuscle s inabdomina l obese contain relativelymor eglycolyti c fasttwitc h fibers (26).Thi smight ,eve n in resting conditions,lea d toa n increased outputo f lactate from skeletal muscles inth eabdomina l obese thani nth enon-abdomina l obese.Lactat ema y thenb e convertedb y the liver intoglucos ea tth eexpens eo f 4mole so fAT P for 1mol e ofglucos e recirculated inthi swa y (23).Thi s recycling of lactate could alsoelevat e restingmetaboli c rate.Finally , itha sbee n reported that the abdominal obesehav e higher levelso fplasm a freetestosteron e in comparisonwit hnon-abdomina l obese (27).Thi sdifferenc e inhormona l profile may cause thedifferenc e inRMR' sadjuste d forage ,fat-fre emas s and fat-mass among thevariou s subgroups of theobese .

-175- Thisstud y joinsother s (25,26)i ndemonstratin g a relationshipbetwee nbod y fatdistributio n and serumlipid san dbloo d glucose.Wit h increasing waist-to-hips girth ratio serum triglycerides,seru m cholesterol and blood glucose levels increased,wherea sHDL-cholestero llevel sdecreased . Fasting blood glucose levels indicatedn omajo r perturbations inglucos e tolerance or insulin resistance inth edifferen tobes egroups .However ,t oevaluat e insulin resistance and glucose tolerance itwoul d havebee nbette r tomeasur e insulin and glucose levels in response toth etes tmeals . Inconclusion ,th epresen t study showstha tDI Tma y show considerable intra-individualday-to-da yvariatio n suggesting that instudie so f small groupso fobes e individuals itma yb enecessar y toasses sDI T repeatedly in each individual.Bod y fatdistributio ndi d nothav e an impacto nDIT .However , RMR'sadjuste d forage ,fat-fre ebod ymas san d fatmas swer e significantly higher inth eabdomina l obese incompariso nwit h thenon-abdomina l obese.Thi s studyals o showed thatadjuste d RMR's inth eabdomina l obesewer e not abnormallyhig h incompariso nwit h theadjuste d RMR's ina non-obes e control group.Ou r findings suggest that thenon-abdomina l obese are theobes ewit h reduced restingmetaboli c ratesand ,a sa corollary ,energ y requirements. These individuals,i nparticula r thegluteal-femora l obese,als o reported a significant earlier onseto fobesit y thanth eabdomina l obese.I t isno t clear fromthi sstud ywhethe r the reduced restingmetaboli c rates inth e non-abdominal obesewome nar e related tothei r earlier onseto f obesity in comparisonwit h theabdomina lobese .

ACKNOWLEDGEMENTS

Theauthor swoul d like tothan k MrMarce lva nLeutere n forpreparin g the manuscript and the subjects for their kind andenthousiasti c participation in the study.

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5.Ravussi n E,Burnan dB ,Schut zY ,Jéquie r E.Twenty-four-hou r energy expenditure and restingmetaboli c rate inobese ,moderatel y obese,an d control subjects.A m JCli nNut r1982;35:566-73 . 6.Bessar d T,Schut zY ,Jéquie r E.Energ yexpenditur e and postprandial thermogenesisi nobes ewome nbefor ean d afterweigh t loss.A m JCli nNut r 1983;38:680-93. 7.Well e SL,Campbel l RG.Norma l thermic effecto fglucos e inobes ewomen . Am J ClinNut r1983;37:87-92 . 8.Pitte tP ,Chappui s P,Acheso n K,d eTechterman n F,Jéquie r E.Thermi c effect ofglucos e inobes e subjects studied bydirec t and indirect calorimetry.B r JNut r1976;35:281-92 . 9. FeligP ,Cunningha m J, LevittM ,Hendle r R,Nade l E.Energ y expenditure inobesit y in fasting andpostprandia l state.A m J Physiol 1983;244:E45-51. 10.Nai r KS,Hallida yD ,Garro wJS .Thermi c response to isoenergetic protein, carbohydrate or fatmeal s inlea nan dobes e subjects.Cli nSe i 1983;65:307-12. 11. SchutzY ,Bessar d T,Jéquie r E.Diet-induce d thermogenesismeasure d over awhol e day inobes e andnon-obes ewomen .A m JCli nNut r1984;40:542-52 . 12.Vansan tG ,De nBeste nC ,Weststrat eJ , Deurenberg P.Bod y fat distribution and theprognosi s forweigh t reduction:preliminar y results. IntJ Obesity ;1988;12:133-40 . 13. DenBeste nC ,Vansan tG ,Weststrat eJA ,Deurenber g P.Restin g metabolic ratean d diet-induced thermogenesis inabdomina l and gluteal-femoral obesewome nbefor e andafte rweigh t reduction.A m JCli nNut r 1988;47:840-7. 14. Seidell JC,Bak xJC ,d eBoe r E,Deurenber g P,Hautvas t JGAJ. Fat distribution ofoverweigh t persons in relation tomorbidit y and subjective health. IntJ Obesit y1985;9:363-74 . 15. Ravussin E,Lillioj a S,Anderso n TE,Christi n L,Bogardu sC . Determinants of24-hou r energyexpenditur e inman .J Cli n Invest1986;78:1568-78 . 16.Acheso n K,Schut zY ,Bessar d T,Ravussi n E,Jéquie r E,Flat tJP . Nutritional influenceso n lipogenesisan d thermogenesis after a carbohydrate meal.A m JPhysio l 1984;:246:E62-E70. 17. Jéquier E,Acheso n K,Schut zY .Assessmen t ofenerg yexpenditur e and fuel utilization inman .An nRe vNut r1987;7:187-208 . 18. Lillioja S,Bogardu s C,Mot t DM,Kenned yAL ,Knowle rWC ,Howar d BV. Relationship between insulin-mediated glucosedisposa l and lipid metabolism.J Cli n Invest1985;75:1106-15 . 19. HartzAJ ,Ruple yDC ,Rim mAA .Th e association ofgirt hmeasurement s with disease in2106 5women .A m JEpidemio l 1984;119:71-80.- 20.Warnic k GR,Benderso n J,Alber sJJ .Dextra n sulfate-Mg precipitation procedure forquantitatio n ofhigh-density-lipoprotei ncholesterol .Cli n Chem1982;28:1379-88 . 21. Sullivan DR,Kruijswij k Z,Wes t CE,Kohlmeie r M, KatanMB .Determinatio n of serum triglyceridesb y anaccurat e enzymaticmetho d notaffecte d by free glycerol.Cli nChe m1985;31:1227-8 . 22. FlattJP ,Ravussi n E.Acheso n KJ,Jéquie r E.Effect so fdietar y fato n postprandial substrate oxidation and oncarbohydrat e and fatbalances .J Clin Invest1985;76:1019-24 . 23. FlattJP .Biochemistr y ofenerg yexpenditure . In:Bra yG , ed.Recen t advances inobesit y research: IILondon :Joh n Libbey,1978:211-8 . 24. Grande F.Energ yexpenditur e oforgan san d tissues.In :Kinne yJM ,ed . Assessment ofenerg ymetabolis m inhealt h anddisease .Columbus :Ros s Laboratories,1980:88-92 .

-177- 25. BjörntorpP .Adipos e tissuedistributio n andmorbidity . In:Berr y EM Blondheim SH,Eliaho uHE ,Shafri r E,eds .Recen tadvance s inobesit y research:V . London:Joh nLibbey ,1986:60-5 . 26. Krotkiewski M,Björntor p P.Muscl e tissue inobesit ywit h different distribution ofadipos e tissue.Effect so fphysica l training. IntJ Obesity1986;10:331-41 . 27. KissebahAH ,Peiri sA ,Evan sDJ .Mechanism sassociatin g body fat distributionwit h theabnorma lmetaboli c profile inobesity . In:Berr y EM,Blondhei m SH,Eliaho uHE ,Shafri r E,eds .Recen t advances inobesit y research:V . London:Joh nLibbey ,1986:54-9 .

-178- Chapter 9. Alcohol and itsacut eeffect s onrestin g metabolic rate and diet-induced thermogenesis

JANA WESTSTRAT EMSC ,INGRI DWUNNIN KBSC ,PAU LDEURENBER GPHD ,JOSEP H GAJHAUTVAS TM D

ABSTRACT

Intw ostudie sth eimpac to falcoho l (ethanol)o nrestin generg y expenditurewa sdetermined .I na firs tstud yth ethermi ceffec to f ethanolo nrestin gmetaboli crat e(RMR )wa sassesse di nte nnon-obes e men,ag e26. 5+ 1. 7 (mean+ sem )years .Energ yexpenditur ewa smeasure d witha ventilate dhoo dsystem .Afte ra ninitia lRM Rmeasurement , subjectsreceive di nrando morde r2 0g o fethano li nthre e concentrations,respectively ,o f7.5 ,1 8an d3 0ml/10 0m lwater .Th e thermiceffec to fethano lwa sthe ncontinuousl ymeasure dfo r9 0minutes . Ethanolha da significan tthermi ceffect ,whic hvaried ,dependin go nit s concentration,betwee n0.2 2kJ/mi n(7.5% )t o0.3 0kJ/mi n (30%).However , nosignifican tdifference si nthermi ceffec tamon gth ethre e concentrationwer eobserved .I na secon dstud yth eimpac to fethano lo n diet-inducedthermogenesi s (DIT)wa sassesse di ntwelv enon-obes emen , age27. 3+ 1. 5years .Afte ra ninitia lRM Rmeasurement ,subject s receivedi nrando morder ,i nduplicate ,eithe ra mea lo ffoo d (2.00MJ , 13percen tprotein ,3 0percen tfat ,5 7percen tcarbohydrates )plu sa n alcoholicaperiti f (20g i n1 8ml/10 0ml )o ra niso-energeti cmea lo f foodalon e (2.55MJ ,1 3percen tprotein ,3 0percen tfat ,5 7percen t carbohydrates)plu sa placeb oaperitif ,simila ri ntast ean dappearanc e toth ealcoho laperitif .DI Twa sthe ncontinuousl ymeasure dfo ra perio d of4 hours .N osignifican tdifference si nDI Tbetwee nth etreatment s wereobserved .Afte rthe .mea lplu salcohol ,substrat eoxidatio nrate so f glucosean dfa twer esignificantl yreduce di ncompariso nwit hth emea l

-179- alone.A t thepostprandia l period duringwhic hethano l oxidationwa s at itsmaximum , therewa s a slightpotentiatio n ofDI Tb yethanol .

Keywords: indirect calorimetry,alcohol ,ethanol ,thermi ceffect , restingmetaboli c rate,diet-induce d thermogenesis,substrat e oxidation, human

INTRODUCTION

Inmos tWester n countries totalpe r capita alcohol consumptionha s increased dramatically over the last fewdecade s (1,2). A recent nation-wide food consumption study inth eNetherland s showed that alcohol contributes onaverag e 3percen t totota ldail yenerg y intake in women and 5.4 percent inme nage d 22-49year s (3).Simila r figureshav e been reported forme nan dwome n inothe rWester n countries (4,5). Thehealt h effectso fexcessiv e alcohol intakear ewel l documented (6-8), but themetaboli c consequencesan d relation toth e incidence of disease ofmoderat e alcoholus e remaint ob edefinitel y assessed. In this respect there isa genera l interest inth eutilizatio n of alcohol asa nenerg ysource .Th e issuestha t receivemos tattentio nar ewhethe r alcohol caloriesar eadde d too r substituted fornon-alcoholi c calories in thedie tan dwhethe r energy intake fromalcoho l is related to increased levelso fadiposit y inth esam ewa ya senerg y intake from non-alcoholic sources. Themajorit yo fobservationa l studies showa strikingphenomenon . Alcohol usuallyprovide sadditiona l energy toth ediet ,bu t itsus e is not associatedwit hhighe r levelso fadiposit y (9-13). In concordance with these findingsar e the resultso fvariou sexperimenta l studies (14-16). In these studies itwa s found that iso-energetic substitution ofnon-alcoholi c caloriesb yalcoho l resulted inweigh t loss in volunteers and thatalcoho l added toth edie tdi dno t result inweigh t gain.Thes e findings suggest thatalcoho l isuse d lessefficien t asa n energy source than,fo rexample ,fat san d carbohydrates,o r that alcohol interfereswit h theefficien tutilizatio no f thesesubstrates . However, results frommetaboli c studiesassessin g the impacto falcoho l ingestiono nenerg yexpenditur e giveequivoca l support for the latter proposition. Inth e classical reporto fAtwate r andBenedic t itwa s

-180- shown thatalcoho lus edi dno t increaseenerg yexpenditur e inma n relative to fatso r carbohydrates (17).Mor e recent studiesdi d observe either an increase in,o rn oeffec to noxyge nconsumptio n after alcohol use (18-21).Th e reasons for thesediscrepan t findings areunclear ,bu t ingenera l theexperimenta ldesign ,ie ,numbe ran d sexo f subjects studied,duratio n of study,amoun to falcoho l givenan dwa y inwhic h alcoholwa sprovide d and thetechniqu euse d tomeasur e energy expenditure differ considerably among thevariou s studies.Thus , in spite of its importance asa nenerg y source inth eaverag e adult individual,littl e isknow nwit h certainty onth e impacto falcoho l on overall energymetabolism . Inparticular ,ther e islittl edat ao n the effect ofalcoho l ondiet-induce d thermogenesis (19,21). Inthi s reportw euse d theventilate d hood technique toasses s the thermic effecto fethano l invaryin g concentrations.I naddition ,w e investigated the impacto fethano l ondiet-induce d thermogenesis.Th e results showa significantthermi ceffec to fethano lo n fastingenerg y expenditure,bu tn o systematic potentiation of diet-induced thermogenesis byethanol .

SUBJECTSAN D METHODS

Thepresen t studyconsiste d of twoparts .I nth e first study the impact of ingestion ofethano l onpostabsorptiv e restingmetaboli c rate (RMR) was studied. Ina second studyw e assessed theeffec to fethano l on diet-induced thermogenesis (DIT).

SUBJECTS

Eighteenmal e subjectswer e invited forparticipatio n inth e studies by anadvertisemen t inth eweekl yperiodica l of theUniversit y and through friendsan d relatives.Fou r subjectsparticipate d inbot hexperiments . Subjectswer e all,excep tone ,student so rmember so f staffo fth e University.Afte r applying forparticipatio n subjects received a letter inwhic h thepurpos e andnatur eo f theexperiment(s )wer e explained. Theyals o received twoquestionnaires ,on e forassessin gpas tan d presenthealt h statusan don e forobtainin g informationo nhabitua l eating,drinking , smoking and exercise habits.Th emedica l questionnaire

-181- wasevaluate db ya physicia n (JGAJH).Afte rth esubject sgav ethei r writtenconsen tt oparticipat ean dwhe nthe yfulfille dth eentr y criteria,subject scoul dparticipat ei nth eexperiments .Subject sha dt o beapparentl yhealth y(n opas to rpresen tevidenc eo fhypo -o r hyperthyroidismo rdiabete smellitus) ,non-obes e (bodyfa tpercentag e below2 5percent )an dha dt ohav emoderat esmokin g(les stha n1 0 cigarettes/d)o rdrinkin ghabit s(n omor etha n5 alcoholi c consumptions/d).Non eo fth esubject suse ddrug sknow nt oaffec tenerg y metabolism,non ewa so na diet ,al lsubject swer econsumin gnorma l balancedmeal san dal lsubject sha dha dweigh tfluctuation swithi n2. 5 kgfo ra tleas tsi xmonth sprio rt oth estar to fth estudy .Th eprotoco l ofth estudie swa ssubmitte dt oan dapprove do fb yth eMedical-Ethica l Committeeo fth eWageninge nAgricultura lUniversity .

METHODS

Experimentaldesig n

Thermiceffec to fethano l

Inthi sstud yte nsubject sreceive do nthre eoccasion sa tdifferen tday s anora ldos eo f2 0g ethano ldissolve di neithe r33 5m lwate r(7. 5 percentsolution) ,14 0m lwate r (18percen tsolution )o r8 5m lwate r(3 0 percentsolution )afte ra ninitia lRM Rmeasurement .T othes esolution s were,respectively ,adde d1. 2g o fa nonnutritiv esweetene r(Natrena , BayerGmbH ,Germany )an d0.01 2m go fanisee doi l (toth e7. 5percen t solution)o r0. 8g o fth esweetene ran d0.00 6m go fanisee doi l(t oth e 18an d3 0percen tsolutions )t oimprov esensor ycharacteristic s (mimickingAnisette) .Th eethano lsolution swer edrun ka ta temperatur e between10-15° Cwithi n1 0minute san dwit hth eventilate dhoo dremove d fromth esubject sheads .Afte rth ealcoho lingestio nth ehoo dwa s replacedan denerg yexpenditur ewa smeasure dcontinuousl yfo ranothe r9 0 minutes.Energ yexpenditur ewa smeasure dwit ha ventilate dhoo dsyste m aspreviousl ydescribe d (22).Subject sreceive dth ethre eethano l solutionseithe rearl yi nth emornin gafte ra novernigh tfas tor ,earl y inth emornin gafte ra fastin gperio do fa tleas t4. 5hour safte ra standardizedbreakfas to fabou t2 MJ .I na separat estud yi n5 subject s

-182- (datano tshown )th ethermi ceffec to fdrinkin g35 0m lwate rplu s1. 2g ofth esweetene rwa sassessed .Wate rplu ssweetene rdi dno tinduc ea thermiceffect .Betwee neac hsessio nwa sa time-interva lo fa tleas ttw o days.Subject sreceive dth ethre eethano lsolution si nrandomize dorder .

Impacto fethano lo ndiet-induce dthermogenesi s

Inthi sstud ytwelv esubject sreceive do nfou rdifferen tday si nth e afternoontw otreatment si nduplicate ,ie ,eithe ra tes ttreatmen t consistingo fth eingestio no fa nalcoholi caperiti f (containing2 0g o f ethanoli na n1 8percen tsolutio n (v/v))an da yoghurt-base dmixe d liquidtes tmea l (containing1.9 6MJ ,1 3percen tprotein ,2 7percen t fat,6 0percen tcarbohydrates )o ra placeb oaperiti f(containin gzer o calories)an da niso-energeti cyoghurt-base dmixe dliqui dtes tmea l (containing2.5 5MJ ,1 3percen tprotein ,2 7percen tfat ,6 0percen t carbohydrates).T obot haperitif sflavoring swer eadde dt omimie k Anisette.T oth ealcoholi caperiti fwa sadde d0. 8g o fa nonnutritiv e sweetener (Natrena,Baye rGmbH ,Germany )an d0.0 6m go fanisee doil .T o theplaceb oaperiti fwer eadde d0. 8g o fth esweetener ,0.0 6m go f aniseedoil ,0.0 3m go fkini nan d0.00 7m go fmaltol .I na separat e controlexperimen ti n8 subject s(dat ano tshown )th ethermi ceffec to f kinini na muc hlarge rdos e (220m gi n35 0m lwater )wa stested .Kini n didno tinduc ea significan tthermi ceffect .Th eimpac to fmalto io n energyexpenditur ewa sno tstudied .Th equantit yuse dwa sextremel ylo w andn oeffect so nenerg yexpenditur ewer eexpected .Th eaperitif swer e givena ta temperatur ebetwee n10-15° Cafte rRM Rha dbee nmeasure dfo r atleas t1 hour ,an dwit hth ehoo dremove dfro mth esubjects .Afte r ingestiono fth eaperiti f (within1 0minutes) ,th ehoo dwa sreplace d overth esubject' shead .Fiftee nminute slate rsubject sreceive dth e liquidtes tmea lthroug ha stra wwhic hwa spasse dthroug hth ehood .Th e mealswer egive na troo mtemperatur ean dwer eeate nwithi n5 minutes . Energyexpenditur ewa sthe ncontinuousl ymeasure dfo r4 hours .Contro l andtes ttreatment swer eassigne dt oth esubject saccordin gt oa Lati n squaredesign . Betweentreatment swa sa time-interva lo fa tleas ttw odays .

-183- Energyexchang e measurements

Energyexpenditur e of subjects resting ina hospita l bed ina supine positionwa smeasure dwit h aventilate d hood system.Detail so f the measurement procedure and conditionshav ebee npreviousl y published (22,23). Spontaneousmovement so f the subjectswer e unobtrusively registered bymean so fa loadcel l (TokyoSokk i Kenkyujo,TKA-200A , Tokyo,Japan )place dunde r one of theleg so f thehospita l bed.Onc e everyhour ,durin g calibrationo f thega sanalyzer swit h freshoutsid e air, subjectswer eallowe d tomov ebriefl y for 2-3minutes . Subjectswhos e energyexpenditur ewa smeasure d inth eafternoo nwer e instructed tohav e a smallbreakfas t (<2 MJ )befor e 8.00 hoursan d to avoid coffeedrinking , sleeping andmoderat e orheav yexercis e onth e morningbefor e thega sexchang emeasurements .Unde r theseconditions , RMR andDI Tmeasure d inth eafternoo nd ono tdiffe r systematically from RMR andDI Tmeasure d inth emornin g (22).Afte r thega sexchang e measurementsurin ewa s collected fordeterminin g urea-nitrogen excretion. Energyexpenditur ewa s calculated according toJéquie r et al. (24).Th e thermic effecto fethano lwa scalculate d asth edifferenc e between the average energyexpenditur e over aperio d of 90minute s and the corresponding baseline RMR.DI Twit ho rwithou tethano lwa s calculated asth edifferenc e between theaverag ehourl yan d total (4hours ) postprandial energyexpenditur e and thecorrespondin g baseline RMR.

Substrate oxidation ratesan dnutrien t balances

Substrate oxidation ratesan dnutrien tbalance swer e calculated as previously described (22-24). Inaddition ,th econtributio n ofethano l oxidation to total oxidationwa sestimated .Alcoho l oxidationwa s assumed tob eequa l toth eaverag eeliminatio n rateo falcoho l fromth e blood, ie,10 0mg/k gbod yweight/hou r(25) . Generally about93-9 8percen t ofa n ingesteddos e ofalcoho l is eliminated fromth ebod yb yoxidatio n andnegligabl e amounts leave the bodyb y respiration and inth eurin e (25).W e calculated theamoun to f oxygenneede d tooxydiz e 1g o fethano l (1.4594 1)an d the amounto f carbondioxide thusproduce d (0.973 1).Tota l oxygen consumptionan d carbondioxide productionwer e subsequently corrected for thevolumin a of

-184- oxygenan d carbondioxide associatedwit hethano l oxidation.Thes e corrected gasvoluminawer euse d inth ecalculatio n of the oxidation rateso f fatsan d carbohydrates.I tshoul d benote d that therema yb e considerable inter-individual difference inth eeliminatio n rateo f alcohol fromth ebloo d (25).However ,sinc e inthi sstud yeac hperso n is measured repeatedlyunde r different conditions suchvariatio nwil l probablyno tbia s conclusions onwithin-perso n comparisonso f treatments.

Body composition

Body fatmas san dbod y fat-freemas swer eestimate d fromtota l body densityusin g Siri's formula (26).Tota lbod ydensit ywa smeasure d in each subjectb yhydrostati cweighin gwit h simultaneousdeterminatio n of residual lungvolum eb ya heliu mdilutio ntechnique .

Statistics

The thermic effecto fethano lwa sevaluate dusin g two-sided paired t-tests. Analysiso fvarianc ewa suse d totes tdifference s among the three treatments (7.5,1 8an d 30percen t solutions)i nthermi c effect.I nth e second study, resultso f theduplicat e measurementswer epoole d and averaged forbot h treatments.Analysi s ofdifference sbetwee nbot h treatmentswer eperforme dusin g two-sidedpaire d t-tests.Correlatio n coefficientswer e calculated asPearson' sproduct-momen t correlation coefficients. Resultsar e expressed asmea n+ sem .

RESULTS

Table 1give s some characteristics ofth e subjectsparticipatin g inth e twoexperiments .

-185- Table1 :Age ,bod yweight ,bod yfa tpercentag ean dhabitua l alcoholus eo fth esubjects .

Study 1 StuoV 2 Thermiceffec to falcoho l Effecto falcoho l ondie t -inducedtherncgenesi s n= 10 n= L2

Mean sem Bange Mean -. Bange

Age (yrs) 26.5 1.7 22 - 41 27.3 1.5 21 - 41 Bodyweigh t (kg) 76.8 3.2 66.2- 102. 0 78.3 3.0 58.8- 101. 8 Bodyfa tconten t(% ) 17.0 1.1 13.0- 24.7 16.0 1.2 7.9- 22. 5 Habitualalcoho lus e 1.2 0.2 0.0- 3 1.4 0.4 0.0- 5

nd« of alcoholic beverages habitually used/ti

The averagewa s2 7+ 1. 5year s (range:21-4 1years) , bodyfa tpercentag ewa s onaverag e16. 5+ 1, 2percen t (range:7.6-24. 7percent) .Thre e subjectsha da bodyfa tpercentag e between2 0an d2 5percent ,bu tnon ewa sobese .Habitua l useo falcoholi c beverageswa so naverag e1- 2units/ d (range: 0-5). Table2 show smetaboli c rate,oxyge nconsumption ,carbondioxid eproductio nan d respiratory quotientsi nsubject sbefor ean dafte r ethanol ingestioni nthre e different concentrations.Non eo fth evariable swa ssignificantl y different amongth ethre e treatments.Alcoho l ingestion induceda significan t increase inmetaboli c ratean di noxyge n consumption,bu tno ti ncarbondioxid e production.A sa consequenc e respiratoryquotient sdecrease d significantly after alcoholuse .O naverage ,metaboli c rate increased4. 4percen t after intakeo f7. 5percen t ethanol solution,4. 8percen tafte r ingestiono fth e1 8 percentethano l solutionan d6. 2percen tafte rus eo falcoho li na 3 0percen t solution.Thes evalue sdi dno tdiffe r significantly. Sincew edi dno tobserv e significantdifference si nthermi c effect among iso-energeticethano l solutionso fvaryin g concentration,th eeffec to f ethanolo nDI Twa sstudie di nth esecon d studyusin gth emoderatel y concentrated solutiono f1 8percent . Table3 give spre -an dpostprandia l metabolic rate,oxyge n consumptionan d carbondioxide productioni nsubject swh oreceive d induplicat e eithera mea l witho rwithou tethano la sa naperitif . Metabolic rate,oxyge nconsumptio nan dcarbondioxid e production increased significantly after ingestiono fth emea leithe rwit ho rwithou t alcohol.N o significantdifferenc ewa sfoun dbetwee nbot h treatmentsi nth eincreas ei n metabolic rateo ri noxyge n consumption.Th epostprandia l risei n carbondioxidewas ,however ,significantl y smallero nth etes ttreatmen ti n

-186- Table2 :Metaboli c rate,oxyge n consumption, carbondioxide productionan d respiratory quotients insubject s beforean dafte r ingestiono fa 2 0g ethano l dosis inthre e different concentrations.

ETHANOL Volune percent 7.5 18 30 Before After Before After Before After

Metabolic rate (kj/tnin) 4.98 + 0.13 5.20 ± 0.16 4.95 + 0.21 5.19 + 0.24Î 4.85 ± 0.22 5.15 ± 0.24 t O Consunption (ml/tnin) 247 + 7.0 261 ± 8.3 245 ± 11.3 260 + 12.st 241 + 11.0 258 ± 12.1 Î CO production (ml/tnin) 206 + 5.7 204 + 6.1 206 + 6.5 203 + 7.4 201 + 9.3 200 + 10.3 Respiratory quotient 0.84 + 0.02 0.78 ± O.Olt 0.85 + 0.02 0.78 ± O.Olr 0.84 ± 0.02 0.77 ± O.orf

Value* s are given as wan + sea Difference between 'before' and 'after' signifirTeltly different fro» zero at p < 0.05 T Bùdae at p < 0.01 $ Ibide» at p < 0.001

Table 3:Metaboli c rate,oxyge n consumption, carbondioxide production, respiratory quotientan ddiet-induce d thermogenesisi nsubject sbefor ean d after ingestiono fa tes tmea lwit ho rwithou ta dos eo f2 0g ethano l givena s an aperitif.

Meal + ethanol Control - irea] t Before After Before After ffetabolic rate (kj/tain) 5.34 + 0.23 6.25 + 0.25 5.34 + 0.26 6.12 + 0.27 0. Consunption (ml/hiin) 267 ± 11 312 ± 12 267 ± 13 300 ±13 00. Production (ml/hiin) 216 + 9 251 ± 11 216 ± 11 263 ± H Respiratory quotient 0.81+ 0.01 0.80 ± 0.01 0.88 ± 0.01 0.88 ± O.Olt DIT (kJ) - 219 ± 14 - 185 ± 20 DIT-% ME (%) - 8.6 ± 0.6 - 7.2 + 0.8

Values axe givan as aean ± see EO* T — diet—induoed thersDganesis; % IC = peccentage of aetabolisable energy intake Bwrgy intake: 2.545 ± 0.005 KT (22.7 pttlUBUL derivad froB etiianol) t Biergy intake: 2.551 ± 0.004 HI f Postprandial respiratory quotients significantly different between treatBBnts (p = 0.002)

comparison toth econtro l treatment.Respirator y quotients remaineda tthei r preprandial level after ingestiono fa mea lwit h ethanol,wherea s after ingestiono fa mea lwithou t ethanol respiratory quotients showed a significant increase.O naverage ,DI Twa sabou t1 6percen t highero nth etes t treatmenti n comparisonwit hth econtro l treatment.Th edifferenc e inDI Tbetwee nth e treatmentswas ,however ,no tsignificant . Figure1 show saverag e hourlyDI Tvalue s separately forbot h treatments.

-187- diet-induced thermogenesis (kJ/min)

1.40-

g MEAL

MEAL»ETHANOL

2 3 4 postprandialtim e(h )

Figure1 .Diet-induce d thermogenesis (mean+ sem)i nsubject safte r ingestion of iso-energeticmeal swit ho rwithou t ethanol.

Duringth esecond , thirdan dfourt hhour ,DI Twa shighe ro nth etes t treatment compared toth econtro l treatment,bu tonl y forth esecon dhou r thedifferenc e reached statistical significance (p=0.03). Figure2 give s respiratoryquotient sfo rRM Ran dfo rth ecours eo fth eDI T measurement separately forbot htreatments . Significant differenceswer e foundbetwee n both treatmentsi nrespirator y quotients duringth efirst ,secon dan dthir dhou ro fth epostprandia l period. Table 4give s energyan dnutrien t intake,an dpostprandia l metabolismo fth e ingested nutrientsfo rbot h treatments.Glucos ean dfa toxidatio n rateswer e significantly lower (p<0.001 )o nth etes ttreatmen ti ncompariso nwit hth e control treatment.Postprandia l protein oxidationwa sno tsystematicall y affectedb yethano l ingestion.O nth econtro l treatment subjectsoxydize do n average about5 0percent o fth ecarbohydrat ean dfa tintake .Durin ga 4 hour s postprandial period,whe n foodwa seate nwit h ethanol,abou t4 0percent o fth e

-188- respiratory quotient gMEAL 0.89- i ^1 MEAL»ETHANO L 0.87-

0.85-

0.83- I 0.81- Z 0.79- I I

0.77- i Ü RMR ~~r~ 1 MEAL before/aftermea lingestio n(h )

Figure2 .Respirator y quotients (mean+ sem)i nsubject sbefor ean dafte r ingestiono fiso-energeti c mealswit ho rwithou t ethanol.

glucose intakean dabou t2 0percen to fth efa tintak ewer e subsequently oxydized. Total energy retentionwa sno tsignificantl y differentbetwee n both treatments. Onth etes t treatment,ne tlipogenesi soccurre d innin e subjectsdurin gth e first postprandial hour (1.9+ 0. 7g )an di neigh t subjectsdurin gth esecon d hour (1.9+ 0. 7 g). Netlipogenesi swa so fnegligabl e importancei nth e subjectso nth econtro l treatment.

-189- Table4 :Nutrien tan denerg y intake,utilizatio nan dstorag ei nth ebod yove r a periodo f4 hour si nsubject safte r ingestiono fa tes tmea lwit ho rwithou t a doseo f2 0ethano l givena sa naperitif .

Energy Carbohydrate Fats Protein Ethanol MfE MfE M+E M-E MŒ M-E »E M-E MfE kJ kJ g g g g g g g

Intake 2543+ 5 2550+ 4 68.7 + 0.1 90.8 + 0.1 14.1 ± 0.0 18.6 + 0.0 15.0 + 0.0 19.8 + 0.0 19.9 + 0.1 Oxidation 29.8 + 3.5 47.5 ± 3.3* 3.1 ± 1.5 9.2 ± 1.6t 17.5 + 1.5 18.8 + 1.4 19.8 ± 0.3 Expenditure 1500 + 60 1469 + 65 Storage 1093 ± 61 1081 + 66 38.9 ± 3.5 43.3 + 3.3 11.0 1.5 9.4 ± 1.6 -2.6 + 1.5 1.0 + 1.4 0.1 + 0.0

Values are gi\en as aean ± sea HI* E= Maal with ethanol; M-E= Baal without ethanol Sionificant difference betwaan MtE and M-Ea t p < 0.01 + Sionificant difference Letweon MfE and M-E at p < 0.05

DISCUSSION

Thermiceffec to fethano l

The resultso fthi s study showtha tethano l ina moderat e amounto f2 0g inducesi nfastin g individualsa significan t increasei noxyge n consumption andmetaboli c rate,bu tno ti ncarbondioxid e production.Th eincreas ei n metabolic ratewa sno tsignificantl y relatedt oth econcentratio no fth e ethanol solution. Ethanol induceda nincreas e inmetaboli c rateo fabou t4- 6percen to fth e ingested caloric load (about0. 6MJ) . Thisi ssimila ro rslightl ylowe r toth e thermic effecto ffat so rcarbohydrate s (27).Th eresult so fthi s study supportth efinding so fPerma n (18)an dRosenber gan dDurni n (19),bu tar ei n contrastwit h theobservation so fStoc kan dStuar t (21)an dBarne se tal . (22). Itis ,however ,difficul tt ocompar e these studies,becaus eo f differencesi nexperimenta ldesig nan di nth etechniqu euse d forassessin g alcohol-induced thermogenesis.I ncontras tt oth epresen t study,non eo fth e aforementioned studiesemploye dth eventilate d hood techniquet oassess energ y expenditure.Thi stechniqu ei sparticularl y suitable formeasurin g energy expenditure accurately overprolonge d periodso ftime ,ie ,ove r severalhours , andha sbee n recommended forus ei nmetaboli c studiesassessin g thermogenesis (28). Inth epresen tstud yw egav e2 0g o fethano ldissolve d invaryin gamount so f waterwit h added flavorings.Thi senable du st ostud yth eimpac to fethano li n

-190- differentconcentration so nenerg yexpenditure .Perma n(18 )an dRosenber gan d Durnin(19 )gav eethanol ,respectively ,i na dosi so f0.2 9gA g dissolvedi n 150m lo fchille dfrui tjuic eo ri na dosi so f2 3g a s23 0m lo fre dwine . Barnese tal . (22)an dStoc kan dStuar t(21 )use dwhisk yi na dosi so f1. 5 ml/kgan di na quantit yo fabou t8 0ml ,respectively .Beside sdifference s amongthes estudie si nth eamoun to fethano lgiven ,th econcentratio no fth e ethanolsolution san dth eamoun to fnon-alcoholi ccalorie svaried .Fo r example,re dwin ean dfrui tjuic ema ycontai ncarbohydrates ,whic hca nprovok e anincreas ei nenerg yexpenditur e(27) . Forth epresen tstud yw efirs tteste da quantit yo f3 0g ethano l (18percent ) andfoun dtha tthi samount ,drun kwithi n1 0minutes ,induce ddrowsines san d symptomso fmil dintoxicatio ni nfastin gindividuals .Sinc edrowsines sma y interferewit ha correc tassessmen to fmetaboli crate ,w eopte dfo rth e smallerdosi so f2 0g .I nfastin gsubject ssuc ha namoun to fethano lshoul d quicklyrais eth ebloo dethano lconcentration st ovalue sbetwee n5 an d1 5 mmol/1(29) .I twa sexpecte dtha tth emor econcentrate dsolution swoul dlea d tohighe rmaxima lbloo dalcoho lcurve s (29).A thighe rbloo dalcoho llevel sa n increasedmetabolis mo fethano lb yth emicrosoma lethano loxydizin gsyste m (MEOS)occur s(14) . Oxidationo fethano lb yMEO Swil lresul ti na les s efficientcouplin g (about2 0percen treductio ni nne tAT Pproduction )o f oxidationt oAT Psynthesi san da nincrease ddeman dfo rNADP Hcompare dt o oxidationb yth ealcohol-dehydrogenas e (ADH)pathway .Th elatte rpathwa y operatesfull ya tlowe rbloo dalcoho lconcentration s (14).Thi sma yaffec t thermogenesisi ntw oways .A relativeincreas ei nethano loxidatio nb yMEO S mayincreas eth erat eo fethano loxidatio nt omee tth etissue sdeman dfo rATP , secondly,th eeventua lNADP Hrepletio nma yincreas eenergy ,ie ,AT Pdemand , fromothe rmetaboli cpathways .Bot hprocesse scoul dlea dt ohighe roxyge n consumptionan dhenc eincrease dthermogenesis .However ,w edi dno tfin da significantrelatio nbetwee nth ethermi ceffec to fethano lan dit s concentration.Nonetheless ,a tlowe rethano lconcentration s (7.5an d1 8 percent)th ethermi ceffec to fethano lwa so naverag e4. 6percen tversu s6. 2 percenta tth ehighes tethano lconcentration .Thi si sa relativ edifferenc eo f about3 0percen tan dcoul db epartl yattribute dt oa nincreas ei nrate 'o f ethanoloxidatio na tth ehighes tethano lconcentratio nt ocompensat efo rth e lessefficien tmetabolis mo fethanol ,ie ,a relativel ygreate rpar tb yMEO S thanb yth eAD Hpathway .However ,th eincreas ei nth ethermi ceffec to f ethanola tth ehighes tethano lconcentratio nwa sno ta systematica lphenomeno n anddi dno treac hstatistica lsignificance .I ti sals opossibl etha tth e

-191- different solutionsdi dno t result inclea rdifference s inbloo d ethanol concentrationsan d hence inethano lmetabolism . Sincebloo d alcohol curves wereno tmeasure dw e canno tpu t thisexplanatio n toempirica ltest . Inthi s study itwa sals o found thatethano l ingestionan d its subsequent oxidation significantly reduced basal respiratoryquotients .I ti swel l known thatethano l oxydizeswit ha respiratoryquotien to f 0.666,whic h ismuc h lower than respiratoryquotient susuall y found inovernigh t fasting individuals(30) .

Impact ofethano l ondiet-induce d thermogenesis

Very fewstudie shav eassesse d theeffec to fethano l ondiet-induce d thermogenesis (19,21).Rosenber g andDurni n (19)foun dn o significant difference between theaverag e increase inmetaboli c rateove r 3hour safte r an iso-energeticmea l of foodo ro f foodplu salcohol .However , inth e latter study,durin g the last 30minute so f the 3hour spostprandia l period the increased oxygen consumptionwa s significantlygreate r after themea l including thealcohol .Stoc k and Stuart (21)foun d thatwhisk y (837kj )take n witha mea l produced a 22percen t increase inoxyge nconsumptio nwhic hwa s significantly greater thanth e 13percen t increase caused by consuming an iso-caloricnon-alcoholi c meal.Thi seffec twa s observed during the entire postprandial period. Inbot h studies,however ,a t theen do f thepostprandia l period energy expenditurewa s stillelevate dwit hmor e than 20percen t above thepremea l baseline energyexpenditure ,indicatin g that the total thermogenic responsewa sno tcompletel ymeasured . Inthi s studyw e compared thermogenesis induced bya iso-energeticmea l of foodalon e and of foodplu salcohol .Alcoho lwa s givena sa naperiti f to minimize effectso f foodo n theabsorptio no fethano l fromth e gastro-intestinal tract.Contro l and testtreatment swer e carried out in duplicate toobtai na mor eprecis eestimat e ofa n individuals response of energymetabolis m to food.I npreviou s studies itwa s showntha tdiet-induce d thermogenesis ispron e toconsiderabl ewithin-perso nvariatio n (22,31). Postprandialmeasurement swer e carried outove r aperio d of 4hours ,whic hwa s sufficientlyprolonge d induratio n toallo wa nalmos t completeassessmen t of the thermogenic response.Thus ,energ yexpenditur e averaged over the fourth hour of thepostprandia lperio dwa so naverag e 4.5 percentelevate dabov e the corresponding premeal baseline energyexpenditur e onth e control treatment and 6.5 percent on the testtreatment .

-192- Inth epresen t studyoveral ldiet-induce d thermogenesiswa sno tpotentiate d by ethanol ingestion.O naverage ,i nparticula rdurin g the lastthre ehour so f thepostprandia l period,DI Tvalue swere ,however ,slightl yhighe r on the test treatment thano n the control treatment.Th emaximu mpotentiatio n ofDI Twa s observed during the second and thirdhou r of thepostprandia l period.Afte r ethanol consumption ittake sabou t 30-45minute s infastin g subjects to reach themaxima l blood ethanol concentrations (29).Dependin g on the total amount of ethanol ingested ethanol,eliminatio nwil l take severalhours .I tca nb e calculated that inthi s studymos to f theethano lwil l have been eliminated betweena perio d of 30-150minute safte r ingestiono f themeal ,whic hwa s given 15minute s after theaperitif .Durin g thisperio d ethanol oxidation contributed onaverag e 3.8 kJ/mint otota lenerg y expenditure.O n average between 55an d 65percen to f total energyexpende dwa sderive d fromethano l oxidation. Analysis of the substrateoxidatio n rates revealed thatethano l was preferentially oxydized incompariso nwit h fatsan d carbohydrates,bu tno t in comparisonwit hproteins .I nth e first 180minute so f thepostprandia l period, glucose and fatoxidatio nwer emarkedl y reduced, respectively,o naverag e by about 22g an d 7g afte r ingestiono fa mea l of foodplu salcoho l in comparisonwit h amea l of foodalone .Thi smean s thatdurin g thisperio d more fatsan d carbohydrateswer e stored onth etes t treatmentwhe n compared to the control treatment.Increase d storageo fnutrient sprio r tooxidatio nafte ra mealwil l increasepostprandia l energyexpenditur e (30).Whe n fatsan d carbohydrates areoxydize dwithou tprio r storage,abou t 2percen t of the energy contento f these substratesha st ob eexpende d toprovid eAT P for handling costs.I ncontrast ,i f fatsar e stored prior tooxidatio n total energydissipate d forhandlin g thisproces s isabou t 4percen t of the energy content of fat.Fo r glucose stored asglycoge no ra s fatth e costs for processing are,respectively ,abou t7 an d 24percen t of theenerg y contento f glucose (30).Th e averagedifferenc e inhandlin g costso f the postprandial processing of thenutrient swa s calculated atabou t0.2-0. 3kJ/mi n for the first, second and third houro f thepostprandia l period. Figure 1 shows that for thisperio ddifference swer e foundbetwee nbot h treatments inenerg y expenditure onaverag e of -0.03, 0.23 and 0.21 kJ/min,respectively . In particular, thevalues for the second and thirdhou r are close towha twa s expected. Probably,ove r the firsthou r of thepostprandia l period differences in storage ofnutrient sbetwee nbot h treatments are small.Thi s could bedu e toa relatively lowethano l oxidationespeciall y inth e firsthal f of this

-193- period. Itha sals obee n found (27)tha tnutrient-induce dthermogenesi scanno t solelyb e explained interm so f thebiochemistr yo fenerg y expenditure associatedwit h theprocessin g of ingested nutrients (30).Par to fnutrient - ordiet-induce d thermogenesis, ie,abou t 30-40percent ,i sthough t tob e mediated by an increased activityo f the sympathetic nervous systemafte r food ornutrien t ingestion (32).Th e latterpar to fth e total thermogenic response isusuall ydesignate dwit h the term facultative thermogenesis, indicating that thispar t of thermogenesismigh tno tb eoperatin g at the same level ineac h individual,o r that itmigh tb e susceptible toenvironmenta l stimuli.I ti s possible thatnutrient-induce d facultative thermogenesis iseffectivel y operative inth eperio d immediatelyafte r food ingestion (0-60minutes )an d thatethano l reduces theamoun to fenerg yexpende d by the facultative mechanism. The resultso f this studyo nth e thermiceffec to fethano l and the impacto f ethanol onDI Td ono t indicate thatmoderat e amountso fethano l are less efficientlyuse d asa nenerg y source incompariso n to fatso rcarbohydrates . We therefore tentatively conclude that theobservatio n that alcohol provides additional energy toth ediet ,bu t that itsus e isno t associatedwit h increased levelso fadiposity , isdu e tomethodologica l problemso f accurately quantifying energy and alcohol intake infree-livin g humans,o ro f the accurate quantitiono fothe r factors thandie tan dalcoho l thataffec t energy balance orbod yweigh t inman ,fo rexample ,o f thedegre e ofphysica l activity. Inconclusio nw e can state thatethano l consumed ina moderat eamoun tha sa significant thermic effect,probably , similar to fatso r carbohydrates.N o potentiationb yethano l ingestiono fdiet-induce d thermogenesiswa s found. Ethanol fed incombinatio nwit ha mixe d liquidmea lha d a substantial impact on substrateoxidatio nan d storage rates,i nparticula r during theperio d at which ethanol oxidationwa sa t itsmaximum .Durin g thisperio d ethanol ingestion slightly increaseddiet-induce d thermogenesis.Overal l postprandial energy retentionwas ,however ,simila r betweena iso-energeticmea l of food plusalcoho l and of food alone.Substitutin gmoderat eamount so f calories from fatsan d carbohydratesb yalcoho l innon-alcoholic s isno texpecte d to decrease energy retention significantly or tob eusefu l asa nadjuvan t in weight reducing regimes.

-194- ACKNOWLEDGEMENTS

Theauthor swan tt othan kth esubject sfo rthei renthousiasti cparticipation . Wear eals oindebte dt oQues tInternationa l (Naarden,th eNetherlands )fo r adviceo nflavorin gth eethano lsolution san dfo rprovidin gth enecessar y flavoringagents .

REFERENCES

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-195- 18.Perma nES .Increas ei noxyge nuptak eafte rsmal lethano ldose si nman . ActaPhysio lScan d1962;55:207-9 . 19.Rosenber gK ,Durni nJVGA .Th eeffec to falcoho lo nrestin gmetaboli c rate.B rJ Nut r1978;40:293-8 . 20.Barne sEW ,Cook eNJ ,Kin gAJ ,Passmor eR .Observation so nth emetabolis m ofalcoho li nman .B rJ Nut r1965;19:485-9 . 21.Stoc kMJ ,Stuar tJA .Thermi ceffect so fethano li nth era tan dman .Nut r Metab1974;17:297-305 . 22.Weststrat eJA ,Wey sPJM ,Poortvlie tEJ ,Deurenber gP ,Hautvas tJGAJ . Diurnalvariatio ni npostabsorptiv erestin gmetaboli crat ean d diet-inducedthermogenesis .A mJ Cli nNut r1989:i npress . 23.De nBeste nC ,Vansan tG ,Weststrat eJA ,Deurenber gP .Restin gmetaboli c ratean ddiet-induce dthermogenesi si nabdomina lan dgluteal-femora l obesewome nbefor ean dafte rweigh treduction .A mJ Cli nNut r 1988;47:840-7. 24.Jéquie rE .Direc tan dindirec tcalorimetry .In :Garro wJS ,Hallida yD , eds.Substrat ean denerg ymetabolism .London :Joh nLibbey ,1985:82-92 . 25.Bouma nWC ,Ran dMJ .Textboo ko fpharmacology .2n ded .Oxford :Blackwel l ScientificPublications ,1980:42.7 . 26.Sir iWE .Th egros scompositio no fth ebody .Ad vBio lMe dPhy s 1956;4:239-80. 27.Jéquie rE .Thermogenesi sinduce db ynutrien tadministratio ni nman . Infusionstherapie1984;4:184-8 . 28.Kinne yJM .Th eapplicatio no findirec tcalorimetr yt oclinica lstudies . In:Kinne yJ Med .Assessmen to fenerg ymetabolis mi nhealt han ddisease . Reporto nth efirs tRos sconferenc eo nmedica lresearch .Columbus :Ros s Laboratories,1980:42-48 . 29.DiPadov aC ,Worne rTM ,Julkune nRJK ,Liebe rCS .Effect so ffastin gan d chronicalcoho lconsumptio no nth efirst-pas smetabolis mo fethanol . Gastroenterology1987;92:1169-73 . 30.Flat tJP .Biochemistr yo fenerg yexpenditure .In :Bra yG ed .Recen t advancesi nobesit yresearch:II .London :Joh nLibbey ,1978:211-28 . 31.Ravussi nE ,Lillioj aS ,Anderso nTE ,Christi nL ,Bogardu sC .Determinant s of24-hou renerg yexpenditur ei nman .J Cli nInves t1986;78:1568-78 . 32.Sim sEAH ,Danfort hE .Expenditur ean dstorag eo fenerg yi nman .J Cli n Invest1987;79:1019-25 .

-196- Chapter 10. Doespalatabilit y effect thepostprandia l response ofenerg y expenditure?

JANA WESTSTFAT EMSC ,THE ODOPHEID EBSC ,LEONIE KROBROC HBSC ,PAU LDEURENBER G PHD,JOSEP HGA JHAUTVAS TM D

ABSTRACT

The impacto fpalatabilit yo ndie t (DIT)-an dsucros e (SIT)-induced thermogenesiswa sstudie d intw oexperiment swit h2 4health yyoun g normal-weight subjects,1 2me nan d1 2women .I nth efirs t study, subjects receiveda trandom ,i nduplicate ,eithe ra norma l liquid testmea l (2000kJ , 12percen t protein,3 3percen t fat,5 5percen t carbohydrates)o ra n iso-energetic unpalatable testmea lwit h kinin.Palatabilit ydi dno thav ea significant impacto npostprandia l metabolism. IntegratedDI Tvalue swere , respectively16 5+ 1 5(mea n+ sem)k Jfo rth econtro l treatmentan d18 5+ 1 4 kJ forth etes t treatment.I nth esecon d study,subject s receiveda trando m eithera palatabl e sucrose solution (900kJ) , aniso-energeti c standard sucrose solutiono ra niso-energeti c unpalatable sucrose solution.Kini nan da citrusflavorwer euse dt ovar ypalatability . Postprandial energy expenditure overa perio do f15 0minute swa sno tsignificantl y affectedb ydifference si n palatability. Inbot h studies,me nha da highe r risei npostprandia l energy expenditure thanwome ndid ,i nparticular ,afte rth econtro lmea lan dafte r theunpalatabl e sucrose solution.A separate control experimentt oasses sth e effecto fkini no nenerg yexpenditur ewa scarrie dou ti n8 subjects .Kini nha d no significanteffec to nenerg yexpenditur e overa perio do f12 0minute s after ingestion.

Keywords: indirect calorimetry,energ yexpenditure ,palatability , diet-induced thermogenesis,postprandial ,sucrose ,human .

-197- INTRODUCTION

Food intake evokesa promp t increase inenerg y expenditure,know na s diet-induced thermogenesis,th e thermiceffec to f foodo r postprandial thermogenesis.Th epostprandia l rise inenerg y expenditure depends largely on theamount o fenerg yan d typeo fnutrien t ingested (1,2). Generally,th e thermic effecto f food increaseslinearl ywit h energy intake (2)an d islarge r forprotein s and carbohydrates thanfo r fats (1).I nth eaverag e individual about 10percen to fth etota ldail yenerg youtpu twoul db eassociate d with food ingestion (3).Thi s represents aconsiderabl e losso fenerg y for the individual. Therefore it isno t surprising that interestaros e indiet-induce d thermogenesis, inparticular ,a t the suggestion thatobes e individualswoul d expend lessenerg y aftermea l ingestionwhe n compared tolea nsubject s(4) . Postprandial energyexpenditur e canb e largelyattribute d toth eenerg yneede d forprocessin g the ingestednutrient s (1).However ,th e totalobserve d thermic response to foodo rnutrient s canno t completelyb eunderstoo d interm so f the biochemistry ofenerg yexpenditur e of thepostprandia l processingo fnutrient s (1,5). Parto f the response,abou t 30-40percent ,i sthough t tob emediate d by a diet-induced increase inth eactivit yo f the sympathethicnervou s system (5). Thislatte rpar to fth e thermogenic response involves facultative thermogenicmechanisms ,whic hma yvar ybetwee n individualso r in response to environmental stimuli.Th edeterminant so fdiet-induce d facultative thermogenesis inma nar elargel yunknown .Degre eo fadiposit y (6),aerobi c capacity (7),typ eo fnutrien t (8)an d sensory characteristics (9)o f foodma y be important.

Information on the impacto f sensory characteristics of food ondiet-induce d thermogenesis isscarce .Mea l ingestion isusuall yassociate dwit holfactive , gustative and cognitive stimulation (10),whic hma yaffec t theactivit y of the sympathetic nervous system. It ispossibl e thatmeal sdifferin g in palatability result indifferen t sensory stimulationand ,hence ,inductio no f metabolic processes involved infacultativ e thermogenesis. Recently,LeBlan c et al. (9)reporte d thatdiet-induce d thermogenesiswa s significantly elevated ona n iso-energeticpalatabl emea l incompariso nwit h anunpalatabl emeal . Inthi sstudy ,however ,th epalatabl ean dunpalatabl e mealvarie d extremely inwate r content,whic h couldhav e caused the observed difference indietar y thermogenesis (11).Difference s indiet-induce d thermogenesisbetwee npalatabl e andunpalatabl emeal shav ebee n associated withdifference s ininsuli n responses (9). After ingestion ofa nunpalatabl e

-198- meal the insulin responsewa s significantly reduced incompariso nwit ha palatable meal (9).Thi sphenomeno nma yb e related todifference s inth e early, ie,pre-absorptiv e orcephali c insulin response (10,12-14).Afte r ingestiono f anunpalatabl e meal the cephalic insulin responsewoul d be reducedo rabsent .Blockin g ofth eearl y insulin responsewa s found to reduce the thermic effecto f infused glucose(15) . Inthi s reportw edescrib e twostudie sassessin g the impacto fpalatabilit y on diet-induced thermogenesis. Inth e first study, iso-energeticmeal s identical inenerg ycontent ,nutrien t compositionan dwate r content,bu tdifferin g in palatabilitywer e compared. Inth e second study, iso-energetic sucrose solutionso fvaryin gpalatabilit ywer e tested.Thi sstud ywa sperforme d to investigate differences inpostprandia l thermogenesisbetwee na palatabl e and unpalatablenutrien t solutionwit ha particula r strong impacto npostprandia l insulinan d glucosehomeostasis . The resultso f these studiessho wtha tpalatabilit ydi dno t significantly affectpostprandia l energyexpenditure .

SUBJECTSAN DMETHOD S

The totalexperimen t consistedo f several studies,tw ometaboli c studies for determining the impacto fpalatabilit y onpostprandia l energy expenditure and a sensoric studyt oasses s thepalatabilit yo f themeal san d sucrose solutions tob eused inth eenerg y expenditure studies.I naddition ,a control studywa s performed tomeasur e the impacto fkini no nenerg yexpenditure .Kini nwa sused fordecreasin g palatability.

SUBJECTS

Subjectswer e recruited forparticipatio n inth e studiesb y an advertisement inth eweekl yperiodica l of theUniversity . Inaddition ,som e subjectswer e contacted forparticipatio n through studentorganisations .I ntota l 36 subjectsapplie d forparticipation .Subject s received a letter informing them on thenatur e andpurpos eo f theexperiments .I naddition ,the y received two questionnaires,on e forobtainin g informationo npas tan dpresen thealt h statusan d one forgettin g information oneating ,drinking , smoking and exercise habits.Th emedica l questionnairewa sevaluate db ya physicia n

-199- (JGAJH).Fo rbot hstudie s2 4subject swer eneeded ,1 2wome nan d1 2men . Subjectswer eselecte dfo rparticipatio nwhe nthe yha da bod yfa tpercentag e lesstha n2 5percen tfo rme no rles stha n3 5percen tfo rwomen ,di dno tsmok e orsmoke dles stha n1 0cigarettes/d ,wer eapparentl yhealthy ,wer econsumin g normalbalance ddiet san dha dha dn omajo rweigh tfluctuation s (moretha n2. 5 kg)withi na perio do fsi xmonth sprio rt oth estudy .I naddition ,th e selectedsubject sha dt osho wconsisten tpreferenc e ratingsi nth esensor y evaluationo fth etes tmeal san dsolutions .Thi smean stha tthe yha dt ob e ablet odiscriminat econsistentl ybetwee nth evariou smeal san dsolutions .Al l subjectswer estudent sa tth eUniversity ,non eo fth esubject swa so na die t oruse ddrug sknow nt oaffec tenerg yexpenditure .Al lsubject sparticipate di n onlyon eenerg yexpenditur estudy .Befor eth estar to fth estudie ssubject s gavethei rwritte ninforme dconsen tt oparticipate .Th eprotoco lo fth estud y wassubmitte dt oan dapprove do fb yth eMedical-Ethica lCommitte eo fth e WageningenAgricultura lUniversity .

METHODS

Experimentaldesig n

Sensoryevaluatio n

Beforeth estar to fth eenerg yexpenditur estudies ,th etast epreferenc eo f eachsubject sfo rth evariou stes tmeal san dsucros esolution swa sevaluate d ina sensori cstudy . Threedifferen ttes tmeal swer eprepared ,identica li nenerg ycontent , nutrientcomposition ,textur ean dwate rcontent . Liquidyoghurt-base dtes t mealswer euse dwit ha nenerg yconten to f4.1 2MJ/k g (12percen tprotein ,3 3 percentfats ,5 5percen tcarbohydrates) .Meal swer evarie di npalatabilit y eitherb yaddin g0.1 0gA g ofa colorin gagen t(E-151 ,brillan tblack )o rb y addingth ecolorin gagen tplu s0.7 9gA g ofa 1 0percen tsolutio no fkini ni n ethanol.A mea lwithou tthes eadde dsubstance sserve da sa referenc emeal . Sucrosesolution s (143g/1 )wer evarie di npalatablit yeithe rb yaddin g 0.63 gAg ofa 1 0percen tkini nsolutio ni nethano lo rb yaddin g0.6 3gA g ofa commerciallyavailabl ecitrusflavo ro nethano lbasi s(Baukje ,Rijssen ,th e Netherlands).A standardsucros esolutio nwa suse dfo rreferenc epurposes . Afterbein ginstructe di nth estandar dsip-and-spi tprocedure ,subject s

-200- received thevariou s stimuli forpairwis e comparison insmal lplasti c cups (approximately 15ml )a t room temperature.Test swer e carried out inth e afternoonwit h the subjects ina 5-6 hourspostabsorptiv e state.Thoroug h rinsingbetwee n the sampleswa semphasized . Foreac h studyal l 6 permutations of the 3 itemswer euse dan d theorde ro fpresentatio no f stimuluspair swa s randomized across subjects.I naddition ,t oth epairwis e comparisons of the various stimuli subjectsha d to rank theirpreferenc e foreac h itemo na standard nine-pointhedoni c preference scale that ranged from 'dislike extremely' (1)t o 'likeextremely ' (9).Result so f the sensory evaluation showed thatth emea lwit hadde dcolorin g agentwa sno t sufficiently disliked in comparisonwit h the standard meal.Therefore ,th e impacto fpalatabilit y on meal-induced thermogenesiswa s investigatedwit h twomea l typesonly , ie,th e standardmea l and themea lwit h coloringplu skinin .Difference s in palatability among thethre e sucrose solutionswer e statistically significant.

Impact ofpalatabilit y onmeal-induce d thermogenesis

Inthi s study 12subjects ,6 male san d 6females ,receive d inth emorning , after anovernigh t fast,o n fourdifferen tday stw o timesa n iso-energetic meals induplicate ,eithe r thepalatabl e standard testmea lwit h an energy content of 2.00 MJ (12percen tprotein ,33. 1percen t fat,54. 9 percent carbohydrates)o r theunpalatabl e testmeal .Meal swer e givena t room temperature after an initialmeasuremen to f restingmetaboli c rateo f at least onehour .Energ yexpenditur ewa sassesse dwit ha ventilate d hood systema s previouslydescribe d (16).Meal swer egive nthroug ha strawwhic hwa spasse d through theventilate d hood.Meal swer e ingestedwithi n fiveminutes .Energ y expenditurewa s thencontinuousl ymeasure d for 3.5 hours.Meal swer e given to the subjects according toa Lati n squaredesig n tobalanc e theorde r of treatmentwit h the typeo f treatment.N oattemp twa smad e tomeasur e thewome n ina specific phase of themenstrua l cycle.I na previou s study (17)w e had not found a significant impacto f the stage of themenstrua l cycle on postprandial energyexpenditure .Measurement swere ,however ,alway s carried outa t least threeday safte r theonse to fmenstruation .Betwee n the measurementswa sa n intervalo fa t least twodays .

-201- Impact ofpalatabilit yo nsucrose-induce dthermogenesi s

In this study 12 subjects,6 me nan d 6women , received on threeday s inth e afternoon three iso-energetic solutions consisting ofa standard sucrose solution (0.90 MJ), ahighl yunplalatabl e sucrose solution ora highl y palatable sucrose solution.Subject swer emeasure d inth eafternoo n for practical reasons.Subject swer e instructed tohav e a smallbreakfas t (<2 MJ ) before 8.00 hoursan d toavoi d coffeedrinking , smoking,sleepin g and moderate orheav yexercis e on themornin gbefor e theenerg yexpenditur emeasurements . Under these conditions restingmetaboli c ratean ddiet-induce d thermogenesis dono t show significantdiurna lvariatio n (16).Sucros e solutionswer e given at room temperature after an initialmeasuremen t ofbaselin e postabsorptive energyexpenditur e ofa t leaston ehour .Sucros e solutionswer e given through a straw,whic hwa spasse d through theventilate d hood andwer edrun kwithi n 5 minutes. Energyexpenditur ewa s thencontinuousl ymeasure d for2-2. 5 hours. Because of the relatively lowenerg y contento f the sucrose solutions (< 1MJ) a shorter duration of thepostprandia l measurementswa s chosen.Simila r toth e other study, subjects recieved sucrose solutions ina randomorde r according toa Lati n squaredesign .Als on oattemp twa smad e tomeasur e thewome n ina particular phaseo f themenstrua l cycle.Betwee n thetest swa sa time interval ofa t least twodays .

Control studyusin g kinin

In8 subjects ,3 me nan d 5women , recruited from studentsan d staffworkin g in thelaboratory ,th e impactwa sassesse d onenerg yexpenditur e of 0.22 go f a 10percen t (v/v)kini n solution inethanol ,give neithe r in 350m l ofwate r or ina gelati n capsule ingestedwit h 350m lo fwater .Restin g metabolic ratewa s measured after anovernigh t fast fora tleas ton ehour .Afte r theRM R measurement subjects received thekini n solution or capsulewit hwate r ina randomorder .Energ yexpenditur ewa s thenassesse d continuously for 120 minutes.Al l subjects considered thekini n solution asextremel yunpleasant . No sensory stimulationwa s induced by the ingestiono f thekini n capsule.Thu s the impacto fkini no nenerg yexpenditur e couldb eassesse d controlling for itssensory-mediate d effects.Betwee n the testswa sa n interval of at least twodays .

-202- Gasexchang e measurements

Energy expenditurewa smeasure dwit ha ventilate d hood system in subjects reclining ina supinepositio n ina hospita l bed.Detail so f themeasuremen t procedurean d conditionshav ebee npublishe dpreviousl y (16).I naddition , movementswer e objectively registeredusin ga load cell (TKA-200A,Toky o Sokki Kenkyujo CoLtd ,Tokyo ,Japan )connecte d toon eo f theleg so f thebed .Onc e everyhou r during calibrationo f theoxyge nanalyze rwit h freshoutsid eair , subjectswer e allowed tomov ebriefl y for 2-3 minutes.Afte r thega sexchang e measurements forassessin g themeal -o r sucrose-induced thermogenesisi n relation topalatability ,urin ewa s collected fordeterminin g urea-nitrogen excretion.

Calculations ofenerg yexpenditur e and substrate oxidation

Energyexpenditur ewa s calculated according toJéquie r et al. (18).Th e thermic effecto f thevariou s testmeal san d solutionswa sdefine d as the integrated increase inpostprandia l energyexpenditur e above the corresponding preprandial baseline energyexpenditur e after ingestiono f the test food. Substrate oxidation ratesan dpostprandia l nutrientbalance swer e calculated aspreviousl ydescribe d(16) .

Body composition

Body fatconten twa s estimated fromtota lbod ydensit yusin g Siri's formula (19). Totalbod ydensit ywa smeasure d ineac h subjectparticipatin g inth e two major studiesb yhydrostati cweighin gwit h simultaneous assessment of residual lungvolum ewit h aheliu mdilutio ntechnique .

Statistics

Pairwise comparisons oftes t stimuliwer e statistically evaluatedusin g the chi-square statistic.Preferenc e ratingsfo reac h stimuliwer e compared using paired t-testswit h a two-tailed regiono f rejectionan d aconfidenc e level of 95percent .Treatment swer e compared inth emea l studyusin gpaire d t-testso n thepoole d average resultso f theduplicat emeasurements .I nth e sucrose study, treatmentswer e comparedusin ganalysi so fvariance .

-203- RESULTS

Table1 give sag ean dsom echaracteristic so fth esubject sparticipatin gi n thevariou sexperiments .Subject sparticipatin gi nth epalatabilit ystudie s

Table1 .Age ,bod yweigh tan dbod yfa tpercentag ei nsubject sparticipatin gi n studies.

Study1 Study2 Study3 palatability- DI T palatability- SI T Control- kini n men women men women men women n= 6 n= 6 n= 6 n= 6 n= 3 n= 5

Age (yrs)22.7+0. 8 21.2+0.8 22.2+0.3 21.8+1.1 35.3+6.8 23.4+0.8 Body weight (kg)71.8+2. 9 60.6+2.0 74.2±1.6 61.4+2.1 71.8+3.1 60.0±0.8 Bodyfa t content (%) 12.0+1.0 29.0+1.1 12.4+1.5 28.0±2.5 16.5+2.8 29.1+2.1

Resultsar egive na smea n+ se m DIT= diet-induce dthermogenesis ;SI T= sucrose-induce dthermogenesi s

hadage sbetwee n1 8an d2 5years ,norma lbod yweight san dbod yfa t percentages.Th eme nwh oparticipate di nth ekini nstud ywer eo naverag e slightlyolde rcompare dt oth eothe rmen ,age swere ,respectively ,28 ,2 9an d 49years ,bu tnon eo fthes eme nwa sobese .

Table2 .Preferenc eratin go fth evariou stes tmeal san dsucros esolutions .

Study1 Study 2 palatability- DIT palatability- SI T Meal type n = 12 Solutiontyp e n = 12

1.standar d meal 1.standar dsucros e 5.5+ 0.3- p<005 2. standard+ color p<0.001 2.standar d+ flavor 6.6 + 0.3 p<0.001 p<0.001 p<0.001 3.standar d+ colo r+ kinin 1.8+ 0.3 3.standar d+ kinin 1.8+ 0.2-

t are given as maan+ sen EOT= diet—induced theraogenesis; STT= sucrose-induced theraogenasis Preferences were rated on a standard nine-polat bédane scale ranging fron 'dislike extremely (1) to 'like extreaely' (9)

-204- Table2 give sth eresult so fth esensor yevaluatio no fth evariou stes tmeal s andsucros esolutions .Fo rth emeal sa significan tdifferenc ei npreferenc e ratingwa sfoun dbetwee nth estandar dmea lan dth emea lwit hadde dcolorin g pluskini nonly .Therefore ,i twa sdecide dt ostud yth eimpac to fpalatabilit y ondiet-induce dthermogenesi swit hthes emea ltype sonly .Preferenc erating s differedsignificantl yamon gth ethre esucros esolutions .Th elarges t differencesoccurre dbetwee nth estandar dsolutio nan dth esolutio nwit h citrusflavoringo nth eon ehand ,an dth esolutio nplu skini no nth eothe r hand.

Table3 .Diet-induce dthermogenesis ,postprandia l respiratoryquotient san d substrateoxidatio nrate si nsubject sreceivin gmeal so fdifferin g palatability.

palatablemea l unpalatablemea l n= 12

DIT (kJ) 166+ 1 5 185+ 1 4 DIT- %/M E (%) 8.3+ 0. 7 9.0+ 0. 7 RQ 0- 60mi n 0.90+ 0.0 1 0.89+ 0.0 1 60- 12 0mi n 0.93+ 0.0 1 0.93+ 0.0 1 120- 18 0mi n 0.86+ 0.0 1 0.84+ 0.0 1 180- 21 0mi n 0.83+ 0.0 1 0.81+ 0.0 1 Glucoseoxidatio n (mg/min .kgFFM) 3.4+ 0.1 7 3.7+ 0.2 3 Fatoxidatio n (mg/min .kgFFM) 0.7+ 0.0 6 0.8+ 0.0 9 Proteinoxidatio n (mg/min .kgFFM) 1.4+ 0.0 9 1.5+ 0.0 9

Resultsexpresse da smea n+ se m DIT= diet-induce dthermogenesis ;%M E= percen to fenerg yintake ; RQ =respirator yquotients ;kgFF M= k gfat-fre emas s

Table3 give sdiet-induce dthermogenesis ,postprandia l respiratoryquotient s andsubstrat eoxidatio nrate si nsubject safte rth epalatabl ean dunpalatabl e testmeal .Non eo fth evariable sdiffere dsignificantl ybetwee nth e treatments.Baselin eenerg yexpenditur erate s(restin gmetabli c rate)an d respiratoryquotient sdi dno tdiffe rsignificantl ybetwee nth etreatments . Restingmetaboli crate san drespirator yquotient swere ,respectively ,4.5 8+ 0.25kJ/mi nan d0.8 3+ 0.0 1befor eingestio no fth epalatabl emea lan d4.6 8+ 0.25kJ/mi nan d0.82+0.0 1befor eth eunpalatabl emeal . Figure1 give sth etim ecours ei nDI Tafte ringestio no fa palatabl eo ra n unpalatablemeal .N osignifican tdifference swer eobserve di nintegrate d hourlyDI Tvalue sbetwee nbot htreatments .

-205- Therewa sa surprisin g sexdifferenc e inDIT .O nbot h treatmentsDI Twa s higher inme ntha ni nwomen , respectively,20 3+ 1 1k J compared to12 8+ 1 6k J (p=0.005)fo rth epalatabl e mealan d21 0+ 1 6k Jversu s15 9+ 1 8k J (p=0.06) forth eunpalatabl e meal.Th edifferenc ebetwee nme nan dwome n occurred entirely inth efirs ttw ohour so fth epostprandia l period.

diet induced thermogenesis (kJ/min) 1.50- f |PALATABL E MEAL I- UNPALATABLEMEA L M 0.90 i^- M

0.30

12 3 4 timeafte rmea lingestio n(h )

Figure1 .Diet-induce d thermogenesis (mean+ sem )ove r 4hour si nsubject s after ingestiono fa palatabl eo runpalatabl emeal .

Table 4give s sucrose-induced thermogenesis,postprandia l respiratory quotientsan dsubstrat e oxidation ratesi nth esubject s after ingestiono fth e three sucrose solutions.Non eo fth evariable sdiffere d significantly among the treatments.Baselin e energy expenditure ratesan drespirator y quotients were similar forth ethre e treatments, respectively, 4.81+ 0.26, 4.95+ 0.3 3 and 5.03+ 0.3 0kJ/min ,an d0.8 1+ 0.01,0.8 0+ 0.0 1an d0.8 0+ 0.01 . Figure2 give sth etim e courseo fth esucrose-induce d thermogenesis forth e various treatments.N odifference s intim e course among treatments were observed.SI Twa sreduce d tozer ofo rmos t subjects after twohours . A significant sexdifferenc e inSI Twa sobserve d onlyfo rth eunpalatabl e sucrose solution,4 3+ 5 k Ji nme nversu s2 6+ 4 k Ji nwome n (p=0.03).

-206- Table4 .Sucrose-induce dthermogenesis ,postprandia l respiratoryquotient san d Substrateoxidatio nrate si nsubject sreceivin gsucros esolution so fdifferin g palatability.

Most Standard Unpalatable palatable solution solution n= 1 2

SIT (kJ) 44+ 9 42+ 1 2 34+ 4 SIT-%ME (%) 4.9+ 1. 0 4.6+ 1. 3 3.8+ 0. 5 RQ 0-60 min 0.88+ 0.0 2 0.93+ 0.0 1 0.91+ 0.0 2 60- 12 0mi n 0.85+ 0.0 1 0.87+ 0.0 1 0.88+ 0.0 1 120- 15 0mi n 0.84+ 0.0 2 0.86+ 0.0 1 0.86+ 0.0 1 Glucoseoxi d(mg/kgFF M min) 3.0+ 0.2 6 3.6+ 0.2 6 3.2+ 0.23 Fatoxi d (mgAgFFM min) 0.9+ 0.1 2 0.6+ 0.1 2 0.6+ 0.0 6 Proteinoxi d {mq/WgFFVi min) 1.6+ 0.1 4 1.5+ 0.1 4 1.5+ 0.0 9

Resultsar eexpresse da smea n± se m SIT- sucrose-induce dthermogenesis ;%M E= percen to fenerg yintake ; RQ= respirator yquotients ;kgFF M= k gfat-fre emas s

sucrose-induced thermogenesis (kJ/min) ffjjjl MOST PALATABLE SOLUTION

0.64- f==j STANDARD SOLUTION

UNPALATABLE SOLUTION 4-

1 2 time after meal ingestion(h )

Figure2 .Sucrose-induce dthermogenesi s (mean+ sem )i nsubject safte r ingestiono fsucros esolution so fdifferen tpalatability .

-207- Table 5.Restin gmetaboli c ratean d respiratoryquotient s insubject sbefor e andafte r kinin ingestion.

Kinin solution Kinin capsule T n =8 n= 8 before after before after

Resting metabolic rate (kJ/min) 4.23 ± 0.36 4.32 ± 0.36 4.05 ± 0.35 4.17 + 0.32 Respiratoryquotien t 0.81 + 0.01 0.79 ± 0.02 0.85 + 0.03 0.81 ±0.02 t

Resultsar eexpresse d asmea n+ sem * Kininwa sgive n ina dosi so f 0.22 g (10%ethano l solutionv/v ) in35 0m lwate r T Kininwa sgive n ingelati n capsule ina dosi so f0.2 2 g (10%ethano l solutionv/v ) + Significant differencea tp <0.0 1 between 'before'an d 'after'

Table 5give s the resultso fth e impacto fkinin ,eithe r insolutio no r in capsule on restingmetaboli c ratean d respiratoryquotient .O naverage , restingmetaboli cwa sno t significantly affected bykini n ingestion.Ther e was,however ,a small increase in restingmetaboli c rateo fo naverag e 2.3 percent.Respirator y quotientsdecline d significantly after kinin ingestion, inparticula r after ingestiono f thekini ncapsul ewit hwater .

DISCUSSION

This studydi dno t showa significant impacto fpalatabilit y ondiet-induce d thermogenesis.Th e thermic effecto f sucrosewas ,albei tno t significantly, slightly increased following ingestiono fa palatabl e sucrose solutionwhe n compared toa nunpalatabl e sucrosedrink .Postprandia l respiratory quotients and substrate oxidation rateswer ever y similar following ingestion of either a palatable orunpalatabl emea l or sucrose solution.A surprising difference was found betweenme nan dwome n indiet-induce d thermogenesis.Me nha d a significantly higher rise inpostprandia l energyexpenditur e thanwome ndid , inparticula r following ingestiono f thepalatabl e mealan d theunpalatabl e sucrose solution. Our findingswit h respect toth eeffec to fpalatablit y ondietar y thermogenesis are incontras twit hobservation s of LeBlanc et al. (9).Th e experimentaldesig nuse d inthi sstud ydiffer sconsiderabl y fromth eon e employed byLeBlan c et al. (9).LeBlan c etal . (9)prepare d the unpalatable -208- meal bymixin g together ina blende r all the ingredients served inth e palatablemea l andpresentin g itt oth e subjects inth e formo fa desiccated biscuit.Th e palatablemea lwa s composed ofa parmesa n fondue,spaghett i with meatballs ,a chocolate eclair anda bottl eo f softdrink .Althoug h no quantitative datao n themas so fbot hmeal sar egive n inLeBlan c etal.' s report, itseem s likely thatth eunpalatabl emea lha d amuc h lowerweigh t than thepalatabl e meal.Thi sma y seriouslyaffec t the interpretation of their results.Recently ,Blondhei man dHir t (11)reporte d thatdiet-induce d thermogenesiswa s considerably smaller aftera n isocaloricmea l of smallmas s thanafte r amea l ofa larger size.Ove ra perio d of 75minute s thedifferenc e between thesemeal s in risei noxyge nconsumptio nafte r ingestiono f themea l was on average about8. 4 percent.Thi s figure isver y similar toth e difference between thepalatabl ean dunpalatabl emea l inpostprandia l oxygen consumption,ie ,8 percent ,LeBlan c et al. report.I t isno tclear ,therefore , whether thedifferenc e indiet-induce d thermogenesisbetwee nmeal so fvaryin g palatablity LeBlanc et al. found,ha st ob eascribe d toth e impacto f palatability per se,o r toth edifferenc e inmas sbetwee n thesemeals . Inthi s studymeal s identical incalori c content,nutrien t composition,wate r contentan dmass ,bu tdifferen t inpalatabilit ywer e investigated onthei r differential effecto ndiet-induce d thermogenesis.Palatabilit y of the test mealswa svarie dwit h kininan d a coloringagent .Sensor y ratings,obtaine d in 5-6 hourspostabsorptiv e subjects,showe d that thesemeal swer e significantly different inpleasantness .Fo r eachmea l type,postprandia lmeasurement swer e carried out induplicat e fora perio d of 2.5 hours.Tota l andhourl y integratedvalue s fordiet-induce d thermogenesiswer eno t significantly differentbetwee n the twomea l types.Sinc ea neffec to fkini no nenerg y expenditure couldno tb eexcluded ,a contro lexperimen twa sperforme d for assessing the impacto fkini no nenerg yexpenditure .Kini n ingestionwa s associatedwit ha slightbu tnon-significan t increase in restingmetaboli c rate.Thi s increasewa sno tmediate d by sensory stimulation since kinin encapsulated caused a similar increase inenerg y expenditure askini n in solution.Whe n theobserve d integrateddiet-induce d thermogenesis is corrected for the slighteffec to fkini no nbaselin e energyexpenditure ,th e resulting averagevalu eo f 163k J isver y similar toth e thermiceffec tobserve d after ingestiono f thepalatabl e testmeal , ie,16 6kJ .Th e resultsclearl y refute

-209- thehypothesi s thatpalatabilit y significantlyaffect sdiet-induce d thermogenesis. Itwa sexpecte d thatafte r ingestiono fa palatablemea l the sympathetic nervous systemactivit ywoul db emor e stimulated thanafte r ingestion ofa revoltingmeal .A palatablemea lwa sals oexpecte d toinduc ea significantly higher cephalic insulin response,and ,hence ,a total insulin response in comparison toa nunpalatabl emea l (10,12-14).Bot heffect scoul d increase postprandial thermogenesis.Firstly ,throug h stimulationo f facultative thermogenicmechanisms ,and ,secondly ,b y stimulationo fpostprandia l glucose metabolism, ie,oxidatio nan d storage.Ou r resultssugges t that these processeswer eno t sufficiently stimulated by ingestiono f thepalatabl e meal, or,alternatively , suppressedb y theunpalatabl e meal, tohav ea majo r impact on the overall rateo fenerg yexpenditure .I naddition , iti spossibl e that ingestiono fa revoltingmea lma yprovok ea stress reaction,which , init s turn,coul d increase the turnover ofcatecholamine s and the rateo f energy expenditure.Consequently ,a maskin go fa differenc e indiet-induce d thermogenesisbetwee nmeal so fdifferin gpalatabilit ywoul d occur.Mor e research isnecessar y to investigate thesepossibilities . Inagreemen twit h the findingso ndiet-induce d thermogenesis, sucrose-induced thermogenesiswa sno t significantly affected bypalatabilit y either.Ther ewa s a trend towarda lower thermic effecto f sucrose following ingestiono f the unpalatable sucrose solution compared toth emor epalatabl e solutions.Th e total thermic effecto f sucosewa s low,ie ,abou t 4percen to fth e ingested caloric load.Thi s isprobabl y related toth e relatively lowenerg y contento f the testdrinks ,ie ,o f less than1 M J (about 55g o f sucrose). Inmos t subjects the thermogenic responsewa s completedafte r 2hours .Th e responsew e foundo f about4 0k J isi naccordanc ewit hdat a givenb yWell ee t al.(20) . The observed thermiceffec t is ingoo dagreemen twit h theenerg yexpende d in thepostprandia l oxidationan d storageo fth e sucrose (21) . At theen d of the experiment abouthal fo f the ingesteddos ewa soxydize d at theexpens e of about 10k J and theothe rhal fwa s stored,predominantl y asglycogen ,a ta n energy costo fabou t 30kJ .Whe n the integrated sucrose-induced thermogenesis iscorrecte d for the impacto fkini no nenerg yexpenditur e thethermi c effect is reduced toabou t 20kJ .Thi svalu e islowe r thanth e thermic effect calculated on thebasi so f theenerg y costso f thepostprandia l processing of sucrose.Thi sma yb e indicativeo fa diminutio no f thethermogeni c response to sucrosewhe n sucrose isgive na sa nunpalatabl e solution.A replicationo f the

-210- experimentwit h sucrose solutionso fhighe r energy content isnecessar y to test thishypothesis . Therewa s aclea r effecto fgende r on the rise inpostprandia l energy expenditure.T oou r knowledge thiseffec tha sno tbee ndescribe d before.Owe n eta l (22)repor tdifference sbetwee nme n andwome n inpostprandia l metabolism,bu tgiv en odat ao nenerg yexpenditure .Th edifferenc e between the sexes in rise inpostprandia l energyexpenditur e followingmea l ingestionwa s present onlydurin g the first twohour so f thepostprandia lperiod . During thisperiod ,i nparticula rdurin g the firsthou r ofth epostprandia l period, menha d significantlyhighe r respiratoryquotient s thanwome ndid .Th e mechanismunderlyin g thedifferenc e indiet-induce d thermogenesisbetwee nme n andwome n isno tclear .Variou s factorsma yb e involved.A higher rateo f digestion and absorptiono f the ingestednutrient sma y result ina greate r fluxo fnutrient s from thegastrointestina l tract into thebloodstrea mpe r unit of time inme nwhe n compared towomen .Th e relatively large availability ofnutrient sma y exceed thecapacit y for immediate oxidation causing nutrients tob e directed into 'energeticallywasteful ' (21)storag eprocesses . Inthi s respect, thepostprandia l processing ofglucos e isinteresting .Th e respiratory quotient indicatestha t the rateo fglucos edigestio nan d absorptionwa shighe r inme n than inwomen ,i nparticula r inth e firsthou r of thepostprandia l period.Mos to f theabsorbe d glucosewil l beoxydize do r stored in thebod ya smuscle -o rhepatic-glycogen . It iswel l known thatmen , atsimila rbod yweights ,hav ea largermuscl emas stha nwomen .Hence ,me nhav e a larger storage capacity forglucos e thanwomen .Th e relatively larger storage capacity for glucose combinedwit ha relatively greater influx of nutrients fromth egastrointestina l tract intoth ecirculatio nma y cause the rise inpostprandia l energy expenditure tob e relativelyenhance d inme n in comparison towomen . Inaddition ,i t ispossibl e that inme n stimulation by food ingestiono f facultative thermogenicmechanism s is relatively larger than inwomen .

Inconclusio nw e can state that inthi s studyn o significant impacto f palatabllty onmeal-o r sucrose-induced thermogenesiswa s found.However ,th e thermic effecto f sucrosewa s slightly reduced following ingestiono f an unpalatable sucrose solution.Me nha do naverag e agreate r rise in postprandial energyexpenditur e compared towome na swel l asafte r ingestion ofmixe dmea l asafte r ingestiono fa nunpalatabl e sucrose solution.

-211- ACKNOWLEDGEMENTS

The authorswan t tothan k the subjects for theirwillin g cooperation and for their courage toconsum e rather largequantititie s of revolting testmeal so r sucrose solutions.

REFERENCES

1. Jéquier E.Thermogenesi sinduce db ynutrien t administration inman . Infusionstherapie 1984;11:184-8 2. D'Alessio DA,Kavl eEC ,Mozzol iMA ,Smalle y KJ,Polansk yM ,Kendric k ZV, OwenLR ,Bushma nMG ,Bode nG ,Owe nOE .Thermi ceffec t of food in leanan d obesemen .J Cli n Invest1988;81:1781-9 . 3. Danforth E.Die tan d obesity.A m JCli nNut r 1985;41:1132-45. 4. KaplanML ,Leveill eGA .Calorigeni c responses inobes e and non-obese women.A m JCli nNut r 1976;29:1108-13. 5. Sims EAH,Danfort h E.Expenditur e and storageo fenerg y inman .J Clin Invest1987;79:1019-25 . 6. Segal KR,Guti nB ,Nyma nAM ,Pi-Sunye r FX.Thermi c effect of food at rest, during exercise,an dafte r exercise inlea nan d obeseme no f similar body weight.J Cli n Invest1985;76:1107-12 . 7. Hill JC,Heymsfiel d SB,McMannu sC .Mea l sizean d thermic response to food inmal e subjectsa sa functiono fmaximu maerobi c capacity Metabolism 1984;33:743-9. 8. Welle S,Lilaviva tU ,Campbel l RG.Thermi c effecto f feeding inman : increased plasmanorepinephrin e levels following glucose butno t protein or fatconsumption .Metabolis m1981;30:953-8 . 9. LeBlanc J,Bronde l L.Rol eo fpalatabilit y onmeal-induce d thermogenesis inhuma n subjects.A m JPhysio l 1985;248:E333-6. 10. PowleyTL ,Berthou d HR.Die tan d cephalic phase insulin responses.A mJ ClinNut r 1985;42:991-1002. 11. Blondheim SH,Hir tR . Relationshipbetwee ndietar y thermogenesis and bulk ofmeal . IsrJ Me d Sei1982;18:969-71 . 12. Louis-Sylvestre J. Relationshipbetwee n two stageso fprandia l insulin release in rats.A m JPhysio l 1978;235:E103-11. 13.Bellisl e F,Louis-Sylvestr e J,Demoza y F,Blaz yD , LeMagne nJ . Cephalic phase of insulin secretionan d food stimulation inhumans :a new perspective.A m JPhysio l 1985;249:E639-45. 14. Bruce DG,Storlie nLH ,Furie r SM,Chishol mDJ .Cephali c phase metabolic responses innorma lweigh t adults.Metabolis m1987;36:721-5 . 15. Calles-Escandon J,Robbin sDC .Los so fearl yphas e of insulin release in humans impairs glucose tolerance andblunt s thermic effecto fglucose . Diabetes1987;36:1167-72 . 16.Weststrat e JA,Wey s PJM,Poortvlie t EJ,Deurenber g P,Hautvas t JGAJ. Diurnalvariatio n inpostabsorptiv e restingmetaboli c ratean d diet-induced thermogenesis.A m JCli nNut r 1989:inpress . 17.Weststrat e JA,Dekke r J, StoelM ,Begheij nL ,Deurenber g P,Hautvas t JGAJ. Restingenerg yexpenditur e inwomen :impac to fobesit yan dbod y fat distribution.Metabolis m 1989:inpress . 18. Jéquier E,Acheso n K,Schut zY .Assessmen t ofenerg y expenditure and fuel utilization inman .An nRe vNut r1987;7:187-208 .

-212- 19.Sir iWE .Th egros scompositio no fth ebody .Ad vBio lMe dPhy s 1956;4:239-80. 20.Well eSL ,Campbel lRG .Norma lthermi ceffec to fglucos ei nobes ewomen .A m JCli nNut r1983;37:87-92 . 21.Flat tJP .Biochemistr yo fenerg yexpenditur ein :Bra yG .ed .Recen t advancesi nobesit yresearch :II .London :Joh nLibbey ,1978:211-28 . 22.Owe nOE ,Mozzol iMA ,Pate lMS ,Bode nG ,Reichar dGA ,Trap pV ,Shuma nCR , FeiigP .Substrate ,hormone ,an dtemperatur eresponse si nmale san d femalest oa commo nbreakfast .Metabolis m1980;29:511-23 .

-213- Chapter 11. Unexpected interaction betweenth e effects ofshort-ter m carbohydrate overfeeding and prior exerciseo nrestin gmetaboli crat e and diet- induced thermogenesis

JANA WESTSTRAT EMSC ,JOSEP HGA JHAUTVAS TM D

ABSTRACT

In1 0youn gnorma lweigh tsubjects ,5 me nan d5 women ,th eeffect swer e investigatedo fcarbohydrat eoverfeedin gan dprio rexercis eo nrestin g metabolic rate(RMR )an ddiet-induce dthermogenesi s (DIT).Subject swer ekep t oncontrolle ddiet si na cross-ove rdesig nfo rtw operiod so f8 days ,wit ha one-week intervali nbetween .Durin gth elas t4 day so feac hperio dsubject s receivedadditiona lcarbohydrate so nto po fthei rdiet .Th ecarbohydrat e overfeedingstarte da t1 5percen ti nexces so fth eestimate denerg y requirementso nda y1 increasin gt o6 0percen to nda y4 .A tth ebeginnin gan d atth een do fth e4-day scarbohydrat eoverfeedin gperiod sRM Ran dDI Twer e measured,respectively ,fo r1 an d3. 5hour susin ga ventilate dhoo dsystem .I n oneo fth e8-day speriods ,o nevening sbefor eth eenerg yexpenditur e measurements,subject sperforme da maximu mwor kcapacit ytes to na cycl e ergometer.Subsequently ,the ycycle dfo ra tota lo fabou t8 0minute sa tfixe d percentages (between30-80% )o fthei rmaximu mwor kcapacity .RM Ran dDI To n controltreatmen twer e4.5 6+ 0.2 2kJ/mi nan d7. 6+ 0. 9percen t (ofenerg y contento ftes tmea l (Ein)), respectively.Afte rexercise ,RM Ran dDI Twer e 5.10+ 0.2 5kJ/mi nan d10. 1+ 0. 8percen t (Ein),respectively .Prio rexercis e onday sbefor eth emeasurement sincrease dRM Ran dDI Tsignificantly , respectively,b y11. 9+ 2. 6percen tan d32. 2+ 13. 7percent .RM Rafte r carbohydrateoverfeedin gwa s4.5 0+ 0.2 8kJ/min ;i ncombinatio nwit hprio r exercise,RM Rwa s4.8 2+ 0.1 7kJ/min .DI Twa s11. 5+ 1. 2percen t (Ein)afte r carbohydrateoverfeedin gan d13. 2+ 1. 4percen t (Ein)afte rcarbohydrat e overfeedingwit hprio rexercise .Carbohydrat eoverfeedin gsystematicall y reducedth eimpac to fprio rexercis eo nRM Ran dDIT .Carbohydrat eoverfeedin g

-214- didno t significantly influenceRMR ,bu t increasedDI Tsystematicall yby , respectively, 50+ 18.6percen twithou tprio r exercisean d by 31.1+ 8.7 percentwit hprio rexercise . It isconclude d thatprio r exercise anda nanteceden tdie thig h in carbohydrates affectsRM R andDIT ,an d that theseeffect sar e interactive.

Keywords: indirect calorimetry, carbohydrates,overfeeding ,exercise , resting metabolic rate,diet-induce d thermogenesis,dietar y trial,huma n

INTRODUCTION

An important area in researcho nhuma nenerg ymetabolis mpertain s toth e quantitationo fth e responseo fenerg yexpenditur e tothermogeni c stimuli,i n particular, tofood . It isno wgenerall yassumed ,tha t inth e average sedentary individual,foo d intake contributesabou t 10percen t totota lenerg y expenditure (1,2). However,th enatur ean ddeterminant s of inter-individual differences inth eproportio n ofdail y energy outputdependin g on food intake remain tob edefinitel y assessed (1,2). The thermic effecto f food,als o referred toa sdiet-induce d thermogenesis (DIT), isprimaril y a functiono f theamoun to fenerg y and type of nutrients ingested (3,4). Inaddition ,th eanteceden tdie tma yaffec tDI Ta swel l(5) . Recently, itwa s showntha ta die thig h incarbohydrate s increased DIT (5).I t was suggested that inthi s respectth e state of thebody' sglycoge n stores would beo fpivota l importance.O na hig hcarbohydrat edie t theamoun to f glycogen stored inlive r and skeletalmuscle s increases (6-8). Inthi sstat e a relatively largerpar to f ingested carbohydrateswoul db econverte d intofat , resulting ina lessefficien tenerg yutilizatio n compared tostorag e of the ingested carbohydrates asglycogen .Thus , theenerg yexpende dwhe n a given amount ofglucos e istransforme d tolipi dan d issubsequentl y oxydized is substantially greater than ifth eglucos e isconverte d intoglycoge n before being oxidized(9) . Inadditio n todiet ,physica l activityca nhav e aparticula r strong impact on the state of thebody' s glycogen stores (6,7). Prolonged high intensity exercise iswel l known todeplet e liver andmuscle -glycoge n (6,7). Whether priorglycogen-depletin g exercise affectsDI T isunclear .Sinc e prolonged exercise reduces thebody' s glycogen stores,i tmigh tb e hypothesized thatDI Twoul db e reduced.Alternatively ,DI Tmigh tb e increased,

-215- since sustained heavyexercis ema y inducea nexces spostexercis e oxygen consumption of relatively highmagnitud e andduratio n (10-12). Inth epresen t study the stateo fth ebody' s glycogen storeswa smodulate d by short-term (4-days)carbohydrat e overfeeding orb yprolonge dprio r heavy exercise bouts.Th epurpos e of the studywa s toasses s theeffect s of these twotreatments ,an d their interaction,o npostabsorptiv e - and postprandial resting energyexpenditur e and substrate oxidationrates .

SUBJECTSAN D METHODS

SUBJECTS

Subjectswer e recruited forparticipatio n inth eexperimen tb yposter s placed inUniversit ybuilding s and studentdormitories . After applying forparticipation , subjects received a letterwit h detailed informationo n thenature ,purpos e andpossibl e inconveniences of the experiment.The yals o received aquestionnair e forobtainin g information on past andpresen thealt h status,sportin g activities,smokin ghabits ,habitua l useo f alcoholicbeverages ,foo daversion san d allergies,and , forwome nonly , use ofora l contraceptives.Th emedica lpar to f thequestionnair e was evaluated bya physicia n (JGAJH).Subject swer e selected for participation when theywer enon-obes e (body fatpercentag e below 25percen t inme nan d below 35percen t inwomen) , apparently healthy (nopas t orpresen t evidence of diabetesmellitu san d thyroid disorders),non-smokers ,consume d less than2 0 alcoholic beverages/Week andwhe n theyha dmoderat e sporting activities (between 1an d 7hours/week) .A totalo f 10subjects ,5 me nan d 5women ,wer e selected forparticipation .Thes e subjectswer eno to na diet ,ha dha d no major, ie,greate r than 2.5 kg,bod yweigh t fluctuationswithi na perio d of6 monthsprio r toth ebeginnin go f the studyan dwer eno tusin g anydrugs .Tw o womenuse d oral contraceptives.Befor e the starto f the study subjectsgav e theirwritte n informed consent toparticipate .Th eprotoco lo fth e studywa s submitted toan d approved ofb y theMedical-Ethica l Committee of the WageningenAgricultura l University.

-216- METHODS

Experimentaldesig n

Thestud yuse da crossove rdesig nan dconsiste do ftw operiod so f8 day swit h aon ewee kinterva lbetwee nbot hperiod st opreven tcarry-ove reffect s(13) . Thetota lstud ylaste d2 4days .Durin gbot hperiod so f8 day ssubject swer eo n controlledexperimenta ldiets .I nth ewee kbetwee nth eexperimenta lperiod s subjectsconsume dthei rhabitua ldiets .A tfixe dtim einterval sdurin gth e study,ie ,o nth emornin go fda y5 ,da y9 ,da y2 0an dda y24 ,energ y expenditurewa smeasure di nsubject si nth efastin gan dpostprandia lstate . Betweenda y1 an dda y5 ,an dbetwee nda y1 6an d2 0subject swer ekep to na normalmixe dweigh tmaintenanc ediet .Betwee nda y5 an dda y9 ,an dbetwee nda y 20an dda y2 4subject sreceive dadditiona lcarbohydrate si nthei rdiet . Carbohydrateoverfeedin gwa sstarte da t1 5percen to fth eenerg yintak edurin g theweigh tmaintenanc eperiod .Carbohydrate swer eadde dcumulativel yt oth e diet,startin gwit h1 5percen to nth efirs tda yan dincreasin gt o6 0percen t onth efourt hday .Hal fo fth esubjects ,randoml ychosen ,ha dexercis ebout s ontw oevening sbefor eth eenerg yexpenditur emeasurement si nth efirs tperio d ofth eexperiment .Th eothe rhal fo fth esubject sperforme dth eexercis ebout s inth esecon dperiod .O nth eevenin g (between19.3 0an d22.3 0hours )o fda y4 andda y8 ,i nth efirs to rsecon dperio dsubject sperforme da maximu mwor k capacitytes to na cycl eergometer .Afte rth edeterminatio no fth emaximu m workcapacit ytes tsubject scycle da tfixe dpercentage so fthei rmaximu mwor k capacityfo ra maximu mo ftw operiod so f4 5minute swit ha fiftee nminute s restingperio di nbetween .

Experimentaldiet san dcarbohydrat eoverfeedin g

Beforeth esubject sstarte di nth estud ythei renerg yrequirement swer e estimated.Energ yrequirement swer eestimate do nth ebasi so fa duplicat e restingmetaboli crat e(RMR )measurement .Overnigh tfastin gsubject swer e takeni nth emornin gb yca rt oth elaborator yfo ra measuremen to fthei rRMR . RMRwa sassesse dwit ha ventilate dhoo dsyste ma spreviousl ydescribe d(14) . Afterth eRM Rmeasurement ,subject sreceive da smal lbreakfas ttailore dt o theirindividua lenerg yneed sbu tno texceedin g2. 0MJ .Subject seithe rstaye d atth elaborator yo rwer ebrough thom eb ycar .O nth esam eda yi nth e afternoonth eRM Rwa smeasure dagain ,no wwit hth esubject si na 4. 5hour s

-217- postabsorptive state.N o systematicdiurna lvariatio n inRM Roccurred . Therefore,th emea no f thetw oRM Rmeasurement swa s takena s themos t precise estimator of a subject'sRMR . Itwa s thenassumed ,tha t insubject swit h 1-3 hours/week sportingactivitie s theRM R represented 70percen to f the total daily energyexpenditure ,ie ,requirements .I nsubject swit h 3-5 hours/week sporting activities,th eRM Rwa s taken to represent 67percen t of the total daily energy outputan d for subjectswit h 5-7 hours/week theRM Rwa s takena s 64percen to f thedail yenerg youtput . It isgenerall y assumed thatth eRM R representso naverag e between 60an d 75 percento f totaldail yenerg youtpu t (1,2). Thisassumptio n hasbee n corroborated by several respirationchambe r studiesan d studiesusin g the doubly labelledwate rmetho d infree-livin g individuals (15,16). Subsequently, energy requirementswer e calculated foreac h individual.Energ ywa sprovide d inth edie t in0. 5 MJ/dmodules ,startin gwit ha basa ldie to f 7.0 MJ/d. For each indivual theexperimenta l dietswer eweighe d outaccordin g toth e estimated energy requirements.Th e experimental dietsconsiste d ofnorma l food stuffs andprovide d breakfast,lunc han ddinne r aswel l as snacksan ddrinks . Subjectswer e instructed toconsum e eachda yonl y the food items that they were providedwith .Subject swer eals oallowe d tohav e one free alcoholic consumptioneac hda yo ron eglas so forang e juice.Coffee ,te a (bothwithou t sugar andmilk )an dminera lwate r orwate r could beconsume d ad libitum. Sugar use inte ao r coffeewa s compensated forb y reducing theamoun to f sugaruse d inothe r foodstuffs.Subject skep ta dail y record ofal l food itemsan d drinks consumed inexces so f the foodan ddrink s they received. Sportingactivities , intercurrent illnessesan dmenstruatio ndate swer e also recorded inthi s notebook. A 4-daysrotatin gmen uwa sgive nt oth e subjects tohav e abalance d number of consumptions ofeac hdail ymen uove r theentir eexperimenta l period. For practical reasons itwa sno tpossibl e toprovid e the subjectswit h an8-day s rotatingmenu ,a 2-day s rotatingmen uwa s considered tob e toomonotonou s for the subjectsan d tocaus eproblem swit hdietar y compliance.Th ediet swer e prepared bya trained dietitianan dnutritionis t and consisted ofo n average 14percen tprotein ,3 5percen t fat (11percen t saturated fats,1 2percen t mono-unsaturated fat,1 2percen tpoly-unsaturate d fat), 51percen t carbohydrates (28'percentmon o- an ddisaccharides ,2 3percen t polysaccharides), 5mg/M J cholesterolan d 5g/M Jdietar y fiber.Energ yan d nutrient intakewer e calculatedusin g the198 5editio no f theDutc h computerized food table (17).Subject s consumed their fooda thome ,ho tmeal s

-218- were provided astake-awa ydeepfroze nmeals . Energy intakewa sadjuste d according to the subject'swishe s inth e beginning of the studywhe n subjectsha dproblem seatin g all the food,whe n they experienced chronic hunger feelingso rwhe nweigh t changedwit hmor e than 0.5 kgdurin g the first fourday so f theexperiment .Sevent ypercen t of the carbohydrate overfeedingwa sperforme dwit hnorma l food stuffs (fruitjuice , raisins,apple-sauce ,marsh-mallows ,Englis h licorice), predominantly containing mono-an ddisaccharides .Thirth ypercen to f thecarbohydrat e load wasgive na sa commerciall y availablemaltodextrin s supplement (Fantomalt, Nutricia,Zoetermeer ,Th eNetherlands) . Duplicate portionswer e takeno fthre e complete 4-daysmenus , respectively, for ahypothetica l subjectwit ha nenerg y intakeo f8-8. 5 MJ/d,8.5- 9 MJ/d or of 9-9.5 MJ/d.Th eduplicat e portionswer e stored at-2 0C befor e analysiso f energyan dnutrien t content.Th eduplicat e portionso feac h 4-daysmen uwer e mixed thoroughly and then freezedried. Theas hconten t andmoistur e levelwer e determined (18)an d thenth emateria lwa s stored at-2 0C .Aliquot swer e analyzed forprotei n (19),tota l fat (20),th eproportio n of individual fatty acids after saponification andmethylatio n (21),dietar y fiber (22)an d cholesterol (23).Mono- ,di -an dpolysaccharide swer eenzymaticall y determined (24). Themea ncompositio n of thediet swa s calculated fromth e duplicate portion analysis.N o chemical analysiswa sperforme d of the foodstuffsuse d for thecarbohydrat e overfeeding treatment.Th emea ncompositio n of the diets during the carbohydrate overfeeding periodswa s calculated fromth e duplicate portion analysesplu s the calculated contribution of theextr a carbohydrates from foodtables.

Maximumwor k capacityan dexercis e tests

On twooccasion sdurin g theexperimen t subjectswer e requested toperfor m a maximumwor k capacity testo na cycleergomete r and,subsequently , to cycle for amaximu m of9 0minute sa tthre e fixedpercentage s of themaximu mwor k capacity.Hal f of the subjects,randoml ychosen ,ha d their exercise andwor k capacity testo n theevenin g ofda y 4an dda y 8, ie,o n theevenin g before the energyexchang emeasurements .Th eothe r halfo n theevenin g ofda y 19an d day 23. Subjects came to thelaborator y at least 2hour safte r their lastmeal . Subjects thenperforme d amaximu mwor k capacity testo na cycleergometer .Th e first fifteenminute sperio do feac h testwa suse d fora warming-up .Durin g thisperio dwome n cycled equalperiod so ftim e at 100,12 5an d 150W . Men

-219- followed the sameprocedur e buta twor k loadso f150 ,17 5o r 200an d 200o r 250W , depending onestimate d physical fitness.Afte r thisperio d thewor k loadwa sgraduall y increased every threeminute swit heithe r 10o r 25W steps until exhaustion occurred.Th e complete testwa salway s finishedwithi n 25 minutes.Afte r theassessmen t ofth emaximu mwor k capacity subjects hada fifteenminute s restperio ddurin gwhic h theycoul d drinkwate r or tea,o r on the second occasiononly ,coul d eat someo f their extra carbohydrates.Afte r this restperiod , subjectsha d tocycl e for 45minutes ,fo requa l periodso f timea ta mor e or less fixedpercentag e of theirmaximu mwor k capacity, startingwit h 70-80percent , followedb y 50-60percen tan d finallyb y 30-40 percent. Some subjects couldno t sustaincyclin ga t 80percen t for the entire period of 15minutes .Fo r these individuals thehighes twor k loadwa s slighlty decreased,bu tneve rbelo w7 0percent .Afte r the firstcyclin g period of 45 minutes subjects could rest for 15minute sdurin gwhic hwate r or teawer e drunk. Subjects then started a secondperio do f 45minute s (orles si f exhaustion occurred prematurely)cyclin gaccordin g toth e sameprotoco l used inth e firstperiod .Th eprotoco l followed on the first cycling eveningwa s repeated on the secondevening .Afte r theexercis e tests (between 22.00an d 22.30hours )th eexhauste d subjects received 4 (women)o r 8 (men)g o f glucose before theywen thom e (bybik emostly) .The ywer e instructed to fastunti l the nextmorning ,no t tosmok e and todrin kwate r only.

Energyexchang emeasurement san d calculations

Energyexpenditur ewa smeasure dwit h aventilate d hood systemo nmornings . Subjects reclined ina supinepositio n ina hospita lbed .Detail s of the measurement procedure and conditionshav ebee npublishe d before(14) . Movementso f the subjectswer e registeredb ymean so fa load cell (TKA-200A, Tokyo Sokki Kenkyujo,Tokyo ,Japan )place dunde r oneo f the legso f the hospital bed.Onc eever yhou r during calibration of theoxyge nanalyze r with freshdrie d outside air,th e subjectswer eallowe d tomov e briefly for 2-3 minutes. Restingmetaboli c rate (RMR)o r premeal baseline energyexpenditure ,wa s continuouslymeasure d inovernigh t fasting individuals fora t least 60 minutes.Afte r theRM Rmeasurement s subjects receiveda liquid yogurt-based testmeal , containingo naverag e 1.34 MJ (13percen tprotein ,3 4percen t fat, 53percen t carbohydrates).Th e liquid testmea lwa sgive n througha straw whichwa spasse d intoth eventilate d hood.Th emea lwa seate nwithi n 5

-220- minutes.Afte r ingestiono f the testmea lpostprandia l energyexpenditur e was measured continuously for 210minutes .Afte r thega sexchang e measurements urinewa s collected fordeterminin gurea-nitroge n excretion.Energ y expenditurewa scalculate d according toJéquie r et al. (25).Diet-induce d thermogenesiswa s calculated asth e increase inpostprandia l energy expenditure above thepremea l baselineenerg yexpenditur e (RMR)afte r ingestiono f the test food.Substrat eoxidatio n ratesan d postprandial nutrientbalance swer e calculated using thenonprotei n respiratoryquotien t and Lusk'sequation s (26).Ne tlipogenesi swa squantitate d aspreviousl y described(14) .

Body composition

Body fatconten twa s estimated fromtota lbod ydensit y aspreviousl y described (14).

Statistics

Analysiso fvarianc ewa suse d toevaluat e theeffect so f the exercise treatment and the carbohydrate overloading onth eRM Ran d thethermi c effect of food (27).I naddition ,pairwis e comparisonswer eperforme d between different treatments.Pairwis e comparisonswer e statistically evaluated using thewithin-perso n t-testwit h atwo-taile d regiono f rejectiona ta confidence level of 95percent .Dat a aregive na smea nvalue s+ sem.

RESULTS

Table 1 showssom echaracteristic s of the subjectsa tentr yo f the study.Al l subjectsha d abod y fatconten twithi na norma l range. Morning RMR'sdi d notdiffe r significantly fromafternoo nRMR's . Energy requirementsestimate d on thebasi so fth eRM Rmeasurement swer eo n average close toenerg y requirements,observe ddurin g thecontro lperiod .Tw o subjects,n o2 an d 6, experienced problemseatin gal l their food. For these subjectsenerg y intakewa s slightly reduced on the fourthda yo f the study with 0.5 MJ/d. Inon e subject,n o 5,energ y requirementswer e underestimated with about 20percent .Thi ssubject' s energy intakewa s increased on the third dayo f theexperimen twit h 1.0 MJ/d and onth e fifthda ywit hanothe r 1.0

-221- MJ/d.I nthi ssubjec tth efirs tenerg yexpenditur emeasurement swer emad eo n themornin go fda y8 .

Table1 :Som echaracteristic so fsubject sa tth ebeginnin go fth estudy .

Subject Sex Age Height Fat Stort H« FNR BMR Estimated Energy requirement noming Afternoon Msan Ho ryr Years Kg % Hrs/W kj/min kJ/inin kj/min H/J/d

1 M 29 66.1 14.5 1 - 2 4.33 4.48 4.41 9.0 - 9.5 2 M 22 65.5 20.1 1 - 2 4.68 4.68 4.68 9.5 - 10.0 3 M 22 83.6 17.5 3 - 4 5.14 5.31 5.22 11.0 - 11.5 4 F 24 62.1 14.6 4 - 5 4.43 4.40 4.42 9.0 - 9.5 5 M 23 69.0 8.8 6 - 7 4.93 4.35 4.64 10.0 - 10.5 6 M 22 81.4 13.8 5 - 6 6.22 6.05 6.14 13.5 - 14.0 7 F 20 51.8 18.6 1 - 2 3.97 4.25 4.11 8.0 - 8.5 8 F 22 53.1 20.0 1 - 2 3.39 3.43 3.41 7.0 - 7.5 9 F 24 55.0 20.8 3 - 4 4.21 4.17 4.19 9.0 - 9.5 10 F 22 54.1 25.1 1 - 2 4.02 4.06 4.05 8.0 - 8.5

Msan + sem 23 + 0.8 64.2+3.6 17.4+1.5 4.53+0.25 4.52+0.23 4.53+0.23

HR= resting •etatxriic rate

Table2 :Mea ndail yenerg yan dmacronutrien tintak ei nsubject sbefor ean d duringperiod so fcarbohydrat eoverfeeding .

Energy Protein Fat Carbohydrates MJ/d g/d g/d g/d

BeforeCH Ooverfeeding : day 1 - 4 / 1 - 19 9.9 + 0.6 85 + 5 93 + 6 293 + 17 During CHO overfeeding: day 5/20 11.4 + 0.7 85 + 5 93 + 6 382 + 23 6/21 12.9 + 0.8 85 + 5 93 + 6 470 + 28 7/22 14.4 + 0.8 85 + 5 93 + 6 559 + 33 8/23 15.9 + 1.0 85+5 93 + 6 652 + 38

Valuesar egive na smea n+ se m CHO:carbohydrat e

Bodyweigh tdi dno tsho wmarke dchange sdurin gth ecours eo fth eexperiment . Bodyweight swere ,respectively ,64. 2+ 3. 6k g (control),64. 5+ 3. 6k g (carbohydrate),63. 8+ 3. 5k g(exercise )an d64. 2+ 3. 6k g

-222- (carbohydrate/exercise). Table2 give senerg yan dmacronutrien tintak ebefor ean ddurin gth e carbohydrateoverloadin gperiods . Meanenerg yintak ewa s9. 9+ 0. 6MJ/ d(14. 6percen tprotein ,34. 9percen tfa t and50. 5percen tcarbohydrates )durin gth efou rday sbefor eth ecarbohydrat e suppletion.Energ yintak eincrease ddurin gth ecarbohydrat eoverfeedin gfro ma meano f11. 4MJ/ d(da y1 )t o15. 9MJ/ d(da y 4). Carbohydrateintak eincrease d duringthi sperio dfro ma naverag eo f29 3g/ dt o65 2g/d .O nth efirs tda yo f thecarbohydrat esuppletio nperiod ssubject sconsume do naverag e9 0g/ d (1.5 MJ/d)i nexces so fthei rintak eo fcarbohydrate so nth efirs tfou rday so fth e treatmentperio drequirements .O nth elas tda yo fth esuppletio nperiod sth e excesscarbohydrat eintak eamounte do naverag et o35 9g/ d (6.1MJ/d) .Mos t subjectsexperience dsom eproblem si ningestin gsuc hlarg eamount so f carbohydrateso nto po fthei rdail ydiet .

Table3 :Maximu mwor kcapacity ,averag ewor kloa dan dexercis eduratio n duringa cycl eergomete rtes ti nsubject sbefor ean da tth een do fa carbohydrateoverloadin gperiod .

Subject Sex Maxinunwor k capacity Average work load Averageexercis e duration No M/F Start de End OC StartCH D Enda c Start an End cm

W W W W Min Min

1 M 270 270 140 140 65 70 2 M 225 225 130 135 65 65 3 M 290 280 170 190 90 90 4 F 220 220 130 130 90 90 5 M 240 245 149 150 68 90 e M 315 330 170 240 90 90 7 F 200 200 120 120 90 90 8 F 205 205 135 138 80 80 9 r 200 210 125 130 90 90 10 F 185 190 110 120 75 75

Maan± sem 235+ 13.7 238+ 13.9 137.9± 6.3 149.3± 11.9 80.3± 3.5 83± 3.1

both subjects had additional exercise before cycle test (running). Ulis HBS conpensated for en the second exercise test by increasing the duration (no 5) or the work load (no 6) of the cycle test QD = f irwwywut*

Table3 show smaximu mwor kcapacity ,averag ewor kloa dan dexercis eduratio n obtainedo ntw ocycl eergomete rtests ,on ea tth estar to fth ecarbohydrate s

-223- suppletionperio dan don ea tth een do fthi speriod .A tth estar to fth e suppletionperio d subjectscycle do naverag efo r8 0minute sa t5 9percen to f theirmaximu mwor k capacity.A tth een do fth ecarbohydrat e suppletionperio d subjectsexercise do naverag efo r8 3minute sa t6 3percen to fthei rmaximu m work capacity.O nbot h occasionsa maximu mwor k capacity testwa sperforme d prior toth eexercis e test.Durin gth eassessmen to fth emaximu mwor k capacity theme ncycle do naverag e2 2mi na t200-20 5W andth ewome n cycled2 0mi na t 140W . Intotal ,subject scycle do nbot hexercis e testso naverag efo rmor e than10 0minutes .

Table4 :Bestin gmetaboli c ratean drespirator yquotient si npostabsorptiv e subjectsa tdifferen tperiod si nth estudy .

Subject Sex Control Ou Exercise Exercise + a© FMR R3 FMR SQ RMR R3 RMR BQ Ito M/F kJ/hrin kj/hiiii kj/tnin kj/inin

1 M 3.97 0.85 3.75 0.90 4.17 0.83 4.70 0.85 2 M 5.33 0.83 4.94 0.92 5.60 0.79 4.98 0.97 3 M 5.39 0.85 5.04 0.81 5.85 0.77 5.40 0.84 4 F 4.35 0.84 4.89 0.91 5.16 0.79 4.69 0.86 5 M 5.01 0.80 5.90 0.90 5.06 0.74 4.71 0.87 6 M 5.46 0.81 5.37 0.89 6.40 0.77 5.86 0.83 7 F 4.58 0.83 4.18 0.99 5.65 0.77 4.73 0.89 8 F 3.75 0.83 3.05 0.93 4.01 0.79 3.75 0.88 9 F 3.61 0.86 3.46 0.95 4.65 0.75 4.80 0.85 10 F 4.16 0.83 4.42 0.94 4.45 0.82 4.60 0.87

HBan ± son 4.56 + 0.2 0.83 + 0.01 4.50 + 0.28 0 91 ± 0.01 5.10 ± 0.25 0.78 + 0.01 4.82 ± 0.17 0.87 + 0.01

IHt = resting •etabolic rate; BQ = respiratory opotients; CHDcarbohydrat e overloading

Table 4show sRMR' san dRQ' si nfastin g subjectsa tdifferen tperiod si nth e study.Prio rexercis eo nth eda ybefor eth emeasurement s significantly increasedRM R(p=0.02) .N osystemati c effecto fth ecarbohydrat e suppletiono n theRM Rwa sobserved .Bot h treatmentsha da significan t impacto nfastin g RQ's. PostabsorptiveRQ' swer eo naverag e1 0percen t highera tth een do fth e carbohydrate suppletionperio d comparedt oth eRQ' sobserve da tth estar to f the suppletionperiod .I ncontrast ,RQ' swer eo naverag e1 2percen t lower after prior exercise comparedt oth eRQ' smeasure d inth enon-exercis e state. Notal lsubject s showedth esam e relativemagnitud e inrespons eo fRM Rt o exercise. Subjectn o5 di dno trespon da tall ,wherea s subjectn o6 increase d herRM R2 9percen ti nrespons et oprio rexercise . -224- Table 5give sDIT' san dpostprandia l RQ'si nsubject sa tdifferen t periodsi n the study.Ther ewa sa significan teffec to fcarbohydrat eoverfeedin go nDIT . Carbohydrate suppletionwithou tprio r exercisewa sassociate dwit ha mea n increase inDI To f5 0percent .Prio r exercisedi dno tsystematicall y affect DIT.DI Tvalue swer e only significantly increasedb y3 3+ 1 4k J, ie ,0.1 6+ 0.07 kJ/min (p=0.035)i nrespons et oexercise ,whe n subjectsdi dno treceiv e extra carbohydrates.DI Tvalue s expresseda sa percentag eo fth eingeste d energy contentwere ,respectively ,7. 6+ 0. 9percen t (control),11. 5+ 1. 2 percent (carbohydrate),10. 1+ 0. 8percen t (exercise)an d13. 2+ 1. 4percen t (carbohydrate/exercise).Th einter-individua l difference inrespons eo fth e postprandial risei nenerg yexpenditur e toprio r exerciseo rcarbohydrat e overloadingwa sconsiderable .Difference si nrespons ewer eno tsystematicall y associatedwit hth ese xo fth esubjec to rwit hth emaximu mwor k capacity.

Table5 :Diet-induce d thermogenesisan dpostprandia l respiratoryquotient si n subjectsa tdifferen tperiod si nth estudy .

Subject Control ac Exercise Exercise+ OC DIT BQ DIT BQ DIT BQ DIT BQ

No M/T kJ kJ kJ KJ

1 M 63 0.90 174 0.89 142 0.82 268 0.93 2 M 131 0.83 145 0.96 188 0.77 176 0.96 3 M 80 0.88 148 0.93 113 0.61 103 0.89 4 F 94 0.90 151 0.91 186 0.79 226 0.89 5 M 146 0.89 280 0.93 130 0.76 178 0.86 6 M 97 0.85 170 0.89 95 0.79 219 0.88 7 F 139 0.85 119 0.99 143 0.79 155 0.93 8 F 44 0.85 118 0.97 127 0.80 164 0.85 9 F 71 0.87 133 0.99 96 0.80 77 0.91 10 F 156 0.92 94 0.97 128 0.82 200 0.92 Mean+ se m 102± 12 0.87± 0.01 153± 1 6 0.94+ 0.01 135± 10 0.79 ± 0.01 177± 18 0.90+ 0.01

DÛT = diet-dniioed thenDgaiesis; HQ = re^irstory ^lotient; OD- cmlxiyltate overloar&iig

Prior exercisean dcarbohydrat e suppletionaffecte d postprandialRQ' si na similarwa ya sthe y influenced thepostabsorptiv e RQ's.Figur e1 show sth e time courseo fth epostprandia l risei nenerg yexpenditur e foreac ho fth e four treatments.Th ehighes t responsewa sobserve d inth eperio dbetwee n3 0

-225- and 120 minutes of the postprandial period.Afte r 210 minutes the thermic effect of food was negligible for all treatments, except for the carbohydrate/ exercise treatment.

diet induced thermogenesis(kJ/min ) U

1 2 3 timeafte rmea l ingestion(h )

Figure 1. Diet-induced thermogenesis in subjects during control treatment (•), after prior excerise (/S.), after short-term carbohydrate overfeeding (0), after short-term carbohydrate overfeeding plus prior exercise (.A.)•

Table 6 give pre-and postprandial substrate utilization rates at different periods in the study. Protein oxidation rateswer e significantly reduced by carbohydrate overfeeding. Also fat oxidation rates, both pre- and postprandially, were systematically decreased by carbohydrate suppletion. Glucose oxidation rates were significantly increased after carbohydrate overloading. Exercise impacted significantly on pre-and postprandial glucose and fat oxidation rates. Postprandial glucose and fat balances were significantly affected by both exercise and carbohydrate overfeeding. Postprandial fat balances were, respectively, 5.2 + 1.0 g (control), 9.9 + 1.1 g (carbohydrate), -4.5 + 1.2 g (exercise) and 6.4 + 0.9 g (carbohydrate/exercise). Postprandial glucose balances were, respectively, for the different treatments, 8.2 ± 2.1 g, -8.0 + 3.1 g, 22.9 + 1.6 g and -4.8 ± 3.6 g.

-226- Table6 .Pre -an dpostprandia l substrateutilizatio n ratesi nsubject sa t different periodsi nstudy .

Preprandial Postprandial Period instud y Period instud y 1 2 3 4 1 2 3 4

Protein (mg/inin) 46+ 4 37+ 3 51± 4 45+ 4 46+ 4 37+ 3 51+ 4 45+ 4 Fat (mg/hon) 49+ 4 22± 6 79± 6 37+ 4 37+ 4 13± 5 84+ 6 30± 4 Glucose (mg/hiin) 100+ 6 165± 12 58+ 7 143 ± 1 1 169+ 1 1 237± 1 5 93+ 8 223+ 1 6 Lipogenesis (mg/tnin) 0 0 0 0 1.1± 0. 9 6.2± 1 8 0 2.8+ 0. 8

Results aie given as seen + sea Patiod 1 = control 2 = caifachythate overloading 3 = exercise 4 = caxbohydrste overloading + exercise

DISCUSSION

In recentyear s thereha sbee n considerable interest indefinin gth e determinantso fa nindividual' s restingmetaboli c rate (RMR)an dthermi c response tofood ,ie ,diet-induce d thermogenesis (DIT)(3,4,28-31) .Th eRM Ro f a subjecti sprimaril ydepende do nth esiz eo fhi so rhe rfat-fre ebod ymas s (28-31).Th ethermi c effecto ffoo di slargel ydepende d onth eamoun to f energyan dtyp eo fmacronutrient s ingested (3,4). Inaddition ,t oassessin g the sizean dnatur eo finter-individua l variationi nRM Ran dDIT ,i ti s importantt oinvestigat ewithin-perso n responseso fRM Ran dDI Tt ovariou s stimuli. Such studiesma ygiv e informationo nth ebiologica l capacityi n humanso fmodifyin g energyus ethroug hmetaboli cprocesse s inrespons et o changing environmentalfactors . Inthi s studyw einvestigate d theimpac to nRM Ran dDI To fa nanteceden t hyper-energetic diethig hi ncarbohydrate san do fprolonge d intense prior exercisebouts .Th epresen t studyals oconfirme sa previou s studyo fthi s laboratoryo nth eabsenc eo fsignifican tdiurna lvariatio n inRM R(14) . Exercisean dcarbohydrat e overfeedingwer e chosenfo rstudyin g their impacto n RMRan dDIT ,sinc e theyar eknow nt ohav e large effectso nth estat eo fth e body's glycogen stores (6-8).Th estat eo fth ebody' s glycogen stores seemso f pivotal importance indeterminin g themetaboli c routingo fingeste d carbohydrates (5,32,33).Thus ,wit hbooste d glycogen stores ingested glucose will,i nadditio nt oimmediat e oxidation,b estore da slipi d rather thana s

-227- glycogen (5,32,33). Incontrast ,i na glycogen-deprive d state ingested glucose will be stored rather asglycoge n thana s fat (5,32,33). Storage of ingested glucose asglycoge no ra s fatinvolve sa highe rAT Pus e thandirec t oxidation of theabsorbe d glucose (9).Thi s implicatestha ta metaboli c state favoring storage ofdietar y carbohydrateseithe r asglycoge no r as fatprio r to oxidationwil l increase thethermi ceffec to f food,compare d toa metaboli c statewher emos to f the ingested carbohydratesar eoxydize dwithou t prior storage. The resultso fth epresen t studysho wa n increasedDI T in response to carbohydrate overloading eitherwit ho rwithou tprio rexercise .W e also found thatprio r exercise increased DIT.Th e systematic effecto fprio r exercise on DITwas ,however ,lackin gdurin g the carbohydrate overloading.Furthermore , prior exercisedi d increase theRMR ,but ,again ,th eeffec to fexercis e onRM R wasmodulate d by carbohydrate overloading.Bot h carbohydrate overloading and prior exerciseha d significant effectso npostabsorptiv e and postprandial substrateutilizatio n rates.Exercis e increased fatoxidatio n ratesan d reduced glucoseoxidation ,wherea s theopposit eeffec twa s observed for the carbohydrates overloading,confirmin g the findingso fpreviou s studies (5,10-12,32,33). Theeffec to fprio r exerciseo nRM Rha sbee nobserve d inothe r studies (10-12,33-35)a swell .Generall y thisphenomeno n isdesignate d excess postexercise oxygenconsumptio n (EPOC)(36) .Severa l studieshave ,however , notobserve d a significant after-effect onRM Ro fprio r exercise (37-39). The reasons for thesediscrepan t findingsar eno t clear.Generally ,ther e is considerablevariatio namon g the studies inth e type,th e intensityan d durationo f theapplie d exercise tests.I naddition ,i nsom e studiesn o strict control over theanteceden tdie twa sperforme d (33-35,38). Iti slikel y that onlyprolonge d intenseglycogen-depletin g exercise affectsth ebody' s energy economy sufficientlywel l tocaus e anenhance d thermogenesis inth e postexercise period. Furthermore,a s this study shows,th eeffec to f exercise onRM R canb e affected by theanteceden tdiet . Thebiochemica l basiso f theexces spostexercis e oxygenconsumptio n isno tye t elucidated (36).Probabl yexces senerg y isexpende d inth epostexercis e period for replenishing thebody' sglycoge n stores,fo rexample ,i nconvertin g glyconeogenic precursors intoglycogen .Exercis ema yals o increase various other energy-demanding processes inth epostexercis e period sucha s increased substrate cyclingan d sodiumpumpin g (36).Th e finding thatexces s carbohydrates reduced theafter-effec to fexercis e onRM R suggests thata

-228- relatively largepart ,ie ,abou t 0.30 kJ/min (5.10- 4.80 kJ/min)o f EPOC mightb e related toenerg yexpende d for refilling thebody' s glycogenstores . The latter process islikel y tob ediminishe d afterprolonge d exercisewit h previous carbohydrate overloading compared toprolonge d exercise without previous carbohydrate suppletion. The effect ofprio r exercise onDI Tha sno tbee n studied before.Onl y one studyassesse d the impacto fprio r exercise on the thermic effect of glucose (40). Inth e studyo fYoun g et al. (40)i twa s found thatprio r exercise potentiated the thermic effecto fglucos eb yabou t 65percent .Th e effecto f exercisewa s found tob e independento f the stateo f training of thesubjects . Inthi s study exercise potentiated DITo naverag e by 33percent .N o significant relationwa s foundbetwee n the response toexercis e ofDI Tan d the maximumwor k capacity.Par to f thepotentiatio n ofDI Tb yprio r exercise can be attributed toth e increased energy costs for storing the ingested glucose asglycogen .Postprandia l nutrientbalance s showed thato n themornin g after exercisewithou t previous carbohydrate overloading onaverag e 22.9g o f glucosewer e retained inth ebody ,presumabl y asglycogen .Th e costo f this nonoxidative storage ofglucos e addsu p to 20kJ .Thi s isalmos t 60percen t of the total increase inDI Tafte r prior exercise.Th e remainingpar t of the increase shouldb e attributed to stimulation byprio r exercise of thermogenic processes, sucha s increased sodiumpumpin g and substrate cycling(36) . Similar to theeffec to fprio r exercise onRM R theeffec to fexercis e on DIT wasmodulate d by carbohydrate overloading.Carbohydrat e overfeeding caused an increase inDI To fo naverag e 50percen t (withoutprio r exercise)an d of 31 percent (withprio r exercise). These resultsar e incontras twit h findingso f Welle et al. (41).However ,a spointe d outb yWell e et al. (41),th e relatively shortduratio n ofDI Tmeasurement s inthei r studypreclude s any major conclusions on the impacto f short-term Carbohydrateoverfeedin g onDIT . The resulto f thepresen t studyar e concordantwit h the findingso fAcheso n et al. (5).Acheso ne tal .observe d a significant increase inDI T in subjects on a high carbohydrate compared toth eDI To n iso-energetic dietshig h in fato r ofnorma l composition.Th e relativedifference s among thevariou sdiet s inDI T were, respectively, 60percen t (highcarbohydrat eversu shig h fat)an d 32 percent (highcarbohydrat e versusmixe ddiet ) inth e studyo fAcheso n et al. Thesedifference s agreewel lwit h the relativedifference s inDI T observed among thevariou s treatments inthi sstudy .

The reason for thepotentiatio n of theDI Tb y thecarbohydrat e overloading is notdirectl y clear.Postprandia l nutrient balances showed thatal l the

-229- ingested carbohydrateswer e oxydized and thatn omajo r net storage occurred. Therewa s somene t lipogenesis,ie ,6 mg/mi no n themornin gwithou t prior exercise and 3mg/mi no n themornin gwit hprio r exercise.W e calculated that at themos t 5-10 percent of the total thermic responsewa sdu e toth ene t conversiono f ingested carbohydrates intolipid . Itshoul d be realized, however,tha t indirect calorimetry isno ta suitable technique formeasurin g total lipogenesis (25).Indirec t calorimetry givesa nestimat e of the amount of lipid synthetized fromglucos e inexces so f theamoun to f fat concomitantly oxydized (25).Thi smean stha t ifsubstantia l fatsynthesi soccur swit h concomitant fatoxidation ,th e simultaneousoccurrenc e of these twoprocesse s would cause an increase inenerg yexpenditur e ofabou t 20-25percen t of the energy contained inth eglucos e channeled intolipogenesis .Calculatin g the amounto fenerg yexpende d for theconversio no fglucos e intone t lipogenesis would, intha tcase ,underestimat e theenerg y costsmad e for total lipogenesis. Iti sno tknow nwhethe r thebod ywoul d simultaneously synthetize and oxydize largequantitie so f fat.Whe n it isassume d that postprandial glucoseoxidatio n rates inexces so f the rateobserve d at the control treatment, representglucos eoxydize da s lipid,then ,th eenerg y costsmad e for lipogenesis could easilyexplai n theobserve ddifference s inDI Tamon g the treatments.Th e amounto fglucos eoxydize d aslipi d corresponds to14. 3g (controlversu s carbohydrate)an d 11.3g (exerciseversu s carbohydrate/exercise).Th eenerg yexpende d forconvertin g theseamount so f glucose into fatare ,respectively , 54k Jan d 42kJ .Thi s isequa l toth e difference inDI Tbetwee n the controlan d carbohydrate treatmentan d toth e difference inDI Tbetwee n theexercis e and carbohydrate/exercise treatment.I t represents,however ,onl y 56percen to f thedifferenc e inDI Tbetwee n the control and carbohydrate/exercise treatment.Thi ssuggest stha tth e combination ofexercis e and carbohydrate overloading resulted ina stimulation ofadditiona l thermogenicmechanism s inth epostprandia l period. Ivye t al. (42)sugges t thata die thig h incarbohydrate s followingexercis e causes an increased insulin response.Insuli nha sbee n shownt o increase circulating norepinephrine concentrations,ie ,t oactivat e the sympathethicnervou s system activity, independento f changes inbloo d glucose (43).Bot h insulin and SNS activity couldaffec t the rateo f influxo f sodium intocell san d thereby stimulate theus e ofAT P toextrud e the sodium fromcell s (44,45). Catecholaminesma yals o increase the rateo f substrate cycling (46)an d the recycling ofglucos e through 3-carboncompound s (47).Tha tprio r exercise can

-230- potentiateth ethermi ceffec to finsuli nwa srecentl yreporte db yDevli nan d Horton(39) . Inconclusion ,th eresult so fth epresen tstud ysho wtha tintens eprolonge d priorexercis eincrease senerg yexpenditur ebot hi nth epostabsorptiv ean dth é postprandialphase .Short-ter mcarbohydrat eoverfeedin genhance sth ethermi c responset ofoo dconsiderably ,bu tdoe sno tincreas erestin gmetaboli crate . Carbohydratesuppletio nseem st oreduc eth eexces slos so fenerg yfro mth e bodyafte rexercise .Th eresult sindicat etha ti nassessin gth ethermi c responset ofoo dcontro love rprio rexercis ema yb ewarranted .Th eresult s alsounderscor eth erol eo fth eanteceden tdie ti ndeterminin ga nindividual' s thermicrespons et ofood .

ACKNOWLEDGMENTS

Theauthor swan tt othan kth esubject sfo rthei renthousiasti cparticipatio n inthi sstudy .W ear eals oindebte dt oMr sSaski aMeyboo mfo rexcellen t dietaryadvic ean dhel pi npreparin gth eexperimenta ldiet sfo rth esubjects .

REFERENCES

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-231- 12.Bah rR ,Ingue sI ,Vaag e0 ,Sejerste d0M ,Newsholm eEA .Effec to fduratio n ofexercis eo nexces spostexcercis e0 ,consumption .J App lPhysio l 1987;62:485-90. 13.Wila nAR ,Pate rJL .Carryove ran dth etwo-perio dcrossove rclinica ltrial . Biometrics1986;42:593-9 . 14.Weststrat eJA ,Wey sPJM ,Poortvlie tEJ ,Deurenber gP ,Hautvas tJGAJ . Diurnalvariatio ni npostabsorptiv e restingmetaboli crat ean d diet-induced thermogenesis.A mJ Cli nNut r1989 :i npress . 15.Ravussi nE ,Burnan dB ,Schut zY ,Jéquie rE .Twenty-four-hou renerg y expenditurean drestin gmetaboli crat ei nobese ,moderatel yobese ,an d controlsubjects .A mJ Cli nNut r1982;35:566-73 . 16.Prentic eAM ,Blac kAE ,Cowar dWA ,Davie sHL ,Goldber gGR ,Murgatroy dPR , AshfordJ ,Sawye rM ,Whitehea dRG .Hig hlevel sof.energ yexpenditur ei n obesewomen .Bri tMe dJ 1986;292:983-7 . 17.Commissi eUCV .UC Vtabel ,' sGravenhage :Voorlichtingsburea uvoo rd e Voeding,1985 . 18.Osborn eDR ,Voog tP .Th eanalysi so fnutrient si nfoods .London :Academi c Press,1978 . 19.William sS .ed .Officia lmethod so fanalysi so fth eassociatio no f officialanalytica lchemists .14t hed .Arlington :VA :ADA CIne ,1984 . 20.Folc hJ ,Lee sM ,Sloan eStanle yGH .A simpl emetho dfo rth eisolatio nan d purificationo ftota llipid sfro manima ltissues .J Bio lChe m 1957;226:497-509. 21.Metcalf eLD ,Schmit zAA ,Pelk aJR .Rapi dpreparatio no ffatt yaci dester s fromlipid sfo rga schromatographi canalysis .Ana lChe m1966;38:514-5 . 22.Prosk yC ,As pNG ,Furd aJ ,d eVrie sJW ,Schweize rTF ,Harlan dBF . Determinationo ftota ldietar yfibe ri nfoods ,foo dproduct san dtota l diets:interlaborator ystudy .J Asso cAna lChe m1984;67:1044-53 . 23.Va nd eBovenkam pP ,Kata nMB .Cholestero lconten to fchicke nskin .J Foo d Sei1981;46:291 . 24.Boehringer .Method so fenzymati cfoo danalysi susin gtest-combinations . Foodanalysis ,Boehringe rMannhei m1983 . 25.Jéquie rE ,Acheso nK ,Schut zY .Assessmen to fenerg yexpenditur ean dfue l utilization.An nRe vNut r1987;7:187-208 . 26.Lus kG .Anima lcalorimetry .Analysi so fth eoxidatio no fmixture so f carbohydratean dfat .J Bio lChe m1924;59:41-2 . 27.Wine rBJ .Statistica lprinciple si nexperimenta ldesig n2n ded .Ne wYork : McGraw-HillBoo kCompany ,1971 . 28.Ravussi nE ,Lillioj aS ,Anderso nTE ,Christi nL ,Bogardu sC .Determinant s of24-hou renerg yexpenditur ei nman .Method san dresult susin ga respiratorychamber .J Cli nInves t1986;78:1568-78 . 29.Bogardu sC ,Lillioj aS ,Ravussi nE ,Abbot tW ,Zawadzk iJK ,Youn gA , KnowlerWC ,Jacobowit zR ,Mol lPP .Familia ldependanc eo fth erestin g metabolicrate .N Eng lJ Me d1986;315:96-100 . 30.Owe nOE ,Holu pJL ,D'Alessi oDA ,Crai gES ,Polansk yM ,Smalle yKJ ,Kavl e EC,Bushman nMC ,Owe nLR ,Mozzol iMA ,Kendric kZV ,Bode nGH .A reappraisal ofth ecalori crequirement so fmen .A mJ Cli nNut r1987;46:875-85 . 31.Owe nOE ,Kavl eE ,Owe nRS ,Polansk yM ,Capri oS ,Mozzol iMA ,Kendric kZV , BushmannMC ,Bode nG .A reappraisalo fcalori erequirement si nhealth y women.A mJ Cli nNut r1986;44:1-9 . 32.Acheso nKJ ,Flat tJP ,Jéquie rE .Glycoge nsynthesi sversu slipogenesi s aftera 50 0gra mcarbohydrat emea li nman .Metabolis m1982;31:1234-40 . 33.Acheso nKJ ,Schut zY ,Bessar dT ,Anantharama nK ,Flat tJP ,Jéquie rE . Glycogenstorag ecapacit yan dd enov olipogenesi sdurin gmassiv e carbohydrateoverfeedin gi nman .A mJ Cli nNut r1988;48:240-7 . 34.Edward sHT ,Thorndik eA ,Dil lDB .Th eenerg yrequirement si nstrenou s muscularexercise .N Eng lJ Me d1935;213:532-5 . -232- 35.D eVrie sHA ,Gra yDA .Afte reffect so fexercis eupo nrestin gmetaboli c rate.Re sQuar t1962;34:314-21 . 36.Passmor eR ,Johnso nRE .Som emetaboli cchange sfollowin gprolonge d moderateexercise .Metabolis m1960;9:452-5 . 37.Gaesse rGA ,Brook sGA .Metaboli cbase so fexces spost-exercis eoxyge n consumption:a review .Me dSe iSport sIn t1984;16:29-43 . 38.Dallos oH ,Jame sWPT .Dietar ythermogenesi san dexercise .Pro cNut rSo c 1981;35A(abstract) . 39.Freedman-Akaba sS ,Col tE ,Kissilef fHR ,Pi-Sunye rFX .Lac ko fsustaine d increasei nVO -followin gexercis ei nfi tan dunfi tsubjects .A mJ Cli n Nutr1985;41:535-9 . 40.Devli nJT ,Horto nES .Potentiatio no fth ethermi ceffec to finsuli nb y exercise:difference sbetwee nlean ,obes ean dnon-insuli ndependen t diabeticmen .A mJ Cli nNut r1986;43:884-90 . 41.Youn gJC ,Treadwa yJC ,Balo nTO, Garra sHP .Ruderma nNB .Prio rexercis e potentiatesth ethermi ceffec to fa carbohydrat eload .Metabolis m 1986;35:1048-53. 42.Well eS ,Campbel lRG .Stimulatio no fthermogenesi sb ycarbohydrat e overfeeding.Evidenc eagains tsympatheti cnervou ssyste mmediation .J Cli n Invest1983;71:916-25 . 43.Iv yJC ,Sherma nWM ,Mille rW ,Farrel lS ,Frishber gB .Glycoge nsynthesis : effecto fdie tan dtraining .In :Knuttge nHG ,Voge lJA ,Poortsma nJ .eds , Biochemistryo fexercise .Huma nKinetic sResearc h1983;13:291 . 44.Feli gP .Insuli ni sth emediato ro ffeeding-relate dthermogenesis :insuli n resistanceand/o rdeficienc yresult si na thermogeni cdefec twhic h contributest oth epathogenesi so fobesity .Cli nPhysio l1984;4:267-77 . 45.Moor eRD .Stimulatio no fNa :H exchang eb yinsulin .Biop hJ 1981;33 : 203-10. 46.Horwit zBA .Metaboli caspect so fthermogenesis :neurona lan dhormona l control.Fe dPro c1979;38:2147-9 . 47.Newsholm eEA .substrat ecycles :thei rmetabolic ,energeti can dthermi c consequencesi nman .Bioche mSo cSym p1978;43:183-205 . 48.DeFronz oRA ,Thori nD ,Felbe rJ ,Simonso nDC ,Thiebau dD ,Jéquie rE ,Gola y A.Effec to fbet aan dalph aadrenergi cblockad eo nglucose-induce d thermogenesisi nman .J Cli nInves t1984;73:633-9 .

-233- Chapter12 . Generaldiscussio n

The studies described inthi s thesiswer e conducted toanswe rth efollowin g questions:

1.Ho wlarg e isth eintra-individua lvariatio n inrestin gmetaboli c ratean d diet-induced thermogenesis?D osex ,degre eo fbod y fatnessan dcontro l ofth e antecedent diet affect thesiz eo fth eintra-individua l variation inrestin g energy expenditure?

2.Wha t isth enatur ean dmagnitud eo finter-individua l differences inrestin g metabolic ratean ddiet-induce d thermogenesis inlea nan dobes e individuals? Doesth etyp eo fbod yfa tdistributio ni nobesit yaffec t these differences?

3.T owha t extent isrestin gmetaboli c rateaffecte db ynon-nutritiona l factors,ie ,previou sexercise ,psychologica l stressan dtim eo fth eday , and towha t degreeb ynutritiona l factors,ie ,ethano l ingestionan dshort-ter m carbohydrate overfeeding?

4.T owha t extent isdiet-induce d thermogenesis affectedb ynon-nutritiona l factors,ie ,previou sexercise ,psychologica l stress,tim eo fth eda yan d palatabilityo ffood ,an dt owha t degreeb ynutritiona l factors,ie ,ethano l ingestionan dshort-ter m carbohydrate overfeeding?

AD1

Twocomponent so fdail yenerg yexpenditur ewer e investigated, resting metabolic rate (RMR)an ddiet-induce d thermogenesis (DIT). These components comprise 70-85percen to ftota l energy expenditure inth eaverag e individual (1). Iti sno tsurprisin g that researcho nhuma n energy expenditureha s focusedo nstudyin g these components inrelatio nt oth epathogenesi so f

-234- obesity.The yar equantitativel y important foroveral l energyexpenditur e and canb e assessed relativelyeas ywit h theus eo f indirect calorimetry.

Inth e studiesdescribe d inthi sthesi sth eventilate d hood techniquewa suse d tomeasur e RMRan dDI T inhumans .Befor e the starto f theexperiment smuc h effortwa s spent inth edevelopmen tan d construction ofa ventilate d hood system.Ther ewa sn oexperienc e inou rDepartmen twit h theassessmen t of energyexpenditur e using theventilate d hood technique.Therefore ,extensiv e studieswer e carried out totes t theaccurac yan d the reproducibility of measurements ofenerg y expenditure.Thes e studiesar edescribe d inchapte r 3. A salient finding of these studies isth e relatively low reproducibility of measurements ofDIT .Intra-individua lcoefficient s ofvariatio nwer e around 30 percent.Whe n a characteristic showsa relatively large intra-individual variation, thisma ypos e seriousproblem s for studies thataim e todetermin e within-person responses totreatment s (2).Also ,distinguishin g groupso f individuals fromanothe rma ybecom edifficul t(2) . Reproducibility data,obtaine d from the studiesdescribe d inchapte r 3,wer e used todetermin e theexperimenta l designs of the studies reported in subsequent chapterso f thisthesis .I nassessin g theeffec t onDI To f non-nutritional andnutritiona l factors,a minimu m sample sizeo f 10 subjects wasused . Inmos t studies repeatedmeasurement s per subjectwer e obtained for each treatment condition.Experimenta l designswer edevise d tohav e apowe r of 80 to9 0percen t indetectin g treatmentseffect so nDI To fabou t 30percent . Chapter 7 reportso nbetween-grou p comparisons ofDIT .Dat ao n between-person variation inDIT ,a sdescribe d inchapte r 3,indicat e that sample sizespe r groupwer e sufficiently large todetect ,wit ha powe r between 80t o9 0 percent, realbetween-grou pdifference s inDI To fabou t 25percent .Thi smean s thatth e failure to reachstatistica l significance intreatmen teffect so r between-groupdifferences ,a s reported insom eo fou r studies, isprobabl y related to theactua l lack ofeffec to f treatment or between-group differences.

Inth e study ofhuma nenerg y expenditure one of themor e controversial issues is,whethe r obeseperson shav e adefectiv e thermogenic response to food in comparison to their lean counterparts (3). A defective thermogenic response to food couldb eo f significance forth epathogenesi s ofhuma n obesity(3) . Whether a relativelyhig h intra-individual variation indiet-induce d thermogenesis hascontribute d to thegenesi so f thiscontroversy , canno tb e

-235- ascertained fromou r studies.Afte r scrutinizing the literature fordat a of other groups on the reproducibility ofDIT ,onl y one reference was found containing this information (4).Ravussi n et al. (4)conclud e thatDI T shows low reproducibility. Our results confirm their conclusions.A survey of the various studies onDI T shows, thatmos t of the studies reporting no systematic difference inDI Tbetwee n the leanan d theobes e had sample sizesprobabl y too small todetec t even relatively largedifference s of about 50percen t (5-17). The absence ofdat a on reproducibility ofDI Tmeasurement s ispuzzling . Reproducibility ofRM R or 24-hour energy expenditure measurements is frequently reported (4,18-21).Whateve r the reason is for the lack ofdat a on reproducibility ofDI Tmeasurements , this thesis shows that,befor e concluding on the existence or absence of systematic treatment effects onDI To r between-group differences inDIT ,investigator s should obtain information on [_within-perso n and between-person variation inDIT . The specific cause for the relatively highwithin-perso n variability in diet-induced thermogenesis wasdifficul t todetermine .Coefficient s of variation were notaffecte d by sex,degre e ofbod y fatconten t or oral contraceptive use.Contro l of the antecedent dietdi d not seem toaffec t the size of the intra-individualvariation . Itwa s concluded that,i n particular, variation.in _siibjectLs_bfihavia ran d inmeasuremen t circumstances, ie,i n motion-pictures,attribute d to thehig hwithin-perso n variability. To assess the effect ofvariatio n inmotion-picture s onRM R and DIT,tw oextrem e types ofmotion-picture s were compared, the romantic-family typeversu s the horror type. DIT increased significantly onhorro r films compared to romantic-family type of films.Thi seffec twa sdu e toa synergistic effect ofmea l ingestion and exposure tohorro r motion-pictures. The results suggest that part of the within-person variation indiet-induce d thermogenesis could have been related tovariatio n among the repeated measurements inth e type of motion-picture thatwa s shown to the subjects.Unde r normal measurement circumstances, however,variatio n inmotion-picture swa sno ta sextrem e asapplie d inth e study described inchapte r 6. The synergistic interaction between the exposure toa stressor and food ingestion onDI T isdifficul t toexplain .Th e results of chapter 5sugges t the existence of facultative diet-induced thermogenesis after food ingestionunde r exposure toa relatively mild stressor. From the absence of aneffec t ofpsychologica l stresso n fasting energy expenditure, it mayb e concluded that food triggers sympathetic nervous system activity(22) , and that additional exposure toa stressor may enhance thisproces s and thereby facultative thermogenesis.

-236- AD 2

Theexten tan dnatur eo f inter-individualvariatio n inRM Ran dDI Twer e determined ina grou po f 154obes e andnonobes epersons . An importantoutcom e of this studywa s the lack ofa systematic difference betweennon-obes e and obese females inth eefficienc yo fenerg yutilization , either in fedo r fasted conditions.Thi sconfirm s recent reports (4,17), but is incontradictio nwit h reportso fvariou sothe rgroup s (3,23-25).W e haven o explanation for thisdiscrepancy .A s stated before,th epresen t studyha d sufficient power todetec teve n relativelymino rdifference s inDI Tbetwee n thenon-obes e and theobes e females.Sinc en oobes eme nwer e studied in this thesis, inferenceso ndifference s inDI Tbetwee n obeseme nan dnon-obes eme n shouldb edon e cautiously. A surprising findingwa s the lower DITo fme n compared tQ_women.Aroun d 25 percent of the thermogenic response tofoo d inme nwa sunaccounte d forb y the energy expended fornutrien toxidatio nan d retention.Thi s indicates thatme n havea facultative thermogenic response of significantmagnitud e after mixed meal ingestion. Do theseobservation s fit inwit h the 'model'o n thenatur eo fDIT ?Me nha d in particular higher postprandial glucoseoxidatio n rateswhe ncompare d towomen . Postprandial glucose oxidation islikel yt ob ea functiono f theamoun to f carbohydrates ingested and subsequentlydigested ,th e insulin response to ingested carbohydratesan d themas so fglucose-metabolizin g cells.Unde r normal resting conditionsafte r carbohydrates ingestion, insulindetermine s largely the intensity of cellular glucosemetabolism . Ihypothetiz e that the difference inDI Tbetwee n the sexes is related toa differenc e in insulin response.Me nma yhav ea relatively larger insulin response,ie ,characterize d by relativelylarg epea kheight so rpostprandia l surfaceareas .A higher insulin response could increase the cellularmetabolis mo fglucos e and, in addition,activat e the sympathetic nervous system (22).Thi sthesi soffer sn o hard data totes t thishypothesis . A difference ininsuli n response between menan dwome n couldpartl yb ea functiono fdifference s inth e rateo f digestionan d absorptiono f carbohydrates.A_highe r rateo f stomach emptying isexpecte d to result ina n increased fluxo f carbohydrates into the small .intestine, resulting ina highe r absorption rateo f carbohydrates,and , consequently, ina highe r insulin response.Thi shypothesi s could be tested by monitoring andvaryin g the rateo f stomach emptying in response todifferen t meals inme nan dwomen .I tpredict s that,a tgive nora ldose so f

-237- carbohydrates,increase d rateso f stomachemptyin g and carbohydrate absorption will result ina highe r DIT.Whe n thishypothesi s canno tb e refuted, itwoul d indicate thatth emode lo f thecurren t theoryo nDI Tshoul db eexpande d by incorporating inth emode l ofDI Tth e rateo fnutrien tdigestio nan d absorption asa determinan t ofDIT . A second surprising findingwa s the significantly lowerRM Rpe runi tweigh t of fat-freemas s inme nwhe ncompare d towomen .Thi sdoe sno tnecessaril y indicate adifferenc e inbasi cenergy-demandin g processesa t the cellular levelbetwee n the sexesa sexplaine d inchapte r 2.Me nma yhav e a relative hypertrophy of fat-free tissueswit h lowmetaboli c activity compared towomen . A combinationo faccurat e inviv o imagingtechnique s forassessin g body composition,direc t or indirectmacrocalorimetr yan dmicrocalorimetr yma y elucidate theprecis e nature ofou r finding. Whereas thedat a onRM R indicate thatme nar emor e efficientuser s of energy compared towomen ,th e resultso f_the_ppstprandia lenerg y expenditure measurements suggest theopposite .A similar anomalybetwee n trained and untrained malesha s recentlybee npublishe d by Poehlmane t al.(26) . The clinicallymos t relevant findingo f the studiesdescribe d inchapter s 7 and 8wa s theobservatio n thatnon-abdomina l obesewome nha d relatively lower RMR'swhe n compared toabdomina l obesewomen ,youn g leanmen ,youn gwome n and non-obesewome nmatche d inage .Th e impacto f thetyp eo fbod y fat distribution onmetaboli c complications ofoverweigh t and obesity is currently the object of intense investigations (27).A relativepredominanc e of fat in theabdomina l regionha s conclusivelybee n shownt ob eassociate d with an increased risk onmetaboli c complications.Whethe r the type ofbod y fat distributionaffect senerg yexpenditur ewa sunknow nwhe nw e started our studies.Th e resultso fou r studies suggest thatth e typeo fbod y fat distributionmigh tb ea predicto ro f long-termbod yweigh tgain . Thequestio nho w tointerprèt eth e relativelylowere d RMR'samon g the non-abdominal obese isintrigueing .Di d these lowered rateso f energy expenditure contribute toth eobesit y inthes ewome no r are theysecondar y to theobes e state?Onl yprospectiv e studieswit hpre-obes eperson s subdivided according totyp e ofbod y fatdistributio n andaccordin g to rateo f energy expenditure cangiv ea definit e answer tothi squestion . Ithink ,however , that the reduced rateso fenerg yexpenditur e among thenon-abdomina l obese contributed tothei r obesity rather thanbein g causedb y it.Th e arguments for thisopinio nar ebase d on three observations.Firstly , thenon-abdomina l obese women, inparticula r thegluteal-femora lobese ,reporte d a childhood

-238- (prepubertal)histor yo fobesity ,wherea s theabdomina l obese reported an adult-onsetobesity .Th eassociatio no fa nearl yonse to fobesit y inth e non-abdominal obese and theoccurrence ,late r inadulthood ,o f reduced rates ofenerg yexpenditur e suggestsa causa l relationshipbetwee nth e reduced rates of energyexpenditur e and theobesity . Secondly, ina nearlie r reporto fou r group (28)i twa sobserve d thatdurin gweigh t lossRMR' s reduced significantly inabdomina l obesewome nbu tno t innon-abdomina l obesewomen .RMR' s inth e non-abdominal obesedi dno tadap tt oth e chronically reduced energyintake . This indicates that the reducedRMR' s inth enon-abdomina l obesedi d contribute toth eobes e state.Thirdly ,th eabdomina l obese,i ncontras t to thenon-abdomina l obese,ar e frequentlycharacterize d byhyperinsulinemi aan d hypertension (27).Insuli nma y increase the sympathetic nervous system activity (22).A n increased sympatheticnervou s systemactivit yma y increase thermogenesis, forexample ,i nprocesse s contributing toRMR . Intha tcase , the relatively increased RMR's inth eabdomina l obese couldb eviewe d asa n adaptation inth eabdomina l obese toexcessiv eenerg y intake.Th e increased thermogenesiswoul d antagonize furtherweigh t gain.Thi smean stha t the relatively increasedRMR' s inth eabdomina l obesewoul d bea consequence of theobes e state,implyin g that the relatively reduced RMR's inth e non-abdominal obesehav eno tadapte d toth eobes e state,ie ,ar e constitutional.

When the reducedRM R inth enon-abdomina l obese are constitutional,coul d it affectoveral l energybalance ?Usin gth e resultso fchapte r 8,w e calculated thata surfeito fabou t 5percen to fdail y energy intake could occur inth e non-abdominal obesecompare d toth eabdomina lobese ,assumin gequa l energy intakes,degree s and intensitieso fphysica l activity inbot hgroups .A n energy retentiono f 5percen to ftota ldail yenerg y intake could certainly contribute toweight gain.I tshoul db e realized,however ,tha ta positive energybalanc e induceschange s inenerg yexpenditur e thataffec tbod yweigh t gain.Thi s implicates thata simple summation intim eo f thecalculate d daily energy surfeitsdivide db y theenerg yequivalen to fweight gai n (29)i sonl y useful for estimating themaximall ypossibl eweigh t gain.I fth e lowered RMR inth enon-abdomina lwome n isconstitutional ,then ,i tmigh tb epredicte d that inparticula r thesewome nwil l bemos t resistent toweight reduction. The typeo fbod y fatdistributio ndi d notaffec tDIT ,suggestin g thata (pre)pubertal history ofobesity ,associate dwit h the typeo fbod y fat distribution, isno ta n importantdeterminan t ofdietar y thermogenesis.

-239- AD 3

Inadditio nt oinvestigation so n the intra-an d inter-individualvariatio n in RMRan d DIT,studie swer e carried outt oasses s the impacto fvariou s non-nutritional andnutritiona l factorso n these components ofenerg y expenditure. Inchapte r 5i twa s shown thatprio r exercisedi dno thav ea significant1 0 hoursafter-effec t onRMR . Inthi s studyenerg yexpenditur ewa sno t continuouslymeasure d afterexercise .I ti spossibl e that theexercis e induced a significant after-effecto nRM R thatlaste d onlya relatively shorttime . The resultso f this studysugges t thatexercis ewoul d not inducea greater energy loss thanth e losstha t could be inferred fromcalculatio no f the crossproduct of theapplie dworkloa d andworkefficiency . Fromthi s studyw e mayconclud e thatexercis ewoul dprobabl yno tb eusefu la sa nadjuvan t for weight control in subjectsoccasionall y engaged in recreational physical activity. Surprisingly,however ,th e studydescribe d inchapte r 11 showed a significant 10-hoursafter-effec t ofexercis e onRMR .Th edifferenc e in resultsbetwee n these two studies isprobabl y related toth edifferenc e in exercise durationan d intensity. Theexercis eperforme d inth e study described in chapter 11wa s of theundul yheav y type.Afte r amaximu mwor k capacity test, subjectsha d tocycl e foranothe r 80minute sa tvaryin gpercentage so f theirmaximu mworkload .Th ecombinatio no fprolonged ,alternatin g intense and less intense exerciseprove d tob ea particula r effective stimuluso f resting metabolic rate.Suc hheav y rateso fexercis e are obviouslyno t frequently encountered under normal conditions.Thus ,th e resultso f chapter 11d ono t invalidate the conclusiono nth eefficac yo foccasiona l recreational acitivity forweigh t controlpurposes .

Another aspectwhic hwa s studied related todiurna lvariatio n inRMR .Garrow , an authority on studieso fenerg ybalanc e andobesit y inhumans ,points ou t thatdiurna lvariatio n inRM Rexist san d that itcoul d seriouslyaffec t the calculation ofDI T (29).A t close inspection it seemstha tGarro wbase shi s opiniono na single reference,th e classical paperb yAschof fan d Pohl, on rhytmicvariation s inenerg ymetabolis m (30).Thi spape r givesa figureo n oxygenuptak eo fa female subjectmeasure d in resting conditionsove r awhol e day in thermoneutral conditions.I tca nno tb e concluded fromthi spape r whether this subjectwa smeasure dunde r strict fastingcondition so r periodically fed. Iti sprematur e toconclud e from thisobservatio n that diurnalvariatio n inRM Rexists .Th e resultso f studiespresente d inchapte r 3

-240- and inchapte r 4clearl ysho wtha ttim eo f theda ydoe sno t significantly impacto nRM R andDIT .Th e resultso f these studiesar eno ta tal l spectacular,bu tthe yhav e importantmethodologica l consequences for the measurement ofRM R andDIT ,a sexplaine d inchapte r 3.

AD 4

Theeffect s onDI To fpalatability ,ethano l ingestion and short-term carbohydrate overfeedingwer e investigated totes tvariou shypothese so n the mechanisms ofpostprandia l energyexpenditure . Mealswit h relativelyhig hpalatabilit ywer eexpecte d to induce a relatively largepre-absorptive , ie,cephalic ,insuli n réponse,and ,consequently ,a higher post-absorptive insulin response.Thi swa s expected tocaus e enhanced storage of carbohydrates and,possibly ,activatio no f the sympathetic nervous system.Bot hprocesse s could lead toincrease dDIT .Thi shypothesi swa s not supported by the findingso fth e studypresente d inchapte r 10.N odifferenc e inDI Toccurre d betweenmeal so fdifferen tpalatability . Power calculations revealed that thedesig nuse d inth e studywa s sufficiently sensitive to detect awithin-perso n change inDI T in response totreatmen to f around 25 percent. Itseems ,therefore ,tha thumans ,i nshort-term ,d ono tprotec t themselvesagains t theundesirabl e effectso nenerg ybalanc e ofa n occasional overindulgence atdinner .Th e absence ofa significanteffec t of palatability onDI Twa s explained byassumin g thathighl yunpalatabl emeal sevok ea stress reaction,tha t increases facultative thermogenesis.Evidenc e for thisnotio n comes from the studyo n the impacto fpsychologica l stresso nDIT .I nth e palatability study twomea l typeswer e compared,a palatabl eversu sa highl y unpalatable meal. The resultso f this studyd ono tpreclud e the possibility that the thermic response tofoo ddiffer sbetwee npalatabl emeal san d ordinary meals. The impacto fethano l onDI Twa sassesse d ina studydescribe d inchapte r 9. Ethanol ingested ina moderat e dosis induced a significant thermiceffect . According toth e literature (32)th e impacto fethano l onenerg y expenditure observed in this thesis issimila r to glucose-o r fat-induced thermogenesis. We did notcompar e the impacto f iso-energetic loadso f thethre e individual macronutrientso nRM R totha to fethanol .Ethano l ingestion immediately prior tomea l ingestion (aperitif)di dno tpotentiat e DIT incompariso nwit h an equalamoun to fcalorie sgive n ina mixe dmea l form.Again ,th eethano l was not compared for itseffect so nDI Twit heac h individualmacronutrient . Itwa s

-241- expected that ethanol ingestionwoul d increase nutrient storage and thereby DIT,no t bya neffec t on insulin response,but ,simply ,becaus e ethanol would act asa n energy source incellula r metabolism, substituting for fat,glucos e orproteins .Evidenc e forthi smechanis m came fromth eobserve d increase in DITafte r ethanol ingestion inth e second and third hour of the postprandial period. Overall,however ,ethano l ingestion did notpotentiat e DIT. This thesis doesno toffe r an explanation for theparadoxica l finding in observational studies that ethanol isusuall y added on topo f thediet ,bu t that itsus e isno t associated with increased levels ofadiposit y (33-37). Three comments should bemade . First,equalit y ofbod ymas s index (weight 2 (kg)/height (m))betwee n drinkers and abstainers ofethano l doesno t mean equality ofmetabolicall y 'active'mas s or,a s acorollary , equality of body fat stores (38).Secondly , ethanol usemigh t covarywit h other lifestyle factors affecting energy expenditure, ie,smoking , coffee consumption, sleeping behavior (39)o rphysica l activity. Ideally, observational studies should adjust the relationship between ethanol intake andbod yweigh t for these potential confounders.Thirdly ,chroni c ethanol use could induce thermogenic processes,fo rexample ,b yeffect s on the autonomic nervous system (40). Finally, inferring about the chronic effects ofa stimulususin g results of acute effects of that stimulus should always bedon e cautiously. Ina last studydescribe d inchapte r 11,th e impactwa s assessed onRM R and DITo fundul y heavy previous exercise and short-term carbohydrate overfeeding. Itwa sexpecte d thatcarbohydrat e overfeedingwoul d result ina n increase of DIT. Thiswa s indeed found; carbohydrate overfeeding increased DIT significantly. The effect of carbohydrate overfeeding onDI Twa s increased by prior exercise.N o effect of carbohydrate overfeeding onRM Rwa s observed. Unduly prior heavy exercise significantly affected RMR and DIT. Surprisingly, the carbohydrate overfeeding reduced the impact ofprio r exercise on RMR. These findings suggest thatpar t of the after-effect ofexercis e on energy expenditure is related to replenishing thebody' s glycogen stores.Suc h a processwoul d be relatively diminished after previous carbohydrate loading. The study described inChapte r 11provide s evidence for theexistenc e of facultative, ie,adaptiv e thermogenesis.Th e increased DIT reduced the energy surfeit caused by the carbohydrate overfeeding. The response after exercise doesno t seem to indicate an adaptive responsewit h respect to restoring energybalance .Bot h stimulus and response tend todecreas e thebody' s energy stores.Thi s indicates that after heavy exercise, restoring the original steady state inth ebody' s glycogen stores iso f greater importance in

-242- comparison tobalancin g short-term fluctuations inth ebody' senerg ystores . Although this studygive sevidenc e for theexistenc eo f the facultative component ofDIT ,i ti sdifficul t toasses s from thisstud y the contribution tooveral l DITo fneuronally-mediate d stimulationo f facultative thermogenic processesand , forexample ,o fa n increased turnover between themobilisatio n and storage formso fa nutrient . General textbook knowledge states that ingested carbohydratesar e oxydized or converted to fat (41).I nth ecarbohydrat e overfeeding study itwa s shown that,eve nafte r carbohydrateoverfeeding ,ne t transformationo fglucos e into fatwa so fnegligibl e importance forth enon-oxidativ e storageo f carbohydrates.Thi sconfirm spreviou sfinding s (42,43)an d supports Björntörp'san dSjöström' sspeculation san dquantitativ e considerationswit h regard tocarbohydrat e storage inme n (44).Onl yafte rmassiv e overfeeding with carbohydratesd enov olipogenesi sbecome sa quantitativel y important pathway for storing ingested carbohydrates (45).I nsuc hcondition sdail y energyexpenditur e increasesmarkedly ,largely ,du e toth eenerg y expended in converting carbohydrates tofa t(45) .

Finally,doe s thewor k described inthi sthesi scontribut e toth e solvingo f someo f themajo r problemso fth e studyo fhuma nenerg yexchange ?

This thesis showstha t individualdifference s in restingenerg y expenditure exist,eve nafte raccountin g statistically fordifference s inage ,bod yweigh t andbod y composition.Whethe r these inter-individual differences are associated withdifference s inth edegre e or intensityo fhabitua l physical activity amongou r subjectsca nno tb e inferred fromth e studiesdescribe d in thisthesis .Secondly ,afte r accounting fordifference sage ,i nbod yweigh t andbod ycompositipn ,thi sthesi sshow s that simple personalcharacteristics , ie, sexan d typeo fbod y fatdistributio n inobesity ,ar e still related to inter-individual differences in restingenerg yexpenditure . Short-termmanipulatio n ofth eplan e ofnutrition ,suc ha s carbohydrate overfeeding,wa s shown toinduc e some formo fa protectiv e response inDI T againstenerg y overnutrition.Whethe r this responsewoul db eo f significance in totaldail yenerg yexpenditure ,and ,whethe r achroni cdietar ymanipulatio n would inducesuc ha response,ca nno tb e concluded fromth e resultso f this thesis.Thes equestion s remainunanswered . Thewor k described inthi sthesi s showstha tobes ewome nwit ha relative predominance ofbod y fato nbuttock san dhip s (non-abdominal typeo f fat

-243- distribution)ar echaracterize db ya relativel y reduced RMR.Ou rstudie sd o not allowt odra wa definit e conclusiono nth eimportanc eo fthi s findingfo r the pathogenesiso fnon-abdomina l obesity. Theyd osugges t that studying energy expenditure inhuma n obesity inrelatio nt obod y fatdistributio nma y prove,tob evaluabl e forunderstandin g thenatur eo fhuma n obesity. This thesis also shows thatth eorigi no fDI Ti sno texclusivel y determinedb y the postprandial processingo fingeste d nutients.Som e simple non-nutritional factors,i nparticula r psychological stress,se xo fa subjec tan dprio r exercise,ca nsignificantl y affectth epostprandia l rise inenerg y expenditure.T oaccoun tfo rth eimpac to fthes e factors,a revised 'model'o f the origino fDI Ti spresente d inFigur e1 .

PRE-ABSORPTIVE NON-NUTRITIONAL FOOD/NUTRIENTS RESPONSES STIMULI

INGESTION - DIGESTION ABSORPTION TRANSPORT

INSULIN < Ç—- OXIDATION —i 44 STORAGE i SNS .,^ — ACTIVITY i i i OBLIGATORY FACULTATIVE OBLIGATORY THERMOGENESIS THERMOGENESIS THERMOGENESIS

DIET-INDUCED THERMOGENESIS

Figure1 .Revise d 'model'o forigi no fdiet-induce d thermogenesis, incorporating theimpac to fnon-nutritiona l factors.Thic k lines indicate established contributions, interrupted lines indicate contributions stilli n doubt (SNS= sympatheti c nervous system).

-244- Non-nutritional factorswer e shown toaffec tdietar y thermogenesis. The precise mechanism of these effects could not be elucidated from thewor k described inthi s thesis.Tentatively , Istat e that the effects of these factors onDI Tcoul d have beenmediate d through pre-absorptive influences on thepost-absorptiv e insulin response,th e rateo fdigestio n and absorption of nutrients, inparticula r of carbohydrates, the routing of ingested nutrients over storage and immediate oxidation, the turnover between storage and mobilisation formso f nutrients and, finally, through adirec t or indirect, ie, through stimulation of the activity of the sympathetic nervous system, stimulatory effect on the rateo f ionpumpin g or substrate cycli.Mor e research isneede d toasses s the contribution of thesevariou s processes to overall DIT.

CONCLUSIONS

This thesis shows that intra-individualvariatio n in diet-induced thermogenesis is relatively large.Thi s finding is important inth edesig n of studies that aime to compare diet-induced thermogenesis between different groups or toquantif y within-person responses ofdiet-induce d thermogenesis to a thermogenic stimulus.Diet-induce d thermogenesis showed a large inter- individual variation,bu tn o evidence wasobtaine d that, inparticular , the obesewoul d have ablunte d thermogenic response to food.Me nha d a significantly higher diet-induced thermogenesis thanwome ndid . Iti s hypothetized that this is related toa differenc e between the sexes in the postabsorptive insulin response,probabl y mediated bya differenc e in carbohydrate digestion and absorption rate.Me nwer e found tohav e lower resting metabolic ratespe runi tweigh t of fat-free masswhe n compared to women.Thi s could indicate ahighe r effiency of fasting energyutilizatio n in men.Alternatively , this finding could be related toa relative hypertrophy in men of components of the fat-free masswit h relatively lowmetaboli c activity. Obesewome nwit h anon-abdomina l typeo fbod y fatdistributio n were found to have reduced resting metabolic rates compared toyoun g non-obese men,youn g non-obese women,age-matche d non-obesewome n and incompariso nwit h obese womenwit h an abdominal type ofbod y fatdistribution . It ishypothetize d that the reduced resting metabolic rates inth enon-abdomina l obesewome n are causally related to their obesity. To test thishypothesi s it isnecessar y to measure energy expenditure ingroup s ofobes ewomen , subdivided according to

-245- type ofbod y fatdistribution ,before ,durin gan d afterweigh t loss,and ,t o compare theirdat a tothos eo fnon-obes econtrols . Thewithin-perso n responseso f restingmetaboli c ratean d diet-induced thermogenesis tovariou s stimuliwer eassessed .Restin gmetaboli c ratewa s not systematically affected bypsychologica l stress,tim eo f theda yo r by moderate toheav ypreviou s exercisebouts .Undul yheav ypreviou s exercise bouts induced a significant rise in restingmetaboli c rate.Moderat e ingestion ofethano l hada significanteffec t on restingmetaboli c rate.However ,thi s effectdi d not indicatea reduced efficiency ofenerg yutilizatio n after ethanol ingestion.Diet-induce d thermogenesiswa s significantly elevated by psychological stress,previou sundul yheav y exercisebout san db y short-term carbohydrate overfeeding. Palatability of fooddi d notaffec t diet-induced thermogenesis.Moderat e prior ethanol ingestiondi dno tpotentiat e subsequent diet-induced thermogenesis. It isconclude d that these results support a role fornon-nutritiona l factors inth eorigi no fdiet-induce d thermogenesis.

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1. SimsEAH ,Danfort h E.Expenditur e and storage ofenerg y inman .J Clin Invest1987;79:1019-25 . 2. LiuK , Stander J, DyerA ,McKeeve r J, McKeever P. Statistical methods to assess andminimiz e the roleo f intra-individualvariabilit y in obscuring the relationshipbetwee ndietar y lipidsan d serumcholesterol .J ChronDi s 1978;31:399-418. 3. Jéquier E.Doe sa thermogenic defectpla ya role inth epathogenesi so f humanobesity ?Cli nPhysio l1983;3:1-7 . 4. Ravussin E,Lillioj a S,Anderso n TE,Christi nL ,Bogardu sC . Determinants of 24-hour energyexpenditur e inman .Method san d resultsusin g a respiration chamber.J Cli n Invest1986;78:1568-78 . 5. Strang JM,McClugag e HB.Th e specific dynamic action of food inabnorma l states ofnutrition .A m JMe d Sei1931;182:49-81 . 6. Bradfield RB,Jorda nMH .Relativ e importance of specific dynamic action in weight-reduction diets.Lance t1973;2:640-3. " 7. ShariefNN ,MacDonal d I.Difference s indietary-induce d thermogenesis with various carbohydrates innorma l andoverweigh tmen .A m JCli nNut r 1982;35:267-72. 8. Nair KS,Hallida yD ,Garro wJS .Thermi c response to isoenergetic protein, carbohydrate or fatmeal s inlea nan dobes e subjects.Cli nSe i 1983;65:307-12. 9. Blaza S,Garro wJS .Thermogeni c response totemperature ,exercis e and food stimuli inlea nan d obesewomen ,studie d by 24h direc t calorietry.B rJ Nutr1983;49:171-80 . 10. SchwartzRS ,Ravussi nE ,Massar iM ,O'Connel l M,Robbin sDC .Th e thermic effect of carbohydrateversu s fatfeedin g inman .Metabolis m 1985;34:285-93. 11.Anton-Kuchl yB ,Lava lM ,Choukrou nML ,Mancie tG ,Roge r P,Varen e P. Postprandial thermogenesis andhormona l release inlea nan d obese subjects.J Physio l (Paris)1985;80:321-9 . -246- 12.D'Alessi oDA ,Kavl eEC ,Mozzol iMA ,Smalle yKJ ,Polansk yM ,Kendric kZV , OwenLR ,Bushma nMC ,Bode nG ,Owe nOE .Thermi ceffec to ffoo di nlea nan d obesemen .J Cli nInves t1988;81:1781-9 . 13.Zahorska-Markiewic zB .Thermi ceffec to ffoo dan dexercis ei nobesity .Eu r JApp lPhysio l1980;44:231-5 . 14.Felbe rJP ,Meye rHU ,Curcho dB ,Iseli nHU ,Rousell eJ ,Maede rE ,Pahu dP , JéquierE .Glucos estorag ean doxidatio ni ndifferen tdegree so fhuma n obesitymeasure db ycontinuou sindirec tcalorimetry .Diabetologi a 1981;20:39-44. 15.Well eSL ,Campbel lRG .Norma lthermi ceffec to fglucos ei nobes ewomen .A m JCli nNut r1983;37:87-92 . 16.Feli gP ,Cunningha mJ ,Levit tM ,Hendle rR ,Nade lE .Energ yexpenditur ei n obesityi nfastin gan dpostprandia lstate .A mJ Physio l1983;244:E45-51 . 17.Owe nOE ,Kavl eE ,Owe nRS ,Polansk yM ,Capri oS ,Mozzol iMA ,Kendric kZV , BushmanMC ,Bode nG .A reappraisalo fcalori crequirement si nhealth y women.A mJ Cli nNut r1986;44:1-19 . 18.Bogardu sC ,Lillioj aS ,Ravussi nE ,Abbot tW ,Zawadzk iJK ,Youn gA , KnowlerWC ,Jacobowit zR ,Mol lPP .Familia ldependenc eo fth erestin g metabolicrate .N Eng lJ Me d1986;315:96-100 . 19.Murgatroy dPR ,Davie sHL ,Prentic eAM .Intra-individua lvariabilit yan d measurementnois ei nestimate so fenerg yexpenditur eb ywhol ebod y indirectcalorimetry .B rJ Nut r1987;58:347-56 . 20.Soare sMJ ,Shett yPS .Intra-individua lvariation si nrestin gmetaboli c rateso fhuma nsubjects .Hu mNutr:Cli nNut r1986;40C:365-9 . 21.Mahadev aK ,Passmor eR ,Wool fB .Individua lvariation si nmetaboli ccos t ofstandardize dexercises :effect so ffood ,age ,se xan drace .J Physio l (London)1953,-121:225-31 . 22.Landsber gL ,Youn gJB .Th erol eo fth esympatheti cnervou ssyste man d catecholaminesi nth eregulatio no fenerg ymetabolism .A mJ Cli nNut r 1983;38:1018-24. 23.Sega lKR ,Guti nB ,Alb uJ ,Pi-Sunye rFX .Thermi ceffect so ffoo dan d exercisei nlea nan dobes eme no fsimila rlea nbod ymass .A mJ Physio l 1987;252:EllO-7. 24.Shett yPS ,Jun gRT ,Jame sWPT .Reduce ddietary-induce dthermogenesi si n obesesubject-befor ean dafte rweigh tloss .Pro cNut rSo c1979;38:87 A (abstract). 25.Schwart zRS ,Halte rJB ,Bierma nEC .Reduce dthermi ceffec to ffeedin gi n obesity:rol eo fnorepinephrine .Metabolis m1983;32:114-7 . 26.Poehlma nET ,Melb yCL ,Badyla kSF .Restin gmetaboli crat ean dpostprandia l thermogenesisi nhighl ytraine dan duntraine dmales .A mJ Cli nNut r 1988;47:793-8. 27.Björntör pP .Adipos etissu edistributio nan dmorbidity .In :Berr yEM , BlondheimSH ,Eliaho uHE ,Shafri rE ,eds .Recen tadvance si nobesit y research:V .London :Joh nLibbey ,1986:60-85 . 28.De nBeste nC ,Vansan tG ,Weststrat eJA ,Deurenber gP .Restin gmetaboli c ratean ddiet-induce dthermogenesi si nabdomina lan dgluteal-femora lobes e womenbefor ean dafte rweigh treduction .A mJ Cli nNut r1988;47:840-7 . 29.Garro wJS .Energ ybalanc ean dobesit yi nman .2n ded .Amsterdam : Elsevier/NorthHollan dBiomedica lPress ,1978;96-7 . 30.Aschof fJ ,Poh lH .Rhythmi cvariation si nenerg ymetabolism .Fe dPro c 1970;29:1541-52. 31.LeBlan cJ ,Bronde lL .Rol eo fpalatabilit yo nmeal-induce d thermogenesis inhuma nsubjects .A mJ Physio l1985;248:E333-6 . 32.Jéquie rE .Thermogenesi sinduce db ynutrien tadministratio ni nman . Infusionstherapie198 4;11:184-8 . 33.Fishe rMA ,Gordo nT .Th erelatio no fdrinkin gan dsmokin ghabit st odiet : thelipi dResearc hClinic sPrevalenc estudy .A mJ Cli nNut r 1985;41:623-30. -247- 34.Hiller sVN ,Masse yLK .Interrelationship so fmoderat ean dhig halcoho l consumptionwit hdie tan dhealt hstatus .A mJ Cli nNut r1985;41:356-62 . 35.Beb bHT ,House rHB ,Witsch iJC ,Littel lAS ,Fulle rRK .Calori ean d nutrientcontributio no falcoholi cbeverage st oth eusua ldiet so f15 5 adults.A mJ Cli nNut r1971;24:1042-52 . 36.Jone sBR ,Barrett-Conno rE ,Criqu iMH ,Holdbroo kMJ .A communit ystud yo f caloriean dnutrien tintak ei ndrinker san dnondrinker so falcohol .A mJ ClinNut r1982;35:135-9 . 37.Grucho wHW ,Sobocinsk iKA ,Barboria kJJ ,Schelle rJG .Alcoho lconsumption , nutrientintak ean drelativ ebod yweigh tamon gU Sadults .A mJ Cli nNut r 1985;42:289-95. 38.Weststrat eJA ,Deurenber gP .Methode nte rbepalin gva nd e lichaamssamenstellingva nd emens .Dee lIII .Indirect emethoden .Voedin g 1988;49:206-14. 39.Baeck eJAH ,Burem aJ ,Frijter sJER ,Hautvas tJGAJ ,va nde rWiel-Wetzel s WAM.Obesit yi nyoun gDutc hadults :II ,dail ylife-styl ean dbod ymas s index.In tJ Obe s1983;7:13-24 . 40.Johnso nRH ,Eisenhofe rG ,Lambi eDG .Th eeffect so facut ean dchroni c ingestiono fethano lo nth eautonomi cnervou ssystem .Dru gAl eDe p 1986;18:319-28. 41.Davidso nS ,Passmor eR ,Broc kJF ,Truswel lAS .Huma nnutritio nan d dietetics.7t hed .Edinburgh :Churchil lLivingstone ,1979:30-31 . 42.Acheso nKJ ,Schut zY ,Ravussi nE ,Jéquie rE ,Flat tJP .Nutritiona l influenceso nlipogenesi san dthermogenesi safte ra carbohydrat emeal .A m J Physiol1984;246:E62-70 . 43.Acheso nKJ ,Flat tJP ,Jéquie rE .Glycoge nsynthesi sversu slipogenesi s aftera 50 0gra mcarbohydrat emea li nman .Metabolis m1982;31:1234-40 . 44.Björntör pP ,Sjöströ mL .Carbohydrat estorag ei nman :speculation san d somequantitativ econsiderations .Metabolis m1978;27:1853-65 . 45.Acheso nKJ ,Schut zY ,Bessar dT ,Anatharama nK ,Flat tJP ,Jéquie rE . Glycogenstorag ecapacit yan dd enov olipogenesi sdurin gmassiv e carbohydrateoverfeeding .A mJ Cli nNut r1988;48:240-7 .

-248- Summary

This thesiscontain s studies inhuman so n individualdifference s in resting metabolic rate (RMR)an ddiet-induce d thermogenesis (DIT),an do n the impact ofvariou snutritiona l andnon-nutritiona l factorso nRM Ran dDIT . RMR and DITaccoun t forbetwee n 70-85percen to f totaldail y energy expenditure inth eaverag e individual.RM R isth eenerg yexpenditur e ofa postabsorptive subject in resting conditions.DI T isth e increase inenerg y expenditure, relative toRMR , ina resting individual after food ingestion. Interest for studying RMRand , inparticular ,DI Taros ea t the suggestion that obese personswoul d havea relatively loweredDI Twhe ncompare d to lean persons.A loweredDI Twa s considered tob e caused bya n increased 'efficiency'o fenerg ymetabolism , contributing toth epathogenesi s of obesity.Opinion so n this issueare ,however ,divergent . Froma differen tpoin to fvie w interest inRM R andDI Tha sals obee n expressed. Itha sbee n suggested that individuals could reduce energyus e throughRM R orDIT ,fo rexample ,durin g energyundernutritio n ordurin g increased energy requirements.Suc ha reductionwoul d result ina mor e 'efficient'energ ymetabolism , resulting indecrease d energy requirements. Little isknow nabou t thenatur e andmagnitud e of individualdifference s in healthyadul t subjectso fRM Ran d DITan do n the impacto fnutritiona l and non-nutritional factorso nRM R andDIT .Th e objective of the studiesdescribe d inthi sthesi s ist oprovid ene wknowledg e on thesematters .

Chapter 1give sa n introduction toth e field of studyan d states thequestion s thatwer e investigated inthi sthesis . InChapte r 2a reviewi sgive no faspect so f the studyo fhuma nenerg y exchange and their relation toproblem so fenerg ybalance .Th e components and determinants of energyexpenditur e arediscussed .Th e control of food (energy) intake is reviewed aswel l asth e control of energybalanc e in relation to energyunder -an dovernutrition . 'Models'o fRMR ,DIT ,th e control of food intake and energybalanc ear epresented . Chapter 3contain smethodologica l studieso n themeasuremen t inhuman s of RMR, DITan d fuelutilizatio nwit h aventilate d hood system. Itwa s found thatRM R measurements showed good reproducibility. DITmeasurement swer e less reproducible.Stric tcontro l over theanteceden tdie t seemed not to reduce the within-personvariabilit y ofDIT .Th e importance ofth e relatively low reproducibility for the studyo fDI T isdiscussed .N o systematic effect of the phase of themenstrua l cycle onRM Ran dDI Twa s observed.DI Twa s positively

-249- associated with theenerg yconten t ofth e testmeal. Formixe dmeal swit h an energy contentbetwee nabou t1.5-2. 5M J DITca nb eassesse dwit h sufficient accuracywithi n threehours . Chapter 4 reportso n the impacto f timeo f theda yo nth emeasuremen t ofRM R and DIT.N o significantdiurna lvariatio n inRM R andDI Twa s observed. Chapter 5contain s a study toasses swhethe r priormoderat e toheav y exercise boutsaffec tRM R in fasting subjects.N o significant 10-hoursafter-effec t of exercise onRM Rwa s found. Chapter 6describe sa studyt omeasur e the impacto fpsychologica l stresso n RMR andDIT .Psychologica l stressdi dno t systematically affectRMR . Surprisingly,psychologica l stress increasedDIT . Chapter 7 reportso n thenatur ean dmagnitud e of inter-individual variation in RMR and DIT inlea nan dobes e subjects.N o significantdifferenc e inDI Twa s found between leanan d obese female subjects.DI Twa s onaverag e 24percen t higher inme ncompare d towomen .Me nha da relatively reducedRM R compared to womenpe runi tweigh t of fat-freemass .Obes ewome nwit ha non-abdomina l type ofbod y fatdistributio n had significantly lower RMR's,adjuste d for body weight,bod y composition andage ,compare d tonon-obes eme nan dwomen ,bu t also, incompariso n toobes ewome nwit ha nabdomina l type ofbod y fat distribution. Chapter 8 reports indetai l on the impacto fbod y fatdistributio n on resting energyexpenditur e inobes ewomen .Thre e groupso fobes ewome nwer e studied. A groupwit h a gluteal-femoral typeo fbod y fatdistribution ,a grou pwit h an intermediate type ofbod y fatdistributio n anda grou pwit h a typical abdominal type ofbod y fatdistribution .A control groupo f age-matched non-obesewome nwa sals o studied.RMR' sadjuste d forbod yweigh t and body compositionwer e similar inth enon-abdomina l (gluteal-femoral/intermediate type ofbod y fatdistribution )obes ewomen .Adjuste d RMR's inth e non-obese and theabdomina l obesewome nwer eno t statistically different.Adjuste d RMR's of the latter groupswer e onaverag e 15percen t higher compared toth eRMR' s of thenon-abdomina l obesewomen .Th enon-abdomina l obese reported a relatively earlier onsetobesity . It isconclude d that the relatively reduced RMR's inth enon-abdomina l obesear eprobabl yo fpathogeneti c significance. The thermic effect ofethano l (alcohol)an d the impacto fethano l onDI Tar e described inChapte r 9. Ethanol induced a significant thermiceffect .N o effect of concentration onth e thermic effecto fethano lwa s found. Ingestion of ethanol didno tpotentiat e DITwhe ncompare d toa n iso-energetic mixed meal.

-250- The impacto f thepalatabilit y of foodo nDI Twa s evaluated ina study described inChapte r 10.Difference s inpalatabilit y amongotherwis e identical mixedmeal s or sucrose solutionsdi d notproduc e systematicdifference s in DIT. Chapter 11describe sa studyo nth e influenceso fprio rundul yheav yexercis e bouts and short-term carbohydrate overfeeding onRM Ran dDIT .Prio r exercise increased RMRan dDI T significantly. Carbohydrate overfeedingdi d not affect RMR,bu t raisedDI T significantly. The impacto fexercis e onRM Rwa s attenuated by carbohydrate overfeeding. Chapter 12contain s agenera ldiscussion .Th e resultso f thevariou s studies arediscussed ,amon gothe r things in relation toth eproblem s of energy balance and toa 'model'o fDIT .

-251- Samenvatting

Ditproefschrif tbeva t studiesbi jd emen snaa r individueleverschille ni n ruststofwisselinge nthermogee neffec tva nvoedin g (Engels,diet-induce d thermogenesis (DIT))alsmed enaa rd einvloe dva nverschillend evoedings -e n andersoortige factoreno pruststofwisselin ge nDIT . Ruststofwisselinge nDI Tdrage nbi jd egemiddeld emen svoo r 70-85procen tbi j aand etotal edagelijks e energie-afgifte.D eruststofwisselin g isd e energie-afgifteva nnuchter epersone ni nrust .DI Ti sd estijgin gva nd e energie-afgiftebi jee npersoo nn avoedselinnemin g tenopzicht eva nhe t nuchtere rustniveau. De interessevoo r onderzoek naard eruststofwisselin g en,me tname ,DI Tna m toen ad esuggestie ,da tvetzuchtig e personenee nverlaagd eDI Tzoude nhebbe n invergelijkin gme tmense nme tee nnormaa lvetpercentag e inhe tlichaam .Ee n verlaagdeDI Tzo uee ngevol g zijnva nee n'efficiëntere 'energiewisseling .Ee n efficiëntereenergiewisselin g zoubijdrage naa nhe tontstaa nva nvetzucht .D e meningen overdez e zaak lopenechte r bepaalduiteen . Vanee nander e invalshoek ise roo kinteress evoo r ruststofwisselinge nDIT . Zoi sgesuggereer dda tmense nhu nenergie-afgift e inruststofwisselin go fDI T zoudenkunne nverlagen ,bijvoorbeeld ,bi jondervoedin go fee nlangduri g verhoogde energiebehoefte.Ee ndergelijk everlagin g zouresultere ni nee n verlaagde energiebehoefte. Overd eaar de ngroott eva nindividuel everschille n inruststofwisselin ge n DITbi jgezond evolwassene n alsmede overd einvloe dva nvoedings -e n andersoortige factoreno pruststofwisselin ge nDI Ti sno gweini g bekend.He t doelva nd estudie si ndi tproefschrif t isove rdez e zakennieuw e kennist e verkrijgen. Hoofdstuk 1schet she talgemen e kaderwaarbinne nhe tonderzoe k valte nbeva t eenopsommin gva nd eonderzoeksvragen . Inhoofdstu k 2word tee noverzich t gegevenva naspecte nva nd estudi eva nd e energiewisselingbi jd emen se nhu nrelati eme tprobleme nva nd e energiebalans.D ecomponente ne ndeterminante nva nd eenergie-afgift e worden besproken.D eregulati eva nd evoedsel(energie)innemin gpasseer td erevu e alsmeded econtrol eva nd eenergiebalan si nrelati eto tonder -e novervoeding . 'Modellen'"va nd eruststofwisseling ,DIT ,d econtrol eva nd e voedsel(energie-)inneminge nd e energiebalansworde n gepresenteerd. Hoofdstuk 3beva t studies,di eingaa no pmethodologisch e aspectenva nmetinge n bijd emen sva nd eruststofwisseling ,DI Te nsubstraatgebrui kme tee n

-252- 'ventilated hood' systeem.Metinge nva nd e ruststofwisselingware n goed reproduceerbaar. Metingenva nDI Tvertoonde n een slechtere reproduceerbaarheid. Strikte controle opd evoedselopnam e leek geen noemenswaardig effect tehebbe n opd ebinnen-persoonsvariati e van DIT.He t belangva nd e relatief slechte reproduceerbaarheid vanDI Tmetinge n voor onderzoek naar DITword tuitgediept .D e faseva nd emenstruel e cyclus had geen significant effect op ruststofwisseling enDIT .DI Tvertoonde~ee n positieve samenhang metd eenergie-inhou d vand e testvoeding. Bijgebrui k van een gemengde testvoeding met eenenergie-inhou d van ongeveer 1.5-2.5M J kan DIT bijee nmeetduu r vandri euu rvoldoend e nauwkeurig gemetenworden . Hoofdstuk 4 rapporteert over de invloed vanhe t tijdstipva nd eda g opd e ruststofwisseling enDIT .Tusse n ochtendmetingen enmiddagmetinge nva n ruststofwisseling enDI Twer d geen systematisch verschil gevonden. Hoofdstuk 5beva tee n studie naar heteffec tva nvoorafgaand e inspannende lichamelijke aktiviteit op ruststofwisseling. Inspannende lichamelijke aktiviteit had na 10uu r geenaantoonbaa r effect opd e ruststofwisseling. Inhoofdstu k 6word t een studie beschreven naar de invloed van psychologische stress op ruststofwisseling enDIT .Psychologisch e stressha d geen invloed op de ruststofwisseling. Verassend genoegverhoogd e psychologische stressd eDIT . Hoofdstuk 7beva t een studie naar de aard engroott eva n individuele verschillen in ruststofwisseling enDI T invetzuchtig e en niet-vetzuchtige personen. DITverschild e niet significant tussenvetzuchtig e en niet-vetzuchtige personen.DI Twa s gemiddeld 24procen t hoger inmanne n in vergelijking metvrouwen .Manne n hadden invergelijkin g metvrouwe n gemiddeld een lagere ruststofwisseling per kilovetvrij emassa .Vetzuchtig e vrouwen met eenniet-abdominal e lichaamsvetverdeling hadden gemiddeld een lagere ruststofwisseling, ook na correctie voorverschille n in lichaamsgewicht, lichaamssamenstelling en leeftijd, invergelijkin g met niet-vetzuchtige personen,maa r ook invergelijkin gme tvetzuchtig e vrouwenme t een abdominale lichaamsvetverdeling. Hoofdstuk 8 rapporteert indetai l overd e invloed vand e lichaamsvetverdeling bijvrouwe nme tvetzuch t op ruststofwisseling enDIT .Dri e groepen vrouwen werden bestudeerd. Een groepme tee ngluteaal-femoraa l typevetverdeling , een groepme t een intermediair typevetverdelin g enee ngroe pme t een typisch abdominale vetverdeling. Een controle groepva nniet-obes e vrouwen, overeenkomend inleeftij d metd evetzuchtig e groep,wer d ook onderzocht.D e ruststofwisseling, gecorrigeerd voorverschille n inlichaamsgewich t en lichaamssamenstelling, was overeenkomstig bijd evrouwe nme t een

-253- niet-abdominalevetverdelin g (gluteaal-femoraal/intermediair type vetverdeling). De ruststofwisselingverschild e niet significant tussen niet-vetzuchtige vrouwen envrouwe nme tabdominal e vetzucht.Gemiddel d lagd e ruststofwisseling bijd eniet-abdominal e vetzuchtige groep 15procen t lager in vergelijking metd e andere groepen.D evetzuch t bijd eniet-abdominal e vrouwen was relatief op jongere leeftijd ontstaan.D e conclusie wordt getrokken dat de lagere ruststofwisseling vand evrouwe nme t een niet-abdominale lichaamsvetverdeling vanwaarschijnlij k pathogenetisch belangis . Het thermogeen effectva n ethanol (alcohol)e nd e invloed vanethano l opd e DITworde n beschreven inhoofdstu k 9. Ethanol had een significant thermogeen effect.He t thermogeen effectwer d niet beïnvloed doorverschille n in concentratie. Ethanol verhoogde deDI Tnie t invergelijkin g met een iso-energetische gemengde testvoeding. De invloed van aangenaamheid vanvoedse l opd eDI Twer d nagegaan inee n studie,di e inhoofdstu k 10 staatbeschreven .Verschille n in aangenaamheid tussenverde r identieke testmaaltijdeno f identieke suikeroplossingen leidden niet tot significante verschillen inDIT . Hoofdstuk 11beschrijf t een studie naar de invloed opd e ruststofwisseling en DITva nvoorafgaand e zeer zware lichamelijke inspannende aktiviteit enva n kortdurende overvoeding met koolhydraten. Zeer inspannende lichamelijke aktiviteit had indez e studie nogn a 10uu r een systematisch verhogend effect op ruststofwisseling enDIT .Overvoedin g metkoolhydrate n verhoogde de DIT, maar had geenduidelij k invloed opd e ruststofwisseling. Het effectva n de aktiviteit opd e ruststofwisseling wasgeringe r wanneer overvoeding met koolhydraten plaatsvond. Hoofdstuk 12beva t eenalgemen e discussie.D e resultaten vand e verschillende studiesworde nbesproken , onder andere in relatieme t problemen van de energiebalans en in relatie totee n 'model'voo rDIT .

-254- Curriculum vitae

JanAdriaa nWeststrat ewer d geboreno p2 3januar i 1959i nKrabbendijke .I n 1977behaald ehi jhe tgymnasium- Bdiplom aaa nd eChistelijk e ScholengemeenschapWalchere nt eMiddelburg .I ndatzelfd e jaarbego nhi jzij n studieaa nd etoenmalig e Landbouwhogeschool teWageningen . Inhe tstudiejaa r 1982/1983wer dee nstageperiod e doorgebrachtbi jhe tDun nClinica l Nutrition Centret eCambridg e inEngeland . Inseptembe r 1984behaald ehi jaa nd e LandbouwuniversiteitWageninge nhe tdiplom ava nlandbouwkundi g ingenieur ind e afstudeerrichting "HumaneVoeding "me tal shoofdvakken ,Human eVoedin ge n Toxicologie,e nal sbijvakken ,Wiskundig eStatistie ke nMethode ne nTechnieke n vanSociaal-Wetenschappelij k Onderzoek.Vana f januari 1985to tseptembe r198 8 washi jverbonde nal swetenschappelij k assistentaa nd eVakgroe p Humane Voedingva nd eLandbouwuniversiteit .Vana fmedi o198 5verrichtt ehi jonderzoe k naard eenergiewisselin ge nlichaamssamenstellin gbi jd emens .He tonderzoe k naard eenergiewisselin gbi jd emen s resulteerde inee nproefschrift .He t onderzoek naard elichaamssamenstellin g leiddeto t9 publicaties ,waarva n6 i n internationalevaktijdschriften .Vana f september 1988i shi jwerkzaa mal s voedingskundigebi jUnileve r ResearchLaboratoriu mi nVlaardingen .

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