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1 This is the pulishe version of pper pulishe in Physiologil Reports. Cittion for the originl pulishe pper (version of reor): Jensen, L., Gejl, K., Ørtenl, N., Nielsen, J., Beh, R. et l. (205) Crohyrte restrite reovery from long term enurne exerise oes not ffet gene responses involve in mitohonril iogenesis in highly trine thletes.. Physiologil Reports, 3(2) Aess to the pulishe version my require susription. N.B. When iting this work, ite the originl pulishe pper. Cretive Commons CC BY 3.0: Permnent link to this version:

2 ORIGINAL RESEARCH Physiologil Reports ISSN X Crohyrte restrite reovery from long term enurne exerise oes not ffet gene responses involve in mitohonril iogenesis in highly trine thletes Line Jensen,2, Ksper D. Gejl, Niels Ørtenl,3, Jko L. Nielsen, Rune D. Beh 4, Tois Nygr 5, Kent Shlin 6 & Ulrik Frnsen Institute of Sports Siene n Clinil Biomehnis, SDU Musle Reserh Cluster, University of Southern Denmrk, Oense, Denmrk 2 Institute of Clinil Reserh, Clinil Pthology, SDU Musle Reserh Cluster, University of Southern Denmrk, Oense, Denmrk 3 Deprtment of Helth Sienes, Sweish Winter Sports Reserh Centre, Mi Sween University, Ostersun, Sween 4 Deprtment of Orthopei Surgery, Oense University Hospitl, Oense, Denmrk 5 Deprtment of Orthopei Surgery, Rigshospitlet, Copenhgen, Denmrk 6 The Astrn Lortory of Work Physiology, GIH, The Sweish Shool of Sport n Helth Sienes, Stokholm, Sween Keywors seline mrna, glyogen epletion, PGC-, type fiers. Corresponene Ulrik Frnsen, Institute of Sports Siene n Clinil Biomehnis, SDU Musle Reserh Cluster, University of Southern Denmrk, Cmpusvej 55, 5230 Oense M, Denmrk. Tel: Fx: E-mil: Ufrnsen@helth.su.k Funing Informtion The stuy ws supporte y The Dnish Ministry of Culture, the Committee on Sports Reserh. Reeive: 7 August 204; Revise: 2 Otoer 204; Aepte: 6 Otoer 204 oi: 0.484/phy2.284 Physiol Rep, 3(2), 205, e284, oi: 0.484/phy2.284 Astrt The im ws to etermine if the metoli pttions, prtiulrly PGC- n ownstrem metoli genes were ffete y restriting following n enurne exerise out in trine enurne thletes. A seon im ws to ompre seline level of these genes to untrine. Elite enurne thletes (VO 2mx 66 2mLkg min, n = 5) omplete 4 h yling t ~56% VO 2mx. During the first 4 h reovery sujets were provie with either or only H 2 O n therefter oth groups reeive. Musle iopsies were ollete efore, fter, n 4 n 24 h fter exerise. Also, resting iopsies were ollete from untrine sujets (n = 8). Exerise erese glyogen y % (from to mmolkg w ) with no ifferene etween groups. Wheres 4 h of reovery with prtly replenishe glyogen, the H 2 O group remine t post exerise level; nevertheless, the gene ws not ifferent etween groups. Glyogen n most gene levels returne to seline y 24 h in oth n H 2 O. Bseline mrna of NRF-, COX-IV, GLUT4 n PPAR- gene trgets were higher in trine ompre to untrine. Aitionlly, the proportion of type I musle fiers positively orrelte with seline mrna for PGC-, TFAM, NRF-, COX-IV, PPAR-, n GLUT4 for oth trine n untrine. restrition uring reovery from glyogen epleting exerise oes not improve the mrna response of mrkers of mitohonril iogenesis. Further, seline gene of key metoli pthwys is higher in trine thn untrine. Introution Crohyrte () is n importnt fuel for skeletl musle oth uring (Bergstrom et l. 967) n following exerise (Bergstrom n Hultmn 967), n epletion of musle glyogen uring prolonge exerise is linke to ftigue n impirment of performne in trining n ompetition (Jos et l. 98). Within the lst ee, new onept of exerising with reue musle glyogen levels ( trin low ) hs een introue in orer to optimize the ption to trining (Pilegr et l. 2002). Exerising with low musle glyogen levels or restrition of intke uring or fter exerise my e wy of inresing trining inue trnsriptionl improvements in musle oxitive pity, whih n potentilly enhne thleti performne. This is n open ess rtile uner the terms of the Cretive Commons Attriution Liense, whih permits use, istriution n reproution in ny meium, provie the originl work is properly ite. Pge

3 Reovery from Glyogen Depleting Exerise L. Jensen et l. In the pioneering stuy y Hnsen n olleges (Hnsen et l. 2005) untrine (UT) men performe kiking-exerise in one leg every y, while the other leg ws exerise twie every other y, the seon out performe with low glyogen vilility. After 0 weeks of trining time to exhustion t 90% power output ws inrese in the twie--y leg only (294 vs. 3%, respetively). Similrly, trine men yling twie every seon y ompre to one every y for 3 weeks, showe inrese glyogen storge, ft oxition n mitohonril enzyme tivity, lthough no improvement in performne ws foun (Yeo et l. 2008). Only few stuies hve investigte the effet of restrition uring reovery. Pilegr et l. (2005) reporte ugmente tivtion of metoli genes following 75 min yling exerise omine with long term (>5 h) low reovery iet when ompre to ontrols. Mthi et l. foun no ifferenes etween high or low iet uring reovery from 2 h yling (Mthi et l. 2008) n Cluerton et l. supporte this fining with high or low iet uring the first 3 h fter 60 min yling (Cluerton et l. 2005). Thus, the effet of mnipultion uring reovery is urrently unertin. It is well-estlishe ft tht exerise inue hnges in gene trnsription n trnsltion regulte protein pttions through istint signling pthwys (Pilegr et l. 2000; Joseph et l. 2006). Consequently, repete enurne exerise les to pttions in oxitive pity, e.g. inrese in size, ensity n effiieny of mitohonri, improve metoli pthwys n inrese pillry ensity (Coffey n Hwley 2007). The importne of peroxisome prolifertor-tivte reeptor gmm o-tivtor -lph (PGC-) orhestrting key ownstrem events in this ontext is well reognize (Srpull 2002; Ojuk 2004; Olesen et l. 200). Both the regultion of mitohonril iogenesis n tivtion of PGC- hve een linke to exerise n vilility through severl pthwys. PGC- oorintes the of oth nuler n mitohonril genes y interting with severl trnsription ftors, inluing nuler respirtory ftor (NRF), whih in turn tivtes mitohonril trnsription ftor A (TFAM) (Srpull et l. 202). Although the role of PGC- in regulting multiple spets of metoli pttions to exerise is well estlishe, the moleulr mehnisms meiting the response re not fully unerstoo, prtiulrly in the highly trine (HT) (Lursen n Jenkins 2002). Yers of trining les to highly pte skeletl musle phenotype (Wittwer et l. 2004) n ility to optimize fuel expeniture (Erlenush et l. 2005), n seline mrna levels potentilly iret gene responses to exerise ifferently in HT. Most stuies evluting the effet of speifi exerising regimes re onute in UT or moertely trine sujets using short (< h), non-pplie exerising protools. After introuing the onepts of trin low n restrition uring reovery, it is importnt to investigte if the vilility/restrition moes introue similr effets in highly enurne trine sujets. Here we onut long term (4 h) enurne exerise in HT sujets refleting rel-life yling session in regrs to intensity n urtion, n exmine the importne of vilility on the of genes involve in metoli pttion. Further, seline levels of these key metoli ell signling pthwys hve not een ompre etween highly enurne trine n ontrols. The speifi im ws to etermine if the metoli pttions, prtiulrly PGC- n ownstrem metoli trgets were ffete y restriting uring the initil 4 h following n enurne exerise out in HT enurne thletes. Further, seline levels of these genes were ompre to untrine. We hypothesize tht: () HT thletes isply higher seline levels of key regultory mitohonril n metoli genes ompre to UT; (2) exhustive yling exerise inue n up-regultion of PGC- n ownstrem key regultory metoli genes in HT thletes; n (3) elye replenishment of glyogen stores uring post exerise reovery further up-regulte these genes. Experimentl Protool Sujets Fifteen helthy HT Dnish trithletes were inlue in the stuy (Tle ). All thletes ompete t ntionl n/or interntionl level. Six trithletes were urrent or former Dnish Ntionl Tem memers, n four were reently ple in the top three t the Europen or Worl Long Distne Trithlon Chmpionships. One to two weeks efore prtiipting in the stuy the sujets visite the experimentl fility to get fmilirize with the exerise protool n to etermine generl nthropometris, mximl oxygen uptke (VO 2mx ) n mximl hert rte (HR mx ). Eight UT ontrol sujets were reruite for purposes of ompring seline gene levels (Tle ), n i not prtiipte in the exerise protool. All sujets were inlue in the stuy fter informe written onsent. The stuy ws pprove y the lol ethil ommittee of the Region of Southern Denmrk (Projet-ID S ) n ws onute oring to the Delrtion of Helsinki. Exerise protool The HT sujets omplete single exerise session onsisting of 4 h of yling t n verge of 73 % of Pge 2

4 L. Jensen et l. Reovery from Glyogen Depleting Exerise Tle. Anthropometri, physiologil n trining hrteristis of prtiipnts. H 2 O HT ( + H 2 O) UT n Age (yers) Height (m) Boy Mss (kg) Boy Ft (%) History of elite trining (yers) None Trining volume (hweek ) VO 2mx (Lmin ) VO 2mx (mlkg min ) * Type I fires (%) * Type II fires (%) * Dt from, H 2 O n UT groups n poole t from ll HT re presente. *Signifintly ifferent from, P < Signifintly ifferent from HT, P < HR mx, (; 74 % n H 2 O; 7 0%) whih, estimte from the su-mximl test, equle pproximtely 56% of VO 2mx. Athletes were supplie with wter only (minimum of ml wterkg h ) to eplete musle glyogen stores uring this session. Athletes were rnomly ssigne to either wter (H 2 O group; n = 7) or provision ( group;.06 g kg h ; n = 8) formulte for optiml loing for the following 4 h reovery perio (Fig. A). Room temperture (~22 C) n humiity (~35%) were mintine stle uring the yling exerise. Sujets yle using personl shoes, pels, n iyles mounte on turo triners (Elite Crono Mg ElstoGel Triner, Fontniv, Itly) t self-selete ene. supplementtion Dietry intke ws ontrolle n orrespone to the reommentions from the Amerin College of Sports Meiine (Roriguez et l. 2009). Clorie intke ws lulte on sis of oy mss. Brekfst onsiste of rih foos (i.e. porrige ots, risins, skimme milk, ornge juie n energy r; 82 kjkg w). Following the prolonge exerise, the group reeive mel onsisting of pst, hiken, vegetles n everge (.07 g kgw h ) uring the initil 2 h of the reovery perio n everge n n energy r (.05 g kgw h ) uring the susequent 2 h of the reovery. The H 2 O group ws provie with wter only uring the initil 4 h of the reovery perio. The remining 20 h of reovery oth groups reeive stnrize -enrihe mels (: inner n rekfst; H 2 O: lunh, sportsr, inner n rekfst) equlizing the totl energy intke etween groups. The time elpse etween rekfst onsumption n olletion of pre n 24 h- iopsies ws.5 h on oth ys. Sujets reeive 264 kjkgw on the first experimentl y orresponing to 7.2 to 22.6 MJ (0 g kgw y ). Smple olletion Musle iopsies were ollete from m. vstus lterlis uner lol nestheti (% lioine; Amgros, Copenhgen, Denmrk) using 5 mm Bergstr om neele with sution. Smples were ollete efore (pre) n pproximtely 5 min fter the exerise out (post), s well s fter 4 h n 24 h of reovery (4, 24 h). For ontrols only the pre smples were ollete. For ll sujets the experimentl proeure ws initite in the morning oring to our stnrize protool. Prtiipnts were Brekfst Musle iopsy Musle iopsy Musle iopsy Musle iopsy 90 min reovery 4 h yling with 4 h reovery with or 20 h reovery with Figure. Exerise protool esigne for glyogen mnipultion uring reovery. Shemti illustrtion of the experimentl esign. All prtiipnts followe the sme proeure, prt from reeiving either wter (H 2 O) or rohyrte () uring the first 4 h of reovery. Pge 3

5 Reovery from Glyogen Depleting Exerise L. Jensen et l. instrute to voi physil tivity (24 h), lohol n ffeine (72 h) efore the experiment n were provie with stnrize iet uring the entire experimentl perio (24 h pre n post exerise). Biopsies were tken in the sme region n epth n re ws tken to voi mging effet of multiple iopsies y lternting legs n seprting inisions y ~5 m (Vissing et l. 2005). Of mg tissue ws exise, quikly issete from ft n onnetive tissue, ivie into multiple piees n either iretly frozen in liqui nitrogen or emee in TissueTek (Skur Finetek, Alphen n en Rijn, the Netherlns) n frozen in preoole isopentne. The smples were store t 80 C for further nlysis. MHC isoform nlysis Whole musle homogente n gel eletrophoresis ws use to etermine the MHC isoform omposition s previously esrie in (Slviti et l. 986) n moifie for humns (Anersen n Agr 2000). Briefly, musle iopsy smples were mnully homogenize in :0 volumes of ie-ol uffer (300 surose mmoll, EDTA mmoll,0mmoll NN3, 40 mmoll Tris-se n 40 mmoll L-histiine, ph 7.8) t 0 C, n susequently frozen in liqui nitrogen. For nlysis, homogentes were mixe with smple-uffer (0% glyerol, 5% 2-merptoethnol n 2.3% SDS, 62.5 mmoll Tris n 0.2% romophenollue, ph 6.8), hete t 00 C for 3 min n for eh three smples (9 mg, 6 mg n 22 mg protein) were loe on SDS-PAGE gel (6% polyrylmie). Gels were run t 80 V for t lest 60 h t 4 C n ns were stine with Coomssie. The gels were snne (Linosn 400 snner, Heielerg, Germny) n MHC ns quntifie ensitometrilly (Phoretix D, nonliner, Newstle, UK) oring to the loe protein mounts. Western lotting ws use to ientify MHC II (Sigm M 4276) with the protool Xell IITM (Invitrogen, Crls, CA). Musle glyogen ontent The t on musle glyogen ontent hve previously een pulishe (Gejl et l. 204). Musle glyogen ws etermine using spetrophotometry (Bekmn DU 650) (Pssonneu n Lowry 993). Freeze rie musle tissue ws oile in 3% w/v of moll HCL for 2.5 h efore it ws oole n entrifuge t 3500 g for 0 min t 4 C. One milliliter of regent solution ( moll tris-uffer, 00 mmoll ATP, moll MgCl 2, 00 mmoll NADP + n G-6-PDH) ws mixe with 40 ll of oile musle smple n the proess ws initite y ing 0 ll of ilute hexokinse. Asorne ws reore for 60 min n musle glyogen expresse s mmolkg w. The nlysis ws unsuessful in four of the musle iopsies, whih fore us to leve out these t points. Perioi i-shiff (PAS) stining with immunofluoresenes Setions were stine with PAS n immunofluoresene for myosin hevy hin I with proeure opte from Shrt et l. (2004). All inutions were rrie out t room temperture (9 22 C). Briefly, musle setions were fixe for 0 min in 4% formlehye with 0.05% Triton X-00, wshe n then trete with % perioi i (Sigm Alrih, Brøny, Denmrk) in wter for 5 min. The slies were inute with Shiff s regent (Sigm Alrih; ontining % prrosniline, 4% soium metisulphte n 0.25 moll hyrohlori i) for 5 min n wshe in wter for 0 min. For immunofluoresene stining, setions were wshe in Wsh Buffer (Dko, Glostrup, Denmrk) n loke in Protein Blok (Dko) efore inution with monolonl nti-myosin hevy hin I (MHC-I), :2000 (Sigm-Alrih) for h n susequent Alex-flour 488 onkey nti-mouse seonry ntioy, :500 for h. The slies were mounte with Pro- Long Gol Antife with pi (Life Tehnologies, Nerum, Denmrk). RNA extrtion n DNA synthesis Tissue smples were weighe n homogenize in tues ontining 0 ermi es n one siliium rystl using the MgnLyzer (Rohe, Hviovre, Denmrk). Susequent, totl RNA ws extrte using TRIzol Regent (Life Tehnologies) oring to the mnuftures iretions n RNA onentrtion ws mesure using the Nno Drop (ND000; Thermo Sientifi, Hviovre, Denmrk) returning 260/280 rtios ove.8 for ll smples. 500 ng of RNA ws onverte into omplementry DNA using High Cpity DNA Reverse Trnsription Kit (Applie Biosystems, Nerum, Denmrk). Rel-time reverse trnsriptse Polymerse Chin Retion Rel-time RT-PCR ws performe using TqMn Low Density Arrys ustom esigne with 46 genes n two ontrols. Eh port on the rs ws loe with DNA equivlent to 25 ng totl RNA mixe with 29 Gene Expression Mstermix n smples were run t 50 yles in uplites on the 7900 Sequene Detetion System (ll regents from Applie Biosystems). Dt were ollete n nlyze using SDS 2.4 softwre (Applie Biosystems). Tehnil uplites were evlute n smples Pge 4

6 L. Jensen et l. Reovery from Glyogen Depleting Exerise exlue when DC t >. Control genes were verifie using GeNorm softwre (Vnesompele et l. 2002) n t were expresse reltive to the referene genes GAPDH n RPLP0 using the qbse+ softwre (Biogzelle, Zwijnre, Belguim). The following TqMn ssys were use; PPARGCA-Hs006724_m, NRF-Hs006026_ m, TFAM-Hs _m, COX4I-Hs _m, PDK4-Hs003772_m, LPL-Hs _m, PPARA- Hs _m, UCP3-Hs006052_m, SLC2A4-Hs _m, RPLP0-Hs _m, GAPDH-Hs _m. PGC- protein Eight lm ryo-setions were fixe in 4% NBF, permelize in 0.5% triton 00-X, loke in protein loker (Dko) n inute with () PGC- lone (K-5, :200; Snt Cruz) (Russell et l. 2003); (2) PGC- together with monolonl nti-myosin hevy hin I (MHC-I, :0.000; Sigm-Alrih) or (3) PGC- together with monolonl nti-myosin hevy hin II (MHC-II, :0.000; Sigm-Alrih) over night. Susequent, setions were inute with Alex-flour 555 onkey nti-mouse n 488 got nti-rit seonry ntioies (Life Tehnologies), :000 for h n mounte with ProLong Gol Antife with pi (Life Tehnologies). Negtive ontrols leving out primry ntioies were inlue. Sttistil nlysis Before performing the sttistil nlysis, ll gene t were logrithmi trnsforme to insure norml istriution n re reporte in the rtile s geometri men SEM (k trnsforme vlues) of the. The remining t re presente s men SE. A twowy ANOVA (SigmStt, 3.5; Systt Softwre, Erkrth, Germny) ws performe etween time n tretment ( or H 2 O) for mrna trgets n glyogen followe y Stuent-Newmn-Keuls post ho test, when signifint intertion ws ientifie. Stuent s t-test ws use to ientify ifferenes in seline mrna, n Persons orreltion ws use for orreltions. Dt were onsiere signifint when P < Grphs were me employing GrphP Prism 5.0 (GrphP Softwre, In., L Joll, CA). Results Fier type istriution The HT thletes isplye n inrese numer of type I fiers ompre to the UT ( % vs %, P = 0.00) with onsequent lower proportion of type II fiers ( % vs %, Tle ). The fier type istriution in the n H 2 O groups were not ientil s h % type I fiers ompre to % in H 2 O(P = 0.00, Tle ). Bseline mrna Compring seline mrna s showe higher level of in HT sujets of nuler respirtory ftor (NRF-) n ytohrome oxise-iv (COX-IV) involve in mitohonril iogenesis (29 n 94%, respetively) (Fig. 2A). Bseline mrna of metoli trgets pyruvte ehyrogense kinse 4 (PDK4) ws lower (63%), while gluose trnsporter type 4 (GLUT4), n peroxisome prolifertor-tivte reeptor lph (PPAR) ws higher (63 n 44%, respetively) in HT ompre to the UT (Fig. 2B). When omining t from the HT n the UT perentge of type I fiers n seline mrna were positively orrelte for PGC-, TFAM, NRF, COX-IV, PPAR- n GLUT4 (P < 0.05, Fig. 3A F). A Reltive mrna seline B Reltive mrna seline HT UT Mitohonril iogenesis ** *** PGC NRF TFAM COX-IV * * Metolism PDK4 LPL PPAR UCP3 GLUT4 Figure 2. Bseline mrna levels of HT vs. UT. Expressions levels of trgets relte to mitohonril iogenesis (A) or metolism (B). Vlues re normlize to the referene genes GAPDH n RPLP0, n reporte s geometri mens SEM. HT; n = 5, UT; n = 8. Signifintly ifferent from UT; *(P < 0.05), **(P < 0.0), ***(P < 0.00). ** Pge 5

7 Reovery from Glyogen Depleting Exerise L. Jensen et l. A B C D E F Figure 3. Bseline mrna orreltes with perentge of type I fiers. Correltions etween iniviul perent of type I fiers n seline mrna (A F). H 2 O: white irle; : white squre; UT: lk ross. Vlues re normlize to the referene genes GAPDH n RPLP0. Glyogen Biohemil etermintion of musle glyogen is given in Tle 2. Perioi i-shiff stinings on trnsverse musle iopsy setions revele mrke reution in musle glyogen with epletion primrily of type I fiers (Fig. 4A). Aitionlly, solute hnges in musle glyogen from oth groups uring epletion (pre-post) n replenishment (post-24 h) orrelte positively with VO 2mx (r 2 = 0.52, P = n r 2 = 0.567, P = for epletion n repletion, respetively; Fig. 4B n C). Gene response to exerise Exerise inue hnges in mrna re epite in Fig. 5. PGC- mrna ws inrese fol immeitely fter the exerise out n remine elevte Tle 2. Musle glyogen levels. Totl musle glyogen onentrtions t pre n post exerise n fter 4 n 24 h of reovery expresse s mmolkg w. Note ifferent glyogen repletion rtes etween H 2 O n fter 4 h reovery. (mmolkg w ) H 2 O (mmolkg w ) Pre Post *** *** 4 h *** 264 3***, 24 h Signifintly ifferent from pre is inite y *** (P < 0.00), n y when signifintly ifferent from t orresponing time point (P = 0.002). for the first 4 h in reovery, while returning to seline levels y 24 h. Likewise, TFAM mrna showe signifint inrese post exerise n t 4 h, while Pge 6

8 L. Jensen et l. Reovery from Glyogen Depleting Exerise A B PAS MHCII Dpi C Δpre-post, Δpre-post, 200 r 2 = 0.52 P = Musle glyogen repletion 800 Δ musle glyogen (mmol kg w ) Δ musle glyogen (mmol kg w ) D Musle glyogen epletion 0 Δpost-24 h, Δpost-24 h, 600 r 2 = P = VO2mx (ml O2 kg w min ) VO2mx (ml O2 kg w min ) Figure 4. Seletive musle glyogen epletion. Doule immunohistohemil stinings show effetive glyogen epletion post exerise, primrily of type I fiers n restortion of glyogen fter 4 n 24 h of reovery. White fiers inite glyogen epletion (top), while type II fiers re lele green n nulei lue (ottom). Imges re from sujet in (A) n group (B). Sle r: 50 lm. Asolut hnges in musle glyogen orrelte with VO2mx for pre-post exerise epletion (y = 9.3x ; r2 = 0.523; P = ; n = 2) n post24 h repletion (y = 8.0x 755.9; r2 = ; P = ; n = 3) (C, D). NRF n COX-IV mrna showe signifint erese t those time points. All three genes returne to pre levels y 24 h (Fig. 5). A signifint inrese in mrna ws foun for PDK4 n lipoprotein lipse (LPL) post n 4 h fter exerise, while PDK4 returne to seline levels LPL ws Pge 7

9 Reovery from Glyogen Depleting Exerise L. Jensen et l. A Reltive mrna 00 0 PGC- α * * * * B Reltive mrna 0 TFAM * * * * C Reltive mrna 0 NRF- * * * D Reltive mrna 0 COX-IV * * * * E Reltive mrna 00 0 PDK4 * * * * F Reltive mrna 0 LPL * * * * * * G PPAR - α H UCP3 I 0 0 Reltive mrna * * Reltive mrna * * Reltive mrna 0 GLUT4 * * * * Figure 5. Mitohonril iogenesis n metoli mrna in n. Vlues re normlize to the referene genes GAPDH n RPLP0, expresse reltive to pre vlues, n reporte s geometri mens SEM, n = 5. Signifintly ifferent from pre is inite y *(P < 0.05). still inrese fter 24 h (Fig. 5). The exerise out i not give rise to n immeite effet on PPAR- or mitohonril unoupling protein 3 (UCP3) mrna post or t 4 h, lthough n inrese n erese, respetively ws foun fter 24 h. GLUT4 mrna ws slightly erese post exerise, ut returne to sl levels uring the reovery perio. Influene of vs. H 2 O on gene Supplementtion with uring 4 h of reovery i not inrese of ny of the mrna trgets fter 4 h ompre to H 2 O group (Fig. 5). PGC- protein Immunohistohemil evlution of PGC- protein showe inonsistent results (Fig. 6). Evlution of PGC- lone revele no positivity, wheres oule lelling with PGC- n MHC-I showe PGC--positive type I fiers, n oule lelling of PGC- n MHC-II showe PGC--positive type II fiers. Negtive ontrols showe no stining. Disussion This stuy is, to our knowlege, the first to investigte the effet of rohyrte intke versus elye replenishment of glyogen stores of more thn 2 h following long term enurne exerise on PGC- n ownstrem metoli gene trgets in HT thletes. The exerise protool inue trnsient in PGC- n severl ownstrem gene trgets; however restrition uring reovery i not inrese the gene response. Musle glyogen stores n gene s levels were ompletely reovere fter 24 h with n Pge 8

10 L. Jensen et l. Reovery from Glyogen Depleting Exerise A MHC-I B PGC-α C Overly D MHC-II E PGC-α F Overly G No ntioy H PGC-α I Overly Figure 6. Immunohistohemil evlution of PGC- protein. Seril setions from the 4 h time point re stine with PGC- n MHC-I (A C), PGC- n MHC-II (D F) or PGC- lone (G I). Sle r: 50 lm. without intke. These mjor finings emonstrte tht (I) elye replenishment of glyogen stores 4 h into reovery oes not ugment the of PGC- n ownstrem metoli gene trgets fter glyogen epleting exerise n (II) withholing the initition of refueling for s long s 4 h oes not ompromise musle glyogen ontent in HT thletes fter 24 h of reovery from long term enurne exerise. These oservtions n influene nutritionl strtegies in trining n ompetition of thletes tht for vrious resons nnot refuel immeitely, n thletes wishing to mnipulte their PGC- response shoul not o so y omining nutritionl n long term exerise strtegies. Bseline omprison HT vs. UT Generlly, the HT isplye n inrese numer of type I fiers, whih n e the result of nturl seletion of thletes with high proportion of type I fiers, ut lso reflet swith towrs fiers with more oxitive profile (Henriksson 992). Interestingly, we oserve tht perentge of type I fiers n seline mrna were positively orrelte, whih supports previous finings (Shmitt et l. 2003; Stepto et l. 2009). Similrly, PGC- mrna ws up-regulte in enurne thletes ompre to norml tive n spinl or injure (Krmer et l. 2006) suggesting type I fiers to proue n ontin more trnsripts potentilly ue to higher mitohonril ensity n seletive tivtion uring enurne exerise. The high sl levels of PPAR n GLUT4 n erese levels of PDK4 in HT thletes ompre to UT suggest tht HT possess highly effiient lipi n gluose metolisms. The fining tht the solute hnges in musle glyogen were positively orrelte with VO 2mx further unerlines the trining-inue pility of musle fing metoli hllenges. Plusile explntions for the vritions in glyogen storge pity oul e ifferenes in musle ontent of e.g. glut-4 protein (Hikner et l. 997), insulin signling (Wittwer et l. 2004) or musle loo flow (Eeling et l. 993) n ultimtely gluose istriution to the musles, ut the inrese proportion of type I fiers in HT might lso influene this fining. Exerise inue gene response The experimentl protool ws esigne to reflet regulr trining out n onsistent with previous stuies (Pilegr et l. 2003; Cluerton et l. 2005; Psilner et l. 203) we foun n inrese in PGC- mrna following exerise, whih returne to seline levels within 24 h of reovery. The protool le to n -fol inrese in PGC- immeitely fter the exerise out, whih is nlogous to the highest inrese reporte in the literture (Wtt et l. 2004; Wng et l. 2009; Hrer et l. 200), suggesting tht long term enurne exerise sessions impt the mitohonril iogenesis sustntilly in Pge 9

11 Reovery from Glyogen Depleting Exerise L. Jensen et l. HT. Furthermore, n ompnying inrese in TFAM inite iret tivtion of mitohonril gene n initition of mitohonril iogenesis. The exerise out le to mple inreses in oth PDK4 n LPL mrna, proposing swith from gluose to ftty i metolism in orer to spre glyogen. This shift hs previously een shown following glyogen epleting exerise in untrine (Shmitt et l. 2003; Pilegr n Neufer 2004), n ppers to e similr in the highly enurne trine musle. Influening PGC- response Following exerise, the musle glyogen levels were very low n withholing intke uring the initil reovery phse inue mrke ifferene in glyogen repletion rtes etween n H 2 O groups prompting potentil ifferene in vilility of irulting sustrtes etween groups (e.g. FFA/gluose, insulin, hormones). Nevertheless, in ontrst to our hypothesis we foun no ifferene in mrna etween n H 2 O. This is in line with other stuies fining no effet of restrition uring reovery from exerise (Cluerton et l. 2005; Mthi et l. 2008; Cohrn et l. 200). Differentiting etween the effets of low glyogen n exerising with low glyogen n e iffiult n might explin some of the ifferene oserve in stuies regring PGC-. In the present stuy low glyogen lone i not ugment PGC- n other stuies supports the nee for omining exerise with low glyogen (Hnsen et l. 2005; Yeo et l. 2008; Psilner et l. 203; Brtlett et l. 203) Durtion n intensity might lso influene the PGC- response. In the present stuy, it is likely tht the exerise urtion lone impts the skeletl musle to suh n extent tht susequent ietry interventions re inple of further tivting PGC-. Severl stuies investigting enurne exerise use protools of min of exerise, n sine PGC- inrese in n intensity-epenent mnner (Egn et l. 200), it is importnt tht the exerise protool reflets regulr work of group of enurne thletes. This is highlighte y the ility of short high intensity intervl trining (HIT) protools in inuing moerte PGC- responses (Little et l. 20). The present stuy ws esigne with the ustome trining regime of enurne thletes in min, s 4 h yling is prts of the weekly trining progrm. If the urtion/intensity-epenent tivte PGC- inrese inepenently of ietry intke, it is questionle if restrition will hve ny prtil effet in the trining regime of the highly enurne trine, who exerises up to 35 h per week. Timing of intke For yers thletes hve een vise to onsume s soon s possile post exerise, n elying the proess might impt the performne within the next 4 8 h (Ivy et l. 988). This stuy showe mrke ifferene in musle glyogen t 4 h reovery with the H 2 O group eing physilly ffete y eprive glyogen levels. However, fter 24 h these ifferenes vnishe suggesting tht restrition i not influene glyogen reovery, s long s suffiient energy ws onsume overll. A previous stuy le to similr onlusion fter refrining from for 2 h following exerise (Prkin et l. 997), n musle glyogen ontent 24 h fter exerise ws ientil etween sujets onsuming few lrge or numerous smll mels (Costill et l. 98; Burke et l. 996). At whih point etween 4 h n 24 h the H 2 O group thes up with the group will e of interest in regr to optimize thleti performne, ut nswers to this question nee to e resse in susequent stuies. Methoologil onsiertions This stuy ws limite in the numer of iopsies tht oul e ollete from eh sujet. In future experiments eluition of the mrna levels t time points etween 4 n 24 h n eyon woul e helpful in unerstning the effet of low reovery. The fier type nlysis revele ifferene in fier type istriution etween n H 2 O espite rnomizing the sujets to either group. Bse on our hypothesis we expete n ugmente inrese in PGC- in H 2 O upon the nutritionl intervention n ompnying metoli stress. The seline orreltion etween type fiers n PGC- inites potentil for higher PGC- in, ut suh ifferene ws not etete following ientil exerise stimulus in the two groups. Further, when normlizing mrna levels for eh sujet to the orresponing iniviul fier type istriution t every time point, it i not hnge the mrna-response, suggesting tht neither ifferent seline potentil nor fier type istriutions ffete the inrese in PGC-. Immunohostohemil evlution of PGC- protein ws unsuessful (Fig. 6) n highlights the prolems rising when trying to exmine of PGC- t the protein level, s no relile ntioies exist. However, generl onsensus exists tht PGC- is minly regulte t the trnsriptionl level (Olesen et l. 200), n mrna is, t lest for now, the only wy to evlute PGC- until new ntioies re evelope. Pge 0

12 L. Jensen et l. Reovery from Glyogen Depleting Exerise Conlusions In summry, the results from the present stuy emonstrte tht restrition uring the first 4 h following glyogen epleting enurne exerise i not influene the metoli gene, inluing PGC-. However, within 24 h ll sujets were le to reover to pre exerise levels in terms of glyogen storge n most mrna trgets, suggesting tht HT thletes possess the ility to reover reltively fst espite emning exerise session followe y longer perio of restrition. This hs importnt potentil for thleti nutritionl plnning in regrs to optimizing trining n ompetition outomes. Finlly, it is importnt for future reports to investigte fier type istriution mong the inlue sujets, s this stuy emonstrtes tht seline mrna vlues re orrelte with fier type istriution. This my e n importnt step unerstning exerise trining physiology n metoli se iseses suh s oesity n type 2 ietes. Conflits of Interest No onflits of interest, finnil or otherwise, re elre y the uthors. Referenes Anersen, J. L., n P. Agr Myosin hevy hin IIX overshoot in humn skeletl musle. Musle Nerve 23: Brtlett, J. D., J. Louhelinen, Z. Iql, A. J. Cohrn, M. J. Gil, W. Gregson, et l Reue rohyrte vilility enhnes exerise-inue p53 signling in humn skeletl musle: implitions for mitohonril iogenesis. Am. J. Physiol. Regul. Integr. Comp. Physiol. 304:R450 R458. Bergstrom, J., n E. Hultmn Synthesis of musle glyogen in mn fter gluose n frutose infusion. At Me. Sn. 82: Bergstrom, J., L. Hermnsen, E. Hultmn, n B. Sltin Diet, musle glyogen n physil performne. At Physiol. Sn. 7: Burke, L. M., G. R. Collier, P. G. Dvis, P. A. Friker, A. J. Snigorski, n M. Hrgreves Musle glyogen storge fter prolonge exerise: effet of the frequeny of rohyrte feeings. Am. J. Clin. Nutr. 64:5 9. Cluerton, L. J., S. L. MGee, R. M. Murphy, n M. Hrgreves Effet of rohyrte ingestion on exerise-inue ltertions in metoli gene. J. Appl. Physiol. 99: Cohrn, A. J., J. P. Little, M. A. Trnopolsky, n M. J. Gil Crohyrte feeing uring reovery lters the skeletl musle metoli response to repete sessions of high-intensity intervl exerise in humns. J. Appl. Physiol. 08: Coffey, V. G., n J. A. Hwley The moleulr ses of trining pttion. Sports Me. 37: Costill, D. L., W. M. Shermn, W. J. Fink, C. Mresh, M. Witten, n J. M. Miller. 98. The role of ietry rohyrtes in musle glyogen resynthesis fter strenuous running. Am. J. Clin. Nutr. 34: Eeling, P., R. Bourey, L. Kornyi, J. A. Tuominen, L. C. Groop, J. Henriksson, et l Mehnism of enhne insulin sensitivity in thletes. Inrese loo flow, musle gluose trnsport protein (GLUT-4) onentrtion, n glyogen synthse tivity. J. Clin. Invest. 92: Egn, B., B. P. Crson, P. M. Gri-Roves, A. V. Chilin, F. M. Srsfiel, N. Brron, et l Exerise intensityepenent regultion of peroxisome prolifertor-tivte reeptor otivtor- mrna unne is ssoite with ifferentil tivtion of upstrem signlling kinses in humn skeletl musle. J. Physiol. 588: Erlenush, M., M. Hu, K. Munoz, S. MConnie, n B. Stillwell Effet of high-ft or high-rohyrte iets on enurne exerise: met-nlysis. Int J Sport Nutr Exer. Met. 5: 4. Gejl, K. D., L. G. Hvi, U. Frnsen, K. Jensen, K. Shlin, n N. Ørtenl Musle glyogen ontent moifies SR C 2+ relese rte in elite enurne thletes. Me. Si. Sports Exer. 46: Hnsen, A. K., C. P. Fisher, P. Plomgr, J. L. Anersen, B. Sltin, n B. K. Peersen Skeletl musle pttion: trining twie every seon y vs. trining one ily. J. Appl. Physiol. 98: Hrer, M. P., A. R. Konopk, B. Jemiolo, S. W. Trppe, T. A. Trppe, n P. T. Reiy Musle protein synthesis n gene uring reovery from eroi exerise in the fste n fe sttes. Am. J. Physiol. Regul. Integr. Comp. Physiol. 299:R254 R262. Henriksson, J Effets of physil trining on the metolism of skeletl musle. Dietes Cre 5:70 7. Hikner, R. C., J. S. Fisher, P. A. Hnsen, S. B. Rette, C. M. Mier, M. J. Turner, et l Musle glyogen umultion fter enurne exerise in trine n untrine iniviuls. J. Appl. Physiol. 83: Ivy, J. L., A. L. Ktz, C. L. Cutler, W. M. Shermn, n E. F. Coyle Musle glyogen synthesis fter exerise: effet of time of rohyrte ingestion. J. Appl. Physiol. 64: Jos, I., P. Kiser, n P. Tesh. 98. Musle strength n ftigue fter seletive glyogen epletion in humn skeletl musle fiers. Eur. J. Appl. Physiol. Oup. Physiol. 46: Joseph, A. M., H. Pilegr, A. Litvintsev, L. Leik, n D. A. Hoo Control of gene n Pge

13 Reovery from Glyogen Depleting Exerise L. Jensen et l. mitohonril iogenesis in the musulr pttion to enurne exerise. Essys Biohem. 42:3 29. Krmer, D. K., M. Ahlsen, J. Norrom, E. Jnsson, N. Hjeltnes, T. Gustfsson, et l Humn skeletl musle fire type vritions orrelte with PPAR lph, PPAR elt n PGC- lph mrna. At Physiol. (Oxf.) 88: Lursen, P. B., n D. G. Jenkins The sientifi sis for high-intensity intervl trining: optimising trining progrmmes n mximising performne in highly trine enurne thletes. Sports Me. 32: Little, J. P., A. Sfr, D. Bishop, M. A. Trnopolsky, n M. J. Gil. 20. An ute out of high-intensity intervl trining inreses the nuler unne of PGC-lph n tivtes mitohonril iogenesis in humn skeletl musle. Am. J. Physiol. Regul. Integr. Comp. Physiol. 300:R303 R30. Mthi, A. S., A. Bonen, C. R. Benton, D. L. Roinson, n T. E. Grhm Rpi exerise-inue hnges in PGC-lph mrna n protein in humn skeletl musle. J. Appl. Physiol. 05: Ojuk, E. O Role of lium n AMP kinse in the regultion of mitohonril iogenesis n GLUT4 levels in musle. Pro. Nutr. So. 63: Olesen, J., K. Kiilerih, n H. Pilegr PGC-lphmeite pttions in skeletl musle. Pflugers Arh. 460: Prkin, J. A., M. F. Crey, I. K. Mrtin, L. Stojnovsk, n M. A. Ferio Musle glyogen storge following prolonge exerise: effet of timing of ingestion of high glyemi inex foo. Me. Si. Sports Exer. 29: Pssonneu, J. V., n O. H. Lowry Enzymti Anlysis, A Prtil Guie. Humn Press In, Totow, NJ. Pilegr, H., n P. D. Neufer Trnsriptionl regultion of pyruvte ehyrogense kinse 4 in skeletl musle uring n fter exerise. Pro. Nutr. So. 63: Pilegr, H., G. A. Orwy, B. Sltin, n P. D. Neufer Trnsriptionl regultion of gene in humn skeletl musle uring reovery from exerise. Am. J. Physiol. Enorinol. Met. 279:E806 E84. Pilegr, H., C. Keller, A. Steenserg, J. W. Helge, B. K. Peersen, B. Sltin, et l Influene of preexerise musle glyogen ontent on exerise-inue trnsriptionl regultion of metoli genes. J. Physiol. 54: Pilegr, H., B. Sltin, n P. D. Neufer Exerise inues trnsient trnsriptionl tivtion of the PGC- lph gene in humn skeletl musle. J. Physiol. 546: Pilegr, H., T. Os, L. T. Anersen, J. W. Helge, B. Sltin, n P. D. Neufer Sustrte vilility n trnsriptionl regultion of metoli genes in humn skeletl musle uring reovery from exerise. Metolism 54: Psilner, N., P. Frnk, M. Flokhrt, n K. Shlin Exerise with low glyogen inreses PGC-lph gene in humn skeletl musle. Eur. J. Appl. Physiol. 3: Roriguez, N. R., N. M. Di Mro, n S. Lngley Amerin College of Sports Meiine position stn. Nutrition n thleti performne. Me. Si. Sports Exer. 4: Russell, A. P., J. Feilhenfelt, S. Shreier, M. Prz, A. Crettenn, C. Goelet, et l Enurne trining in humns les to fier type-speifi inreses in levels of peroxisome prolifertor-tivte reeptor-gmm otivtor- n peroxisome prolifertor-tivte reeptor-lph in skeletl musle. Dietes 52: Slviti, G., R. Betto, D. Dnieli-Betto, E. Bisi, M. Seren, M. Mini, et l Myosin light hins n musle pthology. Neurology 36: Srpull, R. C Nuler tivtors n otivtors in mmmlin mitohonril iogenesis. Biohim. Biophys. At 576: 4. Srpull, R. C., R. B. Veg, n D. P. Kelly Trnsriptionl integrtion of mitohonril iogenesis. Trens Enorinol. Met. 23: Shrt, G., R. P. Hesselink, H. A. Keizer, G. vn Krnenurg, M. R. Drost, n M. K. Hesselink A moifie PAS stin omine with immunofluoresene for quntittive nlyses of glyogen in musle setions. Histohem. Cell Biol. 22:6 69. Shmitt, B., M. Fluk, J. Deomz, R. Kreis, C. Boesh, M. Wittwer, et l Trnsriptionl pttions of lipi metolism in tiilis nterior musle of enurne-trine thletes. Physiol. Genomis 5: Stepto, N. K., V. G. Coffey, A. L. Crey, A. P. Ponnmplm, B. J. Cnny, D. Powell, et l Glol gene in skeletl musle from well-trine strength n enurne thletes. Me. Si. Sports Exer. 4: Vnesompele, J., K. De Preter, F. Pttyn, B. Poppe, N. Vn Roy, A. De Pepe, et l Aurte normliztion of rel-time quntittive RT-PCR t y geometri verging of multiple internl ontrol genes. Genome Biol., 3: RESEARCH0034. Vissing, K., J. L. Anersen, n P. Shjerling Are exerise-inue genes inue y exerise? FASEB J. 9: Wng, L., N. Psilner, M. Tonkonogi, S. Ding, n K. Shlin Similr of oxitive genes fter intervl n ontinuous exerise. Me. Si. Sports Exer. 4: Wtt, M. J., R. J. Southgte, A. G. Holmes, n M. A. Ferio Suppression of plsm free ftty is upregultes peroxisome prolifertor-tivte reeptor (PPAR) lph n elt n PPAR otivtor lph in Pge 2

14 L. Jensen et l. Reovery from Glyogen Depleting Exerise humn skeletl musle, ut not lipi regultory genes. J. Mol. Enorinol. 33: Wittwer, M., R. Billeter, H. Hoppeler, n M. Fluk Regultory gene in skeletl musle of highly enurne-trine humns. At Physiol. Sn. 80: Yeo, W. K., C. D. Pton, A. P. Grnhm, L. M. Burke, A. L. Crey, n J. A. Hwley Skeletl musle pttion n performne responses to one y versus twie every seon y enurne trining regimens. J. Appl. Physiol. 05: Pge 3