Ian P. G. Amaral* and Ian A. Johnston. Scottish Oceans Institute, School of Biology, University of St Andrews, St Andrews, Fife KY16 8LB, UK

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1 2125 Th Journl o Exprimntl Biology 214, Pulish y Th Compny o Biologists Lt oi:1.1242/j RESEARCH ARTICLE Insulin-lik growth tor (IGF) signlling n gnom-wi trnsriptionl rgultion in st musl o zrish ollowing singl-stiting ml In P. G. Amrl* n In A. Johnston Sottish Ons Institut, Shool o Biology, Univrsity o St Anrws, St Anrws, Fi KY16 8LB, UK *Author or orrsponn (ipg@st-nrws..uk) Apt 8 Mrh 211 SUMMARY Ml zrish (Dnio rrio) wr st or 7 ys n to stition ovr 3 h to invstigt th trnsriptionl rsponss to singl ml. Th intstinl ontnt t stity (6.3% oy mss) rs y 5% t 3 h n 95% t 9 h ollowing oo withrwl. Phosphoryltion o th insulin-lik growth tor (IGF) signlling protin Akt pk within 3 h o ing n ws highly orrlt with gut ullnss. Rtin prlogus o IGF hormons gns wr rgult with ing, with ig1 showing pronoun pk in xprssion tr 3 h n ig2 tr 6 h. Ig-I rptor trnsripts wr mrkly lvt with sting, n rs to thir lowst lvls 45 min tr ing. ig1r trnsripts inrs mor quikly thn ig1r trnsripts s th gut mpti. Prlogus o th insulin-lik growth tor ining protins (IGFBPs) wr onstitutivly xprss, xpt or igp1 n igp1 trnsripts, whih wr signiintly lvt with sting. Gnom-wi trnsriptionl rsponss wr nlys using th Agilnt 44K oligonuloti mirorry n slt gns vlit y qpcr. Fsting ws ssoit with th uprgultion o gns or th uiquitin-protsom grtion pthwy, nti-prolirtiv n pro-poptoti gns. Protin hprons (un45, hsp1, hsp5, hsp9.1, hsp9.2) n hpron intrting protins (hs1 n stip1) wr uprgult 3 h tr ing long with gns or th initition o protin synthsis n mrna prossing. Trnsripts or th nzym ornithin roxyls 1 show th lrgst inrs with ing (11.5-ol) n wr positivly orrlt with gut ullnss. This stuy monstrts th st ntur o th trnsriptionl rsponss to ml n provis vin or irntil rgultion o rtin prlogus o IGF signlling pthwy gns. Supplmntry mtril vill onlin t Ky wors: tlost, myotoml musl, ish nutrition, growth, trnsriptomis. INTRODUCTION Growth hormon is synthsiz, stor n srt y spiliz lls in th ntrior pituitry n plys ntrl rol in ontrolling ing hviour, ll growth, osmorgultion n rproution in tlosts (Kwuhi n Sowr, 26). Growth hormon ts irtly on musl through srolmml rptors n inirtly vi th proution o insulin-lik growth tors (IGFs) in th livr n priphrl tissus, whih r rls into th irultion (Woo t l., 25). IGFs r lso prou y prrin pthwys n r stimult y mino i inlux into th musl (Bowr n Johnston, 21). In mmmls, th IGF systm ompriss tn omponnts: two hormons (IGF-I, IGF-II), two rptors (IGF-IR, IGF-IIR) n six ining protins (IGFBP1 6) (Dun t l., 21). IGFBPs hv istint physiologil rols in vlopmnt n rgult IGF rls to tissus in ssoition with spii protss (Dun t l., 21). Bining o IGF-I to its rptor tivts svrl ownstrm signlling ss inluing th P13K AKT TOR n MAP kins pthwys tht r wll onsrv in ish n mmmls (Engrt t l., 1996; Dun t l., 21). Ativtion o P13K AKT TOR stimults phosphoryltion s tht inrss trnsltion n protin synthsis (Engrt t l., 1996; Dun t l., 21) n inhiits protin grtion y th 26S protsom systm (Witt t l., 25). In th zrish (Dnio rrio), no wr thn 16 omponnts o th IGF systm hv n sri (Murs t l., 22; Murs n Dun, 22; Chn t l., 24; Zhou t l., 28; Wng t l., 29; Zou t l., 29; Di t l., 21). Th lrgr numr o IGF omponnts in zrish ompr with mmmls rlts whol gnom uplition (WGD) tht ourr t th s o tlost volution (Jillon t l., 24). It is thought 15% o th uplit gns or prlogus rom this sl WGD hv n rtin in xtnt spis (Jillon t l., 24). Th istint pttrns o tissu xprssion n trnsriptionl rgultion o mny IGF systm prlogus osrv in zrish (Murs t l., 22; Murs n Dun, 22; Chn t l., 24; Zhou t l., 28; Wng t l., 29; Zou t l., 29; Di t l., 21) is onsistnt with ithr suuntionliztion or nountionliztion o ths gns. Fsting-ring protools r ommonly us to invstigt trnsriptionl rgultion in th IGF-systm in tlosts ollowing th trnsition rom toli to noli stts (Chuvign t l., 23; Slm t l., 25; Gillr t l., 26; Rsn t l., 27; Bowr t l., 28). Fing to stition tr prolong st rsults in inrs ing intnsity rltiv to ontinuously ontrols n prio o ompnstory or th-up growth (Niiz n Mtl, 1997). Th trnsriptionl rsponss osrv in suh xprimnts r pnnt on th nutritionl stt o th ish prior to sting, prtiulrly th xtnt o t stors, th urtion o th st, oy siz n tmprtur (Johnston t l., 211). Fish show iurnl rhythms in ing hviour n tivity rivn y ntrl osilltors in th rin tht r synhronis y nvironmntl yls n o-orint with priphrl lok gns rgulting mtolism

2 2126 I. P. G. Amrl n I. A. Johnston (Dvi t l., 29). In quultur, ml tims ntrin iologil rhythms n ry physiologil systms in ntiiption or prossing th oo (Snhz t l., 29). As onsqun, grt r shoul tkn in signing sting-ing xprimnts in orr to in ll xprimntl vrils inluing th rquny n timing o ing in rltion to iurnl yls. Following th igstion n ssimiltion o ml, mtolism hngs rom n ovrll toli to n noli stt, utilizing th nutrints rom th ml to prou nrgy n synthsiz nw moluls, whih is hrtristi o th postprnil prio. To our knowlg thr is no stuy sriing th trnsriptionl hngs uring n ollowing postprnil prio in ish, with most stuis ousing on th hngs in mtoli rt (Clrk t l., 21; Vnll t l., 21) n th plsm lvl o mtolits ollowing ing (Ems t l., 21; Elison t l., 21; Woo t l., 21). W hv invstigt trnsriptionl rgultion in th st myotoml musl o ml zrish in rspons to singl stiting ml livr t irst light. Exprssion o ll 16 gns o th IGF systm ws invstigt y quntittiv polymrs hin rtion (qpcr) n supplmnt with gnom-wi survy o trnsript unn using th Agilnt 44K oligonuloti mirorry. Trnsript unn n th phosphoryltion o th signlling protin Akt wr trmin in rltion to th prsn o oo in th gut s rrn point. Th singl-ml xprimntl sign potntilly provis grtr tmporl rsolution or stuying trnsriptionl rsponss ompr with ontinuous ring whr rly n lt vnts quikly om onoun. Th ims o th stuy wr to tst th hypothsis tht prlogus o IGF-systm gns wr irntilly rgult with ing, n to isovr novl gns ssoit with th sting n stts in skltl musl. MATERIALS AND METHODS Fish n wtr qulity Th F5 gnrtion o wil-ught popultion o zrish [Dnio rrio (Hmilton 1822)] rom Mymnsingh, Bnglsh, ws us in this stuy. All ish wr ult mls g 9 months. Prior to th singl ml xprimnt th ish wr mintin in singl 5 litr tnk t 27.6±.4 C in 12 h:12 h light:rk photoprio. Th ish wr looworms (On Nutrition TM, Essn, Blgium) to stity twi ily or 1 wk. Nitrit ( p.p.m.), nitrt (1 2 p.p.m.), mmoni ( p.p.m.) n ph (7.6±.2) wr tst uring limtion n xprimntl prios using Frshwtr Mstr Tst Kit (Aqurium Phrmutils In., Chlont, PA, USA). Th singl ml xprimnt Two rplit xprimnts wr rri out, 3 months prt, with intil nvironmntl onitions n oo to ount or ny tnkto-tnk vrition in th ing rspons. Th xprimntl protool involv sting ish or 7 ys n thn ing singl ml o looworms livr ovr 3 h prio, tr whih ny untn oo ws rmov rom th tnk y siphoning. In th irst rplit xprimnt svn ish pr tim-point wr smpl n in th son rplit xprimnt six ish pr tim-point wr smpl t th ollowing tims: 156, 24, h (or th ml) n.75, 3, 6, 7.5, 9, 11, 24 n 36 h (tr th ml). Th vrg oy mss in th irst rplit xprimnt ws.46±.2 g n th stnr lngth (rom tip o snout to lst vrtr) o th ish ws 29.8±.4 mm (N 77), n in th son rplit xprimnt ws.53±.16 g n 32.6±.3 mm (N 66), rsptivly (mns ± s..m.). Fish wr kill humnly y n ovros o thyl 3-minonzot mthnsulphont slt (MS-222; Fluk, St Louis, MO, USA). Fst skltl musl ws isst rom th orsl pxil myotoms, lsh rozn in liqui nitrogn n stor t 8 C prior to totl RNA n protin xtrtion. Th igstiv trt ws isst n ix in 4% (m/v) prormlhy or ltr quntiition o intstin ontnt to th nrst milligrm. Fixtion ws nssry to prvnt tissu loss uring isstion n to hiv n urt quntiition o intstin ontnt. Bus th ntur o th tissu n oo wr th sm or ll smpls, ny shrinking us y ixtion shoul proportionl to th mount o mtril n ws not onsir in th intrprttion o th rsults. All xprimnts n niml hnling wr pprov y th Animl Wlr n Ethis Committ, Univrsity o St Anrws n onorm to UK Hom Oi guilins. Protin xtrtion Totl protin ws xtrt rom st skltl musl rom iv rnomly slt ish pr tim-point in h o th inpnnt xprimnts. Tissu (3 mg) ws homogniz in Lysing Mtrix D (MP Biomils, Qiogn, Irvin, CA, USA) in FstPrp mhin (Qiogn) using 35 l o 25 mmol l 1 MES (2-morpholinothnsuloni i monohyrt) ph 6. ontining 1 mol l 1 NCl,.25% (m/v) CHAPS, DNA/RNA nuls (Invitrogn, Crls, CA, USA) n prots inhiitor oktil (Invitrogn). Wstrn lotting Smpls (2 l, ontining 2 g o protin) wr to 6 l o solution ontining 5 l o 5 protin loing ur n 1 l 2 ruing gnt (Frmnts, Vilnius, Lithuni), ht or 5 min t 95 C, lo into NuPAGE Novx 4 12% Bis-Tris gls (Invitrogn) n ltrophors t 12 V. A protin lr rnging rom 1 to 25 kd (Frmnts) n rrn smpl wr lo in ll gls to stimt th molulr mss o protins o intrst n to srv s normliztion smpl, rsptivly. Protins sprt y ltrophorsis wr trnsrr to PVDF Immoilon-P Trnsr Mmrn (Millipor, Billri, MA, USA) t 25 V or 15 min. Sussul protin sprtion n trnsr wr onirm y Ponu S stining (Sigm, St Louis, MO, USA). PVDF mmrns wr lok ovrnight t 1 C using 5% (m/v) nont milk (AppliChm, Drmstt, Grmny) prpr in PBS (Sigm) ontining.1% (v/v) Twn 2 (Sigm). Blok mmrns wr inut ovrnight t 1 C with th ollowing primry ntiois (IgGs): P-Akt [1:1 ilution (v/v), Cll Signling #46, Dnvrs, MA, USA], Akt [1:1 (v/v), Cll Signling #2966], tin [1:2, (v/v), Sigm A266] n GAPDH [1:3, (v/v), Sigm G9545]. Pro mmrns wr inut t 2 C or 1 h with th sonry ntioy ginst mous or rit IgG onjugt to horsrish proxis [oth rom Sigm n us t 1:6, (v/v)]. Atr inution or 1 min with ECL Wstrn Blotting Dttion Rgnts (GE Hlthr, Amrshm, Bukinghmshir, UK) t room tmprtur, mmrns wr xpos to Hyprilm ECL (GE Hlthr). Exprimntl vritions in th ltrophorsis n trnsr wr normliz using rrn smpl ommon to ll mmrns. Th ol-hng in phosphoryltion o Akt t h tim-point ws ompr with th smpls rom 159 h. Furthr inormtion on prliminry optimistion xprimnts, th normlistion strtgy n tsts o P- Akt ntioy spiiity r givn in th Appnix Fig. A1. Totl RNA xtrtion rom skltl musl n irst strn DNA synthsis Totl RNA ws xtrt y homogniztion in Lysing Mtrix D (Qiogn) using 1 ml o TRI rgnt (Sigm) in FstPrp

3 Fing ts on trnsription 2127 Tim (h) Intstin Gut ullnss (%) Antrior rgion Postrior rgion mm Ml i h g Fig. 1. Th ing rspons o ml zrish uring th ours o th singl ml xprimnt. Fish tht h n twi ily wr smpl tr 9, 144 n 168 h o sting ( 156, 24 n h prior to th singl ml) n thn n xss o looworms. Furthr smpls wr tkn, 45 min, 3, 6, 7.5, 9, 11, 24 n 36 h tr th initition o ing. (Uppr pnl) Th miro-isstion o rprsnttiv intstin or h smpl point. Gut ullnss rh mximum tr 45 min, initing stity. (Lowr pnl) Th rltiv intstinl ontnt (prntg mximum ullnss) throughout th xprimnt, togthr with th light:rk yl. Vlus r mns ± s..m., N 13 ish pr smpl. Dirnt lttrs signiy sttistilly irnt mns (P<.5; s txt or tils) Tim (h) Light Drk Light Drk Light mhin (Qiogn). Th RNA onntrtion 26/28 n 26/23 rtios wr msur using NnoDrop sptrophotomtr (ThrmoFishr Sintii, Loughorough, Listrshir, UK) n wr n >2, rsptivly. RNA intgrity ws lso hk y gros gl ltrophorsis. A Quntitt Rvrs Trnsription kit (Qign, Hiln, Grmny) ws us to prou irst-strn DNA rom.6 g o totl RNA ollowing th mnuturr s instrutions. Mirorry xprimnts Mirorry xprimnts wr rri out y n Agilnt-rtii mirorry srvi provir (Mirorry Cntr, Univrsity Hlth Ntwork, Toronto, Cn) using th Dul-Mo Gn Exprssion Anlysis Pltorm (Agilnt Thnologis In., Snt Clr, CA, USA) in 4 44K sli ormt [Zrish (v2) Gn Exprssion Mirorry]. RNA rom th 3 h n 6 h tim-points wr hyriiz with th RNA rom th h smpl to intiy irntilly rgult gns in 7-y st ish ollowing singl stiting ml. Th 3 h smpl oini with 5% o mximum gut ullnss (Fig. 1). Six phnotypi rplits rom h group wr us. R, vrsion 2.9n., with rryqulitymtris_2.2. n limm_2.18. ws us or qulity nlysis o mirorry t. Mirorry rsults wr lso nlys using GnSpring, v7. Th intnsitis o spots mong rrys ws normlis using th AQuntil mtho n intnsitis wr log trnsorm or prorming t-tst using th Bnjmini n Hohrg mtho or multipl tsting orrtion (Bnjmini n Hohrg, 1995). A list o irntilly rgult gns ws prou y srning ginst th ollowing ritri: >twool hng in xprssion, B-vlu sttisti > n n just P-vlu <.5. Blst2GO ( sotwr ws us to nlys th gn ontology (GO) trms o th irntilly xprss gns. GO nrihmnt nlysis o th nnott gns ws prorm with th GOSSIP tool using Blst2GO sotwr. Th Europn Bioinormtis Institut Mim ArryExprss ssion numr or th mirorry xprimnt is E-MEXP Primr sign n loning Trnsript squns rom th Ensml ts (rls 55; wr us to sign primr pirs or h gn list in supplmntry mtril Tl S1. Primrs wr sign using NtPrimr ( inx.html; Prmir BioSot, Plo Alto, CA, USA). Whr possil th primr pirs wr sign so tht t lst on primr spnn n xon xon ounry (othrwis primrs pirs wr in irnt xons), with T m los to 6 C. First-strn DNA rom musl ws us s tmplt to synthsis PCR prouts or loning. Th PCR rtion mixtur ontin 1.5 mmol l 1 MgCl 2, 1 NH 4 ur,.25 l BioTq TM DNA polymrs (Biolin, Lonon, UK),.2 mmol l 1 NTP (Promg, Mison, WI, USA), 1 l DNA n.5 mol l 1 o h primr (Invitrogn) n th ollowing thrml yling onitions wr mploy: nturtion or 5 min t 95 C ollow y 36 yls o 3 s t 95 C, 3 s t 6 C n 3 s t 72 C, inl longtion or 5 min t 72 C. Atr gros gl ltrophorsis th prouts o xpt siz wr xtrt n purii using QIAquik Gl Extrtion Kit (Qign) n lon using StrtClon TM PCR Cloning Kit (Strtgn, L Joll, CA, USA) oring to th mnuturr s instrutions. Positiv lons wr pik tr 16 h o growth t 37 C in LB-gr plts ontining mpiillin (.1 mg ml 1 ) n trnsrr to 96-wlls plts ontining LB roth n mpiillin (.1 mg ml 1 ). Atr 16 h o growth t 37 C olony-pcr ws prorm to onirm th squn o th insrt using Big Dy trmintor squning (Appli Biosystms, Fostr City, CA, USA) t th Univrsity o Oxor (T3 n T7 primrs wr us to onirm th squn in oth irtions). Th lons ring plsmis with th xpt insrt wr grown in 5 ml LB roth n plsmis wr purii using QIAprp Spin Miniprp Kit (Qign). Th plsmi onntrtion, 26/28 n 26/23 rtios wr msur using NnoDrop instrumnt n wr highr thn 1.8 n 2., rsptivly. qpcr All protools n rporting o qpcr ssys hr to Minimum Inormtion or Pulition o Quntittiv Rl-Tim PCR

4 2128 I. P. G. Amrl n I. A. Johnston Exprimnts guilins (Bustin t l., 29). Th qpcr rtion mixtur ontin 7.5 l 2 Brillint II SYBR QPCR Low ROX Mstr Mix (Strtgn), 6 l 4-ol ilut DNA,.25 mol l 1 h primr n nuls-r wtr (Qign) to inl volum o 15 l in 96-wll plts (Strtgn). Th rtions wr prorm in Strtgn MX35P mhin (initil tivtion t 95 C or 1 min, ollow y 4 yls o 3 s t 95 C, 3 s t 6 C n 3 s t 72 C) n th luorsn rsults, ollt tr th longtion stp (72 C), wr ror using th MxPro sotwr v4.1 (Strtgn). Ngtiv ontrols wr inlu n run in uplit, n ontin ithr ll omponnts o th rvrs trnsription mixtur, xpt rvrs trnsripts (no rvrs trnsripts ontrol) or wtr inst o DNA tmplt (no tmplt ontrol). Atr th qpcr, issoition urv (rom 55 to 95 C) ws prou to vriy th prsn o singl pk. Th spiiity o h qpcr ssy ws lso vlit y squning trnsormnts or h qpcr prout. Asolut opy numr o h gn ws lult s on stnr urv o t lst six orrs o mgnitu, prpr with th plsmis. This t ws lso us to nlys th iiny o h primr pir (supplmntry mtril Tl S1). Th thrshol o luorsn (or Rn vlus) us or trmintion o th quntiition yl (C q ) ws st to 1. or ll plts to llow or omprison twn plts. Comprison twn plts ws possil tr normliztion to six smpls lo on ll plts. Six rrn gns slt rom th litrtur (1, rpl13, tin2, 2m, usp5, tp n yp1) n our slt rom th mirorry xprimnt (tomm2, rpl7, lmn2 n is3l2) wr nlys using Gnorm v3.5 (Vnsompl t l., 22) with M st to <1.5. Th two gns with th most stl lvl o xprssion ross th xprimntl onitions wr 1 n lmn2 (M.4). Th xprssion o gns o intrst ws normliz to th gomtri vrg o th two most stl gns n gn xprssion ws rport s ritrry units (.u.). Dt nlysis n sttistis All t wr nlys or norml istriution n qulity o vrin. Normlly istriut t wr nlys using ANOVA ollow y Tuky s post ho tsts using PASW Sttistis 18 (SPSS In., Chigo, IL, USA). Kruskl Wllis non-prmtri tsts ollow y Conovr post ho tsts wr us or th t tht wr not normlly istriut using BrightStt sotwr (Strikr, 28). Thr ws no sttistilly signiint irn in th stnr lngth, oy mss, intstin ontnt or normliz gn xprssion twn th two rplit xprimnts (P>.5). Thror, th rsults rom oth xprimnts wr omin to ilitt thir intrprttion, rsulting in N 13 pr tim-point (i.. svn ish rom th irst rplit xprimnt plus six rom th son rplit). In orr to omin th mrna xprssion t rom oth xprimnts, th rsults o gn xprssion rom th son rplit xprimnt wr normliz to th vrg vlu o svn smpls rom th irst rplit xprimnt, whih wr inlu rom th DNA synthsis stp onwrs. Corrltion o gn xprssion rom oth qpcr n mirorry xprimnts ws nlys y Sprmn s orrltion tst using PASW Sttistis 18 (SPSS In.). Clustring o gn xprssion ws prorm using PrmutMtrix ( RESULTS Fing rspons uring th singl ml xprimnt Fish wr ontinuously to stition n thn st or 7 ys prior to ing singl ml ovr 3 h. Thr smpls wr tkn uring th st: 156, 24 n h, orrsponing to 9 h, 144 h n 168 h tr th lst oo. Only trs o oo rmin in th gut tr 9 h o sting ( 156 h tim-point), orrsponing to.1% o oy mss, with only il osrv with mor prolong sting ( 24 n h timpoints). Th mximum vrg gut ullnss, quivlnt to 6.3% o oy mss, ws osrv t.75 h (45 min) tr oo m vill, initing stity h n rh. Intstinl ontnts h rs y 5% 3 h tr oo ws withrwn n 95% o th oo ingst h n ssimilt or xrt y 9 h (Fig. 1). Phosphoryltion o th IGF signlling protin Akt In st myotoml musl th protin Akt show mrk inrs in phosphoryltion to pk lvls within 3 h o th strt o ing n m stily phosphorylt s th intstin mpti (Fig. 2). Th lvl o th protin lo ontrols GADPH n tin n th phosphorylt Akt i not hng signiintly ovr th ours o th xprimnt. Trnsriptionl rgultion o th IGF systm Th xprssion o ll th prlogus o IGF-systm gns, inluing thos o Ig1, Ig2, Ig rptors n Ig ining protins, ws trmin y qpcr. In mny ss rtin prlogus o IGF systm omponnts show istint pttrns o trnsriptionl rgultion ovr th ours o th xprimnt. Chngs in trnsript lvls oul irtly ttriut to ing us mrk rsponss wr lrgly prsnt in only th irst o two light:rk yls ollowing th ml. IGF hormon gns ig1 xprssion ws orrlt with, ut lgg hin gut ullnss showing istint pk 3 h tr th strt o ing (P<.5; Fol-hng in phosphoryltion o Akt 159 h h h.75 h 3 h 6 h Tim (h) Light Drk Light Drk Light Atin Totl Akt P-Akt Fig. 2. Phosphoryltion o th insulin-lik growth tor (IGF) signlling protin Akt in th st myotoml musl o ml zrish uring th ours o th singl ml xprimnt. (Top) A rprsnttiv wstrn lot with th phosho-akt ntioy. (Bottom) Quntiition o th phosphoryltion o Akt (soli squrs n lk lin). Th gry lin rprsnts th vrg gut ullnss illustrt in Fig. 1. Vlus r mns ± s..m., N 5 ish pr smpl. Dirnt lttrs signiy sttistilly irnt mns (P<.5; s txt or tils). 9 h 12 h 24 h 36 h Gut ullnss (%)

5 Fing ts on trnsription 2129 ig1 gn xprssion (.u.) A Tim (h) Light Drk Light Drk Light 25 2 C Gut ullnss (%) ig2 gn xprssion (.u.) B Tim (h) Light Drk Light Drk Light Gut ullnss (%) Fig. 3. Trnsriptionl rsponss o th IGF systm gns in th st myotoml musl o ml zrish uring th ours o th singl ml xprimnt, trmin y qpcr. (A,B) Hormon trnsripts (soli squrs n lk lins) o Ig1 (A) n Ig2 (B). Th gry lin rprsnts th vrg gut ullnss illustrt in Fig. 1. (C) Copy numr o Ig1, Ig1, Ig2 n Ig2. Vlus r mns ± s..m., N 13 ish pr smpl. Dirnt lttrs signiy sttistilly irnt mns (P<.5; s txt or tils). Copy numr h 3 h 6 h h 3 h 6 h h 3 h 6 h ig1 ig1 ig2 ig2 Trnsript Fig. 3A). Th ig1 prlogu ws not tt in st myotoml musl tr 35 yls o PCR (Fig. 3C). ig2 xprssion ws lso inrs ollowing ing, showing pk xprssion 3 h ltr thn ig1 (Fig. 3B). Th ig2 prlogu ws 3.7 tims mor unnt thn th ig2 trnsripts t h (Fig. 3C) n ws onstitutivly xprss uring th xprimnt with no isrnil pttrn in rltion to ing (not shown). IGF rptor gns Th xprssion o th IGF rptor gn ig1r inrs mor thn 2.7-ol twn 9 h n 168 h o sting, rs to its lowst lvls 45 min tr ing n thn show vril, though still prss, xprssion ovr th nxt 36 h (Fig. 4A). ig1r prlogu xprssion ws invrsly rlt to gut ullnss showing mor thn 3.3-ol rs within 45 min o ing, rturning to lvls not signiintly irnt rom thos tr sting or only 12 h (Fig. 4B). ig1r trnsripts wr mor unnt thn ig1r trnsripts ovr th singl ml xprimnt, with 7.4 mor opis t th strt o ing (Fig. 4D). Trnsripts o th singl ig2r gn show no onsistnt hngs in xprssion ovr th sting ing sting yl (not shown). IGF ining protins gns Th two rtin prlogus o th IGF ining protin 1 gn (igp1 n igp1) show similr hngs in xprssion ovr th xprimnt, with high lvls uring prolong sting ( h), mrk rution in trnsript unn within 45 min o th strt o ing n vril, ut gnrlly low lvls ovr th ollowing 36 h (Fig. 4C). Exprssion o igp1 ws onsirly highr thn igp1 ovr th xprimnt, with 12.4 tims grtr opy numr t h (Fig. 4D). Th rmining IGF ining protin gns (igp2, igp2, igp3, igp5, igp5, igp6 n igp6) show no onsistnt hng in xprssion in rspons to singl stiting ml (not shown). Gnom-wi hngs in gn xprssion with ing In orr to intiy som nutritionlly rsponsiv nit gns or urthr invstigtion, whol gnom mirorry nlysis ws prorm. Totl RNA rom mximlly st ish ( h) ws hyriiz with RNA rom ish smpl 3 h n 6 h tr th initition o ing. Gns wr onsir irntilly rgult i thy show mor thn twool hng in xprssion, B vlu >, n th irn in xprssion ws signiint t P<.5, ollowing orrtion or multipl omprisons. Th hyriiztions o sting n 6 h smpls prou rltivly short list o gns tht wr lso rprsnt in th hyriiztion o sting n 3 h smpls n so wr not onsir urthr. Fst skltl musl rom zrish st or 7 ys h 56 uprgult gns (Tl 1) n 91 ownrgult gns (Tl 2) ompr with ish to stition ovr 3 h. 45 o th uprgult gns in sting n 79 o th uprgult gns with ing h ssoit gn ontology (GO) trms. For th gns uprgult with sting GO trm nlysis rvl signiint nrihmnt o trms ssoit with toli prosss, uiquitin ligs tivity n positiv rgultion o nothlil ll irntition, inluing th gns tg1 n tg2 tht hv nti-prolirtiv proprtis (Winklr, 21) (ull listing in Tl 3). Anlysis o th gns uprgult with ing show nrihmnt or GO trms suh s unol protin ining, protin oling, noplsmi rtiulum lumn, protin mturtion, hpron ining, sromrognsis, myosin ilmnt ssmly, ollgn iosynthsis n rgultion o th JAK-STAT s (ull listing in Tl 3). Th gn showing th lrgst ol hng o 32.2 with sting (Tl 1) o or novl protin with ~23% intity to th mmmlin orthologu o hringr trnsposs riv 1 (hri1), whih is thought to hv nuls tivity (Kpitonov n Jurk, 24). Th list o gns uprgult with ing inlu mny hpron gns (un45, ptgs3 n srpinh1), ht shok protin (hsp9.1, hsp9.2, hsp1 n hsp5) n ht shok protin-ssoit (hs1, lrl n stip1)

6 213 I. P. G. Amrl n I. A. Johnston ig1r gn xprssion (.u.) igp1 gn xprssion (.u.) Tim (h) Light Drk Light Drk Light A C Tim (h) Light Drk Light Drk Light Gut ullnss (%) Gut ullnss (%) ig1r gn xprssion (.u.) Copy numr B Tim (h) Light Drk Light Drk Light D 12 1 h 3 h 6 h h 3 h 6 h h 3 h 6 h h 3 h 6 h ig1r ig1r igp1 igp1 Trnsript Gut ullnss (%) Fig. 4. Trnsriptionl rsponss o Insulin-lik growth tor (IGF) systm gns in th st myotoml musl o ml zrish uring th ours o th singl ml xprimnt trmin y qpcr. (A C) IGF rptors ig1r (A) n ig1r (B) n IGF ining protin, igp1 (C; soli squrs n lk lins) Th gry lins rprsnt th vrg gut ullnss illustrt in Fig. 1. (D) Copy numr o ig1r, ig1r, igp1 n igp1. Vlus r mns ± s..m., N 13 ish pr smpl. Dirnt lttrs signiy sttistilly irnt mns (P<.5; s txt or tils). gns (Tl 2). Fing ws lso ssoit with nrihmnt o th intrlukin-2 rptor ining GO trm (Tl 3) n inrs il34 xprssion (Tl 2). Exprssion n lustring o nit nutritionlly rgult gns A sltion o nit nutritionlly rgult gns omprising ight gns uprgult with sting (ox32, zg:86757, kl11, nr11, it2, 3, zn653 n hs2) n six gns uprgult with ing (o1, kp5, s1, oxo1, hsp9.1 n hsp9.2) plus som ontrtil protin gns (mylz2 n tnni2.4) ws urthr invstigt using qpcr. Th log ol-hng in xprssion o ll th gns ssy y qpcr show goo orrltion with th mirorry xprimnt (R.79; P<.1; N 76; supplmntry mtril Fig. S1). Gns rom th IGF pthwy n thos slt rom th mirorry xprimnt orm iv mjor lustrs (Fig. 5). Clustr I ontin gns uprgult with ing, with low lvls o xprssion uring prolong sting ( 24 n h) n intrmit lvls o xprssion rom 9 to 36 h (Ig1, hsp9.1, o1, oxo1, igp6, Ig2, s1 n kp5). Clustr II ompris gns tht wr ownrgult t 159 h n rom.75 to 9 h, with uprgultion uring prolong ( 24 n h) n rly sting (12 36 h) (ig1r, ig2r, ig1r, xo32, zg:86757, kl11, 3 n zn653). Clustr III ontin gns tht wr only uprgult with prolong sting (144 n 168 h) n just tr th ginning o ing (45 min), with low xprssion t othr tim-points (tnni2.4, hsp9.2, nr11, igp1, hs2 n igp1). Clustr IV ompris gns with high xprssion uring prolong sting, low xprssion whil th intstin ws ull (.75 6 h) n intrmit lvls o xprssion rom 7.5 to 36 h (Ig2, igp3 n it2). Clustr V ompris gns with high xprssion with prolong sting, ut with intrmit lvl o xprssion uring ing n rly sting (mylz2, igp5, igp2, igp5, igp2 n igp6). Gns pirs tht show strong signiint orrltions (R>.7, P<.5, N 126) wr onsir nits or orgult xprssion. Eight strong positiv orrltions wr oun n inlu hs2 vrsus nr11 (R.83; P<.1) n xo32 (R.78; P<.1); 3 vrsus zn653 (R.76; P<.1) n kl11 (R.75; P<.1); xo32 vrsus MURF1 (R.73; P<.1); n 3 vrsus it2 (R.73; P<.1) n xo32 (R.72; P<.1). Th only strong ngtiv orrltion oun ws twn o1 n 3 (lustr II). All ight o th nit gns tht wr oun to uprgult with sting in th mirorry xprimnts wr vlit y qpcr. Th xprssion o th musl-spii uiquitin ligss, xo32 (lso known s MAFx/Atrogin-1) n MURF1 (nnott s zg:86757 in th zrish gnom ssmly) ws highly snsitiv to nutritionl sttus. Trnsripts or xo32 n MURF1 inrs 13.3 n 2.7-ol, rsptivly, twn 9 h n 144 h o sting n wr ownrgult y 55% (xo32) n 77% (MURF1) in th 45 min smpl tr ing, with lowst lvls osrv tr 6 h (Fig. 6A,B). Exprssion o oth gns strt to inrs soon tr oo ws lr rom th intstin n mrna lvls wr t th 168-h sting lvls y 36 h tr th ml (Fig. 6A,B). Four gns (3, it2, zn653 n kl11) h xprssion pttrns tht wr invrsly orrlt with gut ullnss. Trnsript unns wr lowst 45 min to 3 h tr ing n rpily inrs s th gut mpti (Fig. 7A D). nr11 n hs2 mrna lvls inrs ~13- n ~21-ol, rsptivly, twn 9 h n 144 h o sting, n wr strongly ownrgult with ing n show trnsint inrs in lvls 15 h tr th intstin ws mpty (Fig. 7E,F). Thr o th slt gns uprgult with ing in th mirorry xprimnt show xprssion pttrns rlt to gut ullnss. kp5 n o1 show pk o xprssion o-inint with th prsn o oo in th intstin, orrsponing to 5.6- n 17.2-ol inrs in trnsript unn rltiv to

7 Fing ts on trnsription 2131 Tl 1. Filtr list o gns rom th mirorry xprimnt showing trnsripts uprgult with sting in th zrish singl ml xprimnt Gn symol Gn sription Fol hng* Ajust P-vlu 1 Novl gn Novl gn 32.2± zg:86757 MURF1 (musl-spii RING ingr protin 1) 25.4± xo32 (MAFx) F-ox protin ± pk2 pyruvt hyrogns kins ± zp2 zon pllui glyoprotin ± h1m linkr histon H1M 12.8± si:h Novl protin (Si:h ) 12.5± kl11 Kruppl-lik tor ± zg: Hypothtil protin LOC ± BCL2 ining omponnt 3 9.4± si:h211-63o2.5 Hypothtil protin LOC ± ypl3 yipp-lik 3 8.7± it2 Cp/p3-intrting trnstivtor, with Glu/Asp-rih roxy-trminl omin, 2 8.6± nr11 nulr rptor sumily 1, group D, mmr 1 8.5± ng2 ylin G2 7.6± gp glyogn synths kins ining protin 7.6± gpr137 G protin-oupl rptor ± si:ky-42i9.4 B-ll trnslotion gn 1-lik 6.9± zg:192 Hypothtil protin LOC ± LOC muin 3-lik 6.3± zg:55582 myomglin 5.9± sl169 solut rrir mily 16 (monoroxyli i trnsportrs), mmr 9 5.6± r4 RAB4B, mmr RAS onogn mily 5.4± TCEANC trnsription longtion tor A (SII) N-trminl n ntrl omin ontining 5.4± zg:77714 roxy-trminl omin RNA polymrs II polyppti A smll phosphts 2 5.3± stk11ip srin/thronin kins 11 intrting protin 5.3± tg2 B-ll trnslotion gn 2 5.2± LOC79483 pyruvt hyrogns kins 2-lik 5.2± vgll2 vstigil lik 2 5.2± hs2 ht shok tor 2 5.1± zn653 zin ingr protin ± h hs homolog 4.8± pr1 prio homolog 1 4.5± zg:92851 Jun imriztion protin 2 4.4± pmt2 protin-l-isosprtt (D-sprtt) O-mthyltrnsrs omin ontining 2 4.3± LOC Similr to THAP omin ontining, poptosis ssoit protin 1 4.3± rms1l rst nr mtstsis-supprssor 1-lik protin 4.± us1 upstrm trnsription tor 1 3.9± ur1 uiquitin protin ligs E3 omponnt n-rognin 1 3.8± vsg1 vssl-spii 1 3.7± si:ky Hypothtil protin LOC ± sl251 solut rrir mily 25 (mitohonril rrir; itrt trnsportr), mmr 1 3.6± si:h Hypothtil protin LOC ± n4p2 NEDD4 ining protin 2 3.4± xo25 F-ox only protin ± oil-oil omin ontining ± npl N-tylnurmint lys (NALs; EC ) (N-tylnurmini i lols; 3.2±.4.5 N-tylnurmint pyruvt-lys; sili i lys; silt lys; silt-pyruvt lys; sili i lols) 48 zg:1178 Hypothtil protin LOC ± mtus1 mitohonril tumor supprssor 1 homolog A 3.1± zg: Hypothtil protin LOC ± i2 inhiitor o DNA ining 2, ominnt ngtiv hlix-loop-hlix protin, 3.± nr1 nighor o BRCA1 gn 1 3.± gml1 grm ll-lss homolog 1 (Drosophil) 3.± LOC Hypothtil protin 2.9± zg:1633 intiv Um1-spii prots 1 2.9± spns1 spinstr homolog 1 (spinstr-lik protin) 2.8±.2.47 *Vlus r mns ± s..m., N 6. Nms in prnthss r ltrntiv nms. mximl sting vlus, rsptivly (Fig. 8A,B). s1 show ~6.5-ol inrs in xprssion 6 to 9 h tr th strt o th ml n sty lin to lvls not signiintly irnt rom sting vlus y 36 h (Fig. 8C). DISCUSSION Trnsriptionl rgultion o th IGF systm In th sn o inormtion on plsm hormon n mino i lvls in smll tropil ishs, trnsit o oo through th

8 2132 I. P. G. Amrl n I. A. Johnston Tl 2. Filtr list o gns rom th mirorry xprimnt showing trnsripts uprgult with ing in th zrish singl ml xprimnt Gn symol Gn sription Fol hng* Ajust P-vlu 1 o1 ornithin roxyls ± ppt7 PTC7 protin phosphts homolog 1.5± tsll thpsin L, lik 9.9± x5 DEAD (Asp-Glu-Al-Asp) ox polyppti 5 9.8± pip5 protin isuli isomrs-rlt protin 9.5± si:h211-76m11.7 si:h211-76m ± mylk4 myosin light hin kins mily, mmr 4 8.9± ms2 mjor ilittor suprmily omin-ontining protin 2-B 8.± zg:11154 ukryoti trnsltion initition tor 4E-lik 7.9± kp5 FK56 ining protin 5 7.7± rn3 rtiulolin 3, EF-hn lium ining omin 7.5± yrk2 ul-spiiity tyrosin-(y)-phosphoryltion rgult kins 2 7.5± lrl lrtiulin-lik 7.2± hs1 AHA1, tivtor o ht shok protin ATPs homolog 1 7.± i41 ukryoti trnsltion initition tor 4A, isoorm 1A 6.8± kl13l Kruppl-lik tor 13 lik 6.8± LOC Similr to Dul spiiity protin phosphts 13 (Tstis- n 6.6± skltl-musl-spii DSP) (Dul spiiity phosphts SKRP4) 18 sl2525 lium-ining mitohonril rrir protin SCMC-2-A (smll lium-ining 6.5± mitohonril rrir protin 2-A; solut rrir mily 25 mmr 25-A) 19 LOC Aptor-rlt protin omplx 1 ssoit rgultory protin-lik 6.3± mi1ip1 MID1 intrting protin 1 5.9± zg:7323 Hypothtil protin LOC ± hsp9.1 Ht shok protin HSP 9-lph 1 5.7± trl hymotrypsin-lik 5.7± hsp9.2 ht shok protin 9-lph 2 5.6± oxo1 orkh ox O1 5.4± zg:1181 protin phosphts 5, tlyti suunit 5.3± zg: nosylhomoystins 5.3± synripl synptotgmin ining, ytoplsmi RNA intrting protin, lik 5.1± nj4 DnJ (Hsp4) homolog, sumily A, mmr 4 5.1± m69 mily with squn similrity 69, mmr B 5.± s1pr2 sphingosin 1-phospht rptor 2 (S1P rptor 2; S1P2; 5.± sphingosin 1-phospht rptor Eg-5; S1P rptor Eg-5) 32 pis4 protin inhiitor o tivt STAT, 4 5.± lmn1 lmin B1 5.± hmt1 uhromti histon-lysin N-mthyltrnsrs 1 rgmnt 4.9± smy1 SET n MYND omin ontining 1 4.8± LOC1633 novl protin similr to glutmins (Gls) 4.8± hsp1 ht shok 6 kd protin 1 4.7± slmo2 slowmo homolog 2 4.6± ATP-ining sstt, su-mily F 4.6± zg:92429 Hypothtil protin LOC ± kt2 potssium hnnl ttrmristion omin ontining 2 4.5± ppig pptiylprolyl isomrs G 4.5± g3p1 rs-gtps-tivting protin SH3-omin-ining protin 4.4± sl42 solut rrir mily 4, nion xhngr, mmr 2 4.4± LOC Novl protin similr to vrtrt nylt yls 9 (ADCY9) Frgmnt 4.3± sul1 sults 1 4.3± s1 SUMO-tivting nzym suunit 1 (uiquitin-lik 1-tivting nzym E1A) 4.3± zg:8572 Hypothtil protin LOC ± tp-ining sstt, su-mily B (MDR/TAP), mmr 1 4.2± rnp1 rn ining protin 1 4.1± zp1 zp1 protin rgmnt 4.± il34 intrlukin 34 4.± rz rizzl-rlt protin 3.8± zg: SWI/SNF-rlt, mtrix-ssoit tin-pnnt rgultor o hromtin, 3.8±.5.41 sumily, ontining DEAD/H ox 1 55 smr5 SWI/SNF rlt, mtrix ssoit, tin pnnt rgultor o 3.8±.7.49 hromtin, sumily, mmr 5 56 gr2 ntrior grint 2 homolog 3.7± zg: phosphoistrs 3.7± stip1 strss-inu-phosphoprotin 1 (Hsp7/Hsp9-orgnizing protin) 3.7±.6.48 Tl 2. Continu on nxt pg

9 Fing ts on trnsription 2133 Tl 2. Continu Gn symol Gn sription Fol hng* Ajust P-vlu 59 si:h p2.4 soium-oupl nutrl mino i trnsportr 2 (mino i trnsportr A2; systm A mino i trnsportr 2; systm N mino i trnsportr 2; systm A trnsportr 1; solut rrir mily 38 mmr 2) 3.7± zg:17228 ADP-riosyltion tor 6-lik 3.6± zg: trnsmmrn n oil-oil omin mily 1 3.5± zg:15329 oil-oil omin ontining ± zg:11424 Golgi trnsport 1 homolog B 3.5± zg:86751 Hypothtil protin LOC ± ppp4 srin/thronin-protin phosphts 4 tlyti suunit B 3.3±.5.48 (PP4C-B)(EC ) 66 LOC ptor-rlt protin omplx 2, lph 1 suunit-lik 3.3± mpk ump-mp kins (EC ) (ytiylt kins; oxyytiylt kins; 3.3±.5.49 ytiin monophospht kins; uriin monophospht kins; uriin monophospht/ytiin monophospht kins; UMP/CMP kins; UMP/CMPK) 68 si:h h3.1 si:h h ± hsp5 ht shok 7kD protin 5 3.3± sl384 solut rrir mily 38, mmr 4 3.3± nl1 nilin-1 prursor (nistrin-lik protin 1) 3.2± ssrp1 strutur spii rognition protin 1 3.2± zg: rginin-rih, mutt in rly stg tumors 3.2± lm1 lminin, t 1 3.2± zg:77244 potssium hnnl ttrmristion omin ontining 5 3.2± zg: Hypothtil protin LOC ± zg:17163 srin (or ystin) protins inhiitor, l H, mmr 1 3.2± un45 un-45 (C. lgns) rlt 3.1± zg: musl-rstrit ul spiiity phosphts 3.1± s34 spliing tor 3, suunit 4 3.1± tho6 THO omplx 6 homolog (Drosophil) 3.± pl1 pl1 3.± trm1 trnsloting hin-ssoiting mmrn protin 1 3.± zg:565 oreli omin ontining 1 3.± pr2 prio homolog 2 3.± si:h YLP moti ontining 1 2.8± snrnp4 smll nulr rionuloprotin 4 (U5) 2.8± h1 EH-omin ontining 1 2.8± si:h m4.4 xportin-t [trna xportin; xportin(trna)] 2.8± lg9 sprgin-link glyosyltion 9 protin 2.7±.3.5 *Vlus r mns ± s..m., N 6. Nms in prnthss r ltrntiv nms. gstrointstinl systm n phosphoryltion o th IGF-pthwy signlling protin Akt provi mns o intrprting th trnsriptionl rspons to singl stiting ml. In prvious sting ring stuy using rinow trout, Onorhynhus mykiss, mximum plsm insulin n mino i lvls wr ror 3 min n 2.5 h tr ing n wr quikly ollow y phosphoryltion o svrl kinss, initing tivtion o th TOR signlling pthwy (Siliz t l., 28). Amino is n insulin Tl 3. Enrihmnt nlysis o gn ontology trms or iologil prosss ssoit with gns irntilly rgult in rspons to singl-stiting ml, using th 44K Agilnt zrish mirorry V2 GO intiir GO trm Numr o gns irntilly xprss FDR* Enrih with sting GO:43632 Moiition-pnnt mromolul toli pross 8.31 GO:4563 Positiv rgultion o nothlil ll irntition 2.52 Enrih with ing GO:4226 Protin roling 3.4 GO:5164 Protin mturtion 3.35 GO:3241 Skltl musl thik ilmnt ssmly 2.35 GO:34619 Cllulr hpron-mit protin omplx ssmly 2.35 GO:42517 Positiv rgultion o tyrosin phosphoryltion o Stt3 protin 2.35 GO:48769 Sromrognsis 2.35 GO:46425 Rgultion o JAK-STAT s 3.42 GO:45618 Positiv rgultion o krtinoyt irntition 2.45 GO:4566 Positiv rgultion o pirml ll irntition 2.54 GO:796 Mitohonril outr mmrn trnslos omplx ssmly 2.68 *FDR, ls isovry rt. Only th most-spii trms r shown.

10 2134 I. P. G. Amrl n I. A. Johnston Downrgultion <> Ml 159 h 24 h h.75 h 3 h 6 h 7.5 h 9 h 12 h 24 h 36 h ig1 hsp9.1 o1 oxo1 igp6 ig2 s1 kp5 ig1r ig2r ig1r xo32 zg86757 kl11 3 zn653 tnni2.4 hsp9.2 nr11 igp1 hs2 igp1 ig2 igp3 it2 mylz2 igp5 igp2 igp5 igp2 igp5 Uprgultion Clustrs Fig. 5. Hirrhil lustring n ht mp o IGF systm gn trnsripts n nit nutritionlly rgult gns intii rom mirorry xprimnts ovr th tim ours o th singl ml xprimnt. Th Romn numrls init lustrs isuss in th txt. Rows (mrna trnsripts) in th ht mp r stnriz to hv mn o zro n stnr vition o on (i.. stnr sor normliztion). R n grn shing, rsptivly, inits th highst n lowst xprssion lvls, s init in th sl r t th ottom o th igur. Eh lok rprsnts th vrg stnr-sor normliztion or th 13 ish smpl t h tim-point in th xprimnt. lso rpily inrs th lvl o irulting IGFI in rown trout (Bnos t l., 1999). In our xprimnts with zrish, 5% o th oo ingst h n pross n limint within 3 h tr th strt o ing n th gut ws mpty tr 9 h (Fig. 1). Thr ws signiint inrs in phosphoryltion o Akt within 45 min, with pk lvls t 3 h tr ing n phosphoryltion ovr similr tim ours to th limintion o oo rom th gut (Fig. 2). Chngs in th lvls o musl mrnas lso mostly took pl with tim ours o hours n wr gnrlly quikr thn sri in th litrtur or lrgr tmprt ish spis mintin t lowr tmprturs (Chuvign t l., 23; Bowr t l., 28). Th irst im o this stuy ws to tst th hypothsis tht rtin prlogus o IGF-systm gns wr irntilly rgult ollowing trnsition rom toli to n noli stt. Fsting hs n shown to rsult in n uprgultion o musl IGF rptors in numr o tlost spis n is proly rspons to rs in irulting IGF hormon lvls (Chuvign t l., 23; Gillr t l., 26; Bowr t l., 28; Bowr n Johnston, I II III IV V xo32 gn xprssion (.u.) zg:86757 gn xprssion (.u.) A B Tim (h) Light Drk Light Drk Light 21). In rinow trout, prolong sting rsult in n uprgultion o IGFR-I, ut not IGFR-I (Chuvign t l., 23) whrs in zrish oth IGFR prlogus wr uprgult (Fig. 4A,B). Howvr, zrish ig1r inrs mor rpily ollowing gut mptying thn ig1r rhing sting lvls >36 h n <25 h, rsptivly (Fig. 4A,B), initing irntil rgultion o IGFI rptor prlogus. Funtionl hrtristion stuis o IGFIIR in ish r sr n its rol in th rspons to nutrint lvls is not yt lr. In Atlnti slmon, IGFIIR trnsripts wr signiintly uprgult with prolong sting n ownrgult tr 7 ys o ring (Bowr t l., 28). In this stuy thr ws no isrnil pttrn o trnsriptionl rgultion o ig2r ovr th xprimnt. Musl IGF hormon trnsript lvls show istint pks within w hours o ing (Fig. 3A,B). In th s o Ig1 pk vlus wr oun within 3 h o ing n h lin to sting lvls or th gut ws mpti (<5 h; Fig. 3A). In ontrst, Ig2 trnsripts rh pk 6 h tr ing n wr not signiintly irnt to sting lvls y 9 h (Fig. 3B). Ig1 trnsripts wr not tt in st musl whrs th Ig2 prlogu ws onstitutivly xprss n show no onsistnt hng in xprssion ovr th sting-ing-sting yl ssoit with singl ml. In vitro stuis with Atlnti slmon (Slmo slr) myoyts hv shown synrgisti ts o insulin, IGF-I n mino is on musl IGF-I trnsript unn, initing multipl pthwys ling to IGF trnsription (Bowr Fig. 6. Exprssion proils o nit nutritionlly rgult gns in ml zrish intii rom mirorry xprimnts ovr th tim ours o th singl ml xprimnt s trmin y qpcr. Gns uprgult uring sting (soli squrs n lk lins): (A) xo32 n (B) muslspii RING ingr protin 1 MURF1 (zg:86757). Th gry lins rprsnt th vrg gut ullnss illustrt in Fig. 1. Vlus r mns ± s..m., N 13 ish pr smpl. Dirnt lttrs signiy sttistilly irnt mns (P<.5; s txt or tils) Gut ullnss (%) Gut ullnss (%)

11 Fing ts on trnsription gn xprssion (.u.) zn653 gn xprssion (.u.) nr11 gn xprssion (.u.) A C E Tim (h) Light Drk Light Drk Light Gut ullnss (%) Gut ullnss (%) Gut ullnss (%) it2 gn xprssion (.u.) kl11 gn xprssion (.u.) hs2 gn xprssion (.u.) B D F Tim (h) Light Drk Light Drk Light Gut ullnss (%) Gut ullnss (%) Gut ullnss (%) Fig. 7. Exprssion proils o nit nutritionlly rgult gns in ml zrish intii rom mirorry xprimnts ovr th tim ours o th singl ml xprimnt s trmin y qpcr. Gns uprgult uring sting (soli squrs n lk lins): (A) BCL2 ining omponnt 3 (3), (B) Cp/p3-intrting trnstivtor, with Glu/Asp-rih roxy-trminl omin, 2 (it2), (C) zin ingr protin 653 (zn653), (D) Kruppl-lik tor 11 (kl11), (E) nulr rptor sumily 1, group, mmr 1 (nrl1), n (F) ht shok tor 2 (hs2). Th gry lins rprsnt th vrg gut ullnss illustrt in Fig. 1. Vlus r mns ± s..m., N 13 ish pr smpl. Dirnt lttrs signiy sttistilly irnt mns (P<.5; s txt or tils). n Johnston, 21). In mmmls, th ining o IGF to th IGF- IR inus its uto-phosphoryltion rsulting in th tivtion o svrl own-strm signl trnsution ss vi ptor moluls suh s th insulin rptor sustrt 1 (IRS-1), whih hs multipl tyrosin phosphoryltion sits (Dun t l., 21). IGF- I stimults growth vi ts on protin synthsis (Romml t l., 21), myolst prolirtion n irntition ting through istint signlling pthwys (Rn t l., 21). Th tiv onntrtion o IGF in th musl is rgult y six IGFBPs, whih r gr y spii protss to rls th hormon to trgt sits (Woo t l., 25). Evin, lrgly rom mmmls, inits tht IGFBPs n inhiit n/or potntit IGF tions pning on th llulr ontxt n/or nvironmntl onitions (Dun t l., 21). In Atlnti slmon, th trnsition rom mintnn to st growth ws ssoit with onstitutiv uprgultion o IGFBP-4, trnsint inrs in IGFBP-5.1 n ownrgultion o IGFBP-2.1 (Bowr t l., 28). Th two rtin zrish prlogus o igp1 h similr xprssion in th singl ml xprimnt with high trnsript unn uring sting, mrk rution in mrna lvls within 45 min o ing n vril, ut gnrlly low lvl o xprssion ovr th ollowing 36 h (Fig. 4C). Mmmlin stuis init tht in ition to its rol s moultor o IGF1 vilility, IGFBP-1 hs puttiv IGF-inpnnt iologil tivitis through intrtion with llsur intgrins, with puttiv irt ts on th P13K AKT pthwy (Whtrot n Krny, 29). Thr ws no vin or th trnsriptionl rgultion o igp2, igp2, igp3, igp5, igp5, igp6 n igp6 prlogus with ing in our xprimnts. Although xprimntl ontxt my xplin som o ths irns in IGFBP xprssion it is lr tht thr r

12 2136 I. P. G. Amrl n I. A. Johnston kp5 gn xprssion (.u.) o1 gn xprssion (.u.) s1 gn xprssion (.u.) A B C ling-spii irns in IGFBP untion n rgultion within th tlosts. For xmpl, igp4 is not rprsnt in th urrnt Dnio rrio gnom ssmly ( Projts/D_rrio/) n is proly snt rom th zrish ling. Our rsults t lst init tht ution is n in inrring similr untions o IGFBPs twn tlost lings n rtinly twn tlosts n mmmls. Ovrll w onlu tht ollowing WGD som o th rtin prlogus o IGF-systm gns show irntil trnsriptionl rgultion with sting n ring in skltl musl, ut tht omplx pttrns o rgultion hv volv twn n within lings Tim (h) Light Drk Light Drk Light Fig. 8. Exprssion proils o nit nutritionlly rgult gns in ml zrish intii rom mirorry xprimnts ovr th tim ours o th singl ml xprimnt s trmin y qpcr. Gns uprgult with ing (soli squrs n lk lins): (A) FK56 ining protin 5 (kp5) (B) ornithin roxyls 1 (o1) n (C) SUMO-tivting nzym suunit 1 (s1). Th gry lins rprsnt th vrg gut ullnss illustrt in Fig. 1. Vlus r mns ± s..m., N 13 ish pr smpl. Dirnt lttrs signiy sttistilly irnt mns (P<.5; s txt or tils) Gut ullnss (%) Gut ullnss (%) Gut ullnss (%) Gnom-wi trnsriptionl rgultion with toli to noli trnsition Mirorry xprimnts provi snpshot o th st musl trnsriptom uring sting n t th point IGF trnsripts rh thir mximum unn ollowing ing. Th srning ritri us to uil lists o irntilly rgult gns wr pprntly roust us ll 14 gns tst wr vlit y qpcr n wr wll orrlt (R.79; supplmntry mtril Fig. S1). Fsting in zrish (Fig. 6A,B) n Atlnti slmon (Bowr t l., 28; Bowr n Johnston, 21) is ssoit with lrg inrs in th unn o E3 uiquitin ligss MURF1 n xo32 trnsripts. In mmmls, th uiquitin sustrt rognition systm hs n implit in spii grtion o myod (Tintign t l., 25; Finn n Di, 26) n othr promyogni trnsription tors (Finn n Di, 26). Two sustrt rognition omponnts o th uiquitin ligs systm, F-ox only protin 25 (xo25) n RAB4B, mmr RAS onogn mily (r4), wr lso highly uprgult with sting in zrish st musl (Tl 1). Two gns with puttiv rols in utophgy wr oun to uprgult uring oo privtion [mirotuul-ssoit protin 1 light hin 3 t (mp1l3) n nighour o BRCA1 gn 1 (nr1)] (Tl 1). It is known tht myoirillr protins r gr to provi sour o mino is or nrgy mtolism uring prolong sting in tlosts (Johnston n Golspink, 1973). Howvr, th rltiv importn o th uiquitin-protsom grtion pthwy, utophgy n othr lsss o protss in miting protin rkown uring norml protin turnovr n short prios o sting rmins to stlish. Cll yl rrst n poptosis lso sms to n importnt rspons in pting to prios o limit nrgy supply in zrish, s vin y th uprgultion o nti-prolirtiv protin gn trnsripts [Bll trnslotion gn 1 n 2 (tg1 n tg12), THAP ominontining protin 1 (thp1) n pro-poptoti gns (3 n kl11)] (Tl 1). Trnsition to n noli stt 3 h tr th ml rsult in mjor hngs in th musl trnsriptom. Fing ws ssoit with th uprgultion o trnsripts or hpron protins (un45, ppig, pip5, nj n stip1) inluing vrious ht shok protins n ssoit protins (hsp9.1, hsp9.2, hsp1 n hsp5; Tl 2). Molulr hprons r ssntil or oth th oling n mintnn o nwly trnslt protins n th grtion o misol n stiliz protins (Zho n Houry, 27). In zrish, th hprons Hsp9 n Un45 r orgult n involv in th oling o th gloulr h o myosin uring myoirillrgnsis, ssoiting with th Z-lin on myoirillr ssmly is omplt (Etr t l., 28). Th squning o sutrtiv DNA lirris rom st skltl musl o Atlnti slmon ithr mintnn or stiting rtions lso rvl tht xprssion o hpron protins initiv o unol protin rspons (UPR) pthwys suh s DNAJ4, HSPA1B, HSP9A n CHAC1 ws n rly rspons to inrs oo intk n growth (Bowr n Johnston, 21). Aumultion o unol protins n our whn th mounts o nwly synthsiz protins xs tht o th protin oling pity o th noplsmi rtiulum (Ok t l., 22). Tkn togthr ths rsults init tht n inrs in protin hpron gn xprssion n tivtion o UPR pthwys is gnrl rspons o tlost skltl musl ollowing th trnsition rom toli to noli stt. Th lrgst inrs in trnsript unn oun with ing ws or th gn noing th nzym ornithin roxyls 1 (o1; 11.5-ol). Ornithin roxyls, ky mtoli nzym o th polymin iosynthsis pthwy, lso show n inrs in

13 Fing ts on trnsription 2137 tivity tr ing in st rt tissus (Moor n Swnsi, 1983). Polymins hv numr o iologil untions inluing ll growth n poptosis n t in onntrtion-pnnt mnnr (Lrqué t l., 27). Th noli stt ws ssoit with nrihmnt o GO trms or mitohonril trnslos tivity, initition o protin synthsis [.g. ukryoti trnsltion initition tor 4A isoorm 1A (i41), ul-spiiity tyrosin-(y)- phosphoryltion rgult kins 2 (yrk2)] n mrna prossing, mturtion n xport protin gns [.g. DEAD (Asp-Glu-Al-Asp) ox polyppti 5 (x5) n smll nulr rionuloprotin 4 (U5) (snrnp4)]. Furthrmor, pis4 n s1, whih r involv in th post-trnsltionl onjugtion o SUMO (smll uiquitin-lik moiir) to trgt protins, wr uprgult with ing. Sumoyltion o protins my t thir stility, loliztion n tivity (Giss-Frilnr n Mlhior, 27). In ontrst to th uiquitin-onjugt protins, sumo-onjugt protins r not trgt or grtion n in sumoyltion my untion s n ntgonist pthwy to th uiquitin-protsom pthwy (Dstrro t l., 1998; Hy, 25). Fing ws lso ssoit with signiint hngs in th xprssion o gns tht my untion s pignti swiths oing or protins tht moiy hromtin strutur n ltr th xprssion o suits o othr gns. For xmpl, SET n MYND omin ontining 1 (smy1) trnsripts inrs 4.8-ol twn th n st stts (Tls 1 n 2). Smy1 untions s trnsriptionl rprssor in mous ri musl (Gottli t l., 22) n is rquir or norml skltl musl vlopmnt in zrish (Tn t l., 26). In th prsnt stuy ing lso rsult in 4.9-ol inrs in trnsript unn or th uhromti histon-lysin N-mthyltrnsrs 1 (hmt1). Th humn orthologu o Ehmt1 ws oun to prt o th E2F6 omplx n is proly involv in th silning o MYC- n E2Frsponsiv gns (Ogw t l., 22), n is thought to ply rol in G G1 ll-yl trnsition. Th rruitmnt n hyprtrophy o st myotoml musls in zrish ws shown to typil o tlosts (Johnston t l., 29). Th prsnt stuy lso monstrts th utility o th zrish or mhnisti stuis on th rgultion o growth signlling pthwys in tlost musl, proviing th vntgs o squn gnom, ommrilly vill molulr rsours n low husnry osts us o th smll oy siz. Howvr, ution shoul ppli in xtrpolting ll rsults rom mol to quultur ish spis in th light o vin or ling-spii pttrns o prlogu rtntion (Mqun n Johnston, 28; Mqun n Johnston, 28) n IGFBP gn xprssion (this stuy). Th singl ml prigm lso provis n intrsting ltrntiv to th us o ontinuous ing rgims to invstigt trnsriptionl rgultion uring th trnsition rom toli to n noli stt. APPENDIX Dtil inormtion on protin xtrtion n wstrn-lotting ttion xprimntl protools. Protin xtrtion Skltl musl protin rom iv rnom rplits or h timpoint ws xtrt y homogniztion o 3 mg o tissu in 35 l o 25 mmol l 1 MES ph 6. ontining 1 mol l 1 NCl,.25% (m/v) CHAPS, DNA/RNA nuls (Invitrogn) n prots inhiitor oktil (Invitrogn) using Lysing Mtrix D (Qiogn) in FstPrp mhin (Qiogn). Optiml protin loing or SDS sprtion n trnsr Th optiml protin mount to us or ltrophorsis sprtion n trnsr ws mpirilly trmin y pplying rom 1 to 6 g o protin o rrn smpl in uplits n nlysing th nsity o th Ponu S stining (Fig. A1A,B). TotlL sotwr (Nonlinr Dynmis, Nwstl upon Tyn, Tyn n Wr, UK) ws us to nlys th nsity o ns rom Ponu S stining n wstrn lots. Protin sturtion ws osrv whn mor thn 3 g o protin ws lo in th gl. Th optiml mount ws 2 g onsiring oth Ponu-S-stining linrity (Fig. A1B) n totl protin vilility or th stuy. Loing protin ontrol n P-AKT ntioy spiiity Th mmrns us or optiml protin loing trmintion inlu rrn smpl trt with l intstinl lklin phosphts (A2356; Sigm) to onirm tht th ntioy trgt th phosphorylt moity o th protin o intrst (Fig. A1C). Atin intnsity ws ttr orrlt with Ponu S stining whn ompr with GAPDH (Fig. A1C) n ws us to normliz irns in protin loing. Dphosphoryltion o P-AKT signiintly rs its ttion y P-AKT spii ntioy (Fig. A1C) whrs no hng in ttion ws osrv or tin ntioy in th phosphorylt smpl (Fig. A1C), onirming th spiiity o th P-AKT ntioy to th phosphorylt moity o AKT. Wstrn-lotting o trgt protins Th smpls (2 l, ontining 2 g o protin) wr to 6 l o solution ontining 5 l o 5 protin loing ur n 1 l 2 ruing gnt (Frmnts), ht or 5 min t 95 C n lo in NuPAGE Novx 4 12% Bis-Tris gls (Invitrogn) n run t 12 V. A protin lr o 1 25 kd (Frmnts) n rrn smpl wr lo in ll gls to stimt th molulr mss o protins o intrst n srv s normliztion smpl, rsptivly. Protins sprt y ltrophorsis wr trnsrr to PVDF Immoilon-P Trnsr Mmrn (Millipor) t 25 V or 15 min. Sussul protin sprtion n trnsr wr onirm y Ponu S stining (Sigm). PVDF mmrns wr lok ovrnight t 1 C using 5% (m/v) nont milk (AppliChm) prpr in PBS (Sigm) ontining.1% (v/v) Twn 2 (Sigm). Blok mmrns wr inut ovrnight t 1 C with th ollowing primry ntiois (IgGs): P-AKT (1:1 ilution; Cll Signling, #46), AKT (1:1; Cll Signling, #2966), tin (1:2,; Sigm A266) n GAPDH (1:3,; Sigm, G9545). Pro mmrns wr inut t 2 C or 1 h with th sonry ntioy ginst mous or rit IgG onjugt to horsrish proxis (oth rom Sigm n us t 1:6, ilution). Positiv rtions wr ror y xposing Hyprilm ECL (GE Hlthr) to th mmrns tr inution or 1 min with ECL Wstrn Blotting Dttion Rgnts (GE Hlthr) t room tmprtur. Exprimntl vritions in th ltrophorsis n trnsr wr normliz using rrn smpl ommon to ll mmrns. Th ol-hng in phosphoryltion o AKT in h tim point ws ompr with th smpls rom 159 h. GLOSSARY Myotom Tissus within th mryoni somits tht orm th trunk musls. Nountionliztion Gin o nw untion y on prlogu, whil th othr rtins th originl untion prorm y th nstrl gn.