Mitochondrial function is enhanced by thyroid hormones during zebra finch development

This is a sel -a chi ed e sion o an o iginal a icle. This e sion may di e om he o iginal in pagina ion and ypog aphic de ails. Au ho (s): Ti le: Yea : Ve sion: Copy igh : Righ s: Righ s u l: Please ci e he o iginal e sion: CC BY 4.0 h ps://c ea i ecommons.o g/licenses/by/4.0/ Mi ochond ial unc ion is enhanced by hy oid ho mones du ing zeb a inch de elopmen © 2024 he Au ho s Published e sion Oe ele, Ma lene; Hau, Michaela; Ruuskanen, Su i; Casag ande, S e ania Oe ele, M., Hau, M., Ruuskanen, S., & Casag ande, S. (2024). Mi ochond ial unc ion is enhanced by hy oid ho mones du ing zeb a inch de elopmen . Royal Socie y Open Science, 11, A icle 240417. h ps://doi.o g/10.1098/ sos.240417 2024 Mi ochond ial unc ion is enhanced by hy oid ho mones du ing zeb a inch de elopmen Ma lene Oe ele1, Michaela Hau1,2, Su i Ruuskanen3 and S e ania Casag ande1 1E olu iona y Physiology Resea ch G oup, Max Planck Ins i u e o Biological In elligence, Ebe ha d-Gwinne -S asse, Seewiesen 82319, Ge many 2Depa men o Biology, Uni e si y o Kons anz, Kons anz D-78464, Ge many 3En i onmen al Physiology Resea ch G oup, Uni e si y o Jy äskylä, Seminaa inka u 15, Uni e si y o Jy äskylä, Jy äskylä FI-40014, Finland MO,0009-0008-4152-6320 An o ganism’s esponse o i s en i onmen is la gely de e mined by changes in he ene gy supplied by ae obic mi ochond ial me abolism ia adenosine iphospha e (ATP) p oduc ion. ATP is especially impo an unde ene gy- demanding condi ions, such as du ing apid g ow h. I is cu en ly poo ly unde s ood how en i onmen al ac o s in luence ene gy me abolism and mi ochond ial unc ioning, bu ecen s udies sugges he ole o hy oid ho mones (TH). TH a e key egula o s o g ow h and me abolism and can be lexibly adjus ed o en i onmen al condi ions, such as en i onmen al empe a u e o ood a ailabili y. To es whe he TH enhancemen is causally linked o mi ochond ial unc ion and g ow h, we p o ided TH o ally a physiological concen a ions du ing he main g ow h phase in zeb a inch (Taeniopygia gu a a) nes lings ea ed in a challenging en i onmen . TH ea men accele a ed maximal mi ochond ial wo king capaci y—a ai ha e lec s mi ochond ial ATP p oduc ion, wi hou a ec ing g ow h. To ou knowledge, his is he i s s udy o cha ac e ize he egula ion o mi ochond ia by TH du ing de elopmen in a semi-na u alis ic con ex and o add ess implica ions o i ness- ela ed ai s, such as g ow h. 1. In oduc ion O ganisms con inually adjus o hei su oundings, and ene gy dynamics play a pi o al ole in shaping hese esponses. Cen al © 2024 The Au ho (s). Published by he Royal Socie y unde he e ms o he C ea i e Commons A ibu ion License h p://c ea i ecommons.o g/licenses/by/4.0/, which pe mi s un es ic ed use, p o ided he o iginal au ho and sou ce a e c edi ed. Resea ch Ci e his a icle: Oe ele M, Hau M, Ruuskanen S, Casag ande S. 2024 Mi ochond ial unc ion is enhanced by hy oid ho mones du ing zeb a inch de elopmen . R. Soc. Open Sci. 11: 240417. h ps://doi.o g/10.1098/ sos.240417 Recei ed: 12 June 2023 Accep ed: 25 June 2024 Subjec Ca ego y: O ganismal and e olu iona y biology Subjec A eas: e olu ion, ecology, cellula biology Keywo ds: mi ochond ia, hy oid ho mones, cellula espi a ion, g ow h, me abolism Au ho o co espondence: Ma lene Oe ele e-mail: [email p o ec ed].de Elec onic supplemen a y ma e ial is a ailable online a h ps://doi.o g/10.6084/ m9. igsha e.c.7349588. Downloaded om h ps:// oyalsocie ypublishing.o g/ on 08 Augus 2024 o hese dynamics is ae obic mi ochond ial me abolism, which uels cellula unc ions h ough he p oduc ion o adenosine iphospha e (ATP). The signi icance o ATP becomes pa icula ly e iden unde ene gy-in ensi e condi ions, such as pe iods o apid g ow h. Howe e , he in e play be ween en i onmen al ac o s, ene ge ic needs and mi ochond ial pe o mance emains insu icien ly unde s ood. Recen esea ch has begun o shed ligh on his issue, indica ing ha hy oid ho mones (TH) may se e as a link be ween en i onmen al in luences and me abolic adjus men [1,2]. TH plays a cen al ole in e eb a e de elopmen , wi h a signi ican in luence on g ow h [1,3]. They a ec cellula di e en ia ion, p oli e a ion [4] and me abolic egula ion [5], all o which a e essen ial o g ow h. A he cellula le el, unde s anding he in e ac ion be ween TH and mi ochond ia is c ucial because hei sec e ion is lexibly adjus ed o en i onmen al condi ions, such as clima ic condi ions o ood a ailabili y [1,2,6–8]. Ae obic mi ochond ial me abolism—and hence ATP p oduc ion—de e mines an o ganism’s ene gy me abolism [9] and hus he abili y o adjus o di e en ecological condi ions [10]. Mi ochond ia a e known o be impo an d i e s o li e-his o y decisions and adjus men s o ecological challenges [11]. To da e, we know li le abou he mechanisms ha media e en i onmen al in luences on mi ochond ial unc ion, bu i is plausible ha hey a e unde he in luence o TH [5,12–20]. Howe e , we a e s ill missing ecological pe spec i es o his ascina ing biochemical TH–mi ochond ia in e play. The con ol o mi ochond ial unc ion, hence o ene gy p oduc ion, is especially impo an du ing g ow h and de elopmen , a c i ical li e-his o y s age cha ac e ized by high ene ge ic demands. G ow h equi es p o ein syn hesis and cell eplica ion, which a e p ocesses ha necessi a e ene gy in he o m o ATP. In e eb a es, op imal g ow h a e and la ge body size a e ela ed o su i al [21–24] and hus ul ima ely de e mine i ness ou comes o pa en s and o sp ing. Ne e heless, only a ew s udies ha e examined mi ochond ia and TH in an ecological con ex du ing o sp ing de elopmen and mos o hem we e ca ied ou in bi ds. The de elopmen o bi ds, pa icula ly du ing he ea ly li e pe iod, is cha ac e ized by apid g ow h and high me abolic demands [23,25]. A ian species exhibi ema kable pheno ypic plas ici y and adap i e s a egies o cope wi h luc ua ing en i onmen al condi ions du ing de elopmen , hus ep esen ing excellen models o in es iga e he e ec o change in TH on g ow h and me abolism [6]. In g ea i (Pa us majo ) nes lings, a single TH-injec ion in o he eggs did no a ec mi ochond ial espi a ion [26] o mi ochond ial densi y [27] a he ledgling s age. This could mean ha TH ele a ion du ing he emb yonic phase does no ha e a las ing e ec , ha ing disappea ed by he end o a ha chling’s de elopmen . Howe e , in al icial ha chlings, TH le els ha e been shown o ise s eadily in he i s week a e ha ching and each adul le els a ound ledging—in pa allel wi h he ha chlings’ g ow h pa e ns, and hus, pos -ha ching TH and g ow h pa e ns should be s udied [25]. Al hough some s udies ha e add essed he e ec s o TH on g ow h in al icial nes lings, esul s ac oss s udies a e inconclusi e [25–36]. In g ea i s, p ena al TH manipula ions had no e ec on g ow h a any s age [26]. Howe e , in colla ed lyca che s (Ficedula albicollis), p ena al TH-ele a ions lead o la ge chicks and ea lie ha ching, bu no long- e m consequences in he nes ling s age we e obse ed [35]. Ne e heless, he en i onmen al ac o s ha a e known o p omo e ho monal lexibili y a e mani old and i is s ill unknown whe he and how TH luc ua ions du ing he ledging s age a ec mi ochond ia and g ow h. The e ec s o TH in he ledging s a e ha e mos ly been s udied in poul y bu a concen a ions exceeding physiological anges. These s udies show ha bo h hypo- and hype - hy oidal condi ions educe g ow h and de elopmen [6,37–39]. Fu he mo e, in ed-winged blackbi d (Agelaius phoenicius) nes lings, hype - and hypo- hy oidal condi ions led o educed skele al g ow h, compa ed wi h unmanipula ed nes lings [33] and in he zeb a inch (Taeniopygia gu a a), TH-inhibi ion was ela ed o dec eased g ow h [34]. Howe e , i has emained unclea whe he hese ela ionships a e induced by pha macological TH concen a ions o in ac do occu a physiological TH concen a ions as well. To s udy en i onmen ally induced TH esponses, i is c ucial o ope a e wi hin he physiological ange o TH le els, which ep esen he e ol ed le els in esponse o en i onmen al signals. These s udies a e i al o unde s anding he e ec s o en i onmen al challenges on li e-his o y ou comes. Fo example, i is s ill no clea how an o ganism’s me abolism is adjus ed o changing en i onmen al condi ions, like ising empe a u es o a lack o nu ien a ailabili y—bo h o which ha e been shown o in luence TH-sec e ion [1,2,6,7,40]—and wha he consequences a e o i ness and li e-his o y ou comes. G ow h is a aluable ai o s udy because g ow h pa e ns in ea ly li e can de e mine su i al, especially in young passe ines [41]. Fu he mo e, i is impo an o analyse he me abolic consequences media ed by TH, because TH in e ac wi h me abolism, and g ow h and me abolism pe se a e highly in e connec ed. Fo ins ance, TH in luence g ow h ho mone sec e ion, a co e componen o g ow h egula ion [42]. G ow h ho mone 2 oyalsocie ypublishing.o g/jou nal/ sos R. Soc. Open Sci. 11: 240417 Downloaded om h ps:// oyalsocie ypublishing.o g/ on 08 Augus 2024 can boos mi ochond ial e iciency, me abolic a e and biogenesis in andem wi h insulin-like g ow h ac o o mee g ow h’s me abolic demands [43]. Thus, he e ec s o TH on mi ochond ia could be indi ec , o ins ance ia g ow h ho mone. Howe e , TH has also been shown o a ec mi ochond ial unc ion independen ly o g ow h in mul iple ways [44]. Fo example, by p omo ing he ansc ip ion o mi ochond ial enzymes such as cy och ome a and c [15,19,45], ia he ansc ip ion o nuclea genes, by in e ac ing wi h TH-binding si es in he DNA, ia cellula signalling cascades [15,46] o di ec s imula ion o componen s o he mi ochond ial elec on anspo chain, such as complex V [15,19]. Also, TH has been shown o s imula e mi ochond ial me abolic unc ion wi hin minu es h ough mi ochond ial T3 binding si es o by inducing he ansc ip ion o mi ochond ial genes [46]. This p obably enables he o ganism o eac o sudden changes in ene ge ic demands. An example o a sho - e m adjus men like his would be he need o boos he mo egula ion when expe iencing a apid d op in en i onmen al empe a u e. Mi ochond ial he mogenesis in ol es leak espi a ion, whe e ene gy is ans o med in o hea ins ead o ATP, and TH in e ac s wi h mi ochond ial uncou- pling p o eins [19]— he p ima y d i e s o leak [47,48]. While TH is in ol ed in apid mi ochond ial adjus men s such as o he mo egula ion, ano he impo an unc ion o TH is he adjus men o mi ochond ial unc ion o sus ained physiological demands, such as g ow h o long-las ing en i on- men al challenges [19]. These adjus men s can be con eyed h ough epigene ic p og amming by he modi ica ion o me hyla ion pa e ns du ing de elopmen [49,50] o h ough p omo ing he ansc ip- ion o mi ochond ial genes [46]. He e, we expe imen ally es ed he hypo hesis ha TH plays a ole in s imula ing mi ochond ial unc ion du ing de elopmen , in addi ion o augmen ing g ow h. To be able o ans e ou indings o na u al sys ems, we epea edly ele a ed ci cula ing le els o T3 ( iiodo hy onine, he biologically ac i e hy oid ho mone) wi hin physiological anges du ing he mos apid pos -ha ch g ow h pe iod in cap i e zeb a inches o add ess he ollowing ques ions: (i) do ele a ed TH concen a ions enhance g ow h in zeb a inch nes lings? We expec ed TH- ea ed nes lings o ha e as e g ow h a es in bo h skele al size and body mass. As a esul o accele a ed g ow h, TH- ea ed chicks may ei he ha e la ge body sizes a he ime o ledging han con ol chicks, o al e na i ely, each he same inal size as con ol chicks bu a an ea lie age. (ii) Is mi ochond ial unc ion enhanced by sus ained ele a ed TH le els du ing g ow h? We expec ed TH o accele a e mi ochond ial espi a ion a e and oxida i e phospho yla ion, which a e he p ima y de e minan s o ATP p oduc ion a e. 2. Me hods 2.1. Model species and housing Zeb a inches om a local cap i e Seewiesen popula ion we e allowed o b eed o 23 weeks in wo communal indoo a ia ies. The local cap i e zeb a inch popula ion was acqui ed in 2002 (231 indi iduals a he ime) om a colony held since 1985 by T. R. Bi khead a She ield Uni e si y, UK. Fo mo e in o ma ion, see popula ion no. 18 in [51]. Each o he a ia ies was equipped wi h 30 wooden nes boxes and 7−11 open nes ing cups, main ained a a cons an empe a u e o 21°C wi h humidi y a 59%, a a ligh : da k pho ope iod o 14 : 10 h. The bi ds we e p o ided wi h whi e co on and coconu ib es as nes ing ma e ial, comme cial seed mix u e, supplemen a y comme cial egg ood, cu le ish bone, g i and wa e ad libi um. The bi ds we e housed in communal a ia ies whe e access o seeds and egg ood was cons ained by o e ing a 2 : 1 ood- o-husk a io o mimic na u ally challenging ood esou ces [52]. Once pe week, he die was supplemen ed wi h salad and mul i i amins. The husk–seed mix u e was p o ided in open boxes (60 × 40 × 12 cm) and he husk–egg ood mix u e in la pla es. Food dishes we e placed on he loo . Nes s we e checked once pe day o new ha chlings, and new ha chlings we e colou ma ked wi h ood colou ing on hei head o back down ea he s. As pa o a sepa a e s udy, a 1 o 2 days pos -ha ch (dph), each chick was c oss- os e ed o c ea e ei he la ge (six o se en chicks) o small b oods ( wo o h ee chicks). All chicks included in his s udy we e ea ed in enla ged b oods and hus expe ienced a challenging en i onmen , which included a possibili y o ood sho age, he need o enhanced begging a es and compe i ion o space [53]. The mean b ood size du ing he expe imen o he o al du a ion was 5.56, s.d.: 0.699. This numbe is sligh ly lowe han he ini ial b ood size o six o se en chicks, owing o na u ally occu ing dea h o chicks h oughou he nes ling pe iod and asynch onous ledging. 3 oyalsocie ypublishing.o g/jou nal/ sos R. Soc. Open Sci. 11: 240417 Downloaded om h ps:// oyalsocie ypublishing.o g/ on 08 Augus 2024 2.2. S udy design and TH adminis a ion The hy oid gland p oduces mainly hy oxine (T4), which is con e ed o iiodo hy onine (T3) by deiodinase enzymes [54] while main aining a ce ain T3/T4 a io in he plasma. Ou ea men consis ed o T3, as i is he biologically ac i e o m o TH, because o i s gene ally highe ecep o a ini y han T4 [55]. We aimed o ele a e T3 wi hin physiological anges du ing he main g ow h phase; howe e , he daily T3 p oduc ion a e o ha chling zeb a inches is no known. Thus, we conside ed he daily sec e ion a e o 1–3 µg T4 pe 100 g body mass as shown in o he bi d species [56] in combina ion wi h da a on he T3 o T4 a io, which is a ound 0.5, as shown in he ci cula ion o de eloping zeb a inches [57]. We selec ed a ea men dosage o 0.5 µg T3 pe 100 g body mass. We achie ed his dosage by mul iplying he lowe ange o he known sec e ion a e o 1–3 µg T4 pe 100 g body mass wi h he known T3/T4 a io: (1 µg T4 pe 100 g body mass) × (0.5 T3/T4 a io). We decided o use he lowe ange o he known T4 sec e ion a e o ou calcula ion o a oid o e dosing. All con ol chicks ecei ed he ec o solu ion. Since mi ochond ial unc ion is sensi i e o s ess, o example, media ed ia glucoco icoids [58], ou aim was o minimize handling s ess as much as possible. Fo his eason, T3 was adminis e ed e e y second day (i.e. on dph 5, 7, 9, 11 and 13 wi h a 200 µl pipe e, adjus ed o he exac body mass o each chick, ± 0.01 g) o ob ain peaks o ele a ed T3 le els h oughou he main g ow h pe iod be ween 5 and 14 dph [33,59]. Blood sampling was done a 14 dph (ca 20 h a e T3 adminis a ion in he expe imen al g oup) o measu e ci cula ing T3 concen a ions and mi ochond ia unc ion. T3 was adminis e ed in a wi hin-b ood design, hence wi hin each b ood, one andomly chosen chick ecei ed T3 (n = 19). Fo he con ol g oup, ei he a sibling o he TH- ea ed chicks (ha ched in he same nes o o igin), was chosen o accoun o po en ial gene ic simila i ies (sibling pai s be ween TH- ea ed and con ol chicks n = 16), o , because o a lack o a ailabili y, chosen andomly ( o al chicks wi hou gene ic sibling n = 19). Fo an illus a ion o he s udy design, see igu e 1. Each os e nes con ained a a ying numbe o ocal chicks ha a e included in his s udy: 11 nes s con ained one o wo TH-chicks and one– h ee con ol; ou nes s con ained one TH-chick each and no con ols; one nes con ained one con ol chick and no TH-chicks; and wo nes s con ained h ee con ol chicks and no TH-chicks. 2.3. TH- alida ion g oup A sepa a e g oup called ‘TH- alida ion’ (n = 7) was used o con i m ha he ea men led o he desi ed peaks o ele a ed plasma T3 sho ly a e each adminis a ion. In he TH-chicks, he las T3 dose was gi en a noon on 13 dph and blood sampling (see §2.6) was done a 14 dph a a ound 09.00. Hence, i is likely ha T3 gi en on 13 dph had been me abolized in la ge pa s be o e blood sampling [33,59,60], and he e o e T3 plasma le els a ha ime poin do no e lec he ull ex en o which T3 plasma le els whe e aised on mul iple occasions h oughou he ea men pe iod. To con i m ha he ea men did in ac aise T3 plasma le els as expec ed, we added he so-called ‘TH- alida ion’ g oup (n = 7) o ou expe imen . To ou knowledge, he o al up ake a e o T3 in bi ds has no been es ed while in humans T3 plasma le els each hei peak abou 2.5 h a e o al adminis a ion [60]. Since bi ds ha e a as e me abolism han humans—e.g. mass-speci ic basal me abolic a es a e 30–40% highe in bi ds han mammals [61]—we expec ed plasma T3 le els o each peak le els a ound 1 h pos adminis a ion in ou nes lings. To con i m his, he TH- alida ion g oup ecei ed hei las ea men dose on 14 dph, 1 h be o e aking he blood samples, ins ead o on 13 dph, like he es o he bi ds in he s udy. O he han he inal dose lagging o 1 day (14 dph ins ead o 13 dph), he TH- alida ion g oup was ea ed in he same way as he TH-chicks ( he expe imen al g oup). The TH- alida ion g oup was used o con i m he T3- ea men and included in he g ow h cu e analysis, bu was no pa o any o he analysis in his s udy. To e i y ha he ea men ele a ed T3 plasma le els in he TH-chicks as expec ed, a sepa a e eg ession model was un in which plasma T3-le els o he TH- alida ion g oup we e included in he TH-chicks g oup, see §2.9. The TH- alida ion g oup was no conside ed o any o he analysis. 2.4. TH and ec o solu ion Fo he p epa a ion o he ho mone solu ion, c ys alline T3 (3,3′,5- iiodo-L- hy onine, g ea e han 95% HPLC, CAS numbe 6893-02-3, Sigma-Ald ich) was i s dissol ed in 0.1 M NaOH (NaOH eagen g ade, g ea e han o equal o 98%, anhyd ous pelle s, CAS numbe 1310-7-3-2, dilu ed in MilliQ 4 oyalsocie ypublishing.o g/jou nal/ sos R. Soc. Open Sci. 11: 240417 Downloaded om h ps:// oyalsocie ypublishing.o g/ on 08 Augus 2024 wa e ) and hen his T3–NaOH solu ion was dilu ed a 1 : 60 wi h ap wa e [62]. The concen a ion o he solu ion was adjus ed, so ha a 1 µl inal solu ion could be adminis e ed pe g am body mass o he chick [63], wi h a dosage o 0.5 µg T3 pe 100 g body mass. Fo he ec o p epa a ion, 0.1 M NaOH solu ion was dilu ed a 1 : 60 wi h ap wa e . P epa ed ec o and ea men aliquo s we e s o ed a −20°C and kep on ice un il use. Thawed aliquo s we e s o ed in he idge and used wi hin 1 day. 2.5. Chick g ow h In o de o moni o he ha chlings’ de elopmen and o adjus he ea men dosage (see §2.2), body mass (KERN, ± 0.01 o Pesola sp ing scale, ± 0.25 g) and a sus leng h we e aken a 0, 3, 5, 7, 9, 11, 13 and 14 dph. Because TH can a ec ei he skele al g ow h [33], muscula g ow h [64], a deposi ion [65] o po en ially all o hese ac o s, we assessed body condi ion in addi ion o a sus leng h and body mass measu es, o es i he e ec s o TH on ei he o hese g ow h pa ame e s a ec ed body condi ion. To assess body condi ion, we used he scaled mass index (SMI) [66] be ween body mass and a sus leng h a 5, 7, 9, 11, 13 and 14 dph. Pea son’s co ela ion coe icien o body mass and a sus leng hs anged om 0.76 o 0.85, wi h p- alues less han 2.2 × 10−16. G ow h a es we e calcula ed sepa a ely o a sus leng h and body mass and om he e on will be called ‘skele al g ow h a e’ and ‘mass g ow h a e’, espec i ely. G ow h a es we e calcula ed o 5 dph ( he s a o he ea men ), 7, 9, 11 and 13 dph in he ollowing way (see also [67]): (Ta sus( )–Ta sus(s))/( –s), whe e s is he p eceding measu emen o ime poin . The ime poin 14 dph was no included in g ow h a e calcula ions. While all o he measu emen s we e aken a noon, he measu emen a 14 dph was aken in he mo ning sho ly be o e blood sampling. Thus, body mass was expec ed o be lowe a 14 dph compa ed wi h 13 dph because o he ime o he day. Ta sus leng hs did no change be ween 13 and 14 dph. 16 sibling pai s, 19 chicks wi hou gene ic sibling Nes o O igin (n=23) Fos e Nes (5-6chicks pe nes , n=18) Con ol n=25 Con ol T3 TH-chicks n=19 TH- ea ed chicks n=26 TH-Valida ion n=7 Figu e 1. Schema ic s udy design and sample sizes pe g oup. The assignmen o chicks o con ol (blue squa e), TH-chicks (expe imen al g oup, o ange squa e) and TH- alida ion (no included in analysis) g oups is shown, as well as an example o he wi hin-b ood design in he os e nes s. Pho o c edi : Kim Holzmann. 5 oyalsocie ypublishing.o g/jou nal/ sos R. Soc. Open Sci. 11: 240417 Downloaded om h ps:// oyalsocie ypublishing.o g/ on 08 Augus 2024 2.6. Blood sampling Blood samples we e aken wi hin 6 min o en e ing he a ia y by b achial enipunc u e om indi idu- als a 5 dph, o a sepa a e s udy, and a 14 dph, o ob ain plasma samples and mi ochond ial measu es o his s udy. A maximum amoun o 1% o he nes ling’s body mass was aken. Blood was s o ed immedia ely on ice and cen i uged wi hin 2 h a 2000g o 10 min. Plasma was s o ed a −80°C and analysed wi hin six mon hs. Red blood cells (RBCs) we e p ocessed immedia ely o mi ochond ial measu emen s, acco ding o [68]. 2.7. Mi ochond ial measu emen A ian e y h ocy es possess unc ional mi ochond ia [69,70], allowing us o assess mi ochond ial unc ion in in ac RBCs [68]. We used in ac RBCs o analyse mi ochond ial ai s in he na u al physiological s a e o he bi d, wi hou a i icially al e ing hei immedia e en i onmen , e.g. subs a e a ailabili y in he cell. We included he ollowing i e mi ochond ial a iables in his s udy: cellula me abolic a e (CMR)— he o al amoun o oxygen consump ion in he cu en cellula condi ion; p o on leakage (‘Leak’)— he po ion o mi ochond ial espi a ion no used o ATP p oduc ion; oxida i e phospho yla ion (‘OxPhos’, calcula ed by sub ac ing leak espi a ion om CMR)— he po ion o he mi ochond ial O2 consump ion cu en ly used o ATP p oduc ion; maximal wo king capaci y o he elec on anspo sys em (‘ETS’)— he maximum capaci y o he mi ochond ia o p oduce ATP unde he cu en cellula condi ions, including subs a e and elec on chain enzyme a ailabili y; and he lux con ol a ios (FCR) L/CMR ( a io be ween leak and CMR) [68]. Fo assessing mi ochond ial unc ion, a Cla k elec ode high- esolu ion espi ome e (Oxyg aph-2k, O obo os Ins umen s, Innsb uck, Aus ia) was used on in ac RBCs. An aliquo o 7–30 µl RBC (mean 16.33 µl; s.d.: 5.99 µl) was aken a e cen i uga ion o he blood sample om he bo om o he ube wi h a cu - o pipe e ip. All measu emen s we e no malized o he olume o RBC used o he analysis. The RBC aliquo was hen gen ly homogenized in cold espi a o y MiR05 bu e (0.5  mmol l−1 EGTA, 3 mmol l−1 MgCl2, 60  mmol l−1 po assium lac obiona e, 20  mmol l−1 au ine, 10  mmol l−1 KH2PO4, 20  mmol l−1 Hepes, 110  mmol l−1 suc ose, 15  mmol l−1 a y acid- ee bo ine se um albumin, pH 7.1). The RBC-bu e solu ion was cen i uged a 500g o 5 min and he supe na an was emo ed. Washed RBCs we e esuspended in esh Mi 05 bu e a 40°C and added o he espi ome e chambe . Then, RBC oxygen consump ion was quan i ied acco ding o he ollowing p o ocol: (i) CMR, o en called ‘Rou ine’ espi a ion, is he basal espi a ion o he cell; (ii) 1  µg ml−1 oligomycin was added o he chambe o inhibi ATP-dependen oxygen consump ion and o assess p o on leak, he e called ‘leak’ espi a ion; (iii) 1  µmol l−1, i a ion o ca bonyl cyanide m-chlo ophenyl hyd azine, a mi ochond ial uncouple was added, o assess he maximal capaci y o he elec on anspo chain, so-called ‘ETS’; (i ) 5  µmol l−1 an imycin a, o quan i y non-mi ochond ial oxygen consump ion. Non-mi ochond ial oxygen consump ion was sub ac ed om all measu es abo e [68]. 2.8. T3 ex ac ions—liquid ch oma og aphy–mass spec ome y In b ie , 15 µl o plasma was mixed in me hanol and spiked wi h a known amoun o 13C12-T4 (La odan, Sweden) o ack eco e y. We i s pe o med liquid–liquid ex ac ion wi h chlo o o m and me hanol. Samples hen wen h ough solid phase ex ac ion using chlo ide- o m anion exchange esin (Bio-Rad) o pu i ica ion and we e d ied unde N2. The inal p oduc s we e e-dissol ed in 0.01% NH3 and plasma T3 and T4 we e simul aneously measu ed using a alida ed nano- low liquid ch oma og aphy–mass spec ome y (LC-MS) p o ocol, o de ails see [71]. 13C6T3 and 13C6T4 ( om Sigma) we e spiked in each sample as in e nal s anda ds, and T3 and T4 we e quan i ied using peak a ea a ios o sample o i s in e nal s anda d. Se e al quali y con ol samples we e also included. Each sample was analysed once, and ac oss sample ypes, he in e -assay a e age CV% was 6.9 o T3 and 4.4% o T4 [71]. MS da a we e acqui ed au oma ically using The mo Xcalibu so wa e (The mo Fishe Scien i ic) and analysed using Skyline [72]. 2.9. S a is ics All s a is ical analyses we e done in R, e sion 4.2.2 [73]. We used gene alized linea models ia he s an unc ion om he package ‘ s ana m’, e sion 2.21.3 [74] o calcula e pos e io means and 95% 6 oyalsocie ypublishing.o g/jou nal/ sos R. Soc. Open Sci. 11: 240417 Downloaded om h ps:// oyalsocie ypublishing.o g/ on 08 Augus 2024 c edible in e als (C I), om 4000 simula ed alues om he join pos e io dis ibu ion o he model pa ame e s. The p io dis ibu ion o s ana m can be classi ied as weakly in o ma i e, wi h a Gaussian dis ibu ion and scaling based on he dis ibu ion o he da a. The de aul p io o each a iable is a no mal dis ibu ion cen ed a 0. The scale (s.d.) o his p io is calcula ed by he p og am in a da a-dependen way, wi h he scale ha is a unc ion o he esponse a iable (see elec onic supple- men a y ma e ial, able S5, whe e p io s o each dependen a iable ha e been epo ed). P io s ha e a mean o ze o o all g oup di e ences, hus a oiding bias owa ds signals om ei he he expe i- men al o con ol g oup. The mean o he in e cep is pooled om da a om bo h g oups [75]. The speci ic p io pa ame e s can be ound in elec onic supplemen a y ma e ial, able S5. We modelled he esponse a iables pe p edic o s o in e es ia a Gaussian e o dis ibu ion and he ‘Iden i y’ link unc ion. We epo s a is ical unce ain ies o he es ima es using 95% C I and pos e io p obabili ies o speci ic hypo heses o each s a is ical es . Fu he , we calcula ed he pos e io p obabili ies (P), by assessing he p opo ion o simula ed alues in line wi h he hypo hesis, o each model p edic o [76]. Residual dis ibu ion was assessed by using no mal quan ile–quan ile plo s, Tukey–Ascombe plo s and by plo ing esiduals agains le e age. Model con e gence and i we e assessed ia he unc ion ‘shinys an’ in he ‘ s ana m’ package. Fu he mo e, he R2 was calcula ed, as well as he esidual a iance o he model and he a iance o he andom ac o s. To es o he e ec o he T3 ea men on ocal pa ame e s, he a iable ‘T ea men ’ (Con ol: n = 25; TH-chicks: n = 19) was used as a p edic o . In all models, he nes o bi h ‘nes o o igin’ (n = 23) was included as a andom e ec , o con ol o gene ic simila i ies be ween siblings. To con ol o di e en ea ing condi ions ac oss nes s, ‘ os e nes ’ (n = 18) was also included as a andom ac o in all models. To accoun o possible ime e ec s (23 weeks o he expe imen ), we included sampling o de ep esen ing he o e all ha ching o de as a co a ia e in all models. Sampling o de is a con inuous sequence o he o de o bi h o indi iduals, simila o Julian da es. All con inuous p edic o a iables we e s anda dized by compu ing z-sco es ((x-mean)/s.d.). Fo some indi iduals, da a we e missing (e.g. low amoun o blood, ailed mi ochond ial measu emen s, missing da a), hence sample sizes a e sligh ly di e en be ween he models (see elec onic supplemen a y ma e ial, ables S2–S5). Plo s we e gene a ed using he package ‘ggplo 2’ [77]. In o de o assess whe he he T3 plasma le els di e ed be ween he g oups, and whe he his di e ence was enhanced by he TH- alida ion g oup (see §2.3), we an one model in which he TH- alida ion g oup was included in he TH-chicks g oup (Con ol: n = 25; TH-chicks including TH- alida ion g oup: n = 26) and one model wi hou he TH- alida ion g oup (Con ol: n = 25; TH-chicks wi hou he TH- alida ion g oup: n = 19). T3 alues we e log- ans o med o achie e a no mal dis ibu ion o he da a and o ob ain he bes i o he models. F equen is esul s can be ound in elec onic supplemen a y ma e ial, ables S6–S8.2. I one o he andom ac o s (1|nes o o igin) o (1| os e nes ) explained close o 0 a iance in he equen is models, i was emo ed om he model o a oid con e gence issues. Resul s om equen is models a e he same compa ed wi h Bayesian in e e ence ac oss all models. 2.10. Mo phome ics To es he hypo hesis ha he TH- ea men posi i ely a ec ed he a o emen ioned mo phological measu es, we an one model wi h a sus leng h a 14 dph, one wi h body mass a 14 dph and one wi h SMI a 14 dph. We i s es ed o a di e ence in baseline measu es a 5 dph in all h ee a iables, by unning one model pe a iable wi h he baseline measu e a 5 dph as an ou come a iable. As sampling o de did no ha e a meaning ul e ec in he model o a sus leng h a 5 dph, we emo ed i because o con e gence issues. Ta sus leng h a 5 dph, hence be o e he onse o he ea men , was smalle in TH-chicks (es ima e [95% C I], P(ß < 0): −0.59 [−1.028; −0.163], 0.995. See elec onic supplemen a y ma e ial, able S3, igu e S1). The e was no di e ence be ween he g oups in body mass a 5 dph and SMI a 5 dph (see elec onic supplemen a y ma e ial, able S3 and igu es S2 and S3). We con olled o baseline measu es a 5 dph in all h ee models es ing he pa ame e s a 14 dph ( a sus leng h, body mass, SMI), o accoun o he ela i ely high bu na u al a ia ion a 5 dph, which can be seen in he dispe sion o he aw da a (elec onic supplemen a y ma e ial, igu es S1–S3). In his way, we could disen angle he e ec s o he ea men om exis ing p e- ea men di e ences. To es o di e ences in g ow h be ween he g oups, we used gene alized addi i e mixed models (GAMMs) wi h he s ana m unc ion s an_gamm4 (see [67]). We included ‘ ea men ’ as a ixed e ec and a smoo hing e m wi h an in e ac ion be ween age and ea men , o allow o di e en shapes o he cu es in he wo g oups. Fu he , we included andom in e cep s o ID o e Age o accoun o 7 oyalsocie ypublishing.o g/jou nal/ sos R. Soc. Open Sci. 11: 240417 Downloaded om h ps:// oyalsocie ypublishing.o g/ on 08 Augus 2024 di e ences in indi idual g ow h ajec o ies. GAMMs we e calcula ed o skele al and mass g ow h a es, as well as o body mass (g), a sus (mm) and SMI o he ime poin s 5 dph (s a o he ea men ), 7, 9, 11 and 13 dph. 2.11. Mi ochond ia To es o di e ences in he i e mi ochond ial ai s o in e es (CMR, OxPhos, ETS, Leak and FCR L/CMR) we an one model pe ai , wi h he ai being he esponse a iable. CMR, OxPhos, ETS and Leak we e log- ans o med o achie e a no mal dis ibu ion o he da a and o ob ain he bes i o he models. Du ing he ea men , we adminis e ed T3 e e y second day o ob ain peaks o T3 plasma le els, wi h he aim o egula ly ele a ing T3 h oughou he main g ow h phase. In humans, TH ha e been shown o a ec mi ochond ia di e en ly in he sho e m (wi hin minu es o hou s a e ea men ) e sus he long e m (wi hin weeks) [15]. Ou s udy ques ion aimed o add ess he e ec s o ele a ed T3 on mi ochond ia h oughou he main g ow h phase. Acu e le els o T3 a he ime o sampling may di e be ween indi iduals, o example owing o a ying speeds o me abolizing he T3 o he eeding s a us a he ime o adminis a ion. Thus, we included acu e T3 plasma le els as a co a ia e o con ol o immedia e e ec s o T3 on mi ochond ial unc ion ha may di e om he e ec s o p olonged ele a ed T3 le els h oughou he ea men pe iod. Fu he mo e, we included body mass a 14 dph (which was no s a is ically meaning ully a ec ed by he ea men , see elec onic supplemen a y ma e ial, able S3) as a co a ia e, o con ol o body mass di e ences no a ibu ed o he ea men . We e- an he models o mi ochond ial ai s wi hou acu e T3 le els o es whe he he esul s changed, and we did no ind a di e ence in ei he model i o meaning ulness o esponse a iables (see elec onic supplemen a y ma e ial, able S4.1). Ta sus leng h be o e he onse o he ea men (a 5 dph) happened o be smalle in TH-chicks han in con ol chicks, while he g oups did no di e in a sus leng h a 14 dph. To con i m ha mi ochond ial ai s we e no a ec ed by po en ial ca ch-up g ow h, we e- an he models o mi ochond ial ai s wi h a sus leng h a 5 dph as a co a ia e. Body mass a 14 dph was hen emo ed om he models since body mass a 14 dph is expec edly highly dependen on a sus leng h a 5 dph. Ta sus leng h a 5 dph did no ha e a meaning ul e ec on mi ochond ial ai s, no did model i o meaning ulness o ou come a iables change (see elec onic supplemen a y ma e ial, able S4.2 o ull ou pu s o models including a sus leng h a 5 dph). 3. Resul s 3.1. TH- ea men As expec ed, T3 concen a ions 1 h a e adminis a ion in he sepa a e alida ion g oup ‘TH- alida- ion’ (pmol ml−1 mean: 2.568, s.e.: 0.313), and in he expe imen al g oup abou 24 h a e adminis a ion (pmol ml−1 mean: 1.825, s.e.: 0.067) we e inc eased and wi hin he ange o he physiological le els ound in he con ol g oup (pmol ml−1 mean: 1.68, s.e.: 0.991). T3 plasma le els in he TH-chicks anged om 1.315 o 2.25 pmol ml−1, which is wi hin he ange o 0.825 o 2.674 pmol ml−1 o he con ol g oup. As expec ed, T3 le els in he TH-chicks we e s a is ically ma ginally highe han in he con ol g oup (pmol ml−1 es ima e [95% C I], P(ß > 0): 0.127 [−0.016; 0.268], 0.962) and meaning ully highe when TH- alida ion chicks and TH-chicks we e analysed oge he (pmol ml−1 es ima e [95% C I], P(ß > 0): 0.206 [0.066; 0.348], 0.997) (see igu e 2, elec onic supplemen a y ma e ial, able S2). Fu he mo e, T3-le els we e simila o hose obse ed in nes lings o o he passe ine species, ed-winged blackbi ds (Agelaius phoenicius) [33] and g ea i s [78]. 3.2. G ow h TH-chicks had smalle a sus leng hs a 5 dph be o e he onse o he ea men (es ima e [95% C I], P(ß < 0): −0.59 [−1.028; −0.163], 0.995; see elec onic supplemen a y ma e ial, able S3, igu e S1). The las ea men dose was gi en on 13 dph and 1 day a e he las ea men dose, on 14 dph, he TH-chicks did no di e in body size (in a sus leng h o body mass) compa ed wi h he con ol g oup ( a sus leng h in mm es ima e [95% C I], P(ß < 0): −0.006 [−0.327; 0.317], 0.514). Body mass in g ams es ima e [95% C I], P(ß < 0): −0.381 [−0.97; 0.21], 0.906; see elec onic supplemen a y ma e ial, igu es S4 and S5). 8 oyalsocie ypublishing.o g/jou nal/ sos R. Soc. Open Sci. 11: 240417 Downloaded om h ps:// oyalsocie ypublishing.o g/ on 08 Augus 2024 Re e ences 1. Beh inge V, Deimel C, Hohmann G, Neg ey J, Schaebs FS, Deschne T. 2018 Applica ions o non-in asi e hy oid ho mone measu emen s in mammalian dcology, g ow h, and main enance. Ho m. Beha . 105, 66–85. (doi:10.1016/j.yhbeh.2018.07.011) 2. Kees a S, Högq is Tabo V, Al e gne A. 2021 Rein e p e ing pa e ns o a ia ion in human hy oid unc ion: an e olu iona y ecology pe spec i e. E ol. Med. Public Heal h 9, 93–112. (doi:10.1093/emph/eoaa043) 3. 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