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341Mitochondrial
Respiration
and Triiodothyronine
Concentration
in Liver from Postpubertal
and Adult Rats
5. ossa, L. Lionetti, N/].
P [/]ollica,
R. Crescenzo,
N4.
Bottè, C. Liveini
Depanment ol Genefal and Environmenta Phy5iooqy. ì-rnlveEityofNapes "FEDERICO 1",ltaly
I The purpose ofthìs stùdy was to investigate the decline in rat liver mitochondrìa rèspìratìon found in adult rats.ompared toyounqerones, and to find a link between this respi.atory im-pairmentand atissue hypothyroidism statè. To this end. hepatìc .oncentration and serum levels of t.iiodothyronine were mea sured in postpubertèl Ets (60 dèys old)and adult rats (180 days old).In addìtìon, in these rats we measured oxidative phosphoF ylation in homoqenate together with coupled and un.oupled respiration in isolat€d mitochondrìa úsinq succinate or durohy droquinone as substrate. We found that mitochondria from adùlt rats consumed less oxygen compared toyounger rats due to lower electron transport chain and phosphorylating system activity. In addition, we found that in state 4.ondition, mito-chondria from adult rats consumed less oxygen than mitochon-driè from young rats. Finally, we found a decreas€ ìn liver triio dothyronin€ concentration in adult rats. In concllsion, the fe-sults of this study showthat hepatic mitochondria in adult rats have a decrèased AtP synthesis capacity and proton pemeabil-ity, both consisientwith the tissue hypothyroidism found in the
r Key words: Homogenate Respiration - fi4ito.hond.ialProtein Mass - Uncoupled Respiration - Cltochrome Content - SerLrm lriiodothyronìne Level
Thyrcid hormones :re impo|i:nt factorc in lhe regulation of hepatic DretaboÌism and fnitochondrìa coold be consid€red as targets ofthyroid hormone actìon in vìew of their central role in eneryy metabolism. In fact, thyrcìd homones rcgùÌate nany of the úìitochondrial m€tabolìc functions l1l ard tuflu-eDce oxidative phosphorylation I2l. In particular, it has been sbown that in ìiver mitochondria liom hypotby.oid rats, oxy gen consunption and .ATP synthesis are lower tban thole oi liver mitochondria ftonìno|nlal rats l3 ì. Intercstinsly, oùr p.e vìoùs resùlts showed that the rate of FADlinked fespimtioD significandy decìined in mitochondria from 180-day-old rats compa.ed with those fromyoùnger rats {41.In contrast to the above results, the rate of NAD lìnked respiration was foùnd
undiminished withage. It seems thatthe age relateddecrease in nitochondri:1 respiration k sublrate specifìc. Onepossibìe explanation may be that with NAD ìinked sùbst.ares. rhe enrry of metabolites ìnto theeìectrontransportchainis l?te limitirìg lof rcspìration, so tlìat tbe decrease in respifation becom€s masked. Therefore, FAD-link€d respifation allowed us to de-tect at the liver level the fifst age-linked bìochenical impaìr-ment of energy transdùcing parhways.ln ofde. to have infor
n d l o n à b o u l
" p o i b e i T p ì ' c d r i o n o t t \ j r o i d ì ' o - m o r e 5 i n this .espi.atory impairnent, variations iD serum íree tdiodo-tbyronine (Tr) levels werc cbecked h postpubertal (60 days) a n d r d u l r r d t c r l 8 0 d a \ , 1 ( r n c e r \ c u e h y p o . h \ r o r d r s r n I n " y o c -cuf ìn the presence of no.mal o. modestly dec.€ased serum r h y r o r d h o r n o ì é e v e . [ 5 ] . { F r l r o n e r q r e d h e p d r , l , r o l
ìr addition, we were interested hidertiryingthe sites rcspon-sible fof th€ decreas€d fespimtion rate foLnd in isolated mìto chondria fron1 180 day old rats. changes in oxygen consump tìon in isolated mùochondria may essentialìy reflect modìfica tìons in the features ofrespì.atory chaiú and/or ATP synthesis system, as weÌl as in the suppìy of reducjng equivalents into tlìe respiratory chain I6ì. ln this study, we nìeasured tre fes-piration rates using succinate or dùfohydroquinone as b' str:te. Dùrohydroquinone was used tÒ elìminate the regula tion of subfrate sùppÌy, namely of dicarboxylate carier and substrate dehydfogenase. The cyloch.ome content was also d€te|mined in order toverify ifany clìarg€s in tbe compon€nts ofthe electfon chain occurfed. rinally, we assessed uncoupled respifation to evaluate the maximal€lectron flow through the respiratory chain. The respiration measùrements were carfied out ìn liver homogenate and isolated mitochondria from post pubeúal and aduit llts.
Materials and Methods
Male Wistar rats were obtained frol]ì charles River (calco, como, ìtaly). The rats were hoúsed at 24 c únder an artificial cìrcadìan 12 h 1ìght/12 h dark cycle, with rd libihrm access to
H o r m M e t a b R e s 2 0 0 1 ; 3 3 : 3 4 3 - 3 4 7 O Ceo|g Thieme Ve.lag Stùttgart New York ISSN 0018 5043
34ì Hom r\/l€tab R€s 2OOr ll
waterand a standard stock diet (Mucedola 4RF2l, Settino Mi lirDese, Milàn, lta ly) belore ìreiDssacdficed.Two sroups ofnine ratswerc used: postpubertalard ad ult, wbich were 60 and 180 days old àt tiùe ofsacrifice, rcspectively. Annnalcare, horsing, and kjllìng were carrìed out accorljng to the guirlelìnes ofthe ìtaììan HeaÌth Mìnist.y.
Preporatian af honogenotet ond isaloted mitochondrio The rats. withoút any previoùs food deprìvatìon, were anaes
thetised with chìoral hydrate (40m9/100g body weight). BÌood was coÌìectedvia the intè.ior cavavein, and se.um san ples werc storcd at 20'C. Livers werethe.lieed ofblood by "in situ pel.fusioD usins 1s0ml ofa cold solution of220nlM m:nojtol,70mM mcrose, 20nlM Hepes, pH 7.4, 1 oìM EDTA, and 0.1% (wivJ fatty acid free bovine seiùm albumin (BsA). Atter pe.tusìon, Ìive6 we.e qùickly renoved. bìotted, weighed, and aliquots werc in]mediateìy frozen in ìiquid nitfogen fof the dete|nlination ofliverTr concentratior. The rcmaiDdef of the liver was fìnely nnrced, gently homogerìsed with th€ d b o v F r e d n / l : 4 . w A l r d o è r I v p h t e n h o m o g e ì r " set at s00rpm (a strokelmìn), and filtered th.ough ste.jle gauze. Afte. wìthd.awal of aÌiqùots of the fresh homogenate for respiration measurements, ìt was furthef processed fof pfepamtion of isolated nitocbondria as previously repofted l / ì . 1 h è p r o r p r r o ì F r r o ' r 5 è n r r o . h o r . L n a \ U c p e ì c r o n w , , . determined accordìng to the úlethod oiHartree [8ì, using bo vine serum albùmin as the protein standard.
Poloroqrophic neosurement of rcspirotion in livet honogenotes and nitachond a
MitochondriaÌ oxygen consùnption in homogenates was e5ti mated using a Claflctype electrode (Yellow Springs lnstru-ments, Yellow sprinss, oH, UsA), maftaìlled inwater jaclrcted chamber at 30 'C. AliqLrots of the homogenate were added to 3 ml of the respiratory merlium containingS0mM (cl,50 mM H E P E S , p H 7 . 0 , 5 m M K H z P O a , l m M E C T A , 0 . 1 % ( w / v J B S A , a n d state3 and 4 rcspi.atory.ates were measured as prcvioùsìy '€ ported I9l. Oxysen consumptioD in isolated mitochordria was m€asLrfed polarogfaphìcally with tbe above-nlentioned clarlc type elecrrode. kolated mirochondrìa (1 mg prorein) were in cubated in 3ml of the above .espiEtory mediun at 30"C. Measurenents were nade wùhin 2 h foÌlowing the isolation of tbe nìitochonddr. The mitochoDdda were allowed to oxi-dise theif endogenous nrbstrates for few minutes. State 4, coopled oxyg€n consLrmplion (state 3), and Lrncorpled oxygen consùmptionwere then measored in the presence oisrÌccinate 1 0 m M + . o t e n o n e 3 . 7 5 F M o . d ù . o h y d i o q u i n o n e 4 . 5 m M + . o renone 3.75 !M as substrates. State4oxygen consumption fate was det€|mhed by addnrg olìgon]ycio (2$/mg ofpmteinl to preventATP synthesìs. Coupled respiration rate was neasored by adding ADP at a lÌnalconcentratìon oi0.l mM. Uncoupled respiration rate was determìned by adding carbooyì cyanìde Ìr t.iflùoromethoxyphenyìhydrazone IFCCP] at the final con ceDtratior of 1!M iD the presence of olisomycin (2lsillls of prot€in). R€spiratorycontrol ratio (RcR)and ADP/O ratìos werc calcrlated accorling to Estabrook [10ì. ATP prodLrction rate dùring coùpled.espi.ation was measù.ed monito.ing the dis appeafance of inoryanic phosplìorus in the above rcspifatory n ì e d i u m i r t h e p r e s e r c e o f s ù M A D P I 1 1 1 .
Deterninotian of cttochtone content and enzyne a.tivily cytochrcl]lecoDtentolisolated mitochondfìàwas det€rmìned as pf€viously 'epofied [12]. Succinic dehydrogenase (SDH) (E.C.l.l.99.llactivity was measù.€d by the method desc.ibed by Lee and La.dy ll3l in homogenate and isolated
mitochon-Deterninotions of livet 13 concentrotian antl setún free I3 level
To extract T3 Uon ììver sanpÌes, we used the pfocedure of Moreale de Èscobar et al. []4ì. Recovery ofùe extncted iodo-tlryronire was cafied out in n similar w:ìy to tbàt feported by M o r r e a l e d e t s c o b a . e t a l . I l 4 l ( s 0 7 s % ) . T o m e a s ù r e L h e c o n ' " r l . l o r o ' l \ p e r r d , p d . o n n p r r ' d d r o r r n u r o r \ say kit (ìCN PharmaceùtìcaÌs, Diagnostic Divisìon, New Yo ( USAJ was used. The standard cuNe was constructed by using caliblltof nude addiDg pureTj to plìosplìate buffer so that Tl coDcentfation raDges from appfoxnnately 100 to 1000pgiml. The same radioimnonoassay kir was aho rsed for determìna rion olserum free T3 levek. Measu.ements of live. T3 concen t.ation and se.um liee T3 Ìeveìwere carfied out ì. a singÌe as say !o.emove rnrerassayva arons.
Data aregiven as means l SEM fron nine differc.t |?ts. Statis-lical analyses were peformed by two tailed uDpaircd Stu-deDts f-test or two-way aDalysis ofvariance (ANoVAl fof re-p " 3 è d n e ì . l e . . l o b i b r l r y v a r re-p ' l e . ' r l ' re-p ì 0 0 s s F r F r o n side.ed to jndicate a significant difference. AÌì anaÌyses were peúbrmed using CfaphPad Prism (GraphPad Softwarc, SaD Drego, CA).
ADq .otenone, succinate. FCCP, and duroqùinone we.e pùr chased from Sigma Chemicaì Co., St. Louìs, USA. Very hìgh specific activity L25l-labeled Tj was purchased ftom NEN Life Sci€nce Pfoducts, lnc, Boston, USA. All othef reagents were ot the highest prrjty commercialìy available.
Results
Hepatic mitochondfial respìratory rates ìn lìve. homogenates from 60- and 180-day okl rats osing succinate as the súbfrate afe reported ìn Tablel. The measùfements made in Ìjve. homogenates alÌow ùs to link mìtochondriaÌ oxidatrve actrv ities with tbe effective nìitocbondrial prcteir nìass. State 3 d r d n o r ' i r n r f .d n r v , h r n g e . d i í e r p n r d g p . . w \ r F " r . r e 4 c r g nifìcantly decreased in 180 day old rars compared to yoùnger rats.The n1ìtochondrìàl protein ùrass (mg/g liver)was calculat ed by divìdjng the values ot the SDH activùy in lìver homoge nates by the vaìues ofthe SDH activity in isolated liver mito cbondfia. Tbis calcuÌatior caD be doDe sìllce ouf mitochondrial prcparations afe vìrtrlally pLr.e as shown by rhe resùlrs olcon trol experìmenLs, ìn whìch marl(er enzyme actìvìties ofplarma memb.rne and o.ganelÌes have been determined. Mitochon ddalprotein mass was sisnificantly hisheriD 180-ddy-old rats
Livef Tiiodothyronine and Resplfauon
H o m
M e t a b
R e s
2 o 0 l ; x m
Tablèl Liver mltochond ahydrcqenóe (sDH) activity, day od and r8o-day-old rats
respnation in hofroqenate, succlnlc de-and ritochondia proteln mass in 60
E
60'day-old rats '180-day od rats % change
É
'
"
E
6
É 4 u . = ó 3 0 . = c6
E
1 0 0
SDN activity in 9 9 9 2 1 4 3 2 I l 9 l 1 6 6 258!24 8 249:r 8ll 8 6 s r 5 6 ' I 1 . 2 1 0 . 5 22M1' - 1 1 2 1 8vaues rè thÈ nìeèns l SEM of nif,e differcnt Ets.:l .hanqè referred to 6 0 d a y o L d F t s : 'P < 0 0 s . o m p a É d 1 0 6 0 . d a y o d a t s ( t w o t a i e ! s t u d è n r s
ADP- and FCCPrtimuìated respìration (coupled and uncou pled respiration, fespectively) measù.ed ìn isolated mitocbon-dria ùsing succinate as the substEte is presented jn Fjg.la. T b e v a l u e s o f R c R ( 6 - 3 1 0 . 4 a n d 6 - 1 1 0 . 6 i n 6 0 d n d 1 8 0 d a y old l!ts, respectiveÌyl :nd ADP/o ratio (1-910.1 and 1.710.1 in 60- and 180 day oìd .ats, respectìvely) found in this study indicate the bigh quality ofthe mitochondrial pfepaEtion. A sisnificant dec.eas€ (p<0.05)withasewas foùnd in ADP/o ra-tio. As shown in Fig.la, coupled and uncoupìed .espìration sigrincantly dec.eased in 180-day-old rats compared to 60 day-old rats. Uncoupìed respìration was not significantly dif ferent ftoù coupled rcspi.ation in 60 day-old fats. ln 180-day oìd rats, how€v€f, uncoupled respirationwas signìficantly hìgher than coúpled respìration. Fig.1b shows coupled and u l l o u p e d r e s p r r i r o n o b l " . r . e d u . i n 8 d u o \ y d o q u i n o r e a . the substfate. Electrons fÍonl durohydroquinone and sùccinate entef the mitochondnaÌ respiratory chain at tlìe level ofcoen zyme q but the synthetìc sùbstrate dùrohydroquinone is rap idìy transpofted across the nìitocbondriaÌ membrane wjthoui the need ofcarriers. coupled and uncoupled respiration sìgnif-icantly decreased in 18o'day-old rats comparcd to younger mts. ìn addition, uncoùpled respirationwas significandy high erthan coupled rcspiEtion both in 60 day-old ratsand in 180 day old rats. State 4 respiratory rates in isolated mitochoDdda Èom 60 and 180-dayold l?ts using succìnateand drrohydro-quinone as subfrates are pres€nted in Fig.lc. Mitochondria lrom 1S0-day-old fats had significantly lower state 4 respì.a tion than 60-day-old-rats, whateve. the substrate ùtilised. Fig.2 shows that ATP productioD during state 3 respiralion both iD homogenate :nd isolated nitochondria significandy d€creased in 180 day old rats compafed toyoungerones. The cont€nt of cytochromes b,c1 lcomplex IIÌ) was 0.4431 0.026 and 0.43210.032, llìe content oi cytochrom€ c was 0.18710.015 and 0.18310.015; fìDally, the content of cyto-chfomes a,a3 (complex lv) was 0.29410.023 and 0.262! 0.023 fof 60- and 180-day oìd rats, respectively. Tlìe rcsults show that no dìfference due to age was fornd in conplex lll
Fig.1 Coupled and !n.ouplèd respiratory rates in isolated mito-.hondfla frÒm 60 and r80 day o d rats usinq slccÌnate (a) and duró hydróqù noie (b) as substÈtes. State 4 resplfaiofy.ates in isolated m t o . r o n d i a l r o m 6 0 ' r n d 1 8 0 d a y o l d r a i s u s n g s ù . c i n a t e a n d dufohydfoquinon€ as sùbshates (c). Values arc the means l sEM or nlfe different rats. h a and b statistÌcaL siqnifican.€ by ANOVA (p<0.0s): 'effect ol age. +effe.t of lncoupling, qinteractiof. rc:
'p< 0.05 compafed to 60 day old rats (two tailed stùdent's t-test).
6 0 d a y s 1 3 0 d a y s 6 0 d a y s lA o d a y s Fig.2 AfP producuon in isoLated mltochondria núól/(m ntmg po t e i ù a n d in h o m o q e n a t e n m o / ( m i i x 9 l v e r ) . V a l ù e s a r e t h e m e a n s l sEM ol nine different rats. 'p<0.05.ompared to 60-day-od fats (two talied student 5 a-test).
f
g
P
3.
f l c o u p l e dW u n c o u p l e d
346 Horr lvletatr Res 20011 ll
r a b l e 2 S c r u ù ff e e l r e l e s a f d v e r r r c o r . c f n a t i o n s i n 6 0 d a y o d
6 0 d a y o d f J t s 1 8 o - d a y ' o l d f a t s l . h a n g e
rats comp<rred to yoùnge. ones, we foùnd signifìc:ìDtly de c|eased bothcoùpled and uncoLrpl€d rates ofrespifarion. Since the percentage dec.erse in coLrpled respì.atjon wirh durohy droqLrinone and succiD.rte as the sùbstErcs was simiÌar ìn aduÌt rats (abour40%), ìt can be suggefed tbar rhe rate timir-ingstep under these conditions js the same and no! uue ro ae c.eased dicaf boxylate car r ier and/or SDH acrivity.
By à process oi eÌimi. ation, :ìllother factor rhar can be involved io the decline j. coupled fespìration tound in adulr rars when n'ccinate is oxidised is the eìectron rangport chàin acrìvity it-seÌ1, nameìy activity of compÌex lll, cytocbrome c, and complex lV The lower.rctivìtv ofthe above corìplexes coùÌd be due, in princjple, toa change ìn enzyrìe nl.ìss.To assess rhis, rhe cyto cnroDre cont€nt oi nitochondrial preprrations tuonl the two experlmental groups was measured. lhe reÍlts show that r ' r " . o r " n . o l . \ r o c l ' i o n è . b r d r d c v o . ì r o f f È , s " , p r d . i caÌly the sa.re in both mìtochondfia froù 60 and 180 d.rys-oÌd .ais, whereas in mitochond .r tuom okle. rars the content ofcytochfon]e aar tended to decfease. The.efore, the d€cline in succiDate*Lrpported .espifation obse.ved in mìtochond.ial preparations i.om oÌder fats does not appeaf ro be dependent on a decrease in dre mass ofboth enzyùre coIìlplex ÌlÌand cy tochrome c. O. ùe othef hand, a decrease in the mass oicyro-chrcme oxidase may be assocjared widì decreased nitochon-dfial coupled respj.ation ibund in 180 day oÌd fars. lr should be noted that cytoch.ome oxidase is considered an ìnporrant f : ì c t o f i n t h € .e g ù Ì a t i o n o f r ì i t o c h o n d r i a Ì r e s p i l l r i o n I 1 5 1 . As lbr state 4 nitochondriaÌ respjrarion ir sjgnrficantìy de-cli.ed ir hoorogenate and isolated mitochondria ffom 180 day-old mts compa.ed to 60-day old .ats. Stdtc 4 respìraro.y fates in the p.esence of satllrating amounts of oljgonycìn, whjch p.events dDy ATP synthesis by prcton flux th.oùgh AfP synthase. can givean indìrect meas ufelnenr of the prcton te:ìk o r m i t o d ì o n d d a l ì n n e . n e m b f i n e s 1161. T h e r e i o , c , u r e ( e creased fate4 respj.atory rates forlnd in mùocho.dfiì isolar e d fr o m 1 8 0 d a y o Ì d . a t s n l a y i D d i c a t e t h a t t h e f e i s iì r c o u c u o n ir mrtochondriaÌ p.oton leakat thjs developD enta I stage. Thú observation is in liDe with lowered T3 concentfation foùnd j. Ì j v e . fr o m a d u l t ra t s . I n f a c t , i t i s w e l l k r o w n th a t th y r o i d h o F m o n e s t a t u s i n f l o e n c e s m e n ì r f a D e p e n e a b i l i t y . N 4 i t o c h o n d f ì a troll] lryperthyrojd rats have an inùeased Ìeakto proroD across the rnDer membrane. wh ilc hypothyroidisn decreiìses rhe pro t o n le a k [ 1 7 1 .
Taken togedref, tb€ above resuìts indicate rhat in the transitior liom puberty to adolthood, thefe are structu.aland fundiooal changes ir the hepatic mitochondrìal comparrmenr. Mito chondrìa irom older arxìah consùmed significantlv lers oxy gen ùndef coupled and uncoùpÌed conditiol]s, consistentwjttì a Ìowefactivity oftbe elect.on tfa.spolt chàin irom complex tl onward and phosphorylati.g systenl. ln addìtion, s.ce n]rro-chondria ftom adùÌt .ats ìn state 4 conditions co.sunìed sig nificiìntly less oxygen than mitochondna fromyoung arimals, jt can be suggested that mirochondrjal pemeabìliry de crcased. ìote.estirgly the above changes in h€patic mjtocho. driaÌ compaúnleDts are chrradedsrics of hvpothyroid fare 13.171.ln agrcement we have found sìgnificantÌy decreas€d Tr concentratjon in Ììve., whil€ only a tende.cy to decfease was o b s e r v e d i n T r s e f u m c o n c e n t . à t i o n . T h e a c h i e v e m e n t o f a d ù Ì t hood seems iobe.rssociated with rissue bypothyroìdism in the presence of nrodestly dec.eased sefum thyrojd ho.mone lev-( p q / 1 0 0 m ) 246 -26 2 . : 1 4 1 0 . 1 1 ' 2 9 0 : , : l l ) . J 0 - 4 . 2 5 - 1 5 1.1
v:ues re ihe meJú 1sLrJr Òt nn. diffefenr fits. -1 .Drqè tufcf.d t. 60 dJy.! d cr' p<0 05 .onpi ed to 60.dày ! d GG tlvo b ed strdeit I
of cytochrcme c, whjle a tendency to decrease was toù.d in complex lV content tÌom.rdrlt fats compared toyoungef rars. TabÌe2 shows the resuhs obtained neasuring senln f.ee T3 leveìs and liverTr concent.ation. Serun] free Tr levels showed a slight tendency to decrease iD 180-dav old rats compared with yoonger rats. On the othef haDd, a signiiìcant decrease was ioùnd in ìivef lj co|centration in 180 day old rats corr pared to 60 day old fats.
ìD this sturlv, weshowed that:ì decrcase in liver mitochondrial fesprrato.y activity overlaps liver hypothy.oidis.r in ad!h fÀts, which occu6 despitc sefLrm thy.ojd ho.mo.e levelsonlybeing s lìghdy decr eased corìpared toyounger.ats.
As for ùìtochond.ial .espjfation, we foùnd that rhe rates of srlccinaresuppofted coupled respìrarion sìgnifìcanrly de cÌ'n€d rn mitochondfi.r isolated from 180 day old riìrs compar ed to 60 day old rats, ìn agreement wjrh our pfevioùs wo.k [4l.The highef mitochondmaì prctein |lliss lound in aduìr.ats can account for the observation tbnt homagenate state 3 fes-pifahon fates djd not signjlicantìy vary berween the two ex-perinental groùps. On the other hand, a signìfìcanr fall 1r lìomogenàte ATP p.oductior during stare 3 .espir"tion occL|t'-red jn aduìt .ats, despite the hìghe. mitochondrial proreìn n " * l o r , d i r ì r l 4 " r ì r r . I T , r , ! , u l r i d r e r o L n e d e cfeased ATP prodùction and ADP/O r.fio foLrnd rn mrtochon drh isolated trom adùh rats.
MaÌimal mirochondfiìl rcspìratory chain activiry was asses sed by measunng oxygen consùmption in dre pfesence ofex cess ùncoupler FCCP 51nce the vaÌues ofrespifation, mea$ed with succirate as sùbstrate, in uncoupled and coupÌed states afe siNilaf ìn 60 days oÌd fats but significantÌy ditlèrcnt (in-creased uncoùpled respìration conpared to coupled)ìtl adúlr rats, it is conceivable that rheATP sy.rhesis syfem acrivity jn these aninals is lowe.than tÌratofyourgef rats.In fact, wÌriÌe the ATP syrtbesis system activiry in 60-day old rats is .ot |lte-limìtirg forcoupled mitochondn.rl fespiratjon, decreased cou-pled fespiratìon in aduìt rrts lllay b€ partìy due to a decline in this activity. Howev€r the rncoúpÌed.ate jn adulirats is lowe. than that of60-day-old rats, the.efo.ethe d€cfease in respi.a tion tbuDd ir adult rats is not coqterely do€ ro a falÌ in the AfP syrthesis system activity.
To have infbmratior aborr oLhe. iactors thar aìr be involved in the decline ir coupled respi.ation lourd iiì adolt rats, we uri Ìised the aftifìcìal proton donor, durcbydrcquìnone. ln aduh
Liver liiodÒthyronine and Respnauón H o m M e t l b R e s ? o 0 l : 3 3 @ els. since there h abúndant evidence that T3 is tfansported
across the plasma nembrane of target cells by carrier medi-ated processes tbata.e energy d€pendent 15l, we sùggest that the redúced intracellular energy production that occùffed in adult rats c:n low€rT3 uptake ìnto the liver
In conclusion, hepatic mitochondria from adù1r rats are differ-ent fÍomyounger ones in both declined ATP synthesis capacìty and decreased proton perneability ofthe inner mjtochondrial membrane. These features are consistent with a tissue hypo thyroidism found in liver ofaduìt fats.
Acknowledgements
This work was supported by University ofNaples "Federico II."
I Sobolls.Thyroid homone action on mìtochondrial energy trans ler Biodrim Biophys Acta 1993,1144.1-16
2Bmnd MD, Murphy N4P Control olel€ctron flux ihrough the re-spiratory chain in nitochondria and cells. Biol Rev 1987; 62: 141 193
r losa s, Barletta A, Liverinic, The efièct ofthyroìd state and cold exposurc on .at lìveroxidative phosphorylatìon. Mol CelÌEndo-d n o l 1 9 9 1 ; 7 5 : 1 5 - 1 8
4 tosa s, Lionertì1, Mollica MP, BarlettaA,rliverinic. oxidative ac-tìvity in nitochondria isolated from rat liveratdifferent stag€s of deveìopment. Cell Biochem Funct I998; 16: 261 - 268
5 Hennemann c. Eveús ME, delons C, Lìm Cn Krenning EP, Doctel R. The sìgnifìcance oi plasma membrane lranspo in the bio-avaìlability ofthyroid hormone. Clin Endocrinol 1998; 48: l8 6Nicholls DC. Bioeneryetics, an introductìon to the chemiosmotic
th€ory. London, Academic Press 1982
t Liverìni c, lossa S, Barletta A, The efect ofcold exposurc on rat liver mitodrondriaì .espiratory capacìty. comp Bìochem PhysioÌ 1 9 9 1 i 9 8 : 5 8 3 - 5 8 5
3 HaúreeEF Determinationolprotein: a 'nodifìcation ofthe towry methodthatgives a linear photonetric response. Analyt Bioch€m 1972i 48:422-427
elossa s, Lion€tti L, Mollica MP, crescenzo R, Barletta A, LiveiDic. Effect ofÌong term high fat feeding on energy baìanc€ and livel oxidative activily in rats. BritJ Nutr 2000i 84: 377-385 i0 EstabrookRW Milochondrial respi.atory controÌ and the polaro
gfaphic measurement ofADP:o ratios. Meth Enzymol 1967i 10: 4 1 - 4 1
rrFiske cH, subbarow v The colorimerric delermination oiphor phorus. J Biol chem 1925; 66: 375-383
ì, Lionetti L, lossa 5, Liverini C, Brand lv4D, changes in the hepatic mitochondrial respìFtory system ìn the transition iÌom weaning toadulthood in rats. Arch Bìochem Biophys 1998;152:240-246 13 Lee Y, L2rdy HA.lnfluence ofthyroid hormones on L d-gìycero-phosphate dehydrogenase and other dehydrogenases in varioris organ offte.at.l BioìChem 1965,24011427 1436
r! Mo(eale De EscobarC, PastorR, obregon MJ, Escobir Del Rey F EfTect of maternal hypothyroidism ontheweightand thyroid hoÈ none control ofrat embryonic tissues, before and ait€ronset ol ietal thyroid function- Endocrinolosy 1985r 117:1890 1900 15 Paradies C, Ruggìero FM, Petrosillo C, Quagìiariello E. Enhanced
cytochrome oxidase aciivity and modification of lìpids h heart mitochondria iron lryperthyroid rals. Biochim Bioplrys Acta 1994:1225:165 17o
16 Porter RK Joyce ol, Farmer MK Heneghan RTipton KB Andrew JF, McBennett sM, Lund MD,Jensen CH, Melia HP Indirect mea-sur€ment ol nitochondrìaì pfoton l€ak and its aPplication. IntJ o b e s R e l a t M e t a b D i s o r d 1 9 9 9 : 2 3 : 1 2 1 8
17 Brand MD, SteverdìngD, Kadenback B. stevenson P[4, Hain€r RP. The mechanism ofthe inrease ìn mitochonddal proton perme-ability induced by thyroid hormones. EurJ Bìocbem 1992i 2061 775 747
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