ffi
T
he mechanism of stimulation of
respiration in isolated hepatocytes
from rats fed an energy-dense
diet
Lillà Lionetti, Sùsanna
Iossa, Martin D. Brand,* and Giovanna
Liverini
Depaftnenî aJ Ceneral and Enúronmental Physiolo$, UnireîsítJ of NaPles, Naples, hab; arJ *Depaú,nenl oÍ Biochemístry, Unh,ersiù of Cdmbridqe, Tennit Coutî Raad, CB2 lQW
C r n b , , d g . l 4 c l a n d
We heasured a\s.n .òn suhptian in hepatotltes isaL.îed Íian nît f?d an eheryr'.lente díú We cÒn.eptudLLr
divìde.Ì the rctpintÒr! pathwat into bbtkt Òf rcdctions thdî Prola.ed ot conswned ntù.chonrlriùl netnbrcne
potentidL, and then ùeasured rhe kìnerìt rcsPatse oJ rh\e bbcks of led.tions to rhis roîcnlial The resuhs \tlow
increased h.puti. ftspnaîion nîes in raL!.t'ed at enels-dense diet dccounted Jòr b! an ik.reosed nte oJ îhe
subsrrak .idation rcactions aî un:- partìculor nitothondridl hehbrane patenîiul, bul na chonSe in rhe kinPti. rcsponse tÒ potential af îhe tu.îions thdt consun. íî, reru\ìt8 ii a snaLL is. ìn thìs PÒtentialinrhe ra^ Jtd an LnLrg! tlense .liet, Th. ìnoealed hepotic oqg.n únsunPtiai is ù |in. wíîh th. ikcreased enerlt erPenlìh1 that o.cu6 ìaruîs Í.rldn enagr densedlet. O Elsevief Scìcncc lnc L996(J Nùt. Biochen T:571 576.1996.)
Kcywo.ds: heparo.yrcsi enerAy denle dieri oxygcn consunPúìn: top dosn clasticitt anaìysi:
Introduction
P.evious
resuÌts
have shown
that young ruls provided
wìth
an energy-dense
diet become
hyperphagic,
bul fail to exhibit
, {(e ' qercl-r
g ; r r ' th r o L r g h
a f a c u l u r : \ .
i l r r F a . e n qnef!)
expenditure
(diei ìnduced thermogenesis.
DIT).'' The
sanìe
results
were
prcvioùsly
obtained
feeding
rats the caf
eteria diet," ànd brown adipose
tissuc was initially sug
gested
às the main site of DIT.{' However,
a contribution
to
the incrcàsed
energy
expenditure
found ìn hyperphagic
rats
couldbe
given by the 1ive.,
bccause
itconstitutes
only about
4E of the total bodv weishl and consumes
aboùi209ú
of tlìe
r o r d .
o \ ) c e n u * a ' 1 . r n 1 o r g r n . ' m
-
T l t i . h ' p o r h e . . . .
, u p p o n e d
b r I \ , r e u l t ' o l B e | t l c l a l . . - $ h r c h s h o $ e , l
increased
respiration
rales
in hepatocyLcs
from hyperphagic
Iars fed a "cJeteria diel." ln line with this fìnding,
it was
suggered
ihrl an increase
in ljver ùetabolìsm
contnbules
io
ttre
ìnc'casea
energy
expenditure
of hyperphagic
rats.6'
In view of the above
observarion,
it appeared
oÎ interest
to investigate whether chmges in respiratioD rîl.q oi rso-lared heparocytes occur in hyperphagic rats fed an energy-dense die!. Any changes in respirùtion rates in isolated he patocytes may rellect chaùges eilher ìn the leak of prolon across thc nìtochondriai inner mcmbrane or in the activily of the phosphorylaling system (ATP synthase, Pi trans polter, adcnine nucleotide carier, rnd extramitochondrial ATP consùming reaction\) or in the reactions that oxìdize substrates (cellular catabolic reactions. the ciidc acid cycle, and the electron trùnspof chain).'' The application of the top-down elasticily analysis allows tbe identìficatìon of the blocks of reactions, which are sites of àclion of extemal effeclors within netabolic pathways.rL i'?This approacr
"' volves dividing the system under consìderation into blocks of reactions that produce and that consume a common rn temediatc, whìch is dìe milochondrial membrane potentirl (À1Í,,,) in ùe case of respiràlion in isolated hepatoc)r1es." We can then consider thrcc blocks of reactionsr ì) the re-actìons thar produce A{,,., i.e.. the substmte oxidation path-way that consists of all oî the steps from eùdogenoùs sub-strate to the prodùction oî À"Ir,,,i 2) the reactions that
con-. L m e -\ V - tr^dJcrng A lP and J I cel u lacon-. A lP*on'uming reactions, callcd "phosphorylating syslefn," and 3) the rc actions thal consume À'f- withoul ATP synthesis. called ''proton
leàk." The top-down elasticìly analysis invoives
0955 2863/96/5r5.00 PIt 50955 2861{96)00r22'2
AddEs reprinr requèss to Professor Ciovanna Liverini ar Dipúlnento dr Fisiolosù Cènerale ed AmbientÈle. ViaMeTzÒ.annonc 8. r-80Ì34. Napoli, Received rcbùary 2, 1996ì a.cèpted July 25, 1996.
NùtnionaL Biochennq risTl-576, 1996
O Elsevier S.ìen.o lnc Ì996
Resealch Com m un ication s
measuring
the kinetic responsc
of the above
bìocks of re
a , I o n . I o À V , , . T h i s J l p r o a c n
h r ' D e e n
p r e \ i r t u r l )
u ' e d In
, , l e n l i I rh" ,'Cnifrcrr'r
\ r l e . o r . r o ì o l r h ] r u i d
ì ' t _ ' n o r "
in rats on sùbsequently
isolated
hepatocyies"-.
Thcrefore,
wé thought
it would be interesiing
1(r applv
o o - a ^ q n
e l , ' Ù . ' r l : r J ) n ' r o r J e r r ' f )
r h e
t s r o u p '
o f r P J (
-r , o n '
r h o r .
o u l o
b e r l fc . t e J
i ì , ' u l . r e d
h c o J r " c )
r e ' fr o m ra '
tèd an encrgy
dense
dret
M€thods and matedals
Treatment of animals
M r l p \ r J d r d . o . d h o u " J.r' | . P c 'b r e d 'r ' n c ' r i m d l r o u ' e. i i r ' "
o " o - ' * t . ' e . . " n r r , r I r ' r v " r r ' o r ' " m b n J s "
U K \
" " " o ' i o . u ' n ' o ' * o g u p ' $ r L h r h P a n P c d b o J r q e r t " r . , " " . , , r O q , . . , r O n e g o ; p . i D " , , , u c J 1 . . , r r o ì J i è l, ; ; ; " ; .
" '
r " r r t rn c '
t o t ) , u t e
c n e r c 2 0 P 'rern r0 Ò Did
" 1 , - . ò
i L t " . ' , 1 * " r < 8 ò J 8 r
' " e n e
s ) g ' q n c ' - l ( o r ' e l
; ; ; , r D k . r " ò r c . " e e ' s )
c e ' ' o r ' ' 2 8 ' " r " d n r '
i l i i . * " r ' r ' ^ " m e r t r ' 8 r u c r 1 2
o r l p h " c " '
0 a A r \
ì ' , " . " - i ' z " A I \ m , n e - î \ ' J ' p e r ' e n i e o r o r d l m P , " r r o ' , . o . . . n " r r , ) ' r p o r ' 'r ' 5 0 ' p i l n o " d b o h r J _ r e : ; t ; - ; : , o . . e ; ; r , r ' . ' I n " o r ' , É . , - e n r r . P d r' d - ) ' r ì r r . r \ w e r . o u ' c d 'n d r v d d l l ) i n ' u ' p e n J ' d { ' r e b o l " t e a L . : " c - * " - m r r . 3 l f r c . d r " î 1 2 h l i 8 h 2 h ' d J r c ) c L F Bo,lv weiehts ald food inrakes were úonitored drilv 10 rllos ,:,lcilation-s oibodv qcirhi gain and gross enefgv inÎakè' Ai the e ; : ; e , e e n - ; , . , . p ; io J ' r ' " . ' " . " - " d r e d o ' - m ' 8 h ' n d " " ' " ^ " ' r r ò t o r " o , . " o n ' n r f - r e d h e p a u ) e ' O r ' c r m c l c $ r , ; ú ì r . o r n d ; ) . o ' a r ' ' h d h ' R i e t l r a l i a i J ' o ( o ì o r , , r ' ì - " t . n , a . ) 4 " c u l o e d n - r r l r ' c l ' r r ' ' o r l 2 r ì i c f l - /l J i . r " r . , " i . ' . ' t ' " - . . r r ' u . e , , e r ì è p a ,
m è d
^ r c e n e r r r
" " 0 f " ' i . " - * * ' D h \ . , n r o g \ I r r \ P \ i h . l \ " P l P l r ' \ ' T h e n . , $ e r c 1 , . , o e J ,, r o r d o C U p h r . h r e r e ' J P i r h ' r 'h c o ni."i oi"r o. tr," *.gr_o**
diet for 15 davs At thè end of the
experimental pèiiod, these rals weie làtted oveflighl aùd werc used for ererey balance neasÙreúen$
Energ! baldnce meas remenîs
Bo.lv weìehts rnd food ill.kes were noniloÉd dàilv to alÌow . . . i n , - i . o r u o o t J e r P h g J n d d q n c - e r g r In r ' I c D S c J _ iut" .n" er 't.t. ""lnt inru rc(' nr n"ooJ \pilLteì q"' , ' " . ' " - " í i s J u '" ' e ' ; " r t r s ) m e " Ù c J i n r n e r e c e r o m r h e " i " , , " "'* ' * * . a i m e a - ' d n o m d " ' ) u ' d ( ' Ù m l r i o n i l " ' . u " t : , " i i o . p , g ' , \ 4 t , i l i r e s t u "\Pr-' eo a\ orge'rrblc ... ,"' '"1*.e . O.oo ftì. e-in In ene t) td' obra r ed br 'ub-n " . 'i i * ' r , . - * e l ' o 'ub-n r c r r o i . n I n i r È r s o u o 1 \ è n I r l l e d l i r "".",'*" " "i." tt.r nr"È of 'he nuJJ ' lrom hd 'l e'(h ot d,"-ií' .-"".i-*t"t a;oups At úe end of the expeÎimenl the "rl^.ri *"i. -*,r'",ii"o
tv the inlrapenroreal injection of chlo-ral hvdmte 140 ms/100 s BW) and were kiìled bv decaptiatton rrr*1 Xt". gut
"-r"nr ."-oval. Îhe cÙcasses
ÙÈre autoclaled' . h o D o c J i n l o . - " p i e . c ' r h o r l u e h \ m r \ e J x d h o m o g e 4 r / e o I n ; l i ' " . " - " . q * ' ' ' " " e r h e . d c " ' d a s h r ' v i r h r P o l Ù o s " . " r * . ì i ' " . " ! - " ' . . ^ $ e ' r " d p e ' f e ' e n c e d e -' i i i ; J -' , -' " " . U p * a " " n m q - i ' L ' m r r r p r r r e r ' ' r ó o u r 0 0 r c r i e r e m a d e . r " P erere! . o n ' e n l o l r h c n " ì ì e l ' d d h e 8r '\' Prerly content of coútrol d; Èúelev'dense diel rere úeasured {ith a p m aa,aoai .rorime'er ."t btuRd q r , h dD o" '/.,. x-id n.n ,lui,r. rn",$
"^p"nO,*'" ""'
calfuLreJ lrcm rhè o fteren? be' iween ME irtake md energY gain
Prepdration and íncubation of cells
H e n ' r o ( r r e s F r e p r e p m d b ) c m o o j l r c x r r o n ì ' n r 'n P m r ú o d l È - o m d r n e d o ' . - ' t " ' e n r i r ' d c r e r m n P d b \ d q r r { d
572 J. Nulr' Biochem,
1996,
vol' 7' Oclober
hroNn voluúe oÎ cell suspensìon 10 a condant weight at 70"C1
ii". rrr"
""r"",
we iben subracted
úe dry weisht of an equal
' , i ' , . "
" t
i , " r . ' t' . - r . \ l 8 m V N a c l
< À'1
K C l l 2 _ \ ' f
l i " i o ,
- ' r
"
I m M K H P o . . lr ù r M \r H ' O 2 : r M
c i _ r n c i e t t r h d r + . r . u . r p d c ! I n i . o l f i o n - P d um verc* " i i , . " 1 "." r' 'a '"r, rt. In. ubar
r mrù um ' r^b mv '{acl
i ' " r . r
r t r . : ' m \ 4 N . H r O o a l r ì , \ 4
I 4 g \ O . H O l 0 r M
Na-HPO.,
2.5
úM Caclz.
and
2 251,
(v/v) defatted
hovrne
scrum
,ìÀiÀ'tl sb"t se?,
t""i"" serun albuúin was detalted
bv the
*"ir,,"r
"rCrt"t'",t4
Oirtvted
agaìnst
153
nM NlCl dd I I mM
i - i r r , " , ' * r . . r r . u
p . " ' o r ' " r ' - o u P d s r r h r n p a
b l - e r 0 r a ;
$ \ I I n a \ P u b " u e r h ^ \ r r b r ' r ) I n r r n c v - r , . l h e s a . r b o . . r P . c r . u \ p r " o n d ' r 1 r r g r u b ' l o N i ' s . i , , . .na i ( o ro att ^v pqu,l:bii .n or rhr me.l um o r DH
Measurement
oJ resqiration rdle
lncrbaions
of cell sulpensìons
ùeie routinelv
cdìed out !sl'g
" " " , " r ' . " " f t t .t Òl u.I.Fa\ro " Ò l u
o T g d r . \ ' r t h / n l I ' O
m t r . o p p . - o g - ' r ' r " s l l o \ ' " l . t t ' s t ' 1 ' n t ' -n ; r \ f t e , i - P i , , s : o t o t c P l l i e e P e n c r ' J P ' r r r ì - ' i n rric srraU.g rair bath for l0 Ùìn before rhe additìon of anv , " " 1 i ' . ' . J , . - * A r ' c t ' l u f l h r ' l . u b J r r o r P e n o t o 2 0 n n ; ; , ; ; . . - . - p ' - " " r e ' u | c ' r ' a u ó | : ' ' I ' r d t ' q ú ' ' o ' f . i | - . , . p o r . , o " - r ' ú . I arrn)pc n\)Bcn e'Èrrode rR'$ É , " , " " , 1 , e " " , - " . . ì - . ' , d B c u K m ù n r " e d a 1 - ' R"Pi ; , ; , . , . , . , . ' . " " " ' . ' " " " , 1 p r o r ' . - e ' ' r o ) n n ' t e r h e . o d r , o I o r r í e P l l u p e r ' ' ' r o ' - P .h'mber' ie ur'e rd " " r i " . " ' " , . . " " " - ' i " " J \ 4 r r J . n o n " n " ' r c ' o ' r o r + a I . - . ' . o o r . U ' r " . " " e ' n lorFmr'o h o n d r r d l e ' | | r j i o i ' ' ? o r , t " - . r " L , ' " . t ' " ' ó " I e l n d c ' P r m r r ' rh e a r e "' I o n -l i ' o . r ' " n c ' , r o r . ' . n c . u r n n i o r . . e l l . \ e r P I r r r \ i r J \ r r h m j \ r d . l . n n . e n r . l o n . . r n ) \ o r h i r o l I U u M
Medsurenent oÍ miîochoúdrial membrane poîential ( Lq -) T h e . \ { , . ^ d . , n - u r c J r m u h ' . e o u J v n ' r h o \ \ J P n ' o - ' u m p i o ' " , , " e ' " i o ' . " " r r ' r o , I e l r n o p h r ' r c a n n r r r P \ e n \ ' r e h _ i " " ' . " r , " " , . r P \ 4 P i e r e ri o l ' h p b - r { e c r m r ' " h o r d n J 1 ; ; ; ; r e pden,,r ,'n\ '.'ra rP\lP d, r, bl.on "r 1-"' J
Aú' = -61.s log
v " ' a
-Lcrld [rPMP]d a (v.+ v",l
lcrl" [TPMP]" 4 v"
' ]
wherc V : lolune. a = apparent TPMP' actìvitv coofficient' and subscdDts c, n. e, and toi rep|cseni cvlollasm'c + úùcLed' -ll"n*ari"r. extacellùld, and to!41. rcspeciivelv Appropnale . " " ' " i i * . * . " "pp'"4 ' pl. Ìa - e Ì D r a n e P o r e n r È ' ' ) r o l ' " À . - 0 . ' * r ' " ' i , i . . b r n J r r e o r l P M P c " o o h a f a c r o r ' a ' J . * n t " l u , n " p " ' " n d B r d r d V - 0 8 2 v ' - 0 1 8 ' 0 , 4 ì . " U - . d 0 / l ' . \ 4 i r o l h . n d r i d l m e n b r ' n c o o r e n r a " " " i J - r . " r " " r k n o \ i n B n " e \ r e n r J r r h e ".cumuldion o f i P V P r d o r h e w h o l e c e l i l T P f , { P l " / T ? l \ ' { P l - r ' n d i n r Ò r h c ! \ , n D a . m in t e l i r o r , ' . r h c ' \ r e r i m e o u m ' c ' - l J c l - ' . , T h . t s ú l b - r r o n o r c t { î s a F o u r d r o J ' L d e r n e r l l \ m a î c r rr.""" o"t*ti"r.'" Becausè no differences iú plama menbrane " . , " * . 1 t * . o b r r e J ó e r ú e e n c e l ' ' l r J m C D ' 1 o r D ra'' " $ d n . j d \ r l u e o . C l r c . C l | . , n ì 0 ' q - ' " " m e d - - - . ^ , H e D a l o . \ . e . i e . e I n . u b r . d " r " / " c i n r h e f r e ' e n r ' o l r / r u e " " i ' i ó r . . r a n d r ' - l u r B o 6 L J t I a c l m e ú o ' ) r n u l | l v m l t ' . " ^ ' * ' - t " . f P V P a c u m l a ' r o n : - l t ' o ' r r n c . 1 - 1 0 ' B q o ' I . H , T P \ ' f P ' / m lr ì ' r 0 B o o l q l /
Livet respiration and high-far diet: Lionetti et al.
mL ànd l.? r01 Bq of rH:o/nil for measurcmcnr of 36 cl dis'rribuLion. Incùbation with rH2O allowed the tÒtal pellel volume to be detemined, whercas [''C]nethoxyinùlin allowed the calcula don of extracellular voluúe in pelletsr the cellulu volume was thcn calcul ed às the difference belween the total pelle! volufre and îhe extacellùlaf volume. Carie. inulin (0.1 ng/ml-) and TPMP bromide (0.1 pM) sc.e added to allincùbations.zo At the end of each ircubatìon riplicatÈ àliquots (0.70 trl) wete cenÍi fùged fo. 2 min, dnd were procesed accofdjng to Ha.pcr and Bràndr:' to detemine the mdjolctivities ol the supemaLanL and peller. The appùenr volùme ofpcÌlct alailabìe to each isotope (iîs space h pL) vas calculated as dpm in total pelleî divided by d p m É i o l . u o c r a , " n , q n p l e . I e 'Hll PMI rc!urnul-ron t o r o ! l ' P v P 1 , " l P \ . 1 P ' v r \ . J . L r . e d a \ ' . H l f P N 4 P \ p d . ( lraClfrethoryinùlin space)/(3H,o spacc traclmethoryìnulìn space). For grealer pfecision, corcction for to1al !-ellet volune was mnde in erch Deller .onrainins "Cl : rhus. '"Cl distibùnon t . l l,. r - sa. cdhulrrd ": l.-H O.prp - - l ' ( lme'ho\r ! u ,n .pd.cr i tl o .px.e - [ "cì pcce) r H-o .pnr l'"clne1ho{yinùlin space)1.
Top-do n elasîicif,J analrsís
Top-down è]arìcity dalysis is derived from control analysis." It conrains no brord rsumptions lnd ùscs a very simple cntenon of ''what happens to ) rheù we choge r a lú1le bn inlolvrng o.ly the abillty to changc r and io measure, and r before and afleî the change. Elasticiiy anàltsi! siúply measùres ihe ki.etic response of somc catalysl to iomÈ !ùiable. ùd asks if this response is chanscd b! rhè 1er condiîion (he.e dier). If it js. then the test condìrion affects rhe catalyst direcdy, if it ìs nor, ìt doesn'l. In top down ùalysis the catalyst is a groùped sel ol'ènzynes. cúiers. etc.. but thai doesn i aÌter the lheory ànd í úakes the experimenh snnpbr io do and to imerlret.rr'r2
The kìneric responses of the proton lelh phosphorylation sys-lem and sùbfrate oxidation palhways to Aly- were úèasured in isoÌated hclatocytes iìom CD and ÈD rars by ùsing inhibitos (oljgonycin and nyxorhìzol), which ùe very specifìc and have p.jnary sìtès of àcrion wilhin mitochordrial respiatory systein.
Hepatocyres were incùbated ir a modìum withoul glùcose úd without additioù of any substrates. lf we núipulate the mitochon drial p.oton conductànce wilholigomycin (0.025 pglnl) or FCCP ( 1.5 pM), then Lhè relatioúshìp betreen tbe rate of rcspiratioù and Arl- wlll reflect the Knetic response of all of the leactions in-volved in the substrate oxidation pathways to A!',".
The kinetic rcsponsc of the prolon lea.k pathrvay to the A\p-should be obtained b' tìÍaring the actility of ihe respiratory chain with nyrorhiazol in rhe presence of saturating amounts of oligo-myciú 1o pÉlenr ATP synthesis. However, whcn AV- and respi-.ation were tiÍated vith oligomycìn. low dosès of oliSonycin caused ^Vm to nre. bùt when sufficient oligonycin was added to àlúosr cÒnpletely abolish oxidalive phosphorylatìon 4V., began to fall agaiú rnd respiration was fufhef irhibited, prcsumably becaùse there was no gÌycolytÌc ATP to activate the iatly àcids, v h L h ù e p r . u m a b l ) J ì c I n a t o r r u e l -J " , \ e J J r l . . . . r h r r r l ' e É m a l b c n o . u b J , d r e r w , e . p , , r , . " D , É ro h . r ' l i n h e lV
" . it ùas dìfficùlt to exmine tle kinetic response ofthe pfolon leak pathway to the ^V,.. Coùtrol experiments we.e done titràring wíh myxothiazol in ihe prcsence of difièren1 concentations of oli8o mycin to seo when the kinetic curve so obtalned. ovedapped wlth the linetìc .urve oblained útraling with olieoúych (0.05 to 7.5 pg/nl-) (Fi8rle 1). We foùnd ovedalped cures rhen tbc myxo thiuol tilralìo. (0.10 !o 1.0 pM) eas donc jn the presence of l pg/nl- of oligomycin. Tìis means th!ìt startìng froú this concen tranon ofoligomycin, the curvè ob1àìned titrating wlth ollgomycin overlàps wìth tne kinèlic cùne of the prolon lert response to the A{r-. So ihe oveîlapped kineric cùnes obtained can bo assumed to
o 2 1 6 8 1 0 1 2
Jo {ihol O./1or c.ll! .ninl
Figurè 1 ttratón wth myxolhiao in pfesence of dilferent con-cert.arons of o gomycin in solared hepalocytes llom cD (A)a.d ED rats (B) r tlratio. wilh dferenì concenlrations of oligomycn (0.05 ló 7 5 pq/mL); V O tr litral ons w tlr d iterenl concenìrano.s oj myxothlazor (0.I0 to I 0 !M) n presence of e1l-rer 0 I pg/mL lV) or 0.5 !s/mL (O) or 1 0 ug/mL (E) or ol gomyc n Data are irom a sinq e represenìalve expermenl. The non mtochondrla oxygen conslmplon was srbtracted from each vaue.
represent the kineric response of the proton leaÌ pathray to the If the activìty of thc respiratory chain is titrated wnh myxo thiazol (0.10 to 0.25 FM) withoùr oligomycin and corections de laÈr made for the amoùnl of oxygen reqùired to balance the late of ptuton lÈakage at each ^V- measured, the rehnonship betwee! the rare of respiraiion and ÀE- wlll .eflect the kinenc rcsponse of the phosphorylatine system to ^i['-.
Resulfs
Tarle 1 shows Ìhe results of energy b.rlance neasur€ments
in CD andED rats.
Mean
initial andfinalbody
weights
were
noÌ significandy
different
betweeD
the two groùps,
yielding
a daily body weight gain of aboùt
7 g in both groups.
The
ME intake and energy expenditure of ED rats during the
whole per;od of treatncnt were 309, (P < 0.05) and 517ú
(P < 0.05) hieher,
respectively,
than in CD rats (Tdúle
l). ln
- 12o : 1 o o
E r o
o
l o 0 6.0 4.0 1 4 0 120Reseatch Com
n u nications
Table 1 Eneey balance ol cD and ED rats
5 s o
=
R 1 0 0
; ? 5
z z c
tAl
CD FAIS ED RATS E.ergy gain kJ Energy expe.d tlre kJ7 5 + 2 I 5 3 5 a 1 6 3 1 a g r 2 3 3 2 0 1 1 1 r -1000 r 55 2320 r r 65' 3 0 a 3 "
Data
are
lhe means
1S.E
Ì/l of lour dllterent
rats
ME
inlake
energy
ga n and energv
erpendt!re
are refered
1o
llìe
wlroe
p€rlod
ói treatment
(15 davs)
'P< O.Ob
comoared
to CD rats.
Gross
efrcenav
= (body
energv
gain/MF
inlake)
x r00
addition. ÉD rais showed a decreased
gross effìciencv
{ 2 5 ' ú . P - 0 . L r ) i
T ù b t ?
l ) A ' l a r a s r h e rJ l ' b r e d in l h e
a n i m a l
h o u . e
o l l h e D e f d m e n r o l B i " c h e m i ' r r )
U n i v e F
s | y o t a o m b r o g c .
r h e r i u e r e n ^ i g n i l r c a n r
d : " e r e n c e '
i n
d l i l ) b o d . w e i g l ' '
g J i r b e r u e e î
c D r n d t D r d t ' $ h e r e r '
gn iats srioweJa signlricant
indease
in gross
energv
intake
(+,177.,
P < 0.05).
Re\Diratior
rares
siÚìoLr
addition
ol s,rh rrate
or Inhib:
t o . ' r . n a o s . n " u '
t . ' p " " , ' o n r d r e ' r
ú e r e
' : g n i l r c a n r l )
h r 8 ' r c r
, , r O ' , p . O O S r ; n t r e p a l o c v l e '
l r o n L D - a ' c " n p î r e J r o
òl .ut' (rr.t'i".z). rn. non mitochondrial
portion
of oxvgen
consumDtion
was measured
in prescnce
of saturanng
a n r o u n r
o r n \ ( o r h r î / Ù l \ o n n r L o c h o n d - i r l
r e ' p i r a r ' o n
r a r e .
u e r c h i A h e l
l n L D r a t ' r - 2 4 0 . P ' 0 O 5 t tr o h l 2 )
Thc mitochondn pofion of oxvgen
consumption
was cal
culaGd .Ìs the difÈrence
betwcen
the total respimlion
rale
and rhe non-nitochondrial
respiútion
rate: Îdàle 2 shows
r h a l r e - e \ D I a r r o n
' a r e ' . u o b ' r i n c d
u e r ' ' rl higher
I n
h e p a l o c ) l e '
r r o m F D r a r i c o m p J r e a
$ r l l t c D r 3 r ' r - J 2 r Ú
P < 0 . 0 5 ) .
Fisrrc 2 shows
the kinetic
responses
of the proton
leak,
o t o ' o h o ' l l a r i o n
. ] ' t e î . dnd'ub'!ìzlc
o \ i d a r ' o n
p a r h $ a ) '
l o A . l . - i ; i . o t " r e d
h e f a r o c ) ' e .
t r o m c D a n d tD r a r . .
f o r
the a;iysis of the kinetic responses
oî the three blocks
of
r c a c r r o n '
r o lÙ - , 1 ; q a a 1 ' l h e ro n m r l o c h o n d r ' r l
o o n i o n
o f o x \ s e n c u n s u m f l i o n
q a ' ubr'Jcred
i r o Í ì l ' r c r o u l c '
-r u t a -r
ó i r s e . c o n ' , n p r o n t ] l e r c
d r e ìr o :i g n r l r c a n r
d i f f e '
e n . e ' i n ì r , " ' a r e o t ì h e p r o t o n r c a ^
J n d o n u ' p h o D l ' r : n P
system at any given value of ÀÚ- between CD and ED rats
Tabìè
2 Endogenous
resp
ralion
rates
in hepatoc)les
ffom
CD and
ED rals
a 9 1 0 1 1 Jo {nn.t o/lo' c.lb hih} 1 5 0 . t 2 3 1 6 0 lrlo 120 1 0 0 40 3 4 5 6 7Jo limol or,/1or c.llr ' niiÌ
1 1 5 3 r 0 . 2 7 3 2 8 r 0 1 6 e 2 5 ! o 2 - l 1 4 9 8 i 0 . 3 0 ' 4 . 0 8 r 0 1 5 " \23%) CD RAÎS ED RATS F i q u r e 2 o r p a o o 1 p l e ó s p Ó ó o r î ò ' r b ' t è à i , i " ; , ^ , , p o . ó o ' i B ' | o ' o o ! ì o r ' r d r ' o r é ' - ' o \ v I " " * " . r " , " , r D . t " o D r , i " ,
sr;irató oxiOar on to .l!,- rhe mdde po nt represenis the endog_ ènor" ,"spit"tion t"t" th€ pont on the rlghl represents lhe resp ; : ; -
" ' . ; e ; " . { . " ù - c o | . M ; " o | " o , é o r ' e é r ,;;;" ;_ .i ó. 0.Ò ,o. c.ò o oon. i .o o-5
L o r I r B , l r ó i ! ó p o r ' ò o u a a o \ o d ' i d l 1 . 1 , - . . " , " - " ' " , o l o o I ó o - M o r o r o o l c n I L b r l ' d r e o r j . d r ' r q , L \ r ' " e . " , p ó - j r e m o a v _ ì , . o , , ; o . , " on (o o.o 0 ì. rv o -só'd, o, | ì : " o . " , : o ó q o a , o ' d 7 o " a o i r r ó ; , " ; ; ; ; ; : q o - , , é ' p ó n e p ò e - , ó - é ' F V o o i i ò p r è p é e l o d r . o o ó ' o ' ( r r o r o was subtracled fom each vaue
D a t a a r e e r p r e s s e d i n n m o o , / 1 0 6 c e l - l l i i ' ' n d a r e t h e means r S E M. of four diiierenl expenrnems
lhè valles n brackets feprcsenl tlre % conlribLrlon to $e ncreaseo P . O 0 5 c o m P a r e d r o C D r . r s
r / / d / / , 2 8 . c , O n l h e ^ r h e . h a n J . I n c r e i . J c t e a r d r , r e r e n c c r n m e r J r e ot lhe rcrcionr in\ot\ed in rtf , rb.ra." o\ oa r r o r p s r h $ d ) ^ d t r h e \J m , , \ 3 r L e r .,i _\q' hetuFen a D Jnd r D m r r , t r t J c r . lh e p Ì o t ot I to,.nondnn, o \ ) ! e r t . . In r . o l d l e d h e o a t o . \ l e . r o r n L D , J t . " g r , , , . , l t _ ì , * r b r , ; u e - f r o o b l d r . r e d , ! , r h h e t f l o . ) . e . l r o n . c D r d i : , u . h r h J r , ar any grven 41y", the oxygcn consunprion is higher ìn r e p z r o L ) e \ f r o r t D r J r . rtr:r i,r hcnr o. yrr. rron i O .a,s
r t 6 u f . z A t . tht| e5Ltr inJ crrc, th-r ,ubJriri, o\rduuon ( r c r | o r . r',e nrmut:,ed rn nepntiiclte. f,un I D ,u,. .om par€o $ rfn (D fufs
Discussion
I n n e ù e r e n . ,lLo). ene,$ tutdr,e îea\Lrinen.\ rd,e
,l_"-l.i
l.1l ".
fD rdr, MF rîr,rre
rnd I,ner3)
r,pendr.r._
í g n . r r c a n l t ) incrF .ed compffcJ to CD rdr. wirtroL an):ijli:ll
!:")
úo.shr
!d,r,ralv- /, r,, iùd,,,,.r
d,o..
. . : : , : i : fÌ " \ , p r i r i c a î r , ) ,edJ,,ed i r F D 1 . , , 1 a b í t). , r c ( e f e . l l \ t u c , î s r i p , î e s i r h o L r p r e v i . r u . . ú e , o b l J . n r d r î E D m , , e r h r b , l , r C D n . '
O L r r e . u | ( d t . o . d , _ r rre r,ral re Increr5e i 1 e r . r g l e \ p c r di.ùre ruund r1 I D rdlr r s D a r r J d u e 1 , , r n i n . r o * e n h . p r r , . m . , r b o l n r , J $ n o ú n
"1,:ii:'i'::il'v
\ o r a ì e 0o' ^\)gcn
.on."mp
ioî i,
n . p J l , , c ) t c , f t o , n F D r i r . ful./, ), The:rc.cd.. |l ' r v e r ^ \ e . n . u 1 . r r . r p ' : u n i . d J , . b o r t r t o J r i n c r . . u , e ù m r , ù r o î d . r a t o x ) te n \ o r \J r p t i o n . . , ? r , o t o r n i , c r , - ! e d n o n m ì i ( r ì . ' l d . I o\)9. .un.uîor,iin ) \a. . , footc ,t.:ll- T".i1"'..,
1.,po1'.ore
ro, ,he chrnge. n nun
r l a r r c n o dn,ri o\\è.I con\ùnt ro1 3re nor (luq n. nur ney rnay rnvolve attered mjcrosolnrl and,/o. perorúsomal oxida-_ nve pathways.T o , c a i n I n n g h . . , . tnr. m,.ch. n . r n o i . l r î u l d | | i i r 4l n r o c ì o n ú r a t r\ge1 i on,umpr:on r I D rrr, ue dtptrcJ , . p - o ^ u n etd{ cI) Jr,,tJr, ro iden,it) ufi, n g out, oi rea.ùons involved in nirochoÌdriai res;irarion are aitected
.:..T1':1.
:f'l ì
r,urn
FD , .h
.ì
he h gÀer
,",p,r,ror1
rare.
r o u n c J n . h e D r o c \ t e . t r o r L D r J r . . o u i d b , . d J . r o \ T i :lll,l-"-llj
.:i'' '. of pror^n
'e:k pho,r,,o-)
rJri
's.).,em
! n a / o , . u h , l J : r e o \ i d J ' i o î eJ.rion.. ft. re\ut., .ho$ rhorl: i 1.e,,:
,e,I.on
î o. ti,o. hoaJ.rar
nfo.on
r"rk
.o av , i.
13,"lll:li"
,l ,.or:.er
heDb.o.)re,
r,on r_D
...,,/,r;rp
t o n d n o \ " r ? r on I ni.o,hond,irt f r o l e r m r . . ' . i n L D r ] l . . w c . r n e \ c t J o e \ J n d r i o ,, , | , , ì n , r . p e r r l e r b r 0 ot rtre îiro.tond,i inî.r r_nbr ,ìe. \rni_ ' j l ' l ' , 1 . . ' " : | | . ,e,po_,-e,, rt-" Dh,,,ph.-)iJrr.rs \ ) r r e m ru a v , , . r c h , r n t ,
J _ tf r ò r l p 2a. On Ine ortre- I.rnd .he
irTlj,::.ry1,e
o' rì,e.1b..
rre,,\rd-
on
,er,
Lon.
ro
rLr.
r ! - ( r g n r c d r | \ . n c r e 3 , . . J n heDd.oc\.er I | o n L D r J r r r/;8 u . u . t " ,, t n : r , m L , t r r i o n o r r h e ,u b r r à r e o \ i d . , r i o n f J f " ; ) c a n r d L e ln an inrrer.c In rl-e o.,or ^, rc.1o,rtJte. I n t t l ;
L l ' i : i l i - " i 1
1 . $ e I J . ' o \ i d î r i \ e e 1 / ) m e ! . . i \ i r )
B c
( r ! < c rî1. 'ed un cnero)-d. î . e J i e r c \ h i b r ( e J h . g h c r . L t a . r n g ll e e t d J , c r d /f f 4 . r c \ e t . . r g r e d t . r r i u $ , , t f F \ r o ' T r \ e r d r d d n I n . re a { d f t , A d \ ] | r r b r t i r ) t b r . r \ , . r o \ i d d _ * l : : " * : , . r n . a l o . r i J n . r. kno"n ,hJr cJrn rne prr. m ì , , ) r r . n \ t e t u j . f. rhe îrr1 ,eg, t or) .rep of miro.iuu_ o n i r r d | | ) d c r d o | d d r i o n . . r . r,rcr.'a.ed t r l a h r g n _ . a r d r e . :. A n ì n c - . J . e d ^ t t - A o \ i d a ' i o n nJ) ,,onrnbL,"r u r e o . r c e . e r v a | a D r , t ) o , t r p i d . i^r,torrgc in iJipo,e ri-re lnu, coln L e m c r f g L h e d e v e t r { r n ì e n r o t o b r s r r } .
Ltvet resphation and high_fat díet: Lionetti et al.
T h c rppLcrion
o r r o p _ d . . { 1 e t d . r i . r . )
a 1 r , y . r .
a t l , r w ,
J 5
l : . ' d : r l , y
h e ,y b J , . r e
î \ r r r a r , r c
p r r h u r l d\ a p^.\Jbre.i,e
j " * ' * "
' " ' 1 . , n c r c J . e d
r e , p i r d r i o n
r r e. Jound
i 1 .rutarcJ
Depatocytes
lrom ED rars. whereas
any changes
mat may
: : : : : i l l r i
D r " * rerr rnJ pno.phoir.rr,ng
, y , r e n are
I r!gn|lr?nt rn rerî. of he at...,eo
o \ \ g e 1 \or\rmpr]o1
I r e n r h e , e . e l t .
U e r . e .
f e o - r o . . r e ,
r r ó i C D ra . , , n ú l \
r
i m u t a t r o , r
o t l h e rc n , l t o n \ . n J l
o r o d ù c e
A L p i .
b u t n o . o l
i l : l : a . l o n
h d , o , n , J m e
r r e . u n r r g
n , n : n c . c . , e r r
a V
-uÙe Io Ulc e\a _ v r n 0 n - p , r J l r o r "a1.. I n i n . r . I het$o..\rF. " .he rài,ed d Y f . ( r I \ J n -rì ircrF:,e,j f.oron Ddc(nou ac_or. úe F n e r m r , u . t ^ n d i l t mern'.ranf. s h r c h , . nor coupteo \ ^ i , h ( o n r u m o r r o n t o u n d ir L D r r , A . . n r J r m e c t n î . . m ka s q r g p c ( l e d b ) \ o b , \ e ' d I l o r \ p j r i n t h e J n . r e . r , e d , , \ ) g e n c o 1 . L î D r r , , n i r i,oiare,j h e f r . o , y r e . .u t p J , c J w i r h F È À . o ù r r F , L t . . in d care ndr lhe i rcr,.a,ed L v . . r o \ . g e î c o l , u m p r i o n . d e r. .he .ti nu,àLo1 of ,ub,lrdLe o x r o - l r o n p z l h \ \ r ) . . . on||.bu.e. t ^ l h c r n c r e é . c o
e l e r g ) e \ r . . : . l l . I
" f t D r J r . . p d n oi ni. ,nc.ed"ed u x y s e n .o n r î t l r o r . d r b e d i r . . l e d ro hFJr pjod rcr ,,n rrrne, t]1in lo
r r . u ' r n g i n J a.. red,eo merdbo'ic eiii
Acknowledgments
Y:.11:li llr
, BJ,
dn"hdm
,o, e\pen
,c.h,
icJ,
rs,.,Jnce,
m ( r u o n g r r r t-repJr.r,,,n o t l n e h c p J t ^ c ' t c , . drd se are:'-|]1:
"1
'" P'.r...,'Anror
io Bortcr
a roì rrerpir,
cr,cLr.
.':'
: i,ì.'gr.i úd. .upporeo
b) Minr.rero
Dert.
I ni\er
, r r n L . D e , - R : , e , c d S . i c n , . r c a I l e c n u t o g . - d n d ( o n s r g r r 0 N a Í o n r Ì e D e l t r RiceriheReterences
ì Uvc.ini, G. tosjr. S,andBarlcrj.A. (1994).HÒraù.
mrto.hondrial , i : l ' l i ' . I , , r p o h . . . . . \ , . f/ 14, ù rùó,, -
l . ' " . 1 ' , : . r o d . s . t , . 1 e . r , L \ I o . t . . . \ r p . . . î o B J . k , . A i ' 1 " ì l . ì , . 1 ; ' ' " . : ' - ) _ , o r . r , , . .oel .. .r pon.e ,^ .ooJ .1
, 1 . r ' . ' í 1 ' ; ; i : .
8 , ' q d , , , ,
. *ì;l''i ."i;':"i:l :"i:"1::
]i ,l;,',',\,:X
;.
4 t s d l l J \ r : J , r o . ! , r . , . u , A i , t e . . b o { . p o -. , l . . ' . t ' d L r ' , . n o r r n e . . \ / , . . 2 8 r . . rJ . 1 d \ . o . . ^,t.J | ,qÒ, B oL, " - , p , e r , , e n
: " . ,
L l i . ^ 1 - ' : i i . " ; . -
- a \ r a d , m e r, . . . . o L d \ r
, r
" ii:,,",
ì ij . ""ì"'
.jì',11::'
jì : t' ";,r
";;,,,
1 " : ' " ' " i 1 9 . . , . . d . , . . r r fo i m o a - n . . o i à e . ' " _ _ , " ' . 4 t . P \ , . t \ . , a o ! , , 0 , , t " o a : l : - ' - l l : b " B - \ ' o J e o { r v \ ' é B ' , - b , ui ' l : ' ì i , ì l , l i ' "
D A ' r ' "
n o p - o '' ) a
e 8 4 . V \ . , ' \ D L . , . \ ' q e o d U d r J . . p ù . r o b . l. ì : ; " j j : . . , : : : Ì i . , ' . " r ' o o r
ì v . r v \ , d J t o . t J ^ ! 8 0 B o ,; ; i . . : ; " " ; : ' i " : : :
' . r
a " ' l n ' ù , P \ Ò
l0 B.roùn, c'c., hknr Thonìas, p.L.. aM Bmnd, M.D. (19!0) Comrot
: i , ' . 1 , , - ; " " 1 ' i ; ! ,
i t . D . o ' ) , d
' 1 4
" g : g nen brtn BiùLhrn. Brat,l))\ c.rd 726.41 R0" D : a o . r , . , o , o n . e . . r o . . , , , . o . o r J . . , . { f l r / t s ù t ì . , \ ' t T 8 . , , . . r . \ . t , : I V r J B r . , i J ^ t D i o a . .
R osearc
h C o m m u n i c ati o n s
Eff4ts ol Î$'rcid hoÚones on oxid'tile Pnosphorylarìon at"/ì"n
Soc. lm6 21,185 192
ì i - " . v . È - a e . . d M . D . o o r ' r h è q u ú r r E ù v P ' n ' ú i b ú r ' ó n ' "
.:'J'.**a'i,r P,olor led* and ATP nrÓ\er rrr'ot o he
i,'ì"" '.i"-*i' "*
' t'P'oc re' rrcr nb o drr'e*nr 'hv rcÀ;àú' i B'ol Chen 268 14850 1436u
r , r ' l l ì " , 3 , ' ' ' . . i '. t. n"ricr' v P BureLa a . d r o L i v e n n r ' c i ' L i i n . - . . ' ' ' " r r ' o J ' 1 h r p o h r o r d r ' J / r ' / " 4 n o r 4 8 ' ', ' i',:, i'" dd v.cncr'n Kr' re8a- H'sn errt:e " or 'ne sv
i i i ^ i " . . - . r " - i " - ' l - Ò E e r e d É s l e p r a z o ' c 3 ' 1 N t r s1.379-38?
r o i . r ' r , V t . , ' ' r , . n ' A i E I d d e p r ' r i o n " o r . ú o r p h Ó l o 8 h ' u I r r ,* i""L.a o**ry.-.ill( Lon ra.l,ver' Marodr Ea^aor l2' 625-512
r I é."m r'a.'\'
"ro rti" a O ' laoo' H 8l ) eld.Óre-rdion or no_ ' r " J . , . " " o * " t v " c e l l ' A b ' o c h è n ' d Ú d Î r e s n ' ' Ú d rùdv. J C.' ar,/ 43, 50G520 ' " i i " " " r ' t 9 h 7 ' . R e n o t J ' o r h r i ú d f r o m s é r u m d l b u r i n b J '"
it-"ì'"ii""*-, 1 ataL chen z2' 17) t81
Nobè". c.D. dó BEfo. V D. ,q8o A qrr rbuve òse$Trnr or Lhè r)e o oC d'{nb".ior.o ruóuF pìòm' m"nbúreI.?Ùìù
li isr""o t'"*
",i".
Br,-t' Boph;' A 'asa1' 15 t"
H m r r 8 . . N ' . n o l ì . , D . C . . ú d $ i l l ' d m o l J R ' l o 8 0 ' D È e m
na'on ol Lh' miro rooanal pobnrÙ nÈ rorce :n bo'altd hepÙo_
.\Ès J. A@L ChPn 2s5.l4t8-1464
Nob6. C.D.. H.Ì, $ W dd Bffo M D ' lo90' Th' m4\dhn
l'ì"",i*.i
"i *p*'.. W arv "'id'
'r nora ed hepo@! it' .l' Biat, Chen 2ó5' 129\0-12915
c . , r , ' . r , * " c * "
r ' a ' t ì o o j R ' e " l " u " '
' l l " l ^ T l d
.rida.,on ll mffaid Ùver' Aio'htî aioph" A ta Ltnt LL'
241
B r d d v S B , K n @ b e r ' c M d d B a d ) l l " o 8 o . f { e ! d ' c r u r o _
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