Metabolic control of mitochondrial properties by adenine nucleotide translocator determines palmitoyl-coa effects

Transcription

1 Metabolc control of mtochondral propertes by adenne nucleotde translocator determnes palmtoyl-coa effects Implcatons for a mechansm lnkng obesty and type 2 dabetes Jolta Capate 1,5, Stephan J. L. Bakker 2, Mchaela Damant 3, Gerco van Ekenhorst 1, Robert J. Hene 3, Hans V. Westerhoff 1,4 and Klaas Krab 1 1 Department of Molecular Cell Physology, Insttute for Molecular Cell Bology, Faculty of Earth and Lfe Scences, VU Unversty, Amsterdam, the Netherlands 2 Department of Internal Medcne, Unversty of Gronngen and Unversty Medcal Center Gronngen, the Netherlands 3 Department of Endocrnology, Insttute for Cardovascular Research, VU Unversty Medcal Center, Amsterdam, the Netherlands 4 Manchester Centre for Integratve Systems Bology, MIB, Unversty of Manchester, UK 5 Centre of Envronmental Research, Faculty of Nature Scences, Vytautas Magnus Unversty, Kaunas, Lthuana Keywords metabolc control analyss; oxdatve phosphorylaton; palmtoyl-coa; reactve oxygen speces; type 2 dabetes Correspondence J. Capate, Centre of Envronmental Research, Faculty of Nature Scences, Vytautas Magnus Unversty, Kaunas, Vlekos 8, LT-44404, Lthuana Fax: Tel: E-mal: jolta.capate@falw.vu.nl (Receved 21 June 2006, revsed 19 August 2006, accepted 4 October 2006) do: /j x Inhbton of the mtochondral adenne nucleotde translocator (ANT) by long-chan acyl-coa esters has been proposed to contrbute to cellular dysfuncton n obesty and type 2 dabetes by ncreasng formaton of reactve oxygen speces and adenosne va effects on the coenzyme Q redox state, mtochondral membrane potental (Dw) and cytosolc ATP concentratons. We here show that 5 lm palmtoyl-coa ncreases the rato of reduced to oxdzed coenzyme Q (QH 2 Q) by 42 ± 9%, Dw by 13 ± 1 mv (9%), and the ntramtochondral ATP ADP rato by 352 ± 34%, and decreases the extramtochondral ATP ADP rato by 63 ± 4% n actvely phosphorylatng mtochondra. The latter reducton s expected to translate nto a 24% hgher extramtochondral AMP concentraton. Furthermore, palmtoyl- CoA nduced concentraton-dependent H 2 O 2 formaton, whch can only partly be explaned by ts effect on Dw. Although all measured fluxes and ntermedate concentratons were affected by palmtoyl-coa, modular knetc analyss revealed that ths resulted manly from nhbton of the ANT. Through Metabolc Control Analyss, we then determned to what extent the ANT controls the nvestgated mtochondral propertes. Under steadystate condtons, the ANT moderately controlled oxygen uptake (control coeffcent C ¼ 0.13) and phosphorylaton (C ¼ 0.14) flux. It controlled ntramtochondral (C ¼ )0.70) and extramtochondral ATP ADP ratos (C ¼ 0.23) more strongly, whereas the control exerted over the QH 2 Q rato (C ¼ )0.04) and Dw (C ¼ )0.01) was small. Quanttatve assessment of the effects of palmtoyl-coa showed that the mtochondral propertes that were most strongly controlled by the ANT were affected the most. Our observatons suggest that long-chan acyl-coa esters may contrbute to cellular dysfuncton n obesty and type 2 dabetes through effects on cellular energy metabolsm and producton of reactve oxygen speces. Abbrevatons [AMP] out, concentraton of extramtochondral AMP; AMPK, AMP-actvated proten knase; ANT, adenne nucleotde translocator; Ap5A, P 1,P 5 -d(adenosne-5 )-pentaphosphate; ATP n ADP n rato, ATP to ADP rato n the mtochondral matrx; ATP out ADP out rato, extramtochondral ATP to ADP rato; C X m, concentraton control coeffcent, quantfyng control of ntermedate X m by module ; C Jk control coeffcent, quantfyng control of flux J k by module ; Dw, membrane potental,.e. electrcal potental across the nner mtochondral membrane; J 1 h, proton leak flux; J o, oxygen uptake flux; J p, phosphorylaton flux; LCAC, long chan acyl-coa ester; QH 2 Q rato, rato of reduced to oxdzed coenzyme Q; ROS, reactve oxygen speces; S-13, 5-chloro-3-t-butyl-2 -chloro-4 -ntrosalcylanlde., flux 5288 FEBS Journal 273 (2006) ª 2006 The Authors Journal complaton ª 2006 FEBS

2 J. Capate et al. Palmtoyl-CoA and control of mtochondral functon In mammals, mtochondra produce the majorty of ATP requred to drve energy-dependent cellular processes. However, mtochondra also play more ndrect roles. Impared mtochondral functon s emergng as an mportant factor n nsuln-resstant states: less effcent mtochondral oxdatve phosphorylaton has been demonstrated n both the elderly and nsuln-resstant offsprng of patents wth type 2 dabetes compared wth young, healthy controls [1,2]. Although under physologcal condtons nonesterfed fatty acds are an mportant source of fuel for many tssues because they can yeld relatvely large quanttes of ATP, obesty-related persstent oversupply of nonesterfed fatty acds and accumulaton of tracylglycerols n nonadpose tssues s thought to contrbute to the molecular mechansms underlyng both nsuln resstance and b-cell dysfuncton n type 2 dabetes [3,4]. Nonesterfed and esterfed fatty acds nterfere wth mtochondral oxdatve phosphorylaton n vtro [5,6]. Furthermore, an mbalance n fatty acd metabolsm resultng n actvaton of nonoxdatve rather than oxdatve pathways and accumulaton of bologcally actve molecules [e.g. long-chan acyl-coas (LCACs), ceramde, dacylglycerol] could adversely affect cellular functon by drect effects on a varety of enzymes and nducton of apoptoss [4]. Tght regulaton of ntracellular concentratons of free LCACs by acyl-coa-bndng proten can be mpared under pathologcal condtons wth excess lpd supply (e.g. obesty) because of nadequate expresson of the latter [7]. LCACs modulate the actvty of the mtochondral adenne nucleotde translocator (ANT) from both the outer and matrx sdes of the nner mtochondral membrane by compettve dsplacement of the nucleotde from ts bndng ste on the proten [8]. It has been hypotheszed that ncreased concentratons of free LCACs nterfere wth mtochondral functon through nhbton of the ANT, leadng to lower cytosolc ATP and matrx ADP avalablty, ncreased mtochondral membrane potental (Dw), and reducton level of coenzyme Q [9]. The two latter events are expected to promote the formaton of reactve oxygen speces (ROS) [10,11], resultng n mpared cellular functons and cell death. Moreover, ncreased AMP producton by adenylate knase caused by the low cytosolc ATP ADP rato and further breakdown of AMP to adenosne s expected to result n an ncrease n extracellular adenosne concentraton [12]. The latter s a potent vasodlator [13], whch can promote sodum retenton n the kdney and stmulate sympathetc nervous system actvty [14]. Fatty acd-nduced nsuln resstance n lver s one of the man causes of hyperglycema n type 2 dabetes [15], and the role of mtochondra n ths dysfuncton s not fully elucdated. We have shown that, n solated rat lver mtochondra oxdzng succnate, palmtoyl- CoA nhbted the ANT and nduced workng-condton-dependent changes n ntramtochondral and extramtochondral ATP concentratons and Dw [16]. The relatve contrbuton of a partcular enzyme to the control of metabolc pathway flux and concentratons of reacton ntermedates determnes to what extent nhbton of that enzyme would affect pathway flux and ntermedate concentratons. The control can be quanttatvely assessed usng Metabolc Control Analyss [17 19]. The control of fluxes and ntermedates s a system property,.e. t s determned by all enzymes consttutng the pathway. For ths reason here we quanttatvely assessed the control of fluxes and ntermedates of oxdatve phosphorylaton not only by the ANT but also by other components of oxdatve phosphorylaton. Furthermore, we tested parts of the above hypothess by determnng the effects of palmtoyl- CoA on actvely phosphorylatng (state 3) mtochondra oxdzng a more physologcal NADH-delverng substrate,.e. glutamate plus malate. To nvestgate whch mtochondral enzymes are nvolved n the multple effects that we encountered, we mplemented modular knetc analyss. We found that palmtoyl- CoA acts drectly on the ANT, and then ndrectly nduces ROS producton and a concomtant reducton n the extramtochondral ATP ADP rato. The extent to whch palmtoyl-coa affected dfferent mtochondral propertes can largely be explaned by the magntude of the control exerted by the ANT over these propertes. Results Palmtoyl-CoA effects on steady-state fluxes and ntermedate concentratons Table 1 summarzes the effects of 5 lm palmtoyl-coa on the steady-state fluxes and ntermedate concentratons n solated rat lver mtochondra resprng on glutamate plus malate. Palmtoyl-CoA decreased oxygen uptake flux (J o ) by 56 ± 3% and phosphorylaton flux (J p ) by 58 ± 7%, and ncreased proton-leak flux ( Jh 1 ) by 37 ± 6%. The opposte effect was found on the extramtochondral and matrx ATP ADP ratos: the former decreased by 63 ± 4%, and the latter ncreased by 352 ± 34%. The reduced to oxdzed coenzyme Q rato (QH 2 Q) ncreased by 42 ± 9%, and Dw ncreased by 13 ± 1 mv (9%). The QH 2 Q FEBS Journal 273 (2006) ª 2006 The Authors Journal complaton ª 2006 FEBS 5289

3 Palmtoyl-CoA and control of mtochondral functon J. Capate et al. Table 1. Steady-state values of fluxes and ntermedates, as affected by palmtoyl CoA. Values are mean ± SEM from four experments. No Palmtoyl-CoA +5lM Palmtoyl-CoA J o [nmol O 2 Æmn )1 Æ(mg proten) )1 ] 53 ± 3 23 ± 2** J p [nmol ADPÆmn )1 Æ(mg proten) )1 ] 375 ± ± 15** Jh 1 (nmol O 2Æmn )1 Æ(mg proten) )1 ] 3.3 ± ± 0.4** Dw (mv) 150 ± ± 3** ATP n ADP n 0.67 ± ± 0.04** ATP out ADP out 0.16 ± ± 0.01* QH 2 Q 6.7 ± ± 1.3* AMP out (calculated) (lm) *P <0.05 and **P <0.01 versus no palmtoyl-coa. rato n mtochondra resprng on succnate was 4.9 ± 0.3 and 5.4 ± 0.2 n the absence and presence of 5 lm palmtoyl-coa, respectvely. We conclude that palmtoyl-coa affects vrtually all steady-state propertes of these mtochondra, albet to varous extents. Palmtoyl-CoA effects on mtochondral H 2 O 2 producton We have shown that 5 lm palmtoyl-coa caused a sgnfcant ncrease n Dw n actvely phosphorylatng mtochondra (state 3) resprng on succnate [16] and NADH-delverng substrate (Table 1). To test the noton that the palmtoyl-coa-nduced ncrease n Dw would stmulate ROS producton [10], we determned the effect of palmtoyl-coa on H 2 O 2 producton n mtochondra resprng on succnate. Fgure 1A shows that palmtoyl-coa nduced H 2 O 2 producton n state 3 n a concentraton-dependent manner. The palmtoyl-coa-nduced H 2 O 2 producton was partally senstve to protonophore S-13, suggestng dependence on Dw (Fg. 1B). In lne wth ths, nhbton of the ANT wth atractylosde or carboxyatractylosde and ATP synthase wth olgomycn also nduced H 2 O 2 formaton, although to a lower extent (Fg. 1B). To test whether palmtoyl-coa metabolsm va b-oxdaton contrbutes to ncreased H 2 O 2 producton, we determned the effect of palmtoyl-l-carntne (substrate for b-oxdaton) and malonyl-coa (nhbtor of palmtoyl-carntne transferase 1, part of the mtochondral acyl-coa transport system). Palmtoyll-carntne (5 lm) alone and n combnaton wth atractylosde (to test whether the effect of palmtoyl- CoA requres both ts oxdaton and ts nhbton of the ANT) stmulated H 2 O 2 producton rate less than 5 lm palmtoyl-coa (Fg. 1B), suggestng that b-oxdaton was not nvolved. Furthermore, rotenone, an nhbtor of respratory chan complex I, had no sgnfcant effect on palmtoyl-coa-nduced H 2 O 2 producton. However, partal nhbton of palmtoyl-coanduced H 2 O 2 producton wth malonyl-coa (Fg. 1B) suggests that palmtoyl-coa partally exerts ts effect from the matrx sde. A B Fg. 1. Effect of palmtoyl-coa on H 2 O 2 producton n solated mtochondra resprng on succnate. (A) Dependence of H 2 O 2 producton on palmtoyl-coa concentraton. (B) Comparson of the effects of varous nhbtors on H 2 O 2 producton. St 3, State 3; p-coa, palmtoyl-coa (5 lm), protonophore S-13 (0.2 lm); AT, atractylosde (1.5 lm); CAT, carboxyatractylosde (0.1 lm); Olgo, olgomycn (0.5 lm); Ro, rotenone (2 lm); M-CoA, malonyl-coa (0.1 mm); PC, palmtoyl-l-carntne (5 lm). All nhbtors were added n state 3. Values are mean ± SEM from four experments. *P <0.001 versus state 3; #P <0.02 and $P <0.002 versus 5 lm palmtoyl-coa FEBS Journal 273 (2006) ª 2006 The Authors Journal complaton ª 2006 FEBS

4 J. Capate et al. Palmtoyl-CoA and control of mtochondral functon Palmtoyl-CoA effects on extramtochondral AMP concentraton We have shown that 5 lm palmtoyl-coa caused a sgnfcant decrease n the extramtochondral ATP ADP rato (ATP out ADP out ) (Table 1). However, n ths partcular experment t was not possble to determne the effect of decreased extramtochondral ATP avalablty on extramtochondral AMP formaton expermentally, as we used P 1,P 5 -d(adenosne-5 )-pentaphosphate (Ap5A) as nhbtor of adenylate knase to prevent depleton of avalable ATP and ADP and to mantan steady-state respraton. Instead, we dd a theoretcal calculaton of the extramtochondral AMP concentraton ([AMP] out ) expected at dfferent ATP out ADP out ratos. Ths calculaton assumes that the adenylate knase reacton s at equlbrum, whch s a safe assumpton because there s not much net flux expected through ths enzyme under the condtons nvestgated. Fgure 2A shows [AMP] out predcted to be present at dfferent steady-state ATP out ADP out ratos when the total adenylate concentraton s 0.1 mm, wth the assumpton that the proportons of adenne nucleotdes A B are regulated by the adenylate knase equlbrum. In the range of relatvely low ATP out ADP out ratos, a small decrease leads to a large ncrease n [AMP] out, whereas [AMP] out changes relatvely lttle n the range of hgh ATP out ADP out ratos. As ndcated n Fg. 2A, when expermentally obtaned values of ATP out ADP out ratos (Table 1 and [16]) are used n the calculaton, nhbton wth palmtoyl-coa would cause an ncrease n [AMP] out of 17% and 24% wth succnate and glutamate plus malate as substrates, respectvely. However, such a low ATP out ADP out rato (< 0.2) obtaned usng excess hexoknase and low concentraton of total adenylates (.e. 100 lm) s not lkely to be relevant under physologcal condtons. For ths reason we performed an experment wthout adenylate knase nhbtor and wth hgher and more physologcally relevant total adenylate concentraton (2 mm). We determned how palmtoyl-coa (5 and 10 lm) affects the ATP total ADP total rato and [AMP] total n actvely phosphorylatng (state 3) mtochondra resprng on succnate and compared the expermental and calculated values (Fg. 2B). Because the total adenylate concentraton n the medum was hgh (2 mm) and the contrbuton of the matrx adenylates was relatvely low ( 10 lm), we assumed that changes n the ATP total ADP total rato reflect changes n the ATP out ADP out rato. Palmtoyl-CoA caused a sgnfcant concentraton-dependent decrease n the ATP total ADP total rato and ncrease n [AMP] total, whch corresponded qute well to the correlaton of [AMP] out and the ATP out ADP out rato predcted by the calculaton. Palmtoyl-CoA specfcally affects the ANT Fg. 2. Dependence of AMP concentraton on the ATP ADP rato. (A) Dependence of AMP concentraton on the ATP ADP rato when the total concentraton of adenylates s 100 lm. [AMP] was calculated as descrbed n Expermental procedures usng an equlbrum constant for adenylate knase equal to [42]. The ponts on the curve show [AMP] expected to be present at the expermentally obtaned mean values of ATP out ADP out for succnate [16] and glutamate plus malate (Table 1), respectvely, f adenylate knase was not nhbted. (B) Dependence of AMP concentraton on the ATP ADP rato when the total concentraton of adenylates s 2mM. The ponts show expermentally determned dependence of [AMP] total on the ATP total ADP total rato n actvely phosphorylatng (state 3) mtochondra resprng on succnate wth no adenylate knase nhbtor added. The ponts correspond to condtons wth 0, 5 or 10 lm palmtoyl-coa added, and are mean ± SEM from three ndependent experments. *P <0.05 versus no palmtoyl-coa. Succ, Succnate; g + m, glutamate plus malate; p-coa, palmtoyl- CoA. Open symbols, no palmtoyl-coa; closed symbols, + palmtoyl-coa. To dentfy the stes of oxdatve phosphorylaton drectly affected by palmtoyl-coa, we appled modular knetc analyss n two dfferent ways: wth ether Dw or matrx ATP ADP rato (ATP n ADP n ) as an ntermedate. Modular knetc analyss wth Dw as connectng ntermedate revealed that palmtoyl-coa nhbts the phosphorylatng module (Fg. 3A), as the flux through the module (J p ) was sgnfcantly lower n the presence of palmtoyl-coa than n ts absence, when both condtons were compared for the same levels of Dw. The flux through the substrate-oxdaton module was slghtly, although not sgnfcantly, hgher n the presence of palmtoyl-coa (Fg. 3B), ndcatng a tendency of palmtoyl-coa to stmulate the actvty of ths module, possbly va ts effect on b-oxdaton. The proton-leak module was not affected drectly by palmtoyl-coa (Fg. 3C). FEBS Journal 273 (2006) ª 2006 The Authors Journal complaton ª 2006 FEBS 5291

5 Palmtoyl-CoA and control of mtochondral functon J. Capate et al. A B C Fg. 3. Effect of palmtoyl-coa on the knetcs of the oxdatve phosphorylaton modules around Dw. (A) Knetcs of the phosphorylaton module as determned by ttraton of the substrate oxdaton module wth 0 25 nm myxothazol. (B) Knetcs of the substrate oxdaton module determned by ttratng the phosphorylaton module wth lm olgomycn. (C) Knetcs of the proton leak module as determned by ttraton of the substrate oxdaton module wth 0 55 nm rotenone when the phosphorylaton module was blocked wth 0.3 lm olgomycn. J p was calculated as: J p ¼ J o ) J h at the same value of Dw; Jh 1 was measured as J o n the absence of ADP phosphorylaton [46]. Values are mean ± SEM from four experments. Open symbols, no palmtoyl-coa; closed symbols, +5 lm palmtoyl-coa. Further analyss wth the ATP n ADP n rato as an ntermedate showed that the ATP-consumng module (comprsng the ANT and hexoknase) was nhbted by palmtoyl-coa (Fg. 4A), as concluded from lower flux through the module n the presence of palmtoyl- CoA, whle the ATP-producng module was not affected (Fg. 4B). We have shown prevously that hexoknase s not nhbted by palmtoyl-coa [16]. Therefore our current data ndcate that, also n mtochondra resprng on the NADH-delverng substrate, ANT s the only component of oxdatve phosphorylaton affected by palmtoyl-coa, although a stmulatory effect on substrate oxdaton cannot be excluded. Thus the multtude of effects on steady-state fluxes and ntermedate concentratons exerted by palmtoyl-coa s acheved through nhbton of the ANT. A B Metabolc control of mtochondral propertes To determne whether palmtoyl-coa affected the propertes t would be expected to affect for ts drect acton on the ANT, and to see f we could account for the observaton that some propertes were affected more than others, we used the systems bology method of Metabolc Control Analyss. To assess the control of fluxes and ntermedates, we took a modular approach (Fg. 5). Metabolc control of fluxes Fg. 4. Effect of palmtoyl-coa on the knetcs of the modules of oxdatve phosphorylaton around the ntramtochondral ATP ADP rato. (A) Knetcs of the ATP-consumng module as determned by ttraton of the ATP-producng module wth 0 20 nm myxothazol. (B) Knetcs of the ATP-producng module as determned by ttraton of the ATP-consumng module wth lm atractylosde. J p was calculated as: J p ¼ J o ) J h at the same value of Dw and multpled by the ADP O rato [16] (ADP O ¼ 2.7 ± 0.1). Values are mean ± SEM from four experments. Open symbols, no palmtoyl- CoA; closed symbols, +5 lm palmtoyl-coa. Control coeffcents of the sx modules of oxdatve phosphorylaton over the oxygen uptake (J o ) and phosphorylaton flux (J p ) for both respratory substrates are summarzed n Table 2. The dstrbuton pattern of the control over J p among the modules was smlar to that of J o for both substrates used except for the negatve control exerted by the proton leak (because t dsspates Dw whch s needed to drve ADP phosphorylaton and adenne nucleotde translocaton). In all condtons, the control dstrbuton 5292 FEBS Journal 273 (2006) ª 2006 The Authors Journal complaton ª 2006 FEBS

6 J. Capate et al. Palmtoyl-CoA and control of mtochondral functon Fg. 5. Dvson of the oxdatve phosphorylaton nto modules. The modules: 1, Q-reducng module, comprsng dcarboxylate carrer and substrate dehydrogenases (malate and NADH dehydrogenases n the case of glutamate plus malate oxdaton, or succnate dehydrogenase n the case of succnate oxdaton); 2, QH 2 -oxdzng module, comprsng cytochrome bc 1 and cytochrome c oxdase; 3, proton leak module, comprsng passve membrane permeablty to protons and caton cyclng; 4, ATP synthess, comprsng ATP synthase and phosphate carrer; 5, adenne nucleotde translocator; 6, hexoknase. The ntermedates: a, QH 2 Q rato; b, membrane potental (Dw); d, matrx ATP ADP rato (ATP n ADP n ); c, extramtochondral ATP ADP rato (ATP out ADP out ). Arrows marked e, h 1 and p ndcate electron flux, transmembrane proton flux, and ATP flux, respectvely. The dashed arrow h 1 gong from Q-reducng module to Dw s vald only when glutamate + malate s used as a substrate. Table 2. Metabolc control of fluxes. The control coeffcents were calculated from elastcty coeffcents (Supplementary materal, Table S2) and steady-state fluxes (Table 1 and [16] for glutamate plus malate and succnate, respectvely). Values are mean ± SEM from three (succnate) or four (glutamate plus malate) experments (ndcated as subscrpt). Q red, Q-reducng module; QH 2 ox, QH 2 -oxdzng module; Leak, proton-leak module; ATP synth, ATPsynthess module; ANT, adenne nucleotde translocator; Hk, hexoknase; p-coa, palmtoyl-coa. Module, C Jo was as expected for state 3: the bulk of flux control was shared between the respratory chan and the modules nvolved n the producton of extramtochondral ATP (actually glucose 6-phosphate), wth hardly any control by the proton-leak module. The contrbuton C Jp No p-coa + 5 lm p-coa No p-coa + 5 lm p-coa Succnate Q red ** ** QH 2 ox * * Leak ** ) ) ATP synth * * ANT * ** Hk Glutamate plus malate Q red * * QH 2 ox Leak ** ) ) ** ATP synth ANT * ** Hk *P <0.05 and **P <0.01 versus no palmtoyl-coa. of the ANT to the control of J o and J p was moderate and smlar wth both respratory substrates. When the two substrates are compared, usng glutamate plus malate nstead of succnate, control of the fluxes shfts from the respratory chan to ATP synthess. Furthermore, the dstrbuton of the control wthn the respratory chan shfts from the part downstream of coenzyme Q wth succnate to the part upstream of coenzyme Q wth glutamate plus malate. In agreement wth the fact that the ANT s the only target of palmtoyl-coa n the system of oxdatve phosphorylaton under these expermental condtons, we found that, wth both respratory substrates, the control exerted by the ANT over J o and J p sgnfcantly ncreased upon nhbton wth palmtoyl-coa. The control of J o ncreased by 67% and 55% wth succnate and glutamate plus malate, respectvely, whereas the control of J p was affected more strongly: t ncreased by 87% and 83% wth succnate and glutamate plus malate, respectvely. Owng to the summaton property of flux control coeffcents [17,18], an ncrease n the control strength of one component of the system automatcally leads to a decrease n the control strength of other component(s). In our case, an ncrease n the control of fluxes by the ANT was manly compensated for by decreased control by the respratory chan modules (Table 2). Furthermore, the control by the proton-leak module slghtly but sgnfcantly ncreased because palmtoyl-coa ncreases Dw, movng the system to a new steady state that s closer to state 4, where control by proton leak s hgh. FEBS Journal 273 (2006) ª 2006 The Authors Journal complaton ª 2006 FEBS 5293

7 Palmtoyl-CoA and control of mtochondral functon J. Capate et al. Control of the QH 2 /Q rato and Dw Coenzyme Q reducton level and Dw are among the factors that determne ROS producton by the mtochondral respratory chan [11,12]. The control of these ntermedates by the sx modules of oxdatve phosphorylaton s summarzed n Table 3. The sum of all concentraton (also Dw) control coeffcents n a pathway s zero, by defnton [17,18]. Accordngly, the values of the coeffcents can be postve or negatve dependng on whether an enzyme s nvolved n the producton or the consumpton of an ntermedate, respectvely. For both substrates used, the QH 2 Q rato was almost solely controlled by the respratory chan enzymes, wth the coenzyme Q reducng module exertng a postve control and the coenzyme QH 2 oxdzng module exertng a negatve control, whle the control of Dw was shared equally between the Dw-generatng (postve control) and Dw-consumng or Dwconsumpton-stmulatng processes (negatve control) (Table 3). In the case of succnate oxdaton, most of the control of Dw wthn the respratory chan resded n the part downstream of coenzyme Q (cytochrome bc 1 complex and cytochrome c oxdase), whereas, n the case of glutamate plus malate, the part upstream of coenzyme Q (dcarboxylate carrer and substrate dehydrogenases) had slghtly more control of Dw, possbly because NADH dehydrogenase, a proton-pumpng enzyme, becomes actve. Wth both respratory substrates, the ANT exerted negatve control over the QH 2 Q rato and Dw (Table 3). Ths s because actvaton of the ANT stmulates the phosphorylaton branch of oxdatve phosphorylaton, whch consumes Dw. The negatve control over the QH 2 Q rato s explaned smlarly. Palmtoyl-CoA had hardly any effect on the control of the QH 2 Q rato when glutamate plus malate was used as a substrate. Wth succnate, palmtoyl-coa manly affected the control of the QH 2 Q rato by respratory-chan modules: control by both coenzyme Q-reducng and coenzyme QH 2 -oxdzng modules has decreased. Furthermore, palmtoyl-coa had lttle effect on the control of Dw except that the control by the coenzyme Q-reducng and ATP-synthess module sgnfcantly decreased wth glutamate plus malate as substrate, and for both substrates the negatve control exerted by the proton leak slghtly ncreased because of the effect of palmtoyl-coa on Dw. Control of matrx and extramtochondral ATP/ADP ratos The control of the ATP n ADP n rato and ATP out ADP out rato s summarzed n Table 3. For both substrates used, control of the ATP n ADP n rato was shared among all modules of oxdatve phosphorylaton, wth a slght negatve control exerted by the proton leak. The ANT exerted a large negatve control Table 3. Metabolc control of ntermedates. The control coeffcents were calculated from elastcty coeffcents (Supplementary materal, Table S2) and steady-state fluxes (Table 1 and [16] for glutamate plus malate and succnate, respectvely). Values are mean ± SEM from three (succnate) or four (glutamate plus malate) experments (ndcated as subscrpt). Q red, Q-reducng module; QH 2 ox, QH 2 -oxdzng module; Leak, proton-leak module; ATP synth, ATP-synthess module; ANT, adenne nucleotde translocator; Hk, hexoknase; p-coa, palmtoyl-coa. C QH2=Q C Dw C ATPn=ADPn C ATPout =ADPout Module, No p-coa + 5 lm p-coa No p-coa + 5 lm p-coa No p-coa + 5 lm p-coa No p-coa + 5 lm p-coa Succnate Q red * QH 2 ox ) ) * Leak ) ) ) ) ) ) ) ATP synth ) ) ) ) * * ANT ) ) ) ) ) ) * * Hk ) ) ) ) ) ) ) ) * Glutamate plus malate Q red * * QH 2 ox ) ) Leak ) ) * ) * ) ) ) ) ** ATP synth ) ) ) ) * ANT ) ) ) ) ) ) * ** Hk ) ) ) ) ) ) ) ) *P <0.05 and **P <0.01 versus no palmtoyl-coa FEBS Journal 273 (2006) ª 2006 The Authors Journal complaton ª 2006 FEBS

8 J. Capate et al. Palmtoyl-CoA and control of mtochondral functon over the ATP n ADP n rato, because t functons as a consumer of matrx ATP by transportng t from mtochondra to the ntermembrane space. The dstrbuton of control wthn the respratory chan depended on the substrate used: for succnate, the QH 2 -oxdzng module exerted more control than Q-reducng module, whereas, wth glutamate plus malate as substrate, t was the opposte. Palmtoyl-CoA tended to ncrease the postve control of the ATP n ADP n rato by ATP synthess and the negatve control by the proton leak. The negatve control of the ATP n ADP n rato by the ANT ncreased by 100% and 82% wth succnate and glutamate plus malate as substrate, respectvely. For both substrates, hexoknase exerted the hghest negatve control on the ATP out ADP out rato (Table 3). The remander of the control was dstrbuted among the respratory-chan modules, ATP synthess, and the ANT, wth neglgble negatve control exerted by the proton leak. Smlarly to the control of the ATP n ADP n rato, the dstrbuton of the control of the ATP out ADP out rato wthn the respratory chan depended on the substrate used. Comparng the two substrates, ATP synthess exerted less control over the ATP out ADP out rato n the case of succnate oxdaton. Palmtoyl-CoA ncreased the control of the ATP out ADP out rato by ATP synthess and proton leak, and decreased the control by the Q-reducng module wth both respratory substrates, and the control by the QH 2 -oxdzng module and hexoknase wth succnate. The control of the ATP out ADP out rato by the ANT ncreased by 56% and 113% wth succnate and glutamate plus malate as substrate, respectvely. Partal ntegrated responses to palmtoyl-coa Table 4 summarzes ntegrated elastctes to palmtoyl- CoA and partal ntegrated responses of system fluxes and ntermedates to palmtoyl-coa medated through each module of oxdatve phosphorylaton. Wth both respratory substrates, the ANT had the largest elastcty to palmtoyl-coa, n agreement wth the fndng that, under our expermental condtons, the ANT s the man target of palmtoyl-coa n oxdatve phosphorylaton. As a consequence, the response medated through the ANT contrbuted most to the overall response of the system fluxes and ntermedates to palmtoyl-coa,.e. the response through the ANT was responsble for 68% of the decrease n J o, 68% of the decrease n J p, 56% of the ncrease n the QH 2 Q rato, 70% of the ncrease n Dw, 72% of the ncrease n the ATP n ADP n rato, and 59% of the decrease n the ATP out ADP out rato wth succnate as substrate. Smlar results were obtaned when glutamate plus malate was used as substrate: the response through the ANT contrbuted 75% of the reducton n J o, 76% of the reducton n J p, 68% of the ncrease n Dw, 88% of the ncrease n the ATP n ADP n rato, and 69% of the reducton n the ATP out ADP out rato. The excepton was the QH 2 Q rato, where the response through the Table 4. Contrbuton of ndvdual modules of oxdatve phosphorylaton to the overall response of system varables to palmtoyl-coa. The partal ntegrated responses (IR) of each module to 5 lm palmtoyl-coa were calculated usng control coeffcents (Tables 2 and 3) and ntegrated elastcty coeffcents (Ie) of modules to palmtoyl-coa as descrbed n [21]. Values are mean ± SEM from three (succnate) or four (glutamate plus malate) experments (ndcated as subscrpt). Modules: 1, Q reducng; 2, QH 2 oxdzng; 3, proton leak; 4, ATP synthess; 5, ANT; 6, hexoknase. p-coa, Palmtoyl-CoA; OR, overall response. IR Jo p CoA IR Jp p CoA IR QH2=Q p CoA IR Dw p CoA IR ATPn=ADPn p CoA ATPout =ADPout IRp CoA Ie p CoA Succnate 1 ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) OR ) ) ) Glutamate plus malate ) ) ) ) ) ) ) ) ) ) ) ) OR ) ) ) FEBS Journal 273 (2006) ª 2006 The Authors Journal complaton ª 2006 FEBS 5295

9 Palmtoyl-CoA and control of mtochondral functon J. Capate et al. ANT contrbuted only 29% of the overall ncrease n the QH 2 Q rato, most of the rest of the ncrease stemmng from stmulaton of the Q-reducng module (52%). The response of a system varable to an external effector medated through a specfc module s determned by the control exerted by that module over a system varable and the elastcty of that module to the effector [20,21]. Table 4 shows that the overall effects of palmtoyl-coa on system fluxes and ntermedates were manly medated through the ANT and that the propertes that were controlled most strongly by the ANT were affected the most. Dscusson We have shown that palmtoyl-coa nduces ROS producton n actvely phosphorylatng solated rat lver mtochondra. Furthermore, t nfluences the ATP out ADP out rato n such a way that these changes result n ncreased [AMP] out. Ths s n lne wth a mechansm we proposed to underle the assocaton between obesty and type 2 dabetes [9,12]. However, owng to multple nteractons n lvng systems, t can be dffcult to dfferentate whether all the effects relate to one or many prmary effects. To acknowledge ths, usng modular knetc analyss, we establshed that the prmary cause of the effects of palmtoyl-coa was nhbton of the ANT. Assessment of the metabolc control of fluxes and ntermedate concentratons by the ANT then revealed that ths enzyme partally controls many fluxes, concentratons and potentals. Ths then confrmed that an ncrease n AMP concentraton and, at least partly, stmulaton of ROS producton are effects of ANT nhbton. Ths study thereby shows how systems bology methodologes mght help n dssectng the convoluted cause effect chans n multfactoral dseases such as obesty and type 2 dabetes. Both starvaton and ncubaton wth fatty acds have been shown to cause a concomtant decrease n cytosolc ATP ADP ratos and an ncrease n total LCAC concentratons n perfused rat lver and solated hepatocytes, ndcatng that nhbton of the ANT by LCACs may be relevant n vvo [22 24]. It has been suggested that modulaton of ANT actvty by LCACs mght be physologcally sgnfcant n the regulaton of gluconeogeness by fatty acds through effects on the ntramtochondral ATP ADP rato [23]. A decrease n the extramtochondral concentraton of ATP may result n ncreased formaton of AMP by adenylate knase. Ths may subsequently stmulate a cellular response to stress through actvaton of AMPdependent processes [25] or lead to the breakdown of AMP to adenosne and extracellular release of the latter [12]. The prmary mechansm of ntracellular adenosne producton s hydrolyss of AMP by a cytosolc 5 -nucleotdase [26]. Increased concentratons of free ADP and AMP n the cytosol are major determnants of adenosne producton, wth extracellular adenosne release correlatng lnearly wth free cytosolc AMP concentraton [27]. Exogenous adenosne s a potent vasodlator (EC 0.1 lm), and, under physologcal condtons, t facltates tssue recovery after ntensve workload by ncreasng blood flow and supply of oxygen and metabolc substrates. Under pathologcal condtons characterzed by napproprate ntracellular tracylglycerol accumulaton, a low cytosolc ATP ADP rato may persst because of constant nhbton of the ANT leadng to a sustaned ncrease n extracellular adenosne concentratons, resultng n hyperperfuson, hypertenson, ncreased urate producton, and other abnormaltes common to nsuln-resstant states [12]. We have shown that nhbton of the ANT wth palmtoyl-coa results n a sgnfcantly lower ATP out ADP out rato. Wth respect to the nterrelaton between ATP out ADP out ratos and [AMP] out, we have shown here how [AMP] out ncreases wth decreasng ATP out ADP out rato, wth larger ncreases observed at low ratos and smaller changes at hgh ratos. Ths ndcates that the effect of LCACs on AMP producton wll vary dependng on the energy state of the cell. The theoretcal assessment of the correlaton was supported by expermental fndngs showng that nhbton of the ANT wth palmtoyl-coa leads to a sgnfcant palmtoyl-coa concentraton-dependent decrease n the ATP total ADP total rato and a concomtant ncrease n [AMP] total. On the bass of our fndngs, we expect that, n ntact cells, the absolute cytosolc AMP concentraton wll ncrease moderately n response to a decrease n the cytosolc ATP ADP rato n the physologcally relevant range. However, even at low concentratons of AMP, the relatve ncrease n concentraton would stll be substantal and so would the relatve effect on the rate of producton of adenosne; 5 -nucleotdase operates n vvo at substrate concentratons three orders of magntude below ts K m of 1.2 mm [28]. Inhbton denhbton of the ANT dependng on LCAC concentraton may be relevant n the regulaton of cellular metabolsm n vvo va effects on AMP-actvated proten knase (AMPK). Actvaton of AMPK acts as a swtch from anabolc to catabolc metabolsm whch generates ATP (e.g. stmulaton of b-oxdaton) [25]. Thus actvaton of AMPK would seem to be a desrable effect n obesty, as t would promote the 5296 FEBS Journal 273 (2006) ª 2006 The Authors Journal complaton ª 2006 FEBS

10 J. Capate et al. Palmtoyl-CoA and control of mtochondral functon consumpton of excess fat. However, the combnaton of persstent ANT nhbton by LCACs wth constant actvaton of AMPK may have some adverse effects, because stmulaton of b-oxdaton n response to actvaton of AMPK cannot lead to producton of ATP because of lack of mtochondral ADP. As AMPK stmulates cellular fatty acd uptake [29] and the avalablty of crculatng fatty acds s ncreased n obesty, ths may lead to accumulaton of ntracellular tracylglycerols. Furthermore, owng to the decreased flux through the trcarboxylc acd cycle n the case of ANT nhbton, b-oxdaton-derved acetyl-coa may stmulate pyruvate carboxylase, contrbutng to ncreased rates of gluconeogeness, or may be used for ketone body synthess n the lver. Indeed, short-term overexpresson of AMPK n mouse lver has been shown to nduce fatty lver and ncrease ketogeness [30]. Stmulaton of ROS producton s thought to contrbute to dysfuncton of many dfferent cell types, but, n partcular, to b-cell dysfuncton n nsulnresstant states through low expresson of antoxdant enzymes n these cells [31]. Mtochondral Dw and the redox state of coenzyme Q are known to affect ROS formaton [10,11]. We have shown that 5 lm palmtoyl-coa caused a substantal ncrease n Dw (13 mv wth glutamate plus malate and 15 mv wth succnate [16] as substrate, compared wth a total state 3 state 4 dfference of 25 mv) and nduced H 2 O 2 producton n mtochondra resprng on succnate. The senstvty of the palmtoyl-coa-nduced H 2 O 2 producton to protonophore shows that the process s partly Dw-dependent. Ths substantates the part of our hypothess suggestng that LCACs brng about ROS producton through an ncrease n Dw [9]. The effect of palmtoyl-coa on the QH 2 Q rato wth both respratory substrates was less pronounced, castng doubt on the alternatve route by whch palmtoyl-coa may affect ROS producton by the respratory chan. Effects through the more elusve local ubqunone radcal reman an opton. Our results ndcate that the palmtoyl-coa effect on H 2 O 2 producton mght be partly exerted from the matrx sde, but the effect s b-oxdaton-ndependent as the substrate of b-oxdaton, palmtoyl-l-carntne, stmulated H 2 O 2 producton less than dd equal amounts of palmtoyl-coa. Moreover, palmtoyl-coa dd not enhance respraton drectly, as measured by modular knetc analyss. A possblty remans that palmtoyl-coa ncreased the redox level of ntramtochondral NADH and flavoprotens, but t was not able to further stmulate respraton because t was already operatng at V max.in such a case, the most reduced redox potental at the top of the electron-transfer chan mght cause extra ROS producton. Our observaton that atractylosde, a drect nhbtor of the ANT, caused ROS producton that could only partly account for palmtoyl-coanduced ROS producton ndcates that the ANT s only partly nvolved n ths process. It s possble that palmtoyl-coa decreased mtochondral antoxdant capacty by nhbtng ncotnamde nucleotde transhydrogenase [32], an enzyme that provdes NADPH for regeneraton of two mportant antoxdant compounds, glutathone and thoredoxn, n the mtochondra, and n ths way contrbuted to ncreased ROS producton. Our data show that the ANT controls many steadystate concentratons, potentals and fluxes. In agreement wth ths, the specfc effect of palmtoyl-coa on the ANT appears to be consstent wth ts ablty to affect many fluxes, concentratons and potentals. Table 1 shows that palmtoyl-coa affects dfferent mtochondral propertes to dfferent extents. In the lght of these observatons, we asked whether Metabolc Control Analyss could have served to predct the palmtoyl-coa effects. We showed that the ANT controlled Dw and the QH 2 Q rato to the least extent, and ndeed t was the least affected by palmtoyl-coa. J o, J p, ATP n ADP n and ATP out ADP out ratos were more strongly controlled by the ANT, and agan ths corresponded to a stronger effect of palmtoyl-coa. We conclude that the specfc effect of palmtoyl-coa on the ANT and the varyng extent to whch the ANT controls varous mtochondral propertes at steadystate can largely explan the observed palmtoyl-coa effects. The relatvely weak control of the QH 2 Q rato and Dw by the ANT s n agreement wth the fndng that ANT nhbton by palmtoyl-coa and the resultng ncrease n Dw can only partly account for the observed ncrease n ROS producton. We found that, for Dw and the QH 2 Q rato, ther mmedate producers and consumers,.e. the respratory-chan components, exerted the strongest control. Ths ndcates that, f an ncrease n ROS producton s brought about by alteratons n Dw and the QH 2 Q rato, nterference wth respratory-chan functon wll contrbute more than nterference wth any other component of oxdatve phosphorylaton. It has been shown that the control of J o by the ANT s comparable n solated lver mtochondra [33] and solated hepatocytes [34], ndcatng that, at least to a certan extent, results obtaned n solated mtochondra can be extrapolated to the ntact cell. Our results reconfrmed the observaton that, n solated rat lver mtochondra, the ANT has lmted control over FEBS Journal 273 (2006) ª 2006 The Authors Journal complaton ª 2006 FEBS 5297

11 Palmtoyl-CoA and control of mtochondral functon J. Capate et al. J o [19,33,35,36] and that t controls J p more strongly than J o. However, t has been demonstrated that ANT control of J o changes dependng on ntramtochondral and extramtochondral ATP utlzaton [37,38]. Accordngly, the effect of LCACs on ANT control of J o must depend on the ATP elastcty of the ATP-utlzng processes actve at the moment of nhbton, e.g. nhbton of the ANT n rat lver cells wth the specfc nhbtor, atractylosde, decreased glucose synthess to a greater extent than urea synthess, even though both processes requre ATP [24]. In concluson, we have shown that the ANT controlled all nvestgated propertes of the mtochondral oxdatve phosphorylaton to dfferent extents, wth the largest control exerted over the ATP n ADP n and ATP out ADP out ratos. The effects of palmtoyl-coa largely corresponded to ths. Our results suggest that nhbton of the ANT by LCACs may be mportant n the control of cellular energy metabolsm, but t accounts only partly for stmulaton of ROS producton. Expermental procedures Materals Yeast hexoknase was from Roche (Mannhem, Germany). Horseradsh peroxdase, superoxde dsmutase, olgomycn, myxothazol, atractylosde, carboxyatractylosde, rotenone, palmtoyl-coa, malonyl-coa, Ap5A, p-hydroxyphenylacetc acd and coenzyme Q 1 were from Sgma-Aldrch (Zwjndrecht, the Netherlands). Isolaton of mtochondra Lver mtochondra were solated from male Wstar rats ( g) as n [39]. Proten content was determned by the method of Bradford [40], wth BSA as a standard. Measurement of oxygen uptake and Dw Mtochondra were ncubated at 25 C n a closed, strred and thermostatcally controlled glass vessel equpped wth Clark-type oxygen electrode and tetraphenylphosphonum on (TPP + )-senstve electrode as descrbed [16]. The assay medum contaned 25 mm creatne, 25 mm creatne phosphate, 75 mm KCl, 20 mm Trs, 2.3 mm MgCl 2, 5 mm glutamate plus 5 mm malate, 50 lm Ap5A, ph 7.3. An ADP-regeneratng system consstng of excess hexoknase (5.78 UÆmL )1 ), glucose (12.5 mm) and KH 2 PO 4 (5 mm) was used to mantan steady-state respraton rates. ATP at a concentraton of 100 lm was added to ntate state 3 respraton. Determnaton of adenne nucleotde concentratons Adenne nucleotdes were extracted wth phenol as descrbed [41]. Concentratons were measured usng a lucfern lucferase ATP-montorng kt (BoOrbt, Turku, Fnland). ATP concentratons n the medum and the mtochondral matrx were determned from yeast hexoknase knetcs as descrbed [16]. As hexoknase knetcs were determned n medum contanng creatne and creatne phosphate, ths medum was used n all experments wth mtochondra. AMP concentraton was determned spectrophotometrcally usng a standard enzymatc assay [42]. Measurement of coenzyme Q reducton Coenzyme Q reducton levels were determned n a thermostatcally controlled (25 C) vessel equpped wth platnum and oxygen electrodes, by polarographcally measurng the redox state of exogenous coenzyme Q 1 (2 lm) [43]. To calbrate the platnum electrode traces, samples were taken from ncubatons of mtochondra n standard assay medum wthout further addtons (state 1) and mtochondra ncubated wth substrate (10 mm succnate plus 2 lm rotenone, or 5 mm glutamate plus 5 mm malate, state 2). Then 1 ml of sample was quenched wth 3 ml 0.2 m HClO 4 n methanol (0 C), coenzyme Q was extracted wth 3 ml petroleum ether (40 60 C), and reduced and oxdzed coenzyme Q n the samples was determned by HPLC as descrbed [44]. Measurement of H 2 O 2 producton The rate of H 2 O 2 producton was estmated from the rate of p-hydroxyphenylacetc acd oxdaton (exctaton and emsson wavelengths 317 nm and 414 nm, respectvely) as descrbed [45]. Brefly, mtochondra were ncubated at 25 C n 2 ml standard assay medum contanng 1 mm dethylenetramnepenta-acetc acd, 0.2 mm p-hydroxyphenylacetc acd, 10 UÆmL )1 horseradsh peroxdase and 30 UÆmL )1 superoxde dsmutase under the followng condtons: state 3 and state 3 plus nhbtors [palmtoyl-coa (1, 2.5 and 5 lm), 5-chloro-3-t-butyl-2 -chloro-4 -ntrosalcylanlde (S-13, 0.2 lm), atractylosde (1.5 lm), carboxyatractylosde (0.1 lm), olgomycn (0.5 lm), rotenone (2 lm), malonyl-coa (0.1 mm)] or palmtoyl-l-carntne (5 lm). Fluorescence sgnal was quantfed usng H 2 O 2 as standard. Calculaton of extramtochondral AMP concentratons AMP concentraton was calculated as (for dervaton see supplementary data, Appendx S1): 5298 FEBS Journal 273 (2006) ª 2006 The Authors Journal complaton ª 2006 FEBS

12 J. Capate et al. Palmtoyl-CoA and control of mtochondral functon ak eq ½AMPŠ ¼ r 2 ð1þ þ r þ K eq where r s the ATP ADP rato (equal to our expermental extramtochondral ATP ADP rato only when formaton of AMP s blocked by Ap5A), a s the total amount of adenylate (100 lm), and K eq s the equlbrum constant of adenylate knase (K eq ¼ [42]). Modular knetc analyss To localze the stes of acton of palmtoyl-coa, the system of oxdatve phosphorylaton was conceptually subdvded nto a small number of functonal modules nteractng va a lmted number of ntermedates. The knetc response of flux through the modules to changes n the concentraton of the connectng ntermedate was determned n the presence and absence of palmtoyl-coa by ttratng wth specfc nhbtors as descrbed [16,46]. In the frst applcaton, the system was dvded nto a substrate-oxdaton module, a phosphorylaton module, and a proton-leak module, wth Dw as the connectng ntermedate [46]. In the second applcaton, t was dvded nto an ATP-producng module and an ATP-consumng module, wth the matrx ATP ADP rato as the connectng ntermedate [16]. Metabolc Control Analyss Defntons The flux control coeffcent s defned as the fractonal change n the system flux (J k ) at steady-state n response to an nfntesmal change n the rate of an enzyme (module) (v ) [18]: C J k J k v ss The subscrpt ss refers to the steady-state condton and s hereafter omtted, as are the parentheses. The concentraton control coeffcent s defned as the fractonal change n the steady-state concentraton of ntermedate (or rato of concentratons, Dw) (X m ) n response to an nfntesmal drect perturbaton of the enzyme (module) rate (v ) [17,18]: C Xm m X m Effectvely, the value of the control coeffcent of an enzyme ndcates the percentage reducton n a system flux (for flux control coeffcents) or n an ntermedate concentraton (for concentraton control coeffcents) n response to 1% nhbton of the reacton rate of that enzyme. The elastcty coeffcent s defned as the fractonal change n rate v through enzyme (module), caused by the fractonal change n the concentraton of ntermedate X m, when concentratons of other ntermedates are held constant [17,18]: ss ð2þ ð3þ e X v X m ntermedates constant ntermedates constant Calculaton of control coeffcents For analyss of metabolc control, we conceptually subdvded the system of oxdatve phosphorylaton nto sx modules (coenzyme Q-reducng module, coenzyme QH 2 - oxdzng module, proton-leak module, ATP-synthess module, ANT, and hexoknase) connected by four ntermedates: QH 2 Q rato; Dw; ATP n ADP n ; ATP out ADP out (Fg. 5). The control coeffcents of the modules for the oxygen-uptake and phosphorylaton fluxes and concentratons of four ntermedates were calculated from the system fluxes and elastcty coeffcents (.e. coeffcents quantfyng senstvty of flux through the module to changes n concentraton of an ntermedate [17]) usng the matrx method [47]. In the calculaton, we assumed that the coenzyme Q-reducng and coenzyme QH 2 -oxdzng modules are nsenstve to changes n the ATP n ADP n and ATP out ADP out ratos (.e. elastcty coeffcents are zero) [46]; n the case of succnate oxdaton, the coenzyme Q-reducng module s nsenstve to Dw, ATP synthess s senstve only to Dw and the ATP n ADP n rato, the hexoknase rate s senstve only to the ATP out ADP out rato, whereas proton leak s senstve only to Dw [46]; ANT s senstve to all four ntermedates ncludng the QH 2 Q rato [48]. To obtan the elastcty coeffcents that were assumed to have a nonzero value, we used a multple modulaton method [49],.e. each module was ttrated wth a specfc nhbtor (Table 5) and the co-response of the flux and ntermedate concentraton was measured. The co-response coeffcents quantfyng the rato of responses of ntermedate X m and flux J k after perturbaton of module [50] were determned from the slopes of nhbtor ttraton curves at steady-state as: C Xm O Xm J k C J @lnj k Table 5. Modulatons used to determne the co-response coeffcents. Mal, Malonate ( mm); Olgo, olgomycn (0 0.3 lm); Atr, atractylosde (0 1.5 lm); Rot, rotenone (0 30 nm); Myx, myxothazol (0 25 nm); Hk, hexoknase ( UÆmL )1 ). Module Succnate Glutamate + malate Q reducng Myx, Olgo, Atr Myx, Olgo, Atr QH 2 oxdzng Mal, Olgo, Atr Rot, Olgo, Atr Proton leak Mal, Myx Rot, Myx ATP synthess Mal, Myx, Atr Rot, Myx, Atr ANT Mal, Myx, Olgo, Hk Rot, Myx, Olgo, Hk Hk Mal, Myx, Olgo, Atr Rot, Myx, Olgo, Atr ð4þ ð5þ FEBS Journal 273 (2006) ª 2006 The Authors Journal complaton ª 2006 FEBS 5299