149 The Journl of Experimentl Biology 21, 149-165 Pulished y The Compny of Biologists 27 doi:1.1242/je.2628 Chnges in mitochondril oxidtive cpcities during therml cclimtion of rinow trout Oncorhynchus mykiss: roles of memrne proteins, phospholipids nd their ftty cid compositions Edourd Krffe 1,2, *, Ynic Mrty 1 nd Helg Guderley 2 1 Unité mixte CNRS 6521, Université de Bretgne Occidentle, C.S. 93837, 29238 Brest cedex 3, Frnce nd 2 Déprtement de Biologie, Université Lvl, Quéec, G1K 7P4, Cnd *Author for correspondence (e-mil: Edourd.Krffe@univ-rest.fr) Accepted 1 Novemer 26 Chnges in the properties of mitochondri from oxidtive muscle of rinow trout Oncorhynchus mykiss were exmined during wrm (5 C to 15 C) cclimtion. Trout were studied shortly fter the initil therml chnge nd fter 8 weeks cclimtion to 15 C. To identify potentil mechnisms y which oxidtive cpcities chnge, the modifictions of phospholipid composition, memrne proteins nd functionl cpcities of red muscle mitochondri were exmined. Mrked functionl chnges of isolted muscle mitochondri during wrm cclimtion of rinow trout were reflected y host of modifictions in phospholipid composition, ut y few shifts in protein components. Shortly fter trnsfer of trout from 5 C to 15 C, the mximl oxidtive cpcity of mitochondri mesured t 15 C incresed slightly, ut rtes t oth ssy tempertures (5 C nd 15 C) decresed mrkedly fter wrm cclimtion. The increse in cpcity in short-term wrm exposed trout ws most pronounced when rtes t 15 C were expressed reltive to cytochrome nd c 1 levels. Non-phosphorylting (Stte 4) rtes of oxygen uptke incresed with short-term wrm exposure efore returning to initil levels fter wrm cclimtion. Cytochrome c oxidse (CCO) ctivity in the mitochondril preprtions decresed with wrm cclimtion. The therml sensitivity of the ADP ffinity ws mrkedly modified during shortterm wrm exposure, when the ADP/O rtio incresed, ut wrm cclimtion returned these vlues to those oserved initilly. ADP ffinity incresed fter wrm cclimtion. Chnges in the mitochondril content of cytochromes nd denine nucleotide trnslocse (ANT) could not explin these ptterns. On the other hnd, chnges in the proportions of the lipid clsses nd in the cyl chin Summry composition of certin phospholipid clsses mirror the modifictions in functionl properties. Short-term exposure to 15 C decresed the rtio of dicylphosphtidylethnolmine/dicylphosphtidylcholine (dicylpe/dicylpc), wheres wrm cclimtion led to restructuring of ftty cids (FA) nd to increses of plsmlogen forms of PE nd PC. Modifiction of overll memrne unsturtion did not pper to e the primry im of restructuring memrne FA during wrm cclimtion, s totl mitochondril phospholipids nd the mjor phospholipid clsses only showed slight shifts of their cyl composition with wrm cclimtion. On the other hnd, nturl lysophosphtidylcholine (LysoPC) showed drmtic chnges in FA content, s 16: nd 18:1n- 9 douled wheres 22:6n-3 decresed from round 5% to 32% in wrm cclimted trout. Similrly, in crdiolipin (CL), the levels of 16: nd 18:1n-7 hlved while 18:2n-6 incresed to over 2% of the FA with wrm cclimtion. Given the centrl role of CL in modulting the ctivity of CCO, F F 1 -ATPse nd ANT, these chnges suggest tht specific compositionl chnges in CL re importnt modultors of mitochondril cpcities. The mny structurl chnges in memrne lipids contrst with the limited modifictions of the memrne protein components exmined nd support the concept of lipid structure modulting mitochondril cpcities. Key words: mitochondri, therml cclimtion, oxidtive muscle, cytochrome c oxidse, ADP ffinity, therml compenstion, phospholipids, plsmlogens, ftty cids, rinow trout, Oncorhynchus mykiss. Introduction Due to its impct upon ll levels of orgnistion, temperture is crucil determinnt of the performnce of living systems. As temperture ffects the kinetic energy of molecules, it modifies rtes of diffusion, moleculr interctions nd mcromoleculr stility, memrne properties, nd my
15 E. Krffe, Y. Mrty nd H. Guderley chnge the efficcy of metolic regultion. In ectotherms, cellulr function cn cclimte to therml chnge. Unlike the dptive strtegies ctive during evolution, the mechnisms underlying cellulr cclimtistion must e specified y the existing genotype. When thermlly induced chnges in metolic demnd persist, nimls often remodel mitochondril structure nd function to etter ccommodte energetic needs. During cold cclimtion, the commonly oserved increses in muscle eroic cpcity occur t severl levels: mitochondril volume density, criste surfce density, nd ctivity of enzymes in oxidtive pthwys. Increses in mitochondril undnce my compenste for ctlytic or diffusive limittions (for review, see Guderley, 24). Increses in criste density cn enhnce the potentil for oxidtive phosphoryltion without scrificing volume needed for other orgnelles (St Pierre et l., 1998). Altertions in concentrtions of enzymes cn fcilitte metolic djustments during therml cclimtion (Sidell, 1983), while increses in their specific ctlytic cpcity during cold cclimtion my, t lest prtilly, offset cute therml effects on rection rte. For exmple, cold cclimtion of cyprinids increses the specific ctivity of succinte dehydrogense, F F 1 -ATPse nd cytochrome c oxidse (CCO) in muscle (Hzel, 1972; Itoi et l., 23; Wodtke, 1981). Mitochondri from red muscle of rinow trout show sesonl cycles of their pprent ADP ffinity nd its therml sensitivity suggesting modifictions of cpcities of the denine nucleotide trnslocse (ANT) (Blier nd Guderley, 1993; Guderley nd St Pierre, 1999). One fctor these proteins hve in common is tht they occur in the hydrophoic environment of the inner mitochondril memrne. Lipids tht constitute mitochondril memrnes should e regrded s structurl prtners of memrne proteins nd s potentil modultors of mitochondril processes (Dum, 1985). Proper functioning of iomemrnes ecomes difficult elow the trnsition temperture t which the liquid crystlline structure of the cyl core chnges into gel-like structure. Ectotherml orgnsims re thought to dpt their memrnes to temperture decreses y incresing the cyl chin unsturtion of phospholipids to mintin memrne fluidity. This homeoviscous dpttion (HVA) lso includes shifts in the proportions of phospholipid clsses (phosphtidylethnolmine (PE) reltive to phosphtidylcholine (PC) nd cholesterol content (Hzel nd Willims, 199; Hzel, 1995; Crockett nd Hzel, 1995). Modifiction of the cpcity for oxidtive phosphoryltion during therml cclimtion is climed to e due, t lest in prt, to HVA (Vn Der Thillrt nd Modderkolk, 1978; Wodtke, 1981; Wodtke, 1981; Itoi et l., 23; Guderley, 24). On the other hnd, requirements for specific phospholipids in the vicinity of electron trnsport chin components (Hoch, 1992) suggest tht chnges in the FA composition of specific phospholipids my e centrl in the modultion of ctlytic ctivity during therml cclimtion. Thus, due to the complexity of memrne lipid constituents (sterol, 8 min phospholipid clsses nd suclsses, nd more thn 5 ftty cids) nd the elortely suvided sumemrne domins, specific effects my e oscured when exmining the cyl composition of totl phospholipids. Prticulrly in the protein-dense mitochondril memrnes (Hzel nd Willims, 199), only smll numer of lipid molecules is thought to seprte memrne proteins or protein ggregtes. In mmmlin systems, mitochondril memrne proteins cn show specific requirements for phospholipid hed group rrngements nd FA in their proximity (Clndinin et l., 1985). Thus, even minor phospholipid clsses cn hve n importnt functionl impct on memrne-ound enzymes. Crdiolipin (CL), in prticulr, is specificlly locted in the inner mitochondril memrne, nd is key fctor in the control of CCO, F F 1 -ATPse nd ANT (Schlme et l., 2). Loclised chnges of specific memrne phospholipids, like CL, my e criticl in estlishing the ctivities of memrne proteins nd provide powerful mens of djusting ctivity without requiring protein synthesis or modifiction of existing proteins during therml cclimtion. Thus, detiled chrcteristion of memrne lipids is prerequisite to understnding how heterogeneous mixtures of phospholipids interct with memrne proteins to lter iologicl functions. Although therml cclimtion leds to well chrcterised chnges in the undnce of muscle mitochondri nd mitochondril oxidtive cpcities in temperte zone fishes (Guderley, 24), chnges of cpcity of protein nd phospholipid components hve typiclly een studied seprtely. A few exceptions re provided y the clssic studies (Hzel, 1972; Hzel, 1972; Vn den Thillrt nd Modderkolk, 1978; Wodtke, 1981; Wodtke, 1981), in which chnges in overll phospholipid composition rought out y cold cclimtion were linked with chnges in the ctivity of succinte dehydrogense, Stte 3 respirtion nd CCO. Little is known out the time course of therml cclimtion. Modifictions in memrne structure during the time course of therml cclimtion of rinow trout follow defined sequence, s shown y the restructuring of phospholipid composition nd moleculr species in kidney plsm memrne (Hzel nd Lndrey, 1988; Hzel nd Lndrey, 1988). After 8 h of cold cclimtion (2 C to 5 C), the PC/PE rtio decreses. The proportions of sturted nd monounsturted FA chnge fter 16 48 h nd long-chin polyunsturted FA only increse fter 1 21 dys of cold cclimtion. Such sequence of compositionl chnges in memrne phospholipids ws suggested to explin the nonliner time course of mitochondril oxidtive cpcity during wrm nd cold cclimtion of rinow trout (Bouchrd nd Guderley, 23), ut no study hs estlished such reltionship. The present study ims to evlute t the sme time, detiled modifictions of phospholipid composition, levels of memrne proteins nd functionl cpcities of red muscle mitochondri during wrm cclimtion (5 C to 15 C) of rinow trout Oncorhynchus mykiss. After chrcterising 5 Ccclimted trout, we studied trout shortly (3 dys) fter trnsfer to 15 C nd fter 8 weeks cclimtion to 15 C. By exmining mitochondril sustrte oxidtion t 5 C nd 15 C, the
Therml dpttion in trout mitochondri 151 concentrtions nd ctivities of mitochondril components, s well s the phospholipid composition of mitochondril memrnes we sought to identify potentil mechnisms y which oxidtive cpcities chnge. We mesured (1) the levels of denine nucleotide trnslocse (ANT), (2) the concentrtions of cytochromes,, c nd c 1, (3) the pprent ADP ffinity nd (4) the ctivity of CCO. The content of cytochromes, nd c 1 reflect the numers of respirtory chin complexes: cytochrome is present in Complexes II nd III, c 1 occurs in Complex III, nd cytochrome is prt of Complex IV. To evlute the impliction of the lipid environment in the time course of chnges in mitochondril oxidtive cpcities, we determined the proportions of ech mitochondril phospholipid clss nd suclss s well s their specific FA compositions nd the sterol levels t ech smpling time. Mterils nd methods Animls nd the time course of wrm cclimtion Rinow trout Oncorhynchus mykiss Wlum were otined from fish htchery (Ferme Piscicole Richrd Boily, Île d Orléns, Quéec, Cnd) on 19 Mrch 23, when the temperture in their outdoor holding ponds ws round 5 C. Therml cclimtion occurred t the LARSA (Lortoire Régionl des Sciences Aqutiques) t Lvl University in 1-litre tnk. After their rrivl, the fish were mintined t 5 C under the nturl photoperiod. Fish were fed the sme commercil food (Corey Aqufeeds, New Brunswick, Cnd) used t the htchery t mintennce rtions, clculted for their size, numer nd tnk tempertures, once dy. Twelve fish were used to ssess the oxidtive cpcities of these winter-cclimtised trout tht hd een held t 5 C for week efore eginning the study (cold cclimted). Then, wter temperture ws grdully rised to 15 C over 3 dys, nd eight specimens were studied during the susequent 3 dys (shortterm wrm exposed). Finlly, 8 weeks fter the eginning of the therml chnge, when we ssumed cclimtion ws complete (wrm cclimted), 12 trout were used to ssess finl oxidtive cpcities. Isoltion of mitochondri The fish were killed y low on the hed, nd the superficil lterl red muscle ws immeditely removed nd minced. All the mnipultions were crried out on ice except the centrifugtions, which were performed t 4 C. Mitochondri were isolted nd ssyed (Guderley et l., 1997). To optimise the purity of mitochondril pellets, dditionl steps were done during the differentil centrifugtion protocol. Briefly, fter centrifuging the homogente t 9 g for 1 min, the superficil lipid lyer ws removed. The remining superntnt ws recentrifuged t 9 g for 1 min. The superntnt otined ws considered free of unroken cells or cell deris nd ws centrifuged t 9 g. The resulting pellet ws rinsed once to remove MgCl 2 y resuspension in isoltion uffer free of MgCl 2 nd recentrifuged t 9 g. The mitochondril pellet ws resuspended in volume of rection uffer corresponding to one-tenth of the mss of muscle used. Mesurement of mitochondril respirtion Oxygen consumption ws mesured in the rection uffer t 5 C nd 15 C. For ech ssy, mlte ws dded to finl concentrtion of.37 mmol l 1 to sprk the Kres cycle, nd pyruvte ws dded to finl concentrtion of 3.45 mmol l 1. The mximl rte of oxidtive phosphoryltion (Stte 3) ws otined y the ddition of ADP to finl concentrtion of.92 mmol l 1. Stte 4 rtes were mesured fter depletion of ADP, once oxygen uptke rtes hd stilized. The ADP/O rtio ws mesured (Chnce nd Willims, 1956). ADP ffinity determintions The pprent ffinity of mitochondri for ADP (K m ) ws determined polrogrphiclly s descried (Guderley nd St Pierre, 1999). This pproch used the hexokinse rection to mintin constnt ADP levels so tht oxidtion rtes could e otined t low ADP levels. The ssy medium ws supplemented with glucose, MgCl 2 nd hexokinse (yest; Boehringer Mnnheim Biochemicls, Montrel, Queec, Cnd) t finl concentrtions of 38 mmol l 1 glucose, 19 mmol l 1 MgCl 2 nd 3 units l 1 hexokinse. Hexokinse exerts no control over mitochondril respirtion when the rtio of mg mitochondril protein to units of hexokinse is less thn 3 (Jcous et l., 1982). In our study, these rtios were consistently less thn.6. Similr rnges of excess hexokinse were present for the mitochondri from cold nd wrm cclimted trout s well s for short-term wrm exposed nimls. Pyruvte ws the cron sustrte (3.45 mmol l 1 ), with mlte present t.37 mmol l 1. Sturtion curves for mitochondri were determined y sequentil dditions of ADP strting with the lowest concentrtion (pproximtely 3 1 3 mmol l 1 totl ADP), followed y grdul dditions of ADP to ttin sturting concentrtions. Oxygen uptke rtes were determined for t lest 9 s t ech ADP concentrtion. Experiments were crried out t 5 C nd 15 C. We stndrdised the quntity of mitochondril protein t ~.15 mg mitochondril protein ml 1 ssy medium to fcilitte comprison etween the smpling periods. The pprent K m for ADP (K m,pp ) nd V mx (mximl velocity) were clculted using the Mrqurdt itertive serch lgorithm to fit the Michelis Menten eqution using Nonliner Regression nlysis (SttGrphics Plus 5.1). The ADP solutions were clirted spectrophotometriclly using the pyruvte kinse nd lctte dehydrogense rections (Bergmeyer, 1983). Cytochromes nd ANT concentrtions Cytochromes,, c nd c 1 in the mitochondril preprtions were quntified y difference spectr. The electron trnsport chin components in 2% deoxycholte-dispersed mitochondri were reduced y 5 mmol l 1 scorte nd the oxygen in the solution ws eliminted y the ddition of dithionite (Willims, 1964). The reduced smples were red ginst the smples
152 E. Krffe, Y. Mrty nd H. Guderley oxidised with 5 mmol l 1 ferricynide. We used the solution to the simultneous equtions required to ssess the individul cytochrome concentrtions (Willims, 1964). Difference spectr were otined using doule-em UV/Vis spectrophotometer (Vrin-Cry 21, Mississug, Ontrio, Cnd). The concentrtion of denine nucleotide trnslocse (ANT) ws mesured in mitochondril suspensions y titrtion with its noncompetitive irreversile inhiitor, croxytrctyloside (CAT) (Guderley et l., 25). Stte 3 respirtion ws grdully inhiited nd the inhiition ws considered complete when ddition of CAT hd no further effect on oxygen uptke. The quntity of ANT in mitochondril suspensions corresponded to the mount of CAT needed for inhiition. Protein concentrtions The protein concentrtion in mitochondril suspensions ws determined y the icinchoninic cid method (Smith et l., 1985) using ovine serum lumin (BSA) s the stndrd. Before nlysis, n liquot of ech mitochondril preprtion ws resuspended in the rection uffer minus BSA nd centrifuged t 9 g t room temperture for 1 min. The superntnt ws discrded nd the pellet resuspended, wshed nd centrifuged twice more to remove the BSA. These preprtions were conserved t 8 C until protein ssys. Cytochrome c oxidse ctivity CCO ctivity ws mesured t 5 C nd 15 C ccording to pulished methods (Bouchrd nd Guderley, 23) except tht mitochondril suspensions were diluted in phosphte uffer without Triton-X (45 mmol l 1 KH 2 PO 4 nd 3 mmol l 1 K 2 HPO 4, ph 6.8). We used n initil optiml cytochrome c concentrtion of 1 mol l 1. All ssys were run in triplicte using fresh mitochondril preprtions. Activities were clculted using n extinction coefficient of 1 19.1 mmol l 1 cm nd re expressed s mol cytochrome c trnsformed min 1 (first order rection). Memrne lipid nlysis The memrne lipids of mitochondril suspensions were extrcted ccording to the method descried (Folch et l., 1957). Before lipid extrction, the liquot of mitochondril preprtion ws resuspended in the rection uffer minus BSA nd centrifuged t 9 g t room temperture for 1 min. The superntnt ws discrded nd the pellet resuspended, wshed in rection uffer nd centrifuged further two times. The finl extrct ws stored t 8 C under nitrogen fter dding.1% w/v utylted hydroxytoluene (BHT, ntioxidnt). Seprtion of polr lipids on silic gel microcolumns An liquot of the lipid extrct ws evported to dryness nd lipids were recovered with three wshings of 5 l of CHCl 3 /methnol (98:2, v/v) nd deposited t the top of silic gel micro-column (3 mm 5 mm i.d., pcked with Kieselgel 6 (7 23 mesh, Merck, Drmstdt, Germny) previously heted t 45 C nd dectivted with 6 weight% H 2 O (Mrty et l., 1992). Neutrl lipids were eluted with 1 ml of CHCl 3 /methnol (98:2, v/v) nd stored t 2 C for lter cholesterol nlysis. The polr lipid frction ws recovered with 2 ml methnol nd stored t 2 C for lter phospholipid clss seprtion y high performnce liquid chromtogrphy (HPLC) nd FA composition nlysis y gs chromtogrphy (GC). Cholesterol nlysis Cholesterol ws nlysed in gs chromtogrph (Chrompk 92, Middelurg, The Netherlnds) equipped with RTX65 (65% diphenyl, 35% dimethylpolysiloxne) fused silic cpillry column (5 m.32 mm,.2 pm film thickness) using n on-column injection system nd hydrogen s crrier gs, with therml grdient from 6 C to 28 C. Quntifiction of cholesterol ws chieved y dding known quntity of cholestne to smples. Seprtion of memrne lipid clsses nd FA nlysis Seprtion of the phospholipid clsses nd suclsses followed Krffe et l. (Krffe et l., 24) using two successive HPLC seprtions with two different moile phses. This method llowed the seprte nlysis of plsmlogen (1- lkenyl-2-cyl-) nd dicyl suclsses of PE nd PC jointly with crdiolipin (CL), phosphtidylinositol (PI), phosphtidylserine (PS), nd nturl lysophosphtidylcholine (LysoPC). The remining dicyl frction of PE nd PC (dicylpe nd dicylpc) presented nd discussed in this pper likely contined the 1-lkyl-2-cyl- form in ddition to the 1,2- dicyl. Ech frction ws collected nd, fter trnsesterifiction (methnol/bf 3 ), nlysed y GC for FA composition. Ftty cid methyl esters (FAME) otined were identified nd quntified using oth polr (CPWAX 52 CB, Vrin, Middelurg, The Netherlnds; 5 m.25 mm i.d.;.2 m thickness) nd nonpolr (CP-Sil 8 CB, Vrin; 25 m.25 mm i.d.;.25 m thickness) cpillry columns nd C23: FA s n internl stndrd. FA were expressed s molr percentge of the totl FA content of ech clss or suclss. For plsmlogen ethnolmine (PlsmPE) nd plsmlogen choline (PlsmPC) suclsses, the totl percentge ws djusted to 5% to tke into ccount the sence of lkenyl chins of the sn-1 position hydrolysed y the cid moile phse. Clcultion of mounts of phospholipid clsses The quntities of ech clss nd suclss of phospholipid were determined from their respective FA spectrum otined y GC. To otin the molr content of ech nlysed frction, correction fctor ws pplied to their respective totl FA molr contents: 1 for PlsmPE nd PlsmPC frctions nd for the nturl lysopc frction; 1/2 for the dicylpe, dicylpc, PS nd PI frctions, nd 1/4 for the CL frction. Sttisticl nlysis Sttisticl comprisons nd liner regressions were crried out with SttGrphics Plus 5.1 (Sigm Plus Inc., Toulouse, Frnce). One-wy nlysis of vrince (ANOVA) followed y
Therml dpttion in trout mitochondri 153 posteriori Tukey multiple comprisons ws used for intergroup comprisons of the impct of wrm cclimtion on mitochondril chrcteristics of trout. Differences were considered significnt when P.5. Results Overll chrcteristics of the trout The externlly mesurle sttus of the trout ody mss, fork length nd Fulton s condition fctor did not chnge significntly during wrm cclimtion (Tle 1). Mitochondril concentrtions of cytochromes nd ANT After short-term wrm exposure, cytochrome levels were unchnged (Fig. 1). Wrm cclimtion decresed the levels of cytochromes nd c, ut left those of cytochromes nd c 1 unchnged. The protein-specific levels of ANT exceeded those of the cytochromes nd were slightly, ut significntly, higher (P<.5) in short-term wrm exposed trout compred to cold or wrm cclimted trout. Mitochondril mximl oxidtive cpcities during wrm cclimtion The isoltion procedure yielded mitochondri with respirtory control rtios (RCR; Stte 3/Stte 4) rnging from 3 to 9 throughout the experiment. Mximl rtes of pyruvte oxidtion (nmol O min 1 mg 1 mitochondril protein) chnged mrkedly during wrm cclimtion. Rtes were stle fter short-term wrm exposure nd dropped mrkedly with wrm cclimtion t oth ssy tempertures (P<.5) (Fig. 2). Stte 4 rtes (nmol O min 1 mg 1 mitochondril protein) showed iphsic pttern, with rtes, prticulrly t 15 C, incresing during short-term wrm exposure nd returning to initil vlues with wrm cclimtion (Tle 2). These ptterns led the men RCR vlues to decrese from 5 to 3.3 with wrm cclimtion. The therml sensitivity (Q 1 ) of mximl rtes of pyruvte oxidtion did not chnge etween cold cclimted nd shortterm wrm exposed trout (men vlues of 1.36±.12 nd 1.49±.7, respectively), ut incresed in wrm cclimted trout (men vlue of 1.84±.7, P<.5). The phosphoryltion cpcity of mitochondri, expressed y the molr rtio etween dded ADP nd consumed oxygen (ADP/O), reched Tle 1. Generl chrcteristics of experimentl trout Condition fctor Body Length (mss/length 3 ) mss (g) (cm) 1 Cold cclimted 414.9±23.1 33.3±.6 1.12±.3 Short-term wrm exposed 41.8±16.9 33.7±.6 1.5±.2 Wrm cclimted 441.±25.7 34.7±.6 1.5±.3 Vlues re mens ± s.e.m. (N=11 for cold cclimted trout, N=8 for short-term wrm exposed nd N=12 for wrm cclimted trout). No significnt differences were found etween cclimtion sttes (ANOVA nd posteriori tests). [Cytochrome] (nmol mg 1 protein) 4.5 4. 3.5 3. 2.5 2. 1.5 1..5 Cold-cclimted Short-term wrm exposed Wrm-cclimted c 1 c ANT Fig. 1. Concentrtion of mitochondril cytochromes in oxidtive muscle during wrm cclimtion. Cytochrome nd ANT concentrtions re normlised to the protein content in the mitochondril preprtions. Vlues re mens ± s.e.m. (N=9 for cold cclimted trout, N=8 for short-term wrm exposed trout nd N=11 for wrm cclimted trout). Different superscript letters indicte vlues tht differ etween cclimtion sttes (ANOVA nd posteriori test, P.5). significntly higher vlues in mitochondri of short-term wrm exposed trout t oth ssy tempertures compred to cold nd wrm cclimted trout: 5 C (men vlues of 2.8±.1 for cold nd wrm cclimted trout; 3.3±.2 for short-term wrm exposed) nd 15 C (3.1±.1 for cold nd wrm climted trout; 3.8±.1 for short-term wrm exposed). The denomintor typiclly used to stndrdise mitochondril rtes is the mount of protein (mg) in the mitochondril preprtion. This reflects protein locted oth in the mtrix nd in the memrne. As the djustments of memrne composition tht re mjor focus of our study would primrily ffect memrne-ound complexes, we exmined oxidtive cpcities reltive to the concentrtions of cytochromes nd ANT, ll locted in the mitochondril memrne. For Stte 3 rtes mesured t 5 C expressed over cytochrome, c 1 nd ANT, the sme pttern ws oserved s with protein specific rtes: stility during short-term wrm exposure nd decrese with wrm cclimtion (Fig. 2). On the other hnd, t 15 C, use of cytochromes nd c 1 s the denomintor reveled iphsic response, with Stte 3 rtes incresing during shortterm wrm exposure (significnt only for cytochrome c 1 ) nd then decresing during wrm cclimtion. Stte 4 rtes expressed over the cytochromes nd ANT chnged less with wrm cclimtion thn protein-specific Stte 4 rtes (Tle 2). Chnges in cytochrome c oxidse ctivity during wrm cclimtion The time course of wrm cclimtion chnged the ctivity of CCO in similr fshion s the protein-specific cpcities of isolted mitochondri (Fig. 3). CCO ctivity in mitochondril suspensions incresed slightly, ut not significntly, in short-term wrm exposed trout when ssyed t 5 C. Wrm cclimtion significntly decresed CCO ctivity compred to cold cclimted or short-term wrm exposed trout (P<.5) t oth ssy tempertures. The sme
154 E. Krffe, Y. Mrty nd H. Guderley Rte mg 1 protein Stte 3 rte nmol 1 cytochrome 16 14 12 1 8 6 4 2 18 16 14 12 1 8 6 4 2 12 1 8 6 4 2 Protein Cytochrome, Cytochrome c l Cold-cclimted Short-term wrm exposed Wrm-cclimted Rte nmol 1 cytochrome Rte nmol 1 ANT 8 7 6 5 4 3 2 1 5 C 15 C 5 C 15 C 4 35 3 25 2 15 1 5 Cytochrome Cytochrome c Fig. 2. Time course of chnges in mximl rtes of pyruvte oxidtion (nmol O min 1 ) y mitochondri from oxidtive muscle during wrm cclimtion. Stte 3 rtes re expressed s mg 1 mitochondril protein, nmol 1 cytochrome nd nmol 1 ANT. Vlues re mens ± s.e.m. (N=11 for cold cclimted trout, N=8 for short-term wrm exposed trout nd N=11 for wrm cclimted trout). Different superscript letters indicte rtes t given ssy temperture tht differ etween cclimtion sttes (ANOVA nd posteriori test, P.5). 12 1 8 6 4 2 ANT pttern ws found when CCO ctivity ws expressed over cytochrome levels. The Q 1 for CCO remined etween 1.4 nd 1.6 during our experiment. Throughout the experiment, only 4 5% of mitochondril CCO ctivity ws used during mximl rtes of pyruvte oxidtion (Stte 3/CCO ctivity expressed in mu). ADP ffinity During the time course of wrm cclimtion, mitochondri from red muscle chnged their pprent ADP ffinity (K m,pp ), s well s its therml sensitivity (Tle 3). At 5 C, the highest vlue for the K m,pp ws otined for cold cclimted trout with short-term wrm exposed nd wrm cclimted trout hving significntly lower vlues. On the other hnd, t 15 C, shortterm wrm exposed trout hd higher K m,pp thn trout in the other groups. Thus, while oth cold nd wrm cclimted trout show 5% decrese of K m,pp with n increse in ssy temperture, for short-term wrm exposed trout the pprent K m vlues did not differ etween 5 nd 15 C (t-test, P>.5). At the ssy temperture of 5 C, the V mx (mximl velocity) clculted y itertive fitting of the Michelis Menten eqution ws close to the vlues of Stte 3 respirtion (Tle 3) nd showed the sme chnges during the cclimtion protocol. Thus, the ADP-dependence of mitochondril oxidtive cpcities t 5 C ws well descried y the Michelis Menten eqution. At 15 C, the clculted V mx ws considerly lower thn the mesured Stte 3 rtes. Mitochondril compositions of memrne phospholipid clsses nd suclsses during the time course of wrm cclimtion Throughout the study, glycerophospholipids (PE, PC, PS, PI, CL nd LysoPC) were the predominnt phospholipid clsses in mitochondri. Plsmlogen forms were only found in PE (PlsmPE) nd PC (PlsmPC), those in PE eing more prominent. Sphingomyelin ws lwys found in trce mounts, indicting tht the mitochondril memrnes were not significntly contminted y other cellulr memrnes.
Therml dpttion in trout mitochondri 155 Tle 2. Time course of chnges in stte 4 rtes of pyruvte oxidtion y mitochondri from oxidtive muscle during wrm cclimtion Stte 4 rte Cold cclimted Short-term wrm exposed Wrm cclimted 5 C Protein Cytochrome Cytochrome Cytochrome c 1 Cytochrome c ANT 15 C Protein Cytochrome Cytochrome Cytochrome c 1 Cytochrome c ANT 19.6±2.1, 27.8±4.2 93.2±7.3 17.6±2.6 158.4±2. 5.7±.6 29.5±4.9 31.±1.6 117.4±26.3 223.3±58.8 192.7±43.7 8.5±2.3 24.6±.8 28.5±4. 97.9±16.6 185.8±15. 153.1±22.5 5.8±.2 42.2±2.3 36.7±2.9 168.3±36.2 315.4±36.9 273.±52. 9.9±.6 17.7±.9 18.8±2. 11.8±8.2 124.6±8.2 221.9±26.4 5.±.4 33.±2.5, 35.2±4.8 192.±18.7 25.3±2.2 455.2±44.3 1.9±1. Stte 4 rtes of pyruvte oxidtion re expressed s nmol O min 1 mg 1 mitochondril protein, or nmol O min 1 nmol 1 cytochrome or ANT. Vlues re mens ± s.e.m. (N=11 for cold cclimted trout, N=8 for short-term wrm exposed trout ndn=11 for wrm cclimted trout). Different superscript letters indicte rtes t given ssy temperture tht differ with cclimtion stte (ANOVA nd posteriori tests, P.5) During wrm cclimtion, the reltive levels of the phospholipid clsses nd suclsses chnged (Fig. 4), with prticulrly mrked chnges in dicylpe, dicylpc, PlsmPE, PlsmPC nd LysoPC. The proportion of the dicyl form of PE declined during wrm cclimtion from 33.95% in cold cclimted trout to 22.94% in wrm cclimted trout (P<.5). With short-term wrm exposure, dicylpe levels decresed, ut not significntly, to 3.14%. The chnges in PlsmPE showed n inverse pttern over the time course of the experiment compred to dicylpe. Indeed, while vlues of PlsmPE remined stle in short-term wrm exposure, levels douled fter 8 weeks of wrm cclimtion. Thus, wrm cclimted trout mitochondri hve the highest proportion of plsmlogen in PE (35.6%) while this vlue ws only 14.4 nd 16.3%, respectively in cold cclimted nd short-term wrm exposed trout. Although proportions of dicylpc were similr etween cold nd wrm cclimted trout, the level of dicylpc rose significntly in short-term wrm exposed trout concomitnt with decrese, leit not significnt, of lysopc. PlsmPC content reltive to totl phospholipids vried in the sme wy s PlsmPE, remining stle in short-term wrm exposure nd douling in wrm cclimtion. Although the proportion of plsmlogens in PC ws mrkedly lower thn tht in PE, the proportion of plsmlogens in PC lso reched the highest level in wrm cclimted trout (2.7%). The proportions of CL, PS nd PI did not vry significntly etween cclimtion groups. However, LysoPC incresed in mitochondri from wrm cclimted trout (P<.5). Cholesterol nd totl phospholipid contents of mitochondril memrnes Memrnes from trout red muscle mitochondri were primrily composed of phospholipids, with pproximtely tenfold higher levels of phospholipids thn cholesterol (Tle 4). Cholesterol, expressed reltive to mitochondril protein, did not chnge with cclimtion sttus (pproximtely.5 mol mg 1 mitochondril proteins) while totl phospholipids were stle etween cold cclimted nd shortterm wrm exposed trout ut decresed in wrm cclimted trout. Consequently, the reltive levels of cholesterol nd phospholipids (molr rtio) were higher in mitochondril memrnes from wrm cclimted trout (P<.5) (Tle 4). Ftty cyl chin composition of phospholipid clsses nd suclsses during wrm cclimtion Chnges in cyl chin composition during the time course of wrm cclimtion differed when exmined for the totl phospholipids (Tle 5) or for the specific clsses (Tles 6 8). The cyl chin composition of plsmlogen suclsses of PE nd PC lso chnged during wrm cclimtion, suggesting tht FA of these suclsses my influence memrne dynmics. For the overll phospholipids, sturted ftty cids (SFA) were dominted y 16: while 22:6n-3 ws the min unsturted FA. The other mjor FA were 18:, 18:1n-9, 18:2n-6, 2:4n-6, 2:5n-3 nd 22:5n-3. The time course of wrm cclimtion did not gretly ffect the FA composition of totl phospholipids. When FA levels differed, it ws the mitochondri of wrm cclimted trout tht were distinct from those otined from the other sttes. The levels of 22:6n-3 rose slightly, ut not significntly, efore returning to initil vlues with wrm cclimtion. 2:5n-3 decresed with wrm cclimtion, wheres 18:1n-9 nd 18:2n-6 rose. The n-3/n-6 rtio ws decresed (P<.5), monounsturted ftty cids (MUFA) were incresed (P<.5), sturted were decresed (ut not significntly) while polyunsturted (PUFA) were stle t the different stges of cclimtion. Dimethylcetls (DMA), otined from the ftty ldehydes ound s vinyl ethers to the sn-1 position of plsmlogen phospholipids, incresed in long-
156 E. Krffe, Y. Mrty nd H. Guderley U CCO mg 1 protein U CCO nmol 1 cytochrome 4 3.5 3 2.5 2 1.5 1.5 4.5 4 3.5 3 2.5 2 1.5 1.5 Cold-cclimted Short-term wrm exposed Wrm-cclimted Protein Cytochrome 5 C 15 C Fig. 3. Time course of chnges in cytochrome c oxidse ctivity during wrm cclimtion, expressed in U CCO mg 1 mitochondril protein nd U CCO nmol 1 cytochrome (U CCO = mol cytochrome c reduced min 1 ). Vlues re mens ± s.e.m. (N=8 for cold cclimted trout, N=7 for short-term wrm exposed trout nd N=12 for wrm cclimted trout). Different superscript letters indicte rtes t given ssy temperture tht differ etween cclimtion sttes (ANOVA nd posteriori test, P.5). term wrm cclimted trout (P<.5). A modest, ut not sttisticlly significnt, decrese in unsturtion index (UI) ws noted for wrm cclimted trout. Chnges in the FA compositions of the two prominent mitochondril phospholipid clsses, PE nd PC, in oth their dicyl nd plsmlogen forms (Tles 6 nd 7), were similr in mgnitude to those in the totl phospholipids. For dicylpc, the more unsturted FA (2:5n-3 nd 22:6n-3) decresed in wrm cclimtion (P<.5). Correspondingly, 18:1n-9 [Phospholipid] (mol%) 6 5 4 3 2 1 CL Cold-cclimted Short-term wrm exposed Wrm-cclimted PS DicylPE PlsmPE DicylPC PlsmPC LysoPC Fig. 4. Time course of chnges in proportion of phospholipid clsses nd suclsses during wrm cclimtion. Vlues re mens ± s.e.m. (N=4 for ech cclimtion stte). Different superscript letters indicte vlues for clss or suclss tht differ etween cclimtion sttes (ANOVA nd posteriori test, P.5). Phospholipids re expressed s mol% of totl moles of glycerophospholipids, clculted s indicted in Mterils nd methods. Arevitions re defined in List of revitions. incresed with the sme time course. SFA, dominted y 16:, incresed in short-term wrm exposed trout, only to fll to initil levels in wrm cclimted trout. The plsmlogen form of PC showed firly constnt levels of individul FA. However, wrm cclimtion incresed PUFA nd decresed SFA with significnt differences etween wrm nd cold cclimted trout. The FA composition of dicylpe differed from tht of dicylpc in tht 18: ws lso mjor SFA nd in tht 22:6n- 3 nd 2:5n-3 were respectively higher nd lower thn in dicylpc. In dicylpe, ll sttisticlly significnt effects distinguished wrm cclimted trout from the other groups, except for 22:6n-3, for which wrm cclimted trout only differed from wrm exposed trout. Among SFA, 16: incresed slightly nd 18: decresed slightly leding to constnt totl SFA in dicylpe during wrm cclimtion. As oserved for dicylpc, 2:5n-3 nd 22:6n-3 decresed in wrm cclimted trout (P<.5), wheres 18:1n-9 tended to increse. The levels of 22:5n-3 ecme higher thn those of 2:5n-3 in mitochondri from wrm cclimted trout. Interestingly, 22:5n- 3 is the sole PUFA tht incresed significntly during wrm PI Tle 3. Time course of the pprent ffinity for ADP (K m,pp ) nd the mximl velocity (V mx ) for oxygen consumption y mitochondri during wrm cclimtion 5 C 15 C K m V mx K m V mx Cold cclimted.12±.7 72.5±5.4.6±.3 69.2±8.1 Short-term wrm exposed.7±.6 72.1±2.7.97±.16 15.6±1.1 Wrm cclimted.3±.3 c 31.6±5.1.15±.2 c 21.8±2.2 Vlues re mens ± s.e.m. (N=8 for cold cclimted, N=7 for short term wrm exposed nd N=12 for wrm-cclimted trout). Different superscript letters indicte rtes t given ssy temperture tht differ with cclimtion stte (ANOVA nd posteriori tests, P.5). K m vlues re expressed in mmol l 1 ADP, V mx vlues s nmol tom O min 1 mg 1 mitochondril protein.
Therml dpttion in trout mitochondri 157 Tle 4. Time course of chnges in content of cholesterol nd totl phospholipids in mitochondri during wrm cclimtion Cold cclimted Short-term wrm exposed Wrm cclimted Totl phospholipids ( mol mg 1 protein).52±.3,.52±.3.37±.6 Cholesterol ( mol mg 1 protein).51±.6.51±.3.53±.3 Cholesterol/phospholipids ( mol mol 1 ).1±.2.1±.1.14±.1 Vlues re mens ± s.e.m. (N=3 for cold cclimted, N=4 for short-term wrm exposed nd N=3 for wrm cclimted trout). Different superscript letters indicte vlues tht differ etween cclimtion sttes (ANOVA nd posteriori test, P.5). cclimtion. These PUFA showed much the sme ptterns of chnge in PlsmPE. More drmtic modifictions during the time course of wrm cclimtion were noted when considering the FA composition of the minor phospholipid clsses CL nd LysoPC (Tle 8). Tle 5. FA composition of totl phospholipids during the time course of wrm cclimtion Totl phospholipids Cold Short-term Wrm Ftty cids cclimted wrm exposed cclimted 16: 22.2±2.4 22.5±.7 19.4±1. 18: 4.6±.5 4.1±.2 5.2±.3 16:1n-7 1.±.1,.9±.1 1.3±.1 18:1n-9 6.6±.1 6.1±.4 9.6±.8 18:1n-7 1.6±.1 1.6±.1 1.5±.1 2:1n-9 1.±.2 1.±.1,.6±.1 18:2n-6 2.9±.1 2.8±.3 4.1±.4 2:4n-6 1.4±.1 1.3±.1 1.6±.1 2:5n-3 7.5±.7 6.1±.2, 5.±.3 22:4n-6.3±.1.2±.1.3±.1 22:5n-6.7±.1.6±.1.7±.1 22:5n-3 2.5±.2, 2.1±.1 2.8±.2 22:6n-3 39.6±2.3, 42.3±.9 37.9±1.1 Others* 5.9±.4 5.7±.3 5.3±.4 DMA** 2.6±.7 3.±.4 5.3±.4 Totl SFA 28.6±2.3 28.1±.8 25.7±1. Totl MUFA 11.6±.3 11.1±.5 14.3±.8 Totl PUFA 57.1±1.4 57.7±1. 57.8±1.3 n-3/n-6 8.6±.5 9.3±.6 6.4±.5 UI 316.9±1. 322.8±6.1 3.5±7.5 Results re expressed s mol%. Vlues re mens ± s.e.m. (N=4 for ech cclimtion stte). Different superscript letters indicte vlues tht differ etween cclimtion sttes (ANOVA nd posteriori tests, P.5). *Others: totl of 19 ftty cids detectle (14:, 15:, 17:, 16:1n- 5, 18:1n-5, 18:3n-3, 18:3n-6, 2:1n-11, 2:1n-7, 2:2n-6, 2:3n-6, 2:4n-3, 21:5n-3, 22:1n-11, 22:1n-9, 24:1n-11, 24:1n-9, 24:5n-3, 24:6n-3), none of which were more thn 1.%. **Totl of dimetylcetls (minly 16:DMA nd 18:DMA). Arevitions re defined in the List of revitions. Effectively, wrm cclimtion cused more mrked ltertions in cyl composition of these phospholipids thn for PC nd PE, with mny FA eing hlved or douled in their levels. In CL, the decrese in 16: nd monounsturted FA, minly 18:1n- 7, ws ccompnied y mrked increse in 18:2n-6 during wrm cclimtion. The 18:2n-6, ppered to e n importnt ftty cid in CL compred to other phospholipids, nd ccounted for more thn 2 mol% of totl FA of CL in wrm cclimted trout. In contrst, 22:6n-3, which ws the most undnt ftty cid in CL, styed constnt during time course of wrm cclimtion. For LysoPC, the sme PUFA vried s in dicylpc, dicylpe nd PlsmPE, ut the chnges were more drmtic. Mrked decreses in 22:6n-3 (from 53.4% to 32.4%) nd in 2:5n-3 in wrm cclimted trout (P<.5) were ccompnied y virtul douling of 18:1n-9 nd 16: (P<.5). Few modifictions in FA composition were noted for PI nd PS (dt not shown). Discussion To identify potentil mechnisms y which oxidtive cpcities of mitochondri from trout oxidtive muscle chnged during trnsfer from 5 C to 15 C, the present study exmined the modifictions of phospholipid composition (phospholipid clsses nd the ssocited ftty cids), levels of memrne proteins nd functionl cpcities on the sme experimentl preprtions. The chnges in functionl cpcities could not e explined y chnges in the concentrtion of mitochondril memrne proteins, ut rther y specific modifictions in the phospholipid ilyer. To ssess our conclusions, we will compre our results for the functionl cpcities, protein composition nd phospholipid composition with pulished studies tht hd generlly descried these chrcteristics seprtely. Mitochondril properties Role of memrne proteins Wrm cclimtion led to the expected shifts in mitochondril oxidtive cpcities. Trnsfer of trout from 5 C to 15 C led to slight increse in mitochondril mximl oxidtive cpcity mesured t 15 C in short-term wrm exposed trout followed y mrked drop fter wrm cclimtion t oth ssy tempertures The increse in cpcity in short-term wrm exposed trout ws most pronounced for Stte 3 respirtion rtes expressed reltive to cytochrome nd c 1 levels. Nonphosphorylting (Stte 4) rtes of oxygen uptke incresed with
158 E. Krffe, Y. Mrty nd H. Guderley Tle 6. Ftty cid composition of dicyl nd plsmlogen forms of PC during the time course of wrm cclimtion DicylPC* PlsmPC** Cold Short-term Wrm Cold Short-term Wrm Ftty cids cclimted wrm exposed cclimted cclimted wrm exposed cclimted 14: 1.9±.2 1.6±.2, 1.2±.2 Trce Trce Trce 16: 26.7±.7 28.7±.4 25.5±.3 9.9±1.5 9.9±.7 6.9±.7 18: 1.2±.1, 1.1±.1 1.7±.1 1.±2. 6.±1. 5.7±.2 16:1n-7 1.7±.1 1.5±.1 1.7±.3 Trce Trce Trce 18:1n-9 7.±.2 6.5±.3 11.4±1.1 6.1±.4 4.5±.9 5.9±.8 18:1n-7 1.2±.1 1.2±.1 1.4±.1 Trce Trce Trce 2:1n-9.3±.1.4±.1.4±.1.6±.5.4±.3.2±.1 18:2n-6 2.1±.1 1.7±.1 3.±.2 2.±1.1.4±.3 2.4±.6 2:4n-6 1.4±.1 1.3±.1 1.6±.1 2.5±1.5 1.5±1.1.5±.3 2:5n-3 9.7±1.3 8.5±.3 6.5±.6 1.±.6.9±.4 2.4±.7 22:4n-6.1±.1.1±..1±.1 Trce Trce Trce 22:5n-6.5±.1.4±.1.5±.1 Trce Trce Trce 22:5n-3 1.6±.2 1.3±.1 1.7±.1.5±.4.9±.6 1.1±.2 22:6n-3 4.8±.5, 41.4±.5 38.5±1.3 14.4±2.1 17.8±2.8 18.7±1.6 Others*** 3.8±.4 4.1±.2 4.4±.4 3.2±1.9 6.9±1.5 5.2±2. Totl SFA 3.4±.7 32.±.1 28.9±.4 19.8±1.4 17.5±1.1, 13.8±1.5 Totl MUFA 12.1±.1 11.9±.5 17.2±1.3 7.9±1.4 5.2±.9 6.8±.6 Totl PUFA 57.5±.6 56.2±.5, 53.9±1.7 22.3±2.4 27.3±1.8, 29.4±.5 Results re expressed s mol%. Vlues re mens ± s.e.m. (N=4 for ech cclimtion stte). Different superscript letters indicte vlues tht differ etween cclimtion sttes (ANOVA nd posteriori tests, P.5). *DicylPC refer to ftty cids t oth sn-1 nd sn-2 positions of the dicyl form. **PlsmPC refer to sn-2 ftty cyl chins of the plsmlogen form. The totl percent ws djusted to 5% to tke into ccount the sence of the lkenyl chins of the sn-1 position hydrolyzed y the cid moile phse s descried in Mterils nd methods. ***Others: totl of 18 ftty cids detectle (15:, 17:, 16:1n-5, 18:1n-5, 18:3n-3, 18:3n-6, 2:1n-11, 2:1n-7, 2:2n-6, 2:3n-6, 2:4n-3, 21:5n-3, 22:1n-11, 22:1n-9, 24:1n-11, 24:1n-9, 24:5n-3, 24:6n-3), none of which were more thn 1.% in ny suclss. Arevitions re defined in the List of revitions. trnsfer from 5 C to 15 C efore returning to initil levels fter long-term therml cclimtion. These functionl chrcteristics of muscle mitochondri nd their modifiction during wrm cclimtion confirmed the iphsic responses previously found for Stte 3 nd 4 rtes (Bouchrd nd Guderley, 23). Mximl rtes of oxygen uptke ccounted for pproximtely 4% of the mximl cpcity of CCO, s oserved for Arctic chr nd rinow trout muscle mitochondri (Blier nd Lemieux, 21; Bouchrd nd Guderley, 23). The therml sensitivity of the ADP ffinity ws drmticlly modified during short-term wrm exposure, when the ADP/O rtio incresed, ut wrm cclimtion returned these vlues to those oserved in cold cclimted trout. In ddition, n increse in ADP ffinity ws oserved fter wrm cclimtion. A loss of pprent ADP ffinity with decrese in temperture chrcterises rinow trout mitochondri over much of their nnul cycle (Blier nd Guderley, 1993; Guderley nd St Pierre, 1999), ut therml independence of the pprent ADP K m (s for the short-term wrm cclimted trout) occurs t oth the coldest nd the wrmest periods of the yer (Guderley nd St Pierre, 1999). As the chnges in mitochondril functionl properties we oserved were similr to those previously determined, cusl mechnisms uncovered re lso likely to pply rodly. Trout oxidtive muscle mitochondri hd similr cytochrome nd ANT concentrtions nd reltive levels s muscle mitochondri from crp, cne tods, chickens, guine pig nd rts (Willims, 1968; Wodtke, 1981; Guderley et l., 25). The increse of ANT during short-term wrm exposure resemles the chnges oserved previously in trout muscle mitochondri (Bouchrd nd Guderley, 23). Chnges in the reltive levels or concentrtions of electron trnsport chin complexes could modify the ctlytic cpcity of the mitochondri (Sidell, 1983). Differences in cytochrome rtios nd ANT in muscle mitochondri from rts, cne tods nd erded drgon lizrds my contriute to interspecific vriility in oxidtive cpcities (Guderley et l., 25). However, the reltive levels of cytochromes nd ANT were quite constnt during wrm cclimtion of trout, lthough wrm cclimtion of trout decresed the levels of cytochromes nd c. Cytochrome is situted in Complexes II nd III. If the portion ssocited with Complex III ecme less undnt,
Therml dpttion in trout mitochondri 159 Tle 7. Ftty cid composition of dicyl nd plsmlogen forms of PE during the time course of wrm cclimtion DicylPE* PlsmPE** Cold Short-term Wrm Cold Short-term Wrm Ftty cids cclimted wrm exposed cclimted cclimted wrm exposed cclimted 16: 13.7±.4 13.1±.3 1.9±.7 6.2±1.7 5.±.5 4.5±.5 18: 7.1±.5 6.8±.3 9.7±.8 3.2±1. 3.1±.2 3.8±.4 16:1n-7.5±.1.4±.1.4±.1.2±.1.4±.1.5±.1 18:1n-9 5.6±.2 5.6±.3 7.6±.9 3.9±.5 5.4±.4, 7.±.8 18:1n-7 2.±.1 2.±.1 1.7±.1.5±.1.8±.1.8±.1 2:1n-9 2.±.2 2.1±.1 1.4±.1.4±.1.7±.1.4±.1 18:2n-6 2.8±.1 2.5±.2 3.1±.4 1.4±.3 1.3±.2 1.7±.2 2:4n-6 1.±.1 1.1±.1 1.3±.1 1.±.1 1.±.1 1.4±.1 2:5n-3 4.2±.3 3.8±.1 3.1±.2 3.4±.1 3.±.1 2.9±.2 22:4n-6.6±.1.6±.1 1.1±.1.2±.1.2±.1.5±.1 22:5n-6.9±.1.9±.1 1.±.1.5±.1.4±.1.5±.1 22:5n-3 4.±.2 4.1±.2 6.1±.4 1.9±.1 1.7±.1 2.7±.2 22:6n-3 5.2±.6, 51.5±.3 47.8±1.2 25.3±1.8 24.7±.4 21.±1.4 Others*** 4.6±.2 5.±.1 4.4±.2 1.6±.3 2.1±.3 2.2±.4 Totl SFA 21.7±.4 2.7±.4 21.3±1.4 9.6±2.4 8.5±.5 8.6±.9 Totl MUFA 1.9±.5 11.2±.4 11.9±1.2 5.9±.8 8.4±.3 9.8±.9 Totl PUFA 67.4±.8 68.2±.5 66.8±1.8 34.5±1.9 33.1±.6 31.7±1.5 Results re expressed s mol%. Vlues re mens ± s.e.m. (N=4 for ech cclimtion stte). Different superscript letters indicte vlues tht differ etween cclimtion sttes (ANOVA nd posteriori tests, P.5). *DicylPE refer to ftty cids t oth sn-1 nd sn-2 positions of the dicyl form. **PlsmPE refer to sn-2 ftty cyl chins of the plsmlogen form. The totl percent ws djusted to 5% to tke into ccount the sence of the lkenyl chins of the sn-1 position hydrolyzed y the cid moile phse s descried in Mterils nd methods. ***Others: totl of 19 ftty cids detectle (14:, 15:, 17:, 16:1n-5, 18:1n-5, 18:3n-3, 18:3n-6, 2:1n-11, 2:1n-7, 2:2n-6, 2:3n-6, 2:4n- 3, 21:5n-3, 22:1n-11, 22:1n-9, 24:1n-11, 24:1n-9, 24:5n-3, 24:6n-3), none of which were more thn 1.% in ny suclss. Arevitions re defined in the List of revitions. it could reduce the cpcity for pyruvte oxidtion. As cytochrome c is sustrte for Complexes III nd IV, reductions in its vilility could decrese mitochondril oxidtive cpcity (Lesnefsky et l., 21). On the other hnd, the constnt levels of cytochrome, component of Complex IV (CCO), suggest tht the concentrtions of Complex IV were mintined in wrm cclimted trout. Crp cclimted to 3 C lso mintin constnt levels of cytochrome in muscle mitochondri nd decrese levels of c+c 1 (Wodtke, 1981). Since wrm cclimtion cused prllel decreses in mitochondril oxidtive cpcity nd CCO ctivity, nd since cytochrome levels did not chnge, we conclude tht the decreses in oxidtive cpcity were not cused y chnge in the concentrtion of CCO. Role of memrne lipid composition Cholesterol. Our study evluted potentil mens y which mitochondril memrne lipid composition sets mitochondril oxidtive cpcities during therml chnge. To ensure proper functioning of iomemrnes, the physicl nd chemicl environment of the memrne must e regulted to mintin ctivity of memrne-ssocited proteins. Among memrne components, chnges in cholesterol re suggested to ply such role during therml cclimtion (Crockett nd Hzel, 1995). The sic stoichiometry of mitochondril memrnes from trout red muscle, in prticulr the phospholipid to protein rtio, ws in close greement with dt from crp red muscle (Wodtke, 1981), nd similr to the vlues otined for mitochondri from pig hert nd rt liver (Comte et l., 1976; Hovius et l., 199). The rtio of cholesterol to protein ws in the rnge of pulished vlues. Therml cclimtion led to sustntil chnges in overll mitochondril lipid composition, ut left the rtio of cholesterol to protein unchnged. Specificlly, the rtio of totl phospholipids to protein ws significntly decresed in mitochondril memrnes from wrm cclimted trout. As suggested (Wodtke, 1981), these modifictions were most likely due to lower proportion of inner memrnes in mitochondri of wrm cclimted trout. Indeed, lthough less frequently oserved during therml cclimtion thn chnges in mitochondril volume density, n incresed mitochondril criste density occurs during winter cclimtistion in oxidtive muscle of rinow trout (St Pierre et l., 1998). Since cholesterol seems to e minly ssocited with the outer mitochondril memrne (Dum, 1985;
16 E. Krffe, Y. Mrty nd H. Guderley Tle 8. Ftty cid composition of crdiolipin (CL) nd nturl lysophosphtidylcholine (LysoPC) during time course cclimtion CL LysoPC Cold Short-term Wrm Cold Short-term Wrm Ftty cids cclimted wrm exposed cclimted cclimted wrm exposed cclimted 16: 8.9±.7 7.4±.5 3.2±.2 17.4±3.3 18.9±2.2 31.2±1.4 18: 2.±.4 1.3±.1 2.2±.4 2.4±.5 5.2±1.5 4.2±13. 16:1n-7 1.2±.1 1.8±.1 3.2±.4.8±.1.6±.3 1.3±.1 18:1n-9 7.4±.2 7.±.3 6.1±.2 5.7±.4 7.±.6 11.9±.8 18:1n-7 6.4±.5 6.3±.3 2.4±.2 1.±.1 1.1±.2 1.4±.1 2:1n-9 2.4±.2 2.1±.2 1.7±.2.5±.1.4±.2.2±.1 22:1n-11 3.1±.5 3.3±.4 2.2±.2 Trce Trce Trce 18:2n-6 13.4±.5 14.4±.5 21.4±.6 2.±.3 2.2±.6 3.5±.3 18:3n-3 1.2±.4 1.7±.1 3.1±.2 Trce Trce Trce 2:4n-6.5±.1.3±.1.5±.2 1.5±.2 1.7±.1 1.5±.1 2:5n-3 1.2±.1 1.±.1 1.2±.1 9.7±.7 7.9±.6 6.3±.8 22:4n-6 Trce Trce Trce Trce Trce Trce 22:5n-6.6±.1.5±.1.6±.1.8±.1.8±.2.7±.1 22:5n-3 2.1±.1 1.4±.1 1.7±.1 c 2.±.1 1.3±.1 1.7±.2 22:6n-3 44.8±.9 44.5±1.2 43.3±.8 53.4±2.3 5.2±4.5 32.3±1.9 Others*** 5.5±.4 5.9±.2 5.7±.4 1.3±.1 1.1±.9 2.1±.6 Totl SFA 12.3±1.3 9.7±.7 6.8±.4 c 21.1±2.8 25.3±4.1 37.±2.5 Totl MUFA 2.4±.7 21.3±.6 15.9±.2 8.6±.6 9.7±1.2 15.3±.6 Totl PUFA 67.2±.7 69.±1. 77.3±.6 7.3±2.4 65.±1.8 47.6±.5 Results re expressed s mol%. Vlues re mens ± s.e.m. (N=4 for ech cclimtion stte). Different superscript letters indicte vlues tht differ etween cclimtion sttes (ANOVA nd posteriori tests, P.5). ***Others: totl of 18 ftty cids detectle (14:, 15:, 17:, 16:1n-5, 18:1n-5, 18:3n-3, 18:3n-6, 2:1n-11, 2:1n-7, 2:2n-6, 2:3n-6, 2:4n- 3, 21:5n-3, 22:1n-9, 24:1n-11, 24:1n-9, 24:5n-3, 24:6n-3), none of which were more thn 1.%. Arevitions re defined in the List of revitions. Echegoyen et l., 1993), specific modifictions of the proportion of cholesterol in the outer mitochondril memrne with wrm cclimtion seem unlikely. Phospholipid clsses nd suclsses restructuring. Phospholipids tht constitute mitochondril memrnes should e regrded s structurl prtners of memrne proteins nd s potentil modultors of mitochondril processes (Dum, 1985). Among mechnisms of memrne remodeling during therml cclimtion, ltertions in phospholipid hedgroup composition re common response of poikiloterms (Hzel nd Crpenter, 1985; Hzel, 1988; Hzel nd Lndrey, 1988). In trout muscle mitochondri, the proportion of PE nd PC, in prticulr the dicyl forms, responded the most to therml chnge. These two phospholipid suclsses represented more thn 78 mol% of totl phospholipids. These rpid chnges fter short-term temperture chnge hve een descried in vrious cell memrnes nd hve led to the concept tht phospholipid hedgroup ltertions re centrl for regultion of physicl nd functionl properties of cell memrnes during the initil stges of therml chnge (Hzel nd Crpenter, 1985; Hzel, 1995). As reported (Hzel nd Lndrey, 1988) nd reviewed (Lee, 1991), these ltertions in memrne lipid composition during wrm cclimtion re not consistent with HVA ecuse reduction in the numers of PE should decrese, not increse, memrne order. The increse in lysopc in wrm cclimted trout lso would not fvour homeoviscous dpttion, s lysopc increses memrne permeility when incorported into PC ilyers (Kumr et l., 1988). On the other hnd, s PE hs smller hedgroup thn PC, decresed proportions of PE re elieved to offset direct effects of high temperture on phospholipid volume nd mintin n pproprite lnce etween ilyer-stilising (lmellr phse forming, like PC) nd -destilising (hexgonl phse forming, like PE) lipids. Severl lines of evidence suggest the PC/PE rtio is primry determinnt of memrne function (Hzel, 1995). The rpid djustments of PE nd PC found in mitochondril memrnes suggest tht mintining memrne sttus, even temporrily, within the time course of wrm cclimtion is criticl. Our study demonstrted the novel finding tht dicyl nd plsmlogen suclsses of PE nd PC do not vry in prllel. Indeed, the proportion of plsmlogens of oth phosphtides rose only in wrm cclimted trout while the dicyl forms of PE nd PC chnged with short-term wrm exposure, with