Fuel use during glycogenesis in rainbow trout (Oncorhynchus mykiss Walbaum) white muscle studied in vitro

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1 871 The Journl of Experimentl Biology 29, Pulished y The Compny of Biologists 26 doi:1.1242/je.271 Fuel use during glycogenesis in rinow trout (Oncorhynchus mykiss Wlum) white muscle studied in vitro Jennifer C. Km nd C. Louise Millign* Deprtment of Biology, The University of Western Ontrio, London, Ontrio, Cnd N6A 5B7 *Author for correspondence (e-mil: millign@uwo.c) Accepted 4 Jnury 26 The purpose of this study ws to exmine fuel used during muscle glycogenesis in rinow trout Oncorhynchus mykiss using n in vitro muscle slice preprtion to test the hypothesis tht intrcellulr lctte is the mjor glycogenic sustrte nd the muscle relies upon extrcellulr sustrtes for oxidtion. Fish were exhustively exercised to reduce muscle glycogen content, muscle slices were tken from exhusted fish nd incuted for 1 h in medium contining vrious sustrtes t physiologicl concentrtions. 14 C-leled lctte, glycerol or plmitte ws dded nd 14 C incorportion into muscle glycogen nd/or CO 2 ws mesured. Lctte clernce in the sence of net glycogenesis suggests tht when suitle oxidizle extrcellulr sustrtes were lcking, intrcellulr lctte ws oxidized. Only muscle incuted in lctte, glycerol or plmitte synthesized glycogen, with the gretest synthesis in muscle incuted in lctte plus glycerol. The mjor fte of these extrcellulr sustrtes ws oxidtive, with lctte Summry oxidized t rtes 1 times tht of plmitte nd 1 times tht of glycerol. Neither extrcellulr lctte nor glycerol contriuted significntly to glycogenesis, with lctte cron contriuting less thn.1% of the totl glycogen synthesized, nd glycerol less thn.1%. There ws 1 times more extrcellulr lctte-cron incorported into CO 2 thn into glycogen. In the presence of extrcellulr lctte, plmitte or glycerol, intrcellulr lctte ws spred n oxidtive fte, llowing it to serve s the primry sustrte for in situ glycogenesis, with oxidtion of extrcellulr sustrtes driving ATP synthesis. The primry fte of extrcellulr lctte is clerly oxidtive, while tht of intrcellulr, glycolyticlly derived lctte is glycogenic, which suggests intrcellulr comprtmenttion of lctte metolism. Key words: lctte metolism, white muscle, glycogenesis, rinow trout, Oncorhynchus mykiss. Introduction In rinow trout, high intensity, urst-type exercise is powered primrily y white muscle, fueled y glycogenolysis nd neroic glycolysis, nd fish rpidly exhust (e.g. Millign, 1996; Richrds et l., 22; Richrds et l., 22). At exhustion, glycogen, denosine 5 -triphosphte (ATP) nd phosphocretine (PCr) stores in working muscle re reduced nd lctte nd H + levels re elevted (Richrds et l., 22). Clernce of neroic metolites nd the restortion of energy reserves y muscle re necessry to prepre the fish for susequent outs of exercise. The current model for the restortion of muscle energy reserves in fish fter exhustive exercise suggests tht in dult fish glycogen is resynthesized in situ with lctte s the min sustrte (Millign nd Girrd, 1993; Wng et l., 1997; Richrds et l., 22). Glucose, the min glycogenic sustrte in mmmlin muscle, is proly not n importnt glycogenic or oxidtive sustrte for trout white muscle since muscle hs low hexokinse ctivity (Knox et l., 198; Storey, 1991) nd the expression of glucose trnsporters in the muscle memrne is extremely low nd their physiologicl role is uncler (Legte et l., 21; Teerijoki et l., 21; Cpill et l., 22). Clerly, if lctte is the min glycogenic sustrte nd glucose is reltively unimportnt fuel for muscle, other extrcellulr sustrtes must e fuelling glycogenesis nd muscle metolism s whole, during recovery from exercise. The concentrtion of lnine, mjor form of inter-tissue trnsport of mino cid-derived cron (Mommsen et l., 198), increses in oth plsm nd white muscle following exercise (Millign, 1997), though its role in fueling muscle recovery from exercise is not known. Overll it is elieved tht protein does not mke significnt contriution to fuelling eroic metolism, despite it eing plentiful in the white muscle (Luff nd Wood, 1996). Trout white muscle hs considerle intrmusculr lipid stores nd Richrds et l. (Richrds et l., 22) hve suggested prominent role for lipid oxidtion in fueling exercise recovery, thus spring intrcellulr lctte cron for glycogenesis.

2 872 J. C. Km nd C. L. Millign Thus, wheres we hve some insights into wht fuels re proly importnt for supporting muscle glycogenesis, we hve very little understnding of wht fuels re preferred nd how these fuels re used y muscle (i.e. cron for glycogen synthesis or for oxidtion). Therefore, the purpose of this study ws to test the hypothesis tht intrcellulr lctte is the mjor sustrte for trout muscle glycogenesis nd the muscle relies upon extrcellulr sustrtes for oxidtion to provide the ATP necessry to drive glycogen synthesis. We used the in vitro isolted white muscle slice preprtion, which hs een shown to e metoliclly vile, cple of glycogenesis nd ffords the opportunity to control sustrte vilility (Frolow nd Millign, 24). Our ojective ws to determine which sustrtes support glycogenesis nd, using pproprite rdiotrcers, determine how these extrcellulr sustrtes re used y muscle (glycogenic versus oxidtive fte). Mterils nd methods Experimentl nimls Rinow trout (Oncorhynchus mykiss Wlum; 2 3 g) of oth sexes were otined from Rinow Springs Trout Frm (Thmesford, Ontrio, Cnd). Fish were held indoors under flow-through conditions in 1 l lck circulr tnk continuously supplied with erted dechlorinted City of London, ON, Cnd wter mintined t 15±1 C nd photocycle of 12 h:12 h light:drk. Fish were cclimted to holding conditions t lest 3 weeks efore use. During holding, fish were fed dily with 2% ody weight rtion of commercil trout pellets. In order to reduce ny dietry effects on metolism, ll fish were isolted in lck, crylic, 4 l oxes, continuously supplied with erted, wter t 15±1 C nd fsted for 2 dys prior to experimenttion (Tng nd Boutilier, 1991). All holding nd hndling of fish ws in ccordnce with the Cnd Council on Animl Cre Guidelines nd pproved y the University of Western Ontrio Sente Animl Use Sucommittee. Experimentl protocol Fish were exhustively exercised y chsing them in 3 l circulr tnk for 5 min t which point they were unresponsive to further mnul stimultion. Previous studies hve shown tht this form of exercise leds to exhustion nd significnt reduction in muscle glycogen (e.g. Millign, 1996). Fish were trnsferred in wter-filled crylic, 4 l ox to tnk contining.3 g MS-222 (tricine methne sulfonte; Syndel, Vncouver, BC, Cnd) uffered to ph of 7.8 with NHCO 3 in 3 l of dechlorinted tpwter. Fish died within 1 2 min without struggling. Resting fish were killed in the sme mnner ut were not exercised. Following nesthetiztion, 1.5 cm 3 lock of muscle tissue of pproximtely 1.5 g ws rpidly excised from epxil muscle close to the mid-dorsl line of the fish, s descried y Frolow nd Millign (Frolow nd Millign, 24). The lock of muscle tissue ws then plced in ice-cold modified Cortlnd s sline contining 14 mmol l 1 NCl, 3.5 mmol l 1 KCl, 1. mmol l 1 MgSO 4 7H 2 O, 3. mmol l 1 NHPO 4 H 2 O, 4.5 mmol l 1 NHCO 3, 1. mmol l 1 CCl 2, 1. mmol l 1 Hepes nd.3% deftted ovine serum lumin oxygented with humidified 99.5% O 2 /.5% CO 2 nd djusted to ph 7.8. Tissue slices of pproximtely mm in thickness nd weighing 243.5±1.8 mg (N=136) were otined from tissue locks mintined in iced sline using Stdie-Riggs microtome nd tissue slicer lde (Thoms Scientific, Swedesoro, New Jersey, USA). One slice ws immeditely lotted dry nd frozen y freeze clmping etween luminum locks cooled with liquid nitrogen to determine the metolic sttus of the tissue t the time of smpling (referred to s time slice). The remining slices were incuted in 3. ml of modified Cortlnd s sline (s ove) continuously oxygented with humidified 99.5% O 2 /.5% CO 2 for 1 h t 15 C in 25 ml Erlenmeyer flsk in shking wter th. Following incution, tissue slices were removed, lotted dry, nd freezeclmped. Frozen tissue slices were ground to fine powder using n insulted mortr nd pestle cooled with liquid nitrogen nd stored t 8 C until time of nlysis. Experimentl series To quntify the oxidtion of extrcellulr sustrtes, [ 14 C]CO 2 production ws mesured using modified CO 2 trpping system (French et l., 1981) dpted y Wlsh et l. (Wlsh et l., 1988). A closed CO 2 trpping pprtus consisted of glss filter pper, wetted with 75 l of 1 mol l 1 enzylthonium hydroxide, plced in well suspended in 25 ml Erlenmeyer flsk, contining 3. ml of erted incution medium. Muscle slices were dded to incution flsks, the flsks were seled nd either [U- 14 C]lctte (specific ctivity: 5 mci mmol 1 ; 1 Ci= Bq), [U- 14 C]glycerol (specific ctivity: 14 mci mmol 1 ) or [U- 14 C]plmitte (specific ctivity: 4 mci mmol 1 ) (ll from ICN Rdiochemicls, Montrel, PQ, Cnd) ws dded to yield the finl specific ctivities in the incution sline given in Tle 1. The sustrte concentrtions for ech experiment re given in Tle 1 nd re typicl levels seen post-exercise in trout plsm (e.g. Richrds et l., 22). Plmitte ws in the form of sodium plmitte, soluilized ccording the methods descried y Berry et l. (Berry et l. 1991). The flsks were incuted in shking wter th t 15 C for 1 h. In every experiment, flsk contining everything except tissue ws incuted with the experimentl flsks to ccount for spontneous [ 14 C]O 2 production from the universlly leled 14 C sustrtes nd the susequent [ 14 C]O 2 production rtes were corrected ccordingly. After 1 h, 5 l of 7% HClO 4 ws dded to ech flsk to hlt metolism nd lierte 14 CO 2. The seled flsks were vigorously shken t room temperture for 6 min on n oritl shker t 1 revs min 1 to ensure complete collection of [ 14 C]O 2. At the end of this 6 min period, the muscle slices were freeze-clmped nd nlyzed for muscle glycogen nd lctte nd the filter ppers removed nd dded to 5. ml of Redy Sfe (Beckmn, Mississug, Cnd) scintilltion fluid in scintilltion vil nd counted for totl 14 C rdioctivity.

3 Extrcellulr sustrte use during glycogenesis in trout muscle 873 Tle 1. Specific ctivities used to clculte extrcellulr lctte, glycerol or plmitte incorportion into tissue glycogen nd/or CO 2 [ 14 C]lctte d.p.m. g 1 tissue Incution conditions d.p.m. mol 1 sline Glycogen CO 2 Lctte (5 mmol l 1 ) ± ± ±643 Lctte (5 mmol l 1 ) + glycerol (1 mmol l 1 ) ±482 22± ± Lctte (5 mmol l 1 ) + plmitte (.31 mmol l 1 ) ± ± ± Lctte (5 mmol l 1 ) + plmitte (.31 mmol l 1 ) + (1 mmol l 1 ) glycerol ±482 73± ±2 324 Lctte (1 mmol l 1 ) ± ± ±2,497 Lctte (1 mmol l 1 ) + glycerol (1 mmol l 1 ) ± ± ±3566 [ 14 C]glycerol (1 mmol l 1 ) ± ±51 859±17 (1 mmol l 1 ) + lctte (5 mmol l 1 ) ± ± ±34 [ 14 C]plmitte Plmitte (.31 mmol l 1 ) + glycerol (1 mmol l 1 ) ± ± Plmitte (.31 mmol l 1 ) + lctte (5 mmol l 1 ) ± ±8761 Plmitte (.31 mmol l 1 ) + lctte (5 mmol l 1 ) + (1 mmol l 1 ) glycerol ± ±9547 Plmitte (1 mmol l 1 ) ± ±236 Plmitte (1 mmol l 1 ) + glycerol (1 mmol l 1 ) ± ±648 Vlues re mens ± 1 s.e.m. (N=8). Initil experiments using [ 14 C]NHCO 3 (specific ctivity: 5 mci mmol l 1 ; ICN Rdiochemicls) indicted tht the [ 14 C]O 2 trpping efficiency of the system is 84±2% (N=8) nd [ 14 C]O 2 production rtes were corrected ccordingly. To determine incorportion of extrcellulr sustrte cron into glycogen cron, 5 l smple of the tissue glycogen suspension (see elow), ws dded to 5. ml of Redy Sfe fluor nd counted for totl 14 C rdioctivity. For ech experiment, ech tretment ws performed in duplicte on tissue slices otined from the sme fish, nd N in the figures refers to the numer of fish used per experiment. Anlyticl techniques nd clcultions Frozen tissue slices were individully ground to fine powder using n insulted mortr nd pestle cooled with liquid nitrogen. Muscle glycogen content ws ssyed y isolting the glycogen (Hssid nd Arhm, 1959) nd mesuring the free glucose fter digestion of the glycogen with myloglucosidse (Bergmeyer, 1965). In our lortory, we typiclly recovery 95 1% of the glycogen with this method. Muscle concentrtions of lctte, denosine triphosphte (ATP) nd phosphocretine (PCr) were mesured on pproximtely 5 mg of muscle ground to fine powder in liquid N 2 -cooled mortr nd vigorously resuspended in 1 ml 8% HClO 4. Lctte, ATP nd PCr were then mesured in the superntnt neutrlized with 3 mol l 1 KOH (Bergmeyer, 1965). Tissue [ 14 C]glycogen ws determined y dding 5 l of the resuspended glycogen (see ove) (Hssid nd Arhm, 1959) to 5 ml of Beckmn Redy Sfe scintilltion cocktil. The incorportion of extrcellulr sustrte into the muscle glycogen pool ws clculted using the specific ctivities of the sustrte in the sline, nd the specific ctivity of muscle glycogen nd CO 2 (Tle 1) fter Pgnott nd Millign (Pgnott nd Millign, 1991) nd ccording to the eqution: mol sustrte incorported into glycogen or CO 2 g wet tissue d.p.m. in glycogen or CO 2 g 1 wet tissue sustrte specific ctivity in sline where sustrte specific ctivity is in d.p.m. mol 1 in the 3. ml incution sline. The contriution of extrcellulr sustrte to glycogen synthesis ws clculted from the sustrte incorportion nd the mount of glycogen synthesized over the 1 h period. All smples were counted on Pckrd 19TR Liquid Scintilltion Counter using utomtic quench correction. All iochemicls were purchsed from Sigm Chemicl Co. (Mississug, ON, Cnd), Boehringer-Mnnheim Chemicl Co. (Lvl, PQ, Cnd), nd ll enzymes were purchsed from Worthington Biochemicl Corp. (Lkewood, NJ, USA). Sttisticl nlyses Vlues presented re mens ± 1 s.e.m. (N). Sttisticl nlyses were performed using one-wy nlysis of vrince =,

4 874 J. C. Km nd C. L. Millign Tle 2. The influence of sustrte interctions on glycogen synthesis nd lctte clernce in muscle slices from exercised fish Averge mount of glycogen Averge mount of lctte Sustrte(s) synthesized ( mol g 1 ) clered ( mol g 1 ) None, sline only 13.1±2.8 Lctte (5 mmol l 1 ) 2.4±1.1 1±3.2 (1 mmol l 1 ) 2.8±.8 1.7±2.3 Plmitte (.31 mmol l 1 ) 2.7± ±2.8 Lctte (5 mmol l 1 ) + glycerol (1 mmol l 1 ) 5.2± ±1.8 (1 mmol l 1 ) + plmitte (.31 mmol l 1 ) 4.14±.8 9.4±2.9 Lctte (5 mmol l 1 ) + plmitte (.31 mmol l 1 ) 9.8±1.9 Lctte (5 mmol l 1 ) + glycerol (1 mmol l 1 ) + plmitte (.31 mmol l 1 ) 9.2±2.1 Muscle slices were incuted for 1 h in the concentrtion of sustrte indicted. Averge glycogen synthesized ws clculted s the difference etween the verge t Time (immeditely fter exercise) level nd tht t the end of 1 h for only those tretments in which the glycogen content t the end of 1 h ws significntly different from tht t Time (N=8 for ech tretment). Vlues re mens ± 1 s.e.m. (ANOVA) followed y Kruskl Wllis comprison tests or Student s t-test nd significnt differences were ccepted when P<.5. Results At the end of exhustive exercise white muscle glycogen levels were significntly reduced from resting levels of 9.34±.75 mol glycosyl units g wet tissue 1 (N=8) to 2.3±.4 mol glycosyl units g wet tissue 1 (N=8) nd lctte levels incresed from resting levels of 2.6±1.4 mol g wet tissue 1 (N=8) to 22.5±2.4 mol g wet tissue 1 (N=8). Glycogen synthesis ws only seen in muscle slices incuted in lctte, glycerol, plmitte, lctte plus glycerol or glycerol plus plmitte, with the gretest glycogen synthesis seen in the ltter two conditions (Tle 2). When plmitte nd lctte were present together, glycogenesis ws inhiited, nd there ws lso no net glycogenesis in slices incuted in lctte, glycerol nd plmitte (Tle 2). Lctte levels were reduced under ll incution conditions, even in the sence of ny net glycogenesis. However, the inclusion of lctte in the incution mixture ppered to reduce lctte clernce s lctte levels t the end of the 1 h incution period ws consistently higher in those slices incuted in lctte (Tle 2). After 1 h incution in 5 mmol l 1 lctte there ws net glycogenesis (Fig. 1A) nd significnt incorportion of extrcellulr lctte, s indicted from 14 C ctivity, into oth the glycogen nd CO 2 pools. The mount of extrcellulr lctte incorported into CO 2 ws out 1-fold greter thn tht incorported into glycogen (Fig. 1B,C). Further, the contriution of extrcellulr lctte to totl glycogen synthesis ws trivil, ccounting for less thn.1% of the totl glycogen synthesized (compre Fig. 1A nd B). Incuting tissue in glycerol plus lctte incresed oth net glycogen synthesis nd lctte cron incorportion into glycogen twofold over slices incuted in lctte only (Fig. 1A,B). However, the totl contriution of extrcellulr lctte to glycogenesis ws still very smll, with only 3.5 nmol g 1 wet tissue of extrcellulr lctte incorported into the 35 nmol g 1 wet tissue of glycogen synthesized, or out.1% of the totl. The ddition of glycerol lso stimulted lctte oxidtion s lctte incorportion into the CO 2 pool incresed y pproximtely 1.4-fold (Fig. 1C). The comintion of.3 mmol l 1 plmitte nd 5 mmol l 1 lctte did not ffect net glycogen synthesis, or lctte incorportion into glycogen or CO 2 compred to either sustrte lone (Fig. 1). However, incution in plmitte plus glycerol plus lctte eliminted the stimultory effect of glycerol on glycogenesis nd lctte oxidtion (Fig. 1A,B). Incution of muscle slices in glycerol plus lctte consistently stimulted glycogen synthesis compred to tht in tissues incuted in glycerol lone (Fig. 2A). However, the incorportion of extrcellulr glycerol cron into glycogen synthesized ws very smll, with only.34 nmol g 1 entering the glycogen pool, ccounting for less thn.1% of the totl glycogen synthesized (Fig. 2B), which is one tenth of the contriution of lctte cron to glycogen (compre Fig. 1B nd Fig. 2B). Extrcellulr glycerol cron mde greter contriution to oxidtive metolism thn glycogenesis, s there ws tenfold greter incorportion into CO 2 thn into glycogen (Fig. 2B,C). However, the oxidtion of extrcellulr glycerol ws only 1% tht of extrcellulr lctte oxidtion (compre Fig. 1C nd Fig. 2C) nd ws unffected y the presence of lctte. Muscle slices incuted with plmitte synthesized glycogen nd oxidized plmitte (Fig. 3A,B). Plmitte oxidtion ws five times tht of glycerol, ut only t out 1% tht of lctte (Fig. 3B vs Figs 2C nd 1C). Incution of tissue in plmitte plus glycerol resulted in significntly greter glycogen synthesis, ut ws without ffect on plmitte oxidtion (Fig. 3A,B). Incuting tissues in plmitte plus lctte or plmitte plus lctte plus glycerol inhiited glycogenesis, ut did not significntly ffect plmitte oxidtion (Fig. 3A,B). The sustrte concentrtions used in the experiments thus

5 Extrcellulr sustrte use during glycogenesis in trout muscle A Glucosyl units (nmol g 1 ) c 5 B Time Lctte Lctte 4 C Lctte +Plmitte Lctte +Plmitte 14 C Incorportion (nmol g 1 ) ,, Lctte Lctte Lctte +Plmitte Lctte +Plmitte Tretment Lctte Lctte Lctte +Plmitte Lctte +Plmitte Fig. 1. Glycogenic nd oxidtive ftes of extrcellulr lctte during glycogenesis in muscle slices from exercised fish. (A) Muscle glycogen levels in tissue smpled immeditely fter exercise (Time ) nd in tissue incuted for 6 min in the sustrtes indicted. (B,C) The incorportion of 14 C from lctte into the muscle glycogen (B) nd CO 2 produced (C). All vlues re given in nmol g 1 wet mss to fcilitte comprisons etween components. Dt re presented s the men ± 1 s.e.m. for slices from eight different fish (N=8). Columns within the sme pnel with the sme letter re not significntly different from one nother. fr were chosen sed upon in vivo plsm concentrtions postexercise, with up to 15-fold concentrtion difference etween some sustrtes (e.g. lctte vs plmitte). In order to determine whether the oserved differences in sustrte utiliztion reflected ctul preferences y recovering muscle or were merely reflection of concentrtion differences, finl series of experiments ws performed with ll sustrtes t equl concentrtion, ut still within the physiologicl rnge. When ll sustrtes were present t concentrtion of 1 mmol l 1, muscle synthesized glycogen (Fig. 4A) nd the stimultory effects of glycerol on glycogenesis in slices incuted in glycerol plus lctte or glycerol plus plmitte were still evident (Fig. 4A). The net mount of glycogen synthesized ws lso unffected y the ltered sustrte concentrtions (compre Fig. 4A with Figs 1A, 2A nd 3A). An 8% decrese in extrcellulr lctte concentrtion corresponded to n 8% decrese in extrcellulr lctte cron incorportion into the glycogen pool, with only.3 nmol g 1 of lctte cron incorported into the glycogen pool s compred with 1.5 nmol g 1 t the higher concentrtion of lctte (compre Figs 4B, 1B). Lowering the concentrtion of lctte to 1 mmol l 1 decresed lctte incorportion into CO 2 y 95% from out 2 nmol g 1 to 1 nmol g 1 (compre Figs 1C, 4C). However, muscle still exhiited preference for extrcellulr lctte oxidtion, s ten times more lctte cron ws incorported into CO 2 thn from glycerol nd three times more thn tht from plmitte (Fig. 4C). The stimultory effect of glycerol on glycogenesis nd lctte use ws lso independent of concentrtion (Fig. 4A C). Interestingly, incresing the plmitte concentrtion y threefold decresed its oxidtion y 7% (Fig. 3B vs Fig. 4C). As well, in keeping with previous oservtions, incution in glycerol plus plmitte resulted in significnt glycogen synthesis (Fig. 4A) without ltering plmitte oxidtion (Fig. 4C). Discussion The results of the present study clerly demonstrte tht in vitro, rinow trout white muscle is cple of glycogen synthesis, provided it hs the pproprite extrcellulr

6 876 J. C. Km nd C. L. Millign 1 A c 1 A c 14 C Incorportion (nmol g 1 ) Glucosyl units (nmol g 1 ) B Time +Lctte sustrtes to support oxidtive metolism. Lctte, glycerol nd plmitte ll stimulted glycogenesis, ut the gretest mount of glycogen synthesis ws seen in tissues incuted in glycerol plus lctte or glycerol plus plmitte, ut when ll three were together, there ws no net glycogenesis. In ll cses, lctte ws clered, with the gretest mount clered in the sence of net glycogenesis. This is the first study of its kind to exmine in ny detil how fish muscle uses extrcellulr sustrtes to fuel glycogenesis nd in the model descried elow (Fig. 5), we ttempt to integrte these new results with wht is lredy known into model to descrie muscle fuel use during glycogen re-synthesis in rinow trout. Extrcellulr sustrte use during muscle glycogenesis In the sence of ny extrcellulr sustrtes, there ws no net glycogen synthesis, despite clernce of significnt mount of muscle lctte, suggesting tht under these conditions, the mjor fte of endogenous lctte is oxidtion (Fig. 5 #1). It is unlikely tht the disppernce of lctte from the muscle is result of lctte efflux since the white muscle 4 C Tretment +Lctte +Lctte Fig. 2. Glycogenic nd oxidtive ftes of extrcellulr glycerol during glycogenesis in muscle slices from exercised fish. (A) Muscle glycogen levels in tissue smpled immeditely fter exercise (Time ) nd in tissue incuted for 6 min in the sustrtes indicted. (B,C) The incorportion of 14 C from glycerol into the muscle glycogen (B) nd CO 2 produced (C). All vlues re given in nmol g 1 wet mss to fcilitte comprisons etween components. Dt re presented s the men ± 1 s.e.m. for slices from eight different fish (N=8). Columns within the sme pnel with the sme letter re not significntly different from one nother. Glucosyl units (nmol g 1 ) 14 C Incorportion (nmol g 1 ) B Time Plmitte Plmitte Plmitte Plmitte Tretment memrne is reltively imperment to lctte efflux nd s consequence, the ulk of the lctte generted is retined within the muscle (Shrpe nd Millign, 23). The presence of extrcellulr lctte, glycerol or plmitte supported glycogenesis, presumly y serving s oxidtive fuels (Fig. 5#2,#3,#4). Their presence proly reduced the relince upon oxidtion of intrcellulr lctte, spring some of it for glycogenesis (Fig. 5#5). Although there ws net glycogenesis when lctte, plmitte or glycerol ws ville, the reduction in intrcellulr lctte ws in excess of tht ccounted for in the mount of glycogen synthesized, suggesting either some intrcellulr lctte ws still eing oxidized or the missing lctte (5 6 mol g 1 ; Tle 2) ws trpped in glycogenic intermedites. The oservtion tht when tissues were incuted in glycerol plus lctte or glycerol plus plmitte, ll the lctte clered could e ccounted for y the glycogen synthesized could men tht the vilility of oxidizle Plmitte +Lctte Plmitte +Lctte Plmitte +Lctte Plmitte +Lctte Fig. 3. Glycogen synthesis nd plmitte oxidtion during glycogenesis in muscle slices from exercised fish. (A) Muscle glycogen levels in tissue smpled immeditely fter exercise (Time ) nd in tissue incuted for 6 min in the sustrtes indicted. (B) The incorportion of 14 C from plmitte into CO 2 produced. All vlues re given in nmol g wet mss 1 to fcilitte comprisons etween components. Dt re presented s the men ± 1 s.e.m. for slices from eight different fish (N=8). Columns within the sme pnel with the sme letter re not significntly different from one nother.

7 Extrcellulr sustrte use during glycogenesis in trout muscle A c Glucosyl units (nmol g 1 ) ,c 2 1 B Time Plmitte 3 C Lctte Plmitte Lctte d 14 C Incorportion (nmol g 1 ) c Lctte Lctte Tretment Plmitte Lctte Plmitte Lctte Fig. 4. Muscle glycogen. (A) Glycogen levels in muscle slices otined from fish immeditely fter exercise (Time ), or fter 6 min incution in sline contining vrious sustrtes in equl concentrtion. (B,C) Incorportion of rdiolel from the vrious sustrtes into muscle glycogen (B) nd CO 2 produced (C). All vlues re given in nmol g 1 wet mss to fcilitte comprisons etween components. Dt re presented s the men ± 1 s.e.m. for slices from eight different fish (N=8). Columns within the sme pnel with the sme letter re not significntly different from one nother. sustrtes is limiting to glycogenesis nd determines the fte of intrcellulr lctte. Lctte ws the preferred extrcellulr sustrte for oxidtion y muscle during glycogen resynthesis, contriuting s much s 16 3% of the ATP required for the oserved glycogenesis. This suggest tht t lest 7% of the ATP needed for glycogenesis cme from oxidtion of intrcellulr sustrtes, proly ftty cids (Richrds et l., 22). These estimtes ssume tht sustrtes were completely oxidized (llows clcultion of ATP yield) nd tht glycogen ws synthesized from intrcellulr lctte (llows clcultion of ATP requirement). This represents true muscle preference for extrcellulr lctte s n oxidizle sustrte nd not consequence of differences in sustrte concentrtions, ecuse when ll sustrtes were present t the sme concentrtion, lctte ws still preferred over the other fuels. Lctte is clerly tken up y the muscle, despite the fct tht the intrcellulr lctte concentrtion ws four times tht of the extrcellulr concentrtion (2 vs 5 mol g 1 wet tissue); grdient tht should fvor net efflux. Lctte uptke y trout muscle is fcilitted y monocroxylte (MCT)-like trnsporter locted in the srcolemml memrne (Fig. 5#6) (Leree nd Millign, 1999) nd the mjor fte of this extrcellulr lctte is clerly oxidtive (Figs 1C, 5#7). The mount of extrcellulr lctte-derived cron entering the CO 2 pool is out 1 times tht entering the glycogen pool (compre Fig. 1B,C) nd its oxidtion ppers to spre lctte of intrcellulr origin for glycogenesis (Fig. 5#5). This model suggests tht the fte of extrcellulr lctte is seprte from tht of intrcellulr, glycolyticlly derived

8 878 J. C. Km nd C. L. Millign lctte; in other words, lctte metolism is comprtmentlized in trout skeletl muscle. Comprtmenttion of crohydrte metolism hs een descried for vsculr smooth muscle (Lynch nd Pul, 1983) in which extrcellulr glucose ws the sole precursor for eroic glycolysis, wheres glycogenolysis ws the precursor for oxidtive phosphoryltion. Similrly, in insect flight muscle, glycolytic metolites generted from glucose do not mix with those from glycogen (Srere nd Knull, 1998). More recently comprtmenttion of lctte metolism hs een suggested to explin the simultneous influx nd efflux of lctte in the isolted rt hert (Chthm nd Forder, 1996; Chthm et l., 21). The dt from Chthm et l. (Chthm et l., 21), in which they perfused the rt hert with [3-13 C]lctte, indicted tht lctte of extrcellulr origin ws preferentilly oxidized wheres glycolyticlly derived lctte (endogenous) ws Fig. 5. A proposed model descriing extrcellulr sustrte use in support of glycogenesis in trout muscle during recovery from exhustive exercise. Thicker rrows indicte preferred pthwys; wvy rrows indicte diffusion. LA e, lctte of extrcellulr origin; LA i, lctte of intrcellulr, glycolytic, origin; MCT, monocroxylte trnsporter; PYR, pyruvte; LDH, lctte dehydrogense; PDH, pryruvte dehydrogense; GLY, glycerol; GK, glycerol kinse; G3P, glyerol 3-phosophte; DHAP, dihydroxycetone phosphte; PA, plmitte, TCA, tricroxylic cid cycle; CoA, coenzyme A; Ac-CoA, cetyl CoA; NAD +, oxidized nicotinmide-denine dinucleotide; NADH, reduced nicotinmidedenine dinucleotide; FAD, flvin denine dinucleotide; FADH 2, reduced FAD. See text for detils (#, rection numer). preferentilly trnsported out of the crdic muscle. These dt re consistent with the concept of n intrcellulr lctte shuttle, first proposed y Stinsy nd Brooks (Stinsy nd Brooks, 199), then lter refined y Brooks (Brooks, 2) to include the concept of direct mitochondril uptke nd metolism of lctte. Although the evidence for the ltter component of the intrcellulr lctte shuttle is conflicting, there is generl greement tht some type of comprtmenttion of lctte metolism exists in skeletl muscle. Gldden (Gldden, 24), in recent review, puts forwrd the ide of microcomprtmenttion to explin the different ftes of extrcellulr nd intrcellulr, glycolyticlly derived lctte. In this model, the physicl loctions of mitochondri nd glycolysis in the cytosol re distinct, such tht mitochondri re locted primrily t the periphery of the cell, close to the srcolemml memrne, the site of extrcellulr lctte uptke vi MCTs, nd removed from the cytosolic loction of glycolysis. Thus, once extrcellulr lctte is trnsported into the cell, it is redily converted to pyruvte vi cytosolic LDH, which is then trnsported into the mitochondri nd oxidized while the glycolyticlly derived lctte (intrcellulr) is loclized closer to the glycogenic mchinery. Our current understnding of teleost white muscle rchitecture, in which mitochondri re loclized peripherlly nd the glycogen grnules, nd presumly the glycolytic nd glycogenic enzymes s well, re locted etween the myofirils (Sänger nd Stoier, 21) is consistent with this concept of intrcellulr comprtmentliztion of lctte metolism. Certinly, the fct tht the presence of oxidizle extrcellulr sustrtes (e.g. lctte, glycerol nd plmitte) stimultes glycogenesis nd reduces lctte clernce is consistent with the notion tht intrcellulr lctte is spred for glycogenesis nd the ide tht the enzymtic mchinery for glycogenesis is remote from the mitochondri. Clerly, vlidtion of this model wits further experimenttion. Wheres extrcellulr glycerol lone cn support glycogenesis, its contriutions to oth oxidtive metolism (its oxidtion yields only out.4% of the ATP needed for glycogenesis; Fig. 2C) nd glycogenesis (glycerol cron contriuted <.1% to glycogen cron; Fig. 2B) were miniml. This miniml use of glycerol s n oxidtive or glycogenic sustrte my reflect low ctivities of glycerol kinse (GK), which ctlyzes glycerol to glycerol 3-phosphte (Fig. 5#8) or n enhnced role of the glycerol 3-phosphte shuttle in fish muscle. The gretest impct of glycerol on muscle glycogenesis ws its stimultory effect on glycogenesis nd lctte use when tissues were incuted in glycerol plus lctte. The effect ws specific for glycerol on lctte, in tht lctte did not influence glycerol use (Fig. 1B,C vs Fig. 2B,C). The explntion for this stimultory effect of glycerol on lctte use nd glycogenesis is not cler, ut my e relted to need for cytosolic NAD + to support the first step in lctte metolism. The glycerol 3-phosphte shuttle, present within mmmlin muscle (Fig. 5#8) (Rsmussen et l., 23), reoxidizes cytosolic NADH to NAD +, potentilly incresing the vilility of NAD + for lctte metolism (Hettwer et l.,

9 Extrcellulr sustrte use during glycogenesis in trout muscle ) nd the presence of extrcellulr glycerol my enhnce the ctivity of the shuttle. Ftty cid oxidtion hs een shown to sustntilly contriute to fueling glycogen re-synthesis during recovery from high-intensity exercise in rinow trout in vivo (Richrds et l., 22; Richrds et l., 24). Ftty cids of extrcellulr origin cn e used y muscle, s ftty cid uptke hs een shown to e crrier-medited in trout muscle, presumly y ftty cid trnslocse tht hs yet to e chrcterized. Once in the muscle, these ftty cids undergo -oxidtion (Fig. 5#9) (Richrds et l., 24), supplying the ATP necessry to support glycogenesis, gin spring intrcellulr lctte for glycogenesis. Accordingly, in the presence of plmitte, there is net glycogenesis nd the mount of lctte clered is reduced. Clerly trout white muscle is le to tke up nd utilize ftty cids, ut the estimted contriution to fueling glycogenesis is somewht less thn suggested in vivo. In vitro, the contriution of ftty cid oxidtion ppers to e less importnt, s plmitte oxidtion is only one tenth tht of lctte (Fig. 1C vs Fig. 3B), nd t most, contriuted to only 16% of the ATP required to support the ssocited glycogenesis. Nonetheless, in the presence of plmitte, there ws net glycogenesis, n effect tht ws enhnced when tissues were incuted in glycerol plus plmitte. did not stimulte plmitte oxidtion, suggesting tht the effects re independent of one nother. Incuting tissues in lctte plus glycerol plus plmitte hd negtive impct on muscle glycogenesis, ut ws without effect on either lctte or glycerol oxidtion. The slight inhiitory effect of lctte on plmitte oxidtion is consistent with the inhiitory effects of crohydrte on ftty cid oxidtion (e.g. Richrds et l., 22) nd the oservtion from the present study tht lctte is preferred sustrte for muscle oxidtive metolism. However, why the comintion of lctte plus plmitte should inhiit glycogenesis is not t ll cler. There re reports of plmitte hving negtive impcts on glycogen synthesis nd lctte use in mmmlin muscle, ut none re consistent with the current oservtions. For exmple, in cultured skeletl muscle cells from insulin-resistnt dietic humns, the products of plmitte oxidtion hve een shown to inhiit frctionl glycogen synthse ctivity presumly ecuse of ftty cid oxidtion-induced production of glucosmines (Mott et l., 2), wheres in the present study, plmitte lone ctully stimulted glycogenesis. Furthermore, the ddition of extrcellulr plmitte to the isolted perfused hert from dietic rts decreses the rte of lctte uptke nd oxidtion (Chthm et l., 1999), gin, not see in this study. The explntion for these negtive interctive effects of lctte, plmitte nd glycerol on muscle glycogen metolism is elusive. The results from this study hve een very useful in dissecting out the potentil roles nd interctions of vrious extrcellulr sustrtes in fueling trout muscle glycogenesis, however, there is fundmentl difference etween these in vitro nd in vivo oservtions tht deserves mention. Nmely, glycogenesis is more rpid in vitro [up to 1 mol g 1 within 1 h post-exercise (Wng et l., 1997; Frolow nd Millign, 24)] suggesting tht mitigting fctors exist in vivo tht ct to retrd metolic recovery in muscle. Hormones, in prticulr cortisol, re sent in theses in vitro preprtions. In vivo, the elevtion of plsm cortisol levels, typiclly seen following exhustive exercise, ppers to e inhiitory to glycogenesis (Millign, 23) s no net synthesis of glycogen is seen until cortisol levels egin to decline (Pgnott et l., 1994). Similr trends were oserved in vitro in muscle slice preprtions (Frolow nd Millign, 24), though the nture of cortisol s effects re uncler. 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