Repeated bouts of high-intensity exercise and muscle glycogen sparing in the rat

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1 The Journl of Experimentl Biology 26, The Compny of Biologists Ltd doi:1.1242/je Repeted outs of high-intensity exercise nd muscle glycogen spring in the rt Ghzl Rj 1, Lmert Bräu 1, T. Normn Plmer 2 nd Pul A. Fournier 1, * 1 School of Humn Movement nd Exercise Science, The University of Western Austrli, Crwley, Western Austrli 69, Austrli nd 2 Deprtment of Moleculr Biology, Jmes Cook University, Townsville, Queenslnd 4811, Austrli *Author for correspondence (e-mil: fournier@cyllene.uw.edu.u) Accepted 28 Mrch 23 Even in the sence of food intke, severl niml species recovering from physicl ctivity of high intensity cn replenish completely their muscle glycogen stores. In some species of mmmls, such s in rts nd humns, glycogen repletion is only prtil, thus suggesting tht few consecutive outs of high-intensity exercise might eventully led to the sustined depletion of their muscle glycogen. In order to test this prediction, groups of rts with led weight of 1% ody mss ttched to their tils were sujected to either one, two or three outs of high-intensity swimming, ech out eing seprted from the next y 1 h recovery period. Although glycogen repletion fter the first out of exercise ws only prtil, ll the glycogen moilised in susequent outs ws completely replenished during the corresponding recovery Summry periods nd irrespective of muscle fire compositions. The impct of repeted outs of high-intensity exercise on plsm levels of ftty cids, cetocette nd β- hydroxyutyrte suggests tht the metolic stte of the rt prior to the second nd third outs of exercise ws different from tht efore the first out. In conclusion, rts resemle other verterte species in tht without food intke there re conditions under which they cn replenish completely their muscle glycogen stores from endogenous cron sources when recovering from highintensity exercise. It remins to e estlished, however, whether this cpcity is typicl of mmmls in generl. Key words: exercise, fsting, glycogen, lctte, skeletl muscle, recovery, rt. Introduction Over three decdes hve elpsed since the demonstrtion tht muscle glycogen is one of the mjor fuels moilised to support muscle energy demnds during physicl ctivity, not only of high ut lso of moderte intensity (Ivy, 1991). The importnce of this fuel is est illustrted y severl studies tht hve reported tht the depletion of muscle glycogen stores dversely ffects exercise performnce of moderte or high intensity (Ivy, 1991; Blsom et l., 1999). Such fll in n niml s cpcity to engge in high-intensity physicl ctivity due to low glycogen stores cn hve severe consequences s it my impir n niml s ility to survive under conditions eliciting fight-or-flight responses. Fortuntely, even in the sence of food intke, muscles recovering from physicl ctivity hve the cpcity to replenish some or even ll of their glycogen stores (reviewed in Fournier et l., 22). In prticulr, during recovery from mximl sprint effort, muscles replenish t lest prt of their glycogen stores. Under these conditions, lctte is the most likely cron source for glycogen synthesis, either directly or indirectly vi its conversion to glucose (Gleeson, 1996; Plmer nd Fournier, 1997; Fournier et l., 22). This synthesis of muscle glycogen from endogenous cron sources hs een demonstrted not only in humns nd rts (Fournier et l., 22) ut lso in vriety of other nimls including fish (Millign nd Wood, 1986; Pgnott nd Millign, 1991; Girrd nd Millign, 1992; Scrello et l., 1992; Millign, 1996), mphiins (Fournier nd Guderley, 1992), snkes (Grtz nd Hutchison, 1977) nd lizrds (Gleeson, 1982, 1996; Gleeson nd Dlessio, 1989). Although it is generlly the cse tht nimls recovering from high-intensity physicl ctivity cn replenish their muscle glycogen stores in the sence of food intke, it is importnt to note tht most lower vertertes (Grtz nd Hutchison, 1977; Gleeson, 1982, 1996; Millign nd Wood, 1986; Gleeson nd Dlessio, 1989; Pgnott nd Millign, 1991; Fournier nd Guderley, 1992; Girrd nd Millign, 1992; Scrello et l., 1992; Millign, 1996) nd some mmml species (Bräu et l., 1999) hve the cpcity to replenish their muscle glycogen stores completely under these conditions wheres the replenishment of muscle glycogen is only prtil in other species of mmmls, such s humns nd rts (Hermnsen nd Vge, 1977; Astrnd et l., 1986; Bngso et l., 1992, 1997; Choi et l., 1994; Nikolovski et l., 1996; Peters et l., 1996; Ferreir et l., 21). On this sis, one might propose tht in those nimls where the extent of glycogen repletion is only prtil post-exercise, few consecutive outs of high-intensity physicl ctivity might

2 216 G. Rj nd others eventully led to progressive decrese in the levels of muscle glycogen ttined fter ech consecutive recovery period. Unless mechnisms exist to protect muscle glycogen ginst sustined depletion, muscle glycogen would e expected to eventully ttin levels low enough to impir the ility of these nimls to engge in fight-or-flight ehviours (Blsom et l., 1999). Since the rt is one niml species where glycogen repletion post intense exercise is only prtil without food intke, the im of this study ws to ssess whether, s predicted, repeted consecutive outs of high-intensity exercise in the rt will eventully result in the sustined depletion of their stores of muscle glycogen or whether mechnisms exist for the spring of their muscle glycogen stores. Mterils nd methods Mterils Chemicls were purchsed from BDH (British Drug Houses Ltd, Poole, Dorset, UK) nd Sigm (St Louis, MO, USA). Biochemicls nd enzymes were otined from Boehringer Mnnheim (Sydney, NSW, Austrli). All chemicls were of nlyticl grde. Animls Adult mle lino Wistr rts (Rttus norvegicus Berkenhout; g) were otined from the Animl Resource Centre t the University of Murdoch, Western Austrli. Mle rts were used in preference to femles to void the physiologicl chnges ssocited with the oestrous cycle. The rts were kept t pproximtely 2 C on 12 h:12 h light:drk photoperiod nd hd unlimited ccess to wter nd stndrd lortory chow diet (Glen Forrest Stockfeeders, Glen Forrest, Western Austrli: 55% digestile crohydrte, 19% protein, 5% lipid nd 21% non-digestile residue y mss). Before experiments, the rts were fsted for 24 h to deplete most of their stores of liver glycogen (Ferreir et l., 21) so s to prevent heptic glycogen nd food present in the gut from providing cron precursors for the replenishment of muscle glycogen post-exercise. All experiments took plce etween 8. h nd 13. h. This reserch project ws pproved y the Animl Ethics Committee of the University of Western Austrli. Exercise protocol As rts re nturl swimmers, n exercise protocol sed on swimming ws dopted for this study, the intensity of the exercise eing similr for ech exercise out nd determined y the mount of led weight ttched to the se of the til (Ferreir et l., 21). The dvntge of this exercise protocol over one tht uses tredmill is tht prolonged trining period is not required for rts to exercise t ner-mximl intensity nd this protocol results in reproducile glycogen moilistion nd lctte ccumultion (Nikolovski et l., 1996; Ferreir et l., 21). Immeditely efore swimming, ech rt ws weighed nd led weight equivlent to 1% ody mss ws ttched to the se of its til. Ech rt swm for 3 min in plstic tnk (3 cm dimeter, 48 cm depth) filled with wter t 34 C s descried previously (Ferreir et l., 21). With the exception of one group of non-exercised rts, which served s the control group, ll nimls were sujected to either one, two or three outs of exercise, ech out eing seprted from the next one y recovery period of 6 min. The nimls were scrificed either immeditely following ech out of exercise or fter ech of the ssocited 6 min recovery periods during which ech rt recovered lone in cge without ccess to food. Other nimls were sujected to only one out of highintensity exercise nd were llowed to recover individully in seprted cges for either, 1, 2, 4, 6 or 12 min prior to eing scrificed. Tissue smpling The study exmined different muscles selected on the sis of their fire compositions. The white, red nd mixed gstrocnemius muscles were selected ecuse they re ctively recruited during high-intensity swimming nd re rich in fst-twitch white, fst-twitch red nd comintion of oth fire types, respectively, ut re poor in slow-twitch red fires, thus reflecting the composition of the hindlim musculture s whole (Mltin et l., 1989). By contrst, the soleus muscle, which is rich in slow-twitch red fire, ws chosen on the sis tht its glycogen stores re not recruited during highintensity swimming (Nikolovski et l., 1996; Ferreir et l., 21). Rts t rest or t time intervls during the post-exercise recovery period were nesthetised under hlothne s descried previously (Ferreir et l., 1998), nd their tissues, nmely individul muscles (soleus muscle nd red, white nd mixed gstrocnemius muscles), lood nd liver, were smpled. After removl, ech tissue ws immeditely freeze-clmped etween luminium pltes pre-cooled in liquid nitrogen, then wrpped in luminium foil nd stored t 8 C. Blood ws smpled from nesthetised rts y crdic puncture nd processed s descried elow. Extrction of lood nd tissue metolites Immeditely following its removl from the hert, lood ws trnsferred into heprinised Eppendorf microcentrifuge tue nd centrifuged immeditely t 72 g for 5 min. Following centrifugtion, 1 µl of the plsm ws deproteinised in 9 µl of 6% (w/v) perchloric cid nd centrifuged t 2 g for 1 min, while the remining plsm ws stored t 8 C. After centrifugtion, the superntnt ws neutrlised with 2 mol l 1 K 2 CO 3 nd centrifuged t 2 g for 1 min. All smples were kept t 8 C until nlysed. Muscles were weighed nd ground under liquid nitrogen (Lehoux nd Fournier, 1999), nd the powdered tissue ws homogenised with 1 volumes of ice-cold 6% perchloric cid. A portion of the homogente ws used for the determintion of glycogen, while 7 µl liquot ws centrifuged t 2 g for 1 min nd the superntnt removed nd kept on ice. The pellet ws re-extrcted with 35 µl of 6% perchloric cid

3 Glycogen spring post-exercise in rts 2161 efore re-centrifugtion t 2 g for 1 min. Following centrifugtion, the two superntnts were comined, neutrlised with 2 mol l 1 K 2 CO 3 nd centrifuged efore eing stored t 8 C until nlysis of metolites. The levels of glycogen, lctte, glucose, glucose 6-phosphte, glycerol, β- hydroxyutyrte nd cetocette were ssyed s descried y Bergmeyer (1974), nd ftty cid levels were determined using the Wko NEFA C Kit (Wko Pure Chemicls Industries, Osk, Jpn). Expression of results nd sttisticl nlyses All metolite concentrtions in tissues nd plsm re expressed in µmol g 1 wet mss nd mmol l 1, respectively, nd results re expressed s mens ± S.E.M. (N=8 12). The effects of exercise nd post-exercise recovery on the levels of metolites in muscles nd plsm were nlysed with onefctor nlysis of vrince (ANOVA) followed y Fisher protected lest significnt difference posteriori test using Stt View SE + Grphics version 1.3 (Acus Concepts, Berkeley, CA, USA). Results Effect of repeted outs of high-intensity exercise nd recovery on muscle nd liver glycogen levels Repeted outs of high-intensity exercise, ech seprted y 1 h recovery period, resulted in significnt rekdown of glycogen in the red, white nd mixed gstrocnemius muscles (Fig. 1). The extent of glycogen rekdown in response to the first exercise out ws higher thn in response to the susequent outs in these muscles. Irrespective of the muscle exmined, however, glycogen levels reched similr levels in response to ech out of exercise. During the 1 h recovery period following ech out of exercise, significnt replenishment of muscle glycogen took plce in the red, white nd mixed gstrocnemius muscles. During recovery from the first out of exercise, muscle glycogen returned to lower thn pre-exercise levels in these muscles. By contrst, during recovery from the susequent outs of exercise, these muscles replenished their stores of glycogen completely nd thus the post-recovery glycogen levels ttined fter the second nd third recovery periods were not significntly different from those ttined fter the first recovery period. A distinct pttern of glycogen metolism ws oserved in the soleus muscle, with glycogen levels remining reltively stle, lthough cumultive effect of consecutive exercise/recovery episodes resulted in n eventul fll in glycogen levels (Fig. 1). In response to the first sprint, heptic glycogen levels decresed from 15.1±2.6 µmol glucosyl units g 1 wet mss to 3.2±1. µmol glucosyl units g 1 wet mss, nd no further chnges occurred in response to the susequent outs of exercise nd ssocited recovery periods. Glycogen (µmol glucosyl units g 1 wet mss) Mixed gstrocnemius White gstrocnemius Red gstrocnemius Soleus Rest E1 Rec1 E2 Rec2 E3 Rec3 Rest E1 Rec1 E2 Rec2 E3 Rec3 Exercise conditions Effect of repeted outs of highintensity exercise nd recovery on lctte levels in muscle Repeted outs of high-intensity exercise, ech seprted y 1 h recovery period, resulted in significnt increse in muscle lctte levels (Fig. 2). The extent of lctte ccumultion in the soleus muscle nd red, white nd mixed gstrocnemius muscles ws highest in response to the first out of exercise. During the recovery period following ech out of exercise, Fig. 1. The effect of repeted highintensity exercise nd recovery on glycogen levels in the soleus nd white, red nd mixed gstrocnemius muscles. On the x-xis, E refers to exercise nd Rec to recovery. The vlues shown represent mens ± S.E.M. (N=12). Identicl superscripts on different columns indicte the sence of significnt differences, wheres columns without superscript differ significntly from ny column ering superscript (ANOVA followed y Fisher PLSD posteriori test; P<.5).

4 2162 G. Rj nd others lctte levels returned to pre-exercise levels in ll muscles exmined. Effect of repeted outs of high-intensity exercise on plsm metolite levels Ech out of high-intensity exercise resulted in significnt increse in the levels of plsm lctte (Fig. 3). By contrst, the levels of plsm glucose (Fig. 3) nd glycerol (Fig. 4) were not significntly ffected y the first out of exercise, ut glucose levels decresed in response to oth the second nd third exercise outs (Fig. 3). With respect to the levels of plsm ftty cids, cetocette nd β-hydroxyutyrte, distinct pttern of chnges took plce in response to ech out of exercise nd ensuing recovery period. Ech out of exercise resulted in significnt decrese in the levels of plsm ftty cids, cetocette nd β-hydroxyutyrte (Fig. 4). During ech of the recovery periods, these metolites returned to levels comprle or higher thn those efore exercise, with their concentrtions fter the third exercise out eing higher thn sl pre-exercise levels. In prticulr, the levels of β- hydroxyutyrte reched fter ech period of recovery showed grdul increse to levels tht, fter the third recovery period, were well ove those fter the first recovery period (Fig. 4). In response to single out of exercise, glycerol remined t stle levels, wheres the levels of plsm free ftty cids, Lctte (µmol g 1 wet mss) Mixed gstrocnemius Red gstrocnemius Rest E1 Rec1 E2 Rec2 E3 Rec3 Exercise conditions cetocette nd β-hydroxyutyrte decresed significntly (Fig. 5). During recovery, the levels of glycerol remined unchnged while those of ftty cids, cetocette nd β- hydroxyutyrte incresed progressively (Fig. 5). Discussion It is generlly cknowledged tht fsted nimls recovering from physicl ctivity of ner-mximl intensity cn replenish their muscle glycogen stores even in the sence of food intke. In some mmml species, such s in rts nd humns, the extent of this replenishment is only prtil (Hermnsen nd Vge, 1977; Astrnd et l., 1986; Choi et l., 1994; Nikolovski et l., 1996; Peters et l., 1996; Bngso et l., 1997; Ferreir et l., 21; Fournier et l., 22), thus suggesting tht few consecutive outs of high-intensity exercise might eventully led to the progressive depletion of their muscle glycogen stores. In order to test this prediction, groups of rts were sujected to series of three outs of highintensity swims to exhustion, ech seprted from the susequent one y recovery period previously shown to e long enough for muscle glycogen nd lctte to return to stle levels (Ferreir et l., 21). This study shows for the first time tht repeted outs of high-intensity exercise do not led to the eventul sustined depletion of the stores of muscle glycogen in fsted rts, irrespective of whether muscles re rich in fst-twitch red or white fires (Fig. 1). Indeed, lthough glycogen repletion fter one out of exhustive exercise is only prtil, ll the glycogen moilised in response to the susequent exercise outs is completely replenished during the respective recovery periods, nd the levels of muscle glycogen ttined fter recovery from one sprint do not differ from those ttined fter recovery from severl sprints (Fig. 1). Soleus On the sis of our findings, one must mend the view tht rts White gstrocnemius Rest E1 Rec1 E2 Rec2 E3 Rec3 Fig. 2. The effect of repeted highintensity exercise nd recovery on lctte levels in the soleus nd white, red nd mixed gstrocnemius muscles. On the x-xis, E refers to exercise nd Rec to recovery. The vlues shown represent mens ± S.E.M. (N=12). Identicl superscripts on different columns indicte the sence of significnt differences, wheres columns without superscript differ significntly from ny column ering superscript (ANOVA followed y Fisher PLSD posteriori test; P<.5).

5 Glycogen spring post-exercise in rts Lctte Glucose Plsm metolites (mmol l 1 ) Rest E1 Rec1 E2 Rec2 E3 Rec3 Rest E1 Rec1 E2 Rec2 E3 Rec3 Exercise conditions Fig. 3. The effect of repeted high-intensity exercise nd recovery on glucose nd lctte levels in the plsm. On the x-xis, E refers to exercise nd Rec to recovery. The vlues shown represent mens ± S.E.M. (N=8). Identicl superscripts on different columns indicte the sence of significnt differences, wheres columns without superscript differ significntly from ny column ering superscript (ANOVA followed y Fisher PLSD posteriori test; P<.5). recovering from high-intensity exercise differ from most species of lower vertertes in tht, in the sence of food intke, they re incple of replenishing completely their glycogen stores. Here, we show tht there re conditions where the glycogen moilised in response to sprint is completely replenished fter exercise in fsted rts (Fig. 1). Furthermore, the oservtion tht repeted sprints fil to cuse progressive fll in the levels of muscle glycogen ttined fter ech Plsm metolites (mmol l 1 ) Ftty cids β-hydroxyutyrte Glycerol Rest E1 Rec1 E2 Rec2 E3 Rec3 Rest E1 Rec1 E2 Rec2 E3 Rec3 Exercise conditions Acetocette Fig. 4. The effect of repeted high-intensity exercise nd recovery on plsm ftty cids, glycerol, β-hydroxyutyrte nd cetocette levels. On the x-xis, E refers to exercise nd Rec to recovery. The vlues shown represent mens ± S.E.M. (N=8). Identicl superscripts on different columns indicte the sence of significnt differences, wheres columns without superscript differ significntly etween ech other nd from ny column ering superscript (ANOVA followed y Fisher PLSD posteriori test; P<.5). c consecutive recovery period might e tken s evidence tht there is criticl mount of muscle glycogen in the rt tht is protected ginst sustined depletion. In order to support further this interprettion, other studies would e required to show tht glycogen levels post-exercise re protected irrespective of the type, intensity nd durtion of exercise nd vilility of endogenous cron sources. Fortuntely, one such study hs een performed in which fsted Wistr rts were forced to engge for nerly 2.5 h in continuous or intermittent eroic exercise in order to deplete eroiclly most of their muscle glycogen stores (Gesser nd Brooks, 198). This study reported tht during recovery, the stores of muscle glycogen incresed to levels (17.6 µmol g 1 ) comprle with those oserved in the present study in response to either one or severl 3-min sprints, ut with the difference tht endogenous cron sources other thn lctte were involved since these eroic exercise protocols resulted only in mrginl increse in lood lctte level (Gesser nd Brooks, 198). Overll, the oservtions tht in fsted rts muscle glycogen levels return to comprle levels in response to rnge of highly different exercise protocols, such s single 3-min sprint, multiple 3- min sprints, prolonged continuous eroic exercise or prolonged intermittent eroic exercise, provide strong support for the existence in skeletl muscles of set glycogen levels tht re protected ginst c sustined depletion post-exercise. It is interesting to note tht the levels t which muscle glycogen levels re protected ginst sustined depletion in the rt re high enough to support little more thn one out of n intense sprint effort to exhustion. Normlly, during sprint to exhustion, it is the ccumultion of H + nd inorgnic phosphte ions rther thn the depletion of muscle glycogen tht cuses ftigue (Fitts nd Metzger, 1993). However, if muscle glycogen stores were to e protected t much lower levels, the limited supply of glycogen would ecome the min fctor

6 2164 G. Rj nd others limiting n niml s cpcity to engge in n intense sprint to exhustion y cusing premture ftigue (Blsom et l., 1999). Although, under most conditions, sprint would e expected to lst only few seconds, there re extreme conditions ssocited with fight-or-flight ehviour where n niml might hve to engge in sprint to ner exhustion. For these nimls, it would e highly dvntgeous to mintin their muscle glycogen stores t levels high enough so tht the uildup of H + nd inorgnic phosphte ions, rther thn the size of their glycogen stores, limits their cpcity to engge in n intense sprint effort to exhustion. In this regrd, it is noteworthy tht fter sprint most niml species in the fsted stte generlly replenish their muscle glycogen stores to levels such tht they cn engge in t lest one out of intense sprint to exhustion without eing limited y the size of their glycogen stores (Hermnsen nd Vge, 1977; Grtz nd Hutchison, 1977; Gleeson, 1982; Gleeson nd Dlessio, 1989; Astrnd et l., 1986; Millign nd Wood, 1986; Scrello et l., 1992; Fournier nd Guderley, 1993; Girrd nd Millign, 1992; Choi et l., 1994; Millign 1996; Bngso et l., 1997). In light of the ove discussion, the oservtion tht Plsm metolites (mmol l 1 ) , Free ftty cids β-hydroxyutyrte,c c Rest Rest Time of recovery (min),c Glycerol Acetocette Fig. 5. The effect of high-intensity exercise nd recovery on plsm ftty cids, glycerol, β-hydroxyutyrte nd cetocette levels. The vlues shown represent mens ± S.E.M. (N=8). Identicl superscripts on different dt points indicte the sence of significnt differences, wheres dt points without superscript differ significntly etween ech other nd from ny dt point ering superscript (ANOVA followed y Fisher PLSD posteriori test; P<.5). c,c glycogen repletion in the rt is only prtil fter single out of exercise (Fig. 1) my e explined on the sis tht muscle glycogen levels in 24 h-fsted rts re higher thn the miniml criticl levels normlly protected in this species. Along this line of resoning, the oservtion tht in severl verterte species the stores of muscle glycogen re completely replenished fter single out of intense exercise (Grtz nd Hutchison, 1977; Gleeson, 1982, 1996; Millign nd Wood, 1986; Gleeson nd Dlessio, 1989; Pgnott nd Millign, 1991; Fournier nd Guderley, 1992; Girrd nd Millign, 1992; Scrello et l., 1992; Millign, 1996; Bräu et l., 1999) might e explined on the sis tht their muscle glycogen stores re kept t their species-specific protected levels. This rises the novel question of whether the muscle glycogen stores in these niml species would lso e only prtilly replenished post high-intensity exercise if one were to mnipulte their glycogen stores so tht more thn protected glycogen levels were to e stored in their muscles prior to exercise. This is n issue tht remins to e investigted. The cpcity of fsted rts to replenish their muscle glycogen stores etween ech consecutive out of exercise rises the question of the identity of the cron sources moilised for the synthesis of glycogen. Since, in the present study, the nimls were in fsted stte, the synthesis of muscle glycogen hd to depend solely on endogenous sustrtes. The lctte uilt up in response to exercise is likely cndidte (Fig. 3), s it is generlly cknowledged s one of the mjor cron sources for the synthesis of muscle glycogen in mny verterte species (Gleeson, 1996; Plmer nd Fournier, 1997; Fournier et l., 22). Tht lctte is lso likely to e mjor cron source for the replenishment of muscle glycogen during recovery from ech out of exercise in rts is suggested indirectly y the oservtion tht the fll in plsm nd muscle lctte levels is temporlly linked with glycogen synthesis. In this respect, the chnges in lctte levels in the soleus muscles my seem t first surprising, considering the sence of net glycogen rekdown nd synthesis in this muscle, ut, s rgued efore (Bräu et l., 1997; Ferreir et l., 21), it is more thn likely tht the exercise-medited rise nd susequent fll in lctte levels in this muscle result from n exchnge of lctte etween the soleus muscle nd the lood. The stores of heptic glycogen re unlikely to provide net source of glucose for the resynthesis of muscle glycogen stores, since liver glycogen remins t stle levels in response to severl sprints nd recovery periods. It is importnt to stress tht mino cids nd glycerol vi their conversion to glucose y the liver could

7 Glycogen spring post-exercise in rts 2165 contriute to the replenishment of muscle glycogen (Gesser nd Brooks, 198; Fournier et l., 22), ut their reltive contriutions, s well s tht of lctte, remin to e estlished. The mechnisms y which the proportion of muscle glycogen replenished post-exercise differs etween the first nd susequent outs of exercise remin to e elucidted. Any ttempt t explining this finding must tke into considertion the oservtion tht the solute mount of glycogen deposited during recovery from the first exercise out is not different from tht fter ech susequent out (Fig. 1). It is ecuse more glycogen is moilised during the first exercise out thn during the following outs, mye ecuse of higher work output, tht the proportion of muscle glycogen replenished fter the first exercise out is lower thn fter the other two exercise outs. It is interesting to note tht such lower extent of glycogen moilistion nd lctte ccumultion in response to the second nd third outs of high-intensity exercise is not shred y ll nimls species, since, in the rinow trout (Oncorhynchus mykiss), for instnce, the extent of glycogen moilistion nd lctte ccumultion does not differ etween consecutive exercise outs (Scrello et l., 1992). Considering tht in the rt more lctte is eliminted during recovery from the first exercise out thn during susequent outs (Figs 2, 3), with equivlent mounts of muscle glycogen eing replenished, lower proportion of lctte is likely to e converted into glycogen during recovery from the first out of exercise. This interprettion holds s long s lctte is the min cron source for glycogen synthesis, nd this might prtly explin the prtil replenishment of muscle glycogen stores in response to single out of exercise. The differences in the pttern of glycogen repletion etween the first nd susequent outs of exercise rise the question of whether other spects of fuel metolism differ mong these outs of exercise. We hve ddressed this question indirectly y exmining the effects of single out s well s tht of repeted outs of high-intensity exercise on plsm levels of glycerol, free ftty cids, β-hydroxyutyrte nd cetocette. As reported previously y others in humns nd rts (Drury et l., 1941; Blsse et l., 1978; Romijn et l., 1993), the levels of plsm ftty cids nd ketone odies decresed significntly from rest levels in response to single out of high-intensity exercise nd incresed throughout recovery to ttin levels comprle with or higher thn those mesured efore exercise (Figs 4, 5). The impct of repeted outs of high-intensity exercise on the plsm levels of ftty cids, cetocette nd β-hydroxyutyrte suggests tht the metolic stte of the rt prior to the second nd third outs of exercise ws different from tht efore the first out (Fig. 4). Indeed, the levels of plsm ftty cids nd ketone odies ttined fter ech of the recovery periods were higher thn those prior to the first exercise out (Fig. 4). Since the rtes of utilistion of ftty cids nd ketone odies y muscle re prtly determined y their concentrtions (Newsholme nd Leech, 1983), the higher levels of ftty cids nd ketone odies prior to the second nd third outs of high-intensity exercise, s well s during recovery from these outs, would e expected to enhnce their oxidtion y muscles. As result, this would e predicted to provide muscles nd other orgns with n incresed supply of fuels other thn glucose nd lctte to support their energy demnds, prticulrly during recovery from exercise. Assuming lctte is the mjor cron source for glycogen synthesis, this lesser oxidtion of lctte nd of glucose derived from lctte might llow for n incresed proportion of lctte converted into muscle glycogen fter the second nd third outs of exercise. This might lso explin the higher proportion of glycogen replenished during recovery from these exercise outs. Irrespective of whether this explntion holds, our findings clerly suggest tht severl spects of fuel metolism differ etween the first nd susequent outs of exercise nd tht this must e tken into considertion when ttempting to explin the incresed spring of muscle glycogen in response to severl outs of exercise. It is not cler, however, the extent to which these differences might prtly result from possile differences in work output etween the first nd susequent exercise outs. Moreover, different hormonl responses might lso exist etween the first nd susequent exercise outs nd explin lso, t lest in prt, the differences in the pttern of glycogen metolism etween consecutive sprints. In conclusion, our results corroorte erlier findings tht glycogen repletion is only prtil in fsted rts recovering from single out of high-intensity exercise. However, this study indictes tht mechnisms exist to ensure tht muscles from fsted rts cn replenish completely their stores of glycogen if sujected to more thn one out of high-intensity exercise. Rts, therefore, resemle mny other verterte species in tht without food intke they cn lso protect their muscle glycogen ginst sustined depletion y replenishing completely their glycogen stores post-exercise to support the energy demnds ssocited with fight-or-flight ehviour. 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