Comparative Biochemistry and Physiology Part A 135(2003) 347–356

1095-6433/03/$ - see front matter� 2003 Elsevier Science Inc. All rights reserved.PII: S1095-6433Ž03.00089-8

Metabolic priorities during starvation: enzyme sparing in liver andwhite muscle of Atlantic cod,Gadus morhua L.

Helga Guderley *, Dominique Lapointe , Martin Bedard , Jean-Denis Dutila, a a b´

Departement de biologie, Universite Laval, Quebec, Que., Canada G1K 7P4a ´ ´Institut Maurice Lamontagne, Peches et Oceans, Mont Joli, Quebec, Canadab ˆ ´ ´

Received 16 December 2002; received in revised form 18 March 2003; accepted 19 March 2003

Abstract

Atlantic cod,Gadus morhua, respond to starvation first by mobilising hepatic lipids, then muscle and hepatic glycogenand finally muscle proteins. The dual role of proteins as functional elements and energetic reserves should lead to atemporal hierarchy of mobilisation where the nature of a function dictates its conservation during starvation. Weexamined(1) whether lysosomal and anti-oxidant enzymes in liver and white muscle are spared during prolongedstarvation,(2) whether the responses of these enzymes in muscle vary longitudinally. Hepatic contents of lysosomalproteases decreased with starvation, whereas those of catalase(CAT) increased and lysosomal enzymes of carbohydratemetabolism and glutathioneS-transferase(GST) did not change. In white muscle, starvation decreased the specificactivity of lysosomal enzymes of carbohydrate degradation and doubled that of cathepsin D(CaD). The activity of anti-oxidant enzymes and acid phosphatase in muscle was unchanged with starvation. In white muscle neither lysosomalenzymes nor anti-oxidant enzymes varied significantly with sampling position. In cod muscle, antioxidant enzymes, CaDand acid phosphatase are spared during a period of starvation that decreases lysosomal enzymes of carbohydratemetabolism and decreases glycolytic enzyme activities. In cod liver, the anti-oxidant enzymes, CAT and GST, were alsospared during starvation.� 2003 Elsevier Science Inc. All rights reserved.

Keywords: Antioxidant; Cod; Lysosome; Liver; Muscle; Reactive oxygen species; Starvation

1. Introduction

Periods of reduced food availability are commonin the life cycles of temperate zone fishes and leadto considerable fluctuations in energetic status. Forexample, Atlantic cod(Gadus morhua) in thenorth-western Atlantic can encounter prolongedperiods with reduced food availability. In responseto sustained decreases in food availability, fishmay decrease locomotor and metabolic activities(Wieser, 1991; Mendez and Wieser, 1993), modify´

*Corresponding author. Tel.:q1-418-656-3184; fax:q1-418-656-2043.

E-mail address:helga.guderley@bio.ulaval.ca(H. Guderley).

tissue metabolic capacities(Guderley et al., 1996)and degrade muscle protein(Black and Love,1986). Simple condition indices such as Fulton’scondition factor (K), liver somatic index, andmoisture content of muscle and liver change mark-edly with starvation of cod(Lambert and Dutil,1997). Periods of reduced food availability leadcod to mobilise their reserves in a specific order:hepatic lipids are the first to be used, followed byglycogen from liver and white muscle(Black andLove, 1986). Mobilisation of macromolecularcomponents requires the participation of lysosomalenzymes, indicating that they should be sparedduring mobilisation of tissue proteins. Some ofthese enzymes degrade carbohydrates, primarily

348 H. Guderley et al. / Comparative Biochemistry and Physiology Part A 135 (2003) 347–356

glycogen(neutrala-glucosidase(NAG) and acida-glucosidase(AAG), acid phosphatase andb-N-acetyl-D-glucosaminidase(NAc)), while othersbreak down protein(cathepsin D (CaD) andcathepsin L (CaL)). This process leads to anaccumulation of intracellular solutes that can retainintracellular water, in liver as well as in muscle.

The regional heterogeneity of muscle metaboliccapacities in fish is becoming increasingly wellestablished(Leonard, 1999; Martınez et al., 2000,´2003). This heterogeneity may be at least partlybased in regional differences in the contribution ofmuscle during swimming. Phase differencesbetween caudally travelling waves of muscle acti-vation and fish bending cause a change in musclefunction along the body(Altringham et al., 1993).In cod, most of the power required for swimmingis produced in the rostral and central portions ofthe musculature, and is transmitted to the caudalfin (Altringham et al., 1993; Davies et al., 1995;Johnston et al., 1995). This, together with thedecrease in the relative abundance of muscle asone nears the tail may explain longitudinal changesin muscle biochemical properties. Effectively, cau-dal sections of cod white muscle have markedlyhigher levels of glycolytic and mitochondrialenzymes than the central regions that in turn havehigher activities than rostral samples(Martınez et´al., 2000). This longitudinal pattern is most appar-ent in fed fish, with starvation reducing the longi-tudinal differences of glycolytic enzymes(Martınez et al., 2003). A longitudinal heteroge-´neity may be observed in the activity of lysosomalenzymes, given that the need to renew proteins islikely to reflect the metabolic intensity of thefibres.

Cod, as all aerobic organisms, require defencemechanisms protecting against the effects of reac-tive oxygen species(ROS) generated during met-abolic activity. ROS are highly reactiveintermediates, resulting from univalent reductionsof O . The ROS include the superoxide anion2

(O ), hydrogen peroxide(H O ) and hydroxyly2 2 2

radicals(OH ). These intermediates can oxidisey

most cellular components and markedly affect thephysiology of the cell, even leading to mutagenesisor cellular death(Cadenas, 1995). However, anti-oxidants and specialised enzymes, including cata-lase (CAT), glutathione peroxidase(GPOX),glutathione S-transferase(GST) and superoxidedismutase, can neutralise these ROS. CAT is aperoxisomal and mitochondrial enzyme that breaks

down hydrogen peroxide. In the cytosol, the break-down of hydrogen peroxide is largely carried outby GPOX, an enzyme that also occurs in mito-chondria and endoplasmic reticulum. GST conju-gates reduced glutathione to xenobiotics or cellularcomponents damaged by ROS(Storey, 1996).Superoxide dismutase which catabolises super-oxide radicals, is both mitochondrial and cytosolic.The activities of superoxide dismutase and GPOXare greater in organisms with higher aerobic capac-ities (Janssens et al., 2000), suggesting that theseactivities are adjusted according to endogenousrates of ROS production. Thus we predict that, asthe generation of ROS is likely to be most activein regions with high mitochondrial enzyme activ-ities, the activity of anti-oxidant enzymes wouldbe highest in caudal muscle samples. Given theimportance of anti-oxidant systems, we predictthey be spared during generalised protein break-down occurring with advanced starvation.

This paper reports measurements of the activitiesof lysosomal and anti-oxidant enzymes in liverand white muscle of fed and starved cod. Wemeasured NAG, AAG, CaD, CaL, NAc, CAT,GPOX and GST. White muscle was sampled dor-sally, in three longitudinal positions: behind thehead(rostral), in the middle of the body and atthe level of the caudal peduncle to assess thelongitudinal variation in capacities.

2. Material and methods

2.1. Feeding and maintenance of cod

Atlantic cod (Gadus morhua) were captured inthe St. Lawrence Estuary near Matane, during thespring of 1998. Initially, the fish were kept in 13m tanks at the Maurice-Lamontagne Institute.3

These tanks were kept at ambient seawater tem-perature(5.3"0.8 8C), salinity (26.9"0.8‰) andthe natural day length at our latitude. During thisperiod, fish received a maintenance ration of cap-elin (Mallosus villosus). The maintenance rationwas calculated as described by Jobling(1988). Weselected 20 healthy cod from this wild stock,lightly anaesthetised them with methomidate at aconcentration of 3 mg l in seawater and taggedy1

them with a visual implant tag(Northwest MarineTechnology, Shaw Island, Wash) in the first andsecond dorsal fins. We measured the cod for forklength and weighed them before randomly distrib-uting them into two circular 1.5 m tanks(ns103

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Table 1Characteristics of cod under different feeding treatments

Initial Final

Fed Starved Fed Starved

Cod mass(g) 1497"229A1 1554"205A1 1765"213B1 1051"200B2

Mass range(g) 1243–1992 1344–2057 1486–2229 831–1540Cod length(cm) 50.5"1.7A1 50.8"1.7A1 57.5"2.2B1 54.2"1.9B2

Length range(cm) 48.5–53.5 49.0–54.4 54.4–60.5 51.9–58.4Condition factor 0.94"0.09A1 0.96"0.06A1 0.93"0.07A1 0.65"0.06B2

Liver mass 110.0"25.71 10.2"5.82

Hepatosomatic index(%) 6.2"0.91 0.93"0.292

Growth rate in mass(%) 0.23"0.171 y0.39"0.062

Growth rate in length(%) 0.16"0.021 0.08"0.012

Note: Each feeding treatment lasted 12 weeks at 9.9"0.4 8C. Data are shown as mean"standard deviation. Statistical differencesdue to feeding treatment(initial vs. final values) are shown by letters after values in a same line; when values do not differ, they havethe same letter(ANOVA, add contrasts,P-0.05). Statistical differences between groups at the start or the end of the experiment areshown by numbers after values in a same line; when values do not differ, they have the same number(ANOVA, add contrasts,P-0.05). ns10 in each feeding treatment. Condition factorstotal mass=length .y3

each). The fish were gradually brought to theexperimental temperature(9.9"0.4 8C), again atambient salinity (27.5"1.3‰) and natural daylengths. The fish were given 2 weeks of feedingat the maintenance ration to recover from the stressof the tagging. Then, during 12 weeks, one groupwas fed to satiety three times per week withcapelin and the other was starved.

2.2. Tissue sampling

The cod were killed by a blow to the head andtransection of the spinal cord, measured andweighed. Samples(2–5 g) of white muscle weretaken at three sites well above the lateral line, toavoid contamination by pink and red fibres. Thefirst sample was taken behind the head, the secondin the middle of the body and the last at the caudalpeduncle. The liver was dissected and weighed.Samples of white muscle and liver were immedi-ately frozen in liquid nitrogen and stored aty708C before transport on dry ice to Universite Laval,´where they were again stored aty70 8C untilenzyme activities were measured.

2.3. Sample preparation

Liver and white muscle samples were homogen-ised (1:10 (wyv)) with a Polytron homogeniser(Brinkman Instruments, Rexdale, Ontario) forthree 10 s periods(1-min pause between eachperiod) in 50 mM imidazole–HCl, 2 mM MgCl ,2

5 mM ethylene diamine tetra-acetic acid(EDTA),1 mM reduced glutathione(omitted in the GPOX

and GST assays), and 0.1% Triton X-100, pH 7.5.The samples were kept on ice during the entirehomogenisation process.

2.4. Enzyme activity assays

All assays were performed in duplicate and 1unit of enzyme activity represents 1mmol ofsubstrate converted to product per min. Lysosomalenzyme activities were measured using the methodof Peek and Gabbott(1990). Assay conditions foreach enzyme were optimised for liver and whitemuscle. Lysosomal hydrolases were measured byfluorimetry using specific 4-methylumbelliferyl(4-MeUm) substrates for each enzyme in 0.1 Msodium acetate buffer at pH 4.4 for acid phospha-tase, pH 5.7 for NAc and pH 4.5 for AAG. NAGwas measured in 0.1 M sodium cacodylate buffer,pH 7.5. Assays were performed at ambient tem-perature and were stopped 15 min after initiationby addition of 2 ml of glycine–NaOH 50 mM, pH10.4. 4-MeUm fluorescence was measured at anexcitation wavelength of 365 nm and an emissionwavelength of 460 nm(slit-width 5 nm). Theresults were compared to 4-MeUm standards. Weencountered difficulties measuring acid phospha-tase activity in liver from fed cod so the resultsare not shown for this enzyme.

Assays for CaL were performed as described byPeek and Gabbott(1990) with a peptide-bound 7-amino-4-methylcoumarin(7-AMC) substrate, 20mM Z-Phe–Arg–7AMCØHCl at pH 3.5. The bufferwas 300 mM sodium formate, 4 mM EDTA andactivator was 8 mM dithiothreitol. Stopping re-

350 H. Guderley et al. / Comparative Biochemistry and Physiology Part A 135 (2003) 347–356

agent was 100 mM sodium monochloroacetate–30mM sodium acetate–70 mM acetic acid, pH 4.3.7-AMC fluorescence was measured at an excitationwavelength of 380 nm and an emission wavelengthof 460 nm (slit-width 5 nm). The results werecompared to 7-AMC standards.

CaD was measured by spectrophotometry. Sam-ples were incubated at 108C for 3 h with 2 samplevolumes of a 2% solution of haemoglobin inacetate buffer, pH 3.5(1.35 M acetic acid and 20mM ammonium sulfate). The reaction was stoppedby the addition of 2 volumes of TCA 0.3 M. Thesamples were then allowed to sit for 30 min beforecentrifuging at 4000=g for 10 min. NaOH 0.5 Mand Folin-Ciocalteu reagent 33%(vyv) were add-ed to the supernatants before reading the absorb-ance at 750 nm. Values were compared to tyrosinestandards.

Antioxidant enzymes were assayed by spectro-photometry. All assays were performed at roomtemperature. The millimolar extinction coefficientsused for H O , NADH and chloro 2,4 dinitroben-2 2

zene (CDNB) were 0.0394, 6.22 and 10,respectively.

The breakdown of 750 mM H O by CAT in2 2

50 mM KPi (pH 7.0) was monitored at 240 nm.The reaction was initiated by addition of enzymeextract. Blanks were run in the absence of eitherH O or enzyme.2 2

GPOX (selenium-independent) activity wasmonitored by following the breakdown of 0.2 mMNADPH in 50 mM KPi pH 7.4, 1.5 mM EDTA,3 mM sodium azide, 10 mM GSH, 1 unit gluta-thione reductase and enzyme extract. The reactionwas initiated by addition of H O at a final2 2

concentration of 0.5 mM. Blanks were run inabsence of enzyme.

GST activity was measured by monitoring thereaction between 6 mM GSH and 1 mM 1 CDNBin a 50 mM KPi(pH 6.5) buffer containing 1 mMEDTA. The reaction was initiated by addition ofenzyme extract and monitored at 340 nm. Blankswere run in absence of GSH and enzyme.

2.5. Calculated parameters and statistical analysis

Length and total mass were used to calculate acondition factor(wtotal mass=length x=100) toy3

monitor the response of the cod to the feedingtreatment. The following equation was used todetermine the individual growth rates:

w zy1 x |Growth rate(% day )s M yM yM t 100,Ž .2 1 1Ž .y ~

where the initial mass in grams is represented byM , the final mass(g) is represented byM and1 2

the growing period in days is represented byt. Ananalogous calculation determined growth in length.Hepatosomatic index(HSI) was expressed as

HISs100(LM yM)

where LM represents liver mass andM representssomatic mass.

One-way ANOVAs were performed(JMP 3.2.1SAS Institute Inc.; Sall and Lehman, 1996) toexamine the effect of feeding treatment on liverenzyme activities. We used two way nested repeat-ed measures ANOVAs(JMP 3.2.1; SAS InstituteInc., Duxbury Press, USA) to examine the effectof inter-individual variation, site of sampling(withrepeated measures in individual fish) and feedingtreatment (feeding and starvation) on enzymeactivity. In this model, feeding treatment was afactor, site of sampling was nested within fish andfish was nested within feeding treatment. An inter-action term between feeding treatment and site ofsampling was included. Enzyme activities werevariables in separate ANOVAs. Homogeneity ofvariance and normality of data were verified beforethe application of statistical tests. Differences wereconsidered significant atAs0.05.

3. Results

3.1. Impact of the feeding treatment

Fish mass and length were significantly changedby the feeding treatment. Condition factor onlychanged significantly for starved cod(Table 1).Final mass, length, condition factor and growthrates differed significantly between the feedingtreatments(Table 1). Fed cod increased their massand length but did not change their conditionfactor. Starved cod decreased their mass and con-dition factor but nonetheless increased their length.The hepatic mass and HSI of fed cod were signif-icantly higher than those of starved cod(Table 1).

3.2. Hepatic enzyme activities

During starvation of cod, hepatic lipids are thefirst reserve to be used(Black and Love, 1986).

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In the absence of other changes, this reduction oflipid content would increase the specific activitiesof liver enzymes. Accordingly, the specific activi-ties of both the lysosomal and the anti-oxidantenzymes were significantly higher in starved thanin fed cod(one-way ANOVA) (Fig. 1).

While typically increasing enzyme specificactivities, starvation reduced the total hepatic con-tent of most of these enzymes(Fig. 2). Fed codhad significantly higher contents of the lysosomalenzymes NAG, CaD and CaL and the anti-oxidantenzyme GPOX. However, the contents of thelysosomal enzymes, AAG and NAc, and the anti-oxidant enzyme, GST, were not significantly dif-ferent between fed and starved cod. Finally, thehepatic content of CAT was higher in starved thanfed cod.

3.3. Muscle enzyme activities

Starvation can markedly increase the water con-tent in white muscle, which can reach 90% duringprolonged starvation(Lambert and Dutil, 1997).This augmentation is caused by a decrease in thecontent of macromolecules as well as an accumu-lation of intracellular solutes produced in part bylysosomal autolysis. These changes likely explainthe marked reduction of the specific activities ofmost lysosomal enzymes in white muscle withstarvation (Fig. 3), even though the lysosomalenzymes are required for the breakdown of muscletissue. However, the activities of CaD, the prote-olytic enzyme most required for mobilisation ofmuscle proteins, were higher in the muscle ofstarved than fed cod. Interestingly, the activities ofanti-oxidant enzymes changed little withstarvation.

We used repeated measures ANCOVAs to exam-ine the effect of interindividual variation, site ofsampling and starvation on white muscle enzy-matic activities. Interindividual variation was sig-nificant for all enzyme activities, but site ofsampling had no significant effect upon any ofthese enzyme activities. Starvation significantlyaffected the specific activities of NAG, AAG, CaDand NAc, but not those of ACP, CAT, GST andGPOX.

4. Discussion

In fish, morphological, physiological and bio-chemical characteristics are strongly influenced by

biotic and abiotic parameters such as food intakeand temperature(Greer-Walker, 1971; Kiessling etal., 1990; Wieser et al., 1992; Sanger, 1993;¨Guderley, 1998; Martınez et al., 2002). While´starvation typically decreases tissue metaboliccapacities, we show that the activities of certainlysosomal enzymes and antioxidant defences weremaintained or even enhanced after starvation. Thedifferences between the enzyme activities ofstarved and fed cod confirm that cod have specifichierarchies in the mobilisation of macromoleculesduring food deprivation.

4.1. Impact of feeding treatment on hepatic enzymeactivities

All the enzyme specific activities(U g wety1

mass) measured were significantly higher in theliver of starved than fed cod. This increase inspecific activities was most likely due to the drasticreduction of lipid contents as well as the mobilis-ation of glycogen and protein by controlled autol-ysis during starvation(Black and Love, 1986;Lambert and Dutil, 1997). Given the markedchanges in hepatic size and composition withstarvation(Lambert and Dutil, 1997), it is moreinstructive to examine the dynamics of the totalhepatic enzyme levels. Starvation of cod reducedthe total hepatic content of NAG, CaD, CaL andGPOX. On the other hand, the lack of change oreven increase of CAT, GST, NAc and AAG con-tents in the face of the drastic reduction of liversize strongly suggests these enzymes are beingspecifically maintained during the generalisedbreakdown of macromolecules. The tripling ofCAT contents and the maintenance of GST duringstarvation suggests that defences against ROSassume greater importance during periods of star-vation and energy reserve mobilisation. Approxi-mately 1–4% of consumed oxygen is converted tooxygen free radicals at the mitochondrial levelpresumably due to electron leaks(Turrens et al.,1982; Konstantinov et al., 1987; Jenkins, 1988;Sjodin et al., 1990; Tiidus and Houston, 1994;Storey, 1996). The mitochondrial and peroxisomallocation of CAT may facilitate a protective role,particularly during a period when the mobilisationof macromolecules may have rendered cellularstructures more susceptible to damage by ROS.

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Fig. 1. Impact of starvation and feeding upon the specific activity(U g wet mass) of lysosomal and antioxidant enzymes in liver ofy1

Atlantic cod. The enzymes measured were neutrala-glucosidase(NAG), acida-glucosidase(AAG), cathepsin D(CaD), cathepsin L(CaL), N-acetyl-glucosidase(Nac), catalase(CAT), glutathione peroxidase(GPOX) and glutathioneS-transferase(GST).

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Fig. 2. Impact of starvation upon the total hepatic contents(U) of lysosomal and antioxidant enzymes. Enzyme abbreviations are asgiven in the legend to Fig. 1.

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Fig. 3. Effect of starvation and feeding upon the specific activities(U g wet mass) of lysosomal and antioxidant enzymes in whitey1

skeletal muscle of Atlantic cod. Enzyme activities were measured in white muscle sampled dorsally at the three longitudinal positionsindicated. The enzymes measured were NAG, AAG, acid phosphatase(ACP), CaD,N-acetyl-glucosidase(Nac), CAT, GPOX and GST.

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4.2. Impact of feeding treatment on white muscleenzyme activities

Proteolysis in muscle fibres is essential fornormal protein turnover(Obled et al., 1984).Prolonged starvation leads to white muscle degra-dation, as muscle glycogen and protein are usedas energy sources. The breakdown of macromole-cules leads to an accumulation of intracellularsolutes which in turn retains water in muscle fibres.Although lysosomal enzymes are required for theseautolytic processes, the specific activities of theenzymes involved in the mobilisation of carbohy-drates(NAG, AAG and NAc) decreased after 12weeks starvation. Muscle glycogen is mobilisedbefore muscle proteins(Black and Love, 1986).By 12 weeks starvation at 108C, muscle glycogenshould be markedly depleted(Martınez et al.,´2003) making maintenance of lysosomal enzymesinvolved in carbohydrate mobilisation less neces-sary. On the other hand, the levels of CaD weretwofold higher in muscle of starved than fed codand those of acid phosphatase did not differ.Protease activity is clearly central in the mobilis-ation of muscle protein, suggesting that CaD wasspared to facilitate the mobilisation of muscleprotein during starvation. Sockeye salmon thatcease feeding during their prolonged spawningmigration show a similar sparing of CaD activityin skeletal muscle(Mommsen et al., 1980).

Muscle specific activities of the anti-oxidantenzymes, CAT, GPOX and GST, did not differbetween fed and starved cod. Again, antioxidantdefences are spared during periods of food depri-vation presumably to neutralise ROS. Oxidativeactivities of mitochondria are a primary endoge-nous source of ROS. In cod white muscle, thespecific activity of mitochondrial enzymes, likecytochrome-C oxidase and citrate synthase,decreases with starvation(Pelletier et al., 1993;Martınez et al., 2003), suggesting decreased rates´of endogenous ROS production. As suggestedabove, cellular disruption during macromolecularmobilisation may increase the sensitivity to dam-age by ROS, explaining why maintenance ofantioxidant defences, both within and outside themitochondria, would be useful during starvation.

In starved cod, mitochondrial enzymes showtheir highest levels in caudal samples of whitemuscle (Martınez et al., 2003), whereas in fed´cod, regional differences in the aerobic capacity of

white muscle are limited(Martınez et al., 2000).´If the activity of anti-oxidant systems were adjust-ed according to muscle aerobic capacity(andhence rates of endogenous ROS formation), lon-gitudinal differences in the antioxidant activitiesshould be apparent in starved but not in fed cod.Such a trend was apparent for GST and CATactivities, but it was not statistically significant. Ifrates of cell renewal followed regional differencesin metabolic intensity, lysosomal enzyme activitiesshould also have been highest in the caudal sam-ples, particularly in starved cod. Again, such dif-ferences were not statistically significant, but atendency for higher activities in caudal musclesamples from starved cod was apparent for NAcand NAG.

Macromolecules have dual roles in living sys-tems: during periods of positive energy balance,their concentrations are established by their func-tional and structural roles whereas their energeticvalue leads to their degradation during periods ofnegative energy balance. The relative importanceof these roles leads to a temporal hierarchy inmacromolecular mobilisation during starvation. Asglycogen and triglycerides have a primary role asenergy reserves, they are the first to be used duringstarvation whereas proteins, glycoproteins andphospholipids are broken down more slowly. Dur-ing starvation of cod, hepatic lipids are used first,followed by liver and muscle glycogen and thenmuscle proteins(Black and Love, 1986). Ourresults show a temporal hierarchy in the mobilis-ation of specific enzymes during starvation of cod.In red and white muscle, glycolytic enzyme levelsdecrease more than mitochondrial enzyme levelsduring prolonged starvation(Martınez et al.,´2003). Here we show that, in white muscle, lyso-somal enzymes involved in the breakdown ofcarbohydrates were decreased by 12 weeks ofstarvation whereas CaD levels were enhanced.Further, muscle activities of enzymes of anti-oxidant defence were maintained during starvation.Sparing of antioxidant enzymes was also apparentin the liver where total hepatic contents of CATand GST were either increased or unchangedfollowing extensive starvation, and this despite a10-fold decrease in liver size. The relative rates ofmobilisation of proteins during starvation mayreflect their abundance in the system, their protec-tion by intracellular chaperones or their criticalfunctional roles.

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Acknowledgments

This study was supported by grants fromNSERC to H. Guderley as well as by funds fromthe Dept. of Fisheries and Oceans obtained by J.D.Dutil. The technical assistance of Pascal Lemelinis gratefully acknowledged. Many thanks are dueto Mario Peloquin of the Institut Maurice Lamon-´tagne for the careful handling of the fish.

References

Altringham, J.D., Wardle, C.S., Smith, C.I., 1993. Myotomalmuscle function at different locations in the body of aswimming fish. J. Exp. Biol. 182, 191–206.

Black, D., Love, R.M., 1986. The sequential mobilization andrestoration of energy reserves in tissues of Atlantic codduring starvation and refeeding. J. Comp. Physiol. B 156,469–479.

Cadenas, E., 1995. Mechanisms of oxygen activation andreactive oxygen species detoxification. In: Ahmad, S.(Ed.),Oxidative Stress and Antioxidant Defenses in Biology.Chapman & Hall, New York, pp. 1–60.

Davies, M.L.F., Johnston, I.A., Van de Wal, J., 1995. Musclefibers in rostral and caudal myotomes of the Atlantic Cod(Gadus morhua L.) have different mechanical properties.Physiol. Zool. 68, 673–697.

Greer-Walker, M., 1971. Effects of starvation and exercise onthe skeletal muscle fibres of the cod(Gadus virens L.).ICES J. Mar. Sci. 33, 421–426.

Guderley, H., 1998. Temperature and growth rates as modula-tors of the metabolic capacities of fish muscle. In: Portner,¨H.O., Playle, R.C.(Eds.), Cold Ocean Physiology Societyfor Experimental Biology Seminar Series 66. CambridgeUniversity Press, Cambridge, pp. 58–87.

Guderley, H., Dutil, J.-D., Pelletier, D., 1996. The physiologicalstatus of Atlantic cod,Gadus morhua, in the wild and thelaboratory: estimates of growth rates under field conditions.Can. J. Fish Aquat. Sci. 53, 550–557.

Janssens, B.J., Childress, J.J., Baguet, F., Rees, J.-F., 2000.Reduced enzymatic antioxidative defense in deep-sea fish.J. Exp. Biol. 203, 3717–3725.

Jenkins, R.R., 1988. Free radical chemistry: relationship toexercise. Sports Med. 5, 156–170.

Jobling, M., 1988. A review of the physiological and nutri-tional energetics of Cod,Gadus morhua L., with particularreference to growth under farmed conditions. Aquaculture70, 1–19.

Johnston, I.A., Van-Leeuwen, J.L., Davies, M.L.F., Beddow,T., 1995. How fish power predation fast-starts. J. Exp. Biol.198, 1851–1861.

Kiessling, A., Johansson, L., Kiessling, K.-H., 1990. Effectsof starvation on rainbow trout muscle. I. Histochemistry,metabolism, and composition of white and red muscle inmature and immature fish. Acta Agric. Scand. 40, 309–324.

Konstantinov, A.A., Peskin, A.V., Popova, E.Y., Khomutov,G.B., Ruuge, E.K., 1987. Superoxide generation by therespiratory chain of tumor mitochondria. Biochim. Biophys.Acta 894, 1–10.

Lambert, Y., Dutil, J.-D., 1997. Can simple condition indicesbe used to monitor and quantify seasonal changes in theenergy reserves of Atlantic cod(Gadus morhua)? Can. J.Fish Aquat. Sci. 54 (Suppl. 1), 104–112.

Leonard, J.B.K., 1999. Regional variation in muscle metabolicenzymes in individual American shad(Alosa sapadissina).Can. J. Zool. 77, 1322–1326.

Martınez, M., Dutil, J.-D., Guderley, H., 2000. Longitudinal´and allometric variation in indicators of muscle metaboliccapacities in Atlantic Cod(Gadus morhua). J. Exp. Zool.287, 38–45.

Martınez, M., Guderley, H., Nelson, J.A., Webber, D., Dutil,´J.-D., 2002. Once a fast cod, always a fast cod: maintenanceof performance hierarchies despite changing food availabi-lity in cod (Gadus morhua). Physiol. Biochem. Zool. 75,90–100.

Martınez, M., Guderley, H., Dutil, J.-D., Winger, P.D., He, P.,´Walsh, S.J., 2003. Condition, prolonged swimming perform-ance and muscle metabolic capacities of cod(Gadus mor-hua). J. Exp. Biol. 206, 503–511.

Mendez, G., Wieser, W., 1993. Metabolic responses to food´deprivation and refeeding in juveniles ofRutilus rutilus(Teleostei: Cyprinidae). Env. Biol. Fishes 36, 73–81.

Mommsen, T.P., French, C.J., Hochachka, P.W., 1980. Sitesand patterns of protein and amino acid utilization duringthe spawning migration of salmon. Can. J. Zool. 58,1785–1799.

Obled, A., Ouali, A., Valin, C., 1984. Cysteine proteinasecontent of rat muscle lysosomes. Evidence for an unusualproteinase activity. Biochimie 66, 609–616.

Peek, K., Gabbott, P.A., 1990. Seasonal cycle of lysosomalenzyme activities in the mantle tissue and isolated cellsfrom the musselMytilus edulis. Mar. Biol. 104, 403–412.

Pelletier, D., Guderley, H., Dutil, J.-D., 1993. Does the aerobiccapacity of fish muscle change with growth rates? FishPhysiol. Biochem. 12, 83–93.

Sall, J., Lehman, A., 1996. JMP start statistics: a guide tostatistics and data analysis using JMP and JMP in software.Duxbury Press, Belmont California, USA.

Sanger, A.M., 1993. Limits to the acclimation of fish muscle.¨Rev. Fish Biol. Fish. 3, 1–15.

Sjodin, B., Hellsten-Westin, Y., Apple, F.S., 1990. Biochemicalmechanisms for oxygen free radical formation during exer-cise. Sports Med. 10, 236–254.

Storey, K.B., 1996. Oxidative stress: animal adaptations innature. Braz. J. Med. Biol. Res. 29, 1715–1733.

Tiidus, P.M., Houston, M.E., 1994. Antioxidant and oxidativeenzyme adaptations to vitamin E deprivation and training.Med. Sci. Sports Exerc. 26, 354–359.

Turrens, J.F., Freeman, B.A., Levitt, J.G., Crapo, J.D., 1982.The effect of hyperoxia on superoxide production by lungsubmitochondrial particles. Arch. Biochem. Biophys. 217,401–410.

Wieser, W., 1991. Limitations of energy acquisition and energyuse in small poikilotherms: evolutionary implications. Funct.Ecol. 5, 234–240.

Wieser, W., Krumschnaber, G., Ojwand-Ikwor, J.P., 1992. Theenergetics of starvation and growth after refeeding in juve-niles of three cyprinid species. Environ. Biol. Fishes 33,63–71.