Journal of Experimental Marine Biology and Ecology 404 (2011) 40–46

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Journal of Experimental Marine Biology and Ecology

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Glucose metabolism in the hepatopancreas of the crab Neohelice granulatamaintained on carbohydrate-rich or high-protein diets: Anoxia and recovery

Alessandra Marqueze b, Fabiana Ribarcki a, Inajara Kirst a,Luiz Carlos Kucharski a,⁎, Roselis Silveira Martins Da Silva a

a Departamento de Fisiologia, Instituto de Ciências Básicas da Saúde (ICBS), Universidade Federal do Rio Grande do Sul (UFRGS), 90050-170, Porto Alegre, RS, Brazilb Curso de Ciências Biológicas, Centro Universitário LaSalle, RS, Brazil

⁎ Corresponding author at: Depto. de Fisiologia - ICB500, CEP 91050-170 Porto Alegre-RS - Brazil. Tel.: +5551 3308 3656.

E-mail address: kuchars@ufrgs.br (L.C. Kucharski).

0022-0981/$ – see front matter © 2011 Published by Edoi:10.1016/j.jembe.2011.05.003

a b s t r a c t

a r t i c l e i n f o

Article history:Received 26 July 2010Received in revised form 3 March 2011Accepted 5 May 2011Available online xxxx

Keywords:DietAnoxiaRecoveryGlucose metabolismCrustacean

The effects of 1-h anoxia and 3-h recovery periodswere assessed on hepatopancreatic glucosemetabolism in thecrab Neohelice granulata fed on either a carbohydrate-rich (HC) or high-protein (HP) diet. Anoxia increasedpyruvate kinase (PK) and hexokinase (HK) activities in HC-fed animals, coinciding with a decrease in glycogenlevels. No significant changesoccurred inglucoseuptake andglycogensynthesis ineither dietarygroup. InHP-fedcrabs, HK activity decreased and PK activitywas 40% lower than that found in theHC-fed group after anoxia. Also,anoxia decreased 14CO2 production, ATP and glycogen levels in hepatopancreas in both groups. During recovery,hemolymph glucose levels decreased in both groups but glucose uptake remained constant. In the HP group, theincrease in glycogen synthesis represents an important fate of glucose during recovery from anoxia. In HC-fedcrabs, the hepatopancreas glycolytic pathway is involved in the post-anoxia fate of glucose.

S-UFRGS, Rua Sarmento Leite,21 51 3308 3623; fax: +55 21

lsevier B.V.

© 2011 Published by Elsevier B.V.

1. Introduction

Neohelice granulata is a semi terrestrial crab that lives in themidlittoral and supralittoral zones of estuaries along the southernBrazilian coast, where it is an opportunistic feeder (D'Incao et al., 1990).In its habitat, N. granulata is exposed to environmental challenges suchas changes in temperature, photoperiodicity, environmental oxygenpartial pressure, food availability and composition, and salinity. Theseparameters may vary seasonally, and may change metabolic patterns(Chittó et al., 2009; Kucharski and Da Silva, 1991; Oliveira et al., 2004;Schein et al., 2004, 2005).

Glucose concentration in crustaceans hemolymph rises in responseto a number of stress agents such as handling (Bergmann et al., 2001;Paterson and Spanoghe, 1997), emersion (Bernasconi and Uglow, 2008;Speed et al., 2001) salinity (Bianchini et al., 2008; Da Silva andKucharski, 1992), and pollutants (Reddy et al., 1996). As in most otheranoxia-tolerant crustaceans (Henry et al., 1994;Hervant et al., 1997;Hillet al., 1991; Paschke et al., 2010; Santos and Keller, 1993; Spicer et al.,2002), anaerobic metabolism in Neohelice granulata relies on thecoupled fermentation of glycogen, with an accumulation of glucoseand lactate as end products (Maciel et al., 2008; Oliveira et al., 2001;2004). However, upon return to oxygenated water, N. granulata clears

lactate via hepatopancreatic and muscular gluconeogenesis (Maciel etal., 2008; Oliveira et al., 2004).

Previous studies with N. granulata showed that the carbohydrateor protein contents in the diets administered to crabs induce differentmetabolic adjustments during anoxia and post-anoxia recoveryperiod (Marqueze et al., 2006; Oliveira et al., 2001, 2004).

After 1-h anoxia, pyruvate kinase (PK) activity increased in jawmuscle fromN. granulata fed on a high-protein diet (HP); in contrast, ahigh carbohydrate diet (HC) induced a reduction in this enzyme'sactivity (Marqueze et al., 2006). During recovery (3 h), PK activity injawmuscle decreased in crabs fed on an HP diet, and remained low incrabs receiving an HC diet (Marqueze et al., 2006). Anoxia increasedthe 14C-lactate incorporation into 14C-glycogen in the hepatopancreasof crabs fed on an HP diet or an HC diet (Oliveira et al., 2004). In post-anoxia recovery, in HP maintained crabs, the fate of L-lactate washepatopancreas gluconeogenesis. In contrast to HP results, in crabsfed on an HC diet the hepatopancreas gluconeogenesis was notinvolved in L-lactate fate (Oliveira et al., 2004).

The maintenance of large stores of glycogen in all tissues underaerobic conditions is an adaptation feature that allows surviving inhypoxia or anoxia environments, and has been identified in differentcrustacean species (Childress and Seidel, 1998; Hervant et al., 1995;Storey and Storey, 2007). Short periods of anoxia and hypoxia are partof the normal biological cycle of N. granulata, and glycogen is animportant fermentable substrate for this animal (Maciel et al., 2008;Oliveira et al., 2004). In this scenario, the following questions remainto be addressed: Is there a difference in the hepatopancreaticglycolytic flux in response to short periods of anoxia and post-anoxia

41A. Marqueze et al. / Journal of Experimental Marine Biology and Ecology 404 (2011) 40–46

recovery in crabs having different concentrations of fermentablesubstrate in their tissues? How is glucose metabolized in thehepatopancreas during the post-anoxia recovery period in crabswith different initial concentrations of glycogen? To answer thesequestions, we evaluated the effects of short periods of anoxia andrecovery from anoxia on the glycolytic flux, glucose oxidation anduptake and glycogen synthesis in the hepatopancreas of crabs withhigh (HC fed crabs) and low (HP fed crabs) glycogen concentrations.

2. Materials and methods

2.1. Animals

Male Neohelice granulata in stage C of the intermolt cycle,according to the morphological criteria described by Drach andTchernigovtzeff (1967), were collected in Lagoa Tramandaí, (S 29° 58'25.5” W 50° 08' 18.5”). The experimental protocol followed theBrazilian Environmental Law. Every effort was made to minimize thenumber of animals used.

2.2. Experimental procedure

Animals weighing 16±2 g were placed in aquaria at a salinity of20‰, at 25 °C, and a natural light/dark cycle.

Crabs were divided into two groups, one was fed on a high-protein/low-carbohydrate diet (HP, beef), while the other was fed ona carbohydrate-rich diet (HC, boiled rice) according to Kucharski andDa Silva (1991). Both groups were fed daily ad libitum (about 50 g) inthe late afternoon for two weeks before the experiments. There wasno variation in the body mass (16.5±0.5 g) of crabs in either groupduring the experimental period.

For the anoxia study, pools of 14 animals receiving either the HC orthe HP diet were placed in glass aquaria (30×25×30 cm; 20 L) at asalinity of 20‰ and a temperature of 25 °C. The water in which thecrabs were immersed was bubbled with N2 for 40 min until oxygenconcentration reached zero (monitored with an ISO2/Oxel-1, WorldPrecision Instruments). The aquaria were then sealed, and the crabswere submitted to anoxic conditions for 1 h, after which they wereused in the experiments.

The control group was kept under normoxic conditions (PO2

155 Torr) in 20‰ salinity at 25 °C. The oxygen concentration in the N.granulata aquatic habitat ranges from 2.78 mgO2/L to 11.78 mgO2/Lduring the year.

For the recovery experiments, crabs were kept under anoxicconditions for 1 h in aquaria. Then, the deoxygenatedwaterwas replacedwithnormoxicwater. After 3 h in normoxicwater, the recovered animalswereused in the experiments. The crabswere anesthetized by chilling onice for 5 min, for all experimental procedures.

The incubation buffer utilized was (in mM): NaCl 300, KCl 10, CaCl225, MgCl2 10, H3BO3 8.8, plus HEPES 10 and phenylmethylsulphonylfluoride (PMSF) 0.1, equilibrated to pH 7.8 with carbogen. This bufferwas used for in vitro experiments (glucose uptake, glycogen synthesisand 14CO2 production), and the incubations were performed in aDubnoff incubator with constant shaking. Radioactivity was measuredin an LKB Wallac scintillation counter, and the scintillation liquid usedwas toluene–Triton X-100 (2:1)-PPO-POPOP. All counts contained aninternal standard curve for quench correction.

2.3. Glucose uptake

Hepatopancreas (50±2 mg) from each experimental group wereincubated at 25 °C for 60 min in 500 μL incubation buffer with 0.2 μCi1-[14C]-2-deoxy-D-glucose (2-DG) (39 mCi mmol−1; AmershamInternational). Following incubation, the hepatopancreas was rinsedthree times in cold incubation buffer and blotted with filter paper.Glucose uptake was determined according to Machado et al. (1991).

Results are expressed as a tissue/medium (T/M) ratio, that is, isotopedisintegrations per minute (DPM)/mL tissue fluid divided by DPM/mLincubation medium.

2.4. Glycogen synthesis

For measurement of glycogen synthesis, hepatopancreas fractions(50±2 mg) from the different experimental groups were incubated at25 °C for 60 min in 500 μL incubation buffer in 0.2 μCi [U–14C] glucose(230 mCi mmol−1, Amersham International) plus 10 mM glucose.

For 14C-glycogen determination, the hepatopancreas fractions wereremoved, rinsed in cold incubation buffer, blotted with filter paper,immediately transferred to KOH (0.5 N), and boiled for 60 min; proteinswereprecipitated in30% trichloroacetic acid and1 NHCl (2:1, v/v). Aftercentrifugation at 600 g for 10 min, the supernatant (30 μL) was appliedon Whatman 3MM strips. Glycogen was precipitated with 66% ethanolatfinal concentration (Thomas et al., 1968).As ablank, the strip,withoutthe tissue sample, was subjected to the same procedure. The wet stripswere transferred to vials containing 5 mL scintillation liquid. Glycogensynthesis rates are given as nmol of 14C glucose incorporated intoglycogen per mg−1 of tissue min−1.

2.5. 14CO2 production

Glucose oxidation was determined according to Torres et al. (2001).Hepatopancreas samples (50±2 mg) were incubated in flasks sealedwith rubber caps at 25 °C in 1 mL of incubation buffer in 0.2 μCi [U–14C]glucose (230 mCi mmol−1, Amersham International) plus 10 mMglucose for 60 min, according to Marqueze et al. (2006). Saturatingglucose concentrations were used for CO2 production.

In these flasks, small glass wells situated above the level of theincubation medium contained small strips of Whatman 3MM paper.Next, 1 M Hyamine® hydroxide (ammonium, benzyldimethyl (2-(2-(4-(1,1,3,3-tetramethylbutyl) tolyloxy) ethoxy) ethyl)-,chloride) solution inmethanol was injected (0.2 mL) in the central wells to trap 14CO2.Incubationwas stopped by adding 0.2 mL of 50% TCA through the rubbercap. The flaskswere shaken for further 60 min at 25 °C to trap 14CO2. Thecontents of the center well were transferred to vials with scintillationliquid and radioactivity was counted using an LKB counter. Values of14CO2 production were expressed as nmol of 14C glucose incorporatedinto CO2 per mg−1 of tissue min−1.

2.6. Pyruvate kinase activity

Pyruvate kinase (PK) (EC 2.7.1.40) activity in the hepatopancreaswasdetermined according to Feska et al. (2003). Samples of hepatopancreas(500 mg) from the different experimental groups were homogenized(1:5 w/v) in ice-cold buffer (inmM) 0.32 sucrose, 1 mMEGTA (ethyleneglycol tetra acetic acid), 10 Tris–HCl, pH 7.4, and 0.1 PMSF with a Teflonpestle homogenizer. The homogenate was centrifuged at 10,000 g for15 min at 4 °C. The pellet was discarded, and the mitochondria-freesupernatant was used for the PK enzyme assays and determinations ofprotein concentration. Pyruvate kinase activitywas assayed in incubationmedium containing (in mM) 10 M Tris–HCl, 10 MgCl2, 0.16 NADH, 75KCl, 5.0 ADP, 1.0 U of L-lactate dehydrogenase, pH 7.5, 0.1% (v/v) TritonX-100, and 10 μL of the mitochondria-free supernatant in a final volumeof 0.5 mL. Unless stated otherwise, the reaction was using 1.0 mMphosphoenolpyruvate at final concentration. All assays were performedin duplicate at 25 °C. Results were expressed as μmol of transformedsubstrate per mg of protein−1 per min−1.

2.7. Hexokinase activity

Samples of hepatopancreas from the different experimentalgroups were removed and immediately homogenized (1:3, w/v)(Potter-Elvehjem) in ice-cold buffer (in mmol/L): 50 Tris–HCl 5

0

0.2

0.4

0.6

0.8

1

1.2

Control Anoxia Recovery

14C

-2-

Deo

xy-G

luco

se u

pta

ke(T

/M)

HP HC

Fig. 1. Glucose uptake during anoxia and subsequent recovery in hepatopancreas of crabsfed a high-protein (HP) or a carbohydrate-rich (HC) diet. Values represent mean±SEM,n=6–10 animals in each experiment.

42 A. Marqueze et al. / Journal of Experimental Marine Biology and Ecology 404 (2011) 40–46

MgCl2, 1 EDTA, 20 mercaptoethanol, pH 8.2, and 0.1 mM of PMSF.Next, the homogenate was centrifuged at 18,000 g for 20 min at 5 °C.Aliquots of the supernatant were used to determine enzyme activity.

Hexokinase (HK) (EC 2.7.1.1) activity was assayed essentially asdescribedbyBritoetal. (2001). Inaddition to0.1 mLof supernatant, theHKassay medium contained (in mmol/L): 75 Tris–HCl, 7.5 MgCl2, 0.8 EDTA,1.5 KCl, 4.0 mercaptoethanol, 0.4 nicotinamide-adenine dinucleotidephosphate (NADP)+, 2.5 ATP, 10 creatine-phosphate, 1 glucose, andcreatine phosphokinase (100 g, 1.8 units), and glucose-6-phosphatedehydrogenase (10 g, 1.4 units). Controls from which the glucose wasomittedwere run concurrently. The reactionwas startedwith 0.4 mmol/Lof NADP+. All assayswere performed at 25 °C. The resultswere expressedas μmol per min−1 per mg−1 protein.

2.8. Chemical analyses

Protein was determined by Bradford method (1976), using bovinealbumin as standard. Hepatopancreas glycogen was extractedaccording to Van Handel (1965), determined as glucose after acidhydrolysis, and expressed as g of glycogen per 100 g of tissue. Theglucose concentration was determined by the enzymatic oxidasemethod (Labtest kit) and expressed as mmol L−1 of glucose. ATP wasdetermined in hepatopancreas (1 g) from the different experimentalgroups. Samples of hepatopancreas were homogenized (1:5 w/v) inice-cold 6% TCA with an Omni Mixer homogenizer. The homogenatewas centrifuged at 10,000 g for 3 min at 4 °C. The pellet was discarded,and the supernatant was used to determine ATP with a SigmaDiagnosis Kit. ATP values are expressed as μmol ATP mg−1 of tissue.

2.9. Statistical analysis

Results were expressed as means±SEM. All the data passed thetest for normality and variance. Data were analyzed by two-way (forHC and HC groups during normoxia, anoxia or recovery treatment)analysis of variation (ANOVA) followed by Student-Newman-Keuls(SNK) comparison test. Pb0.05 was taken as the criterion ofsignificance. All tests were performed with SPSS for Windows.

3. Results

In all the results there is a statistically significant interactionbetween diets and normoxia, anoxia or recovery treatment.

In the normoxic control group, hemolymph glucose level in HC-fedanimals was 2.8 times higher (PN0.05) than in HP-fed crabs (Table 1).After 1-h anoxia, hemolymph glucose concentrations in HP and HC-fedcrabs increased, and reached levels 7.5 (Pb0.05) and 4 (Pb0.05) timeshigher than control group levels, respectively. In anoxia, the glucoseconcentration in the hemolymph of crabs receiving the HC diet washigher (Pb0.05) than in the HP group. In both dietary groups,hemolymph glucose levels decreased (Pb0.05) after 3 h of recovery ascompared to anoxia group. However, in HC-fed crabs remained higher(Pb0.05) than those of the control group (Table 1).

Table 1Effects of anoxia and post-anoxia recovery on hemolymph glucose concentration (mmol L−

high- protein (HP) or a carbohydrate-rich (HC) diet.

Groups Glucose ATP

HP HC HP

Control 0.4±0.18 1.1±0.07 1.44±0.0Anoxia 3.7±0.53a 4.6±0.23a,⁎ 0.93±0.0Recovery 1.0±0.13b 1.9±0.32a,b,⁎ 1.47±0.0

Values represent mean±SEM, (n=6–10 animals in each experiment).a Mean values are different from the control group (Pb0.05).b Mean values are different from the anoxia group (Pb0.05).⁎ Mean values are different from the HP group (Pb0.05).

The ATP concentration in the hepatopancreas of crabs receiving theHC diet was higher (Pb0.05) than in the HP group. After 1-h anoxia, theATP concentration in the hepatopancreas from both dietary groupsdecreased (Pb0.05) as compared to control animals. In the HC group, theATP concentration was lower (Pb0.05) than in HP-fed animals. Duringthe recovery from anoxia, the ATP content in the hepatopancreas fromboth dietary groups increased (Pb0.05) when compared to the valueobserved in anoxia. In the HP-fed crabs, the ATP content in thehepatopancreas reached a concentration similar to that found in thecontrol group after 3 h in recovery. However, in HC-fed animals the ATPvalue after the recovery period corresponded to 39% of the control groupconcentration (Table 1).

The glycogen concentration in the hepatopancreas of crabsreceiving the HC diet was higher (Pb0.05) than in the HP group.The hepatopancreas glycogen content decreased (Pb0.05) after 1 h inanoxia only in HC-fed group. During the recovery period the glycogenlevel decreases (Pb0.05) in HP group, and remained similar to anoxiagroup in HC-fed crabs. However, the difference (Pb0.05) betweenglycogen levels in the HP and HC groups observed in the control groupwas observed in crabs submitted to anoxia and recovery (Table 1).

Differences in 2DG uptake were not found between crabs fed theHP diet and those maintained on the HC diet (Fig. 1). No differenceswere observed in glucose uptake between the control, anoxia, andrecovery groups, for animals fed on either diet (Fig. 1).

In control crabs, no significant difference was observed in glycogensynthesis in the hepatopancreas between animals fed on the HC and HPdiets (Fig. 2). Anoxia for 1 hdid not significantly affect glycogen synthesisin the hepatopancreas in either experimental group (HP and HC). Incontrast, during recovery, the capacity for glycogen synthesis in the

1), ATP (μmol g of tissue−1) and glycogen (g%) content in hepatopancreas of crabs fed a

Glycogen

HC HP HC

6 1.95±0.16⁎ 1.6±0.08 2.9±0.07⁎

3a 0.7±0.05a,⁎ 1.3±0.11 1.9±0.14a,⁎

7b 1.19±0.04a,b 1.2±0.10a 2.2±0.20a,⁎

0

2

4

6

8

10

12

14

Control Anoxia Recovery

Gly

coge

n S

ynth

esis

(nm

ol o

f 14C

-glu

cose

inco

rpor

ated

. m

g of

tiss

ue-1

. min

-1)

HP HC

a b

*

Fig. 2. Glycogen synthesis during anoxia and subsequent recovery inhepatopancreas of crabsfed a high-protein (HP) or a carbohydrate-rich (HC) diet. Values represent mean±SEM,n=6–10 animals in each experiment. a Mean values are significantly different from thecontrol group (Pb0.05). b Mean values are significantly different from the anoxia group(Pb0.05). * Mean values are significantly different from the HP group (Pb0.05).

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

Pyr

uva

te K

inas

e (µ

mo

l.mg

pro

tein

-1.m

in-1

)

HP HC

A

*

*

*

a

a

a

b

Control Anoxia Recovery

43A. Marqueze et al. / Journal of Experimental Marine Biology and Ecology 404 (2011) 40–46

hepatopancreas of the HP group increased by about 8 (Pb0.05) and 5times (Pb0.05), when compared to those of the control and anoxiagroups, respectively. This significantly increasewasalso compared to thatobserved in the recovery group fed on the HC diet (Fig. 2).

In the control group, the 14C-glucose oxidation in the hepatopancreasof crabs receiving the HC diet was lower (Pb0.05) than in the HP group(Fig. 3). After 1 h in anoxia, 14C-glucose oxidation in the hepatopancreasof the HP group was about 64% (Pb0.05) lower, than in thehepatopancreas of control HP-fed crabs. After 3 h in recovery, nosignificant difference in 14C-glucose oxidation was observed in the HPgroup, as compared to anoxia. In HC-fed crabs, anoxia for 1 h or recovery

Control Anoxia Recovery

14C

O2

Form

atio

n(n

mol

14C

-glu

cose

inco

rpor

ated

. mg

of ti

ssue

-1 .

min

-1)

0.0

0.5

1.0

1.5

2.0

2.5

3.0 HP

HC

a

a*

*

Fig. 3. Effects of anoxia and recovery on 14CO2 formation in hepatopancreas of crabs feda high-protein (HP) or a carbohydrate-rich (HC) diet. Values represent mean±SEM,n=6–10 animals in each experiment. a Mean values are significantly different from thecontrol group (Pb0.05). b Mean values are significantly different from the anoxia group(Pb0.05). ⁎ Mean values are significantly different from the HP group (Pb0.05).

for 3 h did not significantly affect the 14C-glucose oxidation in thehepatopancreas. However, in HC-fed crabs the 14C-glucose oxidation inthe hepatopancreas from crabs submitted to anoxia was significantlyhigher, than in the hepatopancreas of HP-fed animals (Fig. 3).

In the controlHC group, PK activity in hepatopancreaswas81%higher(PN0.05) than in the HP group. After 1 h exposed to anoxia, increased(Pb0.05) PK activity was observed in both diet groups (Fig. 4A).However, in the HC group, PK activity was twice (Pb0.05) as high as inthe HP-fed crabs. PK activity decreased (PN0.05) in HP animals as theywere exposed to recovery. Still, PK activity was similar in the anoxia andpost-anoxia period in HC animals. However, in HC-fed animals was 3.5times higher (PN0.05) than in HP-fed crabs (Fig. 4A).

Fig. 4B shows that in the control group, HK activity wassignificantly higher (Pb0.05) in HC-fed animals than in the HP-fedgroup. After 1 h in anoxia, enzyme activity increased (Pb0.05) in theHC group. However, in HP-fed crabs, HK activity decreased about 66%(Pb0.05) after 1 h in anoxia. In the recovery period, in the HC-fedcrabs, HK activity decreased 40% (Pb0.05) and 53% (Pb0.05) ascompared to the control and anoxia groups, respectively. Conversely,HK activity in the hepatopancreas from HP-fed crabs increased about

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

Hex

oki

nas

e

HP HC

B

a

a bba

**( µ

mo

l.mg

pro

tein

-1.m

in-1

)

Control Anoxia Recovery

Fig. 4. Activities of pyruvate kinase A and hexokinase B during anoxia and subsequentrecovery in hepatopancreas of crabs fed a high-protein (HP) or a carbohydrate-rich(HC) diet. Values represent mean±SEM, n=6–10 animals in each experiment. a Meanvalues are significantly different from the control group (Pb0.05). b Mean values aresignificantly different from the anoxia group (Pb0.05). ⁎ Mean values are significantlydifferent from the HP group (Pb0.05).

44 A. Marqueze et al. / Journal of Experimental Marine Biology and Ecology 404 (2011) 40–46

threefold (Pb0.05) during the recovery period, reaching levels closeto those of the control group (Fig. 4B).

4. Discussion

The present data show that administration of an HC diet results ina marked increase in hepatopancreatic glycolytic flux in normoxic N.granulata, as evidenced by the higher activities of the glycolyticenzymes HK and PK. These findings show that in N. granulatahepatopancreas the HK and PK activities were under the nutritionalregulation. This result agrees with the high PK activity found in jawmuscle of HC-fed N. granulata, when compared to that observed in thegroup fed on the HP diet (Marqueze et al., 2006). Also, in fish fed onhigh starch diet, the hepatic PK activity increased (Enes et al., 2006;Tan et al., 2009), indicating carbohydrate control over the enzymeactivity. Contrasting with our results, the hepatic HK activity in fisheswas not affected by the absence or the presence of starch in the diet(Panserat et al., 2000; Tan et al., 2009). On the other hand, no effect ofdiet composition on hepatopancreatic glucose uptake, and glycogensynthesis was observed in normoxia crabs. In contrast, in the HC-fedgroup the 14CO2 production from 14C-glucose was three times lowerthan in the HP group. This result may be explained by the higher ATPand glycogen levels found in hepatopancreas from HC-fed crabs.

For a variety of animals, including N. granulata, access toenvironmental oxygen is intermittent. This semi terrestrial crabundergoes daily episodes of hypoxia during its forays on land. Inwinter, this crab faces hypoxia or anoxia when remaining in its holesfor long periods with a low oxygen supply (Turcato, 1990).

Like other crustaceans, there was a marked increase in theconcentration of hemolymph glucose after 1 h in anoxia in HC- andHP-fed crabs, suggesting that glucosemobilization in the tissues exceedsits utilization in the glycolytic pathway, causing an accumulation ofglucose in the hemolymph (Anderson et al., 1994; Hervant et al., 1997;Van Aardt, 1988; Zou et al., 1996). Furthermore, the concentration ofhemolymph lactate increases 5–14 times in N. granulata fed an HC or HPdiet and subjected tohypoxic or anoxic stress for different periods of time(Maciel et al., 2008; Oliveira et al., 2004).

Vertebrates and invertebrates show a biphasic response to areduction in oxygen levels in the environment (Lutz and Storey, 1997;Storey and Storey, 2007). The first phase leads to the activation of theglycolytic pathway to maintain or increase ATP production, since theoxygen-dependent production pathways may be significantly reduced.The second phase would lead to the reduction in metabolic rate, whichaffords the gradual use of endogenous reserves and survival forprolonged periods of severe hypoxia or anoxia, as observed in turtlesandmollusks (terrestrial, bivalve, and cephalopod) (Churchill and Storey,1989; Guppy et al., 1994; Seibel and Childress, 2000).

The results of this study demonstrate that the hepatopancreas fromHC crabs were in a transition state, from normoxia to hypoxia,characterized by the increase in PK and HK activities, which led to anincrease in glycolytic flux. In this group, the activation of thehepatopancreatic glycolytic flux during anoxia coincided with thedecline in glycogen levels in this organ. In contrast, 1-h anoxia decreasedPK activity in jawmuscle of N. granulata fed on anHC diet (Marqueze etal., 2006). These results agree with Chang's (2005) observation that inlobster submitted to hypoxia stress the animal's tissues do not respondproportionately to a given stress. This difference may be explained by alate activation in the glycolytic flux in hepatopancreas as compared tomuscles (Marqueze et al., 2006). In muscles of crabs fed on HC diet, thedecrease in PK after 1 h in anoxia was followed by an increase in 14CO2

production from 14C-glucose, suggesting that glucose is diverted fromthe glycolytic pathway to the pentose phosphate pathway, which canoperate to oxidizeglucose completely toCO2 andwater (Marquezeet al.,2006). However, in hepatopancreas from HC-fed crabs the 14CO2

production from 14C-glucose remained unchanged during the anoxiaperiod, suggesting a bypass of the glucose to the glycolytic flux. These

results agree with the marked glycogen mobilization found inhepatopancreas of crabs fed on an HC diet.

In contrast, no significant changes were observed in glucoseuptake and glycogen synthesis in the hepatopancreas of crabs fed onthe HC diet measured after 1 h in anoxia. These results may beexplained by the high glycogen mobilization as a consequence of thehigh glycogen content found in HC-fed hepatopancreas animals.

Unlike the HC-fed crabs, the HP-diet group was already enteringthe second stage of response to reduction in O2 levels in theenvironment. This statement is based on the observation that after1 h in anoxia, in the crabs fed on the HP diet, HK activity decreasedmarkedly and PK activity in hepatopancreas was 40% lower than thatfound in the HC-fed group. Similarly, in hepatopancreas from HP-fedcrabs, the 14CO2 production from 14C-glucose decreased with thereduction in oxygen levels.

In the hepatopancreas from HP-fed group the HK activitydecreased during anoxia, suggesting an increase in the percentageof dissociation HK enzyme from active complex as observed in Otalalactea (Plaxton and Storey, 1986). This mechanism appears, therefore,to be a simple and fast way to change the flux through glycolysis in areadily reversible fashion. In anoxia, the increase in glucoseconcentration in the hemolymph of the HP-fed crabs was 5 timeshigher than that found in HC-fed animals, despite the low glycogencontent and mobilization in hepatopancreas of this dietary (HP)group, suggesting a marked reduction in the utilization of glucose byglycolytic flux during anoxia stress. These results suggest thatglycolysis in HC-fed crabs increased with anoxia, a response possiblyassociated with the diet. In contrast, animals fed on the HP the HKactivity decreased, suggesting that in those crabs the glycolysispathway was not affected by hypoxic conditions.

However, true anoxia did not yet occur in HP-fed crabs, because therates of glucose uptake and glycogen synthesis are similar to thoseobserved in normoxic animals. Oliveira et al. (2001) observed a decreasein glucose uptake and glycogen synthesis only after 2 h in anoxia.

After 1 h in anoxia, the concentrations of ATP in the hepatopancreasof crabs fed on the HP diet decreased about 1.5 times, while in theanimals receiving theHCdiet the reductionwas of about 3 times. On theother hand, the preferential use of phosphagens stores in order tomaintain a high ATP concentration and decrease glycolysis flux in thehepatopancreas during the first hour of anoxia, as observed in othercrustaceans andmollusks (Gäde, 1983, 1984;Hervant et al., 1995; 1997;Isani et al., 1989), may explain the maintenance of high ATP andglycogen contents in HP-fed crabs. On the other hand, the highhemolymphglucose levels could inhibit themobilizationof fuel reservesin the hepatopancreas; or in HP-fed crabs the pool of free amino acidsproduct of the catabolism of the alimentary and/or muscular proteinsduring the first hour of anoxia may be used as an energy source asdemonstrated in Penaeus setiferus juveniles submitted to prolongedhypoxia (Rosas et al., 1999). Ultimately there could be a decrease in thetotal adenine nucleotide pool without a change in the energy state([ATP]/[ADP*Pi] ratio). These are possibilities that need to be investigated.

The post-anoxia recovery process has great functional importance,since it is during recovery that energy reserves are restored and theaccumulated end products are removed from the organism.

During the 3 h of post-anoxia recovery, hemolymph glucose levelsdecreased in both dietary groups, though without reaching the levelsseen in the normoxia group. However, the post-anoxia recovery did notaffect glucose uptake in the hepatopancreas in either diet group. Thus, intheHP-fed animals, alongwith the return of HK activity to levels similarto those of the control group, the increase in the capacity of glycogensynthesis represents an important fate of glucose during recovery fromanoxia. Taking into account that PK activity in the hepatopancreas of therecovery HP-group was similar that found in normoxia group, anotheralternative pathway for glucose fate is the aerobic glycolysis. Thishypothesis is corroborated by increase in ATP concentration in HP-fedcrabs hepatopancreas after the 3-h recovery period. Also, inN. granulata

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fed on an HP diet, the jawmuscle is an important organ in reprocessingglucose in the 3-h post-anoxia recovery period, increasing glucoseoxidation and glycolytic flux (Marqueze et al., 2006).

In N. granulata fed on the HC diet, the glycolytic pathway in thehepatopancreas is one pathway involved in the post-anoxia fate ofglucose. During recovery, HK activity decreased in the group fed onthe HC diet, although this activity level was similar to that observed inthe HP group in recovery. However, in spite of the decline in HKactivity, the contribution of the glycolytic flux to the utilization ofglucose and recovery of the energy reserves cannot be neglected. Inthis diet group, the ATP concentration in the hepatopancreasincreased compared to the anoxia group, suggesting an increase inenergy reserves. The decrease in HK activity may have played a role inthe maintenance of high hemolymph glucose levels and low glycogenconcentration, although no change in glucose uptake was observed inHC-fed crabs.

In conclusion, itwas possible to identify differentmetabolic strategiesused by the hepatopancreas tissue from N. granulata subjected to 1 h ofanoxia and 3 h of post-anoxia recovery, based on the glycogen content inthe tissue. In the crabs fed on the HP diet, the significant reduction in HKandPKactivities,when compared to those observed in theHCgroupafter1 h of anoxia, suggest that the low concentration of hepatopancreaticglycogen observed in the HP group may induce a decrease in glycolyticactivity. In the HC group, which showed high levels of glycogen in thetissues, the glycolytic activity was markedly increased after 1 h anoxia .

In post-anoxia recovery, glycogen synthesis represents an importantfate of glucose in the hepatopancreas of HP-fed crabs, and glycolytic fluxin the HC-fed group.

Acknowledgments

The experiments were performed according to the current Brazilianlaws regarding animal experimentation. Thisworkwas supported by theFundaçãodeAmparoàPesquisadoRioGrandedoSul (FAPERGS), and theConselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq),Brazil. R.S.M.S. and L.C.K. are CNPq fellows. [SS]

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