High pressure and glycolytic flux in the freshwater Chinese crab, Eriocheir sinensis

High pressure and glycolytic flux in the freshwater Chinese crab, Eriocheir sinensis

Comparative Biochemistry and Physiology Part B 126 (2000) 537 – 542 www.elsevier.com/locate/cbpb High pressure and glycolytic flux in the freshwater ...

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Comparative Biochemistry and Physiology Part B 126 (2000) 537 – 542 www.elsevier.com/locate/cbpb

High pressure and glycolytic flux in the freshwater Chinese crab, Eriocheir sinensis Philippe Se´bert a,*, Yann Choquin a, Andre´ Pe´queux b b

a Laboratoire de Physiologie, E.R. 2217, U.F.R. Me´decine, B.P. 815, 29285 Brest Cedex, France Laboratoire de Physiologie Animale, Uni6ersite´ de Lie`ge, 22 Quai 6an Beneden, 4020 Lie`ge, Belgium

Received 1 January 2000; received in revised form 22 March 2000; accepted 27 March 2000

Abstract The hexose part of glycolysis has been studied in the freshwater Chinese crab Eriocheir sinensis exposed to high pressure (101 ATA, i.e. 1000 m depth) at 14°C and in normoxic conditions. Glycolytic fluxes (from glucose, JA and from Glucose 6 Phosphate, JB) have been determined using NADH depletion during the conversion of dihydroxy acetone phosphate into a-glycerol phosphate. Measurements have been performed at 14 and 19°C. Pressure exposure induces an increase of glycolytic flux and a decrease of the time needed for the transition from aerobic to anaerobic glycolysis. As a consequence pressure-exposed crabs have a higher potential to increase glycolytic flux than control animals at atmospheric pressure. It is concluded that high pressure known to alter numerous enzymes individually, can also modify an overall metabolic pathway. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Eriocheir; Glycolysis; Pressure; Temperature; Metabolism; Muscle; Enzymes; Metabolic pathway

1. Introduction Glucose is probably the primary metabolic substrate of crustaceans (Wolvekamp and Waterman, 1960) and glycolysis is the major pathway for its muscle utilization (Santos and Keller, 1993). Although the relative participations of glycolysis and/or hexose mono-phosphate shunt seem dependent on the seasonal temperature (Mauro and Mangum, 1982; Carlos et al., 1991), little is known about glycolytic activity under different physiological and/or environmental conditions. The freshwater Chinese crab Eriocheir sinensis has

* Corresponding author. Tel.: + 33-2-9801-6462; fax: + 332-9801-6313. E-mail address: [email protected] (P. Se´bert).

been shown to be a good model for studying the specific effects of hydrostatic pressure because although never experiencing high pressure during its life cycle, in contrast with the eel, it is able to acclimatize to pressure exposure for long periods, as the eel (Se´bert et al., 1997). Very high pressures (several kbars) are needed to specifically modify enzymes structure and/or function (considering the enzyme as a protein, Balny et al., 1997), but only some tens of atmospheres are sufficient to modify the metabolism by changing the enzyme environment mainly membrane fluidity and the associated processes (Se´bert, 1997). For example, hydrostatic pressure is known to induce a metabolic state resembling histotoxic hypoxia affecting aerobic metabolism, and it is supposed that glycolysis is activated during the first hours under pressure (Se´bert et al., 1993). However,

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measuring only lactate and/or some enzyme activities are not sufficient to prove that glycolysis functions at a higher rate. For this reason, we have chosen to study the glycolytic flux through the hexose portion of glycolysis (Lupianez et al., 1996; Se´bert et al., 1998) in crabs exposed to high pressure for 6 h. Moreover, Eriocheir is known to be very sensitive to temperature increase with a Q10 value of 4 for critical oxygen pressure which supposes an enhanced role of glycolysis in energy production when temperature increases (Se´bert et al., 1995). Thus, measurements of glycolysis activity have been performed at two temperatures considering the glycolytic fluxes (aerobic and anaerobic), the time required for the transition from aerobic to anaerobic mode and the rate of flux change.

2. Material and methods

2.1. Animals Twelve Chinese crabs E. sinensis were used. They were maintained in running tapwater (Tw = 14°C) in polyethylene tanks (40 l) kept in a room open to the outside to maintain natural conditions of temperature and photoperiod.

2.2. Protocol The day before the experiments, six crabs were placed in individual cages introduced in the experimental tank connected to the high pressure water circulation system previously described (Se´bert et al., 1990). This system allows maintenance of animals under pressure for long periods controlling water oxygenation and temperature. The day of the experiment, the hyperbaric chamber was compressed at the rate of 3 atm min − 1 until 101 ATA hydrostatic pressure (ATA=atmosphere absolute) was reached. This pressure was maintained for 6 h, then the chamber was decompressed at a rate of 3 atm min − 1. Immediately after decompression, animals were removed from the tank, pieces of muscles were sampled from the walking legs and immediately frozen in liquid nitrogen. Samples were kept at − 80°C until analysis. The six others crabs were treated exactly in the same way except the pressure; they were used as control animals at 1 ATA (atmospheric pressure).

2.3. Sample preparation Muscle was chopped into very small pieces and homogenized (4°C) with a Potter–Elvehjem homogenizer using a Teflon pestle, at 1 g/5 ml with 50 mM–Hepes buffer, pH 7.4 with 100 mM KCl, 10 mM MgCl2, 10 mM NaH2PO4 and 1 mg/3 ml of Trypsin Inhibitor (Sigma Type III). Homogenates were centrifuged at 105 000× g and 4°C (Beckman L5-65 with rotor Ty70) for 1 h. Supernatants were used immediately for kinetic experiments.

2.4. Measurements The method used is adapted from Easterby (1981) and described in details in a previous paper (Se´bert et al., 1998). Briefly, it consists in studying the hexose part of glycolysis: glucose (or glucose 6 phosphate (G6P)) was converted into triose phosphate by means of the enzymes present in the soluble muscle extract. Activity was measured by recording the NADH decrease at the a-glycerol phosphate dehydrogenase. Triose phosphate isomerase (TPI) and glycerol phosphate dehydrogenase (a-GPDH) were added as auxiliary enzymes to drive the flux towards the a-GPDH reaction. Creatine-phosphate and creatine kinase were added to buffer the ATP concentration (3 mM). NADH depletion was followed using a spectrophotometer at 340 nm (final volume of the cuvette: 0.5 ml). The system containing all reagents except glucose was allowed to record the NADH depletion for a few minutes. When the recording was horizontal, the reaction was triggered by adding 25 ml of 200 mM glucose to reach 5 mM in the incubation mixture. Adding glucose mimics aerobic metabolism because, the system being fed with external free glucose, it is used by red fibres and does not produce lactate; moreover, phophorylation of glucose into G6P is depending on oxydative phosphorylation. After a short lag time, a constant decay of NADH was observed and recorded (Fig. 1). This constant slope (steady state) was registered as JA (flux of aerobic glycolysis, as the system is fed with external glucose). After a sufficient recording time at steady state, the system was stimulated by adding G6P (25 ml of 200 mM stock solution, final concentration of 5 mM); the system reached a new steady state (Fig. 1) with a steeper slope JB, (flux of anaerobic glycolysis fed with phosphorylated glucose

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Table 1 Pressure effects on glycolytic fluxes and related parametersa 14°C

1 ATA (n= 6)

101 ATA (n=6)


JA JB t99 JB/JA r

2.4 9 0.7 387 9 43 25 9 3 243 964 11 93

1.5 9 0.3 512950 18 9 1 3989 71 239 4

NS NS 0.05 NS 0.05

a Mean values ( 9 S.E.M.) of fluxes JA and JB (nmol min−1 −1 g−1 ) in muscle extracts of FW E. sinensis. ww ), t99 (s) and r (s Acclimation temperature = 14°C; measurement temperature = 14°C. Significant differences between 1 and 101 ATA is tested using Student’s t-test.

derived from muscle glycogen). The transient was also recorded. Measurements were performed at 14 and 19°C, using freshly prepared muscles extracts for each temperature.

Fig. 1. Typical recording of an experiment. Fluxes are assayed as NADH decreases at 340 nm. The system is started with glucose as substrate; when the steady state (JA, aerobic flux) is achieved, it is stimulated by adding G6P which produces an increase in flux (JB, anaerobic flux). t99 is the time required to obtain a steady state of JB after adding G6P. The artefactual events on the curve (above glucose and G6P) correspond to the opening of the spectrophotometer for adding the substrates.

2.5. Calculations From the recordings, glycolytic fluxes were calculated in terms of JA and JB using the corresponding dilution factors in order to obtain fluxes from glucose and G6P, respectively. The time needed to reach the steady state value of JB after adding of G6P was named t99 (Fig. 1). Finally, metabolic reprise r values (ratio between fluxes over the response time, i.e. r = (JB/JA)/t99) were calculated in order to characterize the factor of flux increase achieved per unit time. Statistical significance of the results was estimated at the 5% level using the Student’s t-test and ANOVA analysis.

3. Results Glycolytic flux data and related parameters

measured at 14 and 19°C are reported in Tables 1 and 2. ANOVA analysis shows that independently of assay temperature (14 or 19°C) hydrostatic pressure application increases (+ 30%, PB 0.05) JB without effects on JA; the ratio JB/JA is consequently also enhanced (PB 0.005). As t99 decreases, the metabolic reprise r significantly also increases (PB 0.05). Independently of pressure, an increase in assay temperature induces a strong increase of JB (PB0.001), and hence of JB/JA (PB0.001). As t99 is significantly depressed (PB 0.01), the metabolic reprise r is also increased (PB 0.001). JA is not significantly modified by temperature and/or pressure. Note the high temperature sensitivity of glycolysis as estimated from Q10 values in Table 2. Any rise in hydrostatic pressure and/or temperature enhances anaerobic glycolytic flux and depresses metabolic response

Table 2 Effects of a measurement temperature increase on glycolytic fluxes and related parameters, at 1 and 101 ATA (for control data, refer to Table 1)a 19°C

1 ATA (n= 6)


101 ATA (n =6)



JA JB t99 JB/JA r

1.69 0.3 1018 9129 129 1 756 9 136 619 6

0.4 6.9 0.2 9.7 30

0.9 90.2 1349 9170 10 92 1731 9290 195 936

0.4 6.9 0.3 19 72

NS NS NS 0.05 0.01

−1 Mean values (9 S.E.M.) of fluxes JA and JB (nmol min−1 g−1 ) in muscle extracts of FW Eriocheir sinensis. ww ), t99 (s) and r (s Acclimation temperature = 14°C; measurement temperature =19°C. Significant differences between 1 and 101 ATA is tested using Student’s t-test. a


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times in muscle cells of the Chinese crab. This can mean that in vivo, the ability of the crab in changing from aerobic to anaerobic pathway is improved as judged from the values of r.

4. Discussion Glycolysis is the major pathway for glucose in crustaceans muscle where the activity of hexose mono-phosphate shunt is smaller than in other tissues such as hepatopancreas (Mauro and Mangum, 1982; Santos and Keller, 1993). As previously argued by Se´bert et al. (1998) only the first part of the glycolysis and not the complete pathway by measuring lactate concentration has been studied. In fact, the aerobic part of the glycolysis does not produce lactate and the heterogeneity of muscle composition makes that aerobic fibres can use lactate from anaerobic ones as a substrate (Simon et al., 1991). Several variables have been used to quantify the transition between the two steady states A (aerobic) and B (anaerobic) studied in this experiment. The problem is to find an objective criterion for determining that transition step. Recently, Se´bert et al. (1998) have proposed to consider tm, which represents the time needed to reach the maximum acceleration for JB (the starting point being always the addition of G6P to the system). The variable tm being determined from the second derivative of the progress curve is thus the result of a calculation, tm appears as an objective criterion. However, progress curves are not always sufficiently clean to obtain a clear second derivative. Consequently, we decided to refer in this work to time index values t99. This value is accurate because, t99 was systematically determined by two different observers and the results differed by less than 2% of the curves studied. The results reported in this paper on the Chinese crab roughly show the same pattern as those observed on the eel, i.e. a pressure-induced increase of glycolytic flux and a decrease of the delay needed to obtain a steady state of this flux. Hydrostatic pressure, by altering membrane fluidity, induces an impairment of respiratory chain and oxidative phosphorylation (histotoxic hypoxia, see Se´bert et al., 1993) but also other membrane bound enzymes such as hexokinase, which can explain the observed decrease of JA. We can hypothetize that pressure could induce a

‘crabtree like effect’, i.e. an inhibition of respiration balanced by an enhancement of glycolysis (see Santos and Keller, 1993 for review) as shown with the observed JB increase in the pressure group. Such an increase is quite well withstood by E. sinensis and is in agreement with its good tolerance to hemolymph lactate accumulation (Zou et al., 1996). Such a metabolic response could be helped by a neurohormonal stimulation. In fact, nervous system (including eyestalk) is an important site for pressure action. Actually, a ‘diabetogenic factor’ has been identified in the eyestalks of decapods. The crustacean hyperglycemic hormone (CHH) is produced by perikarya in the medulla terminalis ‘X-organ’ and released from the sinus gland. It has been argued that CHH can mobilize glucose from glycogen stores during hypoxia, which in turn would allow glycolysis to proceed at a higher rate (Santos and Keller, 1993). The work of Lupianez et al. (1996) has shown that glycolysis in long-flight birds has a very high basal activity, but its activation is low and very slow. In contrast, glycolysis of the short-distance sprinter birds has a low basal activity, but its activation is large and very rapid, corresponding to a high metabolic reprise r. These results fit with the locomotor behavior of the animal and with the fact that metabolic design is optimized to face its physiological role. Similar considerations have been studied by Se´bert et al. (1998) in the eel, Anguilla anguilla. These authors have raised the hypothesis that the non-migratory yellow eel when acclimated to high pressure (1 month at 101 ATA) could exhibit some of the metabolic features (this is also true for osmoregulation) observed in migrating silver eels. In fact, it appears that high pressure acclimation modifies the metabolic design of yellow eel white muscle in such a way that energy metabolism is adapted to face an energy consuming migratory activity: increase of basic glycolytic flux (aerobic activity for long lasting swimming) but also an increase in activation speed (decrease of t99, increase of r, which are representative of a possible anaerobic activity allowing to escape from predators). Those results allowed us to consider hydrostatic pressure as an ecophysiological signal able to induce biochemical changes, i.e. optimizing the metabolic design in view of energy requirements. However, during its life cycle, the Chinese crab E. sinensis is never exposed to high pressure. Consequently,

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hydrostatic pressure cannot be considered as an ecophysiological signal in this species. Without neglecting the ecophysiological role of pressure in the eel, that has been clearly established (Se´bert, 1997 for review), it must be considered that high hydrostatic pressure per se is able to modify metabolic design and regulation processes of glycolysis. Such an hypothesis in not really new. There is a bulk of studies investigating pressure effects on enzyme activity (for example, see Somero, 1992). However, our study considers several steps of a metabolic pathway as a whole. One of the first papers reporting results obtained from fish (Baldwin et al., 1975) clearly showed that pressure and temperature interacted in determining enzyme reaction rates, with the overall effect dependent on the relative contributions of each parameter. However, if pressure may increase, decrease or have no influence on the rate of reactions (depending on the relative volumes of reactants and activated complex), a rise in temperature will generally tend to increase the reaction rate. Such a temperature effect is clearly shown in Table 2. When measurements are performed at 19°C instead of 14°C, there is an increase of the anaerobic flux (JB) and a decrease of the transition time resulting in a highly significant increase of r. The Q10 values estimated from mean data show very high temperature sensitivities: about 7 for JB, 0.2 for t99 but 30 for r at 1 ATA and about 75 for r at 101 ATA. Although surprising (because rarely published), such high Q10 values are in agreement with the interesting concept of evolved temperature sensitivities of biological processes in ectotherms (Hochachka, 1991). These findings also fit with the results of Seibel et al. (1997) who observed that Q10 values are significantly higher for invertebrates submitted to elevated hydrostatic pressure, which supposes a pressure–temperature interaction. For example, a temperature rise by only 5°C is able to induce a five-fold activation of glycolytic flux within 1 s at 1 ATA but an eight-fold activation at 101 ATA: thus, when submitted to high pressure and high temperature together, crab muscle appears to be quite able to increase its glycolytic flux by about 20 times within 1 s (r increases from 11 at 1 ATA, 14°C to 195 at 101 ATA, 19°C). Such results show the interest to study a metabolic pathway and its resulting flux, rather than a single enzyme when considering the physiological responses to environmental changes.


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