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Radioisotope studies of the energy metabolism of the shore crab Carcinus maenas (L.) during environmental anoxia and recovery
J. Exp. Mar. Biol. Ecol., 150 (1991) 51-62
51
© 1991 Elsevier Science Publishers B.V. 0022-0981/91/$03.50
JEMBE 01612
Radioisotope studies of the energy metabolism of the shore crab Carcinus m a e n a s (L.) during environmental anoxia and recovery A.D. Hill t, R. H.C. Strang 2 and A.C. Taylor ! ~Department of Zoology, 2Department of Biochemistry, University of Glasgow, Glasgow, UK (Received 9 November 1990; revision received 25 February 1991; accepted 9 March 1991) Abstract: Under both normoxic and anoxic conditions, ~4C from D-[UNC]-glucoseinjected into the haemolymph of Carcinus maenas (L.) was rapidly incorporated into various metabolic fractions. Under both conditions, the greatest proportion oflabei was found in the amino acid fraction (> 70%). During normoxia, the steady accumulation of acid labile, volatile ~4C in the water indicated the complete oxidation of the glucose to CO2. Under both normoxia and anoxia, a considerable part of the label appeared in glycogen. The main differences between aerobic and anaerobic conditions were (a) the slower disappearance oflabel from the neutral sugar fraction and (b)the accumulation of label in glycolytic phosphate material under anaerobic conditions. The proportion of label in lactate doubled during the I st h of the recovery period under normoxic conditions, increasing to a maximum of 23 % ofthe non-amino acid radioactivity before decreasing during the rest of the recovery period. Label from L-[Um4C]-lactate injected into crabs immediately before they were returned to normoxic conditions following exposure to anoxia was partly oxidised, appearing as acid labile material in the medium. Most of the injected label, however, was retained within the animal and appeared as glycogen and subsequently in the amino acid fraction. There was no evidence for the excretion of lactate. Key words: Anoxia; Crustacean; L-Lactate; Metabolism; Radioisotope
INTRODUCTION
The anaerobic metabolism of decapod crustaceans during both environmental and functional (exercise) anaerobiosis has been the subject of a number of studies (see Hill et al., 1991). Information on the metabolism of decapods during periods of recovery from anaerobiosis is, however, more limited. In particular, the fate of lactate during the period of recovery is still unclear. The available information suggests that, in most decapods, little if any lactate is excreted (Phillips et al., 1977; Bridges & Brand, 1980; EUington, 1983; G~de et al., 1986). Other studies, however, have found evidence of lactate excretion. Van Aardt (1988), in his study of the freshwater crab Potamonautes warreni, found a significant difference between experimental and control crabs in the amount of acid-stable radioactivity in the incubation water. Similarly, Head & Baldwin Correspondence address" A.D. Hill, Shell Research Ltd, Sittingbourne Research Centre, Sittingbourne, Kent ME9 8AG, UK.
52
A.D. HILL ET AL.
(1986) concluded that, in the crayfish Cherax destructor, most of the lactate produced during exercise anaerobiosis was excreted rather than metabolized. There is growing evidence to indicate that, in the majority of decapods, lactate is metabolized during the recovery period and the occurrence of gluconeogenesis in decapods during recovery from anaerobiosis has now been clearly established (Phillips et al., 1977; G/ide et al., 1986; Van Aardt, 1988). In comparison with vertebrates, however, the rates of gluconeogenesis are very slow and it may take many hours for lactate concentrations in the haemolymph and in the tissues to return to normal levels (Teal & Carey, 1967; Bridges & Brand, 1980; Albert & Ellington, 1985; Lowery & Tate, 1986; Taylor & Spicer, 1987; Van Aardt, 1988). There is still some controversy about the site(s) ofgluconeogenesis. The midgut gland or "hepatopancreas" of decapods has long been thought to function not only as a site for secretion of digestive enzymes but also as a centre for intermediary metabolism of lipids and carbohydrates. It has, therefore, been suggested that this tissue might be the site of gluconeogenesis (Munday & Poat, 1971; Giles et al., 1975) although other authors have suggested that the gills (Thabrew et al., 1971), haemocytes (Johnston et al., 1971, 1973)or even the abdominal muscles (Eichner & Kaplan, 1977)could act in this capacity. This question remains unresolved, however, for Phillips et al. (1977) were unable to demonstrate the conversion of lactate to glucose in in vitro preparations of "hepatopancreas", gill or haemolymph from C. destructor. In addition, G/tde et ai. (1986) found that, when radio-labelled lactate was injected into the crab Menippe mercenaria, the end-products ofgluconeogenesis occurred in both the "hepatopancreas" and muscles. These authors could not establish whether both tissues were capable of gluconeogenesis or whether giuconeogenesis took place elsewhere and the glucose was transported to these tissues for glycogen synthesis. In a previous paper (Hill et al., 1991), the metabolism of Carcinus maenas (L.) during exposure to environmental anoxia and during subsequent recovery was examined. This study extends this work and considers the fate of L-lactate during the recovery period. MATERIALS AND METHODS
C. maenas were collected by hand from the same location described in the previous
paper (Hill et al., 1991) and were maintained in the laboratory under identical conditions. Only male crabs weighing 3-7 g fresh wt were used in these experiments. Crabs of this size were selected since they regularly occur in littoral rock pools in which they may be exposed to hypoxia (see Hill et al., 1991) and, because their carapace is relatively thin, it was possible to inject radio-labelled compounds into the pericardium without the need to previously drill a small hole through the carapace.
ENERGY METABOLISM OF CARCINAS MAENAS
53
EXPERIMENTAL CONDITIONS AND THE INJECTION OF RADIO-LABELLED COMPOUNDS
Teflon gas-chromatography septa (3 mm diameter) were attached to the carapace of the experimental crabs dorsal to the heart by means ofcyanoacrylate adhesive. This was done to prevent leakage of the injected material into the water. After the attachment of the septa, the crabs were left undisturbed for 48 h in fully aerated seawater to allow complete recovery from handling disturbance. The fate of lactate during recovery from anoxia was investigated by injecting radiolabelled glucose and L-lactate into crabs after exposure to anoxia. D-[ U ~4C]-glucose and L-[U~4C]-lactate (Amersham International)were diluted with a physiological saline (Hill, 1989) to give final specific activities of 0.1 and 0.05 #Ci. #1- ~, respectively. 50 #1 of these solutions were administered using a Hamilton syringe to inject the solutions through the applied septa directly into the pericardium of each crab. In the first experiment, 36 crabs were distributed between four plastic tanks (101) and left undisturbed for 24 h before the Po_, ofthe water in the tanks was reduced to < 1 Torr by bubbling N2 through the water. The crabs were subjected to the same pattern of anoxic and normoxic conditions as described previously (Hill et al., 1991), viz., 12 h anoxia followed by 12 h recovery under normoxia. In addition, a further 12 crabs were kept under normoxic conditions throughout the experiment to act as con,'.rols. ~4C glucose was injected into the experimental crabs after 4 h, i.e., at the start of the period of exposure to fully anoxic conditions (control crabs were also injected), and the crabs quickly returned to the water. This was taken as 0 time in these experiments. In a further set of experiments, the same procedures were used but, in this case, ~4C lactate was injected into the crabs after they had been kept under anoxic conditions for 12 h. The crabs were returned to the tanks and the water made normoxic by aeration except in the case of the control crabs which were exposed to anoxia throughout the experiment. In both sets of experiments, groups of four experimental and control crabs were removed from the tanks at intervals after the injection of metabolites and rapidly frozen in liquid N2. All experiments were carried out at 10 °C. PREPARATION OF TISSUES AND FRACTIONATION OF EXTRACTS
Tissue samples were prepared as described previously (Hill et al., 1991). The acid soluble materials were fractionated by a combination of precipitation and ion-exchange methods as shown in Fig. 1. The ion-exchange chromatography was carried out in 3-5 cm columns in 2-ml disposable plastic syringes and the fractions eluted with up to 10 ml of eluant. After chromatography, the cation and anion exchangers were regenerated using 1 M hydrochloric acid and 1 M sodium hydroxide, followed by 1 M acetic acid, respectively. The columns were then washed with distilled water until neutral. Preliminary experiments had confirmed the effectiveness of the fractionation procedures. Mixtures of known amounts of pure compounds, glycogen, glucose, amino
54
A . D . H I L L E T AL.
Grind tissue in li~u,id nitrogen, extract in 0.6M Perchlodc tctd, centrifuge at 12000g for 10 rains. Add ethanol to 75% to the supematant, leave on ice for 2 hours to precipitate glycogen, then centrifuge again.
I
Remove supematant, ethanol ~luhle fraction.
Dissolve the glycogen in I ml of hot water and centrifuge at 12,000 g for $ minutes Remove supematant.
I
GLYCOGEN (Fi) Add to short column of cation exchange rosin (Dowex 50W X8 in the hydrogen form).
Elute neutral and anionic metabolites with 6 ml of water.
Elute cations with $ ml of 3M ammonium hydroxide.
Add to short column of anionic exdtange resin (Dowex IW X8 in the amate form).
AMINO ACIDS (F2)
Elute with 4 ml of water.
Elute with 7 ml of 5M acetic acid.
I
Elute with $ ml I M hydrochloric acid
I
WEAK ACIDS 11:4)
I
STRONG ACIDS (FS)
NEUTRAL COMPOUNDS IF3)
I
I
Precipitate phosphate esters as barium salts with barium acetate in 50% ethanol.
I
Centrifuge at 12,000 g for IO minutes.
I
I-
,
I
Pellet
Supematant
GLYCOLY'I'IC PItOSPtlATF.S (FSa)
KREBS CYCLE ACIDS (FSb)
Fig. !. T h e elution s c h e m e used to f r a c t i o n a t e p a r t i c u l a r c a t e g o r i e s o f labelled metabolites.
ENERGY METABOLISM OF CARCINAS MAENAS
55
acids (glutamine, alanine and arginine), L-lactate, glucose-6-phosphate and phosphoenolpyruvate were passed through the procedure and recoveries estimated by appropriate specific chemical and enzymic methods (see Hill etal., 1991). The results indicated that recoveries of the known compounds in the appropriate fractions ranged from 86~o for the amino acids to 98 ~o for glucose. APPEARANCE OF RADIOACTIVITY IN THE WATER
Samples were taken at regular intervals throughout the experiments to establish the total radioactivity in the incubation water. Additional samples were then acidified with 0.1 M hydrochloric acid and stirred for 2 h at room temperature in a fume cupboard to drive off acid-labile volatile material. The radioactivity remaining in the water samples was then estimated and the contribution of the acid-labile volatile component (assumed to be CO>/HCOf/CO32- ) was calculated by difference. A further series of experiments was carried out to determine the extent of incorporation of radioactivity into COe under normoxic conditions. Crabs which had been injected with radio-labelled glucose were placed individually in small sealed Perspex chambers (500 ml)containing 300 ml water. The water in the chambers was constantly aerated to drive off the CO: produced which was then collected by passing it through a solution of Hyamine hydroxide (10 ~o solution ofmethylbenzethonium in methanol) contained within a boiling tube. Duplicate samples (0.5 ml) were taken from the chamber and from the boiling tube at regular intervals throughout the experiment. One of the water samples was acidified as described above and then the radioactivity in each of the three samples determined. ESTIMATION OF RADIOACTIVITY
The radioactivity in samples (0.5 ml) of the various fractions (up to 1 ml) and in the water samples was estimated by liquid scintillation counting in 5 ml Ecoscint (Nuclear Medical Electronic Systems & Services Ltd, UK). The efficiency of counting was estimated by the channels ratio method, and cpm corrected to dpm. To eliminate differences between individual crabs, and to obtain a complete picture ofthe distribution of label, the results were expressed on an individual basis as the percentage in each fraction of the total amount of label injected. RESULTS UTILIZATION OF 14C GLUCOSE UNDER NORMOXIC CONDITIONS
The results summarized in Table I show that 6 h after injection of the D-[U'4C] glucose it was possible to account for all of the injected label which was distributed between the acid-soluble material in the whole crab and the surrounding medium. 15% of the total was present in the medium in the form of CO2 and its associated anions. After 12 h, the proportion of fully oxidized C had risen to 37%. By this time, the
56
A.D. HILL ET AL. TABLE I
Metabolism of radioactivity from D-[Ut4C]-glucose injected into C. maenas maintained under normoxic conditions. Values for the % radioactivity in the crab tissues are ~ + SD of at least four crabs. Those for the fractions (F~-Fs) present in the surrounding medium are the ~ values of at least five experiments. Proportion of total radioactivity injected
Location
6h Fi F2
Tissue
F3 F4
F5 Total in tissues Acid labile, volatile Non-acid labile P
Medium
Total recovered
12h
! 1.9 + 2.4 69.5 + 5.6 1.6 + 0.5 1.0 + 0.5 0.6 _+0.2
4.3 + 42.4 + 1.4 + 0.4 + 0.2 +
84.6 14.7 0.9
48.7 36.8 1.3
100.2
86.8
1.8 4. I 0.3 0.1 0.02
F~ glycogen; F2 amino acids; F3, neutral (glucose and oligosacchatides); F,, "weak acid" (lactate); Fs, "strong acid" (glycolytic phosphates and Krebs cycle acids).
proportion of the label which could be accounted for had fallen to 86% of the total. No significant quantity of label in non-acid labile, non-volatile material was found in the medium. Fractionation of the acid-soluble material in crab tissues revealed that by far the greatest amount was present in the amino-acid material. Only 1.6~ of the total was present as glucose and glucose-oligosaccharides. As might be expected under aerobic conditions, there was little radioactivity in the weak acid (lactate) fraction. More surprisingly, perhaps, there was even less in the strong acid fraction (glycolytic phosphates and Krebs cycle acids). After 12 h, the total radioactivity in the major fractions had declined with the increase in the amount excreted in the form of CO2. UTILIZATION OF 14C GLUCOSE UNDER ANOXIA AND DURING RECOVERY UNDER NORMOXIC CONDITIONS
It was found that 1 h after injection of the t4C glucose, 73 + 2% of the radioactivity injected was present in the amino-acid fraction and that this did not alter much throughout the duration of the experiment. Since this tended to obscure changes in the amount of radioactivity present in the other fractions, the amino-acid fraction was omitted from the results, and the fractional distribution based on "non-amino acid, acid-soluble radioactivity". The data presented in Table lI indicate the proportions of radioactivity in non-amino acid fractions obtained from crabs under both aerobic and anaerobic conditions at 6 and 12 h after injection. The changes in the amount of radioactivity in each of the fractions during exposure to anoxia and during subsequent
ENERGY METABOLISM OF C A R C I N A S M A E N A S
57
TABLE II A compariosn of the distribution of radioactivity in non-amino acid fractions from the tissues of C. maenas at~er metabolism of D-[UI4C]-glucose under both aerobic and anaerobic conditions. Values are % of the total radioactivity recovered in the tissue fractions excluding the radioactivity in the amino-acid fraction. All values are means of at least four estimates and have been rounded to the nearest whole number. Fractions are as given in Table I except that: Fsa, glycolytic phosphates; and Fsb, Krebs cycle acids. Fraction
Aerobic 6h
Fi
79
F2
.
F3
11 7 4
1=4 Fs~
Anaerobic 12 h 68
.
.
6h
12 h
58
60
24 8 10 1
16 II 11 3
.
22 6 3
F5b
Anoxia
Recovery
100
•
A
-0° .2,80 e :~
t~
60
40 00"20 0,.
0
I
I
4
8
~
12
i
16
Anoxia
"~ o
I
20
i
!
24
28
Recovery
14
B
12
">' 10
o
=o
8 6-
._
4" 2 0 0
!
!
4
8
!
12
|
16
!
20
i
24
!
28
Time (h) Fig. 2. Time course of incorporation of radioactivity from injected D-[UI4C]-glucose into different metabolic fractions from the tissues of C. maenas during anoxia and during normoxic recovery. Data are for glycogen (O), glucose and oligosaccharides ([]) and lactate (O) in A, and for phosphorylated glycolytic intermediates (1"1) and Krebs cycle acids (O) in B. The ordinate represents the proportion of radioactivity in each fraction as a percentage of the total injected (excluding the amino acid fraction). All values ~_+ SD.
58
A.D. HILL ET AL.
recovery under normoxic conditions are shown in Fig. 2a,b. The percentage of radioactivity in the glucose and oligosaccharide fraction declined slowly, but significantly (P < 0.05), from 35.9% after ! h to 15.6~o after 12 h anoxia. This decline was mirrored qualitatively, but not quantitatively, by the increase in the proportion of radioactivity in lactate (P < 0.01) from 1.9 to 10.7% during the same period. As under aerobic conditions, the greatest amount of radioactivity (50-60% of the total) was found in the glycogen fraction and this did not change significantly during the 12-h period. One clear difference from the aerobic metabolism was the highly significant (P < 0.01) increase in the proportion of radioactivity in the phosphorylated glycolytic intermediates, which increased from 3.6 to 11.4 %. Only a small amount of radioactivity was found in the fraction containing Krebs cycle acids and this did not change significantly during the 12 h that the crabs were exposed to anoxia. The return of normoxic conditions resulted in a dramatic increase in the proportion of radioactivity in lactate which more than doubled to 23.4% during the 1st h. This was accompanied by sharp reductions in the proportion of radioactivity present in both the glycolytic intermediates and in the neutral carbohydrate fraction. During the recovery period, the proportion in glycogen increased to account for almost 90% of the radioactivity, while that in lactate declined to 4%. UTILIZATION OF [
14C] L-LACTATE D U R I N G
RECOVERY F ROM ANOXIA
After the injection of labelled L-lactate, some crabs were kept under anoxia for a further 4 h to act as a control. Unfortunately, almost all of them died. In one survivor, however, it was found that injected radioactivity was retained almost entirely ( > 95 ~o) in the lactate fraction, indicating that, in the absence of air, lactate remained unmeTABLE Ill Metabolism of L-[U ~4C]-lactate during recovery of C. m a e n a s from environmental anoxia. Fractions are as in Table I. Values are ~ _+ so. Location
Proportion of total radioactivity injected
(%)
4h Tissue
Medium
Fi F2 F3 F4 F5 Total in tissues Acid labile, volatile Non-acid labile Total recovered
8h
12.8 +_ 4.5 10.3 _+ 2.5 < 1.0 63.3 _+ 6.1 5.2 __ 2.0
9.4 60.0 < 1.0 17.9 1.9
92.6 7.9 0.4
90.2 9.1 0.4
100.9
99.7
+_ 2.8 __ 4.8 _.+3.4 __ 0.6
ENERGY METABOLISM OF C A R C I N / . S M A E N A S
59
14 m .e,..
0
lO .
........
. . . . .
° ..........
0 G) =.g (g
,p.,,"
Zl
(U i1.
0 0
1
2 Time
after injection
4
6
(h)
Fig. 3. Proportion of m4Cfrom the total injected L-[U~4C]-lactate released by C. maenas into the surrounding medium. The ordinate represents the proportion of radioactivity as a percentage of the total injected under normoxic conditions (O) and under normoxic conditions during recovery from 12 h anoxia (0).
tabolized. The percentages of the total radioactivity found in the different fractions obtained from crabs which were returned to normoxic conditions after the injection of radio-labelled lactate are presented in Table III. In these crabs, the proportion of the radioactivity associated with lactate declined steadily during the recovery period until after 8 h it accounted for < 18% of the original label injected. The proportion in glycogen rapidly reached a plateau, while that in the amino-acid fraction increased by the end cf the 8-h period to proportions found when radio-labelled glucose was injected into crabs maintained under normoxic conditions. Almost no radioactivity appeared in the glucose/oligosaccharide fractions. Radioactivity from the injected lactate appeared as CO2 in the medium rapidly at first, but then more slowly during the later stages of the experiment (Fig. 3). Analysis of the water by the fractionation procedure and subsequent determination of radioactivity indicated that 95 ~ of the radioactivity was in the form of CO2 and its derived anions. DISCUSSION
The results from the control aerobic experiments indicated that the injected glucose was rapidly metabolized and that carbon from glucose, either directly or indirectly, quickly appeared in the medium in the form of CO2, bicarbonate and carbonate. There is no reason to doubt that the normal pathways of glycolysis and the Krebs cycle were operating. The very low proportion of the total radioactivity present in the fraction containing mainly glycolytic intermediates and Krebs cycle acids can be accounted for by the very low steady-state concentration of these metabolites. Although figures are not available for their concentrations in the crab tissues, figures from muscle tissue from other animals, both vertebrate and invertebrate (Essen & Kaijser, 1978; Randle &
60
A.D. I~ ILL ET AL.
Tubbs, 1979; Rowan & Newsholme, 1979), suggest that their combined total will be only 1/tmol. g - 1. Thus, the metabolites in these pathways do not constitute a major pool of radioactivity from glucose. It is rather surprising that so much of the radioactivity should be present in the amino acid fraction. The total concentration of those amino acids which may be expected to be in rapid equilibrium with glycolytic and Krebs cycle intermediates (viz., alanine, glutamate and aspartate) is reported here as being 11.7/~mol • g - t. If all the non-essential amino acids are included, then the total is 42.1 #mol .g-~ (present study). The results suggest a high rate of amino-acid metabolism in major tissues in the crab and an associated high level of transaminase activity. Previously reported values for the rates at which t4C from glucose appears as CO2/HCO; in the medium vary considerably. These have been as high as 34% of the total injected in the xiphosuran chelicerate Limulus polyphemus after 3 h (Stetten, 1982) and as low as 9% in the crab Hemigrapsus nudus after 13 h (Hu, 1958). The values obtained during the present study (8-9% in 6 h) are closer to those found by Hu (1958). The time course of incorporation of label into the various fractions during anaerobiosis and during the subsequent recovery period under normoxic conditions was very similar to the results based on the concentrations of specific metabolites reported previously (Hill et al., 1991). For example, the incorporation of radio-label into lactate from ~4C glucose showed the same initial rise, followed by a plateau after a few hours until, on the re-admission of 02 the rate of incorporation rapidly doubled confirming the previous results which showed a pronounced increase in tissue lactate cc.ncentration during the 1st h of the recovery period (Hill et al., 1991). It is interesting that the proportion of radioactivity in the lactate fraction after 6 h was similar under both aerobic and anaerobic conditions, confirming the rather modest Pasteur effect previously postulated. One rather unexpected result was that, although the proportion of radioactivity in glycogen was lower under anaerobic than aerobic conditions, it still represented the largest single part of the non-amino acid radioactivity. This is difficult to explain but may represent an unexpected turnover of glycogen even under anaerobic conditions. During recovery from anaerobiosis, the radioactivity in glycogen increased (by 20%, P < 0.05) in parallel with the recovery of glycogen concentration, while the proportion in lactate declined by an equal amount, suggesting that most of the lactate formed underwent gluconeogenesis with the final formation of glycogen. The lack of any large transient increase in the radioactivity in mono- and oligo-saccharides, suggests that they may not lie on this pathway of gluconeogenesis. The results obtained from the experiments in which radioactive lactate was injected into the crabs showed that almost all of the label was retained in the crab, to appear in glycogen and then in the amino-acid pool, as was found when glucose was injected into crabs maintained under aerobic conditions. Again, with radio-labelled lactate as the precursor, almost no activity appeared in the mono- and oligo-saccharide fraction, a further indication that, during normoxia and during recovery from anoxia, lactate is
ENERGY METABOLISM OF CARCINAS MAENAS
61
fully oxidised and that, as the label becomes distributed in other metabolic pools, the rate of utilization appears to decline. It is interesting that more lactate appeared to be fully oxidised by resting crabs under normoxic conditions than during the discharge of an O~.debt. This agrees with the findings in the first paper that only part of the increased 02 uptake associated with recovery from anoxia, is actually concerned with the removal of accumulated lactate. One of the unexpected findings of the accompanying paper (Hill et al., 1991) was that the concentration oflactate in the tissues increased sharply after the return to normoxic conditions. The distribution of radio-label confirms that some of the precursors of this had accumulated as glycolytic intermediates, the prcTortion of which increased from almost 0 to 11.5 % during anaerobiosis, and then declined rapidly during the recovery period. This accounted for almost half of the total incorporation of radio-label into lactate, the rest apparently coming from free carbohydrates. Overall, the results of the reported work strengthen the conclusions of the previous study (Hill et al., 1991) that the major response of C. maenas to prolonged anoxia is to greatly reduce its metabolic rate, and that this is associated with inhibition at some point in the pathway of glycolysis, causing the accumulation of intermediatez. The recovery under normoxic conditions is accompanied by a removal of this inhibition, with the consequent surge in tissue lactate concentration. During the present study there was little evidence that C. maenas excreted significant amounts of lactate during the period of recovery from anoxia. Instead, in C. maenas as in Menippe mercenaria (Glide et al., 1986), lactate can be fully oxidised or can undergo gluconeogenesis with the eventual formation of glycogen. Further work is needed, however, to establish the site(s) at which this process takes place. ACKNOWLEDGEMENTS
This work was supported by an NERC Research Studentship to A.D. Hill. REFERENCES Albert, J. L. & W.R. Ellington, 1985. Patterns of energy metabolism in the stone crab, Menippe mercenaria, during severe hypoxia and subsequent recovery. J. Exp. Zool., Vol. 234, pp. i 75-183. Bridges, C. R. & A.R. Brand, 1980. The effect of hypoxia on oxygen consumption and blood lactate levels of some marine Crustacea. Comp. Biochem. Physiol., Vol. 65A, pp. 399-409. Eichner, R. D. & N.O. Kaplan, 1977. Catalytic properties of lactate dehydrogenase in Homarus americanus. Arch. Biochem. Biophys., Vol. 181, pp. 501-507. Ellington, W.R., 1983. The recovery from anaerobic metabolism in invertebrates. J. Exp. Zool., Vol. 228, pp. 431-444. Essen, B. & L. Kaijser, 1978. Regulation ofglycolysis in intermittent exercise in man. ,'. Physiol., Vol. 281, pp. 499-511. Giide, G., R.A. Graham & W.R. Ellington, 1986. Metabolic disposition of lactate in the horseshoe crab Limulus polyphemus and the stone crab Menippe mercenaria. Mar. Biol. Berlin., Vol. 91, pp. 473-479. Giles, I.G., P.C. Poat & K.A. Munday, 1975. Regulation of pyruvate kinase from the hepatopancreas of the crab Carcinus maenas. Biochem. Soc. Trans., Vol. 3, pp. 400-402.
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Head, G. & J. Baldwin, 1986. Energy metabolism and the fate of lactate during recovery from exercise in the Australian freshwater crayfish Cherax destructor. Aust. J. Mar. Freshwater Res., Vol. 37, pp. 641-646. Hill, A.D., 1989. The anaerobic metabolism of the common shore crab, Carcinus maenas (L.) Ph.D. Thesis, University of Glasgow, 185 pp. Hill, A.D., A.C. Taylor & R.H.C. Strang, 1991. Physiological and metabolic responses of the shore crab Carcinus maenas (L.) during environmental anoxia and subsequent recovery. J. Exp. Mar. Biol. Ecol., Vol. 150, pp. 31-50. Hu, A.S.L., 1958. Glucose metabolism in the crab, Hemigrapsus nudus. Arch. Biochem. Biophys., Vol. 75, pp. 387-395. Johnston, M.A., P,S. Davies & H.Y. Elder, 1971. Possible hepatic function for crustacean blood cells. Nature (London), Vol. 230, pp. 471-472. Johnston, M.A., H.Y. Elder & P.S. Davies, 1973. Cytolo~ of the hepatopancreas and blood tissue of Carcinus. Comp. Biochem. Physiol., Vol. 46A, pp. 569-581. Lowery, T.A. & L.G. Tare, 1986. Effect of hypoxia on hemolymph lactate and behaviour of the blue crab Cailinectes sapidus Rathbun in the laboratory and field. Comp. Biochem. Physiol., Vol. 85A, pp. 689-692. Munday, K.A. & P.C. Poat, 1971. Respiration and energy metabolism in Crustacea. In, Chemical zoology, Vol. 6, edited by M. FIorkin & B.T. Scheer, Academic Press, New York, pp. 191-211. Phillips, J.W., R.J.W. McKinney, F.J.R. Hird & D.L. Macmillan, 1977. Lactic acid formation in crustaceans and the liver function of the midgut questioned. Comp. Biochem. Physiol., Vol. 56B, pp. 427-433. Randle, P.J. & P.M. Tubbs, 1979. Carbohydrate and fatty acid metabolism. In, Handbook of Physiology: the cardiovascular system, VoL 1, edited by R.M. Berne, American Physiological Society, Bethesda, pp. 805-844. Rowan, A.M. & E.A. Newsholme, 1979. Changes in content of adenine nucleotides and intermediates of glycolysis and citric acid cycle in flight muscle of the locust on flight. Biochem..J., Vol. 178, pp. 209-216. Stetten, M.R., 1982. Metabolism of glucose and glycogen in Limulus polyphemus in vivo. Comp. Biochem. Physiol., Vol. 73B, pp. 803-813. Taylor, A.C. & J.I. Spicer, 1987. Metabolic responses of the prawns Palaemon elegans (Rathke) and P. serratus (Pennant) (Crustacea: Decapoda) to acute hypoxia and anoxia. Mar. Biol., Berlin, Vol. 95, pp. 521-530. Teal, J.M. & F.G. Carey, 1967. The metabolism of marsh crabs under conditions of reduced oxygen pressure. Physiol. Zool., Vol. 40, pp. 83-91. Thabrew, M. I., P.C. Poat & K.A. Munday, 1971. Carbohydrate metabolism in Carcinus maenas gill tissue. Comp. Biochem. Physwl., Vol. 40B, pp. 531-541. Van Aardt, W.J., 1988. Lactate metabolism and glucose patterns in the river crab, Potamonautes warreni Caiman, during anoxia and subsequent recovery. Comp. Biochem. Physiol., Vol. 91A, pp. 299-304.
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© 1991 Elsevier Science Publishers B.V. 0022-0981/91/$03.50
JEMBE 01612
Radioisotope studies of the energy metabolism of the shore crab Carcinus m a e n a s (L.) during environmental anoxia and recovery A.D. Hill t, R. H.C. Strang 2 and A.C. Taylor ! ~Department of Zoology, 2Department of Biochemistry, University of Glasgow, Glasgow, UK (Received 9 November 1990; revision received 25 February 1991; accepted 9 March 1991) Abstract: Under both normoxic and anoxic conditions, ~4C from D-[UNC]-glucoseinjected into the haemolymph of Carcinus maenas (L.) was rapidly incorporated into various metabolic fractions. Under both conditions, the greatest proportion oflabei was found in the amino acid fraction (> 70%). During normoxia, the steady accumulation of acid labile, volatile ~4C in the water indicated the complete oxidation of the glucose to CO2. Under both normoxia and anoxia, a considerable part of the label appeared in glycogen. The main differences between aerobic and anaerobic conditions were (a) the slower disappearance oflabel from the neutral sugar fraction and (b)the accumulation of label in glycolytic phosphate material under anaerobic conditions. The proportion of label in lactate doubled during the I st h of the recovery period under normoxic conditions, increasing to a maximum of 23 % ofthe non-amino acid radioactivity before decreasing during the rest of the recovery period. Label from L-[Um4C]-lactate injected into crabs immediately before they were returned to normoxic conditions following exposure to anoxia was partly oxidised, appearing as acid labile material in the medium. Most of the injected label, however, was retained within the animal and appeared as glycogen and subsequently in the amino acid fraction. There was no evidence for the excretion of lactate. Key words: Anoxia; Crustacean; L-Lactate; Metabolism; Radioisotope
INTRODUCTION
The anaerobic metabolism of decapod crustaceans during both environmental and functional (exercise) anaerobiosis has been the subject of a number of studies (see Hill et al., 1991). Information on the metabolism of decapods during periods of recovery from anaerobiosis is, however, more limited. In particular, the fate of lactate during the period of recovery is still unclear. The available information suggests that, in most decapods, little if any lactate is excreted (Phillips et al., 1977; Bridges & Brand, 1980; EUington, 1983; G~de et al., 1986). Other studies, however, have found evidence of lactate excretion. Van Aardt (1988), in his study of the freshwater crab Potamonautes warreni, found a significant difference between experimental and control crabs in the amount of acid-stable radioactivity in the incubation water. Similarly, Head & Baldwin Correspondence address" A.D. Hill, Shell Research Ltd, Sittingbourne Research Centre, Sittingbourne, Kent ME9 8AG, UK.
52
A.D. HILL ET AL.
(1986) concluded that, in the crayfish Cherax destructor, most of the lactate produced during exercise anaerobiosis was excreted rather than metabolized. There is growing evidence to indicate that, in the majority of decapods, lactate is metabolized during the recovery period and the occurrence of gluconeogenesis in decapods during recovery from anaerobiosis has now been clearly established (Phillips et al., 1977; G/ide et al., 1986; Van Aardt, 1988). In comparison with vertebrates, however, the rates of gluconeogenesis are very slow and it may take many hours for lactate concentrations in the haemolymph and in the tissues to return to normal levels (Teal & Carey, 1967; Bridges & Brand, 1980; Albert & Ellington, 1985; Lowery & Tate, 1986; Taylor & Spicer, 1987; Van Aardt, 1988). There is still some controversy about the site(s) ofgluconeogenesis. The midgut gland or "hepatopancreas" of decapods has long been thought to function not only as a site for secretion of digestive enzymes but also as a centre for intermediary metabolism of lipids and carbohydrates. It has, therefore, been suggested that this tissue might be the site of gluconeogenesis (Munday & Poat, 1971; Giles et al., 1975) although other authors have suggested that the gills (Thabrew et al., 1971), haemocytes (Johnston et al., 1971, 1973)or even the abdominal muscles (Eichner & Kaplan, 1977)could act in this capacity. This question remains unresolved, however, for Phillips et al. (1977) were unable to demonstrate the conversion of lactate to glucose in in vitro preparations of "hepatopancreas", gill or haemolymph from C. destructor. In addition, G/tde et ai. (1986) found that, when radio-labelled lactate was injected into the crab Menippe mercenaria, the end-products ofgluconeogenesis occurred in both the "hepatopancreas" and muscles. These authors could not establish whether both tissues were capable of gluconeogenesis or whether giuconeogenesis took place elsewhere and the glucose was transported to these tissues for glycogen synthesis. In a previous paper (Hill et al., 1991), the metabolism of Carcinus maenas (L.) during exposure to environmental anoxia and during subsequent recovery was examined. This study extends this work and considers the fate of L-lactate during the recovery period. MATERIALS AND METHODS
C. maenas were collected by hand from the same location described in the previous
paper (Hill et al., 1991) and were maintained in the laboratory under identical conditions. Only male crabs weighing 3-7 g fresh wt were used in these experiments. Crabs of this size were selected since they regularly occur in littoral rock pools in which they may be exposed to hypoxia (see Hill et al., 1991) and, because their carapace is relatively thin, it was possible to inject radio-labelled compounds into the pericardium without the need to previously drill a small hole through the carapace.
ENERGY METABOLISM OF CARCINAS MAENAS
53
EXPERIMENTAL CONDITIONS AND THE INJECTION OF RADIO-LABELLED COMPOUNDS
Teflon gas-chromatography septa (3 mm diameter) were attached to the carapace of the experimental crabs dorsal to the heart by means ofcyanoacrylate adhesive. This was done to prevent leakage of the injected material into the water. After the attachment of the septa, the crabs were left undisturbed for 48 h in fully aerated seawater to allow complete recovery from handling disturbance. The fate of lactate during recovery from anoxia was investigated by injecting radiolabelled glucose and L-lactate into crabs after exposure to anoxia. D-[ U ~4C]-glucose and L-[U~4C]-lactate (Amersham International)were diluted with a physiological saline (Hill, 1989) to give final specific activities of 0.1 and 0.05 #Ci. #1- ~, respectively. 50 #1 of these solutions were administered using a Hamilton syringe to inject the solutions through the applied septa directly into the pericardium of each crab. In the first experiment, 36 crabs were distributed between four plastic tanks (101) and left undisturbed for 24 h before the Po_, ofthe water in the tanks was reduced to < 1 Torr by bubbling N2 through the water. The crabs were subjected to the same pattern of anoxic and normoxic conditions as described previously (Hill et al., 1991), viz., 12 h anoxia followed by 12 h recovery under normoxia. In addition, a further 12 crabs were kept under normoxic conditions throughout the experiment to act as con,'.rols. ~4C glucose was injected into the experimental crabs after 4 h, i.e., at the start of the period of exposure to fully anoxic conditions (control crabs were also injected), and the crabs quickly returned to the water. This was taken as 0 time in these experiments. In a further set of experiments, the same procedures were used but, in this case, ~4C lactate was injected into the crabs after they had been kept under anoxic conditions for 12 h. The crabs were returned to the tanks and the water made normoxic by aeration except in the case of the control crabs which were exposed to anoxia throughout the experiment. In both sets of experiments, groups of four experimental and control crabs were removed from the tanks at intervals after the injection of metabolites and rapidly frozen in liquid N2. All experiments were carried out at 10 °C. PREPARATION OF TISSUES AND FRACTIONATION OF EXTRACTS
Tissue samples were prepared as described previously (Hill et al., 1991). The acid soluble materials were fractionated by a combination of precipitation and ion-exchange methods as shown in Fig. 1. The ion-exchange chromatography was carried out in 3-5 cm columns in 2-ml disposable plastic syringes and the fractions eluted with up to 10 ml of eluant. After chromatography, the cation and anion exchangers were regenerated using 1 M hydrochloric acid and 1 M sodium hydroxide, followed by 1 M acetic acid, respectively. The columns were then washed with distilled water until neutral. Preliminary experiments had confirmed the effectiveness of the fractionation procedures. Mixtures of known amounts of pure compounds, glycogen, glucose, amino
54
A . D . H I L L E T AL.
Grind tissue in li~u,id nitrogen, extract in 0.6M Perchlodc tctd, centrifuge at 12000g for 10 rains. Add ethanol to 75% to the supematant, leave on ice for 2 hours to precipitate glycogen, then centrifuge again.
I
Remove supematant, ethanol ~luhle fraction.
Dissolve the glycogen in I ml of hot water and centrifuge at 12,000 g for $ minutes Remove supematant.
I
GLYCOGEN (Fi) Add to short column of cation exchange rosin (Dowex 50W X8 in the hydrogen form).
Elute neutral and anionic metabolites with 6 ml of water.
Elute cations with $ ml of 3M ammonium hydroxide.
Add to short column of anionic exdtange resin (Dowex IW X8 in the amate form).
AMINO ACIDS (F2)
Elute with 4 ml of water.
Elute with 7 ml of 5M acetic acid.
I
Elute with $ ml I M hydrochloric acid
I
WEAK ACIDS 11:4)
I
STRONG ACIDS (FS)
NEUTRAL COMPOUNDS IF3)
I
I
Precipitate phosphate esters as barium salts with barium acetate in 50% ethanol.
I
Centrifuge at 12,000 g for IO minutes.
I
I-
,
I
Pellet
Supematant
GLYCOLY'I'IC PItOSPtlATF.S (FSa)
KREBS CYCLE ACIDS (FSb)
Fig. !. T h e elution s c h e m e used to f r a c t i o n a t e p a r t i c u l a r c a t e g o r i e s o f labelled metabolites.
ENERGY METABOLISM OF CARCINAS MAENAS
55
acids (glutamine, alanine and arginine), L-lactate, glucose-6-phosphate and phosphoenolpyruvate were passed through the procedure and recoveries estimated by appropriate specific chemical and enzymic methods (see Hill etal., 1991). The results indicated that recoveries of the known compounds in the appropriate fractions ranged from 86~o for the amino acids to 98 ~o for glucose. APPEARANCE OF RADIOACTIVITY IN THE WATER
Samples were taken at regular intervals throughout the experiments to establish the total radioactivity in the incubation water. Additional samples were then acidified with 0.1 M hydrochloric acid and stirred for 2 h at room temperature in a fume cupboard to drive off acid-labile volatile material. The radioactivity remaining in the water samples was then estimated and the contribution of the acid-labile volatile component (assumed to be CO>/HCOf/CO32- ) was calculated by difference. A further series of experiments was carried out to determine the extent of incorporation of radioactivity into COe under normoxic conditions. Crabs which had been injected with radio-labelled glucose were placed individually in small sealed Perspex chambers (500 ml)containing 300 ml water. The water in the chambers was constantly aerated to drive off the CO: produced which was then collected by passing it through a solution of Hyamine hydroxide (10 ~o solution ofmethylbenzethonium in methanol) contained within a boiling tube. Duplicate samples (0.5 ml) were taken from the chamber and from the boiling tube at regular intervals throughout the experiment. One of the water samples was acidified as described above and then the radioactivity in each of the three samples determined. ESTIMATION OF RADIOACTIVITY
The radioactivity in samples (0.5 ml) of the various fractions (up to 1 ml) and in the water samples was estimated by liquid scintillation counting in 5 ml Ecoscint (Nuclear Medical Electronic Systems & Services Ltd, UK). The efficiency of counting was estimated by the channels ratio method, and cpm corrected to dpm. To eliminate differences between individual crabs, and to obtain a complete picture ofthe distribution of label, the results were expressed on an individual basis as the percentage in each fraction of the total amount of label injected. RESULTS UTILIZATION OF 14C GLUCOSE UNDER NORMOXIC CONDITIONS
The results summarized in Table I show that 6 h after injection of the D-[U'4C] glucose it was possible to account for all of the injected label which was distributed between the acid-soluble material in the whole crab and the surrounding medium. 15% of the total was present in the medium in the form of CO2 and its associated anions. After 12 h, the proportion of fully oxidized C had risen to 37%. By this time, the
56
A.D. HILL ET AL. TABLE I
Metabolism of radioactivity from D-[Ut4C]-glucose injected into C. maenas maintained under normoxic conditions. Values for the % radioactivity in the crab tissues are ~ + SD of at least four crabs. Those for the fractions (F~-Fs) present in the surrounding medium are the ~ values of at least five experiments. Proportion of total radioactivity injected
Location
6h Fi F2
Tissue
F3 F4
F5 Total in tissues Acid labile, volatile Non-acid labile P
Medium
Total recovered
12h
! 1.9 + 2.4 69.5 + 5.6 1.6 + 0.5 1.0 + 0.5 0.6 _+0.2
4.3 + 42.4 + 1.4 + 0.4 + 0.2 +
84.6 14.7 0.9
48.7 36.8 1.3
100.2
86.8
1.8 4. I 0.3 0.1 0.02
F~ glycogen; F2 amino acids; F3, neutral (glucose and oligosacchatides); F,, "weak acid" (lactate); Fs, "strong acid" (glycolytic phosphates and Krebs cycle acids).
proportion of the label which could be accounted for had fallen to 86% of the total. No significant quantity of label in non-acid labile, non-volatile material was found in the medium. Fractionation of the acid-soluble material in crab tissues revealed that by far the greatest amount was present in the amino-acid material. Only 1.6~ of the total was present as glucose and glucose-oligosaccharides. As might be expected under aerobic conditions, there was little radioactivity in the weak acid (lactate) fraction. More surprisingly, perhaps, there was even less in the strong acid fraction (glycolytic phosphates and Krebs cycle acids). After 12 h, the total radioactivity in the major fractions had declined with the increase in the amount excreted in the form of CO2. UTILIZATION OF 14C GLUCOSE UNDER ANOXIA AND DURING RECOVERY UNDER NORMOXIC CONDITIONS
It was found that 1 h after injection of the t4C glucose, 73 + 2% of the radioactivity injected was present in the amino-acid fraction and that this did not alter much throughout the duration of the experiment. Since this tended to obscure changes in the amount of radioactivity present in the other fractions, the amino-acid fraction was omitted from the results, and the fractional distribution based on "non-amino acid, acid-soluble radioactivity". The data presented in Table lI indicate the proportions of radioactivity in non-amino acid fractions obtained from crabs under both aerobic and anaerobic conditions at 6 and 12 h after injection. The changes in the amount of radioactivity in each of the fractions during exposure to anoxia and during subsequent
ENERGY METABOLISM OF C A R C I N A S M A E N A S
57
TABLE II A compariosn of the distribution of radioactivity in non-amino acid fractions from the tissues of C. maenas at~er metabolism of D-[UI4C]-glucose under both aerobic and anaerobic conditions. Values are % of the total radioactivity recovered in the tissue fractions excluding the radioactivity in the amino-acid fraction. All values are means of at least four estimates and have been rounded to the nearest whole number. Fractions are as given in Table I except that: Fsa, glycolytic phosphates; and Fsb, Krebs cycle acids. Fraction
Aerobic 6h
Fi
79
F2
.
F3
11 7 4
1=4 Fs~
Anaerobic 12 h 68
.
.
6h
12 h
58
60
24 8 10 1
16 II 11 3
.
22 6 3
F5b
Anoxia
Recovery
100
•
A
-0° .2,80 e :~
t~
60
40 00"20 0,.
0
I
I
4
8
~
12
i
16
Anoxia
"~ o
I
20
i
!
24
28
Recovery
14
B
12
">' 10
o
=o
8 6-
._
4" 2 0 0
!
!
4
8
!
12
|
16
!
20
i
24
!
28
Time (h) Fig. 2. Time course of incorporation of radioactivity from injected D-[UI4C]-glucose into different metabolic fractions from the tissues of C. maenas during anoxia and during normoxic recovery. Data are for glycogen (O), glucose and oligosaccharides ([]) and lactate (O) in A, and for phosphorylated glycolytic intermediates (1"1) and Krebs cycle acids (O) in B. The ordinate represents the proportion of radioactivity in each fraction as a percentage of the total injected (excluding the amino acid fraction). All values ~_+ SD.
58
A.D. HILL ET AL.
recovery under normoxic conditions are shown in Fig. 2a,b. The percentage of radioactivity in the glucose and oligosaccharide fraction declined slowly, but significantly (P < 0.05), from 35.9% after ! h to 15.6~o after 12 h anoxia. This decline was mirrored qualitatively, but not quantitatively, by the increase in the proportion of radioactivity in lactate (P < 0.01) from 1.9 to 10.7% during the same period. As under aerobic conditions, the greatest amount of radioactivity (50-60% of the total) was found in the glycogen fraction and this did not change significantly during the 12-h period. One clear difference from the aerobic metabolism was the highly significant (P < 0.01) increase in the proportion of radioactivity in the phosphorylated glycolytic intermediates, which increased from 3.6 to 11.4 %. Only a small amount of radioactivity was found in the fraction containing Krebs cycle acids and this did not change significantly during the 12 h that the crabs were exposed to anoxia. The return of normoxic conditions resulted in a dramatic increase in the proportion of radioactivity in lactate which more than doubled to 23.4% during the 1st h. This was accompanied by sharp reductions in the proportion of radioactivity present in both the glycolytic intermediates and in the neutral carbohydrate fraction. During the recovery period, the proportion in glycogen increased to account for almost 90% of the radioactivity, while that in lactate declined to 4%. UTILIZATION OF [
14C] L-LACTATE D U R I N G
RECOVERY F ROM ANOXIA
After the injection of labelled L-lactate, some crabs were kept under anoxia for a further 4 h to act as a control. Unfortunately, almost all of them died. In one survivor, however, it was found that injected radioactivity was retained almost entirely ( > 95 ~o) in the lactate fraction, indicating that, in the absence of air, lactate remained unmeTABLE Ill Metabolism of L-[U ~4C]-lactate during recovery of C. m a e n a s from environmental anoxia. Fractions are as in Table I. Values are ~ _+ so. Location
Proportion of total radioactivity injected
(%)
4h Tissue
Medium
Fi F2 F3 F4 F5 Total in tissues Acid labile, volatile Non-acid labile Total recovered
8h
12.8 +_ 4.5 10.3 _+ 2.5 < 1.0 63.3 _+ 6.1 5.2 __ 2.0
9.4 60.0 < 1.0 17.9 1.9
92.6 7.9 0.4
90.2 9.1 0.4
100.9
99.7
+_ 2.8 __ 4.8 _.+3.4 __ 0.6
ENERGY METABOLISM OF C A R C I N / . S M A E N A S
59
14 m .e,..
0
lO .
........
. . . . .
° ..........
0 G) =.g (g
,p.,,"
Zl
(U i1.
0 0
1
2 Time
after injection
4
6
(h)
Fig. 3. Proportion of m4Cfrom the total injected L-[U~4C]-lactate released by C. maenas into the surrounding medium. The ordinate represents the proportion of radioactivity as a percentage of the total injected under normoxic conditions (O) and under normoxic conditions during recovery from 12 h anoxia (0).
tabolized. The percentages of the total radioactivity found in the different fractions obtained from crabs which were returned to normoxic conditions after the injection of radio-labelled lactate are presented in Table III. In these crabs, the proportion of the radioactivity associated with lactate declined steadily during the recovery period until after 8 h it accounted for < 18% of the original label injected. The proportion in glycogen rapidly reached a plateau, while that in the amino-acid fraction increased by the end cf the 8-h period to proportions found when radio-labelled glucose was injected into crabs maintained under normoxic conditions. Almost no radioactivity appeared in the glucose/oligosaccharide fractions. Radioactivity from the injected lactate appeared as CO2 in the medium rapidly at first, but then more slowly during the later stages of the experiment (Fig. 3). Analysis of the water by the fractionation procedure and subsequent determination of radioactivity indicated that 95 ~ of the radioactivity was in the form of CO2 and its derived anions. DISCUSSION
The results from the control aerobic experiments indicated that the injected glucose was rapidly metabolized and that carbon from glucose, either directly or indirectly, quickly appeared in the medium in the form of CO2, bicarbonate and carbonate. There is no reason to doubt that the normal pathways of glycolysis and the Krebs cycle were operating. The very low proportion of the total radioactivity present in the fraction containing mainly glycolytic intermediates and Krebs cycle acids can be accounted for by the very low steady-state concentration of these metabolites. Although figures are not available for their concentrations in the crab tissues, figures from muscle tissue from other animals, both vertebrate and invertebrate (Essen & Kaijser, 1978; Randle &
60
A.D. I~ ILL ET AL.
Tubbs, 1979; Rowan & Newsholme, 1979), suggest that their combined total will be only 1/tmol. g - 1. Thus, the metabolites in these pathways do not constitute a major pool of radioactivity from glucose. It is rather surprising that so much of the radioactivity should be present in the amino acid fraction. The total concentration of those amino acids which may be expected to be in rapid equilibrium with glycolytic and Krebs cycle intermediates (viz., alanine, glutamate and aspartate) is reported here as being 11.7/~mol • g - t. If all the non-essential amino acids are included, then the total is 42.1 #mol .g-~ (present study). The results suggest a high rate of amino-acid metabolism in major tissues in the crab and an associated high level of transaminase activity. Previously reported values for the rates at which t4C from glucose appears as CO2/HCO; in the medium vary considerably. These have been as high as 34% of the total injected in the xiphosuran chelicerate Limulus polyphemus after 3 h (Stetten, 1982) and as low as 9% in the crab Hemigrapsus nudus after 13 h (Hu, 1958). The values obtained during the present study (8-9% in 6 h) are closer to those found by Hu (1958). The time course of incorporation of label into the various fractions during anaerobiosis and during the subsequent recovery period under normoxic conditions was very similar to the results based on the concentrations of specific metabolites reported previously (Hill et al., 1991). For example, the incorporation of radio-label into lactate from ~4C glucose showed the same initial rise, followed by a plateau after a few hours until, on the re-admission of 02 the rate of incorporation rapidly doubled confirming the previous results which showed a pronounced increase in tissue lactate cc.ncentration during the 1st h of the recovery period (Hill et al., 1991). It is interesting that the proportion of radioactivity in the lactate fraction after 6 h was similar under both aerobic and anaerobic conditions, confirming the rather modest Pasteur effect previously postulated. One rather unexpected result was that, although the proportion of radioactivity in glycogen was lower under anaerobic than aerobic conditions, it still represented the largest single part of the non-amino acid radioactivity. This is difficult to explain but may represent an unexpected turnover of glycogen even under anaerobic conditions. During recovery from anaerobiosis, the radioactivity in glycogen increased (by 20%, P < 0.05) in parallel with the recovery of glycogen concentration, while the proportion in lactate declined by an equal amount, suggesting that most of the lactate formed underwent gluconeogenesis with the final formation of glycogen. The lack of any large transient increase in the radioactivity in mono- and oligo-saccharides, suggests that they may not lie on this pathway of gluconeogenesis. The results obtained from the experiments in which radioactive lactate was injected into the crabs showed that almost all of the label was retained in the crab, to appear in glycogen and then in the amino-acid pool, as was found when glucose was injected into crabs maintained under aerobic conditions. Again, with radio-labelled lactate as the precursor, almost no activity appeared in the mono- and oligo-saccharide fraction, a further indication that, during normoxia and during recovery from anoxia, lactate is
ENERGY METABOLISM OF CARCINAS MAENAS
61
fully oxidised and that, as the label becomes distributed in other metabolic pools, the rate of utilization appears to decline. It is interesting that more lactate appeared to be fully oxidised by resting crabs under normoxic conditions than during the discharge of an O~.debt. This agrees with the findings in the first paper that only part of the increased 02 uptake associated with recovery from anoxia, is actually concerned with the removal of accumulated lactate. One of the unexpected findings of the accompanying paper (Hill et al., 1991) was that the concentration oflactate in the tissues increased sharply after the return to normoxic conditions. The distribution of radio-label confirms that some of the precursors of this had accumulated as glycolytic intermediates, the prcTortion of which increased from almost 0 to 11.5 % during anaerobiosis, and then declined rapidly during the recovery period. This accounted for almost half of the total incorporation of radio-label into lactate, the rest apparently coming from free carbohydrates. Overall, the results of the reported work strengthen the conclusions of the previous study (Hill et al., 1991) that the major response of C. maenas to prolonged anoxia is to greatly reduce its metabolic rate, and that this is associated with inhibition at some point in the pathway of glycolysis, causing the accumulation of intermediatez. The recovery under normoxic conditions is accompanied by a removal of this inhibition, with the consequent surge in tissue lactate concentration. During the present study there was little evidence that C. maenas excreted significant amounts of lactate during the period of recovery from anoxia. Instead, in C. maenas as in Menippe mercenaria (Glide et al., 1986), lactate can be fully oxidised or can undergo gluconeogenesis with the eventual formation of glycogen. Further work is needed, however, to establish the site(s) at which this process takes place. ACKNOWLEDGEMENTS
This work was supported by an NERC Research Studentship to A.D. Hill. REFERENCES Albert, J. L. & W.R. Ellington, 1985. Patterns of energy metabolism in the stone crab, Menippe mercenaria, during severe hypoxia and subsequent recovery. J. Exp. Zool., Vol. 234, pp. i 75-183. Bridges, C. R. & A.R. Brand, 1980. The effect of hypoxia on oxygen consumption and blood lactate levels of some marine Crustacea. Comp. Biochem. Physiol., Vol. 65A, pp. 399-409. Eichner, R. D. & N.O. Kaplan, 1977. Catalytic properties of lactate dehydrogenase in Homarus americanus. Arch. Biochem. Biophys., Vol. 181, pp. 501-507. Ellington, W.R., 1983. The recovery from anaerobic metabolism in invertebrates. J. Exp. Zool., Vol. 228, pp. 431-444. Essen, B. & L. Kaijser, 1978. Regulation ofglycolysis in intermittent exercise in man. ,'. Physiol., Vol. 281, pp. 499-511. Giide, G., R.A. Graham & W.R. Ellington, 1986. Metabolic disposition of lactate in the horseshoe crab Limulus polyphemus and the stone crab Menippe mercenaria. Mar. Biol. Berlin., Vol. 91, pp. 473-479. Giles, I.G., P.C. Poat & K.A. Munday, 1975. Regulation of pyruvate kinase from the hepatopancreas of the crab Carcinus maenas. Biochem. Soc. Trans., Vol. 3, pp. 400-402.
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A.D. HILL El" AL.
Head, G. & J. Baldwin, 1986. Energy metabolism and the fate of lactate during recovery from exercise in the Australian freshwater crayfish Cherax destructor. Aust. J. Mar. Freshwater Res., Vol. 37, pp. 641-646. Hill, A.D., 1989. The anaerobic metabolism of the common shore crab, Carcinus maenas (L.) Ph.D. Thesis, University of Glasgow, 185 pp. Hill, A.D., A.C. Taylor & R.H.C. Strang, 1991. Physiological and metabolic responses of the shore crab Carcinus maenas (L.) during environmental anoxia and subsequent recovery. J. Exp. Mar. Biol. Ecol., Vol. 150, pp. 31-50. Hu, A.S.L., 1958. Glucose metabolism in the crab, Hemigrapsus nudus. Arch. Biochem. Biophys., Vol. 75, pp. 387-395. Johnston, M.A., P,S. Davies & H.Y. Elder, 1971. Possible hepatic function for crustacean blood cells. Nature (London), Vol. 230, pp. 471-472. Johnston, M.A., H.Y. Elder & P.S. Davies, 1973. Cytolo~ of the hepatopancreas and blood tissue of Carcinus. Comp. Biochem. Physiol., Vol. 46A, pp. 569-581. Lowery, T.A. & L.G. Tare, 1986. Effect of hypoxia on hemolymph lactate and behaviour of the blue crab Cailinectes sapidus Rathbun in the laboratory and field. Comp. Biochem. Physiol., Vol. 85A, pp. 689-692. Munday, K.A. & P.C. Poat, 1971. Respiration and energy metabolism in Crustacea. In, Chemical zoology, Vol. 6, edited by M. FIorkin & B.T. Scheer, Academic Press, New York, pp. 191-211. Phillips, J.W., R.J.W. McKinney, F.J.R. Hird & D.L. Macmillan, 1977. Lactic acid formation in crustaceans and the liver function of the midgut questioned. Comp. Biochem. Physiol., Vol. 56B, pp. 427-433. Randle, P.J. & P.M. Tubbs, 1979. Carbohydrate and fatty acid metabolism. In, Handbook of Physiology: the cardiovascular system, VoL 1, edited by R.M. Berne, American Physiological Society, Bethesda, pp. 805-844. Rowan, A.M. & E.A. Newsholme, 1979. Changes in content of adenine nucleotides and intermediates of glycolysis and citric acid cycle in flight muscle of the locust on flight. Biochem..J., Vol. 178, pp. 209-216. Stetten, M.R., 1982. Metabolism of glucose and glycogen in Limulus polyphemus in vivo. Comp. Biochem. Physiol., Vol. 73B, pp. 803-813. Taylor, A.C. & J.I. Spicer, 1987. Metabolic responses of the prawns Palaemon elegans (Rathke) and P. serratus (Pennant) (Crustacea: Decapoda) to acute hypoxia and anoxia. Mar. Biol., Berlin, Vol. 95, pp. 521-530. Teal, J.M. & F.G. Carey, 1967. The metabolism of marsh crabs under conditions of reduced oxygen pressure. Physiol. Zool., Vol. 40, pp. 83-91. Thabrew, M. I., P.C. Poat & K.A. Munday, 1971. Carbohydrate metabolism in Carcinus maenas gill tissue. Comp. Biochem. Physwl., Vol. 40B, pp. 531-541. Van Aardt, W.J., 1988. Lactate metabolism and glucose patterns in the river crab, Potamonautes warreni Caiman, during anoxia and subsequent recovery. Comp. Biochem. Physiol., Vol. 91A, pp. 299-304.