Marine Biology (2001) 139: 335±342 DOI 10.1007/s002270100569
G.T. Oliveira á I.C. Rossi á R.S.M. da Silva
Carbohydrate metabolism during anoxia and post-anoxia recovery in Chasmagnathus granulata crabs maintained on high-protein or carbohydrate-rich diets
Received: 22 September 2000 / Accepted: 19 February 2001 / Published online: 12 May 2001 Ó Springer-Verlag 2001
Abstract We assessed the eects of anoxia exposure and recovery on glycogen synthesis and mobilization, glucose uptake, and on the enzymes that control carbohydrate metabolism in the hepatopancreas of Chasmagnathus granulata crabs receiving either a carbohydrate-rich (HC) or a high-protein diet (HP). In both dietary groups, anoxia led to a reduction in glucose uptake and in glycogen synthesis, and to an increase in hepatopancreas glycogen mobilization and in hemolymph glucose concentration. During the ®rst 4 h of exposure to anoxia, total glycogen phosphorylase (GPT) and a form activity increased in HP and HC crabs, leading to a decrease in hepatopancreas glycogen concentration. During recovery, HP and HC crabs rapidly restored the hemolymph glucose levels to pre-anoxia concentrations. In HC crabs, incorporation of 14C from glucose into glycogen increased gradually after 12 h in normoxia, leading to restoration of glycogen concentration. Also during recovery, the ratio of glycogen synthase I (GSI) to glycogen phosphorylase a (GPa) increased in the HC group. In turn, recovering HP crabs had two peaks of glycogen synthesis, related with two peaks in the ratio of GSI to GPa. Consequently, no mobilization of 14C-glycogen occurred in recovering HP animals. Anoxia in C. granulata induces a marked decrease in the synthesis of carbohydrate reserves that is accompanied by an increase in glycogen mobilization and in circulating glucose levels. During the recovery period, there is an activation of endergonic processes which cause a decrease in hemolymph glucose levels. In C. granulata, glycogen metabolism seems to be controlled by the ratio of the GSI form to the GPa form. In ®eld conditions, theses changes in the Communicated by O. Kinne, Oldendorf/Luhe G.T. Oliveira á I.C. Rossi á R.S.M. da Silva (&) Department of Physiology, Instituto de CieÃncias BaÂsicas da SauÂde, Universidade Federal do Rio Grande do Sul, Rua Sarmento Leite, 500, 90050-170 Porto Alegre, RS, Brazil E-mail:
[email protected] Fax: +55-51-3163166
metabolic pattern may result from environmental PO2 availability. In the winter, C. granulata stays in its holes, where environmental PO2 falls to zero. The carbohydrate or protein content of the diets administrated to the crabs seem to induce dierent metabolic adjustments during anoxia and recovery.
Introduction The ecophysiological peculiarities of crustaceans show diverse adaptive metabolic mechanisms. Three modes of adaptation that permit survival in environmental hypoxia or anoxia have been identi®ed in dierent crustacean species: (1) maintenance of large stores of glycogen in all tissues under aerobic conditions; (2) utilization of anaerobic pathways to produce ATP and to maintain redox balance in anaerobic conditions; and (3) reduction of metabolic rates (Hervant et al. 1995; Childress and Seidel 1998). The amphipod species Niphargus rhenorhodanensis and Gammarus fossarum have a high survival time under severe hypoxia (Hervant et al. 1995; Hervant 1996); this seems to result from a great concentration of glycogen and phosphogen in tissues, from a low metabolic rate in normoxia, from the reduction in metabolic and glycolytic rates during hypoxia, and from rapidly re-synthesized glycogen stores during post-hypoxia recovery (Hervant et al. 1999a,b). The duration and eectiveness of recovery are of great functional importance, since during this period energy reserves are restored and accumulated end products are completely removed. Glycogen synthesis and mobilization, glucose uptake, and regulation of enzymes involved in the control of carbohydrate metabolism during anoxia and recovery have received relatively little attention in crustaceans. In contrast to other invertebrates, crustaceans utilize only one basic pathway of anaerobic glycolysis, that is, fermentation of glycogen into lactate (Bridges and Brand 1980; Aardt 1988; Hervant et al. 1995, 1997, 1999a,b).
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Previous studies showed that, as in other crustaceans, in C. granulata anoxia stress induces a marked increase in L-lactate hemolymph concentration, and a slow return to basal levels during the recovery period (Oliveira 1998). In C. granulata crabs adapted to a carbohydrate-rich diet, the levels of hepatopancreas and muscle glycogen and the levels of hemolymph glucose have been observed to be higher than in animals fed with a high-protein diet (Kucharski and da Silva 1991). Moreover, the response of carbohydrate metabolism to fasting and hyposmotic stress also varies according to the composition of the diet previously received by the animals (Vinagre and da Silva 1992; da Silva and Kucharski 1992). C. granulata is a semiterrestrial crab that lives in the mesolittoral and supralittoral zones of estuaries along the southern Brazilian coast (Botto and Irigoyen 1980). In ®eld conditions, the average temperature is 19°C in spring, 22°C in fall, 26°C in summer and 15°C in winter. PO2 ranges from 2.78 to 11.78 mg O2 l±1 throughout the year. However, in winter, when C. granulata stays in its holes for longer periods, environmental PO2 may to fall to zero (Turcato 1990). When C. granulata is exposed to atmospheric air (Santos et al. 1987; Schmitt and Santos 1993), the hemolymph glucose levels of this species have been shown to rise quickly and reach a peak level 60 min after air exposure (Santos and Colares 1986). According to Santos et al. (1987), there is a signi®cant reduction in O2 consumption in C. granulata, and a decrease of 94% in the locomotory activity 2 h after crabs emerge. The present study assesses the eects of anoxia exposure and of the recovery period on glycogen synthesis and mobilization, glucose uptake, and on the enzymes involved in the control of glycogen metabolism in the hepatopancreas of C. granulata fed with carbohydraterich or high-protein diets. The purpose of the two diets was to obtain a group of crabs with high glycogen levels in the hepatopancreas and high hemolymph glucose concentration (carbohydrate-rich diet) and another with low glycogen and glucose concentrations (high-protein diet). Hemolymph glucose concentration and hepatopancreas glycogen levels were determined under these dierent conditions.
Materials and methods Animals Male Chasmagnathus granulata crabs in stage C of the intermolt cycle, according to the morphological criteria described by Drach and Tchernigovtze (1967), were collected in Lagoa TramandaõÂ , a lagoon in the state of Rio Grande do Sul, Brazil (Turcato 1990). The animals were used with permission from the authorities of the Brazilian Federal Environmental Agency IBAMA (Instituto Brasileiro de Meio Ambiente e dos Recursos Naturais RenovaÂveis) (license 138/91-DEVIS). Experimental procedure Animals weighing 15±17 g were placed in aquaria at a salinity of 20&, at a temperature of 22°C and a 12 h light:12 h dark cycle. The experiments were performed throughout the year.
The crabs were divided into two groups. One group was fed a high-protein diet (beef, HP) (protein 21.59%; carbohydrate 0.03%; fat 6.71%; ash 0.35%; ®bers 0.31%; humidity 71.01%), while the other group received a carbohydrate-rich diet (boiled rice, HC) (protein 3.34%; carbohydrates 34.56%; fat 0.45%; ash 0.02%; ®bers 0.30%; humidity 61.33%). The HC diet had approximately the same caloric content as the HP diet. Protein and carbohydrate contents of the crab food constituents were determined by the Food Technology Institute at Universidade Federal do Rio Grande do Sul (UFRGS). Both groups of crabs were fed once daily (50 g) ad libitum in the late afternoon for 2 weeks before being used in the experiments. The body weight of animals within each group did not vary signi®cantly (16.50.7 g) during the experimental period. For the anoxia study, pools of ten animals receiving either the HC or the HP diet were introduced in glass aquaria (20 l) at a salinity of 20& and temperature of 22°C. The air in the incubation ¯ask was replaced with N2 gas for 40 min until PO2 (monitored with an Oxel-1/ISO2, World Precision Instruments) reached 0%. The ¯asks were then sealed, and the crabs were submitted to anoxia conditions for 2, 4 and 8 h. After that, the crabs were used in in vitro experiments. The control group (time zero) was kept under normoxic conditions (PO2 18.95%) at a salinity of 20& and a temperature of 22°C. For the recovery experiments, crabs were kept under anoxia conditions for 8 h, in incubation ¯asks. Then, the deoxygenated water was replaced with normoxic water. After 12, 18, 24 and 30 h in normoxic water the recovered animals were used in in vitro experiments. After the experimental procedures, the crabs were anesthetized by chilling (5 min). For glucose uptake, glycogen synthesis or mobilization experiments, the hepatopancreas was rapidly removed and placed on a Petri dish containing cool incubation buer containing 481 mM NaCl, 12.2 mM KCl, 11 mM CaCl2, 93 mM MgCl2áH2O, 31 mM NaHCO3 , 278 mM Na2SO4, 5.8 mM NaBr, plus 10 mM HEPES and 0.1 mM phenylmethylsulphonyl ¯uoride (PMSF), pH 7.8. Oliveira (1998) has shown that glucose uptake and the rate of incorporation remain linear for the duration of the incubation period. Glucose uptake Hepatopancreas fractions (70 mg) from each experimental group were incubated at 25°C with constant shaking in 250 ll incubation buer, in a Dubno incubator for 90 min in the presence of 0.2 lCi 1-[14C]-2-deoxy-D-glucose ([14C]DG) (39 mCi mmol±1; Amersham International). After this period, the [14C]DG uptake was determined according to Machado et al. (1991). Results are expressed as a tissue/medium (T/M) ratio (in dpm ml±1 tissue ¯uid per dpm ml±1 incubation medium). Glucose synthesis and mobilization For glycogen synthesis measurement, hepatopancreas fractions (70 mg) from the dierent experimental groups were incubated at 25°C for 60 min with constant shaking in 500 ll incubation buer equilibrated to pH 7.8 with 5% CO2:95% O2, in the presence of 0.5 lCi glucose-U-14C (230 mCi mmol±1, Amersham International) and with a ®nal glucose concentration of 1.12 lM. Incubation was performed in a Dubno incubator. For determination of glycogen breakdown, two samples of hepatopancreas fractions from the dierent experimental groups were obtained. One sample was used for the determination of the prelabeled glycogen in each experimental group. The other prelabeled glycogen sample was immediately transferred to the incubation buer (500 ll) without glucose, and incubated for 60 min in a Dubno incubator. After this period, the 14C-glycogen in tissue samples was determined. For 14C-glycogen determination, the hepatopancreas fractions were withdrawn, rinsed in cold incubation buer, blotted with ®lter paper, immediately transferred to KOH (0.5 N) and boiled for
337 60 min. The proteins were precipitated by addition of 30% trichloroacetic acid and 1 N HCl (2:1, v/v). After centrifugation at 600 g for 10 min, the supernatant (30 ll) was applied on Whatman 3MM strips. The glycogen was precipitated by ethanol 66%. The wet strips were then transferred to vials containing toluene-triton (2:1)-PPO-POPOP. Radioactivity was measured in an LKB Wallac scintillation counter. The values of glycogen synthesis are given as micrograms of 14C-glucose incorporated into glycogen per milligram of protein per hour.
total phosphorylase (GPT) activity, the assay mixture contained 100 mM NaF, 5 mM EDTA, 20 mM sodium citrate, 2 mM AMP, pH 6.5 (GPT). For phosphorylase a activity (GPa), the assay was carried out in the presence of 0.5 mM caeine. The hepatopancreas homogenate centrifugation (50 ll) was incubated at 30°C for 30 min, with constant shaking in 100 ll of reaction mixture to GPT or GPa. The reaction was started by adding 50 mM glucose1-phosphate, and interrupted by adding 200 ll cold TCA 10%. Enzyme activity is reported in nmoles of Pi liberated to glucose1-phosphate per milligram of protein per minute.
Enzyme assays Glycogen synthase (EC 2.4.1.11) The glycogen synthase (GS) activity was measured according to Nuttal and Gannon (1989). Hepatopancreas fractions (n=4±6) from the dierent experimental groups were immediately transferred to ice-cold Potter±Elvehjem tubes and homogenized with a motor-driven pestle in 3 vol of 10 mM EDTA, 100 mM KF and 0.1 mM PMSF, pH 7.0 (1:3, w/v). The homogenate was centrifuged for 10 min at 800 g and 4°C. Total glycogen synthase (GST) activity (GSD+GSI) was assayed at 30°C using a ®nal concentration of 6.7 mg rabbit muscle glycogen/ml±1, 500 mM Tris, 200 mM EDTA, 250 mM KF, 10 mM UDP-glucose, 10 mM glucose-6-P and 0.02 lCi UDPglucose-U-14C (319 mCi mmol±1, Amersham International), pH 8.5. GSI activity was measured at a pH of 7.0 in the same reaction mixture, except that the Tris buer was replaced with 500 mM imidazole and glucose-6-P was absent. The supernatant (25 ll) resulting from the centrifugation of hepatopancreas homogenate was incubated at 30°C for 30 min with constant shaking in 50 ll of reaction mixture to GST or GSI. After incubation, 50 ll of this reaction mixture plus the samples were applied on Whatman 3MM strips and processed as described above for 14C-glycogen determination. Enzyme activity is reported in nanomoles of UDP-glucoseU-14C converted into glycogen per milligram of protein per minute. Glycogen phosphorylase (EC 2.4.1.1) For determination of glycogen phosphorylase (GP) activity, hepatopancreas fractions (n=4±6) from dierent experimental groups were transferred to ice-cold Potter±Elvehjem tubes and homogenized with a motor-driven pestle in 3 vol of 5 mM EDTA, 50 mM KF, 0.1 mM PMSF, glycerol 60%, pH 6.5 (1:3, w/v). The homogenate was centrifuged for 10 min at 800 g and 4°C, and the resulting supernatant was used for the activity assay. Glycogen phosphorylase activity was assayed in the direction of glycogen synthesis by measuring the Pi liberated from glucose-1-phosphate, according to Scarpin and Di Giuseppe (1993). For determination of Fig. 1 Chasmagnathus granulata. Uptake of [14C]deoxyD-glucose during anoxia and subsequent recovery in hepatopancreas fractions from crabs fed a high-protein (HP) or a carbohydrate-rich diet (HC). Data are given as meanSEM of six observations aMean values are signi®cantly dierent from those for zero hour (P
