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Hepatopancreas gluconeogenesis and glycogen content during fasting in crabs previously maintained on a high-protein or carbohydrate-rich diet
Comparative Biochemistry and Physiology Part A 137 (2004) 383–390
Hepatopancreas gluconeogenesis and glycogen content during fasting in crabs previously maintained on a high-protein or carbohydrate-rich diet Guendalina T. Oliveiraa, Isabel Cristina Rossia, Luiz C. Kucharskib, Roselis S.M. Da Silvab,* a
ˆ ´ ˆ ´ ´ Departamento de Ciencias Fisiologicas, Faculdade de Biociencias, Pontifıcia Universidade Catolica do Rio Grande do Sul, Av. Ipiranga, 6681 pd.12, 90619-900 Porto Alegre, RS, Brazil b ˆ ´ ´ Departamento de Fisiologia, Instituto de Ciencias Basicas da Saude, Universidade Federal do Rio Grande do Sul, Rua Sarmento Leite 500, 90050-170 Porto Alegre, RS, Brazil Received 20 June 2003; received in revised form 21 October 2003; accepted 22 October 2003
Abstract The present study assessed the effect of different fasting times on the in vitro gluconeogenic capacity of Chasmagnathus granulata crabs previously adapted to a high-protein (HP) or carbohydrate-rich (HC) diet using the incorporation of wU-14CxL-lactate or wU-14CxL-alanine into glucose. We also recorded haemolymphatic glucose and hepatopancreatic glycogen levels. In the HP group, on the third day of fasting there were decreases in the synthesis of glucose from 14Calanine and in haemolymph glucose. After 15 days of fasting, haemolymph glucose and hepatopancreatic glycogen levels were maintained by an increase in the conversion of 14C-alanine into glucose. However, after 21 days of fasting the gluconeogenic capacity was decreased and hepatopancreas glycogen concentration was reduced. In the HC group, hepatopancreatic glycogen was the energy source during the first 6 days of fasting. Gluconeogenesis from 14C-lactate decreased after 6 days of fasting, remaining low until 21 days of fasting. The conversion of 14C-alanine into glucose was increased after 15 days fasting and hepatopancreatic glycogen was raised in relation to that present after a 6-day fasting. In both dietary groups the stabilization in the levels of haemolymph glucose after 21 days fasting may result from a reduction in metabolic rate during restricted feeding. 䊚 2003 Elsevier Inc. All rights reserved. Keywords: Crab; Diet composition; Fasting; Gluconeogenesis; Glycogen; Hepatopancreas; Haemolymphatic glucose
1. Introduction The crab Chasmagnathus granulata lives in the mesolittoral and supralittoral zones of estuaries along the southern coast of Brazil (Botto and Irigoyen, 1980) where it is an opportunistic feeder (D’Incao et al., 1990). In their natural habitat these crabs are exposed to a great number of environmental variables such as temperature, *Corresponding author. Fax: q55-51-3316-3453. E-mail address: [email protected] (R.S. Da Silva).
humidity, salinity, dissolved oxygen in water, photo-periodicity and variation of the items in their diet—all of which can produce changes in behavior, feeding and metabolism (D’Incao et al., 1990; Turcato, 1990; Kucharski and Da Silva, 1991a; Cervino et al., 1996; Brogim and Lana, 1997). In winter, when C. granulata stay in their burrows, the frequency of dietary items in the stomach is low, indicating a reduced availability of energy substrate, and the crabs reduce their metabolic rate (D’Incao et al., 1990). Therefore, C. granulata is
1095-6433/04/$ - see front matter 䊚 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpb.2003.10.017
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adapted to mobilize body constituents and survive natural fasting periods such as those encountered during the winter. Hepatopancreas and muscle glycogen seems to be the largest source of energy in spring and summer, whereas muscle lipids are used as an energy substrate in fall and winter (Kucharski and Da Silva, 1991a). Data of many studies on the influence of starvation on protein, fat and carbohydrate metabolism in crustaceans show great interspecies variability (Marsden et al., 1973; Vinagre and Da Silva, 1992; Hervant et al., 1999c; Hardy et al., 2000; Vinagre and Da Silva, 2002; Hervant and Renault, 2002). Apart from differences in the analytical methods utilized by different authors and the period of starvation, multiple factors linked to biological and ecological peculiarities of the different species probably contribute to the observed diversity. One factor, which appears not to have been taken into account in the published research on starvation in crustaceans, is the protein or carbohydrate content of the food to which they were adapted before food deprivation. Previous studies have shown that C. granulata crabs fed a high-protein (HP)ylow-carbohydrate, or a carbohydrate-rich (HC), diet have a characteristic pattern of metabolic adjustment of carbohydrates and lipids, which depends on the carbohydrate and protein levels in their diet (Kucharski and Da Silva, 1991b). Moreover, the response of carbohydrate metabolism to food restriction also varies according to the composition of the diet to which these crabs have previously adapted (Vinagre and Da Silva, 1992). Crabs fed an HP had a lower haemolymph glucose level and a lower hepatopancreas and muscle glycogen level than the animals fed an HC diet. During 8 weeks of fasting, haemolymph glucose levels remained constant and the hepatopancreas glycogen concentration was little affected by food deprivation in these crabs. However, in crabs fed an HC diet after 8 weeks fasting the hepatopancreas glycogen was hardly detectable, and the haemolymph glucose concentration was reduced to approximately 43% of fed values. On the other hand, muscle glycogen content decreased during the fasting period in both dietary groups (Vinagre and Da Silva, 1992). The occurrence of gluconeogenesis, glyconeogenesis and the activity of phosphoenolpyruvate carboxykinase (PEPCK) have been demonstrated in crustaceans. However, the gluconeogenic and
glyconeogenic capabilities, location and enzymes activities in crustaceans show great interspecies variability and are still uncertain (Van Aardt, 1988; Walsh and Henry, 1990; Hill et al., 1991; Lallier and Walsh, 1991, 1992; Henry et al., 1994; Hervant, 1996; Hervant et al., 1999a,b). It has been suggested that the hepatopancreas might be the site of gluconeogenesis (Van Aardt, 1988; Hill et al., 1991; Lallier and Walsh, 1992; Oliveira and Da Silva, 1997), although others have indicated gills (Thabrew et al., 1971), hemocytes (Johnston and Davies, 1972) and muscles (Lallier and Walsh, 1991; Vinagre and Da Silva, 2002) as other sites of glucose synthesis in crustaceans. In C. granulata, gluconeogenic capacity and the PEPCK activity have previously been demonstrated in the hepatopancreas, anterior and posterior gills and muscle (Oliveira and Da Silva, 1997; ´ 2000; Vinagre and Da Silva, 2002). Chitto, In C. granulata fed an HC or HP diet, hepatopancreas gluconeogenesis is one of the pathways implicated in the metabolic adjustment of the amino acid pool during hyposmotic stress (Oliveira and Da Silva, 2000). Moreover, during the postanoxia recovery the fate of L-lactate is the hepatopancreatic gluconeogenesis in crabs fed an HP diet (Oliveira et al., in press). In C. granulata fasting for 3 weeks and then fed for 48 h with raw beef was found an increase in gluconeogenic capacity only in muscle (Vinagre and Da Silva, 2002). The hepatopancreas from C. granulata presented glucose-6-phosphatase activity (1.35"0.06 nmoles mgy1 of protein miny1) (unpublished data), confirming the complete gluconeogenic pathway by the capacity to release free glucose into the haemolymph (Nordlie et al., 1999; Corssmit et al., 2001). The intrinsic capacity of hepatopancreas for glucose synthesis from alanine or lactate and PEPCK activity do not seem to be affected by dietary composition in C. granulata (Oliveira and Da Silva, 1997). However, in other crustaceans ( juveniles Litopenaeus vannamei), the gluconeogenic capacity in the hepatopancreas can be modulated by dietary carbohydrates, with a high PEPCK activity in shrimp fed low carbohydrate concentration (Rosas et al., 2001). To obtain more information on the gluconeogenic capacity of crabs previously adapted to an HP or HC diet, we conducted in vitro experiments on the effects of different fasting periods on the rate
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of gluconeogenesis in the hepatopancreas of C. granulata using the incorporation of L-alanine-U14 C or L-lactic acid-14C into glucose. We also report on the concentration of glucose in the haemolymph and the glycogen content of the hepatopancreas of these crabs. 2. Materials and methods 2.1. Animals Male crabs C. granulata in stage C of the intermolt cycle according to the morphological criteria described by Drach and Tchernigovtzeff ´ a (1967) were collected in Lagoa Tramandaı, lagoon in the state of Rio Grande do Sul, Brazil. 2.2. Experimental procedure Immediately after the crabs arrived at the laboratory they were weighed (16–18 g) and placed in aquaria containing water with a salinity of 15‰ and a temperature of 25 8C under a lightydark cycle of 12Ly12D. The animals were divided into two groups, one of which was fed an HPylowcarbohydrate diet (HP, beef: protein 21.59%, carbohydrate 0.03%, fat 6.71%), while the other was fed an HC diet (HC, boiled rice: carbohydrate 34.56%, protein 3.34%, fat 0.45%) approximately isocaloric to 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 were fed daily ad libitum (;50 g) in the late afternoon for 2 weeks before being used in the experiments. There was no variation in the body mass (17.5"0.5 g) of the crabs in either of the groups during the experimental period. After this period of adaptation, haemolymph glucose and glycogen content and gluconeogenic capacity of the hepatopancreas were determined in a sample of crabs from the HP and HC groups, constituting the control group. The rest of the crabs were kept individually in closed polythene cages (10=10=10 cm3) inside the aquaria to avoid cannibalism and fasted for 3, 6, 15 and 21 days. After these periods of food deprivation, the same measurements carried out in the control group were performed in crabs submitted to fasting. No crab mortality or body-mass variation was observed in four groups during the experimental period.
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Samples of haemolymph (0.1 ml) were taken from the blood sinus of the claws of individual crabs with a silicon-treated syringe; afterward the crabs were anesthetized by chilling (5 min), and the tissue samples were removed. The hepatopancreas of individual crabs was rapidly removed and cut into three fragments, one (150 mg) was used to determine glycogen content and the other fragments (70 mg each) were used to determine gluconeogenic capacity. For the determination of the glucose synthesis, the hepatopancreas was rapidly removed and placed on a Petri dish containing cold incubation buffer adapted to C. granulata: 481 mM NaCl, 12.2 mM KCl, 11 mM CaCl2, 93 mM MgCl2ØH2O, 31 mM NaHCOy 3 , 278 mM Na2SO4, 8.8 mM H3BO3, plus 10 mM HEPES and 0.1 mM phenylmethylsulfonyl fluoride, pH 7.8, and cut into 70-mg fractions. The fractions (70 mg) were then incubated at 25 8C with constant shaking in 500 ml of incubation buffer, pH 7.8, equilibrated with O2:CO2 (95:5, vyv) in the presence of 0.2 mCi wU-14CxL-lactate (157 mCi mmoly1 Amersham International) or wU14 x C L-alanine (171.70 mCi mmoly1 Amersham International), plus 20 mM of unlabeled L-alanine or L-lactate for 120 min in accordance to Oliveira and Da Silva (1997). After addition of the unlabeled substrates, the incubation medium pH was determined. Following incubation, the rate of glucose synthesis by hepatopancreas fractions from the different experimental groups was obtained by measuring the incorporation of wU-14Cx-L-lactate or wU-14Cx-L-alanine into medium 14C-glucose according to Oliveira and Da Silva (1997). Glucose synthetic activity is expressed as micromoles of 14C-lactate or 14C-alanine converted to glucose per gram of tissue per hour. 2.3. Chemical analyses Haemolymph glucose (mgØ100 mly1) and glucose from glycogen hydrolysis were determined by a glucose-oxidase method (Kit Labtest). Hepatopancreatic glycogen was extracted essentially according to Van Handel (1965), and determined as glucose after acid hydrolysis and expressed as percentage gram of glycogen. 2.4. Statistical analyses Data from the experiments were used to compare the effects of the HP and HC diets by two-
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Fig. 1. Effects of different fasting times on glucose synthesis from 14 C-alanine in the hepatopancreas of C. granulata crabs previously fed different diets (HP, high-protein diet; HC, high-carbohydrate diet). Values are means"standard error of the mean (S.E.M.). The superscript (a) indicates that the mean values are significantly different (P-0.05) from those of the non-fasted control group. Number of crabs used in each experiment is indicated in parentheses.
way analysis of variance (ANOVA). To compare the difference between the experimental conditions to which the two groups were submitted, we used a one-way ANOVA and Duncan’s multiple range test at a significance level of P-0.05. All tests were performed with the Statistical Package for the Social Sciences (SPSS, version 10 for Windows). 3. Results The effects of food deprivation on the rate of glucose synthesis from 14C-alanine in hepatopancreas fractions from crabs previously adapted to an HP or HC diet are shown in Fig. 1. No difference was observed in the gluconeogenic capacity from 14C-alanine in hepatopancreas of fed crabs under the two different diets. In the fasted HC group hepatopancreatic gluconeogenesis from 14C-alanine did not differ significantly from that in fed crabs until 6 days after the start of fasting. However, after 15 days fasting it had increased by 35% (P-0.01), while after 21 days fasting it had returned to the values similar at the control group. As compared to control group, the fasted HP group showed a significant reduction (P-0.01) in the rate of hepatopancreatic gluconeogenesis from 14C-alanine after 3 days fasting.
After 15 days fasting this had increased by approximately 25% (P-0.01), but after 21 days had declined to approximately 38% (P-0.05) of the 15-day value for group fasted crabs and 23% (P0.01) of the 15-day value for fed crabs (Fig. 1). In the fasted HC group, there was a significant decrease (P-0.05) in the rate of hepatopancreatic gluconeogenesis from 14C-lactate in the first 6 days of fasting. After 15 and 21 days fasting, gluconeogenesis from 14C-lactate was reduced to approximately 33% of the values found in fed crabs (Fig. 2). In fasted crabs in the HP group the rate of glucose synthesis from 14C-lactate did not differ significantly from that of fed crabs (Fig. 2). After 21 days of food deprivation, hepatopancreatic glycogen concentration decreased by 51% (P-0.05) in crabs previously maintained on an HP diet. In the HC control group, glycogen levels were higher (P-0.01) than that in the HP group. Food deprivation induced a progressive decline (45%, P-0.05) in glycogen concentration in crabs fed HC diet (Table 1), and after 21 days of fasting, hepatopancreas glycogen was reduced to approximately 64% (P-0.01) of the fed value (Table 1). The fasted HC group had significantly higher (P-0.01) levels of haemolymph glucose than those in the fasted HP group (Table 2). Fasting in the HC group induced a reduction to approximately
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Fig. 2. Effects of different fasting times on glucose synthesis from 14 C-lactate in the hepatopancreas of C. granulata crabs previously fed different diets (HP, high-protein diet; HC, high-carbohydrate diet). Values are means"standard error of the mean (S.E.M.). The superscript (a) indicates that the mean values are significantly different (P-0.05) from those of the non-fasted control group. Number of crabs used in each experiment is indicated in parentheses.
76% (P-0.01) in glucose haemolymph concentration after 3 days. However, after 6 days of fasting the glucose haemolymph values increased approximately 92% (P-0.01) in comparison to 3 days fasting values; the levels then showed a tendency to stabilize until the end of fasting period (21 days). In the fasted HP group, haemolymph glucose levels decreased (P-0.05) in the first 3 days of fasting, while after 6 days of fasting the levels
of glucose did not differ significantly from that in non-fasted crabs.
Table 1 Glycogen concentration in the hepatopancreas of fasted C. granulata crabs previously fed on a HP or HC diet
Table 2 Glucose concentration in the haemolymph of fasted C. granulata crabs previously fed on a HP or HC diet
Fasting (days)
Fasting (days)
Control 3 6 15 21
Glycogen (g%) HC
HP
2.82"0.33 (31) 2.94"0.52 (5) 1.55"0.21 (6) 2.41"0.22 (7) 1.01"0.24a (6)
1.71"0.14 (33) 2.30"0.32 (5) 1.07"0.16 (6) 1.33"0.16 (7) 0.83"0.16a (6)
Values are means"standard error of the mean (S.E.M.). The superscript (a) indicates that the mean values are significantly different (P-0.05) from those of the non-fasted control group. Number of crabs used in each experiment is indicated in parentheses.
4. Discussion The pattern of the adjustments of carbohydrate metabolism to fasting in crabs adapted to an HC diet differs from that observed in crabs fed an HP diet before food deprivation. Previous studies have also demonstrated the influence of diet on the
Control 3 6 15 21
Glucose (mg dly1) HC
HP
25.10"3.41 (17) 6.02"0.66a (6) 11.57"1.57a (6) 11.19"1.67a (7) 11.45"2.30a (6)
11.06"1.93 (18) 4.35"0.68a (6) 6.79"1.31 (6) 10.37"1.51 (7) 9.60"1.29 (6)
Values are means"standard error of the mean (S.E.M.). The superscript (a) indicates that the mean values are significantly different (P-0.05) from those of the non-fasted control group. Number of crabs used in each experiment is indicated in parentheses.
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regulation of carbohydrates and lipid metabolism and their mobilization during food restriction in C. granulata (Kucharski and Da Silva, 1991b; Vinagre and Da Silva, 1992). In the fasted HP group the amount of 14Calanine incorporated into glucose after 3 days of fasting was lower than that in non-fasted crabs. This decrease in gluconeogenesis capacity coincides with a 60% decrease in haemolymph glucose without any significant alteration in hepatopancreatic glycogen. The fact that 3 days fasting decreases the haemolymph glucose concentration in crabs maintained on an HP diet indicates that the gluconeogenic pathway sustained, via the exogenous amino acids delivered by the diet to the hepatopancreas, the haemolymph glucose in non-fasted crabs. In the fed state, C. granulata maintained an HP diet that had lower levels of haemolymph glucose and lower glycogen concentration in tissues than the HC-fed group (Kucharski and Da Silva, 1991b; Vinagre and Da Silva, 1992). In C. granulata previously fed an HP diet and fasted for 1 week, the muscular glycogen content decreases approximately 35% (Vinagre and Da Silva, 1992), the hepatopancreas glycogen levels remain constant and the haemolymph glucose level increases to approximately 56% in relation to 3 days fasting. Hence, the maintenance of gluconeogenic capacity for the conversion of 14C-lactate into glucose seems to indicate that, in crabs previously fed on HP diet, the lactate derived from muscle polysaccharide is an import source of carbon chains for hepatopancreas gluconeogenesis after 6 days fasting. After 15 days of food deprivation, the ability of the HP group to maintain the concentration of hepatopancreatic glycogen and raise the levels of haemolymph glucose to near the values found in the non-fasted controls probably results from an increase in gluconeogenic capacity from 14C-alanine. Supporting this hypothesis a reduction in hepatopancreas glycogen concentration was found when the gluconeogenic capacity from 14C-alanine decreased (61%) after 21 days of fasting, while the conversion of 14C-lactate to glucose remained similar to that of the non-fasted controls. The 3 days of fasting decreased the glucose values in the haemolymph by approximately 75% in crabs adapted to the HC diet and any significant alteration in hepatopancreatic glycogen and in the rate of conversion of 14C-alanine to glucose was occurred, suggesting that in fed crabs dietary
carbohydrates maintain the value of haemolymph glucose 127% higher than that found in crabs on an HP diet. After 6 days of fasting, hepatopancreatic glycogen had dropped to 46%, while haemolymph glucose concentration had risen 92% in relation to the amount present after 3 days of fasting. Even so, the glucose values in the haemolymph were still 54% lower than that of the non-fasted controls. Since the muscle glycogen is not mobilized during this period (Vinagre and Da Silva, 1992) and the conversion of 14C-lactate into glucose dropped 33% in relation to the non-fasted control group and the incorporation of 14C from L-alanine into glucose showed no significant variation, the hepatopancreatic glycogen may be the principal source of glucose in the HC-fed crabs. After 15 days fasting, hepatopancreatic glycogen in the HC group of crabs rose 55% in relation to its value on day 6 of fasting, and it appears that maintenance of haemolymph glucose levels was probably a result of the increase in gluconeogenesis activity from 14C-alanine seen in this group of crabs. However, after 21 days of fasting, the conversion of 14C-alanine into glucose declined 30% in relation to the rate of conversion after 15 days fasting, the values for the conversion of 14Clactate into glucose were low and hepatopancreatic glycogen was reduced by 71%. The fact that haemolymph glucose concentration was stable suggests that glycogen was the principal energy source in the HC group after 21 days of fasting. In both, the HC and HP groups, the tendency towards the stabilization in the levels of haemolymphatic glucose after 21 days fasting may result from a reduction in metabolic rate during restricted feeding, as has been observed in other crustaceans (Marsden et al., 1973; Regnault, 1981; Hervant et al., 1999c; Hervant and Renault, 2002). In conclusion, it is possible to identify several features of the carbohydrate metabolism in crabs fed an HP diet, which contrast with those observed in crabs fed a diet rich in carbohydrates (HC): in the HP group the level of haemolymph glucose was principally maintained by effective gluconeogenesis from 14C-alanine and, probably, the degradation of muscle polysaccharides (Vinagre and Da Silva, 1992), while in the HC group hepatopancreatic glycogen appeared to be the principal energy source during food deprivation, although after 15 days of fasting there was an increase in the production of glucose from 14C-alanine.
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Hepatopancreas gluconeogenesis and glycogen content during fasting in crabs previously maintained on a high-protein or carbohydrate-rich diet Guendalina T. Oliveiraa, Isabel Cristina Rossia, Luiz C. Kucharskib, Roselis S.M. Da Silvab,* a
ˆ ´ ˆ ´ ´ Departamento de Ciencias Fisiologicas, Faculdade de Biociencias, Pontifıcia Universidade Catolica do Rio Grande do Sul, Av. Ipiranga, 6681 pd.12, 90619-900 Porto Alegre, RS, Brazil b ˆ ´ ´ Departamento de Fisiologia, Instituto de Ciencias Basicas da Saude, Universidade Federal do Rio Grande do Sul, Rua Sarmento Leite 500, 90050-170 Porto Alegre, RS, Brazil Received 20 June 2003; received in revised form 21 October 2003; accepted 22 October 2003
Abstract The present study assessed the effect of different fasting times on the in vitro gluconeogenic capacity of Chasmagnathus granulata crabs previously adapted to a high-protein (HP) or carbohydrate-rich (HC) diet using the incorporation of wU-14CxL-lactate or wU-14CxL-alanine into glucose. We also recorded haemolymphatic glucose and hepatopancreatic glycogen levels. In the HP group, on the third day of fasting there were decreases in the synthesis of glucose from 14Calanine and in haemolymph glucose. After 15 days of fasting, haemolymph glucose and hepatopancreatic glycogen levels were maintained by an increase in the conversion of 14C-alanine into glucose. However, after 21 days of fasting the gluconeogenic capacity was decreased and hepatopancreas glycogen concentration was reduced. In the HC group, hepatopancreatic glycogen was the energy source during the first 6 days of fasting. Gluconeogenesis from 14C-lactate decreased after 6 days of fasting, remaining low until 21 days of fasting. The conversion of 14C-alanine into glucose was increased after 15 days fasting and hepatopancreatic glycogen was raised in relation to that present after a 6-day fasting. In both dietary groups the stabilization in the levels of haemolymph glucose after 21 days fasting may result from a reduction in metabolic rate during restricted feeding. 䊚 2003 Elsevier Inc. All rights reserved. Keywords: Crab; Diet composition; Fasting; Gluconeogenesis; Glycogen; Hepatopancreas; Haemolymphatic glucose
1. Introduction The crab Chasmagnathus granulata lives in the mesolittoral and supralittoral zones of estuaries along the southern coast of Brazil (Botto and Irigoyen, 1980) where it is an opportunistic feeder (D’Incao et al., 1990). In their natural habitat these crabs are exposed to a great number of environmental variables such as temperature, *Corresponding author. Fax: q55-51-3316-3453. E-mail address: [email protected] (R.S. Da Silva).
humidity, salinity, dissolved oxygen in water, photo-periodicity and variation of the items in their diet—all of which can produce changes in behavior, feeding and metabolism (D’Incao et al., 1990; Turcato, 1990; Kucharski and Da Silva, 1991a; Cervino et al., 1996; Brogim and Lana, 1997). In winter, when C. granulata stay in their burrows, the frequency of dietary items in the stomach is low, indicating a reduced availability of energy substrate, and the crabs reduce their metabolic rate (D’Incao et al., 1990). Therefore, C. granulata is
1095-6433/04/$ - see front matter 䊚 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpb.2003.10.017
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adapted to mobilize body constituents and survive natural fasting periods such as those encountered during the winter. Hepatopancreas and muscle glycogen seems to be the largest source of energy in spring and summer, whereas muscle lipids are used as an energy substrate in fall and winter (Kucharski and Da Silva, 1991a). Data of many studies on the influence of starvation on protein, fat and carbohydrate metabolism in crustaceans show great interspecies variability (Marsden et al., 1973; Vinagre and Da Silva, 1992; Hervant et al., 1999c; Hardy et al., 2000; Vinagre and Da Silva, 2002; Hervant and Renault, 2002). Apart from differences in the analytical methods utilized by different authors and the period of starvation, multiple factors linked to biological and ecological peculiarities of the different species probably contribute to the observed diversity. One factor, which appears not to have been taken into account in the published research on starvation in crustaceans, is the protein or carbohydrate content of the food to which they were adapted before food deprivation. Previous studies have shown that C. granulata crabs fed a high-protein (HP)ylow-carbohydrate, or a carbohydrate-rich (HC), diet have a characteristic pattern of metabolic adjustment of carbohydrates and lipids, which depends on the carbohydrate and protein levels in their diet (Kucharski and Da Silva, 1991b). Moreover, the response of carbohydrate metabolism to food restriction also varies according to the composition of the diet to which these crabs have previously adapted (Vinagre and Da Silva, 1992). Crabs fed an HP had a lower haemolymph glucose level and a lower hepatopancreas and muscle glycogen level than the animals fed an HC diet. During 8 weeks of fasting, haemolymph glucose levels remained constant and the hepatopancreas glycogen concentration was little affected by food deprivation in these crabs. However, in crabs fed an HC diet after 8 weeks fasting the hepatopancreas glycogen was hardly detectable, and the haemolymph glucose concentration was reduced to approximately 43% of fed values. On the other hand, muscle glycogen content decreased during the fasting period in both dietary groups (Vinagre and Da Silva, 1992). The occurrence of gluconeogenesis, glyconeogenesis and the activity of phosphoenolpyruvate carboxykinase (PEPCK) have been demonstrated in crustaceans. However, the gluconeogenic and
glyconeogenic capabilities, location and enzymes activities in crustaceans show great interspecies variability and are still uncertain (Van Aardt, 1988; Walsh and Henry, 1990; Hill et al., 1991; Lallier and Walsh, 1991, 1992; Henry et al., 1994; Hervant, 1996; Hervant et al., 1999a,b). It has been suggested that the hepatopancreas might be the site of gluconeogenesis (Van Aardt, 1988; Hill et al., 1991; Lallier and Walsh, 1992; Oliveira and Da Silva, 1997), although others have indicated gills (Thabrew et al., 1971), hemocytes (Johnston and Davies, 1972) and muscles (Lallier and Walsh, 1991; Vinagre and Da Silva, 2002) as other sites of glucose synthesis in crustaceans. In C. granulata, gluconeogenic capacity and the PEPCK activity have previously been demonstrated in the hepatopancreas, anterior and posterior gills and muscle (Oliveira and Da Silva, 1997; ´ 2000; Vinagre and Da Silva, 2002). Chitto, In C. granulata fed an HC or HP diet, hepatopancreas gluconeogenesis is one of the pathways implicated in the metabolic adjustment of the amino acid pool during hyposmotic stress (Oliveira and Da Silva, 2000). Moreover, during the postanoxia recovery the fate of L-lactate is the hepatopancreatic gluconeogenesis in crabs fed an HP diet (Oliveira et al., in press). In C. granulata fasting for 3 weeks and then fed for 48 h with raw beef was found an increase in gluconeogenic capacity only in muscle (Vinagre and Da Silva, 2002). The hepatopancreas from C. granulata presented glucose-6-phosphatase activity (1.35"0.06 nmoles mgy1 of protein miny1) (unpublished data), confirming the complete gluconeogenic pathway by the capacity to release free glucose into the haemolymph (Nordlie et al., 1999; Corssmit et al., 2001). The intrinsic capacity of hepatopancreas for glucose synthesis from alanine or lactate and PEPCK activity do not seem to be affected by dietary composition in C. granulata (Oliveira and Da Silva, 1997). However, in other crustaceans ( juveniles Litopenaeus vannamei), the gluconeogenic capacity in the hepatopancreas can be modulated by dietary carbohydrates, with a high PEPCK activity in shrimp fed low carbohydrate concentration (Rosas et al., 2001). To obtain more information on the gluconeogenic capacity of crabs previously adapted to an HP or HC diet, we conducted in vitro experiments on the effects of different fasting periods on the rate
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of gluconeogenesis in the hepatopancreas of C. granulata using the incorporation of L-alanine-U14 C or L-lactic acid-14C into glucose. We also report on the concentration of glucose in the haemolymph and the glycogen content of the hepatopancreas of these crabs. 2. Materials and methods 2.1. Animals Male crabs C. granulata in stage C of the intermolt cycle according to the morphological criteria described by Drach and Tchernigovtzeff ´ a (1967) were collected in Lagoa Tramandaı, lagoon in the state of Rio Grande do Sul, Brazil. 2.2. Experimental procedure Immediately after the crabs arrived at the laboratory they were weighed (16–18 g) and placed in aquaria containing water with a salinity of 15‰ and a temperature of 25 8C under a lightydark cycle of 12Ly12D. The animals were divided into two groups, one of which was fed an HPylowcarbohydrate diet (HP, beef: protein 21.59%, carbohydrate 0.03%, fat 6.71%), while the other was fed an HC diet (HC, boiled rice: carbohydrate 34.56%, protein 3.34%, fat 0.45%) approximately isocaloric to 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 were fed daily ad libitum (;50 g) in the late afternoon for 2 weeks before being used in the experiments. There was no variation in the body mass (17.5"0.5 g) of the crabs in either of the groups during the experimental period. After this period of adaptation, haemolymph glucose and glycogen content and gluconeogenic capacity of the hepatopancreas were determined in a sample of crabs from the HP and HC groups, constituting the control group. The rest of the crabs were kept individually in closed polythene cages (10=10=10 cm3) inside the aquaria to avoid cannibalism and fasted for 3, 6, 15 and 21 days. After these periods of food deprivation, the same measurements carried out in the control group were performed in crabs submitted to fasting. No crab mortality or body-mass variation was observed in four groups during the experimental period.
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Samples of haemolymph (0.1 ml) were taken from the blood sinus of the claws of individual crabs with a silicon-treated syringe; afterward the crabs were anesthetized by chilling (5 min), and the tissue samples were removed. The hepatopancreas of individual crabs was rapidly removed and cut into three fragments, one (150 mg) was used to determine glycogen content and the other fragments (70 mg each) were used to determine gluconeogenic capacity. For the determination of the glucose synthesis, the hepatopancreas was rapidly removed and placed on a Petri dish containing cold incubation buffer adapted to C. granulata: 481 mM NaCl, 12.2 mM KCl, 11 mM CaCl2, 93 mM MgCl2ØH2O, 31 mM NaHCOy 3 , 278 mM Na2SO4, 8.8 mM H3BO3, plus 10 mM HEPES and 0.1 mM phenylmethylsulfonyl fluoride, pH 7.8, and cut into 70-mg fractions. The fractions (70 mg) were then incubated at 25 8C with constant shaking in 500 ml of incubation buffer, pH 7.8, equilibrated with O2:CO2 (95:5, vyv) in the presence of 0.2 mCi wU-14CxL-lactate (157 mCi mmoly1 Amersham International) or wU14 x C L-alanine (171.70 mCi mmoly1 Amersham International), plus 20 mM of unlabeled L-alanine or L-lactate for 120 min in accordance to Oliveira and Da Silva (1997). After addition of the unlabeled substrates, the incubation medium pH was determined. Following incubation, the rate of glucose synthesis by hepatopancreas fractions from the different experimental groups was obtained by measuring the incorporation of wU-14Cx-L-lactate or wU-14Cx-L-alanine into medium 14C-glucose according to Oliveira and Da Silva (1997). Glucose synthetic activity is expressed as micromoles of 14C-lactate or 14C-alanine converted to glucose per gram of tissue per hour. 2.3. Chemical analyses Haemolymph glucose (mgØ100 mly1) and glucose from glycogen hydrolysis were determined by a glucose-oxidase method (Kit Labtest). Hepatopancreatic glycogen was extracted essentially according to Van Handel (1965), and determined as glucose after acid hydrolysis and expressed as percentage gram of glycogen. 2.4. Statistical analyses Data from the experiments were used to compare the effects of the HP and HC diets by two-
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Fig. 1. Effects of different fasting times on glucose synthesis from 14 C-alanine in the hepatopancreas of C. granulata crabs previously fed different diets (HP, high-protein diet; HC, high-carbohydrate diet). Values are means"standard error of the mean (S.E.M.). The superscript (a) indicates that the mean values are significantly different (P-0.05) from those of the non-fasted control group. Number of crabs used in each experiment is indicated in parentheses.
way analysis of variance (ANOVA). To compare the difference between the experimental conditions to which the two groups were submitted, we used a one-way ANOVA and Duncan’s multiple range test at a significance level of P-0.05. All tests were performed with the Statistical Package for the Social Sciences (SPSS, version 10 for Windows). 3. Results The effects of food deprivation on the rate of glucose synthesis from 14C-alanine in hepatopancreas fractions from crabs previously adapted to an HP or HC diet are shown in Fig. 1. No difference was observed in the gluconeogenic capacity from 14C-alanine in hepatopancreas of fed crabs under the two different diets. In the fasted HC group hepatopancreatic gluconeogenesis from 14C-alanine did not differ significantly from that in fed crabs until 6 days after the start of fasting. However, after 15 days fasting it had increased by 35% (P-0.01), while after 21 days fasting it had returned to the values similar at the control group. As compared to control group, the fasted HP group showed a significant reduction (P-0.01) in the rate of hepatopancreatic gluconeogenesis from 14C-alanine after 3 days fasting.
After 15 days fasting this had increased by approximately 25% (P-0.01), but after 21 days had declined to approximately 38% (P-0.05) of the 15-day value for group fasted crabs and 23% (P0.01) of the 15-day value for fed crabs (Fig. 1). In the fasted HC group, there was a significant decrease (P-0.05) in the rate of hepatopancreatic gluconeogenesis from 14C-lactate in the first 6 days of fasting. After 15 and 21 days fasting, gluconeogenesis from 14C-lactate was reduced to approximately 33% of the values found in fed crabs (Fig. 2). In fasted crabs in the HP group the rate of glucose synthesis from 14C-lactate did not differ significantly from that of fed crabs (Fig. 2). After 21 days of food deprivation, hepatopancreatic glycogen concentration decreased by 51% (P-0.05) in crabs previously maintained on an HP diet. In the HC control group, glycogen levels were higher (P-0.01) than that in the HP group. Food deprivation induced a progressive decline (45%, P-0.05) in glycogen concentration in crabs fed HC diet (Table 1), and after 21 days of fasting, hepatopancreas glycogen was reduced to approximately 64% (P-0.01) of the fed value (Table 1). The fasted HC group had significantly higher (P-0.01) levels of haemolymph glucose than those in the fasted HP group (Table 2). Fasting in the HC group induced a reduction to approximately
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Fig. 2. Effects of different fasting times on glucose synthesis from 14 C-lactate in the hepatopancreas of C. granulata crabs previously fed different diets (HP, high-protein diet; HC, high-carbohydrate diet). Values are means"standard error of the mean (S.E.M.). The superscript (a) indicates that the mean values are significantly different (P-0.05) from those of the non-fasted control group. Number of crabs used in each experiment is indicated in parentheses.
76% (P-0.01) in glucose haemolymph concentration after 3 days. However, after 6 days of fasting the glucose haemolymph values increased approximately 92% (P-0.01) in comparison to 3 days fasting values; the levels then showed a tendency to stabilize until the end of fasting period (21 days). In the fasted HP group, haemolymph glucose levels decreased (P-0.05) in the first 3 days of fasting, while after 6 days of fasting the levels
of glucose did not differ significantly from that in non-fasted crabs.
Table 1 Glycogen concentration in the hepatopancreas of fasted C. granulata crabs previously fed on a HP or HC diet
Table 2 Glucose concentration in the haemolymph of fasted C. granulata crabs previously fed on a HP or HC diet
Fasting (days)
Fasting (days)
Control 3 6 15 21
Glycogen (g%) HC
HP
2.82"0.33 (31) 2.94"0.52 (5) 1.55"0.21 (6) 2.41"0.22 (7) 1.01"0.24a (6)
1.71"0.14 (33) 2.30"0.32 (5) 1.07"0.16 (6) 1.33"0.16 (7) 0.83"0.16a (6)
Values are means"standard error of the mean (S.E.M.). The superscript (a) indicates that the mean values are significantly different (P-0.05) from those of the non-fasted control group. Number of crabs used in each experiment is indicated in parentheses.
4. Discussion The pattern of the adjustments of carbohydrate metabolism to fasting in crabs adapted to an HC diet differs from that observed in crabs fed an HP diet before food deprivation. Previous studies have also demonstrated the influence of diet on the
Control 3 6 15 21
Glucose (mg dly1) HC
HP
25.10"3.41 (17) 6.02"0.66a (6) 11.57"1.57a (6) 11.19"1.67a (7) 11.45"2.30a (6)
11.06"1.93 (18) 4.35"0.68a (6) 6.79"1.31 (6) 10.37"1.51 (7) 9.60"1.29 (6)
Values are means"standard error of the mean (S.E.M.). The superscript (a) indicates that the mean values are significantly different (P-0.05) from those of the non-fasted control group. Number of crabs used in each experiment is indicated in parentheses.
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regulation of carbohydrates and lipid metabolism and their mobilization during food restriction in C. granulata (Kucharski and Da Silva, 1991b; Vinagre and Da Silva, 1992). In the fasted HP group the amount of 14Calanine incorporated into glucose after 3 days of fasting was lower than that in non-fasted crabs. This decrease in gluconeogenesis capacity coincides with a 60% decrease in haemolymph glucose without any significant alteration in hepatopancreatic glycogen. The fact that 3 days fasting decreases the haemolymph glucose concentration in crabs maintained on an HP diet indicates that the gluconeogenic pathway sustained, via the exogenous amino acids delivered by the diet to the hepatopancreas, the haemolymph glucose in non-fasted crabs. In the fed state, C. granulata maintained an HP diet that had lower levels of haemolymph glucose and lower glycogen concentration in tissues than the HC-fed group (Kucharski and Da Silva, 1991b; Vinagre and Da Silva, 1992). In C. granulata previously fed an HP diet and fasted for 1 week, the muscular glycogen content decreases approximately 35% (Vinagre and Da Silva, 1992), the hepatopancreas glycogen levels remain constant and the haemolymph glucose level increases to approximately 56% in relation to 3 days fasting. Hence, the maintenance of gluconeogenic capacity for the conversion of 14C-lactate into glucose seems to indicate that, in crabs previously fed on HP diet, the lactate derived from muscle polysaccharide is an import source of carbon chains for hepatopancreas gluconeogenesis after 6 days fasting. After 15 days of food deprivation, the ability of the HP group to maintain the concentration of hepatopancreatic glycogen and raise the levels of haemolymph glucose to near the values found in the non-fasted controls probably results from an increase in gluconeogenic capacity from 14C-alanine. Supporting this hypothesis a reduction in hepatopancreas glycogen concentration was found when the gluconeogenic capacity from 14C-alanine decreased (61%) after 21 days of fasting, while the conversion of 14C-lactate to glucose remained similar to that of the non-fasted controls. The 3 days of fasting decreased the glucose values in the haemolymph by approximately 75% in crabs adapted to the HC diet and any significant alteration in hepatopancreatic glycogen and in the rate of conversion of 14C-alanine to glucose was occurred, suggesting that in fed crabs dietary
carbohydrates maintain the value of haemolymph glucose 127% higher than that found in crabs on an HP diet. After 6 days of fasting, hepatopancreatic glycogen had dropped to 46%, while haemolymph glucose concentration had risen 92% in relation to the amount present after 3 days of fasting. Even so, the glucose values in the haemolymph were still 54% lower than that of the non-fasted controls. Since the muscle glycogen is not mobilized during this period (Vinagre and Da Silva, 1992) and the conversion of 14C-lactate into glucose dropped 33% in relation to the non-fasted control group and the incorporation of 14C from L-alanine into glucose showed no significant variation, the hepatopancreatic glycogen may be the principal source of glucose in the HC-fed crabs. After 15 days fasting, hepatopancreatic glycogen in the HC group of crabs rose 55% in relation to its value on day 6 of fasting, and it appears that maintenance of haemolymph glucose levels was probably a result of the increase in gluconeogenesis activity from 14C-alanine seen in this group of crabs. However, after 21 days of fasting, the conversion of 14C-alanine into glucose declined 30% in relation to the rate of conversion after 15 days fasting, the values for the conversion of 14Clactate into glucose were low and hepatopancreatic glycogen was reduced by 71%. The fact that haemolymph glucose concentration was stable suggests that glycogen was the principal energy source in the HC group after 21 days of fasting. In both, the HC and HP groups, the tendency towards the stabilization in the levels of haemolymphatic glucose after 21 days fasting may result from a reduction in metabolic rate during restricted feeding, as has been observed in other crustaceans (Marsden et al., 1973; Regnault, 1981; Hervant et al., 1999c; Hervant and Renault, 2002). In conclusion, it is possible to identify several features of the carbohydrate metabolism in crabs fed an HP diet, which contrast with those observed in crabs fed a diet rich in carbohydrates (HC): in the HP group the level of haemolymph glucose was principally maintained by effective gluconeogenesis from 14C-alanine and, probably, the degradation of muscle polysaccharides (Vinagre and Da Silva, 1992), while in the HC group hepatopancreatic glycogen appeared to be the principal energy source during food deprivation, although after 15 days of fasting there was an increase in the production of glucose from 14C-alanine.
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