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Metabolic changes in fish liver during the starved-to-fed transition
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Camp. Eiochem.Fh~riol.Vol.98A. No. 2, pp. 329-331, 1991 Printed in Great B&XI
e 1991Petgamon Press plc
METABOLIC CHANGES IN FISH LIVER DURING THE STARVED-TO-FED TRANSITION PABLO GARciA DE FRUTOS,LLUISBONAMUSA and ISABELV. BAANANTE* Unitat de Bioquimica. Facultat de Farmicia, Universitat de Barcelona, Plaza Pio XII sin, Barcelona 08028, Spain. Telephone: (93) 330.79.63 (Received 9 May 1990) Abstract-i,
In contrast to the rat, fish starved for 24 hr maintain high levels of fructose 2,6-bisphosphate in the liver. 2. After refeeding, these levels are decreased and kept low for the next 4 hr. 3. The changes observed in fru-2,6-P, correlate with those in the phosphofructo 2-kinase activity ratio, suggesting an accurate regulation of the process. 4. Measurements of glycogen and hexose 6-phosphate indicate that dietary carbohydrates are utilized by fish in a different way from mammals.
INTRODUCTION
The effect of fructose 2,6-bisphosphate as a potent simulator of glycolysis in mammalian tissues has been well established (Hue and Rider, 1987; Pilkis et al., 1987). The levels of this metabolite play a key role in the regulation of hepatic glucose metabolism, depending on the glucose levels in serum (Sugden et al., 1989). Since the studies of Newgard et al. (1983), several groups have supported the idea that the gfycogen stored in liver is not directly derived from dietary glucose but mainly from 3-carbon compounds via the gluconeogenic pathway (for review, see McGarry et al., 1987). According to this idea, levels of fructose 2,6-bisphosphate remain low in the transition from fasted to fed state until glycogen level has been restored. Then, fru-2,6-Pz levels rise preventing further glycogen deposition and directing the liver metabolism to the synthesis of lipids (Sugden et al., 1989). Little information is available on the changes involved in starved-to-fed transition in animal species other than mammals. Studies with the Japanese quail suggest that the regulation of the pathway is completely different from that in mammals, and in this species there is no indirect pathway (Riesenfeld et al., 1981). Fish metabolism is different to that of mammals (Christiansen and Klugseryr, 1987). Prolonged starvation is required before any glycogen depletion is observed in the liver. Also, it appears that they have a limited capacity for using carbohydrates from the diet which, in carnivorous fish, are often substituted by amino acids as precursors of energetic reserves (Walton and Cowey, 1979). Interestingly, Renaud and Moon (1980) found that in physiological conditions lactate is a better precursor than amino acids for gluconeogenesis. This work was undertaken to determine the molecular mechanisms involved in fish during
*Author to whom all correspondence
should be addressed.
starved-to-fed transition, whether fish metabolism fits an “indirect” model of utilizing carbohydrates from diet and, where possible, why carbohydrates are poorly used by carnivorous fish. This paper presents the changes in hepatic metabolism in fish liver during the starved-to-fed transition. MATERIALS AND METHODS Spans aumtu (gilth~d sea bream) were used in this study. Groups of 20-25 fish, weighing 12-20 g were held in 1001 tanks provided with filtered aerated sea-water, in a closed system with a UV-lamp, the temperature was kept at 19 + 1°C and the dark/light cycle was 12: 12 hr. Fish were fed once a day with 0.5 g of food per fish in pellet form (46% protein, 7% lipids, 24% carbohydrates, 15% salts). A 7-10 day a~limat~~tion period was provided before experimental work was begun. After 24 hr of starvation, the Sparus were gently netted from the holding tank and anaesthetized with MS-222 (1: 1000). The liver was dissected out immediately after decapitation, and freeze-clamped in liquid nitrogen. Blood samples were drawn from the open heart. Several samples were combined to obtain enough serum for glucose and lactate assays. Other starved fish were allowed to refeed and were killed at various times after being fed. Measurements of fru-2,6-P, and PP, : PFK purification from potato tubers were performed according to the method of Van Schaftingen (Bergmeyer, 1984). Fructose 6-phosphate 2-kinase activity was measured in its active and total forms, as described by Bartrons et al. (1983). The rest of metabolites were measured s~ctrophotometricaliy by established enzymatic procedures in neutralized perchloric acid extracts (Bergmeyer, 1984). Glycogen was measured using the antrone reactive method. RESULTS
Figure 1A shows the changes in serum concentrations of glucose and lactate in fish that have been refed after 24 hr of starvation. Glucose concentration rose quickly until 2 hr after food intake, where the levels are three times the initial values. Thereafter, glucose began to fall, reaching 50% of the maximum values 6 hr later. Similar behaviour was observed for 329
330
PABLO GARciA DE FRUTOS et al.
was no significant variation (Table 2). Only a slight decrease was seen in UDPG levels after refeeding. This correlates with a limited active glycogen synthesis while glucose levels were maintained high in serum. As described above (Zhivkov, 1971 in Cowey and Walton, 1989), UDPG levels in liver of S~arus uuratu are also lower than in rat. Changes in hexose 6-phosphate followed a similar pattern to that of fru-2.6-P, (Table 2). Their levels decreased until 2 hr after refeeding, and then rose to initial values. The fructose 6-P level after 2 hr of food supply was 40% of the 24-hr-starved value. Lactate levels in the liver did not show significant differences after food administration, although they were greater than those found in mammals (Claus ct al.. 1984).
(A) 2.0 2.50 3 E \ z 5
2.25
0
(6) 50
0.8 3 0
40
30
20 0.2
10
L
Oh 012345678
Hours
Fig.
1. Changes
after
refeeding
in serum and liver parameters
after refeed-
ing 24-hr starved fish. (A) Changes in serum glucose (m) and lactate (0). (B) Changes in hepatic levels of fru-2,6-P, (0) and active to total mean + SEM of four difference from starved **p <
PF2K ratio. (0). Each point is the to eight animals. Significance of fish by Student’s t-test. *P < 0.05, 0.01. ***p < 0.001.
lactate although with smaller variations, rising to 2.3 pmol/ml. The highest value was found 4 hr after refeeding, and at 8 hr values were not significantly above initial levels. In striking contrast to rat, fru-2,6-P, levels in liver of Sparus au~ata remain high after 24-hr starvation but, after refeeding, the values fell to 50% of the initial levels. No evident change was observed for the next 4 hr (Fig. 1B). At 8 hr after food administration, the fru-2,6-P, levels were restored back to the level of 24-hr starved fish. Interestingly, an essentially similar pattern was followed by changes in active/total PF2K activity ratios (Table I). A high correlation between decreases in active/total ratio and in fru-2,6-P, levels was found throughout the period of study (Fig. IB). Provision of food to the 24-hr starved fish did not lead to significant changes in glycogen storage for the next 8 hr. As the initial levels of glycogen in the liver were high, differences between time intervals were smaller than differences between individuals, so there
DISCUSSION
Recently, it has been suggested that the source of glycogen carbon after refeeding follows an indirect pathway from 3-carbon compounds derived from the metabolism of extrahepatic tissues which are converted to glucose &phosphate by gluconeogenesis (reviewed by Kurland and Pilkis, 1989). Fructose 2.6-P2 levels remain low during glycogen synthesis, which indicates that glycogen is being produced from these precursors (Claus et al., 1984). In animals other than mammals, there is only scant information as to whether this indirect pathway occurs or not. In these experiments, it was observed that, although glycogen levels are high, levels of fru-2,6-P, diminish, even when serum glucose concentration is increased by refeeding (Fig. 1). This could be partially explained because the levels of the substrate of PF2K, fructose 6-P, are decreased. PFZK total activity does not change significantly during this period (Table 1). However, when the active form of the kinase is measured, the decrease in activity observed correlates with that of the fru-2,6-P, level. These results suggest than an accurate regulation of fru-2,6-P, levels is involved in liver of Spat-us auratu, probably by phosphorylation of the enzyme, as it is in rat. Although fru-2,6-P, has been poorly studied in fish, this metabolite activates PFlK and is regulated by diet (Foster et al., 1989; Garcia de Frutos et (II., 1990). When fru-2,6-P, levels are low, the gluconeogenie flux increases in liver (Hue and Rider, 1987). Nagai and Ikeda (in Cowey and Walton, 1989) found that after 6-[‘4-C]g1ucose loading intraperitoneally, it was randomized appreciably, supporting the fact that, in these conditions, the gluneogenic pathway is active in fish. In these conditions, the production of hexose 6-phosphate will increase and then is diverted to different pathways (Suarez and Mommsen, 1987).
Table I. Levels of PFZK activities (mU:g wet wt) in liver of refed fish after 24-hr slarvation. Values are the mean f SEM of the results obtained from four animals. The active form of PFZK is measured at 6.5 pH and I mM fructose 6-P. whereas total activity is measured at 8.5 pH and 5 mM fructose 6-P. Significance is as in Fig. I Hours after refeeding PFZK Total Active Active/total
Ohr
I hr
2 hr
4 hr
8 hr
22.7 k I.1 18.8 + I.1 0.83 + 0.02
21.2 + 3.1 10.3 + 2.9” 0.50 + 0.09’
26.6 + 4.7 15.5 + 3.6* 0.51 + 0.06*
20.4 f 3.6 10.4 f oJ3** 0.65 + 0.02”
21.1 _t 0.7 I9 2 _t 3.7 0.91 +o.is
Starved-to-fed transition in fish
331
Table 2. Changes in hepatic metabolites after refeeding 24-hr starved fish. Values are given in nmol/g glycogen (mg/g) as mean k SEM of six to eight animals. Significance is as in Fig. 1
liver. except
Hours after refeeding 1 hr
0 hr Glycogen (mgig) UDPG Glucose 6-P Fructose 6-P Lactate
154 i_ 13 1X4*22 419 + 90 141 i42 862 + 201
185 141 227 83.7 I045
* * i k *
31 14’ 43’** 16* 157
Mobilization of glycogen stores is rather slow in fish, given that 24-hr starvation does not produce a significant decrease in glycogen liver levels (Morata et al., 1982 and Garcia de Frutos et al., 1990), as it does in mammals (Bois-Joyeux et al., 1986). When the glycogen stores is almost replenished, hexose 6-phosphate could only be partially diverted to glycogen. Furthermore, the levels of UDPG only fall to 85% of the values in fish starved for 24 hr, whereas the UDPG falls to 50% in the rat (Claus et al., 1984). In mammals, much of the hexose 6-phosphate pool is diverted to glycogen synthesis, and this produces a decrease in its concentration (Bois-Joyeux et al., 1986). In fish, the decrease in hexose 6-phosphate could be explained by an activation of the pentose phosphate pathway, as this is one of the main ways of metabolizing sugars in fish liver (Berg and Buth, 1984). Glucose 6-P dehydrogenase and 6-P-gluconate dehydrogenase activities will furnish NADPH for lipogenesis. As lipids are stored in the liver, high levels of NADPH will be required for de noco fatty acid synthesis (Yamauchi et al., 1975). This model would explain why glucose is a poor precursor in fish liver metabolism (Christiansen and Klungsoyr, 1987). As the intake of food diminishes the glycolytic capacity in liver, decreasing fructose 2,6-bisphosphate levels, glucose would be metabolized by extrahepatic tissues and 3-carbon compounds could, by gluconeogenesis, be directed to glucose-glycogen. The excess of glucose is then slowly metabolized to more reduced reserves such as fatty acids in the liver but from 3-carbon precursors. The gluconeogenic pathway should also provide enough substrate to keep the pentose phosphate pathway activated and produce NADPH for lipogenesis. to Dr F. to this work. We thank the “Zoo de Barcelona” for use of the Aquarium facilities. We acknowledge NIDO Industrial SA for providing us with the diets used and partial economic support. We also thank the SAL (Language Advisory Service) for correction of the manuscript. P.G. de F. received a fellowship from CIRIT (Generalitat de Catalunya). Ac,kno~~k~dRemenrs-The
authors
are
grateful
Fernandez and his research group for contributions
REFERENCES
R., Hue L., Van Schaftingen E. and Hers H. G. (1983) Hormonal regulation of fru-2.6-P? concentrations in isolated hepatocytes. Eioclient. J. 214, 829-837. Berg W. J. and Buth D. G. (1984) Glucose dehydrogenase in teleost: tissue distribution properties. Camp. Biochem. Physiol. 778, 285-288. Bergmeyer H. U. (1984) Merliod.v sf Enzymatic Anal.vsis. Academic Press, New York. Bartrons
2 hr 172 141 156 61.1 917
2 k k + F
47 24 23*** 8.1** 83
4 hr 163 i 17 123*31 168 k 47”’ 85.0 k 16. 720F51
8 hr 160+25 152 + I2 281 5 69 163 k 28 805 * 104
Bois-Joyeux B., Chanez M., Azzout B. and Peret J. (1986) Studies on the early changes in rat hepatic fru-2,6-P, and enzymes in response to a high protein diet. J. Nufr. 116, 446454. Cowey C. B. and Walton M. J. (1989) In Fish Nulrition (Edited by Halver J. E.), pp. 260-331. Academic Press, New York. Christiansen D. C. and Klungseyr L. (1987) Metabolic utilization of nutrients and the effects of insulin in fish. Comp. Biochem. Physiol. I38B, 701l711. Claus T. H., Nyfeler F., Muenkel H. A., Burns M. G. and Pilkis S. J. (1984) Changes in fru-2,6-Pz levels and after glucose loading of starved rats. Biochem. Biophys. Res. Commun. 122, 529-534. Foster G. D., Storey K. B. and Moon T. W. (1989) The regulation of fructose 6-phosphate- 1 kinase by insulin and glucagon in isolated hepatocytes of the American eel. Cm. camp. Endocrinol. 13, 382-389. Garcia de Frutos P., Bonamusa L., Fernandez F. and Baanante 1. V. (1990) Fru-2,6-P, in liver of Sparus aurata: influence of nutritional state. Comp. Biochem. Physiol. 96B, 6345. Hue L. and Rider M. H. (1987) Role of fru-2,6-P? in the control of glycolysis in mammalian tissues Biochem. J. 254, 3 133324. Kurland I. J. and Pilkis S. J. (1989) Indirect versus direct routes of hepatic glycogen synthesis. FASEB J. 3, 2271-2281. McGarry J. D., Kuwajima M., Newgard C. B., Foster D. W. and Katz J. (1987) From dietary glucose to liver glycogen: the full circle around. Ann. Rev. Nufr. 7, 51-73. Morata P., Faus M. J., Perez-Palomo M. and SanchezMedina F. (1982) Effect of stress on liver and muscle glycogen phosphorylase in rainbow trout. Comp. Biothem. Physiol. 72B, 421425. Newgard C. B., Hirsch L. J., Foster D. W. and McGarry J. D. (1983) Studies on the mechanism by which exogenous glucose is converted into liver glycogen in rat. J. biol. Chem. 258, 8046-8052. Pilkis S. J., El-Maghrabi R. and Claus T. H. (1988) Hormonal regulation of hepatic gluconeogenesis and glycolysis. Ann. Rev. Biochem. 57, 755-783. Renaud J. M. and Moon T. W. (1980) Characterization of gluconeogenesis in hepatocytes isolated from the American eels Anguilla rostrata. J. camp. Physiol. 135B, 115~120. Riesenfeld G., Wals P. A., Golden S. and Katz J. (1981) Glucose, amino-acids and lipogenesis in hepatocytes of Japanese quail. J. hioi. Chem. 256, 9973-9980. Suarez R. K. and Mommsen T. P. (1987) Glucose in teleost fish. Can. J. Zool. 65, 186991882. Sugden M. C.. Holness M. J. and Palmer T. N. (1989) Fuel selection and carbon flux during starved-to-fed transition. Biochem. J. 263, 313-323. Walton M. J. and Cowey C. B. (1979) Gluconeogenesis from Serine in rainbow trout (Salmo nardnieri) liver. Coma. Biochem. Physiol. 62B, 497499. _ Yamauchi T.. Stegeman J. J. and Goldberg E. (1975) The effect of starvation and temperature acclimatation on phosphogluconato-dehydrogenase in brook trout liver. Arch. Biochem. Biophys. 167, 13-20.
Camp. Eiochem.Fh~riol.Vol.98A. No. 2, pp. 329-331, 1991 Printed in Great B&XI
e 1991Petgamon Press plc
METABOLIC CHANGES IN FISH LIVER DURING THE STARVED-TO-FED TRANSITION PABLO GARciA DE FRUTOS,LLUISBONAMUSA and ISABELV. BAANANTE* Unitat de Bioquimica. Facultat de Farmicia, Universitat de Barcelona, Plaza Pio XII sin, Barcelona 08028, Spain. Telephone: (93) 330.79.63 (Received 9 May 1990) Abstract-i,
In contrast to the rat, fish starved for 24 hr maintain high levels of fructose 2,6-bisphosphate in the liver. 2. After refeeding, these levels are decreased and kept low for the next 4 hr. 3. The changes observed in fru-2,6-P, correlate with those in the phosphofructo 2-kinase activity ratio, suggesting an accurate regulation of the process. 4. Measurements of glycogen and hexose 6-phosphate indicate that dietary carbohydrates are utilized by fish in a different way from mammals.
INTRODUCTION
The effect of fructose 2,6-bisphosphate as a potent simulator of glycolysis in mammalian tissues has been well established (Hue and Rider, 1987; Pilkis et al., 1987). The levels of this metabolite play a key role in the regulation of hepatic glucose metabolism, depending on the glucose levels in serum (Sugden et al., 1989). Since the studies of Newgard et al. (1983), several groups have supported the idea that the gfycogen stored in liver is not directly derived from dietary glucose but mainly from 3-carbon compounds via the gluconeogenic pathway (for review, see McGarry et al., 1987). According to this idea, levels of fructose 2,6-bisphosphate remain low in the transition from fasted to fed state until glycogen level has been restored. Then, fru-2,6-Pz levels rise preventing further glycogen deposition and directing the liver metabolism to the synthesis of lipids (Sugden et al., 1989). Little information is available on the changes involved in starved-to-fed transition in animal species other than mammals. Studies with the Japanese quail suggest that the regulation of the pathway is completely different from that in mammals, and in this species there is no indirect pathway (Riesenfeld et al., 1981). Fish metabolism is different to that of mammals (Christiansen and Klugseryr, 1987). Prolonged starvation is required before any glycogen depletion is observed in the liver. Also, it appears that they have a limited capacity for using carbohydrates from the diet which, in carnivorous fish, are often substituted by amino acids as precursors of energetic reserves (Walton and Cowey, 1979). Interestingly, Renaud and Moon (1980) found that in physiological conditions lactate is a better precursor than amino acids for gluconeogenesis. This work was undertaken to determine the molecular mechanisms involved in fish during
*Author to whom all correspondence
should be addressed.
starved-to-fed transition, whether fish metabolism fits an “indirect” model of utilizing carbohydrates from diet and, where possible, why carbohydrates are poorly used by carnivorous fish. This paper presents the changes in hepatic metabolism in fish liver during the starved-to-fed transition. MATERIALS AND METHODS Spans aumtu (gilth~d sea bream) were used in this study. Groups of 20-25 fish, weighing 12-20 g were held in 1001 tanks provided with filtered aerated sea-water, in a closed system with a UV-lamp, the temperature was kept at 19 + 1°C and the dark/light cycle was 12: 12 hr. Fish were fed once a day with 0.5 g of food per fish in pellet form (46% protein, 7% lipids, 24% carbohydrates, 15% salts). A 7-10 day a~limat~~tion period was provided before experimental work was begun. After 24 hr of starvation, the Sparus were gently netted from the holding tank and anaesthetized with MS-222 (1: 1000). The liver was dissected out immediately after decapitation, and freeze-clamped in liquid nitrogen. Blood samples were drawn from the open heart. Several samples were combined to obtain enough serum for glucose and lactate assays. Other starved fish were allowed to refeed and were killed at various times after being fed. Measurements of fru-2,6-P, and PP, : PFK purification from potato tubers were performed according to the method of Van Schaftingen (Bergmeyer, 1984). Fructose 6-phosphate 2-kinase activity was measured in its active and total forms, as described by Bartrons et al. (1983). The rest of metabolites were measured s~ctrophotometricaliy by established enzymatic procedures in neutralized perchloric acid extracts (Bergmeyer, 1984). Glycogen was measured using the antrone reactive method. RESULTS
Figure 1A shows the changes in serum concentrations of glucose and lactate in fish that have been refed after 24 hr of starvation. Glucose concentration rose quickly until 2 hr after food intake, where the levels are three times the initial values. Thereafter, glucose began to fall, reaching 50% of the maximum values 6 hr later. Similar behaviour was observed for 329
330
PABLO GARciA DE FRUTOS et al.
was no significant variation (Table 2). Only a slight decrease was seen in UDPG levels after refeeding. This correlates with a limited active glycogen synthesis while glucose levels were maintained high in serum. As described above (Zhivkov, 1971 in Cowey and Walton, 1989), UDPG levels in liver of S~arus uuratu are also lower than in rat. Changes in hexose 6-phosphate followed a similar pattern to that of fru-2.6-P, (Table 2). Their levels decreased until 2 hr after refeeding, and then rose to initial values. The fructose 6-P level after 2 hr of food supply was 40% of the 24-hr-starved value. Lactate levels in the liver did not show significant differences after food administration, although they were greater than those found in mammals (Claus ct al.. 1984).
(A) 2.0 2.50 3 E \ z 5
2.25
0
(6) 50
0.8 3 0
40
30
20 0.2
10
L
Oh 012345678
Hours
Fig.
1. Changes
after
refeeding
in serum and liver parameters
after refeed-
ing 24-hr starved fish. (A) Changes in serum glucose (m) and lactate (0). (B) Changes in hepatic levels of fru-2,6-P, (0) and active to total mean + SEM of four difference from starved **p <
PF2K ratio. (0). Each point is the to eight animals. Significance of fish by Student’s t-test. *P < 0.05, 0.01. ***p < 0.001.
lactate although with smaller variations, rising to 2.3 pmol/ml. The highest value was found 4 hr after refeeding, and at 8 hr values were not significantly above initial levels. In striking contrast to rat, fru-2,6-P, levels in liver of Sparus au~ata remain high after 24-hr starvation but, after refeeding, the values fell to 50% of the initial levels. No evident change was observed for the next 4 hr (Fig. 1B). At 8 hr after food administration, the fru-2,6-P, levels were restored back to the level of 24-hr starved fish. Interestingly, an essentially similar pattern was followed by changes in active/total PF2K activity ratios (Table I). A high correlation between decreases in active/total ratio and in fru-2,6-P, levels was found throughout the period of study (Fig. IB). Provision of food to the 24-hr starved fish did not lead to significant changes in glycogen storage for the next 8 hr. As the initial levels of glycogen in the liver were high, differences between time intervals were smaller than differences between individuals, so there
DISCUSSION
Recently, it has been suggested that the source of glycogen carbon after refeeding follows an indirect pathway from 3-carbon compounds derived from the metabolism of extrahepatic tissues which are converted to glucose &phosphate by gluconeogenesis (reviewed by Kurland and Pilkis, 1989). Fructose 2.6-P2 levels remain low during glycogen synthesis, which indicates that glycogen is being produced from these precursors (Claus et al., 1984). In animals other than mammals, there is only scant information as to whether this indirect pathway occurs or not. In these experiments, it was observed that, although glycogen levels are high, levels of fru-2,6-P, diminish, even when serum glucose concentration is increased by refeeding (Fig. 1). This could be partially explained because the levels of the substrate of PF2K, fructose 6-P, are decreased. PFZK total activity does not change significantly during this period (Table 1). However, when the active form of the kinase is measured, the decrease in activity observed correlates with that of the fru-2,6-P, level. These results suggest than an accurate regulation of fru-2,6-P, levels is involved in liver of Spat-us auratu, probably by phosphorylation of the enzyme, as it is in rat. Although fru-2,6-P, has been poorly studied in fish, this metabolite activates PFlK and is regulated by diet (Foster et al., 1989; Garcia de Frutos et (II., 1990). When fru-2,6-P, levels are low, the gluconeogenie flux increases in liver (Hue and Rider, 1987). Nagai and Ikeda (in Cowey and Walton, 1989) found that after 6-[‘4-C]g1ucose loading intraperitoneally, it was randomized appreciably, supporting the fact that, in these conditions, the gluneogenic pathway is active in fish. In these conditions, the production of hexose 6-phosphate will increase and then is diverted to different pathways (Suarez and Mommsen, 1987).
Table I. Levels of PFZK activities (mU:g wet wt) in liver of refed fish after 24-hr slarvation. Values are the mean f SEM of the results obtained from four animals. The active form of PFZK is measured at 6.5 pH and I mM fructose 6-P. whereas total activity is measured at 8.5 pH and 5 mM fructose 6-P. Significance is as in Fig. I Hours after refeeding PFZK Total Active Active/total
Ohr
I hr
2 hr
4 hr
8 hr
22.7 k I.1 18.8 + I.1 0.83 + 0.02
21.2 + 3.1 10.3 + 2.9” 0.50 + 0.09’
26.6 + 4.7 15.5 + 3.6* 0.51 + 0.06*
20.4 f 3.6 10.4 f oJ3** 0.65 + 0.02”
21.1 _t 0.7 I9 2 _t 3.7 0.91 +o.is
Starved-to-fed transition in fish
331
Table 2. Changes in hepatic metabolites after refeeding 24-hr starved fish. Values are given in nmol/g glycogen (mg/g) as mean k SEM of six to eight animals. Significance is as in Fig. 1
liver. except
Hours after refeeding 1 hr
0 hr Glycogen (mgig) UDPG Glucose 6-P Fructose 6-P Lactate
154 i_ 13 1X4*22 419 + 90 141 i42 862 + 201
185 141 227 83.7 I045
* * i k *
31 14’ 43’** 16* 157
Mobilization of glycogen stores is rather slow in fish, given that 24-hr starvation does not produce a significant decrease in glycogen liver levels (Morata et al., 1982 and Garcia de Frutos et al., 1990), as it does in mammals (Bois-Joyeux et al., 1986). When the glycogen stores is almost replenished, hexose 6-phosphate could only be partially diverted to glycogen. Furthermore, the levels of UDPG only fall to 85% of the values in fish starved for 24 hr, whereas the UDPG falls to 50% in the rat (Claus et al., 1984). In mammals, much of the hexose 6-phosphate pool is diverted to glycogen synthesis, and this produces a decrease in its concentration (Bois-Joyeux et al., 1986). In fish, the decrease in hexose 6-phosphate could be explained by an activation of the pentose phosphate pathway, as this is one of the main ways of metabolizing sugars in fish liver (Berg and Buth, 1984). Glucose 6-P dehydrogenase and 6-P-gluconate dehydrogenase activities will furnish NADPH for lipogenesis. As lipids are stored in the liver, high levels of NADPH will be required for de noco fatty acid synthesis (Yamauchi et al., 1975). This model would explain why glucose is a poor precursor in fish liver metabolism (Christiansen and Klungsoyr, 1987). As the intake of food diminishes the glycolytic capacity in liver, decreasing fructose 2,6-bisphosphate levels, glucose would be metabolized by extrahepatic tissues and 3-carbon compounds could, by gluconeogenesis, be directed to glucose-glycogen. The excess of glucose is then slowly metabolized to more reduced reserves such as fatty acids in the liver but from 3-carbon precursors. The gluconeogenic pathway should also provide enough substrate to keep the pentose phosphate pathway activated and produce NADPH for lipogenesis. to Dr F. to this work. We thank the “Zoo de Barcelona” for use of the Aquarium facilities. We acknowledge NIDO Industrial SA for providing us with the diets used and partial economic support. We also thank the SAL (Language Advisory Service) for correction of the manuscript. P.G. de F. received a fellowship from CIRIT (Generalitat de Catalunya). Ac,kno~~k~dRemenrs-The
authors
are
grateful
Fernandez and his research group for contributions
REFERENCES
R., Hue L., Van Schaftingen E. and Hers H. G. (1983) Hormonal regulation of fru-2.6-P? concentrations in isolated hepatocytes. Eioclient. J. 214, 829-837. Berg W. J. and Buth D. G. (1984) Glucose dehydrogenase in teleost: tissue distribution properties. Camp. Biochem. Physiol. 778, 285-288. Bergmeyer H. U. (1984) Merliod.v sf Enzymatic Anal.vsis. Academic Press, New York. Bartrons
2 hr 172 141 156 61.1 917
2 k k + F
47 24 23*** 8.1** 83
4 hr 163 i 17 123*31 168 k 47”’ 85.0 k 16. 720F51
8 hr 160+25 152 + I2 281 5 69 163 k 28 805 * 104
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