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Metabolic changes in Brycon cephalus (Teleostei, Characidae) during post-feeding and fasting
Comparative Biochemistry and Physiology Part A 132 (2002) 467–476
Metabolic changes in Brycon cephalus (Teleostei, Characidae) during post-feeding and fasting b ´ M.L. Figueiredo-Garuttia, *, I. Navarrob, E. Capillab, R.H.S. Souzac, G. Moraesd, J. Gutierrez , M.L.M. Vicentini-Paulinoe
b
a ´ ˜ Jose´ do Rio Preto, Sao ˜ Paulo, Brazil Rua Frei Valerio Kirch, 78, 15054-070 Sao Departament de Fisiologia, Facultat de Biologia, D. III, Universitat de Barcelona, Avinguda Diagonal 645, 08028 Barcelona, Spain c ´ ˜ Paulo, Brazil Laboratorio de Fisiologia, Centro Nacional de Pesquisa de Peixes Tropicais, IBAMA. 13630-000 Pirassununga, Sao d ¸˜ ´ ´ ´ Laboratorio de Bioquımica Adaptativa, Departamento de Genetica e Evolucao, ˜ Carlos. 13560-000 Sao ˜ Carlos, Sao ˜ Paulo, Brazil Universidade Federal de Sao e ˆ ˜ Paulo, Departamento de Fisiologia, Instituto de Biociencias, Universidade Estadual Paulista, UNESP, 18600-000 Botucatu, Sao Brazil
Received 17 August 2001; received in revised form 17 January 2002; accepted 3 March 2002
Abstract Metabolic changes during the transition from post-feeding to fasting were studied in Brycon cephalus, an omnivorous teleost from the Amazon Basin in Brazil. Body weight and somatic indices (liver and digestive tract), glycogen and glucose content in liver and muscle, as well as plasma glucose, free fatty acids (FFA), insulin and glucagon levels of B. cephalus, were measured at 0, 12, 24, 48, 72, 120, 168 and 336 h after the last feeding. At time 0 h (the moment of food administration, 09.00 h) plasma levels of insulin and glucagon were already high, and relatively high values were maintained until 24 h post-feeding. Glycemia was 6.42"0.82 mM immediately after food ingestion and 7.53"1.12 mM at 12 h. Simultaneously, a postprandial replenishment of liver and muscle glycogen reserves was observed. Subsequently, a sharp decrease of plasma insulin occurred, from 7.19"0.83 ngyml at 24 h of fasting to 5.27"0.58 ngyml at 48 h. This decrease coincided with the drop in liver glucose and liver glycogen, which reached the lowest value at 72 h of fasting (328.56"192.13 and 70.33"14.13 mmolyg, respectively). Liver glucose increased after 120 h and reached a peak 168 h post-feeding, which suggests that hepatic gluconeogenesis is occurring. Plasma FFA levels were low after 120 and 168 h and increased again at 336 h of fasting. During the transition from post-feeding to fast condition in B. cephalus, the balance between circulating insulin and glucagon quickly adjust its metabolism to the ingestion or deprivation of food. 䊚 2002 Elsevier Science Inc. All rights reserved. Keywords: Post-feeding; Starvation; Glycemia; FFA; Insulin; Glucagon; Hepatic and muscle glycogen; Hepatic and muscle glucose; Fish; Brycon cephalus
1. Introduction Fish have diverse strategies to deal with periods of food deprivation. The degree of glucose homeo*Corresponding author. Tel.: q55-17-2249627; fax: q5517-2249627. E-mail address: [email protected] (M.L. Figueiredo-Garutti).
stasis during fast varies with species. Long-term responses to food deprivation have been extensively studied. No changes in plasma glucose levels were observed after several weeks of fast in the hagfish (Myxine glutinosa) (Falkmer and Matty, 1966), or even months in species such as Anguilla rostrata (Suarez and Mommsen, 1987) or Clarias ´ lazera (Navarro and Gutierrez, 1995). In Rhamdia
1095-6433/02/$ - see front matter 䊚 2002 Elsevier Science Inc. All rights reserved. PII: S 1 0 9 5 - 6 4 3 3 Ž 0 2 . 0 0 0 9 4 - 6
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hilarii, a 50% decrease of glycemia occurs after 30 days of starvation (Machado et al., 1988). However, it remains unclear whether glycemia control is related to the nutritional preferences of fish (herbivorous or carnivorous species). Our knowledge of metabolic strategies of omnivorous or herbivorous teleost remains limited compared to the data available for carnivorous species. Brycon cephalus is an omnivorous fish from the Amazon Basin in Brazil. In its natural habitat it ingests high quantities of fruits and seeds, and when the food becomes scarce, it eats insects and small fish. Recently, it has been reported that omnivorous species that are more tolerant to diet glucose show different insulin response to dietary carbohydrate and capacity of liver in phosphorylating glucose than carnivorous species (Panserat et al., 2000). Furthermore, muscle insulin receptors are more numerous in species with a higher degree ´ of glucose tolerance (Parrizas et al., 1995). Nevertheless, few data are available on the fasting metabolism of herbivorous or omnivorous fishes ´ (Navarro and Gutierrez, 1995). The maintenance of glycemia during food deprivation is directly related to the capacity of mobilization on hepatic glycogen, at least during the initial stages of fasting, and also depends on posterior activation of the hepatic gluconeogenesis (Higuera and Cardenas, 1984) and the reduction in the rate of glucose utilization (Moon and Foster, 1995). In most fish species liver glycogen is mobilized early during fasting but the degree of glycogen depletion varies greatly between species, ranging from rapid glycogenolysis to partial or almost complete protection of glycogen reserves with fast (Sheridan and Mommsen, 1991). The increased gluconeogenic capacity of the liver may partially be due to the changes in plasma glucoregulatory hormone concentrations, i.e. insulin and glucagon, andyor changes in hepatocyte responsiveness to these hormones (Pereira et al., 1995). Hormone changes and effects in fish are not always similar to that of mammals (Foster and Moon, 1987). In some species, hepatocyte response to insulin is altered with fast or stress, favoring glycogen conservation (Vijayan et al., 1993, 1994). Plasma insulin levels generally decrease with fasting in most fish species (Plisetskaya et al., 1986). In comparison with insulin, the role of glucagon during fasting in fish has been studied in relatively few species. The glucagon response
changes according to the species or length of the fasting period. This hormone decreased in Oncorhynchus mykiss (Moon et al., 1989) and Gadus morhua (Hemre et al., 1990) after 6 weeks and 7 days of fast, respectively. In Dicentrarchus labrax and Salmo trutta few days of fasting provoked ´ increases in circulating glucagon (Gutierrez et al., 1991; Navarro et al., 1992). In contrast with the abundant literature on the metabolic responses to long-term fasting in fish, studies on the metabolic changes during, postfeeding and early fasting periods are not commonly ´ found (Navarro and Gutierrez, 1995). Moreover, there is little information on the biology of Brycon cephalus, especially on the effects of fasting on intermediary metabolism and no data referring to insulin and glucagon in this species. The search for parameters on the nutritional and physiological situation of B. cephalus may help to improve culture conditions of this species, which has a great potential for aquaculture because of its rapid growth. We test the hypothesis that omnivorous B. cephalus possess more flexibility in glucose metabolism than carnivorous species and enhanced ability to modulate metabolism in order to match changes in nutritional state. Here, we examine in B. cephalus the absorption and mobilization of metabolites, especially carbohydrates, during the post-feeding period until 14 days of fasting and their relation with changes in plasma pancreatic hormones. 2. Material and methods 2.1. Animals and experimental procedure The experiments were performed in November. Adult B. cephalus (ns40) bred by induced reproduction with an average initial weight of 876.0"13.6 g were used (a voucher specimen has been identified and deposited at the Museum of ˜ Paulo—MZUSP Zoology of the University of Sao 54008). The animals were distributed in eight homogenous groups and kept in open circuit tanks of 3000-l capacity and provided with well-aerated water. The average water temperature was 27.43"0.02 8C. Fish were fed commercial tropical ´ ´ fish pellets (Socil pro-pecuaria SyA), composed of 6% water, 30% crude protein, 8% crude fat, 7% crude fiber, and 9% mineral mixture. The food was administered ad libitum at 09.00 h daily, the
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habitual time of administration. Tissue and blood samples were collected at 0, 12, 24, 48, 72, 120, 168 and 336 h after feeding. Time 0 corresponded to the moment of food administration (09.00 h). At each time of sampling, five fish were netted from a different tank. The animals were anaesthetized with 2-phenoxyethanol (0.5 mlyl). Blood was taken from the dorsal aorta into heparinized syringes. Aliquots of plasma were frozen and stored at y20 8C until assay. Fish were quickly killed by a blow to the head, and weighed. Then, the viscera were examined and the livers were removed, weighed, frozen immediately on dry ice and stored at y20 8C for later assay. A sample of white epaxial muscle located between dorsal and adipose fins was obtained and similarly stored until analyses. Liver, stomach and intestine were weighed to obtain the hepatic somatic index wHSIs(hepatic weight=100)ybody wt.x, full stomach somatic index w f SSIsfull stomach weight=100)ybody wt.x, and full intestine somatic index w f ISIsfull intestine weight=100)ybody wt.x.
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3. Results 3.1. Body weight and somatic indexes Food deprivation induced a progressive reduction in body weight from an initial value of 876.0"13.6–646.0"71.6 g at the end of the experiment, corresponding to 73.7% of the initial weight. The hepatic somatic index increased from an initial value of 0.75"0.06–1.22"0.18% at 48 h after feeding and then it decreased progressively during the experimental period. The values obtained from 72 h of fast to the end of the experiment were not significantly different. The average stomach somatic index decreased in the first 24 h from 9.17"1.29% to 1.29"0.51% and maintained low values until the end of the experiment. The intestine somatic index decreased from 1.38"0.18% to 0.55"0.10% at 72 h after feeding, and then remained with minimal changes (Fig. 1). 3.2. Plasma glucose, free fatty acids, glucagon and insulin
2.2. Analytical methods Plasma insulin levels were measured by radioimmunoassay (RIA) using bonito insulin as the standard and rabbit anti-bonito insulin as antiserum ´ according to methodology proposed by Gutierrez et al. (1984). Glucagon levels were determined by heterologous RIA validated for fish plasma (Navarro et al., 1995). Validation of the assays for B. cephalus was confirmed by plasma dilution tests for this species, previously performed for both insulin and glucagon RIAs. Plasma glucose concentration was estimated by glucose oxidaseperoxidase (Sigma Chemical Company kit). Plasma free fatty acids (FFA) were measured by the method of Dole and Meinertz (1960). Glycogen content was estimated in the liver and muscle by the method of Duboie et al. (1956), modified by Bidinotto et al. (1997), and the liver and muscle glucose was determined by the method of Duboie et al. (1956). Results are presented as mean"standard deviation (S.D.). Statistical differences were estimated by one-way analysis of variance (ANOVA); and the difference among means was estimated by the Tukey test. The significance level adopted was 0.05.
Immediately after food ingestion, the mean plasma glucose concentration was 6.42"0.82 mM and 12 h later, it increased to 7.53"1.12 mM. At 24 h of fast, glucose levels returned to initial values (6.31"0.78 mM). Subsequently, plasma glucose decreased and no significant changes were observed thereafter. The free fatty acids (FFA) increased from 1.20"0.04 to 1.77"0.06 mM at 48 h of fasting. FFA plasma levels were lower after 120 and 168 h and increased again at 336 h (Fig. 2). The highest levels of plasma insulin levels were found through the first 24 h post-feeding, being already high at 0 h (6.75"1.00 ngyml) with elevated values until 24 h (7.19"0.83 ngyml). Later, circulating insulin decreased to 5.27"0.58 ngyml at 48 h of fast. The lowest values were found at the end of the experiment, after 336 h, with a fall in insulin of 35% (with respect to 24 h post-feeding). Circulating glucagon levels were high at 0 time (10.42"1.49 ngyml), and although they declined, were relatively high at 12 and 24 h (3.06"1.04 ngyml). Glucagon levels continued to decrease until 120 h of fast when the value was 0.59"0.25 ngyml. These low values were maintained until the end of the experiment.
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Fig. 1. Body weight (wt.), hepatic somatic index (HSI), full stomach somatic index ( f SSI) and full intestine somatic index ( f ISI) in B. cephalus (ns5) subjected to fasting. Time indicates hours (h) after feeding, where hour 0 corresponds to the feeding moment (09.00 h). Letters represent statistical differences (PF0.05) by the Tukey test. The values presented are mean"standard deviation.
3.3. Liver and muscle glycogen and glucose The liver glycogen increased from 112.33"47.52 to 183.99"48.97 mmolyg in 12 h after food ingestion and subsequently, the concentration decreased to a minimum of 70.33"14.13 mmolyg at 72 h. The glycogen level was recovered after 120 h and the value presented at 336 h after feeding was not different from the initial one. Liver glucose, whose initial value was 444.6"108.9 mmolyg, presented an initial profile similar to that observed in liver glycogen, with an increase at 12 h after feeding, with a value of 664.3"97.9 mmolyg, followed by a sharp decline reaching a nadir (328.6"192.1 mmolyg), at 72 h of fasting. Liver glucose was higher again with a value of 687.5"181.1 mmolyg at 168 h of fasting (Fig. 3). The content of muscle glycogen, which was 15.29"0.66 mmolyg immediately after feeding, showed a progressive increase until 48 h postfeeding (22.62"1.30 mmolyg). At 72 h post-
feeding, muscle glycogen was 19.72"2.07 mmoly g, which did not change until the end of experiment. Muscle glucose concentration, that initially was 16.77"2.81 mmolyg, decreased until 24 h post-feeding, when it reached its lowest value, 10.68"1.30 mmolyg. Later, muscle glucose recovered, attaining values of 18.28"1.95 and 18.31"0.82 mmolyg, at 120 and 168 h of fasting, respectively. 4. Discussion In the present study metabolic alterations involved in fed-to-fasting transition and circulating pancreatic hormones are analyzed in B. cephalus for the first time. The progressive decrease in stomach somatic index suggests that a period of 12–24 h is needed to complete the gastric emptying. Consequently, the transit of digested food through the intestine extended until 48–72 h as suggested by intestine somatic index. Although the meal size, composi-
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Fig. 2. Plasma glucose, free fatty acids (FFA), insulin and glucagon in B. cephalus (ns5) subjected to fasting. Time indicates hours (h) after feeding, where hour 0 corresponds to the feeding moment (09.00 h). Letters represents statistical differences (PF0.05) by the Tukey test. The values presented are mean"standard deviation.
tion of food and animal size has to be taken into account to compare rates of gastric evacuation, it seems that the digestive process of B. cephalus is slightly delayed in comparison with other teleost species with natural omnivorous regime, which tend to present shorter emptying times than carniv¨ orous fishes (Fange and Grove, 1979). For example, data obtained at temperatures of 20–25 8C in Tilapia noctiluca, an herbivore, the same parameter ranged from 15 to 27 h (Moriarty, 1973) while in different species of the genus Ictalurus, carnivorous, showed emptying ranging from 48–84 h (Lane and Jackson, 1969). The postprandial increase of glycemia observed 12 h post-feeding, together with the return of glucose levels to initial values after 24 h paralleled gastrointestinal transit. This moderate rise in postfeeding plasma glucose (33% increase at 12 h in relation to 24 h levels) and the posterior quick decline in glycemia until 72 h suggests an efficient uptake of glucose by peripheral tissues. Postfeeding increases in glycemia vary with the diet
carbohydrate content and glucose tolerance of the species, but the postprandial profile observed in B. cephalus is similar to that found in other teleost ´ species (Navarro et al., 1993; Medale et al., 1999). In many fish species, liver glycogen is one of the first energy reserves to be consumed after several days of fasting, but the moment when the carbohydrate metabolism shifts from a postabsorptive to fast condition is not well documented. The first glycogen increase observed in the present study (12 h after food ingestion) is in concordance with the increase in glucose that arrives to the liver during the absorptive period. In fact, profiles of plasma glucose, liver glucose and hepatic glycogen are very similar in the first experimental period (from 0 to 72 h). At 72 h, these three parameters decreased, suggesting that carbohydrate metabolism changed from postabsorptive to fasting condition. If so, the liver would act as a glucose donor for extra hepatic tissues at the expense of glycogen, as in other fish species (Moon and Foster, 1995). This process
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Fig. 3. Liver and muscle glycogen and glucose in B. cephalus (ns5) subjected to fasting. Time indicates hours (h) after feeding, where hour 0 corresponds to the feeding moment (09.00 h). Letters represent statistical differences (PF0.05) by the Tukey test. The values presented are mean"standard deviation.
seems to be faster than in other fish in which significant decreases in both plasma glucose and glycogen reserves are seen after several days of ´ food deprivation (Navarro and Gutierrez, 1995). Although we cannot infer metabolic fluxes from changes in metabolite concentration (Haman et al., 1997), the partial recovery of liver glycogen after 120 h of fast suggests an increase in glucose production from hepatic gluconeogenesis that exceeds glucose demand. The increase in liver glycogen with fast is reported in fasted Oncorhynchus nerka (Walton and Cowey, 1982) or rainbow trout fasted for 30 days (Higuera and Cardenas, 1984). The glucose oxidation is diminished in trout hepatocytes during fasting, but the capacity for glyconeogenesis in not altered or even enhanced (Pereira et al., 1995). Increase in gluconeogenic enzymes has been reported in the eel,
rainbow trout or yellow perch after long term fast ´ (review by Navarro and Gutierrez, 1995). An increase in gluconeogenesis already at 120 h fast indicates a rapid change of metabolism in response to food deprivation, in agreement with its omnivorous condition. Carnivorous fish liver do not seem to respond quickly with changes in gluconeogenic rates, that are constantly activated irrespective of the nutritional situation, in contrast to omnivorousy herbivorous fishes (Pereira et al., 1995). The pattern of plasma glucose observed in B. cephalus agrees with the metabolic changes in the liver, since glucose levels are maintained after 120 h. It seems that this omnivorous species, although sensitive to food deprivation, quickly adapts its carbohydrate metabolism in response to the absence of exogenous glucose. Depression of met-
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abolic rate with fast, as observed in other fish species, may help to maintain glucose homeostasis. The percentage of carbohydrate reserves in white muscle is usually low (Bone and Marshall, 1982), but the contribution of this tissue as an organ of uptake or storage is high since the total mass of white muscle represents approximately 50% of the body weight. In this study, initial increase in muscle glycogen level indicates a postfeeding replenishment of reserves, which occurs simultaneously to a decrease in muscle glucose together with high plasma glucose and insulin levels. Muscle glucose content corresponds to both interstitial and intracellular glucose (total glucose), although very specific measurements of free intracellular muscle glucose of mammals have given very low values (Roussel et al., 1998). Decreases in total muscle glucose concentration of rats occur in responses to simultaneous increases in plasma insulin and glucose (Halseth et al., 2001). The fall in muscle glucose observed initially suggest that the rate of removal of interstitial glucose and its subsequent phosphorylation exceed the rate at which the glucose is replenished. These observations are in accordance with an increase in the rate of insulin-stimulated glucose uptake by muscle. Studies on the regulation of fish glucose transporters by insulin may help us to understand the regulation of glucose fluxes into insulin-sensitive tissues (Planas et al., 2000). The subsequently decrease of muscle glycogen (48–72 h) was parallel to the increase in glucose in this tissue. This glucose originates either from muscle glycogen or from a decrease in the rate of glucose uptake by this tissue concomitant to a decrease in plasma insulin values. Muscle glycogen may not contribute directly to homeostasis of blood glucose because the muscular tissue itself probably uses this reserve when it is needed. In fish, glucose production by the Cori Cycle apparently does not occur as it does in mammals (Batty and Wardle, 1979; Cornish and Moon, 1985; Milligan and McDonald, 1988). A significant decrease of glycogen in muscle of fasted Boleophthalmus boddaerti was considered an indication that this substrate could be the first fuel to be utilized for muscle activities (Lim and Ip, 1989). In B. cephalus, the posterior stabilization of glycogen and glucose values of white muscle (after 72 h) indicates that muscle glycogen stores are not primarily used during the period of fast.
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In relation to lipid metabolism, the peak of plasma FFA in B. cephalus observed 48 h after feeding could be related to a characteristic delayed absorption of lipids in comparison with other metabolites. Although an increase in plasma free fatty acids is considered a sign of lipolytic activity in mammals, this is not well established in fish. Plasma FFA increased with prolonged fasting in Carassius auratus, Anguilla anguilla, and Oncorhynchus mykiss (Larsson and Lewander, 1973; Wiegand and Peter, 1980; Leatherland and Nuti, 1981), but decreased in fasting Opsanus tau and Oncorhynchus nerka (Tashima and Cahill, 1965; McKeown et al., 1975). The maintenance of high levels of FFA during the fast period and the increase observed in this study, 336 h after last meal suggest a significant contribution of fat in providing fuel for B. cephalus. Lipid storage utilization may explain the decrease in body weight observed at the end of the experiment. To our knowledge, the levels of plasma insulin and glucagon in B. cephalus were determined for the first time in the present study. Post-feeding glucagon values were high, especially when compared with other species of fish. For instance, postprandial value of 3.0 ngyml has been reported in Oncorhynchus kisutch and Salmo trutta (Sheridan and Mommsen, 1991; Navarro et al., 1995). Furthermore, the highest glucagon levels were found at time 0, but insulin levels were also high. An early pre-feeding response of pancreatic hormones, possibly neurally mediated, has been described in fish (Papatryphon et al., 2001). It has been assumed that in mammals early increases in plasma insulin and glucagon optimize and improve nutrient assimilation, and the release of opposite hormones may maintain balance in glucose homeostasis (Nicolaidis, 1977; Teff and Engelman, 1996). B. cephalus might be especially sensitive to pancreatic hormone response just before eating, which would favor carbohydrate digestion and metabolism. In the present study, post-feeding insulin levels were maintained elevated (from 0 to 24 h) after ingestion of food in concordance with somatic indexes and glucose profiles. As in other fish species, high circulating insulin levels may favor the uptake of glucose by liver and especially by muscle, and the repletion of glycogen stores (Mommsen and Plisetskaya, 1991). In comparison with brown trout, in which plasma insulin levels were high during 1–5 h post-feeding and decreased
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after 8–11 h (Navarro et al., 1993), a prolonged post-feeding insulin profile is observed in B. cephalus. Increases in glucagon levels with short-term fast have been described only in two species of fish, both of them carnivorous. In Dicentrarchus labrax an increase in glucagon levels was observed after ´ 4 days of fasting (Gutierrez et al., 1991), and a similar but less pronounced response was observed in Salmo trutta fario upon fasting for 5 days: this has been explained as a signal to shift carbohydrate metabolism towards degradation of stores (Navarro et al., 1992). However, the levels of both pancreatic hormones decreased with prolonged fast in those and other teleost species, contrary to the response in mammals, where glucagon is maintained (Petersen et al., 1987). Circulating levels of insulin and glucagon were depleted in the plasma of Oncorhynchus kisutch fasting 1 and 3 weeks (Sheridan and Mommsen, 1991). Nevertheless, in some cases the glucagonyinsulin (GyI) molar ratio is maintained at high levels (values of 0.5) with prolonged fast in order to activate fish reserves mobilization (Mommsen and Plisetskaya, 1991; Navarro et al., 1992) especially in situations of natural fast (anorexia during life cycle) (Navarro et al., 1995). Another response to fast is the depression in metabolic rate (Foster and Moon, 1991). The pattern of plasma changes during fasting in B. cephalus is similar to that found in brown trout, but high glucagon levels are maintained only during very short-term fast (48 h) with a posterior decline. The relative high values of plasma glucagon together with a fall in plasma insulin at 48–72 h (which gives GyI ratios of 0.7) would be enough to trigger the supply of glucose by the liver and to switch on gluconeogenic pathways. The decline in insulin with time of fasting (35% at the end of the experiment) coincides with the profile found in other fish species ´ (Navarro and Gutierrez, 1995) and may also contribute to the mobilization of lipid reserves. In conclusion, B. cephalus quickly adapts its carbohydrate metabolism to change from diet carbohydrate supply to the absence of energy from food. This flexibility in glucose metabolism, which characterize an omnivorous species, is related to changes in pancreatic hormones that permit gradual metabolic adjustments that occur during the transition from post-feeding to fast condition.
Acknowledgments ´ Bidinotto who We wish to thank Paulo Maurıcio provided logistical support with glycogen and glucose assays. We thank Mr Robin Rycroft for his help in the review of the English version of the manuscript. This study was supported by grants from the Program of Social Demand CAPES BEX2006y98-7, 1998 from the Brazilian government, Contract Q5RS-2000-30068 from the European Union, DGICY (AGF98-0325 and PB97-0902) from Spain, and CIRIT (2000 SGR0040). References Batty, R.S., Wardle, C.S., 1979. Restoration of glycogen from lactic acid in the anaerobic swimming muscle of plaice, Pleuronectes platessa L. J. Fish Biol. 15, 509–519. Bidinotto, P.m., Souza, R.H.S., Moraes, G., 1997. Hepatic glycogen in eight tropical freshwater teleost fish: a procedure ´ for field determinations of micro samples. Boletim Tecnico CEPTA 10, 53–60. Bone, Q., Marshall, N.B., 1982. Biology of Fishes, Chapter 3.2. Chapman & Hall, New York, pp. 43–45. Cornish, I.M.E., Moon, T.W., 1985. Glucose and lactate kinetics in American eel Anguilla rostrata. Am. J. Physiol. 249, R67–R72. Dole, V.P., Meinertz, H., 1960. Microdetermination of longchain fatty acids in plasma and tissues. J. Biol. Chem. Baltimore 235, 2595–2599. Duboie, M., Gilles, K.A., Hamilton, J.K., Roberts, P.A., Smith, F., 1956. Colorimetric method for determination of sugars and related substances. Anal. Chem. 28, 350–358. Falkmer, S., Matty, A.I., 1966. Blood sugar regulation in the hagfish Myxine glutinosa. Gen Comp. Endocrinol. 6, 334–346. ¨ Fange, R., Grove, D., 1979. Digestion. In: Hoar, W.S., Randall, D.J., Brett, J.R. (Eds.), Fish Physiology, vol. VIII. pp. 161–260. Foster, G.D., Moon, T.W., 1987. Metabolism in sea raven (Hemitripterus americanus) hepatocytes: the effects of insulin and glucagon. Gen. Comp. Endocrinol. 66, 102–115. Foster, G.D., Moon, T.W., 1991. Hypometabolism with fasting in the yellow perch (Perca flavescens): a study of enzymes, hepatocyte metabolism, and tissue size. Physiol. Zool. 64, 259–275. ´ Gutierrez, J., Zanuy, S., Carrillo, M., Planas, J., 1984. Daily rhythms of insulin and glucose plasma levels in sea bass Dicentrarchus labrax after experimental feeding. Gen. Comp. Endocrinol. 55, 393–397. ´ ´ Gutierrez, J., Perez, J., Navarro, I., Zanuy, S., Carrillo, M., 1991. Changes in plasma glucagon and insulin associated with fasting in sea bass (Dicentrarchus labrax). Fish Physiol. Biochem. 9, 107–112. Halseth, A.E., Bracy, D.P., Wasserman, D.H., 2001. Functional limitations to glucose uptake in muscles comprised of different fiber types. Am. J. Physiol., Endocrinol. Metab. 280, E994–E999.
M.L. Figueiredo-Garutti et al. / Comparative Biochemistry and Physiology Part A 132 (2002) 467–476 Haman, F., Zwingelstein, G., Weber, J.-M., 1997. Effects of hypoxia and low temperature on substrate fluxes in fish: plasma metabolite concentrations are misleading. Am. J. Physiol. 273 (Regul. Integrative Comp. Physiol.) 42, R2046–R2054. Hemre, G.-I., Lie, O., Lambertsen, G., Sundby, A., 1990. Dietary carbohydrate utilization in cod (Gadus morhua). Hormonal response of insulin, glucagons and glucagons-like peptide to diet and starvation. Comp. Biochem. Physiol. A 97, 41–44. Higuera, M. de La, Cardenas, P., 1984. Influence of dietary composition on gluconeogenesis from L-(U-14C) Glutamate in rainbow trout (Salmo gairdneri). Comp. Biochem. Physiol. A 81, 391–395. Lane, T.H., Jackson, H.M., 1969. Voidance time of 23 species of fish. Invest. Fish Control 33. Larsson, A., Lewander, K., 1973. Metabolic effects of starvation in the eel, Anguilla anguilla L. Comp. Biochem. Physiol. A 44, 367–374. Leatherland, J.F., Nuti, R.N., 1981. Effects of bovine growth hormone on plasma FFA concentrations and liver, muscle and carcass lipid content in rainbow trout, Salmo gairdneri Richardson. J. Fish Biol. 19, 487–498. Lim, A.L.L., Ip, Y.K., 1989. Effect of fasting on glycogen metabolism and activities of glycolytic and gluconeogenic enzymes in the mudskipper Boleophthalmus boddaerti. J. Fish Biol. 34, 349–367. Machado, C.R., Garofalo, M.A.R., Roselino, J.E.S., Kettelhut, I.C., Migliorini, R.H., 1988. Effects of starvation, refeeding and insulin on energy-linked metabolic processes in catfish (Rhamdia hilarii) adapted to a carbohydrate-rich diet. Gen. Comp. Endocrinol. 71, 429–437. McKeown, B.A., Leatherland, J.F., John, T.M., 1975. Circadian rhythm of plasma prolactin, growth hormone, glucose and free fatty acid levels in juvenile kokanee salmon, Oncorhynchus nerka. Comp. Biochem. Physiol. A 47, 821–828. ´ ´ F., Blanc, D., 1999. Comparaison Medale, F., Ploi, J.M., Vallee, ´ ´ de l’utilisation digestive et metabolique d’un regime riche en glucides par la carpe a` 188C et 258C. Cybium 23, 139–152. Milligan, C.L., McDonald, D.G., 1988. In vivo lactate kinetics at rest and during recovery from exhaustive exercise in coho salmon (Oncorhynchus kisutch) and starry flounder (Platichthys stellatus). J. Exp. Biol. 135, 119–131. Mommsen, T.P., Plisetskaya, E.M., 1991. Insulin in fishes and agnathans: history, structure, and metabolic regulation. Rev. Aquat. Sci. 4, 225–259. Moon, T.W., Foster, G.D., Plisetskaya, E.M., 1989. Changes in peptide hormones and liver enzymes in the rainbow trout deprived of food for six weeks. Can. J. Zool. 67, 2189–2193. Moon, T.W., Foster, G.D., 1995. Tissue carbohydrate metabolism, gluconeogenesis and hormonal and environmental influences. In: Hochachka, P.W., Mommsen, T.P. (Eds.), Biochemistry and Molecular Biology of Fishes, vol. 4. Elsevier Science B.V, pp. 393–434. Moriarty, D.J.W., 1973. The physiology of digestion of bluegreen algae in the cichlid fish Tilapia noctiluca. J. Zool. 171, 25–39. ´ Navarro, I., Gutierrez, J., Planas, J., 1992. Changes in plasma glucagon, insulin and tissue metabolites associated with prolonged fasting in Brown trout (Salmo trutta fario). Comp. Biochem. Physiol. A 102, 401–407.
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´ Navarro, I., Carneiro, M.N., Parrizas, M., Maestro, J.L., Planas, ´ J., Gutierrez, J., 1993. Post feeding levels of insulin and glucagon in trout (Salmo trutta fario). Comp. Biochem. Physiol. A 104, 389–393. ´ Navarro, I., Gutierrez, J., 1995. Fasting and starvation. In: Hochachka, P.W., Mommsen, T.P. (Eds.), Biochemistry and Molecular Biology of Fishes, vol. 4. Elsevier Science B.V, pp. 393–434. ´ Navarro, I., Gutierrez, J., Planas, J., 1995. Estimates of fish glucagon by heterologous radioimmunoassay: antibody selection and cross-reactivities. Comp. Biochem. Physiol. C 110, 313–319. Nicolaidis, S., 1977. Sensory-neuroendocrine reflexes and their anticipatory and optimizing role on metabolism. In: Kare, M., Maller, O. (Eds.), Chemical Senses and Nutrition. Academic Press, New York, pp. 123–140. ` Panserat, S., Medale, F., Blin, C., Breque, J., Vachot, C., Plagnes-Juan, E., Gomes, E., Krisnamoorthy, R., Kaushik, S., 2000. Hepatic glucokinase is induced by dietary carbohydrates in rainbow trout, gilthead seabream, and common carp. Am. J. Physiol., Regul. Integrative Com. Physiol. 278, R1164–R1170. Papatryphon, E., Capilla, E., Navarro, I., Soares, J.H., 2001. Early insulin and glucagon response associated with food intake in a teleost, the striped bass (Morone saxatilis). Fish Physiol. Biochem. 24, 31–39. ´ ´ Parrizas, M., Planas, J., Plisetskaya, E.M., Gutierrez, J., 1995. Insulin receptor and its tyrosine kinase activity in skeletal muscle of carnivorous and omnivorous fish. Am. J. Physiol. 266, 1944–1950. Pereira, C., Vijayan, M.M., Moon, T.W., 1995. In vitro hepatocyte metabolism of alanine and glucose and the response. J. Exp. Zool. 271, 425–431. Petersen, T.D., Hochachka, P.W., Suarez, R.K., 1987. Hormonal control of gluconeogenesis in rainbow trout hepatocytes. Regulatory role of pyruvate-kinase. J. Exp. Zool. 243, 173–180. ´ Planas, J.V., Capilla, E., Gutierrez, J., 2000. Molecular identification of a glucose transporter from fish muscle. FEBS Lett. 481, 266–270. Plisetskaya, E.M., Dickhoff, W.W., Paquette, T.L., Gorbman, A., 1986. The assay of salmon insulin in homologous radioimmunoassay. Fish Physiol. Biochem. 1, 37–43. Roussel, R., Garlier, P.G., Robert, J.-J., Velho, G., Bloch, G., 1998. 13Cy31P NMR studies of glucose transport in human skeletal muscle. Proc. Natl. Acad. Sci. 95, 1313–1318. Sheridan, M.A., Mommsen, T.P., 1991. Effects of nutritional state on in vivo lipid and carbohydrate metabolism of coho salmon, Oncorhynchus kisutch. Gen. Comp. Endocrinol. 81, 473–483. Suarez, R.K., Mommsen, T.P., 1987. Gluconeogenesis in teleost fishes. Can. J. Zool. 65, 1869–1882. Tashima, L., Cahill, G.F., 1965. Fat metabolism in fish. In: Renold, A.E., Cahill, G.F. (Eds.), Handbook of physiology, Adipose Tissue. American Physiology Society, Washington D.C, pp. 55–58. Teff, K.L., Engelman, K., 1996. Palatability and dietary restraint: effect of cephalic phase insulin release in women. Physiol. Behav. 60, 567–573. Vijayan, M.M., Maule, A.G., Schreck, C.B., Moon, T.W., 1993. Hormonal control of hepatic glycogen metabolism in
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food-deprived, continuously swimming coho salmon (Oncorhynchus kisutch). Can. J. Fish Aquat. Sci. 50, 1676–1682. Vijayan, M.M., Pereira, C., Moon, T.W., 1994. Hormonal stimulation of hepatocyte metabolism in rainbow trout following an acute handling stress. Comp. Biochem. Physiol. C 108, 321–329.
Walton, M.J., Cowey, C.B., 1982. Aspects of intermediary metabolism in salmonid fish. Comp. Biochem. Physiol. B 73, 59–79. Wiegand, M.D., Peter, R.E., 1980. Effects of testosterone, estradiol-17b and fasting on plasma free fatty acids in the goldfish, Carassius auratus. Comp. Biochem. Physiol. A 66, 323–326.
Metabolic changes in Brycon cephalus (Teleostei, Characidae) during post-feeding and fasting b ´ M.L. Figueiredo-Garuttia, *, I. Navarrob, E. Capillab, R.H.S. Souzac, G. Moraesd, J. Gutierrez , M.L.M. Vicentini-Paulinoe
b
a ´ ˜ Jose´ do Rio Preto, Sao ˜ Paulo, Brazil Rua Frei Valerio Kirch, 78, 15054-070 Sao Departament de Fisiologia, Facultat de Biologia, D. III, Universitat de Barcelona, Avinguda Diagonal 645, 08028 Barcelona, Spain c ´ ˜ Paulo, Brazil Laboratorio de Fisiologia, Centro Nacional de Pesquisa de Peixes Tropicais, IBAMA. 13630-000 Pirassununga, Sao d ¸˜ ´ ´ ´ Laboratorio de Bioquımica Adaptativa, Departamento de Genetica e Evolucao, ˜ Carlos. 13560-000 Sao ˜ Carlos, Sao ˜ Paulo, Brazil Universidade Federal de Sao e ˆ ˜ Paulo, Departamento de Fisiologia, Instituto de Biociencias, Universidade Estadual Paulista, UNESP, 18600-000 Botucatu, Sao Brazil
Received 17 August 2001; received in revised form 17 January 2002; accepted 3 March 2002
Abstract Metabolic changes during the transition from post-feeding to fasting were studied in Brycon cephalus, an omnivorous teleost from the Amazon Basin in Brazil. Body weight and somatic indices (liver and digestive tract), glycogen and glucose content in liver and muscle, as well as plasma glucose, free fatty acids (FFA), insulin and glucagon levels of B. cephalus, were measured at 0, 12, 24, 48, 72, 120, 168 and 336 h after the last feeding. At time 0 h (the moment of food administration, 09.00 h) plasma levels of insulin and glucagon were already high, and relatively high values were maintained until 24 h post-feeding. Glycemia was 6.42"0.82 mM immediately after food ingestion and 7.53"1.12 mM at 12 h. Simultaneously, a postprandial replenishment of liver and muscle glycogen reserves was observed. Subsequently, a sharp decrease of plasma insulin occurred, from 7.19"0.83 ngyml at 24 h of fasting to 5.27"0.58 ngyml at 48 h. This decrease coincided with the drop in liver glucose and liver glycogen, which reached the lowest value at 72 h of fasting (328.56"192.13 and 70.33"14.13 mmolyg, respectively). Liver glucose increased after 120 h and reached a peak 168 h post-feeding, which suggests that hepatic gluconeogenesis is occurring. Plasma FFA levels were low after 120 and 168 h and increased again at 336 h of fasting. During the transition from post-feeding to fast condition in B. cephalus, the balance between circulating insulin and glucagon quickly adjust its metabolism to the ingestion or deprivation of food. 䊚 2002 Elsevier Science Inc. All rights reserved. Keywords: Post-feeding; Starvation; Glycemia; FFA; Insulin; Glucagon; Hepatic and muscle glycogen; Hepatic and muscle glucose; Fish; Brycon cephalus
1. Introduction Fish have diverse strategies to deal with periods of food deprivation. The degree of glucose homeo*Corresponding author. Tel.: q55-17-2249627; fax: q5517-2249627. E-mail address: [email protected] (M.L. Figueiredo-Garutti).
stasis during fast varies with species. Long-term responses to food deprivation have been extensively studied. No changes in plasma glucose levels were observed after several weeks of fast in the hagfish (Myxine glutinosa) (Falkmer and Matty, 1966), or even months in species such as Anguilla rostrata (Suarez and Mommsen, 1987) or Clarias ´ lazera (Navarro and Gutierrez, 1995). In Rhamdia
1095-6433/02/$ - see front matter 䊚 2002 Elsevier Science Inc. All rights reserved. PII: S 1 0 9 5 - 6 4 3 3 Ž 0 2 . 0 0 0 9 4 - 6
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hilarii, a 50% decrease of glycemia occurs after 30 days of starvation (Machado et al., 1988). However, it remains unclear whether glycemia control is related to the nutritional preferences of fish (herbivorous or carnivorous species). Our knowledge of metabolic strategies of omnivorous or herbivorous teleost remains limited compared to the data available for carnivorous species. Brycon cephalus is an omnivorous fish from the Amazon Basin in Brazil. In its natural habitat it ingests high quantities of fruits and seeds, and when the food becomes scarce, it eats insects and small fish. Recently, it has been reported that omnivorous species that are more tolerant to diet glucose show different insulin response to dietary carbohydrate and capacity of liver in phosphorylating glucose than carnivorous species (Panserat et al., 2000). Furthermore, muscle insulin receptors are more numerous in species with a higher degree ´ of glucose tolerance (Parrizas et al., 1995). Nevertheless, few data are available on the fasting metabolism of herbivorous or omnivorous fishes ´ (Navarro and Gutierrez, 1995). The maintenance of glycemia during food deprivation is directly related to the capacity of mobilization on hepatic glycogen, at least during the initial stages of fasting, and also depends on posterior activation of the hepatic gluconeogenesis (Higuera and Cardenas, 1984) and the reduction in the rate of glucose utilization (Moon and Foster, 1995). In most fish species liver glycogen is mobilized early during fasting but the degree of glycogen depletion varies greatly between species, ranging from rapid glycogenolysis to partial or almost complete protection of glycogen reserves with fast (Sheridan and Mommsen, 1991). The increased gluconeogenic capacity of the liver may partially be due to the changes in plasma glucoregulatory hormone concentrations, i.e. insulin and glucagon, andyor changes in hepatocyte responsiveness to these hormones (Pereira et al., 1995). Hormone changes and effects in fish are not always similar to that of mammals (Foster and Moon, 1987). In some species, hepatocyte response to insulin is altered with fast or stress, favoring glycogen conservation (Vijayan et al., 1993, 1994). Plasma insulin levels generally decrease with fasting in most fish species (Plisetskaya et al., 1986). In comparison with insulin, the role of glucagon during fasting in fish has been studied in relatively few species. The glucagon response
changes according to the species or length of the fasting period. This hormone decreased in Oncorhynchus mykiss (Moon et al., 1989) and Gadus morhua (Hemre et al., 1990) after 6 weeks and 7 days of fast, respectively. In Dicentrarchus labrax and Salmo trutta few days of fasting provoked ´ increases in circulating glucagon (Gutierrez et al., 1991; Navarro et al., 1992). In contrast with the abundant literature on the metabolic responses to long-term fasting in fish, studies on the metabolic changes during, postfeeding and early fasting periods are not commonly ´ found (Navarro and Gutierrez, 1995). Moreover, there is little information on the biology of Brycon cephalus, especially on the effects of fasting on intermediary metabolism and no data referring to insulin and glucagon in this species. The search for parameters on the nutritional and physiological situation of B. cephalus may help to improve culture conditions of this species, which has a great potential for aquaculture because of its rapid growth. We test the hypothesis that omnivorous B. cephalus possess more flexibility in glucose metabolism than carnivorous species and enhanced ability to modulate metabolism in order to match changes in nutritional state. Here, we examine in B. cephalus the absorption and mobilization of metabolites, especially carbohydrates, during the post-feeding period until 14 days of fasting and their relation with changes in plasma pancreatic hormones. 2. Material and methods 2.1. Animals and experimental procedure The experiments were performed in November. Adult B. cephalus (ns40) bred by induced reproduction with an average initial weight of 876.0"13.6 g were used (a voucher specimen has been identified and deposited at the Museum of ˜ Paulo—MZUSP Zoology of the University of Sao 54008). The animals were distributed in eight homogenous groups and kept in open circuit tanks of 3000-l capacity and provided with well-aerated water. The average water temperature was 27.43"0.02 8C. Fish were fed commercial tropical ´ ´ fish pellets (Socil pro-pecuaria SyA), composed of 6% water, 30% crude protein, 8% crude fat, 7% crude fiber, and 9% mineral mixture. The food was administered ad libitum at 09.00 h daily, the
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habitual time of administration. Tissue and blood samples were collected at 0, 12, 24, 48, 72, 120, 168 and 336 h after feeding. Time 0 corresponded to the moment of food administration (09.00 h). At each time of sampling, five fish were netted from a different tank. The animals were anaesthetized with 2-phenoxyethanol (0.5 mlyl). Blood was taken from the dorsal aorta into heparinized syringes. Aliquots of plasma were frozen and stored at y20 8C until assay. Fish were quickly killed by a blow to the head, and weighed. Then, the viscera were examined and the livers were removed, weighed, frozen immediately on dry ice and stored at y20 8C for later assay. A sample of white epaxial muscle located between dorsal and adipose fins was obtained and similarly stored until analyses. Liver, stomach and intestine were weighed to obtain the hepatic somatic index wHSIs(hepatic weight=100)ybody wt.x, full stomach somatic index w f SSIsfull stomach weight=100)ybody wt.x, and full intestine somatic index w f ISIsfull intestine weight=100)ybody wt.x.
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3. Results 3.1. Body weight and somatic indexes Food deprivation induced a progressive reduction in body weight from an initial value of 876.0"13.6–646.0"71.6 g at the end of the experiment, corresponding to 73.7% of the initial weight. The hepatic somatic index increased from an initial value of 0.75"0.06–1.22"0.18% at 48 h after feeding and then it decreased progressively during the experimental period. The values obtained from 72 h of fast to the end of the experiment were not significantly different. The average stomach somatic index decreased in the first 24 h from 9.17"1.29% to 1.29"0.51% and maintained low values until the end of the experiment. The intestine somatic index decreased from 1.38"0.18% to 0.55"0.10% at 72 h after feeding, and then remained with minimal changes (Fig. 1). 3.2. Plasma glucose, free fatty acids, glucagon and insulin
2.2. Analytical methods Plasma insulin levels were measured by radioimmunoassay (RIA) using bonito insulin as the standard and rabbit anti-bonito insulin as antiserum ´ according to methodology proposed by Gutierrez et al. (1984). Glucagon levels were determined by heterologous RIA validated for fish plasma (Navarro et al., 1995). Validation of the assays for B. cephalus was confirmed by plasma dilution tests for this species, previously performed for both insulin and glucagon RIAs. Plasma glucose concentration was estimated by glucose oxidaseperoxidase (Sigma Chemical Company kit). Plasma free fatty acids (FFA) were measured by the method of Dole and Meinertz (1960). Glycogen content was estimated in the liver and muscle by the method of Duboie et al. (1956), modified by Bidinotto et al. (1997), and the liver and muscle glucose was determined by the method of Duboie et al. (1956). Results are presented as mean"standard deviation (S.D.). Statistical differences were estimated by one-way analysis of variance (ANOVA); and the difference among means was estimated by the Tukey test. The significance level adopted was 0.05.
Immediately after food ingestion, the mean plasma glucose concentration was 6.42"0.82 mM and 12 h later, it increased to 7.53"1.12 mM. At 24 h of fast, glucose levels returned to initial values (6.31"0.78 mM). Subsequently, plasma glucose decreased and no significant changes were observed thereafter. The free fatty acids (FFA) increased from 1.20"0.04 to 1.77"0.06 mM at 48 h of fasting. FFA plasma levels were lower after 120 and 168 h and increased again at 336 h (Fig. 2). The highest levels of plasma insulin levels were found through the first 24 h post-feeding, being already high at 0 h (6.75"1.00 ngyml) with elevated values until 24 h (7.19"0.83 ngyml). Later, circulating insulin decreased to 5.27"0.58 ngyml at 48 h of fast. The lowest values were found at the end of the experiment, after 336 h, with a fall in insulin of 35% (with respect to 24 h post-feeding). Circulating glucagon levels were high at 0 time (10.42"1.49 ngyml), and although they declined, were relatively high at 12 and 24 h (3.06"1.04 ngyml). Glucagon levels continued to decrease until 120 h of fast when the value was 0.59"0.25 ngyml. These low values were maintained until the end of the experiment.
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Fig. 1. Body weight (wt.), hepatic somatic index (HSI), full stomach somatic index ( f SSI) and full intestine somatic index ( f ISI) in B. cephalus (ns5) subjected to fasting. Time indicates hours (h) after feeding, where hour 0 corresponds to the feeding moment (09.00 h). Letters represent statistical differences (PF0.05) by the Tukey test. The values presented are mean"standard deviation.
3.3. Liver and muscle glycogen and glucose The liver glycogen increased from 112.33"47.52 to 183.99"48.97 mmolyg in 12 h after food ingestion and subsequently, the concentration decreased to a minimum of 70.33"14.13 mmolyg at 72 h. The glycogen level was recovered after 120 h and the value presented at 336 h after feeding was not different from the initial one. Liver glucose, whose initial value was 444.6"108.9 mmolyg, presented an initial profile similar to that observed in liver glycogen, with an increase at 12 h after feeding, with a value of 664.3"97.9 mmolyg, followed by a sharp decline reaching a nadir (328.6"192.1 mmolyg), at 72 h of fasting. Liver glucose was higher again with a value of 687.5"181.1 mmolyg at 168 h of fasting (Fig. 3). The content of muscle glycogen, which was 15.29"0.66 mmolyg immediately after feeding, showed a progressive increase until 48 h postfeeding (22.62"1.30 mmolyg). At 72 h post-
feeding, muscle glycogen was 19.72"2.07 mmoly g, which did not change until the end of experiment. Muscle glucose concentration, that initially was 16.77"2.81 mmolyg, decreased until 24 h post-feeding, when it reached its lowest value, 10.68"1.30 mmolyg. Later, muscle glucose recovered, attaining values of 18.28"1.95 and 18.31"0.82 mmolyg, at 120 and 168 h of fasting, respectively. 4. Discussion In the present study metabolic alterations involved in fed-to-fasting transition and circulating pancreatic hormones are analyzed in B. cephalus for the first time. The progressive decrease in stomach somatic index suggests that a period of 12–24 h is needed to complete the gastric emptying. Consequently, the transit of digested food through the intestine extended until 48–72 h as suggested by intestine somatic index. Although the meal size, composi-
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Fig. 2. Plasma glucose, free fatty acids (FFA), insulin and glucagon in B. cephalus (ns5) subjected to fasting. Time indicates hours (h) after feeding, where hour 0 corresponds to the feeding moment (09.00 h). Letters represents statistical differences (PF0.05) by the Tukey test. The values presented are mean"standard deviation.
tion of food and animal size has to be taken into account to compare rates of gastric evacuation, it seems that the digestive process of B. cephalus is slightly delayed in comparison with other teleost species with natural omnivorous regime, which tend to present shorter emptying times than carniv¨ orous fishes (Fange and Grove, 1979). For example, data obtained at temperatures of 20–25 8C in Tilapia noctiluca, an herbivore, the same parameter ranged from 15 to 27 h (Moriarty, 1973) while in different species of the genus Ictalurus, carnivorous, showed emptying ranging from 48–84 h (Lane and Jackson, 1969). The postprandial increase of glycemia observed 12 h post-feeding, together with the return of glucose levels to initial values after 24 h paralleled gastrointestinal transit. This moderate rise in postfeeding plasma glucose (33% increase at 12 h in relation to 24 h levels) and the posterior quick decline in glycemia until 72 h suggests an efficient uptake of glucose by peripheral tissues. Postfeeding increases in glycemia vary with the diet
carbohydrate content and glucose tolerance of the species, but the postprandial profile observed in B. cephalus is similar to that found in other teleost ´ species (Navarro et al., 1993; Medale et al., 1999). In many fish species, liver glycogen is one of the first energy reserves to be consumed after several days of fasting, but the moment when the carbohydrate metabolism shifts from a postabsorptive to fast condition is not well documented. The first glycogen increase observed in the present study (12 h after food ingestion) is in concordance with the increase in glucose that arrives to the liver during the absorptive period. In fact, profiles of plasma glucose, liver glucose and hepatic glycogen are very similar in the first experimental period (from 0 to 72 h). At 72 h, these three parameters decreased, suggesting that carbohydrate metabolism changed from postabsorptive to fasting condition. If so, the liver would act as a glucose donor for extra hepatic tissues at the expense of glycogen, as in other fish species (Moon and Foster, 1995). This process
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Fig. 3. Liver and muscle glycogen and glucose in B. cephalus (ns5) subjected to fasting. Time indicates hours (h) after feeding, where hour 0 corresponds to the feeding moment (09.00 h). Letters represent statistical differences (PF0.05) by the Tukey test. The values presented are mean"standard deviation.
seems to be faster than in other fish in which significant decreases in both plasma glucose and glycogen reserves are seen after several days of ´ food deprivation (Navarro and Gutierrez, 1995). Although we cannot infer metabolic fluxes from changes in metabolite concentration (Haman et al., 1997), the partial recovery of liver glycogen after 120 h of fast suggests an increase in glucose production from hepatic gluconeogenesis that exceeds glucose demand. The increase in liver glycogen with fast is reported in fasted Oncorhynchus nerka (Walton and Cowey, 1982) or rainbow trout fasted for 30 days (Higuera and Cardenas, 1984). The glucose oxidation is diminished in trout hepatocytes during fasting, but the capacity for glyconeogenesis in not altered or even enhanced (Pereira et al., 1995). Increase in gluconeogenic enzymes has been reported in the eel,
rainbow trout or yellow perch after long term fast ´ (review by Navarro and Gutierrez, 1995). An increase in gluconeogenesis already at 120 h fast indicates a rapid change of metabolism in response to food deprivation, in agreement with its omnivorous condition. Carnivorous fish liver do not seem to respond quickly with changes in gluconeogenic rates, that are constantly activated irrespective of the nutritional situation, in contrast to omnivorousy herbivorous fishes (Pereira et al., 1995). The pattern of plasma glucose observed in B. cephalus agrees with the metabolic changes in the liver, since glucose levels are maintained after 120 h. It seems that this omnivorous species, although sensitive to food deprivation, quickly adapts its carbohydrate metabolism in response to the absence of exogenous glucose. Depression of met-
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abolic rate with fast, as observed in other fish species, may help to maintain glucose homeostasis. The percentage of carbohydrate reserves in white muscle is usually low (Bone and Marshall, 1982), but the contribution of this tissue as an organ of uptake or storage is high since the total mass of white muscle represents approximately 50% of the body weight. In this study, initial increase in muscle glycogen level indicates a postfeeding replenishment of reserves, which occurs simultaneously to a decrease in muscle glucose together with high plasma glucose and insulin levels. Muscle glucose content corresponds to both interstitial and intracellular glucose (total glucose), although very specific measurements of free intracellular muscle glucose of mammals have given very low values (Roussel et al., 1998). Decreases in total muscle glucose concentration of rats occur in responses to simultaneous increases in plasma insulin and glucose (Halseth et al., 2001). The fall in muscle glucose observed initially suggest that the rate of removal of interstitial glucose and its subsequent phosphorylation exceed the rate at which the glucose is replenished. These observations are in accordance with an increase in the rate of insulin-stimulated glucose uptake by muscle. Studies on the regulation of fish glucose transporters by insulin may help us to understand the regulation of glucose fluxes into insulin-sensitive tissues (Planas et al., 2000). The subsequently decrease of muscle glycogen (48–72 h) was parallel to the increase in glucose in this tissue. This glucose originates either from muscle glycogen or from a decrease in the rate of glucose uptake by this tissue concomitant to a decrease in plasma insulin values. Muscle glycogen may not contribute directly to homeostasis of blood glucose because the muscular tissue itself probably uses this reserve when it is needed. In fish, glucose production by the Cori Cycle apparently does not occur as it does in mammals (Batty and Wardle, 1979; Cornish and Moon, 1985; Milligan and McDonald, 1988). A significant decrease of glycogen in muscle of fasted Boleophthalmus boddaerti was considered an indication that this substrate could be the first fuel to be utilized for muscle activities (Lim and Ip, 1989). In B. cephalus, the posterior stabilization of glycogen and glucose values of white muscle (after 72 h) indicates that muscle glycogen stores are not primarily used during the period of fast.
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In relation to lipid metabolism, the peak of plasma FFA in B. cephalus observed 48 h after feeding could be related to a characteristic delayed absorption of lipids in comparison with other metabolites. Although an increase in plasma free fatty acids is considered a sign of lipolytic activity in mammals, this is not well established in fish. Plasma FFA increased with prolonged fasting in Carassius auratus, Anguilla anguilla, and Oncorhynchus mykiss (Larsson and Lewander, 1973; Wiegand and Peter, 1980; Leatherland and Nuti, 1981), but decreased in fasting Opsanus tau and Oncorhynchus nerka (Tashima and Cahill, 1965; McKeown et al., 1975). The maintenance of high levels of FFA during the fast period and the increase observed in this study, 336 h after last meal suggest a significant contribution of fat in providing fuel for B. cephalus. Lipid storage utilization may explain the decrease in body weight observed at the end of the experiment. To our knowledge, the levels of plasma insulin and glucagon in B. cephalus were determined for the first time in the present study. Post-feeding glucagon values were high, especially when compared with other species of fish. For instance, postprandial value of 3.0 ngyml has been reported in Oncorhynchus kisutch and Salmo trutta (Sheridan and Mommsen, 1991; Navarro et al., 1995). Furthermore, the highest glucagon levels were found at time 0, but insulin levels were also high. An early pre-feeding response of pancreatic hormones, possibly neurally mediated, has been described in fish (Papatryphon et al., 2001). It has been assumed that in mammals early increases in plasma insulin and glucagon optimize and improve nutrient assimilation, and the release of opposite hormones may maintain balance in glucose homeostasis (Nicolaidis, 1977; Teff and Engelman, 1996). B. cephalus might be especially sensitive to pancreatic hormone response just before eating, which would favor carbohydrate digestion and metabolism. In the present study, post-feeding insulin levels were maintained elevated (from 0 to 24 h) after ingestion of food in concordance with somatic indexes and glucose profiles. As in other fish species, high circulating insulin levels may favor the uptake of glucose by liver and especially by muscle, and the repletion of glycogen stores (Mommsen and Plisetskaya, 1991). In comparison with brown trout, in which plasma insulin levels were high during 1–5 h post-feeding and decreased
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after 8–11 h (Navarro et al., 1993), a prolonged post-feeding insulin profile is observed in B. cephalus. Increases in glucagon levels with short-term fast have been described only in two species of fish, both of them carnivorous. In Dicentrarchus labrax an increase in glucagon levels was observed after ´ 4 days of fasting (Gutierrez et al., 1991), and a similar but less pronounced response was observed in Salmo trutta fario upon fasting for 5 days: this has been explained as a signal to shift carbohydrate metabolism towards degradation of stores (Navarro et al., 1992). However, the levels of both pancreatic hormones decreased with prolonged fast in those and other teleost species, contrary to the response in mammals, where glucagon is maintained (Petersen et al., 1987). Circulating levels of insulin and glucagon were depleted in the plasma of Oncorhynchus kisutch fasting 1 and 3 weeks (Sheridan and Mommsen, 1991). Nevertheless, in some cases the glucagonyinsulin (GyI) molar ratio is maintained at high levels (values of 0.5) with prolonged fast in order to activate fish reserves mobilization (Mommsen and Plisetskaya, 1991; Navarro et al., 1992) especially in situations of natural fast (anorexia during life cycle) (Navarro et al., 1995). Another response to fast is the depression in metabolic rate (Foster and Moon, 1991). The pattern of plasma changes during fasting in B. cephalus is similar to that found in brown trout, but high glucagon levels are maintained only during very short-term fast (48 h) with a posterior decline. The relative high values of plasma glucagon together with a fall in plasma insulin at 48–72 h (which gives GyI ratios of 0.7) would be enough to trigger the supply of glucose by the liver and to switch on gluconeogenic pathways. The decline in insulin with time of fasting (35% at the end of the experiment) coincides with the profile found in other fish species ´ (Navarro and Gutierrez, 1995) and may also contribute to the mobilization of lipid reserves. In conclusion, B. cephalus quickly adapts its carbohydrate metabolism to change from diet carbohydrate supply to the absence of energy from food. This flexibility in glucose metabolism, which characterize an omnivorous species, is related to changes in pancreatic hormones that permit gradual metabolic adjustments that occur during the transition from post-feeding to fast condition.
Acknowledgments ´ Bidinotto who We wish to thank Paulo Maurıcio provided logistical support with glycogen and glucose assays. We thank Mr Robin Rycroft for his help in the review of the English version of the manuscript. This study was supported by grants from the Program of Social Demand CAPES BEX2006y98-7, 1998 from the Brazilian government, Contract Q5RS-2000-30068 from the European Union, DGICY (AGF98-0325 and PB97-0902) from Spain, and CIRIT (2000 SGR0040). References Batty, R.S., Wardle, C.S., 1979. Restoration of glycogen from lactic acid in the anaerobic swimming muscle of plaice, Pleuronectes platessa L. J. Fish Biol. 15, 509–519. Bidinotto, P.m., Souza, R.H.S., Moraes, G., 1997. Hepatic glycogen in eight tropical freshwater teleost fish: a procedure ´ for field determinations of micro samples. Boletim Tecnico CEPTA 10, 53–60. Bone, Q., Marshall, N.B., 1982. Biology of Fishes, Chapter 3.2. Chapman & Hall, New York, pp. 43–45. Cornish, I.M.E., Moon, T.W., 1985. Glucose and lactate kinetics in American eel Anguilla rostrata. Am. J. Physiol. 249, R67–R72. Dole, V.P., Meinertz, H., 1960. Microdetermination of longchain fatty acids in plasma and tissues. J. Biol. Chem. Baltimore 235, 2595–2599. Duboie, M., Gilles, K.A., Hamilton, J.K., Roberts, P.A., Smith, F., 1956. Colorimetric method for determination of sugars and related substances. Anal. Chem. 28, 350–358. Falkmer, S., Matty, A.I., 1966. Blood sugar regulation in the hagfish Myxine glutinosa. Gen Comp. Endocrinol. 6, 334–346. ¨ Fange, R., Grove, D., 1979. Digestion. In: Hoar, W.S., Randall, D.J., Brett, J.R. (Eds.), Fish Physiology, vol. VIII. pp. 161–260. Foster, G.D., Moon, T.W., 1987. Metabolism in sea raven (Hemitripterus americanus) hepatocytes: the effects of insulin and glucagon. Gen. Comp. Endocrinol. 66, 102–115. Foster, G.D., Moon, T.W., 1991. Hypometabolism with fasting in the yellow perch (Perca flavescens): a study of enzymes, hepatocyte metabolism, and tissue size. Physiol. Zool. 64, 259–275. ´ Gutierrez, J., Zanuy, S., Carrillo, M., Planas, J., 1984. Daily rhythms of insulin and glucose plasma levels in sea bass Dicentrarchus labrax after experimental feeding. Gen. Comp. Endocrinol. 55, 393–397. ´ ´ Gutierrez, J., Perez, J., Navarro, I., Zanuy, S., Carrillo, M., 1991. Changes in plasma glucagon and insulin associated with fasting in sea bass (Dicentrarchus labrax). Fish Physiol. Biochem. 9, 107–112. Halseth, A.E., Bracy, D.P., Wasserman, D.H., 2001. Functional limitations to glucose uptake in muscles comprised of different fiber types. Am. J. Physiol., Endocrinol. Metab. 280, E994–E999.
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