Temporal changes in liver carbohydrate metabolism associated with seawater transfer in Oreochromis mossambicus

Comparative Biochemistry and Physiology Part B 119 (1998) 721 – 728

Temporal changes in liver carbohydrate metabolism associated with seawater transfer in Oreochromis mossambicus Kazumi Nakano a,*, Masatomo Tagawa b, Akihiro Takemura c, Tetsuya Hirano a a

Ocean Research Institute, Uni6ersity of Tokyo, Nakano, Tokyo 164, Japan b Department of Fisheries, Kyoto Uni6ersity, Kyoto 606 -01, Japan c Sesoko Station, Tropical Biosphere Research Center, Uni6ersity of the Ryukyus, Okinawa 905 -02, Japan Received 31 July 1997; received in revised form 23 December 1997; accepted 20 January 1998

Abstract The metabolic aspects of ionic and osmotic regulation in fish are not well understood. The objective of this study was to examine changes in carbohydrate metabolism during seawater (SW) acclimation in the euryhaline tilapia (Oreochromis mossambicus). Hepatic activities of three key enzymes of the intermediary metabolism, phosphofructokinase, glycogen phosphorylase and glucose 6-phosphate dehydrogenase, together with glycogen content and plasma glucose concentration were measured at 0, 0.5, 1, 2, 3, 6, 12, 24, 48 and 96 h after the direct transfer of tilapia from fresh water (FW) to 70% SW. Plasma growth hormone, prolactin177 and prolactin188, Na + and Cl − concentrations were also measured. Plasma Na + and Cl − levels were highest at 12 h, but returned to FW levels at 24 h after transfer, suggesting the tilapia were able to osmoregulate within 24 h after transfer. Plasma glucose levels were significantly higher in 70% SW than in FW during the course of acclimation, especially in the early stages. Hepatic enzyme activities and glycogen content did not change significantly during the acclimation period. Our results suggest the possibility that glucose is an important energy source for osmoregulation during the acclimation to hyperosmotic environments in O. mossambicus. © 1998 Elsevier Science Inc. All rights reserved. Keywords: Carbohydrate metabolism; Enzyme activities; Euryhaline tilapia; Liver; Osmoregulation; Plasma glucose; Plasma hormones

1. Introduction Based on the changes in oxygen consumption related to changes in water salinity, estimates of the cost of osmoregulation both in fresh water (FW) and seawater (SW), have been studied in rainbow trout (Oncorhynchus mykiss) (20% of its metabolic rate in FW, 27% in SW) [29]; tilapia, O. niloticus (19% in FW and 29% in SW) [11]; and striped mullet (Mugil cephalus) (negligible in FW and high in SW) [26]. The activity-related osmoregulatory cost measured as * Corresponding author. Present address. Department of Animal Science, University of British Columbia, Suite 208-2357, Main Mall, Vancouver, B.C. V6T 1Z4, Canada. Tel.: + 1 604 8224910; fax: +1 604 8224400; e-mail: [email protected] 0305-0491/98/$19.00 © 1998 Elsevier Science Inc. All rights reserved. PII S0305-0491(98)00048-0

oxygen consumption in swimming hybrid tilapia (O. mossambicus and O. hornorum) was 16% of the total metabolic rate in FW and 12% in SW [12]. Morgan et al. [24] showed that the average oxygen consumption rate of tilapia (O. mossambicus) 4 days after transfer was significantly (20%) higher in SW than in FW or isosmotic salinity (12 ppt). These data suggest that the energetic cost for osmoregulation can be expensive in fish moving from FW to SW. However, alterations in intermediary metabolism related to osmoregulation is not fully understood in fish, especially the metabolic adjustments during the course of acclimation to osmotic changes in the environment. Tilapia is euryhaline fish, and their morphological and biochemical changes during SW acclimation and the hormonal control of osmoregulation have been

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extensively studied [2,3,6,8 – 10,15,16,28]. Tilapia exhibit species difference in salinity tolerance; O. mossambicus can adapt to more than 100% SW, while O. niloticus is less euryhaline [6,31,32]. In our previous study, these species differed in their metabolic responses to hyperosmotic water; O. niloticus in 50% SW had higher liver enzyme activities and plasma glucose levels compared with FW, whereas there was no difference in O. mossambicus [25]. The difference in hepatic metabolic capacity between the two species perhaps reflects differences in their salinity tolerance. However hepatic metabolism was compared between the two species only at the completely acclimated state, 4 weeks after transfer from FW to 100 and 160% SW in O. mossambicus, and to 50% SW in O. niloticus. Thus, very little is known about the metabolic reorganization of the liver during SW acclimation. The objective of the present study was to examine the temporal change in liver metabolic capacity during the course of acclimation from FW to 70% SW in O. mossambicus. Plasma ions (Na + and Cl − ), hormones (GH, PRL177 and PRL188) and glucose levels, and liver glycogen content and selected enzyme activities were measured in order to describe the metabolic responses of liver tissue.

2. Materials and methods

2.1. Fish Euryhaline tilapia, O. mossambicus (100 – 200 g) were caught in Okushu River in Kin, Okinawa. Male fish were kept in outdoor FW tanks (1500 l) for more than 2 months before the experiment at Sesoko Station, Tropical Biosphere Research Center, University of the Ryukyus at 309 2°C under natural photoperiod. They were fed tilapia pellet (Kumiai Haigo Shiryo, Japan) at 1% body weight per day, and feeding was withheld for 24 h before the experiment. In a preliminary experiment, five out of ten fish died when directly transferred from FW to 100% SW, and therefore, fish were transferred directly to 70% SW (23 ppt) in this experiment. There was no mortality by the transfer to, and rearing in, 70% SW. All experiments were conducted in July 1995. In the short term experiment, 48 fish were transferred from a stock tank (1500 l, FW) to FW and 70% SW tanks (250 l; maintained as above) after 6 fish were sampled from the stock tank (0 time samples). In FW and 70% SW six fish each were sampled at 0.5, 1, 2 and 3 h after transfer. In the long term experiment, 48 fish were transferred from stock tank to FW and 70% SW, after six fish were sampled from the stock tank (0 time

samples). In each FW and 70% SW six fish were sampled at 3, 6, 12 and 24 h after transfer. A total of 24 fish were transferred to the other tanks and sampled at 48 and 96 h after transfer.

2.2. Sampling Fish were anesthetized with 0.1% phenoxyethanol, weighed and bled by caudal puncture into syringe. The liver was quickly removed and frozen on dry ice. The whole procedure on each fish was carried out on ice and the total time of bleeding and collecting liver samples did not exceed 5 min. The blood was centrifuged at 11700 × g for 5 min at 4oC. The plasma and liver were kept frozen at − 80oC until analyses.

2.3. Tissue preparation Liver was processed for enzyme assays following Mommsen et al. [21] and Moon et al. [23] with minor modifications. Frozen liver was homogenized with a Polytron homogenizer (Model K, Kinematika AG) at maximum speed for 1 min in 5 vol of ice-cold ‘stopping buffer’ (50 mM imidazole, 15 mM b-mercaptoethanol, 100 mM KF, 5 mM EDTA-2K, 5 mM EGTA, and 0.1 mM phenylmethylsulfonyl fluoride, pH 7.5). Homogenates were centrifuged at 14000× g for 25 min at 4oC, and 2.5 ml of the supernatants were desalted to remove possible interfering metabolites by passing them through Sephadex G-25 M PD10 column. The elutes were kept frozen at − 80oC until analyses.

2.4. Enzyme assays Enzyme activities were measured following Mommsen et al. [21] and Moon et al. [23] with minor modifications. Phosphofructokinase (PFK, EC 2.7.1.11): The assay buffer contained 50 mM Tris–HCl, 175 mM KCl, 17.5 mM MgCl2 (pH 7.8). Enzyme/cofactor solution contained 0.88 mM NADH, 20 U/ml aldolase, 100 U/ml triosephosphate isomerase, and 100 U/ml a-glycerophosphate dehydrogenase. Substrate solution contained 280 mM fructose 6-phosphate and 28 mM ATP (omitted for control) to measure the maximum activity. Glycogen phosphorylase (GPase, EC 2.4.1.1): The buffer contained 50 mM K2PO4 (pH 7.0), 0.25 mM EDTA-2K, and 15 mM MgSO4. The enzyme/cofactor solution contained 1.75 mM NADP, 35 mM glucose 1,6-bisphosphate, 20 U/ml phosphoglucomutase, 50 U/ ml glucose 6-phosphate dehydrogenase, and 20 mg/ml glycogen (omitted for control). AMP solution contained 28 mM AMP. Glucose 6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49): The assay buffer contained 50 mM imidazole

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Fig. 1. Changes in plasma Na + (A and B) and Cl − (C and D) levels after the transfer to 70% SW (X)and to FW (control, X). Vertical bars represent S.E.M. (n =6). Error bars were with the symbol for the mean. Significant treatment effects between FW and 70% SW at same time are shown with an asterisk (PB0.05, two-way ANOVA) .Significant time effects are shown as a, b and c (PB 0.05, two-way ANOVA); values with the same alphabet are not significantly different. Significant differences between the mean values in FW and those in 70% SW in the experiments are shown as 70% SW\FW (PB 0.05).

and 7 mM MgCl2 (pH 7.5). Cofactor solution contained 1.4 mM NADP. Substrate solution contained 14 mM glucose 6-phosphate (omitted for control). In all of the assays, 25 ml of the desalted sample (diluted appropriately) were added to 150 ml of assay buffer in a 96-well microplate, followed by 100 ml of enzyme/cofactor solution. A volume of 20 ml of substrate solution (or assay buffer for control) were added after incubation for 10 min at the rearing temperature of the fish, e.g. at 30°C. Absorbance at 340 nm was measured using a microplate reader (MTP-120, Corona Electric, Japan), at 5-min intervals for 25 min to monitor the consumption of NADH or the production of NADPH. The enzyme activity was calculated by subtracting the slope of control from that of the sample with substrate, and expressed as nmol min − 1 mg − 1 protein. All biochemicals were purchased from Sigma. The protein contents of the desalted samples were measures using a protein assay kit (Bio-Rad).

2.6. Plasma hormones and ions Plasma levels of two prolactins (PRL177 and PRL188) and growth hormone (GH) were measured using homologous radioimmunoassays (RIAs) developed by Ayson et al. [4]. Plasma Na + concentration was determined by atomic absorption spectrophotometly (Hitachi 180–50, Hitachi, Japan), and Cl − concentration was determined by a chloridometer (Haake Buchler, USA).

2.7. Statistical analysis Statistical analyses were conducted using a two-way ANOVA followed by Student-Newman-Keuls test. Results are presented as means9 S.E.M. Significance was accepted at the 5% level.

3. Results

2.5. Plasma glucose and li6er glycogen Plasma glucose concentrations were determined using a commercial kit (Glucose CII-Test, WAKO, Wako Pure Chemical Industries, Japan). Glycogen contents of the liver homogenate were determined after amyloglucosidase hydrolysis according to Keppler and Decker [17].

When fish were transferred to 70% SW, significant increases in plasma ion concentrations were first observed 1 h after the transfer both for Na + and Cl − (Fig. 1A and C). Both the ion concentrations reached peak levels at 12 h, then decreased to levels slightly but significantly higher than the FW levels 24 h after the

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Fig. 2. Changes in plasma growth hormone (GH, A and B), prolactin188 (PRL188, C and D) and PRL177 (E and F) levels after the transfer to 70% SW (X) and to FW (control, X). Vertical bars represent S.E.M. (n = 6). Error bars were with the symbol for the mean. Significant treatment effects between FW and 70% SW at same time are shown with an asterisk (PB0.05, two-way ANOVA). Significant time effects are shown as a, b and c (P B 0.05, two-way ANOVA); values with the same alphabet are not significantly different. Significant differences between the mean values in FW and those in 70% SW in the experiments are shown as 70% SW\ FW or FW\70% SW (P B0.05).

transfer, and maintained the same levels thereafter (Fig. 1B and D). Thus the mean plasma levels of both ions in the experiments were significantly higher in 70% SW than in FW (70% SW\ FW, P B0.05). There was no significant difference in plasma GH between FW and 70% SW groups during the 1st 3 h after transfer (Fig. 2A). Fish in 70% SW showed significantly higher GH concentration 12 h after the transfer compared with FW level (Fig. 2B). Levels of PRL188 and PRL177 were significantly and continuously lower in 70% SW group from 1 to 3 h after transfer (Fig. 2C and E). The same trend (FW \ 70% SW) was also seen in the long-term experiment (Fig. 2D and F). In the 70% SW group, a significantly higher plasma glucose level, compared with the FW group, was first observed 2 h after transfer (Fig. 3A) and the same tendency was maintained until the end of the long-term experiment (70% SW\FW, P B0.05, Fig. 3B). Liver glycogen contents became significantly higher in FW compared with pre-transferred FW from the 24 h on. However, there was no clear trend in liver glycogen

contents in 70% SW over time (Fig. 3C and D). Mean values of phosphofructokinase (PFK) activity in the experiment were significantly higher in 70% SW than in FW during the first 3 h after transfer (70% SW \FW, PB 0.05, Table 1). However there was no clear tendency from 3 h until 96 h after transfer (Table 2). The activities of GPase and G6PDH showed no significant difference between FW and 70% SW during the whole experiment (Table 1 and Table 2).

4. Discussion This study is the first attempt to examine the change in liver carbohydrate metabolism during the course of seawater acclimation in fish. The euryhaline tilapia, O. mossambicus, used in this study acclimated well to the hyperosmotic environment, as indicated by the plasma ion and hormone levels that are consistent with previous reports on this species, O. mossambicus [36].

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Fig. 3. Changes in plasma glucose levels (A and B) and liver glycogen contents (C and D) after the transfer to 70% SW (X) and to FW (control, X). Vertical bars represent S.E.M. (n=6). Error bars were with the symbol for the mean. Significant treatment effects between FW and 70% SW at same time are shown with an asterisk (P B0.05, two-way ANOVA). Significant time effects are shown as a, b and c (P B0.05, two-way ANOVA); values with the same alphabet are not significantly different. Significant differences between the mean values in FW and those in 70% SW in the experiments are shown as 70% SW\ FW (P B 0.05).

The significantly elevated plasma glucose levels in 70% SW compared with in FW at 2 and 3 h after transfer (Fig. 3A) indicate that the transfer from FW to 70% SW affects glucose dynamics in O. mossambicus. Assem and Hanke also showed that plasma glucose levels in O. mossambicus adapted in 71% SW elevated during the 1st 24 h after transfer [1]. Plasma cortisol level increases within 6 h after the transfer of O. mossambicus from FW to 80% SW [14]. Fish respond to stressors, including change of environmental salinity, by releasing stress hormones such as cortisol which is known to enhance glucose production in fish [33,34]. Vijayan et al. [35] showed higher levels of cortisol and glucose in plasma in the fish stressed within 2 or 24 h with confinement and also in the fish with cortisol implantation in O. mossambicus. Their results suggest that cortisol, either directly and/or indirectly, contributes to the regulation of glucose metabolism in fish according to increased energy demand caused by stress. In addition, Leung et al. [19] showed that the serum glucose level was significantly higher in the fish injected with bovine GH. In our results, higher GH concentration in 70% SW than in FW was observed only at 12 h (Fig. 2B), while higher glucose concentrations were observed at 2 and 3 h after transfer (Fig. 3A). Studies on turnover rate of GH may be required during the SW adaptation in tilapia. Cortisol levels were not measured in this study, because of possible acute elevation of the

levels by handling stress at sampling. However, the differences in glucose level as well as other factors between 70% SW and FW groups were considered to be caused by the difference in environment. Even if the overall stress level was greater in 70% SW, the excess stress in 70% SW should be included in the cost for adaptation to 70% SW. It is possible that elevated glucose levels in 70% SW compared with FW is related to either GH and/or cortisol, at least partially. Increased plasma glucose levels could be due to glycogen breakdown or gluconeogenesis. There was no decrease in liver glycogen content in 70% SW compared with FW (Fig. 3C and D), hence gluconeogenesis appeared to be the likely candidate for increased plasma glucose levels in this study. Glucose is the preferred substrate for ATP production in fish gills [22]. When fish are transferred to SW, morphological and physiological changes occur in the gills, such as the development and differentiation of chloride cells associated with the increased Na + , K + -ATPase [20]. In SW acclimated tilapia (O. mossambicus), greater number of chloride cells and higher activity of Na + , K + -ATPase, compared to fish in FW, were observed in cell suspension prepared from the gills [27]. Higher plasma glucose levels may be required for these changes in the gills as a substrate after the transfer to 70% SW. The tendency of higher plasma glucose levels in 70% SW compared with in FW lasted rather longer than the

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Table 1 Changes in liver phosphofructokinase (PFK) glycogen phosphorylase (Gpase) and glucose 6-phosphate dehydrogenase (G6PDH) activities after the transfer to 70% SW and to FW (control) in a short term experiment (0 – 3 h) Enzyme

Treatment

PFK: 70% SW\FW (PB0.05) FW 70% SW GPase FW 70% SW G6PDH FW 70% SW

Time after transfer (h) 0

0.5

1.0

2.0

3.0

10.6 9 1.0 (a)

12.49 1.1 13.6 9 1.9 (ab) 90.199.9 76.097.8 (abc) 76.1 9 6.0 72.9 9 6.3 (ab)

10.4 91.2 13.8 91.0 (ab) 93.7 96.8 102.0 96.3 (b) 81.0 98.88 81.2 912 (b)

12.0 9 1.1 18.29 2.1 (ab) 90.5 9 7.3 101.0 9 4.8 (ab) 74.7 9 6.2 97.1 9 12 (ab)

12.9 91.0 14.1 91.0 (ab) 72.8 94.9 79.5 96.4 (c) 84.6 93.8 69.8 93.8 (b)

81.89 3.8 (ac) 64.19 3.60 (a)

All enzyme activities are represented in nmol min−1 mg−1 protein. Values are mean 9 S.E.M. (n= 6). Significant time effects are shown as a, b and c (PB0.05, two-way ANOVA); values with the same letter of the alphabet are not significantly different. Significant difference between the mean values inFW and those in 70% SW in the experiment are shown as 70% SW\FW (PB0.05).

plasma ionic imbalance of fish; the mean values in 70% SW in the experiment were higher than those in FW in the long-term experiment (Fig. 3B), while O. mossambicus attained a steady state of plasma ion levels at 24 h after the transfer. Chloride cell differentiation and increase in Na + , K + -ATPase activity seem to be both the most important morphological and physiological adjustments during acclimation to SW in fish [5,18]. The results of Foskett et al. [13] suggest that chloride cell density and their diameter may still be changing 21 days after transfer from FW to SW in O. mossambicus. Therefore, although O. mossambicus can competently ion-regulate in 70% SW 24 h after the transfer, the morphological reorganization of the gills might take a longer time. This may hence caused plasma glucose levels to tend to be higher in 70% SW than in FW thereafter in this study as an energy source for the reorganization in the gills. Liver maximal enzyme activities showed no clear trend in this experiment. Activities of GPase and G6PDH were not significantly different between FW and 70% SW through the whole experiment, even though the mean values of PFK activity in the experiment was tended to be higher in 70% SW than in FW in the short-term experiment (Table 1) but not in the long-term experiment (Table 2). These results are in contrast with those in less euryhaline O. niloticus, in which these three enzymes showed significantly higher activities even in completely adapted state (4 weeks after transfer) in 50% SW in previous study [25]. Borski et al. [6] suggested the involvement of GH in SW adaptation of O. mossambicus from their observation of higher activity of GH cells in the pituitary in SW fish than FW, and from stimulatory effect of GH on gill Na + , K + -ATPase activity in vivo. Hypophysectomy in this species decreased the Na + , K + -ATPase activity, which is restored by GH treatment [30], also suggesting the involvement. On the other hand in O. niloticus,

involvement of GH in hypoosmoregulation seems absent, since the direct transfer of the fish from FW to blackish water (20 ppt) did not increase the plasma GH levels, and moreover, GH injection to hypophysectomized fish had no effect on SW adaptability [2]. Cioni et al. reported more marked increases of gill chloride cell density and area to SW in O. mossambicus than in O. niloticus [7]. In present study, plasma GH level was significantly higher at 12 h after transfer in 70% SW than in FW in O. mossambicus. Elevated plasma GH, which may affect chloride cell differentiation, and increases in Na + , K + -ATPase activity in the gills during the acclimation to 70% SW in O. mossambicus, was perhaps related to less disturbance to liver metabolic state in this study than in O. niloticus in previous study [25]. In conclusion, the data in present study point to the likelihood that O. mossambicus requires higher plasma glucose levels by being transferred to hypersaline water, probably as a energy source in order to reorganize the osmoregulatory mechanisms suitable for hyperosmotic environment. However, the clear elevation of plasma glucose levels were observed rather temporally, especially in the early stage of acclimation, with a small change of metabolic state of liver, probably reflecting the high ability of SW acclimation of O. mossambicus.

Acknowledgements We are greatly indebted to Dr Toyoji Kaneko, Dr Sho Kakizawa and Ms Sanae Hasegawa, Laboratory of Physiology, Ocean Research Institute, University of Tokyo, Japan, and Dr Felix G. Ayson in Southeast Asian Fisheries Development Center, Philippines, for invaluable discussions and kind assistance in ion analyses and hormone labeling for RIAs. We are grateful to Professor George K. Iwama and Dr Mathilakath M.

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Table 2 Changes in liver phosphorfructokinase (PFK), glycogen phosphorylase (GPase) and glucose 6-phosphate dehydrogenase (G6PDH) activities after the transfer to 70% SW and to FW (control) in a long term experiment (0 – 96 h) Enzyme

Treatment

Time after transfer (h) 0

3

6

12

24

48

96

11.091.3 9.35 90.85 (ab) 79.3911 91.299.6 (b) 76.397.7 74.7 93.1 (b)

6.56 90.3 11.691.8* (a)

12.2 91.2 11.3 91.5 (ab)

12.3 9 1.1 10.89 1.3 (ab)

15.2 91.2 11.69 0.9 (b)

11.9 91.7 10.29 1.3 (ab)

59.894.2 71.8 9 4.1 (ac) 67.6 9 4.1 86.39 6.2 (ab)

61.1 97.3 62.7 95.0 (ac) 79.0 94.7 86.2 98.1 (ab)

52.6 9 3.1 63.89 3.6 (ac) 76.9 9 5.9 84.798.1 (ab)

62.1 94.5 47.99 4.8 (c) 85.7 9 5.6 84.09 6.2 (ab)

70.2 96.4 62.5 95.1 (ac) 91.1 916 81.99 3.4 (ab)

PFK

FW 70% SW

9.82 91.2 (ab)

GPase

FW 70% SW FW 70% SW

73.9 94.7 (ab)

G6PDH

95.1 95.6 (a)

All enzymes are represented in nmol min−1 mg−1 protein. Values are mean 9S.E.M. (n= 6). Significant time effects are shown as a, b and c (PB0.05, two-way ANOVA). Values with the same letter of the alphabet are not significantly different. * Significant treatment effects between FW and 70% SW are PB0.05, two-way ANOVA.

Vijayan, University of British Columbia for critical discussions. Thanks are also due to Professor Mark A. Sheridan, North Dakota State University, for his generous guidance and active discussion on enzyme assays. We are also indebted to Professor Kazunori Takano and the staffs at the Tropical Biosphere Research Center, University of the Ryukyus for their kind acceptance to conduct the experiment using their facilities. This study was supported in part by grants-in-aid from the Ministry of Education, and from the Fisheries Agency, Japan to M.T. and T.H. M.T. and T.H. are affiliate researchers of the Tropical Biosphere Research Center, University of the Ryukyus.

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