Muscle metabolism of freshwater fish, Tilapia

Muscle metabolism of freshwater fish, Tilapia mossambica (Peters), during acute exposure and acclimation to sublethal acidic water V. KRISHNA MURTHY,P. REDDANNA, M. BHASKAR, AND S. GOVINDAPPA Fish Physiology Division, Department of Zoology, Sri Venkateswara University, Tirupati, 51 7 502 (A.P.)India Received December 12, 1980

MURTHY, V. K., P. REDDANNA, M. BHASKAR, and S. GOVINDAPPA. 1981. Muscle metabolism of freshwater fish, Tilapia Can. J. Zool. Downloaded from www.nrcresearchpress.com by MCGILL UNIVERSITY on 12/11/14 For personal use only.

mossambica (Peters), during acute exposure and acclimation to sublethal acidic water. Can. J. Zool. 59: 1909-1915. Freshwater fish, Tilapia mossambica (Peters), were subjected to acute exposure and acclimation to sublethal acid water (pH 4.0), and the muscle metabolism was investigated. Differential patterns of carbohydrate metabolism were witnessed in the red and white muscles in response to both acute exposure and acclimation. The glycogen content of red muscle was elevated whereas that of white muscle was depleted on acute exposure. But on acclimation, both the muscles had elevated glycogen content. The red muscle seems to mobilize carbohydrates into both hexose mono- and di-phosphate pathways, but white muscle does so only into the hexose monophosphate pathway on acclimation. In general, both the muscles exhibited suppressed glycolysis and elevated oxidative phase leading to elevated glycogen level. The muscle metabolism was oriented towards conservation of carbohydrates and lesser production of organic acids on acclimation, as a possible metabolic adaptive mechanism of the fish, enabling them to counteract the imposed acid stress.

MURTHY, V. K., P. REDDANNA, M. BHASKAR et S. GOVINDAPPA. 1981. Muscle metabolism of freshwater fish, Tilapia mossambica (Peters), during acute exposure and acclimation to sublethal acidic water. Can. J. Zool. 59: 1909-1915. Des Tilapia mossambica (Peters), poissons d'eau douce, ont kt6 exposks et acclimatks a de l'eau acide en concentration sublktale (pH = 4,O) afin d'ttudier le mktabolisme musculaire dans de telles conditions. Le mktabolisme des hydrates de carbone des muscles rouges et blancs est affect6 de f a ~ o diffkrente n chez les poissons soumis a une exposition intense et chez les poissons acclimatks l'eau acide. L'exposition intense a l'eau acide augmente la concentration de glycogkne du muscle rouge et diminue la concentration de glycogkne du muscle blanc. Cependant, chez les poissons acclimatks, les deux types de muscles ont des concentrations de glycogtne plus klevkes que la normale. Chez les poissons acclimatks, le muscle rouge semble utiliser les hydrates de carbone par la voie des monophosphates et des diphosphates d'hexose, alors que le muscle blanc de ces poissons n'utilise les hydrates de carbone que par la voie des monophosphates. En gknkral, les deux types de muscles subissent une dirninutionde la glycolyse et une augmentation de la phase d'oxydation, ce qui entraine une augmentation du glycogtne. Chez les poissons acclimatks, le mktabolisme musculaire vise 8 conserver les hydrates de carbone et 8 produire moins d'acides organiques; il est possible que ce soit 18 un m6canisme mktabolique d'adaptation visant 8 rkduire les effets du stress causk par l'eau acide. [Traduit par le journal]

Introduction Fish may encounter acidic waters in big lakes, streams draining from peatlands (Dunson and Martin 1973), tropical forests on nutrient-poor soils (Janzen 1974), hot springs, and volcanic lakes (Mashiko et al. 1973; Watanabe et al. 1973). Acid mine drainage, industrial effluents, air pollutants, and resultant production of acid rains lead to unnatural acidification of freshwater lakes and streams (Beamish and Harvey 1972; Likens et al. 1972; Dovland et al. 1976; Oden 1976; Dillon et al. 1978). These extreme alterations in pH are often cited as being responsible for progressive depletion of wild fish populations in many rivers and lakes of Norway (Jensen and Snekvik 1972), Sweden (Hultberg and Stenson 1970; Anderson et al. 1971; Almer 1972a, 1972b; Almer et al. 1974), Canada (Beamish and Harvey 1972), United States (Schofield 1975), and India (Karuppasamy 1979). Several investigations have been conducted on different aspects of exposure of fish to altered pH media:

tolerance studies (Beamish 1972; Daye and Garside 1975, 1977, 1979; Robinson et al. 1976; Craig and Bakshi 1977; Trojnar 1977a, 1977b; Murthy et al. 1981), tissue histopathological studies (Daye and Garside 1976, 1980a, 1980b), and physiological aspects (Lloyd and Jordan 1964; Packer and Dunson 1970, 1972; Lievestad and Muniz 1976; Neville 1979). However, there has been little work on the tissue metabolic compensatory changes and adaptability of fish to environmental acidification. The tissue metabolic pathways have been influenced by the process of acclimation to various stress conditions (Ekberg 1958; Das and Prosser 1967; Govindappa and Rajabai 1976). In our earlier work, induced proteolysis in fish muscles exposed to altered pH media (Bhaskara Haranath et al. 1978) and modulations in hepatic metabolism of freshwater fish acclimated to acidic pH (Murthy et al. 1981) were elucidated. Muscle metabolism is important for it constitutes major bulk of the body of the fish, aids in movements, and forms im-

0008-4301/81/101909-07$01.00/0 01981 National Research Council of CanadaIConseil national de recherches du Canada

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CAN. J. ZOOL. VOL. 59, 1981

portant site of organic acid production. The purpose of the present study is to understand the metabolic compensatory adjustments of red and white muscles in response to acute exposure and acclimation to sublethal acidic pH.

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Materials and methods

*

Freshwater fish, Tilapia mossambica (Peters), 10 1g were collected from the ponds near Tirupati. They were kept in large aquaria with continuously flowing dechlorinated water to acclimat~zethem to laboratory conditions (25"C, pH 7.0 k 0.2; and light period of 12 h). They were fed with commercial fish pellets. The extreme sublethal acidic pH medium (pH 4.0) to the test fish was prepared and maintained as reported earlier (Murthy ctal. 1981). The fish were divided in10 three groups: p controls. acute exposed, and acclimated. Control ~ o u was maintained at pH 7.0 5 0.2 and the second and third groups of fish at pH 4.0 0.I for 1 day (acute exposed) and for 15 days (acclimated) respectively. Since the fish ar pH 4.0 exhibited stabilized oxygen consumption from day 12 onwards, the period of 15 days was taken as the acclimation period. The fish were sacrificed separately and red and white muscles were isolated and rapidly chilled by placing them in an ice chamber. The white muscle was collected from the region dorsal to the lateral line and the red muscle from along the lateral line. These tissues were used for biochemical analysis. Glycogen (Carroll et al. 1956), pyruvic acid (Friedemann and Hangen 1942), and lactic acid (Barker and Summerson 1941, modified by Huckabee 1956) were estimated in red and white muscles of control and experimental fish. The activities of phosphorylase "a" and "ab" were estimated in the direction of glycogen synthesis (Con et al. 1955). Homogenate (5%) was prepared in an aqueous medium contalning 0.037 M ethylene diarnine tetraacetic acid (EDTA) and 0.1 M sodium fluoride, pH 6.5, as recommended by Guillory and Mommaerts (1960). The homogenate was centrifuged for 15 min at 2500rpm and the supernatant was diluted four times with cysteine (0.03 M), P-glycerophosphate (0.015 M) buffer, pH 6.5. The diluted enzyme (0.4 mL) was added to 0.2 mL of 2% glycogen and incubated for 20 min at 35'C. The reaction was started by the addition of 0.2 mL of 0.016 M glucose-lphosphate (G- 1-P) to one tube (phosphorylase a ) , 0.2 mL of G-1-P and 0.004 M adenosine-5-monophosphateto the other (phosphorylase ab). After incubation for 15 min for phosphorylase ab (total) and 30 min for phosphorylase a (active) activities, the reaction was stopped by the addition of 10% sulphuric acid. The inorganic phosphate (Pi) liberated was estimated by the method of Taussaky and Shorr (1953) and protein by the method of Lowry et al. (1951). Aldolase activity was estimated by the method of Bruns and Bergmayer (1965). The activity levels of succinate dehydrogenase (SDH), NAD glutamate dehydrogenase (GDH), and NAD - lactate dehydrogenase (LDH), and NAD - malate dehydrogenase (MDH) were estimated by the method described earlier (Reddanna and Govindappa 1978). The activity level of NADP glucose-6phosphate dehydrogenase (G-6-PD) was estimated by the method of Georg and Waller (1965).

*

freshwater fish subjected to acute exposure and acclimation to sublethal acid water. Glycogen content was depleted (20.6%) in white muscle on acute exposure. Red muscle had 31.6% elevation in the glycogen content. Phosphorylase ab was highly depleted in both red (26.1%) and white (46.8%) muscles. Phosphorylase a was inhibited (25.8%) in white muscle with no significant change in red muscle. Phosphorylase "b" (inactive) was depleted by 52.6% in white muscle and 31.77% in red muscle. Aldolase activity was depleted in white (81.7%) and red (21.34%) muscles, whereas NAD - lactate dehydrogenase was elevated by 76% in white muscle and 33% in red muscle. In white muscle, pyruvic acid content was elevated by 16.95% and lactic acid content showed no significant change. Red muscle showed no significant change in pyruvic acid content, whereas lactic acid was depleted (17.95%). Glucose-6phosphate dehydrogenase (G-6-PD) activity was decreased by 9.2% in white muscle and 25.7% in red muscle. Glutamate dehydrogenase was activated in both the muscles. Both succinate dehydrogenase and malate dehydrogenase activities were elevated in white and red muscles. The white and red muscles of acclimated fish showed significant elevation of the glycogen content, the maximum increase being in red muscle. The activity levels of phosphorylase ab and b were significantly depleted in both the muscles, but phosphorylase a was highly elevated. Aldolase activity was inhibited by 89% in white muscle, but was increased by 21.56% in red muscle. The levels of pyruvic and lactic acids were decreased in both the muscles. NAD - lactate dehydrogenase and glucose-6-phosphate dehydrogenase activity levels were highly elevated in both the muscles. The activity levels of glutamate dehydrogenase succinate dehydrogenase and malate dehydrogenase were significantly elevated in both the muscles on acclimation.

Discussion White and red muscles of freshwater fish exhibited differential patterns of carbohydrate metabolism on exposure to sublethal acid pH. Since the acid medium forms a stress condition to the animal, the glycogen might have been actively mobilized towards the blood glucose under different stress conditions as suggested by several workers (Bhaskara Haranath 1979; Reddanna and Govindappa 1979) leading to depleted glycogen levels in white muscle on actue exposure. Depressed activities of phosphorylase ab (total), a (active), and b (inactive) in this muscle were suggestive of regulation imposed at the metabolic level to prevent further glycogen degradation. Depletion of Results aldolase activity was also suggestive of a decreased rate The data presented in Tables 1 to 4 illustrate the of glycolysis in this muscle. However, the increased metabolic variations in white and red muscles of pyruvic acid level suggests higher formation of pyruvic

MURTHY ET AL.

TABLE1. Levels of glycogen, pyruvic acid and lactic acid, and the activity levels of phosphorylase and aldolase in red muscles of control and experimental fish. Each value represents the mean of eight observations. Mean 5 SD; + and - indicate percentage increase or decrease over control Experimental Control

Component

Acute exposed

Acclimated


Glycogen (mg . g wet tissue-') Phosphorylase a (Kmol Pi formed. mg protein-' . h-I)

3.45k0.225

Phosphorylase ab (Frnol Pi formed mg protein-' . h-I)

16.04'0.78

Phosphorylase b (Krnol Pi formed mg protein-'

12.59k0.64

. h-I)

Aldolase (pmol FDPcleaved . mgprotein-'

. h-I)

46.38'2.4

Pyruvic acid ( ~ r n o.l g wet tissue-') Lactic acid (mg g wet tissue-')

acid in the tissue. The elevated level of activity of NAD - lactate dehydrogenase (which is involved in the formation of pyruvic acid from lactic acid), observed in the present study might be responsible for the increased pyruvic acid content. Increased activity of alanine amino transferase (AIAT) in the muscles exposed to acid stress conditions (+39% in red muscle and +278% in white muscle, authors' unpublished data) might also suggest mobility of amino acids towards the formation of pyruvic acid. These observations indicate decreased mobilization of glycogen into hexose-diphosphatepathway. Since glucose-6-phosphatedehydrogenase activity was highly depleted, decreased mobilization of glycogen into the hexose monophosphate (HMP) pathway can be envisaged. The activity levels of succinate (SDH) and malate (MDH) dehydrogenases were elevated, suggesting stepped-up oxidative metabolism in the tissues. This elevation in the oxidative phase might be due to mobilization of amino acids as revealed by elevated glutamate dehydrogenase activity and pyruvic acid as indicated by increased lactate dehydrogenase activity. SDH:GDH and SDH:LDH ratios were decreased in these muscles suggesting that the extent of elevation in succinate dehydrogenase (SDH) was not to

the tune of elevation of either glutamate dehydrogenase (GDH) or lactate dehydrogenase (LDH) suggesting the possibility of diversion of TCA cycle intermediaries. Hence the white muscle had inhibited glycolysis with depleted glycogen content in response to acute exposure to sublethal acid water. However, the red muscle showed a different trend, in that it had a significant elevation in the glycogen content on acute exposure. Since NAD - lactate dehydrogenase (NAD-LDH) and glutamate dehydrogenase (GDH) activities were highly elevated, active mobilization of lactic acid and amino acids into the oxidative metabolism can be envisaged. The red muscle metabolism was also oriented towards inhibited glycolysis, as indicated by decreased aldolase activity leading to elevated glycogen content. In spite of the operation of similar metabolic events in white and red muscles, the red muscle had elevated glycogen content, which might be due to participation of tissue lipid components in metabolism. The free fatty acid content was depleted in the muscle (-26%, authors' unpublished data) suggesting the participation of free fatty acids in tissue oxidations. Such a possibility of fatty acid oxidations might be responsible for the suppressed glycolysis

CAN. 1. ZOOL. VOL. 59, 1981

TABLE2. Levels of glycogen, pymvic acid and lactic acid, and the activity levels of phosphorylase and aldolase in white muscles of control and experimental fish. Each value represents the mean of eight observations. Mean 2 SD; + and - indicate percentage increase or decrease over control Experimental Component

Control

Acute exposed

Acclimated


Glycogen (mg . g wet tissue-') Phosphorylase a (pmol Pi formed . mg protein-1 . h-') Phosphorylase ab (kmol Pi formed . mg protein-' . h-') Phosphorylase b (pmol Pi formed . mg protein-'

. h-')

Aldolase (pmol FDP cleaved mg protein-') Pyruvic acid (pmol . g wet tissue-') Lactic acid (mg . g wet tissue-')

(Rennie et al. 1976) leading to elevation in the glycogen content. Since red muscle in fish forms a reserve site for the nutrients of white muscle, increased glycogen content in this tissue suggests the building up of glycogen reserves for general muscular activity. The muscle metabolism seems to be different in acclimated and acute exposed fish in that both white and red muscles had elevated glycogen content, the maximum percentage increase being in red muscle. In white muscle, inhibited aldolase activity in the presence of activated phosphorylase suggests the mobilization of glycogen towards pathways other than glycolysis. Since the activity level of glucose-6-phosphate dehydrogenase (G-6-PD) was considerably elevated, the possible mobilization of glycogen and glucose towards the hexose monophosphate pathway can be visualized. This might be an adaptation towards decreasing the formation of lactic and pyruvic acids and thus aiming for decreased acid production in the muscles. Hence this was a clear case of tissue compensation at metabolic level towards the induced acid stress in the medium. Elevated activity levels of lactate and glutamate dehydrogenases (LDH

and GDH) were suggestive of increased mobilization of lactic acid and amino acids into the oxidative metabolism. Consequent upon such changes, there was not only a depleted lactic acid level in the tissues but also an elevated oxidative phase of metabolism as indicated by succinate and malate dehydrogenase (SDH and MDH) activities. Red muscle metabolism was oriented towards mobilizing glycogen into both hexose di- and monophosphate pathways since phosphorylase, aldolase, and glucose-6-phosphate dehydrogenase (G-6-PD) activities were elevated. Hence both white and red muscles in acclimated fish have regulated the carbohydrate metabolism towards decreasing the production of metabolic acids. From these observations it can be inferred that the red and white muscle metabolism was oriented towards lower production of metabolic acids, increased carbohydrate reserves, with a switching over to aerobic phase during acclimation to sublethal acid water. These metabolic tissue compensatory mechanisms might lead to adaptive changes providing positive survival value to the fish in acid waters.

MURTHY

ET

AL.

TABLE 3. Activity levels of G-6-PD, LDH, GDH, SDH, and MDH in red muscles of control and experimental fish. Each value represents the mean of eight observations. Mean 2 SD; + and indicate percentage increase or decrease over control Experimental Component -

-

Control -

Acute exposed

Acclimated

-


G-6-PD (pmol formazan formed . mg protein-' . h-') LDH (prnol formazan formed mg

rotei in-' .h-')

GDH (pmol formazan formed - rng protein-' h-') SDH (pmol formazan formed. mg protein-' . h-') MDH (prnol formazan formed . rng protein-' h-') SDHIGDH

TABLE 4. Activity levels of G-6-PD, LDH, GDH, SDH, and MDH in white muscles of control and experimental fish. Each value represents the mean of eight observations. Mean -C SD; + and indicate percentage increase or decrease over control Experimental Component G-6-PD (pmol formazan formed. mg

Control

rotei in-' . h-')

LDH (pmol formazan formed - mg protein-' . h-') GDH (prnol formazan formed . mg protein-' h-') SDH (pmol formazan formed . mg protein-' h-') MDH (prnol formazan formed mg protein-' .h-') SDHIGDH

Acute exposed

Acclimated

1914

CAN.


Acknowledgements

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VOL. 59. 1981

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