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Effect of nutritional factors and cortisol on tyrosine aminotransferase activity in liver of brook trout, Salvelinus fontinalis mitchill
Comp. Biochem. Physiol., 1977. Vol. 58B, pp. 189 to 193. Peroamon Press. Printed in Great Britain
EFFECT OF NUTRITIONAL FACTORS A N D CORTISOL ON TYROSINE AMINOTRANSFERASE ACTIVITY IN LIVER OF BROOK TROUT, SALVELINUS FONTINALIS MITCHILL S. J. WHITING* AND A. J. WIGGS Department of Biology, University of New Brunswick, Fredericton, New Brunswick, E3B 5A3, Canada
(Received 18 January 1977) Abstract--1. Starvation for over 50 days increased the activity of Tyrosine aminotransferase (TAT) from brook trout liver and decreased liver glycogen. 2. Cortisol (6-40mg/100g body wt) increased TAT activity, optimally 72hr post-injection; liver glycogen response to cortisol was variable while tissue water either increased or remained constant. 3. Fish fed high-protein/low-carbohydrate had greater TAT activity than fish fed low-protein/high carbohydrate. There was food-induced peak of activity indicating a "diurnal rhythm" analogous to that of rat TAT. 4. TAT activity in brook trout appears to be correlated with protein catabolism.
INTRODUCTION
In teleost fish, steroids identical to the glucocorticoids (i.e. cortisol and cortisone) are synthesized by the adrenal gland homologue (for review see Butler, 1973). Only recently have data been presented to suggest these steroids are involved in gluconeogenesis in fish (Butler, 1968; Freeman & Idler, 1973). In the mammal the demonstration of a cortisol-induced increase in tyrosine aminotransferase (L-tyrosine:2oxoglutarate aminotransferase, E.C. 2.6.1.5, TAT) activity has been considered evidence for a glucocorticoid action of cortisol. Failures to find a cortisolinduced increase in TAT in fish (Chan & Cohen, 1964 and Fellman et al., 1971) have led to the proposals that cortisol acts only as a mineralocorticoid in lower vertebrates (Chan & Cohen, 1964; Janssens, 1970) and that the TAT response to cortisol occurs only above the evolutionary level of amphibians (Nemeth & Jurani, 1974). However, activities of transaminases other than TAT increase with glucocorticoid treatment in fish. For example, the enzyme alanine aminotransferase is increased after cortisol injection in Salvelinusfontinalis (Mann, 1972; Freeman & Idler, 1973) and Carassius auratus (Storer, 1967). Dietary factors are also known to affect TAT and gluconeogenesis in mammals. Fasting produces increases in the enzymes of amino acid degradation in mammals (Freedland & Szepesi, 1971), including TAT (Goswami & Chatagner, 1966): No work has been reported on fish liver TAT after periods of starvation but both alanine aminotransferase (Storer, 1967; Mann, 1972; Creach & Serfaty, 1974) and aspartate aminotransferase (Larsson & Lewander, 1973; Creach & Serfaty, 1974) increase in starved fish. In mammals, high-protein diets increase the amount of glucose manufactured from amino acids (Freedland & Szepesi, 1971) and can induce TAT (Reynolds et al., 1971). * Present Address: Department of Nutrition, University of Guelph, Guelph, Ontario, Canada. c.B.p.58/2B F~
The cyclic intake of protein by ad libitum fed rats appears to be the cause of the diurnal rhythm in liver TAT (Francesconi et al., 1972). There are few reports of diet-induced increases in TAT in nonmammalian species. Ohisalo & Pispa (1975) found increased TAT in Rana temporaria fed a high-protein diet. Frog and tadpole TAT were found to exhibit diurnal rhythms (Lane et al., 1970). A study of tyrosine aminotransferase in brook trout (Salvelinus fontinalis Mitchill) under conditions known to stimulate gluconeogenesis in mammals, i.e. starvation, high-protein feeding, and cortisol treatment was undertaken with a view to evaluating TAT as a gluconeogenic enzyme. Secondarily evidence for the view that cortisol has glucocorticoid function in fish is presented.
MATERIALS AND METHODS
Animals Yearling brook trout (Salvelinusfontinalis Mitchill) were obtained from the Department of Environment Fish Hatchery, Florenceville, N.B., Canada. Fish were kept in aerated, 20gal flow-through tanks (61 tap water/rain). Photoperiod was adjusted to correspond to natural conditions. Water temperature varied seasonally; the lowest temperature, 9°C, occurred in December and the highest, 14.5°C, occurred in July. Fish were fed 15 g Purina Trout Chow pellets/100g body wt on alternate days at 1600hr (unless otherwise stated). Sampling took place at least 42 hr after the last feeding except when samples for the "diurnal rhythm" experiments were taken.
Samplin9 Fish were killed by a sharp blow on the head and body measurements were quickly taken and the liver and gonad removed. Liver was cut so that a central piece (0.25g) was placed in a cold homogenizing tube for TAT assay and a peripheral piece (0.05g) was placed in 3 ml 1N NaOH for glycogen determination. Epaxial muscle samples (ventral to the dorsal fin) were placed in tared vials for muscle water determination. 189
190
S. J. WHITING AND A. J. W]GGS
Injection procedures Fish were injected with cortisol or saline intramuscularly (i.m.) at one or more sites in the caudal area, no more than 0.1 ml per site. There was some leakage of injected material. Fish were injected 17 hr after their last feeding. Cortisol was injected as either hydrocortisone hemisuccinate (Sigma) in 0.72% NaCI or undiluted Solucortef (Upjohn). The amount injected is given as the free alcohol equivalent. There was some mortality when the highest dose (40 mg/100 g body wt cortisol) was given.
they are designated low protein/high-carbohydrate and high-protein/low-carbohydrate (respectively).
Statistics Comparisons were made using one-way analysis of variance tests (Sokal & Roll, 1969). Slopes of the lines of best fit for data of the starvation experiment run OctoberDecember were found by regression analysis. Differences between experimental and control means were considered significant if P < 0.05. Values are expressed as mean + standard error of the mean (S.E.M.).
T A T assay Liver for TAT assay was homogenized for 30 sec in 13 vol of cold (4°C) 0.15 M KCI (containing 10 -3 M EDTA and brought to pH 7.8 with 10N KOH). Homogenates were centrifuged at 28,000 g for 35 min at 4°C. The enzyme preparation consisted of 1 ml supernatant (28,000 g) diluted 1:1 with phosphate buffer (0.1 M potassium phosphate containing 10 -3 dithiothreitol, pH 7.7). Tyrosine aminotransferase activity was measured using the Modified Brigg's method (Canellakis & Cohen, 1956). The assay mix contained 48.4mole tyrosine, 5.55#mole ketoglutarate, 8.35/~mole pyridoxal-5'-phosphate (PLP) and 30pmole diethyldithiocarbamate. Reagents were added, in the specified order, to each assay tube incubating at 20°C: l m l of phosphate buffer; 0.5ml of 1.11 M ct-ketoglutarate and 16.7 mM PLP in 0.1 M dibasic potassium phosphate; 2 ml of 0.024 M tyrosine in 0.2 M dibasic potassium phosphate containing 0.48% 10N KOH. Duplicates of each enzyme preparation (0.2 ml) were added at time zero and each tube was gently inverted once to mix the contents. After 10rain at 20°C, 0.25 ml of 100% trichloracetic acid (TCA) was added to stop the reaction and each tube was mixed vigorously. Sample blanks were treated similarly except that the TCA was added before the enzyme preparation. All tubes were centrifuged for 10min at 2000 x g and 2.0ml portions of supernatant were transferred to tubes containing 1.0ml of 1~o monobasic potassium phosphate. After mixing, 1 ml of 3% ammonium molybdate (in 1.4 N HC1) was added and tubes were again mixed. After 30 min incubation at room temperature, absorbance at 850nm was read. p-Hydroxyphenylpyruvate (HPP) was substituted in the reaction for the enzyme and the relation of HPP concentration to OD 850 was found. Enzyme units were calculated using the following equation:
RESULTS AND DISCUSSION
Effect of starvation Starvation resulted in increased T A T activity in yearling b r o o k trout of two experiments (Figs 1 and 2). Unfortunately it was not possible to control all experimental factors and differences existed in the two studies. These variables--i.e, time of the year (fall vs spring), water temperature (9-11°C vs 11-14°C), genetic b a c k g r o u n d (stocks were unavoidably d i f f e r e n t ) ~ may account for the different patterns of response of T A T to starvation in the two experiments. Brook trout starved between October and December (Fig. 1) exhibited a gradual rise in T A T activity, during which time liver glycogen levels declined and remained low ( < 2.5mg/100mg). Fish starved between M a y and July exhibited high TAT activity only after 40 days of starvation, by which time liver glycogen was almost depleted ( < 1.0 mg/100 mg). These results contrast with mammalian work in which a starvation induced increase
8o iver0Yc°en \\
~ 4,0 8
TAT units = No. #moles HPP formed per gram liver (wet wt) per hr at 20°C. Brook trout hepatic TAT was specific for ct-ketoglutarate as the amino acceptor; transamination of tyrosine did not occur if an equal concentration of pyruvate or oxaloacetate was used in place of ct-ketoglutarate, Vm~xand the apparent K,, (tyrosine) were found to 50#mole/hr per g and 3.2 c 10- 3 M (respectively) and the pH optima was in the range 8-9. The apoenzyme of brook trout TAT was inactive. Removal of the coenzyme PLP was achieved by dialysis against 5% ct-ketoglutarate and TAT activity could not be restored even after the addition of 80#mole of PLP to the assay mix.
0-0 Starved
I
O~
-]-~
I
I
[
J
I
TAT
I
¢~ I 5c ~k
Other measurements Liver glycogen was measured by the method of Roe & Dailey (1966) and is expressed as mg/100 mg liver (wet wt). Per cent muscle water was found by drying epaxial muscle samples to a constant weight at 60°C. The following formula was used: 9/o water = (1.0 - (dry wt/wet wt)) x 100%, Two experimental diets, patterned after Zeitoun et al. (1973) contained 30 and 55% protein, 15 and 14% lipid, 34 and 3% carbohydrate and 5138 and 5403 calories/g;
M S'ta rved ~ . 0 Fed
I
Oci" I0
i
20
I
30 Days
J
40
I
50
i
60
Dec.
Fig. 1. Effect of starvation on liver glycogen concentration and tyrosine aminotransferase (TAT) activity in yearling brook trout sampled between October and December (water temperature decreased from 11 to 9°C). For each point n is given. Vertical bars represented ±S.E.M. Slope of starved TAT (1.012) significantly greater than the slope of fed TAT (0.286). Number of days of starvation given.
Effect of nutritional factors and cortisol
E
Table 1. Effect of feeding 2 isocaloric diets, high-protein/ low-carbohydrate and low-protein/high-carbohydrate, on TAT activity in brook trout fasted 4 days prior to feeding
6o
Amt Diet constituents (~)~ fedb Protein CHO Lipid Days (g)
>,
~o _J ~> g
2o
c--o Starved
"~
-
~ 5 _
o
; ~
5 150 --
~: t
30 55 30 55 30 55
34 3 34 3 34 3
15 14 15 14 15 14
3 3 8 8 lI 12
40 40 30 30 30 30
TAT activity" 91,3 ± 11.5(5)
98,0 +_ 16.5(5) 81.6 _+ 10.7(6) 121.3 ± 5.7(6)* 66.4 ± 6.0(6) 107.4 ± 11.5(5)*
" ~ Dry wt. b Amount fed/t00 g fish. c Moles HPP formed/hr per g liver at 20°C; mean + S.E.M. (n). *P < 0.01.
100
"~
191
50
/ =--o ~te~rved I
I
I
Io
30
40
= /
After 8 days of feeding the experimental diets, the fish fed the high protein diet had significantly inDays creased (P < 0.01) TAT activity compared to that in Fig. 2. Effect of starvation on liver glycogen concentration the fish fed the low protein diet (Table l). At 11 and and tyrosine aminotransferase (TAT) activity in yearling 12 days of feeding, TAT levels in both groups had brook trout sampled between May and July (water tem- declined from the 8-day values but TAT in the highperature increased from 11 to 14°C). For each point, n = 8 protein group (107 units) was still greater (P < 0.01) except where noted. Vertical bars represent +S.E.M. than TAT in the low-protein group (66 units). The Number of days of starvation given. reason for the decline is not known, although it is possible that the low-protein group may have experiin rat liver TAT occurs in 48 hr (Goswami & Cha- enced some carbohydrate repression of TAT as found tagner, 1966). Janssens (1970) suggested that in lower by Ohisalo & Pispa (1975) in glucose-fed frogs. Brook trout TAT activity was found to vary during vertebrates TAT activity and gluconeogenesis are normally at a high level and that little increase occurs the day. Although different feeding times (Fig. 3) were with starvation. In order to survive the long periods used in the two studies the peak of activity occurred of starvation inherent in their life-styles, fish would 7-8 hr after feeding in both studies. These peaks corbe expected to have protein sparing mechanisms to related with feeding time rather than photoperiod. In counteract excessive protein loss through catabolism. rats the peak of activity of liver TAT occurs 4 hr after Under these circumstances fish would not be expected feeding (Zigmond et al., 1969). The longer lag time of 8 hr observed in brook trout may reflect the lower to exhibit a major increase in TAT activity. The results (Figs 1 and 2) suggest that low liver metabolic rate in fish kept at low (11-13°C) temperaglycogen levels are correlated with increased protein tures. The influence of feeding and dietary protein on catabolism as indicated by increased TAT activity. The decline in liver glycogen of control fish of the TAT activity is suggestive of substrate induction. fall experiment (Fig. 1) may be a seasonal effect. Valtonen (1974) found similar seasonal variations in liver glycogen in whitefish. Older fish ( > 3 yr) did not show an increase in TAT activity even after 100 days of starvation at l l ° C (Whiting, 1976). These large trout, having relatively lower maintenance requirements than yearlings (Frost & Brown, 1967), may also have had different energy reserves than the smaller fish. For example, Stimpson (1965) found that young goldfish (Carassius auratus) depleted liver glycogen while old goldfish depleted liver lipid on starvation. I-
Moy
20
50
July
Effect of feedin 9 Fish were fed experimental diets, either a highprotein/low-carbohydrate diet or a low-protein/highcarbohydrate diet, after 4 days of food deprivation. Fish were initially fed 40g of their respective diet, per 100g (wet wt). After 3 days of feeding, TAT activities (measured 40 hr post-feeding) were compai'able in the two groups (Table 1). On the fourth day rations were reduced to 30 g/100 g as fish fed the lower level of protein were not consuming all their ration.
1600
0
0800
1600
T i m e of doy
Fig. 3. Daily variation in brook trout tyrosine aminotransferase (TAT) activity. Two groups of fish were fed either the normal ration (15 g/100g)or twice the normal ration, at the time indicated by their respective arrows. For each point n = 5 (normal ration) and n = 4 (2 x ration). Vertical bars represent ±S.E.M. Black bars represent time in darkness.
192
S.J. WHITINGAND A. J. WXGGS
Table 2. TAT activity, liver glycogen and ~ tissue water in brook trout injected with cortisol, in two experiments (July and September, 1975). Data expressed as mean 4- $.E.M. (n). Comparisons are made against saline-injected controls Treatment July 1975 : uninjected saline cortisol (40mgc) uninjected saline cortisol (40 mgc) uninjected saline cortisol (40 mgc) uninjected saline cortisol (40 mgc) Sept. 1975: uninjected saline cortisol (6 mgc) cortisol (12 mgc) cortisol (25 mgc) cortisol (40 mgc)
Hours post-injection
Hours post-feeding
24 24 24 48 48 48 72 72 72 96 96 96
42 42 42 66 66 66 90 90 90 114 114 114
57.4 _ 5.0(8)~ 90.0 4- 5.9 (8) 113.1 4- 7.1 (8)* 71.3 4- 3.6 (8) 88.4 4- 7.4 (8) 130.3 +_ 6.5 (8)~ 65.6 4- 6.9 (8)t 95.2 + 7.0 (8) 137.0 4- 13.4 (8)I" 65.0 4- 3.1 (8)* 96.2 4- 8.7 (8) 137.2 4- 12.0 (8)t
72 72 72 72 72 72
90 90 90 90 90 90
64.3 4- 3.4 (5) 71.4 4- 4.5 (6) 111.6 4- 6.4 (6)~ 132.7 4- 9.7 (6):~ 115.6 4- 2.9 (4)~: 127.4 4- 7.7 (6)$
TAT activity"
Liver glycogen b
~o Tissue water
4.9 4- 0.7 (8) 3.4 4- 0.5 (8) 1.6 4- 0.6(8)* 77.30 4- 0.65 (6)t 75.35 4- 0.28 (6) 78.17 4- 0.42 (6)t 4.4 4- 0.6 (8) 3.1 4- 0.5 (8) 2.9 4- 0.6 (8) 75.54 4- 0.40 (6) 75.70 4- 0.29 (6) 78.50 + 0.50 (6)t 4.2 4.3 4.4 5.4 2.8
4- 0.3 (6) 4- 0.6 (6) 4- 0.5 (6) 4- 0.2 (4)* 4- 0.6 (6)
77.41 4- 0.19 (6)
77.81 + 0.01 (6)
Moles HPP formed/hr per g liver at 20°C. b mg/100 mg liver. ¢ Dose/100 g fish. * P < 0.05. t P < 0.01. P < 0.001. Szepesi & Freedland (1967) have presented evidence that in the mammal that protein per se is not responsible. Diurnal rhythm studies have implicated quinolinic acid, a metabolite of tryptophan, as a possible control agent (Hardeland, 1973). Evidence also exists to exclude cortisol as regulating agent of cyclic changes in TAT. Effect o f cortisol Increase of brook trout TAT after cortisol treatment required a period of at least 24 hr, considerably longer than the response time of 5 hr seen in rat (Lin & Knox, 1957). On the other hand a single cortisol injection in brook trout produced a high level of TAT activity which lasted for several days (Table 2) while in rats, induction of TAT by cortisol (after intraperitoneal injection) lasts only several hours unless injection is repeated (Kenny et al., 1968). The prolonged effect in brook trout may be attributed in part to the intramuscular injection of hormone (which may have resulted in a slow absorption of cortisol into systemic circulation over time) as well as to the high dose used. The dose of cortisol used in this study to increase TAT in brook trout must be considered pharmacological. The lowest amount which caused a maximal increase in TAT activity was 6mg/100g body wt (Table 2). Other workers, using comparable concentrations in fish and amphibians, (Chan & Cohen, 1964; Fellman et al., 1971; Ohisalo & Pispa, 1975) were unable to increase TAT activity. Saline injected controls generally exhibited increased levels of TAT relative to uninjected controls. Although the increase was significant in some cases, it did not equal the effect of cortisol injections (Table
2). TAT in rats is known to be stimulated by a number of nonspecific agents provided an intact adrenal gland was present (Litwack & Diamondstone, 1962). It is possible that saline injection was acting as a nonspecific stressing agent. As liver glycogen deposition is considered a primary gluconeogenic action of glucocorticoids in mammals (cf. Glenn et al., 1961), it was surprising to find instances of significantly reduced levels of liver glycogen in cortisol-treated fish (Table 2). There may be a time-dependent, dose-dependent response operating since liver glycogen was significantly decreased at 24hr (40 mg/100 g) yet significantly increased at 72hr (25 mg/100g). (Table 2). Glycogen deposition and transaminase induction are the result of different effects of glucocorticoids. The activation of glycogen synthetase by cortisol is not obligatory for gluconeogenesis in mammals (DeWulf & Hers, 1967). A similar relationship could occur in fish explaining the apparent discrepancy. Cortisol injection in brook trout increased the water content of muscle (Table 2). Butler (1973) suggests that cortisol increases protein catabolism. In starved fish the catabolism of protein is correlated with increased tissue water (Love, 1970) so that the rise in tissue water may indicate a fall in tissue protein as a result of cortisol action. Cortisol treatment induces weight loss in brook trout (Freeman & Idler, 1973), Since cortisol increases tissue water the weight loss must indicate a loss of some other tissue component. In the brook trout as in the mammal TAT activity increases after feeding, in response to dietary protein, and during starvation. This correlation with conditions when protein catabolism is prevalent lends sup-
Effect of nutritional factors and cortisol port to the idea that T A T is involved in protein catabolism a n d gluconeogenesis in fish as well as m a m mals. The further observation of cortisol stimulation of T A T activity is consistent with the hypothesis a n d further suggest that cortisol acts as a glucocorticoid in fish as in mammals. REFERENCES
BUTLER D. B. (1968) Hormonal control of gluconeogenesis in the North American eel (Anguilla rostrata). Gen. comp. Endocrin. 10, 85-91. BUTLER D. G. (1973) Structure and function of the adrenal gland in fishes. Am. Zool. 13, 839-879. CANELLAglS Z. N. & COHEN P. P. (1956) Purification studies of tyrosine-~-ketoglutaric acid transaminase. J. biol. Chem. 222, 53-62. CHAN S-K. & COHEN P. P. (1964) A comparative study of the effect of hydrocortisone injection on tyrosine transaminase activity of different vertebrates. Archs Biochem. Biophys. 104, 335-337. CIVEN M., ULRICH R., TRIMMER B. M. & BROWN C. B. (1967) Circadian rhythms of liver enzymes and their relationship to enzyme induction. Science, N.Y. 157, 1563-1564. CREACH Y. & SEREATY A. (1974) Le jefine et la r6alimentation chez la carpe (Cyprinus carpio L.). J. Physiol., Paris 68, 245-260. DEWULF H. & HERS H. B. (1967) The stimulation of glycogen synthesis and of glycogen synthetase in the liver by glucocorticoids. Eur. J. Biochem. 2, 57-60. FELLMAN J. C., ROTH E. S. & FUJITA T. S. (1971) Study of tyrosine aminotransferase in developing salmon. Comp. Biochem. Physiol. 40B, 241-247. FRANCESCONI R. P., COTTER R. 8z MAGER M. (1972) Starvation and refeeding: effects on the periodicity of tryptophan and tyrosine metabolism in mice. J. Nutr. 102, 597-602. FREEDLAND R. A. & SZEPESI B. (1971) Control of enzyme activity: nutritional factors. In Enzyme Synthesis and Degradation in Mammalian Systems (Edited by RECHEIGL M.), pp. 103-140. University Park Press, Baltimore. FREEMAN H. C. & IDLER D. R. (1973) Effects of corticosteroids on liver transaminases in two salmonids, the rainbow trout (Salmo gairdnerii) and the brook trout (Salvelinus fontinalis). Gen. comp. Endocrinol. 20, 69-75. FROST W. E. & BROWN M. E. (1967) The Trout. N.M.N. Collins, London. GLENN E. M., BOWMAN B. J., BAYER R. B. & MEYER C. E. (1961) Hydrocortisone and some of its effects on intermediary metabolism. Endocrinol. 68, 386-410. GOSWAMI M. N. D. & CHATAGNER F. (1966) Starvationinduced adaptation of rat liver tyrosine transaminase and serine dehydrase. Experientia 22, 370-371. HARDELAND R. (1973) Diurnal variations in inducibility of hepatic tyrosine aminotransferase. Int. J. Biochem. 4, 35%364. JANSSENS P. A. (1970) The evolution of corticosteroid function. Steroidologia 1, 308-320.
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LANE E. A., MAVRIDESC. & D'IORIO A. (1970) A comparative study of the daily rhythm of liver tryosine aminotransferase in various vertebrates. Int. J. Biochem. 1, 343-348. LARSSON A. & LEWANDER D. (1973) Metabolic effects of starvation in the eel, Anguilla anguilla L. Comp. Biochem. Physiol. 44A, 367-374. LINE. C. C. & KNOX W. E. (1957) Adaptation of the rat liver tyrosine-ct-ketoglutarate transaminase. Biochim. biophys. Acta 26, 85-88. L1TWACK G. & DIAMONDSTONE T. I. (1962) Nonspecific stimulation of tyrosine-~-ketoglutarate transaminase activity in vivo. J. biol. Chem. 237, 469-472. LOVE R. M. (1970) The Chemical Biology of Fishes. Academic Press, New York. MANN O. E. (1972) The effect of starvation and cortisol on the glutamate-pyruvate transaminase of brook trout, Salvelinus fontinalis (Mitchill). B.Sc. Thesis, University of New Brunswick, Fredericton, N.B. Canada. NEMETH S. & JURANI M. (1974) Hepatic tyrosine aminotransferase (TAT) in stressed trouts and rats. Gen. comp. Endocrinol. 22, 388 (abstr.). OHISALO J. J. & PISPA J. P. (1975) Regulation of hepatic tyrosine aminotransferase in the frog Rana temporaria, Biochim. biophys. Acta 397, 94-100. REYNOLDS R. D., POTTER V. R. & PITOT H. C. (1971) Response of several hepatic adaptive enzymes to a shift from low to high protein diet in intact and adrenalectomized rats. J. Nutr. 101, 797-802. ROE J. H. & DAILEY R. E. (1966) Determination of glycogen with the Anthrone reagent. Analyt. Biochem. 15, 245-250. SOKAL R. R. & ROHLF F. J. (1966) Biometry. Freeman, San Francisco. STIMPSON J. H. (1965) Comparative aspects of the control of glycogen utilization in vertebrate liver. Comp. BiDchem. Physiol. 15, 187-197. STORER J. H. (1967) Starvation and the effects of cortisol in the goldfish (Carassius auratus L.). Comp. Biochem. Physiol. 20, 939-948. SZEPESI B. & FREEDLAND R. A. (1967) Alterations in the activities of several rat liver enzymes at various times after initiation of a high protein regimen. J. Nutr. 93, 301-306. VALTONEN T. (1974) Seasonal and sex-bound variation in the carbohydrate metabolism of the liver of the whitefish. Comp. Biochem. Physiol. 47A, 713-72. WmTING S. J. (1976) Control of hepatic tyrosine aminotransferase in brook trout (Salvelinus fontinalis Mitchill) by cortisol, estrogen and dietary factors. M.Sc. Thesis, University of New Brunswick, Fredericton, N.B., Canada. ZEITOUN I. H., TACK P. I., HALVER J. E. & ULDREY D. E. (1973) Influence of Salinity on protein requirements of rainbow trout (Salmo 9airdnerii) fingerlings. J. Fish. Res. Bd Can. 30, 1867-1873. ZIGMOND M. J., SHOEMAKERW. J., LARIN F. ~ WURTMAN R. J. (1969) Hepatic tyrosine transaminase rhythm: interaction of environmental lighting, food consumption and dietary protein content. J. Nutr. 98, 71-75.
EFFECT OF NUTRITIONAL FACTORS A N D CORTISOL ON TYROSINE AMINOTRANSFERASE ACTIVITY IN LIVER OF BROOK TROUT, SALVELINUS FONTINALIS MITCHILL S. J. WHITING* AND A. J. WIGGS Department of Biology, University of New Brunswick, Fredericton, New Brunswick, E3B 5A3, Canada
(Received 18 January 1977) Abstract--1. Starvation for over 50 days increased the activity of Tyrosine aminotransferase (TAT) from brook trout liver and decreased liver glycogen. 2. Cortisol (6-40mg/100g body wt) increased TAT activity, optimally 72hr post-injection; liver glycogen response to cortisol was variable while tissue water either increased or remained constant. 3. Fish fed high-protein/low-carbohydrate had greater TAT activity than fish fed low-protein/high carbohydrate. There was food-induced peak of activity indicating a "diurnal rhythm" analogous to that of rat TAT. 4. TAT activity in brook trout appears to be correlated with protein catabolism.
INTRODUCTION
In teleost fish, steroids identical to the glucocorticoids (i.e. cortisol and cortisone) are synthesized by the adrenal gland homologue (for review see Butler, 1973). Only recently have data been presented to suggest these steroids are involved in gluconeogenesis in fish (Butler, 1968; Freeman & Idler, 1973). In the mammal the demonstration of a cortisol-induced increase in tyrosine aminotransferase (L-tyrosine:2oxoglutarate aminotransferase, E.C. 2.6.1.5, TAT) activity has been considered evidence for a glucocorticoid action of cortisol. Failures to find a cortisolinduced increase in TAT in fish (Chan & Cohen, 1964 and Fellman et al., 1971) have led to the proposals that cortisol acts only as a mineralocorticoid in lower vertebrates (Chan & Cohen, 1964; Janssens, 1970) and that the TAT response to cortisol occurs only above the evolutionary level of amphibians (Nemeth & Jurani, 1974). However, activities of transaminases other than TAT increase with glucocorticoid treatment in fish. For example, the enzyme alanine aminotransferase is increased after cortisol injection in Salvelinusfontinalis (Mann, 1972; Freeman & Idler, 1973) and Carassius auratus (Storer, 1967). Dietary factors are also known to affect TAT and gluconeogenesis in mammals. Fasting produces increases in the enzymes of amino acid degradation in mammals (Freedland & Szepesi, 1971), including TAT (Goswami & Chatagner, 1966): No work has been reported on fish liver TAT after periods of starvation but both alanine aminotransferase (Storer, 1967; Mann, 1972; Creach & Serfaty, 1974) and aspartate aminotransferase (Larsson & Lewander, 1973; Creach & Serfaty, 1974) increase in starved fish. In mammals, high-protein diets increase the amount of glucose manufactured from amino acids (Freedland & Szepesi, 1971) and can induce TAT (Reynolds et al., 1971). * Present Address: Department of Nutrition, University of Guelph, Guelph, Ontario, Canada. c.B.p.58/2B F~
The cyclic intake of protein by ad libitum fed rats appears to be the cause of the diurnal rhythm in liver TAT (Francesconi et al., 1972). There are few reports of diet-induced increases in TAT in nonmammalian species. Ohisalo & Pispa (1975) found increased TAT in Rana temporaria fed a high-protein diet. Frog and tadpole TAT were found to exhibit diurnal rhythms (Lane et al., 1970). A study of tyrosine aminotransferase in brook trout (Salvelinus fontinalis Mitchill) under conditions known to stimulate gluconeogenesis in mammals, i.e. starvation, high-protein feeding, and cortisol treatment was undertaken with a view to evaluating TAT as a gluconeogenic enzyme. Secondarily evidence for the view that cortisol has glucocorticoid function in fish is presented.
MATERIALS AND METHODS
Animals Yearling brook trout (Salvelinusfontinalis Mitchill) were obtained from the Department of Environment Fish Hatchery, Florenceville, N.B., Canada. Fish were kept in aerated, 20gal flow-through tanks (61 tap water/rain). Photoperiod was adjusted to correspond to natural conditions. Water temperature varied seasonally; the lowest temperature, 9°C, occurred in December and the highest, 14.5°C, occurred in July. Fish were fed 15 g Purina Trout Chow pellets/100g body wt on alternate days at 1600hr (unless otherwise stated). Sampling took place at least 42 hr after the last feeding except when samples for the "diurnal rhythm" experiments were taken.
Samplin9 Fish were killed by a sharp blow on the head and body measurements were quickly taken and the liver and gonad removed. Liver was cut so that a central piece (0.25g) was placed in a cold homogenizing tube for TAT assay and a peripheral piece (0.05g) was placed in 3 ml 1N NaOH for glycogen determination. Epaxial muscle samples (ventral to the dorsal fin) were placed in tared vials for muscle water determination. 189
190
S. J. WHITING AND A. J. W]GGS
Injection procedures Fish were injected with cortisol or saline intramuscularly (i.m.) at one or more sites in the caudal area, no more than 0.1 ml per site. There was some leakage of injected material. Fish were injected 17 hr after their last feeding. Cortisol was injected as either hydrocortisone hemisuccinate (Sigma) in 0.72% NaCI or undiluted Solucortef (Upjohn). The amount injected is given as the free alcohol equivalent. There was some mortality when the highest dose (40 mg/100 g body wt cortisol) was given.
they are designated low protein/high-carbohydrate and high-protein/low-carbohydrate (respectively).
Statistics Comparisons were made using one-way analysis of variance tests (Sokal & Roll, 1969). Slopes of the lines of best fit for data of the starvation experiment run OctoberDecember were found by regression analysis. Differences between experimental and control means were considered significant if P < 0.05. Values are expressed as mean + standard error of the mean (S.E.M.).
T A T assay Liver for TAT assay was homogenized for 30 sec in 13 vol of cold (4°C) 0.15 M KCI (containing 10 -3 M EDTA and brought to pH 7.8 with 10N KOH). Homogenates were centrifuged at 28,000 g for 35 min at 4°C. The enzyme preparation consisted of 1 ml supernatant (28,000 g) diluted 1:1 with phosphate buffer (0.1 M potassium phosphate containing 10 -3 dithiothreitol, pH 7.7). Tyrosine aminotransferase activity was measured using the Modified Brigg's method (Canellakis & Cohen, 1956). The assay mix contained 48.4mole tyrosine, 5.55#mole ketoglutarate, 8.35/~mole pyridoxal-5'-phosphate (PLP) and 30pmole diethyldithiocarbamate. Reagents were added, in the specified order, to each assay tube incubating at 20°C: l m l of phosphate buffer; 0.5ml of 1.11 M ct-ketoglutarate and 16.7 mM PLP in 0.1 M dibasic potassium phosphate; 2 ml of 0.024 M tyrosine in 0.2 M dibasic potassium phosphate containing 0.48% 10N KOH. Duplicates of each enzyme preparation (0.2 ml) were added at time zero and each tube was gently inverted once to mix the contents. After 10rain at 20°C, 0.25 ml of 100% trichloracetic acid (TCA) was added to stop the reaction and each tube was mixed vigorously. Sample blanks were treated similarly except that the TCA was added before the enzyme preparation. All tubes were centrifuged for 10min at 2000 x g and 2.0ml portions of supernatant were transferred to tubes containing 1.0ml of 1~o monobasic potassium phosphate. After mixing, 1 ml of 3% ammonium molybdate (in 1.4 N HC1) was added and tubes were again mixed. After 30 min incubation at room temperature, absorbance at 850nm was read. p-Hydroxyphenylpyruvate (HPP) was substituted in the reaction for the enzyme and the relation of HPP concentration to OD 850 was found. Enzyme units were calculated using the following equation:
RESULTS AND DISCUSSION
Effect of starvation Starvation resulted in increased T A T activity in yearling b r o o k trout of two experiments (Figs 1 and 2). Unfortunately it was not possible to control all experimental factors and differences existed in the two studies. These variables--i.e, time of the year (fall vs spring), water temperature (9-11°C vs 11-14°C), genetic b a c k g r o u n d (stocks were unavoidably d i f f e r e n t ) ~ may account for the different patterns of response of T A T to starvation in the two experiments. Brook trout starved between October and December (Fig. 1) exhibited a gradual rise in T A T activity, during which time liver glycogen levels declined and remained low ( < 2.5mg/100mg). Fish starved between M a y and July exhibited high TAT activity only after 40 days of starvation, by which time liver glycogen was almost depleted ( < 1.0 mg/100 mg). These results contrast with mammalian work in which a starvation induced increase
8o iver0Yc°en \\
~ 4,0 8
TAT units = No. #moles HPP formed per gram liver (wet wt) per hr at 20°C. Brook trout hepatic TAT was specific for ct-ketoglutarate as the amino acceptor; transamination of tyrosine did not occur if an equal concentration of pyruvate or oxaloacetate was used in place of ct-ketoglutarate, Vm~xand the apparent K,, (tyrosine) were found to 50#mole/hr per g and 3.2 c 10- 3 M (respectively) and the pH optima was in the range 8-9. The apoenzyme of brook trout TAT was inactive. Removal of the coenzyme PLP was achieved by dialysis against 5% ct-ketoglutarate and TAT activity could not be restored even after the addition of 80#mole of PLP to the assay mix.
0-0 Starved
I
O~
-]-~
I
I
[
J
I
TAT
I
¢~ I 5c ~k
Other measurements Liver glycogen was measured by the method of Roe & Dailey (1966) and is expressed as mg/100 mg liver (wet wt). Per cent muscle water was found by drying epaxial muscle samples to a constant weight at 60°C. The following formula was used: 9/o water = (1.0 - (dry wt/wet wt)) x 100%, Two experimental diets, patterned after Zeitoun et al. (1973) contained 30 and 55% protein, 15 and 14% lipid, 34 and 3% carbohydrate and 5138 and 5403 calories/g;
M S'ta rved ~ . 0 Fed
I
Oci" I0
i
20
I
30 Days
J
40
I
50
i
60
Dec.
Fig. 1. Effect of starvation on liver glycogen concentration and tyrosine aminotransferase (TAT) activity in yearling brook trout sampled between October and December (water temperature decreased from 11 to 9°C). For each point n is given. Vertical bars represented ±S.E.M. Slope of starved TAT (1.012) significantly greater than the slope of fed TAT (0.286). Number of days of starvation given.
Effect of nutritional factors and cortisol
E
Table 1. Effect of feeding 2 isocaloric diets, high-protein/ low-carbohydrate and low-protein/high-carbohydrate, on TAT activity in brook trout fasted 4 days prior to feeding
6o
Amt Diet constituents (~)~ fedb Protein CHO Lipid Days (g)
>,
~o _J ~> g
2o
c--o Starved
"~
-
~ 5 _
o
; ~
5 150 --
~: t
30 55 30 55 30 55
34 3 34 3 34 3
15 14 15 14 15 14
3 3 8 8 lI 12
40 40 30 30 30 30
TAT activity" 91,3 ± 11.5(5)
98,0 +_ 16.5(5) 81.6 _+ 10.7(6) 121.3 ± 5.7(6)* 66.4 ± 6.0(6) 107.4 ± 11.5(5)*
" ~ Dry wt. b Amount fed/t00 g fish. c Moles HPP formed/hr per g liver at 20°C; mean + S.E.M. (n). *P < 0.01.
100
"~
191
50
/ =--o ~te~rved I
I
I
Io
30
40
= /
After 8 days of feeding the experimental diets, the fish fed the high protein diet had significantly inDays creased (P < 0.01) TAT activity compared to that in Fig. 2. Effect of starvation on liver glycogen concentration the fish fed the low protein diet (Table l). At 11 and and tyrosine aminotransferase (TAT) activity in yearling 12 days of feeding, TAT levels in both groups had brook trout sampled between May and July (water tem- declined from the 8-day values but TAT in the highperature increased from 11 to 14°C). For each point, n = 8 protein group (107 units) was still greater (P < 0.01) except where noted. Vertical bars represent +S.E.M. than TAT in the low-protein group (66 units). The Number of days of starvation given. reason for the decline is not known, although it is possible that the low-protein group may have experiin rat liver TAT occurs in 48 hr (Goswami & Cha- enced some carbohydrate repression of TAT as found tagner, 1966). Janssens (1970) suggested that in lower by Ohisalo & Pispa (1975) in glucose-fed frogs. Brook trout TAT activity was found to vary during vertebrates TAT activity and gluconeogenesis are normally at a high level and that little increase occurs the day. Although different feeding times (Fig. 3) were with starvation. In order to survive the long periods used in the two studies the peak of activity occurred of starvation inherent in their life-styles, fish would 7-8 hr after feeding in both studies. These peaks corbe expected to have protein sparing mechanisms to related with feeding time rather than photoperiod. In counteract excessive protein loss through catabolism. rats the peak of activity of liver TAT occurs 4 hr after Under these circumstances fish would not be expected feeding (Zigmond et al., 1969). The longer lag time of 8 hr observed in brook trout may reflect the lower to exhibit a major increase in TAT activity. The results (Figs 1 and 2) suggest that low liver metabolic rate in fish kept at low (11-13°C) temperaglycogen levels are correlated with increased protein tures. The influence of feeding and dietary protein on catabolism as indicated by increased TAT activity. The decline in liver glycogen of control fish of the TAT activity is suggestive of substrate induction. fall experiment (Fig. 1) may be a seasonal effect. Valtonen (1974) found similar seasonal variations in liver glycogen in whitefish. Older fish ( > 3 yr) did not show an increase in TAT activity even after 100 days of starvation at l l ° C (Whiting, 1976). These large trout, having relatively lower maintenance requirements than yearlings (Frost & Brown, 1967), may also have had different energy reserves than the smaller fish. For example, Stimpson (1965) found that young goldfish (Carassius auratus) depleted liver glycogen while old goldfish depleted liver lipid on starvation. I-
Moy
20
50
July
Effect of feedin 9 Fish were fed experimental diets, either a highprotein/low-carbohydrate diet or a low-protein/highcarbohydrate diet, after 4 days of food deprivation. Fish were initially fed 40g of their respective diet, per 100g (wet wt). After 3 days of feeding, TAT activities (measured 40 hr post-feeding) were compai'able in the two groups (Table 1). On the fourth day rations were reduced to 30 g/100 g as fish fed the lower level of protein were not consuming all their ration.
1600
0
0800
1600
T i m e of doy
Fig. 3. Daily variation in brook trout tyrosine aminotransferase (TAT) activity. Two groups of fish were fed either the normal ration (15 g/100g)or twice the normal ration, at the time indicated by their respective arrows. For each point n = 5 (normal ration) and n = 4 (2 x ration). Vertical bars represent ±S.E.M. Black bars represent time in darkness.
192
S.J. WHITINGAND A. J. WXGGS
Table 2. TAT activity, liver glycogen and ~ tissue water in brook trout injected with cortisol, in two experiments (July and September, 1975). Data expressed as mean 4- $.E.M. (n). Comparisons are made against saline-injected controls Treatment July 1975 : uninjected saline cortisol (40mgc) uninjected saline cortisol (40 mgc) uninjected saline cortisol (40 mgc) uninjected saline cortisol (40 mgc) Sept. 1975: uninjected saline cortisol (6 mgc) cortisol (12 mgc) cortisol (25 mgc) cortisol (40 mgc)
Hours post-injection
Hours post-feeding
24 24 24 48 48 48 72 72 72 96 96 96
42 42 42 66 66 66 90 90 90 114 114 114
57.4 _ 5.0(8)~ 90.0 4- 5.9 (8) 113.1 4- 7.1 (8)* 71.3 4- 3.6 (8) 88.4 4- 7.4 (8) 130.3 +_ 6.5 (8)~ 65.6 4- 6.9 (8)t 95.2 + 7.0 (8) 137.0 4- 13.4 (8)I" 65.0 4- 3.1 (8)* 96.2 4- 8.7 (8) 137.2 4- 12.0 (8)t
72 72 72 72 72 72
90 90 90 90 90 90
64.3 4- 3.4 (5) 71.4 4- 4.5 (6) 111.6 4- 6.4 (6)~ 132.7 4- 9.7 (6):~ 115.6 4- 2.9 (4)~: 127.4 4- 7.7 (6)$
TAT activity"
Liver glycogen b
~o Tissue water
4.9 4- 0.7 (8) 3.4 4- 0.5 (8) 1.6 4- 0.6(8)* 77.30 4- 0.65 (6)t 75.35 4- 0.28 (6) 78.17 4- 0.42 (6)t 4.4 4- 0.6 (8) 3.1 4- 0.5 (8) 2.9 4- 0.6 (8) 75.54 4- 0.40 (6) 75.70 4- 0.29 (6) 78.50 + 0.50 (6)t 4.2 4.3 4.4 5.4 2.8
4- 0.3 (6) 4- 0.6 (6) 4- 0.5 (6) 4- 0.2 (4)* 4- 0.6 (6)
77.41 4- 0.19 (6)
77.81 + 0.01 (6)
Moles HPP formed/hr per g liver at 20°C. b mg/100 mg liver. ¢ Dose/100 g fish. * P < 0.05. t P < 0.01. P < 0.001. Szepesi & Freedland (1967) have presented evidence that in the mammal that protein per se is not responsible. Diurnal rhythm studies have implicated quinolinic acid, a metabolite of tryptophan, as a possible control agent (Hardeland, 1973). Evidence also exists to exclude cortisol as regulating agent of cyclic changes in TAT. Effect o f cortisol Increase of brook trout TAT after cortisol treatment required a period of at least 24 hr, considerably longer than the response time of 5 hr seen in rat (Lin & Knox, 1957). On the other hand a single cortisol injection in brook trout produced a high level of TAT activity which lasted for several days (Table 2) while in rats, induction of TAT by cortisol (after intraperitoneal injection) lasts only several hours unless injection is repeated (Kenny et al., 1968). The prolonged effect in brook trout may be attributed in part to the intramuscular injection of hormone (which may have resulted in a slow absorption of cortisol into systemic circulation over time) as well as to the high dose used. The dose of cortisol used in this study to increase TAT in brook trout must be considered pharmacological. The lowest amount which caused a maximal increase in TAT activity was 6mg/100g body wt (Table 2). Other workers, using comparable concentrations in fish and amphibians, (Chan & Cohen, 1964; Fellman et al., 1971; Ohisalo & Pispa, 1975) were unable to increase TAT activity. Saline injected controls generally exhibited increased levels of TAT relative to uninjected controls. Although the increase was significant in some cases, it did not equal the effect of cortisol injections (Table
2). TAT in rats is known to be stimulated by a number of nonspecific agents provided an intact adrenal gland was present (Litwack & Diamondstone, 1962). It is possible that saline injection was acting as a nonspecific stressing agent. As liver glycogen deposition is considered a primary gluconeogenic action of glucocorticoids in mammals (cf. Glenn et al., 1961), it was surprising to find instances of significantly reduced levels of liver glycogen in cortisol-treated fish (Table 2). There may be a time-dependent, dose-dependent response operating since liver glycogen was significantly decreased at 24hr (40 mg/100 g) yet significantly increased at 72hr (25 mg/100g). (Table 2). Glycogen deposition and transaminase induction are the result of different effects of glucocorticoids. The activation of glycogen synthetase by cortisol is not obligatory for gluconeogenesis in mammals (DeWulf & Hers, 1967). A similar relationship could occur in fish explaining the apparent discrepancy. Cortisol injection in brook trout increased the water content of muscle (Table 2). Butler (1973) suggests that cortisol increases protein catabolism. In starved fish the catabolism of protein is correlated with increased tissue water (Love, 1970) so that the rise in tissue water may indicate a fall in tissue protein as a result of cortisol action. Cortisol treatment induces weight loss in brook trout (Freeman & Idler, 1973), Since cortisol increases tissue water the weight loss must indicate a loss of some other tissue component. In the brook trout as in the mammal TAT activity increases after feeding, in response to dietary protein, and during starvation. This correlation with conditions when protein catabolism is prevalent lends sup-
Effect of nutritional factors and cortisol port to the idea that T A T is involved in protein catabolism a n d gluconeogenesis in fish as well as m a m mals. The further observation of cortisol stimulation of T A T activity is consistent with the hypothesis a n d further suggest that cortisol acts as a glucocorticoid in fish as in mammals. REFERENCES
BUTLER D. B. (1968) Hormonal control of gluconeogenesis in the North American eel (Anguilla rostrata). Gen. comp. Endocrin. 10, 85-91. BUTLER D. G. (1973) Structure and function of the adrenal gland in fishes. Am. Zool. 13, 839-879. CANELLAglS Z. N. & COHEN P. P. (1956) Purification studies of tyrosine-~-ketoglutaric acid transaminase. J. biol. Chem. 222, 53-62. CHAN S-K. & COHEN P. P. (1964) A comparative study of the effect of hydrocortisone injection on tyrosine transaminase activity of different vertebrates. Archs Biochem. Biophys. 104, 335-337. CIVEN M., ULRICH R., TRIMMER B. M. & BROWN C. B. (1967) Circadian rhythms of liver enzymes and their relationship to enzyme induction. Science, N.Y. 157, 1563-1564. CREACH Y. & SEREATY A. (1974) Le jefine et la r6alimentation chez la carpe (Cyprinus carpio L.). J. Physiol., Paris 68, 245-260. DEWULF H. & HERS H. B. (1967) The stimulation of glycogen synthesis and of glycogen synthetase in the liver by glucocorticoids. Eur. J. Biochem. 2, 57-60. FELLMAN J. C., ROTH E. S. & FUJITA T. S. (1971) Study of tyrosine aminotransferase in developing salmon. Comp. Biochem. Physiol. 40B, 241-247. FRANCESCONI R. P., COTTER R. 8z MAGER M. (1972) Starvation and refeeding: effects on the periodicity of tryptophan and tyrosine metabolism in mice. J. Nutr. 102, 597-602. FREEDLAND R. A. & SZEPESI B. (1971) Control of enzyme activity: nutritional factors. In Enzyme Synthesis and Degradation in Mammalian Systems (Edited by RECHEIGL M.), pp. 103-140. University Park Press, Baltimore. FREEMAN H. C. & IDLER D. R. (1973) Effects of corticosteroids on liver transaminases in two salmonids, the rainbow trout (Salmo gairdnerii) and the brook trout (Salvelinus fontinalis). Gen. comp. Endocrinol. 20, 69-75. FROST W. E. & BROWN M. E. (1967) The Trout. N.M.N. Collins, London. GLENN E. M., BOWMAN B. J., BAYER R. B. & MEYER C. E. (1961) Hydrocortisone and some of its effects on intermediary metabolism. Endocrinol. 68, 386-410. GOSWAMI M. N. D. & CHATAGNER F. (1966) Starvationinduced adaptation of rat liver tyrosine transaminase and serine dehydrase. Experientia 22, 370-371. HARDELAND R. (1973) Diurnal variations in inducibility of hepatic tyrosine aminotransferase. Int. J. Biochem. 4, 35%364. JANSSENS P. A. (1970) The evolution of corticosteroid function. Steroidologia 1, 308-320.
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LANE E. A., MAVRIDESC. & D'IORIO A. (1970) A comparative study of the daily rhythm of liver tryosine aminotransferase in various vertebrates. Int. J. Biochem. 1, 343-348. LARSSON A. & LEWANDER D. (1973) Metabolic effects of starvation in the eel, Anguilla anguilla L. Comp. Biochem. Physiol. 44A, 367-374. LINE. C. C. & KNOX W. E. (1957) Adaptation of the rat liver tyrosine-ct-ketoglutarate transaminase. Biochim. biophys. Acta 26, 85-88. L1TWACK G. & DIAMONDSTONE T. I. (1962) Nonspecific stimulation of tyrosine-~-ketoglutarate transaminase activity in vivo. J. biol. Chem. 237, 469-472. LOVE R. M. (1970) The Chemical Biology of Fishes. Academic Press, New York. MANN O. E. (1972) The effect of starvation and cortisol on the glutamate-pyruvate transaminase of brook trout, Salvelinus fontinalis (Mitchill). B.Sc. Thesis, University of New Brunswick, Fredericton, N.B. Canada. NEMETH S. & JURANI M. (1974) Hepatic tyrosine aminotransferase (TAT) in stressed trouts and rats. Gen. comp. Endocrinol. 22, 388 (abstr.). OHISALO J. J. & PISPA J. P. (1975) Regulation of hepatic tyrosine aminotransferase in the frog Rana temporaria, Biochim. biophys. Acta 397, 94-100. REYNOLDS R. D., POTTER V. R. & PITOT H. C. (1971) Response of several hepatic adaptive enzymes to a shift from low to high protein diet in intact and adrenalectomized rats. J. Nutr. 101, 797-802. ROE J. H. & DAILEY R. E. (1966) Determination of glycogen with the Anthrone reagent. Analyt. Biochem. 15, 245-250. SOKAL R. R. & ROHLF F. J. (1966) Biometry. Freeman, San Francisco. STIMPSON J. H. (1965) Comparative aspects of the control of glycogen utilization in vertebrate liver. Comp. BiDchem. Physiol. 15, 187-197. STORER J. H. (1967) Starvation and the effects of cortisol in the goldfish (Carassius auratus L.). Comp. Biochem. Physiol. 20, 939-948. SZEPESI B. & FREEDLAND R. A. (1967) Alterations in the activities of several rat liver enzymes at various times after initiation of a high protein regimen. J. Nutr. 93, 301-306. VALTONEN T. (1974) Seasonal and sex-bound variation in the carbohydrate metabolism of the liver of the whitefish. Comp. Biochem. Physiol. 47A, 713-72. WmTING S. J. (1976) Control of hepatic tyrosine aminotransferase in brook trout (Salvelinus fontinalis Mitchill) by cortisol, estrogen and dietary factors. M.Sc. Thesis, University of New Brunswick, Fredericton, N.B., Canada. ZEITOUN I. H., TACK P. I., HALVER J. E. & ULDREY D. E. (1973) Influence of Salinity on protein requirements of rainbow trout (Salmo 9airdnerii) fingerlings. J. Fish. Res. Bd Can. 30, 1867-1873. ZIGMOND M. J., SHOEMAKERW. J., LARIN F. ~ WURTMAN R. J. (1969) Hepatic tyrosine transaminase rhythm: interaction of environmental lighting, food consumption and dietary protein content. J. Nutr. 98, 71-75.