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Epinephrine effect on glycogen phosphorylase activity in catfish liver and muscles
GENERAL AND COMPARATIVE
Epinephrine
ENDOCRINOLOGY
(1986)
Effect on Glycogen Phosphorylase Liver and Muscles
CELESTINA
Institute
61, 469-475
OTTOLENGHI,
of Gener-ul
Physiology.
A. CRISTINA PLJVIANI, AND LUIGI BRIGHENTI llni~emity
of Frrmrrr.
Activity
M. EMILIA
Via L. Borscrri
in Catfish
GAVIOLA,
46, 44100
Fermru.
Itu!\
Accepted September 8. 1985 In catfish, the percentage of the active (a) form of glycogen phosphorylase with respect to the total (a + b) form varied in control slices from about 90% to 6.54 and 20%. respectively for liver, red. and white muscles. Epinephrine added to the incubation medium of liver and white muscle slices caused a significant increase in the specific activity of phosphorylase
Inc
Metabolic response to hormones in fish is characterized by a slow, delayed, and long-lasting effect, when compared with mammals. Murat et al. (1981) think that this phenomenon cannot be explained by the simple temperature-related Arrhenius plot, and that it is an intrinsic property of lower vertebrates. They state that the hormonal regulation of metabolism is less critical in these animals, which consequently allows them to withstand better longlasting perturbation of the milieu inte’rieur. In previous studies on catfish we demonstrated that insulin effect in vivo on glycogen level and on blood glucose (Ottolenghi et al., 1982) lasted until 48th hr, and that epinephrine effect in vivo (Ottolenghi et al., 1984a), and in isolated and perfused liver (Ottolenghi et al., 1984b), continued for a long period (6 hr or more). The hyperglycemic effect of epinephrine in teleost fish is well documented by in viva experiments (Mazeaud, 1964; Young and Chavin, 196.5; Nakano and Tomlinson, 1967; Demael-Suard and Garin, 1970; Perrier et al., 1971; Murat and Serfaty, 1975; DeRoos and DeRoos, 1978; Ottolenghi et al., 1984a); some authors found that the increase in blood glucose is accompanied by
a decrease in hepatic glycogen concentration (Stimpson, 1965; Demael-Suard and Garin, 1970; Ottolenghi et al., 1984a), but others (Kumar et al., 1966: Young, 1968) did not observe any change in the polysaccharide level. Differences of hormonal effect on fish glycogen metabolism have also been observed in in vitro experiments, and it has been pointed out that they are related to fish species. In fact, Birnbaum et al. (1976) have shown that hepatocytes isolated from goldfish (Carassius auratus) respond to epinephrine treatment by increasing glucose release to the medium via the glycogenolytic pathway. Morata et al. (1982), on the other hand, found that liver slices of rainbow trout (Salmo gairdneri) respond to epinephrine treatment by increasing glucose release to the medium during incubation with no decrease, and sometimes with an increase in the glycogen content. On the basis of glycogen phosphorylase activity found in catfish liver slices (Ottolenghi et al., 1984b). we suggested that the effect of epinephrine in viva and in vitro on glycogen breakdown and on glucose output (Ottolenghi et ml., 1984a) depends on a phosphorolytic mechanism. Glycogen phosphorylase is also present
469 0016-6480186
$1.50
Copyright 0 1986 by Academic Press. Inc. All rights of reproduction in an) form resewed
470
OTTOLENGHI
in muscles of vertebrates. In fish the muscular tissue forms a larger part of the body mass than in other vertebrates (Bone, 1978), and catecholamines are liberated under stress, during attack escape reaction, and during hard muscular exercise (Nakano and Tomlinson, 1967). Therefore in the present work we have extended the study to cover the effect of epinephrine on phosphorylase in both white and red muscles, and have compared the effect with that obtained on liver. MATERIALS
AND METHODS
Animals. Adult catfish (Icfctlurus me/us), weighing approximately 100-200 g, were purchased from a local dealer. Fish were transferred to the laboratory storehouse, and placed in a group of 10 in tanks containing 200 liter of well-aerated dechlorinated and continuously depurated tap water, at the environmental temperature (18-24”). Animals were starved for some days until the experiment. Treatment. Fish were lightly anaesthetized with chloretone (1. I, I-trichlor-2-methyl-2-propanol. 60 ml of a saturated solution in 1 liter water). Liver and lateral white and red muscle slices weighing 100-200 mg. and of about 0.5 mm thickness. were obtained with a Stadie Riggs apparatus. The slices were placed in 25-ml Erlenmeyer flasks containing 3 ml of teleost Ringer solution. pH 7.4, in the presence or in absence of epinephrine bitartrate (S pgiml, corresponding to 1.7 x IO- 5 M, unless otherwise indicated); flasks were aerated with a OZ/C02, 95:s (v/v) gas mixture. and incubated in a shaking bath at the environmental temperature for 1 hr. Other slices were immediately homogenized in 10 vol of ice-cold buffer containing TABLE EFFEU
OF EPINEPHRINE
ON GLYCOGEN
ET AL.
Tris 0.05 M; EDTA 0.005 M; NaF 0.05 M; 2-mercaptoethanol 0.04 M; serum albumin 1 mgiml, pH 6.8. At the end of incubation, the tissue slices were blotted dry and homogenized in the same buffer. Other slices were used for glycogen determinations. Analysis. For glycogen phosphorylase activity all homogenates were centrifuged for 5 min at 5000 rpm in a refrigerated centrifuge and the enzyme assay was made on the supernatant in absence or in presence of 2 mM AMP for n and total (a + h) form respectively. as described by Morata ef ul. (1982). The mixture was incubated at 30” for 10 min, then 4 ml of ice-cold 10% trichloracetic acid was added; the protein precipitate was centrifuged at 5000 rpm for 5 min, and removed. The inorganic phosphate was determined on I ml of supernatant using the Fiske and SubbaRow (1925) method. The specific activity of hepatic and muscle glycogen phosphorylase was expressed as micromoles phosphate per gram of fresh tissue per minute. Statistical analysis was performed by the paired Student’s t test.
RESULTS
The epinephrine effect on liver glycogen phosphorylase activity in catfish is shown in Table 1. The table indicates that there was little, but significant difference between the total and the active a form. Incubation of control liver slices resulted in a significant decline in both the phosphorylase activities. When epinephrine was added to the incubation medium, the activity of phosphorylase u increases. The increase was always significantly greater in those experiments in which the enzyme activity declined the most in control, as in1
PHOSPHORYLASE
ACTIVITY
IN LIVER SLICES OF CATFISH
Enzyme Time (min)
-AMP
test
+ AMP pmol
- AMP/
+ AMP
(A) Control (B) Control (C) Epinephrine
0 60
12.88 7.03
2 0.66 i 0.63**
Pig fresh tissueimin 13.95 2 0.66* 9.12 2 0.79*.*”
0.922 0.770
t t
0.120 0.017t
(5 &ml)
60
10.19
t
II .40 t
0.894
t
0.0153
0.67**.**”
0.73”.**.***
Note. Mean values ? SEM of 16 experiments. Levels of significance (paired Student’s t test): *P < 0.01 with at 0 and 60 min. respect to values in the absence of AMP; ** and ***P < 0.01 with respect to values of control respectively; i and $P < 0.01 with respect to ratios of control at 0 and 60 min, respectively. Correlation coeffiand r = -0.643, respeccient between the values of percentage variation B/A versus C/B were: r = -0.669 tively, for the form (I (~ AMP) and the total form ( + AMP) of the enzyme, with levels of significance of P i 0.01.
EPINEPHRINE
ON CATFISH
dicated by the correlation coefficients (Table 1). In tive different experiments the level of epinephrine was gradually increased from 0.001 to 5 kg/ml (3.5 x lO-9 to 1.7 X 10-j M) in the incubation medium. Figure 1 shows that the effect was still present when the hormone level was 0.01 pgiml, but ceased at the epinephrine concentration of 0.001 pg/ml. To verify if the epinephrine effect was related to an inhibition of phosphorylase inactivation, or to an activation of the enzyme, we tested the phosphorylase a activity in liver slices ever 1.5 min for 1 hr. At the 30th min epinephrine was added. Figure 2 shows that in control slices the enzyme activity decreased until the 30th min of incubation, then it remained constant: the epinephrine addition at the 30th min increased the enzyme activity by about 60%. Table 2 shows the effect of epinephrine (5 kg/ml) on phosphorylase n and total form
EPINEPHRINE
(us/ml)
1. Dose effect of epinephrine on the active form of glycogen phosphorylase. Each curve represents one experiment. The enzyme activity was tested after 60 min incubation. FIG.
471
PHOSPHORYLASE 15
r ---
i
CONTROL EPINEPHRINE
EPINEPHRINE ADDITION
L
i
0
30 TIME
60
(min)
FK,. 2. Time-course of glycogen phosphorylase (I activity and the effect of epinephrine. Epinephrine (5 kg/ml) was added after 30 min incubation. Mean values t SEM of four experiments. Level of significance (paired Student’s t test): l P < 0.05: “P i 0.01 with respect to zero time: *P < 0.05 with respect to correspondent control.
in white muscle slices. Unlike the liver, the white muscle phosphorylase N form was much lower than the total form. Incubation of white muscle slices decreased the phosphorylase a activity by about 50% with respect to the same form of enzyme found at zero time. Epinephrine significantly enhanced the phosphorylase (1 activity in white muscle slices compared with the correspondent control slices activity. The total form of the enzyme was slightly. but significantly reduced by incubation and epinephrine had no effect on this decrease. The level of phosphorylase activity in red muscle slices was greater than in white muscle, chiefly in the active form. which represents about two-thirds of the total activity (Table 3). Incubation decreased the activity of the (1 form only, and the addition
472
OTTOLENGHI
ET AL.
TABLE 2 EFFECT OF EPINEPHRINE
(A) Control (B) Control (C) Epinephrine (5 d-nl)
ON GLYCOGEN
PH~SPHORYLASE
ACTIVITY
IN WHITE
MUSCLE SLICES OF CATFISH
Enzyme test
Time (min)
-AMP
+ AMP
- AMP/ + AMP
0 60
2.69 k 0.24 1.18 k 0.22***
pmol P,ig fresh tissueimin 14.02 L 0.61;” 12.36 k 0.51”,**”
0.195 t 0.016 0.101 t 0.020$
60
1.96 t 0.45**.t
12.75 k 0.48’
0.159 2 0.033
Note. Mean values t SEM of nine experiments. Levels of significance (paired Student’s I test): *P < 0.01 with respect to values in the absence of AMP; ** and ***P < 0.05 and P < 0.01 with respect to values of control at 0 min; tP < 0.05 with respect to values of control at 60 min; SF’ < 0.01 with respect to ratio of control at 0 min.
of epinephrine to incubation medium had no effect on either form of the enzyme.
samples, could be greater than in viro animal, and that the decrease observed in control samples during incubation, takes DISCUSSION place owing the disappearance of catecholIn a previous study (Ottolenghi et al., amine from the system. Janssens et al. 1984b), we found that in catfish liver slices (1983) obtained constant phosphorylase LI the level of enzyme activity at zero time is activation during 60-min incubation, using higher than that found in 60 min incubated in vitro cultures of amphibian liver. Under control for both active and total phosphorthese experimental conditions the zero ylase forms. We have now found the same time samples are well washed, so that any pattern in red and white muscle slices. A catecholamines present are removed. Exsimilar decrease, following incubation, was periments on isolated and well-washed catalso observed by Umminger and Benziger fish hepatocytes (unpublished data) show in brown bullhead liver slices (197.5). The that during incubation, the enzyme activity stress of handling and killing animals may of control never decreases, but it remains constant or sometimes increases. cause a local release of catecholamines As far as epinephrine effect is conwhich would stimulate the activation of phosphorylase. If this is the case, it is pos- cerned, Sutherland (1956) stated that, in sible that the high level of glycogen phos- mammals, epinephrine does not increase phorylase activity found in zero time the enzyme activity originally present in TABLE EFFECT OF EPINEPHRINE ON GLYCOGEN
3
PHOSPHORYLASE ACTIVITY
Enzyme test
Time (min)
-AMP
0 60
11.22 2 0.92 5.48 2 0.82**
60
5.76 2 0.91**
+ AMP pmol
(A) Control (B) Control (C) Epinephrine (5 t&ml)
IN RED MUSCLE SLICES OF CATFISH
-AMP/
+ AMP
P,ig fresh tissueimin 16.42 rt 0.70* 15.82 + 0.70*
0.682 -t 0.039 0.343 t 0.039***
15.88 k 0.64*
0.360 k 0.053***
Note. Mean values 2 SEM of seven experiments. Levels of significance (paired Student’s t test): *P < 0.01 with respect to values in the absence of AMP; **P < 0.01 with respect to values of control at 0 min; ***P < 0.01 with respect to ratio of control at 0 min.
EPINEPHRINE
ON CATFISH
the tissue, but rather prevents or retards the decrease of enzyme activity seen in the control. However, under our experimental conditions epinephrine seems to activate enzyme activity, as shown in Fig. 2. AMP increases the active form of glycogen phosphorylase in mammals (Sutherland and Wosilait, 1956; Verdetti and Piery, 197O), in frog (Piery and Grably, 1969), and in fish (Nagayama, 1961: Yamamoto. 1968: Umminger and Benziger, 197.5). The ratio of phosphorylase activity without AMP to phosphorylase with AMP represents the percentage of total phosphorylase present in the active form. In control liver (Table I) we found that at zero time and after 60 min incubation, 92 and 77%, respectively, of total phosphorylase was present in the active form. In white muscle the pattern was completely different, the active form being only 19% of total form at zero time, and 10% after 60-min incubation. The ratio between a and total form of enzyme activity in red muscle was 68% at zero time control and 34% after incubation. ATP competes with AMP at the same phosphorylase site, and its binding prevents the binding of AMP by failing to induce the activating conformation change: hence ATP is considered as an inhibitor (White et al., 1973). On the other hand, ATP/AMP ratios represent the amount of energy available to the liver tissue. If this assumption is true for catfish liver too, where high level of phosphorylase u is present, then it is possible that glycogen breakdown may occur even if the liver depends to a lesser extent than other tissues on glucose metabolism for its energy requirements. Then the glucose could enter the blood stream and not be burned in the liver for ATP formation. In white muscle the conditions are completely different. The active form of phosphorylase was very low (19% of the total form). White muscle is used up by fish during rapid swimming (Bone, 1966), and usually this tissue utilizes efficient and eco-
PHOSPHORYLASE
473
nomical metabolites such as glucose, although it is well known that the free fatty acids derived from triglycerides are also important fuel source for energy metabolism in fish (Driedzic and Hochachka, 1978). The red muscle, which represents about S-20% of the total muscle mass, consists of aerobic slow fibers. In this tissue the level of glycogen phosphorylase active form, both at zero time and in 60 min incubated control slices, was between the values found in liver and in white muscle. The level of glycogen was also higher (about five times) than that found in white muscle. In a previous work (Ottolenghi et al., 1983). glucose-6-phosphatase activity was seen to be present in red muscle, whereas it was negligible in white muscle. Taking into account this fact, together with the high level of glycogen phosphorylase active form in red muscle in controls, it could follow that the red muscle may contribute to general metabolism by supplying glucose to environment. In white muscle epinephrine enhanced the active form, while the total form of the enzyme was practically unaffected. Epinephrine release takes place under hard exercise (Nakano and Tomlinson, 1967). so our results, although obtained under different experimental conditions, are comparable to those of Morata et al. (1982). who found similar results in rive after stress. Driedzic and Hochachka (1976. 1978) studying the stress conditions caused by strenuous exercise. found that ATP concentration in white muscle was reduced by about 65% in fatigued animals: ADP level also decreased by a small but significant amount. However, AMP concentration remained low and unchanged, and there was an increase in IMP concentration following the action of S’-AMP-deaminase. Thus the total free adenylate pool decreased, and both ATP/ADP and ATP/AMP ratios also diminished. In the light of these findings our results on the phosphorylase activity in
474
OTTOLENGHI
white muscle, could indicate that in the presence of the hormone, as under stress, the decrease in ATP/AMP ratio, and the increase in IMP concentration cause the phosphorylase inactive b form to be converted to active a form. Both forms of glycogen phosphorylase in red muscle were unaffected by epinephrine, which does not even change the glycogen level in this tissue. As far as the dose of epinephrine is concerned, Janssens et al. (1983) reported that in amphibian liver the maximal activity of epinephrine was seen at 10-h M, while 10~” M was without effect. We found that the effect of epinephrine in catfish liver slices was present from a concentration of 3.5 x 1O-8 M, which comes close to the hormone level after stress in some fish (Nakano and Tomlinson, 1967; Dashow et al., 1982; Plisetskaya et al., 1984). In conclusion, results of our experiments could explain the events occurring in carbohydrate metabolism on catfish in the presence of epinephrine. The hormone promotes hyperglycemia chiefly by activation of glycogenolysis in liver, although an activation of gluconeogenetic processes cannot be excluded. The main pathway of glycogen breakdown is the phosphorolytic process, even if other enzymatic mechanisms, such as the amylolytic activity are possibly involved (Plisetskaya et al., 1973; Murat, 1976; Picukans and Umminger, 1979). Epinephrine increases the glucose release from the liver, where the levels of glycogen and glycogen phosphorylase active form are high even under normal conditions. The red muscle has a relatively high level of glycogen and of glycogen phosphorylase activity, and could supply glucose to the blood since a some glucose6-phosphatase activity is present in this muscle. But in red muscle, unlike in liver, epinephrine does not increase glycogen breakdown, probably because this tissue is not involved in quick swimming. In white muscle, low level of the a form of phos-
ET AL.
phorylase and of glycogen are present, so that under rest conditions only a small amount of glycogen is broken down in this muscle. Under stress conditions and in rapid swimming the released epinephrine significantly increases the active form of phosphorylase, so that in the white muscle its own glucose becomes available together with glucose from the liver. ACKNOWLEDGMENT This work was partially supported by a grant for Scientific Research (40%) from Minister0 della Pubblica Istruzione (1983).
REFERENCES Birnbaum, M. J., Shultz, J., and Fain. J. N. (1976). Hormone stimulated glycogenolysis in isolated goldfish hepatocytes. Amer. J. Physiol. 231, 191-197. Bone, Q. (1966). On the function of the two types of myotomal muscle fiber in elasmobranch fish. J. Mar.
Biol.
Ass.
46, 321-349.
Bone, Q. (1978). Locomotor muscle. In “Fish Physiology” (Hoar, W. S., and Randall, D. J., eds.), vol. VII, pp. 361-424. Academic Press. New York. Dashow, L., Epple A., and Nibbio, B. (1982). Catecholamines in adult lampreys: baseline levels and stress induced changes with a note on cardiac cannulation. Gen. Comp. Endocrinol. 46, 500-504. Demael-Suard, A., and Garin, D. (1970). L’interaction entre I’adrtnaline et I’insuline dans la regulation du metabolisme glucidique de la tanche (Tincu tinca). C.R. Seances Sot. Biol. 164, 1505-1510. DeRoos, R., and DeRoos, C. C. (1978). Elevation of plasma glucose levels by catecholamines in Elasmobranch fish. Gen. Comp. Endocrinol. 34, 447-452. Driedzic. W. R.. and Hochachka, P. W. (1976). Control of energy metabolism in carp white muscle. Amer.
J. Physiol.
230, 578-582.
Driedzic, W. R., and Hochachka, P. W. (1978). Metabolism in fish during exercise. In “Fish Physiology” (Hoar, W. S., and Randall, D. J., eds.), vol. VII, pp. 503-543, Academic Press, New York. Fiske, C. H., and SubbaRow, Y. (1925). The colorimetric determination of phosphorus. J. Biol. Chem.
66, 375-400.
Janssens, P. A., Caine, A. G., and Dixon. J. E. (1983). Hormonal control of glycogenolysis and mechanism of action of adrenaline in amphibian liver in vitro.
Gen.
Comp.
Endocrinol.
49, 477-484.
EPINEPHRINE
Kumar, M., Sundararaj, B. I., Narasimkam, P. V., Prasad, M. R. N., and Venkitasubramanian, T. A. (1966). Effect of hormones on liver and saccus vasculosus in Notopterus notopterus (Teleostei). Indian J. Exper. Biol. 4, 4-6. Mazeaud, F. (1964). Vitesse de production de l’hyptrglycemie adrtnalinique en fonction de la tbmperature chez la carpe. Intensite de la response en fonction de la dose d’hormone. C.R. Seances Sot.
Biol.
158, 36-40.
Morata, P., Fans, M. J., Perez Palomo, M., and Sanchez Medina, F. (1982). Effect of stress on liver and muscle glycogen phosphorylase in rainbow trout (Salmo gairdneri). Comp. Biochem. Physiol.
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421-425.
Murat, J-C. (1976). Studies on glycogenolysis in carp liver: Evidence for an amylase pathway for glycogen breakdown. Comp. Biochem. Physiol. SSB, 461-465. Murat, J-C., and Serfaty, A. (1975). Effects de l’adrenaline, du glucagon et de l’insuline sur le metabolisme glucidique de la carpe. C.R. Seances Sot. Biol.
169, 228-232.
Murat, J-C., Plisetskaya, E. M., and Woo, N. Y. S. (1981). Endocrine control of nutrition in cyclostomes and fish. Camp. Biochem. Physiol. 68A, 149-
158.
Nagayama, E (1971). Enzymatic studies on the glycolysis in fish muscle. 1. Activity of phosphorylase. Bull.
Jap.
Sot.
Sci. Fish.
23, 260-264.
Nakano, Y., and Tomlinson, N. (1967). Catecholamines and carbohydrate concentration in rainbow trout (Salmo gairdneri) in relation to physical disturbance. J. Fish Res. Bd. Canad. 24, 1701-1715. Ottolenghi, C., Puviani, A. C., Baruffaldi, A., and Brighenti, L. (1982). In vivo effects of insulin on carbohydrate metabolism of catfish (Ictalurus melas).
Camp.
Biochem.
Physiol.
72A,
35-41.
Gttolenghi, C., Puviani, A. C., Baruffaldi, A., and Brighenti, L. (1983). Glucose-6-phosphatase and carbohydrate metabolism in muscles and liver of catfish (Ictalurus me/as). Mol. Physiol. 4, 255-264. Ottolenghi, C., Puviani, A. C., Baruffaldi, A., and Brighenti, L. (1984a). Epinephrine effect on carbohydrate metabolism in catfish (Ictaiurus melas).
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Camp.
Endocrinol.
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ON CATFISH PHOSPHORYLASE
55, 378-386.
Gttolenghi, C., Puviani, A. C., Gavioli, M. E., and Brighenti, L. (1984b). Epinephrine effect on carbohydrate metabolism in isolated and oerfused catfish liver. Gen. Comp. Endocrinol. 59, 219-229. Perrier, H., Penier, C., and Gars, J. (1971). Etude de l’hyperglyctmie adrenalinique chez la truite arc-
en-ciel d’elevage (Salmo gairdneri), action des substances adrtnolitiques. C.R. Seances Sot. Biol. 165, 2141-2144. Picukans, I., and Umminger, B. L. (1979). Comparative activities of glycogen phosphorylase and amylase in liver of carp (Cyprinus carpio) and goldfish (Carassius auratus). Comp. Biochem. Physiol.
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455-457.
Piery, Y., and &ably, S. (1969). Phosphorylase du coeur de grenouille: variation d’activite induite par l’adrenaline et le travail. C.R. Seances SW. Biol.
136, 870-872.
Plisetskaya, E. M., Rich, A. A., Dickoff, W. W., and Gorbman, A. (1984). A study of triiodothyronine-catecholamine interaction: Their effect on plasma fatty acids in Pacific hagfish (Eptatretus stout;).
Camp.
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78A,
767-772.
Plisetskaya, E. M., and Zheludkova, Z. P. (1973). The effect of epinephrine on amylase activity in the liver and muscles of the lamprey (Lampetra fluviatilis). J. Evol. Biochem. Physiol. (in Russian). 9, 611-613. Stimpson, J. H. (1965). Comparative aspects of the control of glycogen utilization in vertebrate liver. Camp.
Biochem.
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15, 187-
197.
Sutherland, E. W. (1956). Hormonal regulatory mechanisms. In “Proceedings of the 3rd Intern. Congr. Biochem.“, pp. 318-327, Academic Press, New York. Sutherland, E. W.. and Wosilait, W. D. (1956). The relationship of epinephrine and glucagon to liver phosphorylase. 1. Liver phosphorylase preparation and properties. J. Biol. Chem. 218, 459-468. Umminger, D. L., and Benziger, D. (1975). In vitro stimulation of hepatic glycogen phosphorylase activity by epinephrine and glucagon in the brown bullhead (Ictalurus nebolosus). Gen. Comp. Endocrinol. 25, 96-104. Verdetti, J., and Piery, Y. (1970). Increase in heart glycogen phosphorylase b under the effect of adrenaline. J. Physiol. (Paris) 62 (Suppl. 3). 460. White, A., Handler, P., and Smith, E. L. (1973). fn “Principles of Biochemistry,” McGraw-Hill, Kogakusha, p. 472. Yamamoto, M. (1968). Fish muscle glycogen phosphorylase. Canad. J. Biochem. 46, 423-432. Young, J. E. (1968). “Hormonal controls of serum glucose levels and glycogen stores in goldfish (Carassius auratus).” Ph.D. dissertation, Wayne State University, Detroit. (quoted by Umminger, D. L., and Benziger, D., 1975). Young, J. E., and Chavin, W. (1965). Effects of glucose, epinephrine or glucagon upon serum glucose levels in goldfish (Carassius aurarus). Amer. Zooi. 5, 688-689.
Epinephrine
ENDOCRINOLOGY
(1986)
Effect on Glycogen Phosphorylase Liver and Muscles
CELESTINA
Institute
61, 469-475
OTTOLENGHI,
of Gener-ul
Physiology.
A. CRISTINA PLJVIANI, AND LUIGI BRIGHENTI llni~emity
of Frrmrrr.
Activity
M. EMILIA
Via L. Borscrri
in Catfish
GAVIOLA,
46, 44100
Fermru.
Itu!\
Accepted September 8. 1985 In catfish, the percentage of the active (a) form of glycogen phosphorylase with respect to the total (a + b) form varied in control slices from about 90% to 6.54 and 20%. respectively for liver, red. and white muscles. Epinephrine added to the incubation medium of liver and white muscle slices caused a significant increase in the specific activity of phosphorylase
Inc
Metabolic response to hormones in fish is characterized by a slow, delayed, and long-lasting effect, when compared with mammals. Murat et al. (1981) think that this phenomenon cannot be explained by the simple temperature-related Arrhenius plot, and that it is an intrinsic property of lower vertebrates. They state that the hormonal regulation of metabolism is less critical in these animals, which consequently allows them to withstand better longlasting perturbation of the milieu inte’rieur. In previous studies on catfish we demonstrated that insulin effect in vivo on glycogen level and on blood glucose (Ottolenghi et al., 1982) lasted until 48th hr, and that epinephrine effect in vivo (Ottolenghi et al., 1984a), and in isolated and perfused liver (Ottolenghi et al., 1984b), continued for a long period (6 hr or more). The hyperglycemic effect of epinephrine in teleost fish is well documented by in viva experiments (Mazeaud, 1964; Young and Chavin, 196.5; Nakano and Tomlinson, 1967; Demael-Suard and Garin, 1970; Perrier et al., 1971; Murat and Serfaty, 1975; DeRoos and DeRoos, 1978; Ottolenghi et al., 1984a); some authors found that the increase in blood glucose is accompanied by
a decrease in hepatic glycogen concentration (Stimpson, 1965; Demael-Suard and Garin, 1970; Ottolenghi et al., 1984a), but others (Kumar et al., 1966: Young, 1968) did not observe any change in the polysaccharide level. Differences of hormonal effect on fish glycogen metabolism have also been observed in in vitro experiments, and it has been pointed out that they are related to fish species. In fact, Birnbaum et al. (1976) have shown that hepatocytes isolated from goldfish (Carassius auratus) respond to epinephrine treatment by increasing glucose release to the medium via the glycogenolytic pathway. Morata et al. (1982), on the other hand, found that liver slices of rainbow trout (Salmo gairdneri) respond to epinephrine treatment by increasing glucose release to the medium during incubation with no decrease, and sometimes with an increase in the glycogen content. On the basis of glycogen phosphorylase activity found in catfish liver slices (Ottolenghi et al., 1984b). we suggested that the effect of epinephrine in viva and in vitro on glycogen breakdown and on glucose output (Ottolenghi et ml., 1984a) depends on a phosphorolytic mechanism. Glycogen phosphorylase is also present
469 0016-6480186
$1.50
Copyright 0 1986 by Academic Press. Inc. All rights of reproduction in an) form resewed
470
OTTOLENGHI
in muscles of vertebrates. In fish the muscular tissue forms a larger part of the body mass than in other vertebrates (Bone, 1978), and catecholamines are liberated under stress, during attack escape reaction, and during hard muscular exercise (Nakano and Tomlinson, 1967). Therefore in the present work we have extended the study to cover the effect of epinephrine on phosphorylase in both white and red muscles, and have compared the effect with that obtained on liver. MATERIALS
AND METHODS
Animals. Adult catfish (Icfctlurus me/us), weighing approximately 100-200 g, were purchased from a local dealer. Fish were transferred to the laboratory storehouse, and placed in a group of 10 in tanks containing 200 liter of well-aerated dechlorinated and continuously depurated tap water, at the environmental temperature (18-24”). Animals were starved for some days until the experiment. Treatment. Fish were lightly anaesthetized with chloretone (1. I, I-trichlor-2-methyl-2-propanol. 60 ml of a saturated solution in 1 liter water). Liver and lateral white and red muscle slices weighing 100-200 mg. and of about 0.5 mm thickness. were obtained with a Stadie Riggs apparatus. The slices were placed in 25-ml Erlenmeyer flasks containing 3 ml of teleost Ringer solution. pH 7.4, in the presence or in absence of epinephrine bitartrate (S pgiml, corresponding to 1.7 x IO- 5 M, unless otherwise indicated); flasks were aerated with a OZ/C02, 95:s (v/v) gas mixture. and incubated in a shaking bath at the environmental temperature for 1 hr. Other slices were immediately homogenized in 10 vol of ice-cold buffer containing TABLE EFFEU
OF EPINEPHRINE
ON GLYCOGEN
ET AL.
Tris 0.05 M; EDTA 0.005 M; NaF 0.05 M; 2-mercaptoethanol 0.04 M; serum albumin 1 mgiml, pH 6.8. At the end of incubation, the tissue slices were blotted dry and homogenized in the same buffer. Other slices were used for glycogen determinations. Analysis. For glycogen phosphorylase activity all homogenates were centrifuged for 5 min at 5000 rpm in a refrigerated centrifuge and the enzyme assay was made on the supernatant in absence or in presence of 2 mM AMP for n and total (a + h) form respectively. as described by Morata ef ul. (1982). The mixture was incubated at 30” for 10 min, then 4 ml of ice-cold 10% trichloracetic acid was added; the protein precipitate was centrifuged at 5000 rpm for 5 min, and removed. The inorganic phosphate was determined on I ml of supernatant using the Fiske and SubbaRow (1925) method. The specific activity of hepatic and muscle glycogen phosphorylase was expressed as micromoles phosphate per gram of fresh tissue per minute. Statistical analysis was performed by the paired Student’s t test.
RESULTS
The epinephrine effect on liver glycogen phosphorylase activity in catfish is shown in Table 1. The table indicates that there was little, but significant difference between the total and the active a form. Incubation of control liver slices resulted in a significant decline in both the phosphorylase activities. When epinephrine was added to the incubation medium, the activity of phosphorylase u increases. The increase was always significantly greater in those experiments in which the enzyme activity declined the most in control, as in1
PHOSPHORYLASE
ACTIVITY
IN LIVER SLICES OF CATFISH
Enzyme Time (min)
-AMP
test
+ AMP pmol
- AMP/
+ AMP
(A) Control (B) Control (C) Epinephrine
0 60
12.88 7.03
2 0.66 i 0.63**
Pig fresh tissueimin 13.95 2 0.66* 9.12 2 0.79*.*”
0.922 0.770
t t
0.120 0.017t
(5 &ml)
60
10.19
t
II .40 t
0.894
t
0.0153
0.67**.**”
0.73”.**.***
Note. Mean values ? SEM of 16 experiments. Levels of significance (paired Student’s t test): *P < 0.01 with at 0 and 60 min. respect to values in the absence of AMP; ** and ***P < 0.01 with respect to values of control respectively; i and $P < 0.01 with respect to ratios of control at 0 and 60 min, respectively. Correlation coeffiand r = -0.643, respeccient between the values of percentage variation B/A versus C/B were: r = -0.669 tively, for the form (I (~ AMP) and the total form ( + AMP) of the enzyme, with levels of significance of P i 0.01.
EPINEPHRINE
ON CATFISH
dicated by the correlation coefficients (Table 1). In tive different experiments the level of epinephrine was gradually increased from 0.001 to 5 kg/ml (3.5 x lO-9 to 1.7 X 10-j M) in the incubation medium. Figure 1 shows that the effect was still present when the hormone level was 0.01 pgiml, but ceased at the epinephrine concentration of 0.001 pg/ml. To verify if the epinephrine effect was related to an inhibition of phosphorylase inactivation, or to an activation of the enzyme, we tested the phosphorylase a activity in liver slices ever 1.5 min for 1 hr. At the 30th min epinephrine was added. Figure 2 shows that in control slices the enzyme activity decreased until the 30th min of incubation, then it remained constant: the epinephrine addition at the 30th min increased the enzyme activity by about 60%. Table 2 shows the effect of epinephrine (5 kg/ml) on phosphorylase n and total form
EPINEPHRINE
(us/ml)
1. Dose effect of epinephrine on the active form of glycogen phosphorylase. Each curve represents one experiment. The enzyme activity was tested after 60 min incubation. FIG.
471
PHOSPHORYLASE 15
r ---
i
CONTROL EPINEPHRINE
EPINEPHRINE ADDITION
L
i
0
30 TIME
60
(min)
FK,. 2. Time-course of glycogen phosphorylase (I activity and the effect of epinephrine. Epinephrine (5 kg/ml) was added after 30 min incubation. Mean values t SEM of four experiments. Level of significance (paired Student’s t test): l P < 0.05: “P i 0.01 with respect to zero time: *P < 0.05 with respect to correspondent control.
in white muscle slices. Unlike the liver, the white muscle phosphorylase N form was much lower than the total form. Incubation of white muscle slices decreased the phosphorylase a activity by about 50% with respect to the same form of enzyme found at zero time. Epinephrine significantly enhanced the phosphorylase (1 activity in white muscle slices compared with the correspondent control slices activity. The total form of the enzyme was slightly. but significantly reduced by incubation and epinephrine had no effect on this decrease. The level of phosphorylase activity in red muscle slices was greater than in white muscle, chiefly in the active form. which represents about two-thirds of the total activity (Table 3). Incubation decreased the activity of the (1 form only, and the addition
472
OTTOLENGHI
ET AL.
TABLE 2 EFFECT OF EPINEPHRINE
(A) Control (B) Control (C) Epinephrine (5 d-nl)
ON GLYCOGEN
PH~SPHORYLASE
ACTIVITY
IN WHITE
MUSCLE SLICES OF CATFISH
Enzyme test
Time (min)
-AMP
+ AMP
- AMP/ + AMP
0 60
2.69 k 0.24 1.18 k 0.22***
pmol P,ig fresh tissueimin 14.02 L 0.61;” 12.36 k 0.51”,**”
0.195 t 0.016 0.101 t 0.020$
60
1.96 t 0.45**.t
12.75 k 0.48’
0.159 2 0.033
Note. Mean values t SEM of nine experiments. Levels of significance (paired Student’s I test): *P < 0.01 with respect to values in the absence of AMP; ** and ***P < 0.05 and P < 0.01 with respect to values of control at 0 min; tP < 0.05 with respect to values of control at 60 min; SF’ < 0.01 with respect to ratio of control at 0 min.
of epinephrine to incubation medium had no effect on either form of the enzyme.
samples, could be greater than in viro animal, and that the decrease observed in control samples during incubation, takes DISCUSSION place owing the disappearance of catecholIn a previous study (Ottolenghi et al., amine from the system. Janssens et al. 1984b), we found that in catfish liver slices (1983) obtained constant phosphorylase LI the level of enzyme activity at zero time is activation during 60-min incubation, using higher than that found in 60 min incubated in vitro cultures of amphibian liver. Under control for both active and total phosphorthese experimental conditions the zero ylase forms. We have now found the same time samples are well washed, so that any pattern in red and white muscle slices. A catecholamines present are removed. Exsimilar decrease, following incubation, was periments on isolated and well-washed catalso observed by Umminger and Benziger fish hepatocytes (unpublished data) show in brown bullhead liver slices (197.5). The that during incubation, the enzyme activity stress of handling and killing animals may of control never decreases, but it remains constant or sometimes increases. cause a local release of catecholamines As far as epinephrine effect is conwhich would stimulate the activation of phosphorylase. If this is the case, it is pos- cerned, Sutherland (1956) stated that, in sible that the high level of glycogen phos- mammals, epinephrine does not increase phorylase activity found in zero time the enzyme activity originally present in TABLE EFFECT OF EPINEPHRINE ON GLYCOGEN
3
PHOSPHORYLASE ACTIVITY
Enzyme test
Time (min)
-AMP
0 60
11.22 2 0.92 5.48 2 0.82**
60
5.76 2 0.91**
+ AMP pmol
(A) Control (B) Control (C) Epinephrine (5 t&ml)
IN RED MUSCLE SLICES OF CATFISH
-AMP/
+ AMP
P,ig fresh tissueimin 16.42 rt 0.70* 15.82 + 0.70*
0.682 -t 0.039 0.343 t 0.039***
15.88 k 0.64*
0.360 k 0.053***
Note. Mean values 2 SEM of seven experiments. Levels of significance (paired Student’s t test): *P < 0.01 with respect to values in the absence of AMP; **P < 0.01 with respect to values of control at 0 min; ***P < 0.01 with respect to ratio of control at 0 min.
EPINEPHRINE
ON CATFISH
the tissue, but rather prevents or retards the decrease of enzyme activity seen in the control. However, under our experimental conditions epinephrine seems to activate enzyme activity, as shown in Fig. 2. AMP increases the active form of glycogen phosphorylase in mammals (Sutherland and Wosilait, 1956; Verdetti and Piery, 197O), in frog (Piery and Grably, 1969), and in fish (Nagayama, 1961: Yamamoto. 1968: Umminger and Benziger, 197.5). The ratio of phosphorylase activity without AMP to phosphorylase with AMP represents the percentage of total phosphorylase present in the active form. In control liver (Table I) we found that at zero time and after 60 min incubation, 92 and 77%, respectively, of total phosphorylase was present in the active form. In white muscle the pattern was completely different, the active form being only 19% of total form at zero time, and 10% after 60-min incubation. The ratio between a and total form of enzyme activity in red muscle was 68% at zero time control and 34% after incubation. ATP competes with AMP at the same phosphorylase site, and its binding prevents the binding of AMP by failing to induce the activating conformation change: hence ATP is considered as an inhibitor (White et al., 1973). On the other hand, ATP/AMP ratios represent the amount of energy available to the liver tissue. If this assumption is true for catfish liver too, where high level of phosphorylase u is present, then it is possible that glycogen breakdown may occur even if the liver depends to a lesser extent than other tissues on glucose metabolism for its energy requirements. Then the glucose could enter the blood stream and not be burned in the liver for ATP formation. In white muscle the conditions are completely different. The active form of phosphorylase was very low (19% of the total form). White muscle is used up by fish during rapid swimming (Bone, 1966), and usually this tissue utilizes efficient and eco-
PHOSPHORYLASE
473
nomical metabolites such as glucose, although it is well known that the free fatty acids derived from triglycerides are also important fuel source for energy metabolism in fish (Driedzic and Hochachka, 1978). The red muscle, which represents about S-20% of the total muscle mass, consists of aerobic slow fibers. In this tissue the level of glycogen phosphorylase active form, both at zero time and in 60 min incubated control slices, was between the values found in liver and in white muscle. The level of glycogen was also higher (about five times) than that found in white muscle. In a previous work (Ottolenghi et al., 1983). glucose-6-phosphatase activity was seen to be present in red muscle, whereas it was negligible in white muscle. Taking into account this fact, together with the high level of glycogen phosphorylase active form in red muscle in controls, it could follow that the red muscle may contribute to general metabolism by supplying glucose to environment. In white muscle epinephrine enhanced the active form, while the total form of the enzyme was practically unaffected. Epinephrine release takes place under hard exercise (Nakano and Tomlinson, 1967). so our results, although obtained under different experimental conditions, are comparable to those of Morata et al. (1982). who found similar results in rive after stress. Driedzic and Hochachka (1976. 1978) studying the stress conditions caused by strenuous exercise. found that ATP concentration in white muscle was reduced by about 65% in fatigued animals: ADP level also decreased by a small but significant amount. However, AMP concentration remained low and unchanged, and there was an increase in IMP concentration following the action of S’-AMP-deaminase. Thus the total free adenylate pool decreased, and both ATP/ADP and ATP/AMP ratios also diminished. In the light of these findings our results on the phosphorylase activity in
474
OTTOLENGHI
white muscle, could indicate that in the presence of the hormone, as under stress, the decrease in ATP/AMP ratio, and the increase in IMP concentration cause the phosphorylase inactive b form to be converted to active a form. Both forms of glycogen phosphorylase in red muscle were unaffected by epinephrine, which does not even change the glycogen level in this tissue. As far as the dose of epinephrine is concerned, Janssens et al. (1983) reported that in amphibian liver the maximal activity of epinephrine was seen at 10-h M, while 10~” M was without effect. We found that the effect of epinephrine in catfish liver slices was present from a concentration of 3.5 x 1O-8 M, which comes close to the hormone level after stress in some fish (Nakano and Tomlinson, 1967; Dashow et al., 1982; Plisetskaya et al., 1984). In conclusion, results of our experiments could explain the events occurring in carbohydrate metabolism on catfish in the presence of epinephrine. The hormone promotes hyperglycemia chiefly by activation of glycogenolysis in liver, although an activation of gluconeogenetic processes cannot be excluded. The main pathway of glycogen breakdown is the phosphorolytic process, even if other enzymatic mechanisms, such as the amylolytic activity are possibly involved (Plisetskaya et al., 1973; Murat, 1976; Picukans and Umminger, 1979). Epinephrine increases the glucose release from the liver, where the levels of glycogen and glycogen phosphorylase active form are high even under normal conditions. The red muscle has a relatively high level of glycogen and of glycogen phosphorylase activity, and could supply glucose to the blood since a some glucose6-phosphatase activity is present in this muscle. But in red muscle, unlike in liver, epinephrine does not increase glycogen breakdown, probably because this tissue is not involved in quick swimming. In white muscle, low level of the a form of phos-
ET AL.
phorylase and of glycogen are present, so that under rest conditions only a small amount of glycogen is broken down in this muscle. Under stress conditions and in rapid swimming the released epinephrine significantly increases the active form of phosphorylase, so that in the white muscle its own glucose becomes available together with glucose from the liver. ACKNOWLEDGMENT This work was partially supported by a grant for Scientific Research (40%) from Minister0 della Pubblica Istruzione (1983).
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