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Studies on glycogenolysis in carp liver: Evidence for an amylase pathway for glycogen breakdown
Comp. Biochent Physiol., 1976,VoL 55B,pp. 461 to 465. PergamonPress Printedin Great Britain
STUDIES O N GLYCOGENOLYSIS IN CARP LIVER: EVIDENCE FOR AN AMYLASE PATHWAY FOR GLYCOGEN BREAKDOWN JEAN-CLAUDEMURAT Laboratoire d'Ecophysiologie animale (Directeur A. Serfaty), Universit6 Paul Sabatier, 38, rue des 36 Ponts, 31400 Toulouse, France (Received 20 January 1976)
Abstract--1. Glycogen-phosphorylase seems to be lacking in the carp liver. This enzymatic defect bears a resemblance to glycogen storage disease type VI, described in humans. 2. Carp liver homogenates exhibit an important 3'-amylase (~t-glueosidase, EC 3213) activity. By its pH curve and distribution in subceUular fractions of liver, this enzyme could be, to a large extent, of lysosomal origin. 3. During the strong hepatic glycogenolysis, which is induced in carp by insulin injections, the 3,-amylase pathway could offer an explanation for glycogen breakdown in a tissue where glycogen phosphorylase is supposed to be absent.
INTRODUCTION
In the carp (Cyprinus carpio L.), as in some other poikilotherms, the physiological pattern of carbohydrate mobilization appears to be different, in some respects, from what is classically described in mammals. Despite seasonal variations, carp liver glycogen content reaches values up to 15-20rag per 100rag of wet tissue (Plisetskaya, 1975), which are 3 × higher than the maximum values found in the liver of fed rats. When starved in natural or artificial conditions, carp maintain a relatively high glycogen level in tissues, including liver (Wittenberger & Vitca, 1966) for several months. Similar observations have been made in other cold-blooded species, such as goldfish (Stimpson, 1965) or frog (Hanke & Neumarm, 1972). Comparatively, a short period of starvation (24 hr) is enough to deplete the whole carbohydrate reserve in rat liver (Fister et al., 1970). In starved carp, tissue proteins are easily metabolized and are believed to supply most of the energy requirements for a long time (Stimpson, 1965; Nagai & Ikeda, 1972; Gas, 1972; Creach & Serfaty, 1974). According to Blazka (1958) and Hochachka et al., (1973), this preservation of carbohydrate stores could be related to the alleged long survival of carp in natural anoxic environment (ice-locked ponds), since it is known that carbohydrates are indispensable in anaerobic metabolism. We have elsewhere reported that epinephrine and glueagon, when injected in carp, induce important hyperglycaemia, but slight glycogenolysis (Murat & Seffaty, 1975). Since methoxyindole-carboxylic-acid (inhibitor of gluconeogenesis), when previously injected in fish, signififantly reduces the hypergiycaemic responses to these hormones, we think that increase of blood glucose could come, to a large extent, from gluconeogenesis. O n the contrary, hypoglycaemia and simultaneous disappe~_m_aaee of liver glycogen occur in 24 hr after a single insulin injection (2 i.u./100g body weight) 461
(Murat et al., 1974). This glycogenolysis, limited to liver, is observed even if glucose is injected in carp, in order to counterbalance hypoglycaemia (Murat et al., 1975). Decrease in liver glycogen after insulin injection has also been observed in rabbit (Bridge, 1948), in dog (Cahn, 1956), in Clarias lazera (Yanni, 1964), in scorpion-fish (Leibson & Plisetskaya, 1968) and in tenth (Demael, 1971), but the phenomenon appears to be particularly intense in the carp. The liver glycogenolysis induced by insulin in carp is much greater than that observed after injection of large amount of epinephrine or glucagon. Thus, classical interpretations (release of endogenous epinephrine or glucagon, presence of glueagon as a contaminant in injected insulin .... ) seem to be insufficient. Besides, these findings cannot be dearly understood in accordance with classical pattern of glycogen metabolism. In earlier studies (Murat et al., 1972, 1973), we observed that no glycogen phosphorylase activity (EC 2411) was detected in homogenates or histological slices of carp liver, when measured by Cori's method (release of inorganic phosphorus from glucose-l-phosphate). However the enzyme was found to be present and active in other tissues of the fish. The possibility of a technical artefact, involving for instance the disappearance of liberated phosphorus in homogenates, was considered. In the present paper, we report further investigation dealing with the detection of glycogen phosphorylase in the carp liver, using a method in which the enzyme is working in the physiological direction (release of Glucose-l-phosphate from glycogen). Furthermore, we give some data related to an amylase pathway which could offer an explanation for the strong glycogenolysis induced in carp by insulin injections. MATERIALS AND METHODS
We used common carp (Cyprinus carpio L.) weighing 200-300 g and maintained in tanks with running water at
462
JEANCLAUDEMURAT
18-20°C. All fishes were starved 8 days before experiments. Insulin (of bovine origin) is injected in the coelomic cavity. Blood samples are rapidly withdrawn from the cardiac ventricle with a heparinized syringe. Blood glucose, as well as glucose liberated in incubation mediums by amylase action, is measured by glucose-oxydase, with a Beckman Glucose Analyzer. Tissue glycogen content is determinated by a direct enzymatic method (Murat & Serfaty, 1974). Assay of glycogen phosphorylase (EC 2411) In order to measure the activity of this enzyme working in the physiological direction, we use an adaptation of the method described by Bueding et al., (1962). Tissue samples (200-300 mg), rapidly excised, are homogeneized in 5ml of ice-cold buffer (Tris 0.05M; EDTA 0.005 M; NaF 0.05 M; 2-mercaptoethanol 0.04 M; serumalbumine l mg/ml; pH7,1). After light centrifugation (3 min, 1500 g) 0.1 ml of supernatant is mixed with 1.5 ml of incubation medium (PO4KH2 0.02 M; MgC12 0.06 M; NADP 0.0015M; Glycogen lmg/ml; 5'AMP 0.001M; pH 7.1). 0.0l ml of G6PDH (Boehringer Mannheim solution, 1 mg/ml) and 0.01 ml of PGMutase (Boehringer Mannheim solution, 2 mg/ml) are added to the medium in a 1.0 cm light path cuvette, at 25°C. Optical density at 340nm is recorded during a 5 min period. (See Fig. 1). Assay of ~-amylase (EC 3213) Gamma-amylase activity is estimated according to the method of Rosenfeld & Popova (1962), Ruttloff et al., (1967), and Bergmann et al., (1973). Liver samples (about
Glycogen
~'~"G ~-
P04
I'riss~
....
Ihomogena~
I ...... NADP
PG-mu'tase
C-6P
1 0.4-
/
E
P 0.2 - -
i/ I
Heo7
o.~-
,/
/
O.I-
•
0
I
, /
2
3
4
Time of incubation,
,
5
6
7
rain
Fig. 1. Activity of glycogen phosphorylase in liver and heart of carp, measured in the physiological direction (glycogen breakdown). With carp liver homogenate, no increase of optical density is observed, indicating that no glucose-l-phosphate is released from glycogen. When G1P is added to the incubation medium, optical density increases quickly, showing that only the first step of reaction was impaired.
1 g) are excised and immediately homogenized in 3 ml of ice-cold saline solution (NaC1 8 p. 1000; NaF 0.03 M, mercaptoethanol 0.04 M). Each assay mixtures are preincubated with EDTA (0.01 M) at 55°C for I 0 min, in order to destroy digestive ~-amylase, then incubated in phosphate buffer (0.1 M, pH 5.0) containing 5 mg per ml of glycogen. Glucose release is estimated by glucose-oxydase, as mentioned above. Gamma-amylase activity is also measured by the same procedure in subcellular fractions of hepatocytes, which are separated by ultra-centrifugation following the procedure of Pequin et al., (1969). Liver samples are placed in 10ml of ice-cold buffer (Sucrose 0.25M; Tris 0.05M; MgC12 0.005 M; KCI 0.025 M; pH 7.5) and homogeneized in a glass tube using 4 strokes of a prechilled, motordriven, Teflon pestle at a speed of 800 rev/min. The ultra centrifugation of this homogenate is carried out at 0°C with a Martin-Christ machine, model Omikron, Rotor No. 9440, as follows: 10 min, 700 g 20 min, 6500 9 35 min, 38000 g 120 rain, 105000 g
= = = =
Nuclei and damaged cells Heavy mitochondria fraction Light mitochondria fraction Microsomes fractions and last supernatant
RESULTS AND DISCUSSION Figure 1 is representative of the results obtained in more than 30 experiments. It shows that no glycogen phosphorylase activity is found in the carp liver. If glucose-l-phosphate is added to the incubation medium, a rapid increase of optical density is observed, indicating that only the first step of the reaction (glycogen phosphorolysis) was impaired. Comparatively, Fig. 1 indicates that heart homogenate is able to release glucose-l-phosphate from glycogen, demonstrating that glycogen-phosphorylase does work in our technical conditions, when a tissue other than liver is tested. This result confirms our previous findings (Murat et al., 1973). Of course, we cannot definitively assert that there is no glycogen phosphorylase activity in the living fish liver, but the hypothesis of a genetical abnormality in carp could be considered in this lowactive poikilothermic animal, in which proteins are the main energy fuel (Nagai & Ikeda, 1972). Hence, an interesting comparison can be made with glycogen storage disease type VI described in humans (Schmid, 1964; Hug et al., 1974). In this disease, which is not severe, a hepatic glycogenosis and a lack or a very low activity of glycogen-phosphorylase in liver are observed. In the patients, as in carp, the hyperglycaemic response to glucagon or adrenaline is subnormal and supposed to be due to the activation of gluconeogenesis (Schmid, 1964). It is puzzling to note that such an enzymatic defect does not obviate glycogen breakdown in the carp liver, since it can be induced for example by insulin injection. Thus, search for an alternative pathway for glycogenolysis is justified. A lot of studies carried out on mammals and on cold-blooded animals have shown that, despite imprecise identification, amylase (or glucosidase) activities do exist in tissues (Rosenfeld & Popova, 1962; Jeffrey et al., 1970; Palmer, 1971; Gamklou & Schersten, 1972; Lundquist, 1972; Plisetskaya & Zheludkova, 1973; Alemany & Rosell-Perez, 1973). Contrary to ~-amylase which splits polysaccharides into dextrins,
Studies on glycogenolysis in carp liver
463
Table 1. ),-Amylase activity in Carp liver homogenates Control animals (Glycogen content: 15.1 _ 2.2%) + p-chioromercuribenzoate (0.08 mM)
g
1.03 + 0.24
I00
f
Q
0.66 + O.19
P < 0.025 ._>
Insulin treated animals (4 UI/100 g), 15 hours after injection (Glycogen content: 3.5 + 2.7%)
o
50 1.51 _ 0.36
P < 0.025
¢ k
Mean values ___Standard Deviation (N = 6), compared with Controls by Student's test. (expressed as mg of glucose liberated from glycogen/100mg wet tissue/30 min, at 25°C, pH 5.0). Each assay was pre-incubated 10min with EDTA at 55°C, in order to destroy ~-amylase. these enzymes liberate glucose directly and are believed to play a role in regulating the size and quantity of glycogen particle in the cells (Hers, 1972), or even in supplying glucose from carbohydrates stores (Rosenfeld, 1964, Alemany & Rosell-Perez, 1973). Table 1 shows that carp liver homogenates exhibit important y-amylase activity, enough to hydrolyse the whole glycogen content of the liver cells within a few hours. When added in the incubation medium, p-chloromercuribenzoate (0.08 mM) reduces the activity, as it could be expected from data of Rosenfeld & Popova (1962) and Palmer 0971).
c e
oControls
I0-
8 _~ ~
o. 5
.>_
"
o
II
I
®
8
E
._>
0.02
3
_J
o
=
60
0.02
1
3 • I
2o -
II
0 I
I
3
I
6
hr after
I
I0
0.01
I
24
insulin I n j e c t i o n
Fig. 2. ),-amylase activity in carp liver during glycogenolysis induced by insulin (4 i.u./100 g, injected in the coelomic cavity, at the beginning of experiments). Enzyme activity i s expressed as mg of glucose liberated from glycogen/100mg wet tissue/30min, at 25°C and pH5. Blood glucose is expressed as rag/100 ml plasma and liver glycogen is expresseGas mg/10Onig wet tissue. On the Fig. are mentioned the P values (Student's test) which indicate the significance of difference between controls and insulin injected animals. Each plots represent mean values + S.D. (N = 5).
o
I
4
I
5
I
6
I
7
I
8
pH
Fig. 3. Influence of pH on the activity of carp liver ),-amylase. Figure 2 i n d ~ that in liver ~ e n a t e s of carp receiving i n ~ inje~tlons, y-amylase ~ v R y is significantly enhanced when glycogenolysis begins. We note that in controls, the enzyme activity, measured in vitro, is not very low, as it could be expected. In fact, we suppose that the control values do not represent the real physiological activity: a large part of the latent activity may be liberated post-mortem during the preparation of tissue samples. Anyway, the activity of "free" amylase is always greater when insulin glycogenolysis is induced. The fading of the enzyme activity, which appears early in liver homogenates from both controls and insulin-treated fishes, is more difficult to understand• Since this phenomenon is occurring together with the stress-induced hyperglycaemia, we think that it could he also related to the stress of handling and to a strengthening of lysosomal system (see further), but we cannot offer a more precise explanation at the moment. In our experimental conditions, the T-amylase of carp liver works in a rather wide pH range, with maximum activity around pH 5 (Fig. 3). From these data, one cannot conclude the existence of an acid and neutral form of the enzyme, as described in some other species (Popova et al., 1964; Jeffrey et al., 1970). However, the pH-curves of enzymes found in human liver by Gamklou & Schersten (1972), and in mouse tissues by Lundquist (1972), are very similar to the carp's one. Figure 4 shows that amylase activity is found chiefly in the supernatant (105,000 g, 2 hr, 0°C) at the first time of incubation, but after longer incubation, activity also develops in mitochondrial fractions, which contain most of lysosomes (Pequin et al., 1969). No activity is found in microsomes; carp liver amylase is therefore different from the amylase described by Brosemer & Rutter (1961), in rat liver, which is localizated in microsomal fraction and acts as an ~-a/nylase.
We have reported elsewhere that after insulin injections, when glycogenolysis is found to be intense, electron micrographs of carp liver show numerous lysosoreal vesicles with signs of great activity (Murat et al., 1975). All these data argue for at least a partial lysosomal origin of the amylase, and are, in this respect,
464
JEAN CLAUDE MURAT
8o~NUclei + damaged cells /
7
lHeavy mitochondria
801"--.,- h,~=^~°=
4
E o
8o~-+iysosomes 401-Microsomes 4¢
Time of incubation,
hr
Fig. 4. Time-dependence and distribution pattern of ~-amylase in subcellular fractions of carp hepatocytes. Enzyme activity is expressed as /~g of glucose liberated from glycogen/hr/fraction issuing from 2 mg of wet liver (25°C, pH 5). in good accordance with results obtained by Hers (1963), Gamklou & Schersten (1972), and Lundquist (1972), with tissues of mammals. According to Palmer (1971), the biochemical and biophysical properties of lysosomal amylase are very complex: this enzyme may be regarded as possessing at least 3 substrate-binding sites, subject to reciprocal interactions, involving changes in inhibitor effects and optimum pH. Moreover, this enzyme could work as transglucosylase and incorporate glucose in the glycogen molecule. Further studies to identify more accurately and comparatively the amylasic system found in carp liver would be of interest, but our aim in this paper, was only to point out the existence of an alternative pathway for glycogen breakdown in a tissue where glycogen-phosphorylase is supposed to be absent. Since the lysosomal system has been shown to be very sensitive to changes in the intra- and extracellular environment, one could consider the hypothesis that glycogenolysis occurs in carp liver via hn amylaselysosomal pathway in some cases which induce cellular changes. Besides, there could be found an explanation for the glycogenolytic effect of insulin injections. REFERENCES
ALEMANY M. & ROSELL-PEREZM. (1973) Two different amylase activities in the sea mussel. Rev. Esp. Fiziol. 29, 21%22. BERGMANN M., GAIDA P., KUBSCH J. & STEUER E. (1973) Tierexperim¢nteUe und klinische chemische Untersuchungen iiber die 7-amylase. Vorl/iufige Mitteilung. Z. yes. Inn. Med. 28. 146-154. BLAZKA P. (1958) The anaerobic metabolism of fish. Physiol. Zoi~l. 31, 11%128.
BRIDGE E. M. (1938) The action of insulin on glycogen reserves. Bull. John Hopkins Hosp. 62. 408-21. BROSEMER R. W. & RUTTER W. J. (1961) Liver amylase: cellular distribution and properties, physiological role. J. biol. Chem. 236, 1253-63. BUEDING E., BUELBRING E., KURIYAMA H. & GERCKEN G. (1962) Lack of activation of phosphorylase by adrenaline during its physiological effect on intestinal smooth muscle. Nature (Lond.) 196, 944-947. CAHN T. (1956) La rdgulation des processus mdtaboliques dans roroanisme. Presses Universitaires de France, Paris. CRFACH Y. & SERFATYA. (1974) Le je6ne e t l a r6alimentation chez la carpe. J. Physiol., Paris 68, 245-260. DEMnEL A. (1971) Le m6tabolisme de la tanche au cours de l'hypoxie: mise en 6vidence et 6tude des r6gulations adr6nalinique et insulinique. Th~se Doct. Etat, Lyon. ULSTER V., LUTKIC A. & ROSA J. (1970) Hepatic muscular and myocardial glycogen content in ten-day starved and cortisol treated rats. J~oslav. Physiol. Pharmacol. Acta 6. 113-118. GAMKLOU R. & SCHERSTEN T. (1972) Activity of c~-amylase and ~ 1-4-glucosidase in human liver tissue. Scand. J. Clin. Lab. Invest. 30, 201. GAS N. (1972) Alt6rations structurales du muscle blanc de carpe au cours du jefine prolong6. C.r. hebd. Sdane. Acad. Sc., Paris 275. 1403-1406. HANKE W. & NEUMANN U. (1972) Carbohydrate metabolism in Amphibia. Gen. comp. Endocr. suppl. 3. 198-207. HERS H. G. (1963) Alpha-glucosidase deficiency in generalized glycogen storage disease (Pompe's disease). Bigchem. J. 86. 11-16. HERS H. G. (1972) The role of lysosomes in the pathogeny of storage diseases. Biochimie 54, 753-756. HOCHACHKAP. W., FIELD J. & MUSTAEAT. (1973) Animal life without oxygen: basic biochemical mechanisms. Am. Zooloyist 13, 543-555. HUG G., CHUCKG., WALLINGL. & SCHUBERTW. L. (1974) Liver phosphorylase deficiency in glycogenosis type VI. J. Lab. clin. Med. 84, 26-35. JEFFREY P. L., BROWN D. H. & ILLINGWORTH B. (1970) Studies of lysosomal ~-glucosidase. Biochemistry, N.Y. 9, 1416-1422. LEIBSONL. G. & PLISETSKAYAE. M. (1968) Effect of insulin on blood sugar level and glycogen content in organs of some cyclostomes and fish. Gen. comp. Endocr. 11, 381-392. LUNOQUlST I. (1972) Acid amyloglucosidase and carbohydrate regulation. Horm. Metab. Res. 4, 245-249. MURAT J. C., PARENT J. P. & BALAS D. (1972) Quelques donn~es sur l'activit6 de la glycog6ne phosphorylase de T616ost6ens. J. Physiol., Paris 65. 458. MURAT J. C., PARENT J. P. & SEREATYA. (1973) Etude de l'activit6 de la glycog6ne-phosphorylase de la carpe. Experientia 29, 36. MURATJ. C. & SERFATYA. (1974) Simple enzymatic determination of polysaccharide (Glycogen) content of animal tissues. Clin. Chem. 20, 1576-1577. MURAT J. C., GAS N., DEPEYRE J. & CASTILLA C. (1974) Effets hormonaux sur le m6tabolisme glucidique de la carpe. J. Physiol. Paris 69. 280 A. MURAT J. C., CASTILLAC. ~,~ DEPEYRE J. (1975) Effets de l'insuline sur le tissu h6patique de la carpe. J. Physiol., Paris 71, 341-342 A. MURAT J. C. & SERFATY A. (1975) Effets de l'adr6naline, du glucagon et de l'insuline sur le m6tabolisme gtucidique de la carpe. C.r. S~ane. Soc. Biol. 169. 228-232. NAGAI M. & IKEDA S. (1972) Carbohydrate metabolism in fish. III. Effect of dietary composition on metabolism of Glucose U 14C and glutamate U t*C in carp. Bull. Jap. Soc. Fish. 38, 137-143. PALMERT. N. (1971) The maltase, glucomylase and transglucosylase activities of acid c~-glucosidase from rabbit muscle. Biochem. J. 124, 713-724.
Studies on glycogenolysis in carp liver PEQUIN L., V~LAS F. & BOUCHEG. (1969) La glutamine synth6tase chez la carp¢. Arch. Sci. Physiol. 23, 469-480. PLISETSKAY^ E. M. & J~UDKOVA Z. P. (1973) Effet de l'adr6naline sur l'activit6 de l'amylase dans le foie et les muscles de la lamproie (en Russe). J. evol. biokhem. Fiziol. 9, 611-613. PLISETSKAYAE. M. (1975) R dgulation hormonale du mJtabolisme olucidique chez les vertdbrJs inf~rieurs (en Russe). Izdatelstvo "Nauka", Leningradskoe otdelenie, Leningrad. POPOVAI. A., VINNITSKAYAA. I. & ROSENFELDE. L. (1964) Purification et fractionnement de la T-amylase provenant de divers organes d'animaux (en Russe). Biokhimiya 29. 312-321. ROSENFELDE. L. & POPOVA J. A. (1962) L'exopolyglucosidase (~,-amylase) du foie. Bull. Soc. Chim. Biol. 44, 129-139.
465
ROSENFELD E. L. (1964) Animal tissue ~-amylase and its role in the metabolism of glycogen. In Control of Glycogen Metabolism. pp. 176-189. (Edited by WHELAN & CAMERON)Churchill, London. RUTTLOFF H., NO^CK R. & FRIES R. Uber polysaccharidspaltende Enzyme im Biirstensaum der RattenMukosa unter besonderer Berticksichtigung der ~,-Amylase. Acta Biol. Med. Germ. 19, 831-839. SCHMID R. (1964) Glycogen storage diseases. In Control of Glycogen metabolism, pp. 305-320. (Edited by WHELAN & CAMEROn)Churchill, London. STIMPSONJ. H. (1965) Comparative aspects of the control of glycogen utilization in vertebrate liver. Comp. BIOchem. Physiol. 15, 187-197. WITTENBERGERC. & VITCA E. (1966) Variation of the glycogen content in the tissues of the carp, during a work in isolated muscles and during starvation. Stud. Univ. Babes. Bolgai, Ser. Biol. Roum. ll. 117-123.
STUDIES O N GLYCOGENOLYSIS IN CARP LIVER: EVIDENCE FOR AN AMYLASE PATHWAY FOR GLYCOGEN BREAKDOWN JEAN-CLAUDEMURAT Laboratoire d'Ecophysiologie animale (Directeur A. Serfaty), Universit6 Paul Sabatier, 38, rue des 36 Ponts, 31400 Toulouse, France (Received 20 January 1976)
Abstract--1. Glycogen-phosphorylase seems to be lacking in the carp liver. This enzymatic defect bears a resemblance to glycogen storage disease type VI, described in humans. 2. Carp liver homogenates exhibit an important 3'-amylase (~t-glueosidase, EC 3213) activity. By its pH curve and distribution in subceUular fractions of liver, this enzyme could be, to a large extent, of lysosomal origin. 3. During the strong hepatic glycogenolysis, which is induced in carp by insulin injections, the 3,-amylase pathway could offer an explanation for glycogen breakdown in a tissue where glycogen phosphorylase is supposed to be absent.
INTRODUCTION
In the carp (Cyprinus carpio L.), as in some other poikilotherms, the physiological pattern of carbohydrate mobilization appears to be different, in some respects, from what is classically described in mammals. Despite seasonal variations, carp liver glycogen content reaches values up to 15-20rag per 100rag of wet tissue (Plisetskaya, 1975), which are 3 × higher than the maximum values found in the liver of fed rats. When starved in natural or artificial conditions, carp maintain a relatively high glycogen level in tissues, including liver (Wittenberger & Vitca, 1966) for several months. Similar observations have been made in other cold-blooded species, such as goldfish (Stimpson, 1965) or frog (Hanke & Neumarm, 1972). Comparatively, a short period of starvation (24 hr) is enough to deplete the whole carbohydrate reserve in rat liver (Fister et al., 1970). In starved carp, tissue proteins are easily metabolized and are believed to supply most of the energy requirements for a long time (Stimpson, 1965; Nagai & Ikeda, 1972; Gas, 1972; Creach & Serfaty, 1974). According to Blazka (1958) and Hochachka et al., (1973), this preservation of carbohydrate stores could be related to the alleged long survival of carp in natural anoxic environment (ice-locked ponds), since it is known that carbohydrates are indispensable in anaerobic metabolism. We have elsewhere reported that epinephrine and glueagon, when injected in carp, induce important hyperglycaemia, but slight glycogenolysis (Murat & Seffaty, 1975). Since methoxyindole-carboxylic-acid (inhibitor of gluconeogenesis), when previously injected in fish, signififantly reduces the hypergiycaemic responses to these hormones, we think that increase of blood glucose could come, to a large extent, from gluconeogenesis. O n the contrary, hypoglycaemia and simultaneous disappe~_m_aaee of liver glycogen occur in 24 hr after a single insulin injection (2 i.u./100g body weight) 461
(Murat et al., 1974). This glycogenolysis, limited to liver, is observed even if glucose is injected in carp, in order to counterbalance hypoglycaemia (Murat et al., 1975). Decrease in liver glycogen after insulin injection has also been observed in rabbit (Bridge, 1948), in dog (Cahn, 1956), in Clarias lazera (Yanni, 1964), in scorpion-fish (Leibson & Plisetskaya, 1968) and in tenth (Demael, 1971), but the phenomenon appears to be particularly intense in the carp. The liver glycogenolysis induced by insulin in carp is much greater than that observed after injection of large amount of epinephrine or glucagon. Thus, classical interpretations (release of endogenous epinephrine or glucagon, presence of glueagon as a contaminant in injected insulin .... ) seem to be insufficient. Besides, these findings cannot be dearly understood in accordance with classical pattern of glycogen metabolism. In earlier studies (Murat et al., 1972, 1973), we observed that no glycogen phosphorylase activity (EC 2411) was detected in homogenates or histological slices of carp liver, when measured by Cori's method (release of inorganic phosphorus from glucose-l-phosphate). However the enzyme was found to be present and active in other tissues of the fish. The possibility of a technical artefact, involving for instance the disappearance of liberated phosphorus in homogenates, was considered. In the present paper, we report further investigation dealing with the detection of glycogen phosphorylase in the carp liver, using a method in which the enzyme is working in the physiological direction (release of Glucose-l-phosphate from glycogen). Furthermore, we give some data related to an amylase pathway which could offer an explanation for the strong glycogenolysis induced in carp by insulin injections. MATERIALS AND METHODS
We used common carp (Cyprinus carpio L.) weighing 200-300 g and maintained in tanks with running water at
462
JEANCLAUDEMURAT
18-20°C. All fishes were starved 8 days before experiments. Insulin (of bovine origin) is injected in the coelomic cavity. Blood samples are rapidly withdrawn from the cardiac ventricle with a heparinized syringe. Blood glucose, as well as glucose liberated in incubation mediums by amylase action, is measured by glucose-oxydase, with a Beckman Glucose Analyzer. Tissue glycogen content is determinated by a direct enzymatic method (Murat & Serfaty, 1974). Assay of glycogen phosphorylase (EC 2411) In order to measure the activity of this enzyme working in the physiological direction, we use an adaptation of the method described by Bueding et al., (1962). Tissue samples (200-300 mg), rapidly excised, are homogeneized in 5ml of ice-cold buffer (Tris 0.05M; EDTA 0.005 M; NaF 0.05 M; 2-mercaptoethanol 0.04 M; serumalbumine l mg/ml; pH7,1). After light centrifugation (3 min, 1500 g) 0.1 ml of supernatant is mixed with 1.5 ml of incubation medium (PO4KH2 0.02 M; MgC12 0.06 M; NADP 0.0015M; Glycogen lmg/ml; 5'AMP 0.001M; pH 7.1). 0.0l ml of G6PDH (Boehringer Mannheim solution, 1 mg/ml) and 0.01 ml of PGMutase (Boehringer Mannheim solution, 2 mg/ml) are added to the medium in a 1.0 cm light path cuvette, at 25°C. Optical density at 340nm is recorded during a 5 min period. (See Fig. 1). Assay of ~-amylase (EC 3213) Gamma-amylase activity is estimated according to the method of Rosenfeld & Popova (1962), Ruttloff et al., (1967), and Bergmann et al., (1973). Liver samples (about
Glycogen
~'~"G ~-
P04
I'riss~
....
Ihomogena~
I ...... NADP
PG-mu'tase
C-6P
1 0.4-
/
E
P 0.2 - -
i/ I
Heo7
o.~-
,/
/
O.I-
•
0
I
, /
2
3
4
Time of incubation,
,
5
6
7
rain
Fig. 1. Activity of glycogen phosphorylase in liver and heart of carp, measured in the physiological direction (glycogen breakdown). With carp liver homogenate, no increase of optical density is observed, indicating that no glucose-l-phosphate is released from glycogen. When G1P is added to the incubation medium, optical density increases quickly, showing that only the first step of reaction was impaired.
1 g) are excised and immediately homogenized in 3 ml of ice-cold saline solution (NaC1 8 p. 1000; NaF 0.03 M, mercaptoethanol 0.04 M). Each assay mixtures are preincubated with EDTA (0.01 M) at 55°C for I 0 min, in order to destroy digestive ~-amylase, then incubated in phosphate buffer (0.1 M, pH 5.0) containing 5 mg per ml of glycogen. Glucose release is estimated by glucose-oxydase, as mentioned above. Gamma-amylase activity is also measured by the same procedure in subcellular fractions of hepatocytes, which are separated by ultra-centrifugation following the procedure of Pequin et al., (1969). Liver samples are placed in 10ml of ice-cold buffer (Sucrose 0.25M; Tris 0.05M; MgC12 0.005 M; KCI 0.025 M; pH 7.5) and homogeneized in a glass tube using 4 strokes of a prechilled, motordriven, Teflon pestle at a speed of 800 rev/min. The ultra centrifugation of this homogenate is carried out at 0°C with a Martin-Christ machine, model Omikron, Rotor No. 9440, as follows: 10 min, 700 g 20 min, 6500 9 35 min, 38000 g 120 rain, 105000 g
= = = =
Nuclei and damaged cells Heavy mitochondria fraction Light mitochondria fraction Microsomes fractions and last supernatant
RESULTS AND DISCUSSION Figure 1 is representative of the results obtained in more than 30 experiments. It shows that no glycogen phosphorylase activity is found in the carp liver. If glucose-l-phosphate is added to the incubation medium, a rapid increase of optical density is observed, indicating that only the first step of the reaction (glycogen phosphorolysis) was impaired. Comparatively, Fig. 1 indicates that heart homogenate is able to release glucose-l-phosphate from glycogen, demonstrating that glycogen-phosphorylase does work in our technical conditions, when a tissue other than liver is tested. This result confirms our previous findings (Murat et al., 1973). Of course, we cannot definitively assert that there is no glycogen phosphorylase activity in the living fish liver, but the hypothesis of a genetical abnormality in carp could be considered in this lowactive poikilothermic animal, in which proteins are the main energy fuel (Nagai & Ikeda, 1972). Hence, an interesting comparison can be made with glycogen storage disease type VI described in humans (Schmid, 1964; Hug et al., 1974). In this disease, which is not severe, a hepatic glycogenosis and a lack or a very low activity of glycogen-phosphorylase in liver are observed. In the patients, as in carp, the hyperglycaemic response to glucagon or adrenaline is subnormal and supposed to be due to the activation of gluconeogenesis (Schmid, 1964). It is puzzling to note that such an enzymatic defect does not obviate glycogen breakdown in the carp liver, since it can be induced for example by insulin injection. Thus, search for an alternative pathway for glycogenolysis is justified. A lot of studies carried out on mammals and on cold-blooded animals have shown that, despite imprecise identification, amylase (or glucosidase) activities do exist in tissues (Rosenfeld & Popova, 1962; Jeffrey et al., 1970; Palmer, 1971; Gamklou & Schersten, 1972; Lundquist, 1972; Plisetskaya & Zheludkova, 1973; Alemany & Rosell-Perez, 1973). Contrary to ~-amylase which splits polysaccharides into dextrins,
Studies on glycogenolysis in carp liver
463
Table 1. ),-Amylase activity in Carp liver homogenates Control animals (Glycogen content: 15.1 _ 2.2%) + p-chioromercuribenzoate (0.08 mM)
g
1.03 + 0.24
I00
f
Q
0.66 + O.19
P < 0.025 ._>
Insulin treated animals (4 UI/100 g), 15 hours after injection (Glycogen content: 3.5 + 2.7%)
o
50 1.51 _ 0.36
P < 0.025
¢ k
Mean values ___Standard Deviation (N = 6), compared with Controls by Student's test. (expressed as mg of glucose liberated from glycogen/100mg wet tissue/30 min, at 25°C, pH 5.0). Each assay was pre-incubated 10min with EDTA at 55°C, in order to destroy ~-amylase. these enzymes liberate glucose directly and are believed to play a role in regulating the size and quantity of glycogen particle in the cells (Hers, 1972), or even in supplying glucose from carbohydrates stores (Rosenfeld, 1964, Alemany & Rosell-Perez, 1973). Table 1 shows that carp liver homogenates exhibit important y-amylase activity, enough to hydrolyse the whole glycogen content of the liver cells within a few hours. When added in the incubation medium, p-chloromercuribenzoate (0.08 mM) reduces the activity, as it could be expected from data of Rosenfeld & Popova (1962) and Palmer 0971).
c e
oControls
I0-
8 _~ ~
o. 5
.>_
"
o
II
I
®
8
E
._>
0.02
3
_J
o
=
60
0.02
1
3 • I
2o -
II
0 I
I
3
I
6
hr after
I
I0
0.01
I
24
insulin I n j e c t i o n
Fig. 2. ),-amylase activity in carp liver during glycogenolysis induced by insulin (4 i.u./100 g, injected in the coelomic cavity, at the beginning of experiments). Enzyme activity i s expressed as mg of glucose liberated from glycogen/100mg wet tissue/30min, at 25°C and pH5. Blood glucose is expressed as rag/100 ml plasma and liver glycogen is expresseGas mg/10Onig wet tissue. On the Fig. are mentioned the P values (Student's test) which indicate the significance of difference between controls and insulin injected animals. Each plots represent mean values + S.D. (N = 5).
o
I
4
I
5
I
6
I
7
I
8
pH
Fig. 3. Influence of pH on the activity of carp liver ),-amylase. Figure 2 i n d ~ that in liver ~ e n a t e s of carp receiving i n ~ inje~tlons, y-amylase ~ v R y is significantly enhanced when glycogenolysis begins. We note that in controls, the enzyme activity, measured in vitro, is not very low, as it could be expected. In fact, we suppose that the control values do not represent the real physiological activity: a large part of the latent activity may be liberated post-mortem during the preparation of tissue samples. Anyway, the activity of "free" amylase is always greater when insulin glycogenolysis is induced. The fading of the enzyme activity, which appears early in liver homogenates from both controls and insulin-treated fishes, is more difficult to understand• Since this phenomenon is occurring together with the stress-induced hyperglycaemia, we think that it could he also related to the stress of handling and to a strengthening of lysosomal system (see further), but we cannot offer a more precise explanation at the moment. In our experimental conditions, the T-amylase of carp liver works in a rather wide pH range, with maximum activity around pH 5 (Fig. 3). From these data, one cannot conclude the existence of an acid and neutral form of the enzyme, as described in some other species (Popova et al., 1964; Jeffrey et al., 1970). However, the pH-curves of enzymes found in human liver by Gamklou & Schersten (1972), and in mouse tissues by Lundquist (1972), are very similar to the carp's one. Figure 4 shows that amylase activity is found chiefly in the supernatant (105,000 g, 2 hr, 0°C) at the first time of incubation, but after longer incubation, activity also develops in mitochondrial fractions, which contain most of lysosomes (Pequin et al., 1969). No activity is found in microsomes; carp liver amylase is therefore different from the amylase described by Brosemer & Rutter (1961), in rat liver, which is localizated in microsomal fraction and acts as an ~-a/nylase.
We have reported elsewhere that after insulin injections, when glycogenolysis is found to be intense, electron micrographs of carp liver show numerous lysosoreal vesicles with signs of great activity (Murat et al., 1975). All these data argue for at least a partial lysosomal origin of the amylase, and are, in this respect,
464
JEAN CLAUDE MURAT
8o~NUclei + damaged cells /
7
lHeavy mitochondria
801"--.,- h,~=^~°=
4
E o
8o~-+iysosomes 401-Microsomes 4¢
Time of incubation,
hr
Fig. 4. Time-dependence and distribution pattern of ~-amylase in subcellular fractions of carp hepatocytes. Enzyme activity is expressed as /~g of glucose liberated from glycogen/hr/fraction issuing from 2 mg of wet liver (25°C, pH 5). in good accordance with results obtained by Hers (1963), Gamklou & Schersten (1972), and Lundquist (1972), with tissues of mammals. According to Palmer (1971), the biochemical and biophysical properties of lysosomal amylase are very complex: this enzyme may be regarded as possessing at least 3 substrate-binding sites, subject to reciprocal interactions, involving changes in inhibitor effects and optimum pH. Moreover, this enzyme could work as transglucosylase and incorporate glucose in the glycogen molecule. Further studies to identify more accurately and comparatively the amylasic system found in carp liver would be of interest, but our aim in this paper, was only to point out the existence of an alternative pathway for glycogen breakdown in a tissue where glycogen-phosphorylase is supposed to be absent. Since the lysosomal system has been shown to be very sensitive to changes in the intra- and extracellular environment, one could consider the hypothesis that glycogenolysis occurs in carp liver via hn amylaselysosomal pathway in some cases which induce cellular changes. Besides, there could be found an explanation for the glycogenolytic effect of insulin injections. REFERENCES
ALEMANY M. & ROSELL-PEREZM. (1973) Two different amylase activities in the sea mussel. Rev. Esp. Fiziol. 29, 21%22. BERGMANN M., GAIDA P., KUBSCH J. & STEUER E. (1973) Tierexperim¢nteUe und klinische chemische Untersuchungen iiber die 7-amylase. Vorl/iufige Mitteilung. Z. yes. Inn. Med. 28. 146-154. BLAZKA P. (1958) The anaerobic metabolism of fish. Physiol. Zoi~l. 31, 11%128.
BRIDGE E. M. (1938) The action of insulin on glycogen reserves. Bull. John Hopkins Hosp. 62. 408-21. BROSEMER R. W. & RUTTER W. J. (1961) Liver amylase: cellular distribution and properties, physiological role. J. biol. Chem. 236, 1253-63. BUEDING E., BUELBRING E., KURIYAMA H. & GERCKEN G. (1962) Lack of activation of phosphorylase by adrenaline during its physiological effect on intestinal smooth muscle. Nature (Lond.) 196, 944-947. CAHN T. (1956) La rdgulation des processus mdtaboliques dans roroanisme. Presses Universitaires de France, Paris. CRFACH Y. & SERFATYA. (1974) Le je6ne e t l a r6alimentation chez la carpe. J. Physiol., Paris 68, 245-260. DEMnEL A. (1971) Le m6tabolisme de la tanche au cours de l'hypoxie: mise en 6vidence et 6tude des r6gulations adr6nalinique et insulinique. Th~se Doct. Etat, Lyon. ULSTER V., LUTKIC A. & ROSA J. (1970) Hepatic muscular and myocardial glycogen content in ten-day starved and cortisol treated rats. J~oslav. Physiol. Pharmacol. Acta 6. 113-118. GAMKLOU R. & SCHERSTEN T. (1972) Activity of c~-amylase and ~ 1-4-glucosidase in human liver tissue. Scand. J. Clin. Lab. Invest. 30, 201. GAS N. (1972) Alt6rations structurales du muscle blanc de carpe au cours du jefine prolong6. C.r. hebd. Sdane. Acad. Sc., Paris 275. 1403-1406. HANKE W. & NEUMANN U. (1972) Carbohydrate metabolism in Amphibia. Gen. comp. Endocr. suppl. 3. 198-207. HERS H. G. (1963) Alpha-glucosidase deficiency in generalized glycogen storage disease (Pompe's disease). Bigchem. J. 86. 11-16. HERS H. G. (1972) The role of lysosomes in the pathogeny of storage diseases. Biochimie 54, 753-756. HOCHACHKAP. W., FIELD J. & MUSTAEAT. (1973) Animal life without oxygen: basic biochemical mechanisms. Am. Zooloyist 13, 543-555. HUG G., CHUCKG., WALLINGL. & SCHUBERTW. L. (1974) Liver phosphorylase deficiency in glycogenosis type VI. J. Lab. clin. Med. 84, 26-35. JEFFREY P. L., BROWN D. H. & ILLINGWORTH B. (1970) Studies of lysosomal ~-glucosidase. Biochemistry, N.Y. 9, 1416-1422. LEIBSONL. G. & PLISETSKAYAE. M. (1968) Effect of insulin on blood sugar level and glycogen content in organs of some cyclostomes and fish. Gen. comp. Endocr. 11, 381-392. LUNOQUlST I. (1972) Acid amyloglucosidase and carbohydrate regulation. Horm. Metab. Res. 4, 245-249. MURAT J. C., PARENT J. P. & BALAS D. (1972) Quelques donn~es sur l'activit6 de la glycog6ne phosphorylase de T616ost6ens. J. Physiol., Paris 65. 458. MURAT J. C., PARENT J. P. & SEREATYA. (1973) Etude de l'activit6 de la glycog6ne-phosphorylase de la carpe. Experientia 29, 36. MURATJ. C. & SERFATYA. (1974) Simple enzymatic determination of polysaccharide (Glycogen) content of animal tissues. Clin. Chem. 20, 1576-1577. MURAT J. C., GAS N., DEPEYRE J. & CASTILLA C. (1974) Effets hormonaux sur le m6tabolisme glucidique de la carpe. J. Physiol. Paris 69. 280 A. MURAT J. C., CASTILLAC. ~,~ DEPEYRE J. (1975) Effets de l'insuline sur le tissu h6patique de la carpe. J. Physiol., Paris 71, 341-342 A. MURAT J. C. & SERFATY A. (1975) Effets de l'adr6naline, du glucagon et de l'insuline sur le m6tabolisme gtucidique de la carpe. C.r. S~ane. Soc. Biol. 169. 228-232. NAGAI M. & IKEDA S. (1972) Carbohydrate metabolism in fish. III. Effect of dietary composition on metabolism of Glucose U 14C and glutamate U t*C in carp. Bull. Jap. Soc. Fish. 38, 137-143. PALMERT. N. (1971) The maltase, glucomylase and transglucosylase activities of acid c~-glucosidase from rabbit muscle. Biochem. J. 124, 713-724.
Studies on glycogenolysis in carp liver PEQUIN L., V~LAS F. & BOUCHEG. (1969) La glutamine synth6tase chez la carp¢. Arch. Sci. Physiol. 23, 469-480. PLISETSKAY^ E. M. & J~UDKOVA Z. P. (1973) Effet de l'adr6naline sur l'activit6 de l'amylase dans le foie et les muscles de la lamproie (en Russe). J. evol. biokhem. Fiziol. 9, 611-613. PLISETSKAYAE. M. (1975) R dgulation hormonale du mJtabolisme olucidique chez les vertdbrJs inf~rieurs (en Russe). Izdatelstvo "Nauka", Leningradskoe otdelenie, Leningrad. POPOVAI. A., VINNITSKAYAA. I. & ROSENFELDE. L. (1964) Purification et fractionnement de la T-amylase provenant de divers organes d'animaux (en Russe). Biokhimiya 29. 312-321. ROSENFELDE. L. & POPOVA J. A. (1962) L'exopolyglucosidase (~,-amylase) du foie. Bull. Soc. Chim. Biol. 44, 129-139.
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ROSENFELD E. L. (1964) Animal tissue ~-amylase and its role in the metabolism of glycogen. In Control of Glycogen Metabolism. pp. 176-189. (Edited by WHELAN & CAMERON)Churchill, London. RUTTLOFF H., NO^CK R. & FRIES R. Uber polysaccharidspaltende Enzyme im Biirstensaum der RattenMukosa unter besonderer Berticksichtigung der ~,-Amylase. Acta Biol. Med. Germ. 19, 831-839. SCHMID R. (1964) Glycogen storage diseases. In Control of Glycogen metabolism, pp. 305-320. (Edited by WHELAN & CAMEROn)Churchill, London. STIMPSONJ. H. (1965) Comparative aspects of the control of glycogen utilization in vertebrate liver. Comp. BIOchem. Physiol. 15, 187-197. WITTENBERGERC. & VITCA E. (1966) Variation of the glycogen content in the tissues of the carp, during a work in isolated muscles and during starvation. Stud. Univ. Babes. Bolgai, Ser. Biol. Roum. ll. 117-123.