Study on properties and regulation peculiarities of glycogenolysis enzymes in fish skeletal muscles depending on peculiarities of the species motor activity

The activity values, properties and peculiarities of activation of glycogen phosphorylase (GP, EC 2.4.1.1) and glycogen phosphorylase kinase (GPK, EC 2.7.138) were studied in the white skeletal muscle of fishes differing in motor behavior. No differences in the GP and GPK activity levels were revealed in porgy Diplodus annularis (L.), horse mackerel Trachurus mediterraneus ponticus, trout Salmo trutta morphario, scorpionfish Scorpaena porcus, flounder Scophtalnus maeoticus maeoticus, and carp Cyprinus carpio; however, properties of the isolated enzymes and peculiarities of formation of their activated forms during swimming in a hydrodynamic tube are determined by functional peculiarities of the muscle tissue and are associated with the motor activity character of the species. The more rapid ion regulation prevails in fishes capable for the burst swimming type (scorpionfish, trout). The glycogenolysis hormonal regulation leading to a change of the GPK activity index has been found in other species.     

Peculiarities of Activation of Glycogenolysis Enzymes in Skeletal Muscles of the Trout Salmo trutta morpha Fario

In skeletal muscles of the trout, a fish that intensively swims and is capable for sharp sprinting movements, an active form of ATP: phosphorylase b phosphotransferase (EC 2.7.1.38, glycogen phosphorylase kinase; GPK) and partially active 1,4-D-glucan:orthophosphate glucosyltransferase (EC 2.4.1.1, glycogen phosphorylase; GP) are revealed in the state of a relative rest. The isolated GP ab has a higher affinity to substrates (glucose-1-phosphate and glycogen) than GP b and is able to split glycogen without pre-activation with AMP or GPK. The presence of the activated forms of GPK and GP in resting muscles of the trout provides an opportunity for the very fast Ca²⁺-activation of glycogenolysis, coupled with activation of muscle contraction. This seems to be a biochemical mechanism of adaptation for the energy supply of intense muscle activity in this fish species inhabiting rapid cataracted rivers.     

Insulin and glucagon family peptides in relation to activities of hepatic hexokinase and other enzymes in fed and starved Atlantic salmon (Salmo salar) and cod (Gadus morhua).

1. Plasma levels of insulin, glucagon, and glucagon-like peptide (Glp) were all reduced by starvation of salmon and cod. In the salmon the drop in Glp was larger than in insulin and glucagon. 2. After starvation the activity of hexokinase (EC 2.7.1.1) was increased in salmon liver, but decreased in cod liver. The salmon hepatic hexokinase activity was inversely correlated with the Glp/insulin ratio. 3. Activities of hepatic glycogen phosphorylase (EC 2.4.1.1) and phosphofructokinase (EC 2.7.1.11) were increased in starved as compared to fed salmon. In cod, starvation resulted in decreased or unchanged activity of phosphorylase. This discrepancy may be related to different degrees of environmental and handling stress. 4. Intraperitoneal injection of human insulin in salmon gave increased hepatic phosphorylase and hexokinase activities and reduced plasma levels of glucagon, Glp and endogenous fish insulin at sampling after 30 hr. 5. No differences in hepatic hexokinase activities or plasma hormone levels were observed between cod fed low and high carbohydrate diets. Apparently, regulation of glucose phosphorylation by dietary carbohydrate does not occur.     

Seasonality of glycogen phosphorylase activity in crucian carp (<Emphasis Type="Italic">Carassius carassius</Emphasis> L<Emphasis Type="Italic">.</Emphasis>)

Seasonal changes in the activity of glycogen phosphorylase (GP), a rate-limiting enzyme of glycogen degradation, were examined in an anoxia-tolerant fish species, the crucian carp (Carassius carassius L.). In muscle and brain, the activity of GP remained constant throughout the year when tested at 25°C. In contrast, the activities of liver and heart GP displayed striking increases in summer. When seasonal temperature changes are taken into account, the activity of GP during the anoxic mid-winter is only 4–6% of its summer time activity in the muscle, heart and liver, and 13% in brain. In winter-acclimatized fish, experimental anoxia (1–6 weeks) caused sustained depression of the GP activity in heart and gills. In liver and muscle, a transient depression of GP activity occurred during the first week of anoxia but later GP activity recovered back to the normoxic level. GP of the brain was completely resistant to anoxia. In all studied tissues, the constitutive activity of GP is more than sufficient to degrade glycogen deposits during winter anoxia without anoxia-induced activation of GP. The seemingly paradoxical summer-time increase in the activity of liver and heart GP could be related to active life-style of the summer-acclimatized fish (growth, reproduction), the increased demand of energy and molecular precursors of anabolic metabolism being satisfied by preferential degradation of glycogen. The high glycogen content of winter-acclimatized crucian carp is not associated with the elevated GP activity or anoxic activation of GP.     

Phosphorylase activity and glycogen content in cerebral and myocardium tissues in rats of different age

The phosphorylase activity (EC 2.4.1.1) and the glycogen content were determined in the brain and myocardium of adult and old rats. In the cortex and while substance of great cerebral hemispheres, the phosphorylase a activity decreases and the phosphorylase b activity increases, while the total enzyme activity remains unchanged with aging. In the myocardium, the activity of phosphorylase a increases, while that of phophorylase b decreases against a background of an insignificant decrease in the total phosphorylase activity. During aging, the glycogen content decreases sharply in the myocardium, whereas in the cerebral tissue it remains unchanged. Age peculiarities of the adrenaline effect upon myocardial phosphorylase activity are presented.     

Schistosoma haematobium: histochemistry of glycogen, glycogen phosphorylase a and glycogen branching enzyme in niridazole-treated females.

The body posterior to the ovary of Schistosoma haematobium females was investigated. Glycogen, glycogen phosphorylase a (EC 2.4.1.1) and glycogen branching enzyme (EC 2.4.1.18) activities were detected in the subtegumental muscle system, parenchyma and mature vitelline cells, whereas no activities were detected in the tegument and immature vitelline cells of the parasite. Administration of a single niridazole dose of 250 mg kg-1 to the pouched mouse (Saccostomus camestris) produced the following changes in S. haematobium females: a relatively rapid depletion of glycogen stores due to disruption of the absorptive surface of the parasite, and to an increase in the activity of glycogen phosphorylase a; a reduction in the phosphorylase a to phosphorylase b-conversion capacity of glycogen phosphorylase phosphatase (EC 3.1.3.17); a decrease in glycogen branching enzyme activity; and a relatively rapid degeneration of parasite cells possibly due to their loss of endogenous energy reserves.     

The swimming performance of brown trout and whitefish: the effects of exercise on Ca2 + handling and oxidative capacity of swimming muscles

The swimming performance of two fish species, the brown trout and whitefish, having initially different swimming strategies, was measured after nine different training programs in order to relate the effects of exercise on Ca²⁺ handling and oxidative capacity of swimming muscles. The time to 50% fatigue was measured during the training period, and compared with the density of dihydropyridine (DHP) and ryanodine (Ry) receptors and succinate dehydrogenase (SDH) and phosphorylase activity determined by histochemical analysis of the swimming muscles. Overall, both trained brown trout and whitefish had superior swimming performance as compared to control ones. Interestingly, the training programs had different effect on the two species studied since brown trout achieved the highest swimming performance, swimming against the water flow velocity of 2 BL s⁻¹ while among whitefish the best efficiency was seen after training with lower swimming velocities. Training also induced a significant increase in DHP and Ry receptor density in both species. Generally, in brown trout the most notable increase in the receptor densities was observed in red muscle sections from the fish swimming for 6 weeks against water currents of 1 BL s⁻¹ (DHPR 176.5 ± 7.7% and RyR 231.4 ± 11.8%) and white muscle sections against 2 BL s⁻¹ (DHPR 129.6 ± 12.4% and RyR 161.9 ± 15.5%). In whitefish the most prominent alterations were noted in samples from both muscle types after 6 weeks of training against water current of 1.5 BL s⁻¹ (DHPR 167.1 ± 16.9% and RyR 190.4 ± 19.4%). Finally, after all the training regimens the activity of SDH increased but the phosphorylase activity decreased significantly in both the species. To conclude, our findings demonstrate an improved swimming performance and enhanced Ca²⁺ regulation and oxidative capacity after training. Moreover, there seems to be a connection between the swimming performance and receptor levels, especially in white swimming muscles of different fish species, regardless of their initially deviant swimming behaviours. However, depending on the training regimen the divergent swimming behaviours do cause a different response, resulting in the most prominent adaptational changes in the receptor levels of red muscle samples with lower swimming velocities in brown trout and with higher ones in whitefish.     

Effect of acute activation of 5'-AMP-activated protein kinase on glycogen regulation in isolated rat skeletal muscle.

5'-AMP-activated protein kinase (AMPK) has been implicated in glycogen metabolism in skeletal muscle. However, the physiological relevance of increased AMPK activity during exercise has not been fully clarified. This study was performed to determine the direct effects of acute AMPK activation on muscle glycogen regulation. For this purpose, we used an isolated rat muscle preparation and pharmacologically activated AMPK with 5-aminoimidazole-4-carboxamide-1-beta-D-ribonucleoside (AICAR). Tetanic contraction in vitro markedly activated the alpha(1)- and alpha(2)-isoforms of AMPK, with a corresponding increase in the rate of 3-O-methylglucose uptake. Incubation with AICAR elicited similar enhancement of AMPK activity and 3-O-methylglucose uptake in rat epitrochlearis muscle. In contrast, whereas contraction stimulated glycogen synthase (GS), AICAR treatment decreased GS activity. Insulin-stimulated GS activity also decreased after AICAR treatment. Whereas contraction activated glycogen phosphorylase (GP), AICAR did not alter GP activity. The muscle glycogen content decreased in response to contraction but was unchanged by AICAR. Lactate release was markedly increased when muscles were stimulated with AICAR in buffer containing glucose, indicating that the glucose taken up into the muscle was catabolized via glycolysis. Our results suggest that AMPK does not mediate contraction-stimulated glycogen synthesis or glycogenolysis in skeletal muscle and also that acute AMPK activation leads to an increased glycolytic flux by antagonizing contraction-stimulated glycogen synthesis.     

Nitration of a critical tyrosine residue in the allosteric inhibitor site of muscle glycogen phosphorylase impairs its catalytic activity

Muscle glycogen phosphorylase (GP) is a key enzyme in glucose metabolism, and its impairment can lead to muscle dysfunction. Tyrosine nitration of glycogen phosphorylase occurs during aging and has been suggested to be involved in progressive loss of muscle performance. Here, we show that GP (in its T and R form) is irreversibly impaired by exposure to peroxynitrite, a biological nitrogen species known to nitrate reactive tyrosine residues, and to be involved in physiological and pathological processes. Kinetic and biochemical analysis indicated that irreversible inactivation of GP by peroxynitrite is due to the fast (k(inact)=3 x 10(4) M(-1) s(-1)) nitration of a unique tyrosine residue of the enzyme. Endogenous GP was tyrosine nitrated and irreversibly inactivated in skeletal muscle cells upon exposure to peroxynitrite, with concomitant impairment of glycogen mobilization. Ligand protection assays and mass spectrometry analysis using purified GP suggested that the peroxynitrite-dependent inactivation of the enzyme could be due to the nitration of Tyr613, a key amino acid of the allosteric inhibitor site of the enzyme. Our findings suggest that GP functions may be regulated by tyrosine nitration.     

Control of rat skeletal-muscle phosphorylase phosphatase activity by adrenaline.

Administration of adrenaline to an isolated rat hindlimb preparation rapidly decreased muscle phosphorylase phosphatase (EC 3.1.3.17) activity and increased heat-stable and trypsin-labile phosphatase inhibitor activity. This was associated with increased tissue cyclic AMP concentrations, phosphorylase (EC 2.4.1.1) activation and glycogen synthase (EC 2.4.1.11) inactivation.     

Phosphorylase and gluco-6-phosphatase activity and glycogen level in tissues of vertebrates

The activity of phosphorylase (EC 2.4.1.1), glucose-6-phosphatase (EC 3.1.3.9) and the content of glycogen have been determined in tissues of fish, amphibians, reptiles, mammalians. No differences in the activity of phosphorylase and glucose-6-phosphatase in the liver, myocardium, and brain of animals of the phylogenetic groups under study are found. The activity of glucose-6-phosphatase in the anaerobic muscles of poikilothermal animals is found to be rather high. The share of phosphorylase a in the skeletal muscles and brain as well as the glycogen content in the brain of these animals is essentially higher than that of adult mammalians.     

Histochemical fibre types in the lateral muscle of fishes in fresh, brackish and salt water

The histochemical pattern of red, pink and white muscle of fish living in fresh, brackish, and salt water is reported. The muscle fibres were stained routinely during the year for lactate dehydrogenase (LDH), menadione α-glycerophosphate dehydrogenase (Mα—GPDH), succinic dehydrogenase (SDH), myosin adenosine triphosphatase (myosin ATPase), phosphorylase, lipids and glycogen. The pink and red muscles contain more glycogen and lipids and have a higher SDH activity, which is in accord with their aerobic metabolism and function in sustained swimming activity. The acid labile myosin ATPase activity characteristic of fast twitch fibres is present in the white fibres of most species, however in the white muscle of Gobius paganellus the enzyme activity is stable to both acid and alkali and, in addition, there is a scattered distribution of different fibre types in red and, especially, pink muscle. A study of seasonal variation patterns of myosin ATPase in white muscle of mugilidae over a period of two years has demonstrated, in late summer, the appearance of new small diameter fibres, with a high acid stable enzyme activity, that develop into the large diameter acid labile fibres.     

The crystal structure of Escherichia coli maltodextrin phosphorylase provides an explanation for the activity without control in this basic archetype of a phosphorylase.

In animals, glycogen phosphorylase (GP) exists in an inactive (T state) and an active (R state) equilibrium that can be altered by allosteric effectors or covalent modification. In Escherichia coli, the activity of maltodextrin phosphorylase (MalP) is controlled by induction at the level of gene expression, and the enzyme exhibits no regulatory properties. We report the crystal structure of E. coli maltodextrin phosphorylase refined to 2.4 A resolution. The molecule consists of a dimer with 796 amino acids per monomer, with 46% sequence identity to the mammalian enzyme. The overall structure of MalP shows a similar fold to GP and the catalytic sites are highly conserved. However, the relative orientation of the two subunits in E. coli MalP is different from both the T and R state GP structures, and there are significant changes at the subunit-subunit interfaces. The sequence changes result in loss of each of the control sites present in rabbit muscle GP. As a result of the changes at the subunit interface, the 280s loop, which in T state GP acts as a gate to control access to the catalytic site, is held in an open conformation in MalP. The open access to the conserved catalytic site provides an explanation for the activity without control in this basic archetype of a phosphorylase.     

Glucagon control of glycogenolysis in catfish tissues

1. In catfish (Ictalurus melas) after glucagon treatment blood glucose increased until 150 min, then it gradually decreased towards control values at the 5th hr. 2. In glucagon treated fish, liver glycogen levels were significantly lower then in controls 30 min after hormone administration; thereafter, liver glycogen levels returned rapidly to initial values. Glucagon did not induce any significant effect on the glycogen content in white and red muscles. 3. In liver slices, the addition of glucagon to the incubation medium significantly enhanced the glycogen phosphorylase activity and decreased the level of glycogen. Both phosphorylase activity and glycogen content of white and red muscle slices were practically unaffected by glucagon.     

The cyclin-dependent kinase (CDK) inhibitor flavopiridol inhibits glycogen phosphorylase

Flavopiridol has been shown to induce cell cycle arrest and apoptosis in various tumor cells in vitro and in vivo. Using immobilized flavopiridol, we identified glycogen phosphorylases (GP) from liver and brain as flavopiridol binding proteins from HeLa cell extract. Purified rabbit muscle GP also bound to the flavopiridol affinity column. GP is the rate-limiting enzyme in intracellular glycogen breakdown. Flavopiridol significantly inhibited the AMP-activated GP-b form of the purified rabbit muscle isoenzyme (IC50 of 1 microM at 0.8 mM AMP), but was less inhibitory to the active phosphorylated form of GP, GP-a (IC50 of 2.5 microM). The AMP-bound GP-a form was poorly inhibited by flavopiridol (40% at 10 microM). Increasing concentrations of the allosteric effector AMP resulted in a linear decrease in the GP-inhibitory activity of flavopiridol suggesting interference between flavopiridol and AMP. In contrast the GP inhibitor caffeine had no effect on the relative GP inhibition by flavopiridol, suggesting an additive effect of caffeine. Flavopiridol also inhibited the phosphorylase kinase-catalyzed phosphorylation of GP-b by inhibiting the kinase in vitro. Flavopiridol thus is able to interfere with both activating modifications of GP-b, AMP activation and phosphorylation. In A549 NSCLC cells flavopiridol treatment caused glycogen accumulation despite of an increase in GP activity, suggesting direct GP inhibition in vivo rather than inhibition of GP activation by phosphorylase kinase. These results suggest that the cyclin-dependent kinase inhibitor flavopiridol interferes with glycogen degradation, which may be responsible for flavopiridol's cytotoxicity and explain its resistance in some cell lines.     

Enzyme alteration in skeletal muscle of mice with muscular dystrophy.

1. Developmental enzyme alterations were investigated in skeletal muscle of the hereditary progressive muscular dystrophy (PMD) mice of C57BL/6J strain. 2. Enzymes examined were classified into three groups according to changes of activities in dystrophy muscle during ageing. Activities of creatine kinase (EC 2.7.3.2), pyruvate kinase (EC 2.7.1.40), glycogen phosphorylase (EC 2.4.1.1), and fructose-biphosphate aldolase (EC 4.1.2.13), each of which had the respective muscle specific isoenzyme of extremely high activity in normal adult skeletal muscle, decreased rapidly in dystrophy muscle from the early stage of the disease with ageing. Activities of glycogen synthase (EC 2.4.1.11) and hexokinase (EC 2.7.1.1) were higher in dystrophy muscle in the early stage but decreased gradually to lower levels than those in the control with ageing. Activities of glucose-6-phosphate dehydrogenase (EC 1.1.1.49) were always much higher in dystrophy muscle than in the control, with no relation to ageing. 3. Isoenzymes of creatine kinase, pyruvate kinase and phosphorylase in dystrophy muscle were mainly the muscle types, indicating that muscle differentiation was not blocked profoundly even in dystrophy muscle. In limited cases, especially in the early stage of the disease, very weak activities of the non-muscle fetal type isoenzymes of creatine kinase and phosphorylase were detected, apparently associated with partial muscle regeneration in dystrophy muscle.     

Role of the sympathoadrenal system in the regulation of glycogen metabolism in resting and exercising skeletal muscles.

The purpose of the present study was to characterize the role of catecholamines in the regulation of skeletal muscle glycogen metabolism during exercise. Using the rat hindlimb perfusion technique we have measured skeletal muscle glycogen content, glycogen phosphorylase and synthase activities in sympathectomized and/or demedullated rats under epinephrine treatment (10(-7) M) at rest and during muscle contraction. When epinephrine and/or norepinephrine deficiency was induced, muscle contraction resulted in a decrease in glycogen content (-63%) despite a decrease in glycogen phosphorylase activity ratio (0.25 to 0.11; p less than 0.001) and an increase in glycogen synthase activity ratio (0.13 to 0.27; p less than 0.001). Under these conditions, epinephrine treatment further reduced glycogen content while blunting the changes in the activity ratio of the rate-limiting enzymes. These data indicate that catecholamines do not play a primary role in skeletal muscle glycogen breakdown during acute exercise and suggest that allosteric regulators may be of prime importance.     

Glycogen and glycogen phosphorylase associated with sarcoplasmic reticulum: effects of fatiguing activity.

The purpose of the present study was to investigate the effects of fatiguing muscular activity on glycogen, glycogen phosphorylase (GP), and Ca(2+) uptake associated with the sarcoplasmic reticulum (SR). Tetanic contractions (100 ms, 75 Hz) of the gastrocnemius and plantaris muscles, elicited once per second for 15 min, significantly reduced force to 26.5 +/- 4.0% and whole muscle glycogen to 23% of rested levels. SR glycogen levels were 415.4 +/- 76.6 and 20.4 +/- 2.1 microg/mg SR protein in rested and fatigued samples, respectively. The optical density of GP from SDS-PAGE was reduced to 21% of control, whereas pyridoxal 5'-phosphate concentration, a quantitative indicator of GP content, was significantly reduced to 3% of control. GP activity after exercise, in the direction of glycogen breakdown, was reduced to 4% of control. Maximum SR Ca(2+) uptake rate was also significantly reduced to 81% of control. These data demonstrate that glycogen and GP associated with skeletal muscle SR are reduced after fatiguing activity.     

Glycogenolysis during recovery from muscular work. The time course of phosphorylase activity is dependent on Pi concentration in the abdominal muscle of the shrimp Crangon crangon

1) Glycogen is degraded in the abdominal muscle of the shrimp Crangon crangon (Decapoda, Crustacea) during the recovery period following work. The regulation of post-exercise glycogen breakdown and the properties of glycogen phosphorylase (EC 2.4.1.1) have been studied: 2) Glycogen phosphorylase exists as unphosphorylated b-form and phosphorylated a-form, the latter contains 1 molecule phosphate/subunit. Both forms of phosphorylase are dimers, isoenzymes have not been detected. 3) The purified b-form is inactive in absence of AMP and has very low affinities for AMP and Pi. For half-maximum activation 0.33 +/- 0.04 mM AMP is necessary, and the Km-value for Pi at 1 mM AMP is 48 +/- 5 mM. IMP does not affect the activity of the b-form. 4) The a-form is active without effectors, its Km-value for Pi is 5.3 +/- 1.5 mM. The proportion of phosphorylase a increases in vivo, from about 25% at rest, to approximately 90% upon work and remains at this high level during the first minutes of recovery. 5) It is concluded that the glycogenolytic flux in the abdominal muscle of the shrimp even during post-exercise periods depends on the level of the a-form the activity of which is restricted in time and extent by the cytoplasmic Pi concentration (Kamp, G. & Juretschke, H. P. (1987) Biochim. Biophys. Acta 929, 121-127).