Identification of two forms of glycogen phosphorylase in Dictyostelium

It has been known for 20 years that during cellular differentiation of Dictyostelium discoideum, glycogen is degraded to provide the glucose precursors that are required for the synthesis of the end-products of development. Because this pathway provided a distinct developmentally regulated event, a number of laboratories have investigated the regulation of the first step in glycogen degradation, glycogen phosphorylase. Of particular interest was the possible regulation of this enzyme by cAMP. Cyclic AMP is know to act as a signal in this organism for both chemotaxis and cell differentiation. The phosphorylase activity was found to increase during development and, therefore, it has been used in many studies as a marker for late stage development. However, only one form of the phosphorylase was found, and therefore it was concluded that cAMP was not involved in regulation of this key step in the developmental pathway. Here we report the discovery of a second form of the enzyme. This form is completely dependent on AMP for activity and is found only in the undifferentiated stage. This second form contains several of the properties of the nonphosphorylated enzyme that occurs in systems that are regulated by cAMP. This result and the recent discovery of a cAMP-dependent protein kinase has rekindled the possibility that at least one of the effects of cAMP in this organism occurs via a cAMP-dependent cascade of phosphorylation; that is, the activation of glycogen phosphorylase and subsequent production of the precursors for the end-products of development.     

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.     

A hypersensitivity of glycogen phosphorylase activation in hearts of diabetic rats

This study was initiated to determine whether glycogen phosphorylase activation was defective in hearts of alloxan diabetic rats. When hearts were perfused by gravity flow for 1 to 10 min with various concentrations of epinephrine, activation of glycogen phosphorylase in the diabetic was significantly greater at every time and epinephrine concentration than that seen in the normal. Cyclic AMP accumulation and protein kinase activation by epinephrine in the diabetic were not appreciably different or were lower than the normal responses to the hormone. The effects of epinephrine on cAMP and protein kinase were blocked in both normal and diabetic hearts by propranolol. While the beta blocker prevented phosphorylase activation in the normal hearts, it did not block phosphorylase activation by epinephrine in the diabetic hearts. Likewise, the alpha agonist phenylephrine activated phosphorylase in the diabetic but not in the normal hearts. While glucagon produced the same phosphorylase hypersensitivity in diabetic hearts, the cAMP and protein kinase responses were not altered by diabetes. Phosphorylase phosphatase activity was found to be unaltered by either epinephrine or diabetes, whereas phosphorylase kinase activation by epinephrine in the diabetic was double the normal response. These data are consistent with a diabetes-related unmasking of an alpha effect on cardiac phosphorylase activation and an unexplained increase in the sensitivity of phosphorylase kinase activation by protein kinase.     

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.     

5-Aminoimidazole-4-carboxamide 1-beta -D-ribofuranoside (AICAR) stimulates myocardial glycogenolysis by allosteric mechanisms

We tested the hypothesis that activation of AMP-activated protein kinase (AMPK) promotes myocardial glycogenolysis by decreasing glycogen synthase (GS) and/or increasing glycogen phosphorylase (GP) activities. Isolated working hearts from halothane-anesthetized male Sprague-Dawley rats perfused in the absence or presence of 0.8 or 1.2 mM 5-aminoimidazole-4-carboxamide 1-beta-d-ribofuranoside (AICAR), an adenosine analog and cell-permeable activator of AMPK, were studied. Glycogen degradation was increased by AICAR, while glycogen synthesis was not affected. AICAR increased myocardial 5-aminoimidazole-4-carboxamide 1-beta-d-ribofuranotide (ZMP), the active intracellular form of AICAR, but did not alter the activity of GS and GP measured in tissue homogenates or the content of glucose-6-phosphate and adenine nucleotides in freeze-clamped tissue. Importantly, the calculated intracellular concentration of ZMP achieved in this study was similar to the K(m) value of ZMP for GP determined in homogenates of myocardial tissue. We conclude that the data are consistent with allosteric activation of GP by ZMP being responsible for the glycogenolysis caused by AICAR in the intact rat heart.     

Activation of protein kinase and glycogen phosphorylase in isolated rat liver cells by glucagon and catecholamines.

In liver cells isolated from fed female rats, glucagon (290nM) increased adenosine 3':5'-monophosphate (cyclic AMP) content and decreased cyclic AMP binding 30 s after addition of hormones. Both returned to control values after 10 min. Glucagon also stimulated cyclic AMP-independent protein kinase activity at 30 s and decreased protein kinase activity assayed in the presence of 2 muM cyclic AMP at 1 min. Glucagon increased the levels of glycogen phosphorylase a, but there was no change in total glycogen phosphorylase activity. Glucagon increased glycogen phosphorylase a at concentrations considerably less than those required to affect cyclic AMP and protein kinase. The phosphodiesterase inhibitor, 1-methyl-3-isobutyl xanthine, potentiated the action of glucagon on all variables, but did not increase the maximuM activation of glycogen phosphorylase. Epinephrine (1muM) decreased cyclic AMP binding and increased glycogen phosphorylase a after a 1-min incubation with cells. Although 0.1 muM epinephrine stimulated phosphorylase a, a concentration of 10 muM was required to increase protein kinase activity. 1-Methyl-3-isobutyl xanthine (0.1 mM) potentiated the action of epinephrine on cyclic AMP and protein kinase. (-)-Propranolol (10muM) completely abolished the changes in cyclic AMP binding and protein kinase due to epinephrine (1muM) in the presence of 0.1mM 1-methyl-3-isobutyl xanthine, yet inhibited the increase in phosphorylase a by only 14 per cent. Phenylephrine (0.1muM) increased glycogen phosphorylase a, although concentrations as great as 10 muM failed to affect cyclic AMP binding or protein kinase in the absence of phosphodiesterase inhibitor. Isoproterenol (0.1muM) stimulated phosphorylase and decreased cyclic AMP binding, but only a concentration of 10muM increased protein kinase. 1-Methyl-3-isobutyl xanthine potentiated the action of isoproterenol on cyclic AMP binding and protein kinase, and propranolol reduced the augmentation of glucose release and glycogen phosphorylase activity due to isoproterenol. These data indicate that both alpha- and beta-adrenergic agents are capable of stimulating glycogenolysis and glycogen phosphorylase a in isolated rat liver cells. Low concentrations of glucagon and beta-adrenergic agonists stimulate glycogen phosphorylase without any detectable increase in cyclic AMP or protein kinase activity. The effects of alpha-adrenergic agents appear to be completely independent of changes in cyclic AMP protein kinase activity.     

Activation of endogenous phosphorylase kinase in liver glycogen pellet by cAMP-dependent protein kinase

Liver glycogen phosphorylase associated with the glycogen pellet was activated by a MgATP-dependent process. This activation was reduced by 90% by ethylene glycol bis(beta-aminoethyl ether)N,N,N',N'-tetraacetic acid, not affected by the inhibitor of the cAMP-dependent protein kinase, and increased 2.5-fold by the catalytic subunit of cAMP-dependent protein kinase. Low levels of free Ca2+ (8 x 10(-8) M) completely prevented the effects of the chelator. The activation of phosphorylase by MgATP was shown not to be due to formation of AMP. DEAE-cellulose chromatography of the glycogen pellet separated phosphorylase from phosphorylase kinase. The isolated phosphorylase was no longer activated by MgATP in the presence or absence of the catalytic subunit of cAMP-dependent protein kinase. The isolated phosphorylase kinase phosphorylated and activated skeletal muscle phosphorylase b and the activation was increased 2- to 3-fold by the catalytic subunit of cAMP-dependent protein kinase. Mixing the isolated phosphorylase and phosphorylase kinase together restored the effects of MgATP and the catalytic subunit of cAMP-dependent protein kinase on phosphorylase activity. These findings demonstrate that the phosphorylase kinase associated with liver glycogen has regulatory features similar to those of muscle phosphorylase kinase.     

Effects of catecholamines and glucagon on glycogen metabolism in human polymorphonuclear leukocytes.

Addition of 10 micron of the alpha-adrenergic agonist phenylephrine to polymorphonuclear leukocytes suspended in glucose-free Krebs-Ringer bicarbonate buffer (pH 6.7) activated phosphorylase, inactivated glycogen synthase R maximally within 30 s, and resulted in glycogen breakdown. Phenylephrine increased 45Ca efflux relative to control of 45Ca prelabelled cells, but did not affect cyclic adenosine 3',5'-monophosphate (cAMP) concentration. The effects of phenylephrine were blocked by 20 micron phentolamine and were absent in cells incubated at pH 7.4. The same unexplained dependency of extracellular pH was observed with 2.5 nM--2.5 micron glucagon, which activated phosphorylase and inactivated synthase-R, but in addition caused a 30-s burst in cAMP formation. 25 nM glucagon also increased 45Ca efflux. The activation of phosphorylase by phenylephrine and possibly also by glucagon are thought mediated by an increased concentration of cytosolic Ca2+ activating phosphorylase kinase. The effects of 5 micron isoproterenol or 5 micron epinephrine were independent of extracellular pH 6.7 and 7.4 and resulted in a sustained increase in cAMP, an activation of phosphorylase and inactivation of synthase-R within 15 s, and in glycogenolysis. The effects of both compounds were blocked by 10 micron propranolol, whereas 10 micron phentolamine had no effect on the epinephrine action. The efflux of 45Ca was not affected by either isoproterenol or epinephrine. The beta-adrenergic activation of phosphorylase is consistent with the assumption of a covalent modification of phosphorylase kinase by the cAMP dependent protein kinase. Phosphorylation of synthase-R to synthase-D can thus occur independently of increase in cAMP, but the evidence is inconclusive with respect to the cAMP dependent protein kinase also being active in this phosphorylation.     

Control of phosphorylase kinase in the isolated glycogen particle by Ca2+-Mg2+ synergistic activation and cAMP-dependent phosphorylation

The isolated glycogen particle provides a means to examine the regulation of glycogen metabolism with the components organized in a functional cellular complex. With this system, we have studied the control of phosphorylase kinase activation by Ca2+ and cAMP. Contrary to a previous report (Heilmeyer, L. M. G., Jr., Meyer, F., Haschke, R. H., and Fisher, E. H. (1980) J. Biol. Chem. 245, 6649-6656), phosphorylase kinase became activated during incubation of the glycogen particle with MgATP2- and Ca2+. Part of this activation could be attributed to the action of the cAMP-dependent protein kinase; however, it was not possible to quantitatively correlate activation with phosphorylation in the presence of Ca2+ and Mg2+ due to a large, but uncertain, contribution of synergistic activation caused by these ions. This latter activation had properties similar to those described by King and Carlson (King, M. M., and Carlson, G. M. (1980) Arch. Biochem. Biophys. 209, 517-523) with the purified enzyme, and its occurrence also explains why phosphorylase kinase activation in the glycogen particle was not observed previously. The cAMP-dependent activation of phosphorylase kinase in the glycogen particle has been characterized. It occurred in a similar manner when either the cAMP-dependent protein kinase or cAMP was added, thus indicating that the phosphorylation sites of phosphorylase kinase complexed in the glycogen particle were accessible to endogenous or exogenous enzyme. In the glycogen particle, both the alpha and beta subunits were phosphorylated by the cAMP-dependent protein kinase, but the alpha subunit dephosphorylation appeared to be preferentially regulated by Ca2+. The activity of phosphorylase kinase in the glycogen particle is regulated by the phosphorylation of both the alpha and beta subunits.     

Hormonal regulation of hepatic glycogen synthase phosphatase

Perfusion of livers from fed rats with medium containing glucagon (2 x 10(-10) or 1 x 10(-8) M) resulted in both time- and concentration-dependent inactivation of glycogen synthase phosphatase. Expected changes occurred in cAMP, cAMP-dependent protein kinase, glycogen synthase, and glycogen phosphorylase. The effect of glucagon on synthase phosphatase was partially reversed by simultaneous addition of insulin (4 x 10(-8) M), an effect paralleled by a decrease in cAMP. Addition of arginine vasopressin (10 milliunits/ml) resulted in a similar inactivation of synthase phosphatase and activation of phosphorylase, but independent of any changes in cAMP or its kinase. Phosphorylase phosphatase activity was unaffected by any of these hormones. Synthase phosphatase activity, measured as the ability of a crude homogenate to catalyze the conversion of purified rat liver synthase D to the I form, was no longer inhibited by glucagon or vasopressin when phosphorylase antiserum was added to the phosphatase assay mixture in sufficient quantity to inhibit 90-95% of the phosphorylase a activity. These data support the following conclusions: 1) hepatic glycogen synthase phosphatase activity is acutely modulated by hormones, 2) hepatic glycogen synthase phosphatase and phosphorylase phosphatase are regulated differently, 3) the hormone-mediated changes in synthase phosphatase cannot be explained by an alteration of the synthase D molecule affecting its behavior as a substrate, and 4) glycogen synthase phosphatase activity is at least partially controlled by the level of phosphorylase a.     

Modulation of glycogen phosphorylase activity affects 5-phosphoribosyl-1-pyrophosphate availability in rat hepatocyte cultures

The effect of modulation of the rate of glycogenolysis on the availability of 5-phosphoribosyl-1-pyrophosphate (PRPP) was investigated in rat hepatocyte cultures. Dibutyryl cyclic AMP (dbcAMP), forskolin and glucagon, activating glycogen phosphorylase through activation of protein kinase A (PKA), were found to raise PRPP availability by 44%-56%. Arg-vasopressin and phenylephrine, activating glycogen phosphorylase through the phosphoinositide cascade, did not affect PRPP availability. dbcAMP, but not phenylephrine, increased the degradation of pre labeled glycogen by 57%. Caffeine and CP-91149, inhibitors of glycogen phosphorylase, decreased PRPP availability by 33% and 43%, respectively. The finding that induction of glycogenolysis enhances, and inhibition of glycogenolysis decelerates PRPP generation suggests that glycogenolysis is a major contributor to PRPP generation in liver tissue in the basal (postabsorptive) state.     

Cellular regulation by protein phosphorylation

A historical account of the discovery of reversible protein phosphorylation is presented. This process was uncovered in the mid 1950s in a study undertaken with Edwin G. Krebs to elucidate the complex hormonal regulation of skeletal muscle glycogen phosphorylase. Contrary to the known activation of this enzyme by AMP which serves as an allosteric effector, its hormonal regulation results from a phosphorylation of the protein by phosphorylase kinase following the activation of the latter by Ca²⁺ and ATP. The study led to the establishment of the first hormonal cascade of successive enzymatic reactions, kinases acting on kinases, initiated by cAMP discovered by Earl Sutherland. It also showed how two different physiological processes, carbohydrate metabolism and muscle contraction, could be regulated in concert.     

Amylin activates glycogen phosphorylase and inactivates glycogen synthase via a cAMP-independent mechanism.

Although the novel pancreatic peptide amylin has been shown to induce insulin resistance and decrease glucose uptake, the mechanism of amylin's actions is unknown. The following study evaluated the effect of amylin on glycogen metabolism in isolated soleus muscles in the presence and absence of insulin (200 microU/ml). Total glycogen, glycogen phosphorylase and glycogen synthases activities, and cAMP levels were measured. Total glycogen levels were significantly decreased by amylin (100 nM) in fed or fasted muscles under conditions of insulin stimulation. Amylin (100 nM) activated glycogen phosphorylase by as much as 100% and decreased glycogen synthase activity by over 60%, depending on the metabolic state of the muscles. These effects where comparable to those of the beta adrenergic agonist isoproterenol. A lower concentration of amylin (1 nM) did not significantly affect glycogen levels, glycogen phosphorylase, or glycogen synthase activity. Cyclic AMP levels were increased two-fold by isoproterenol but were unaffected by amylin. In conclusion, amylin induces glycogenolysis by decreasing glycogen synthesis and increasing breakdown. The effect of amylin on enzyme activity is consistent with a phosphorylation-dependent mechanism. It is likely that these events are mediated via a cAMP independent protein kinase.     

Bioactivity of glycogen phosphorylase inhibitors that bind to the purine nucleoside site

The bioactivity in hepatocytes of glycogen phosphorylase inhibitors that bind to the active site, the allosteric activator site and the indole carboxamide site has been described. However, the pharmacological potential of the purine nucleoside inhibitor site has remained unexplored. We report the chemical synthesis and bioactivity in hepatocytes of four new olefin derivatives of flavopiridol (1-4) that bind to the purine site. Flavopiridol and 1-4 counteracted the activation of phosphorylase in hepatocytes caused by AICAR (5-aminoimidazole-4-carboxamide 1-beta-D-ribofuranoside), which is metabolised to an AMP analogue. Unlike an indole carboxamide inhibitor, the analogues 1 and 4 suppressed the basal rate of glycogenolysis in hepatocytes by allosteric inhibition rather than by inactivation of phosphorylase, and accordingly caused negligible stimulation of glycogen synthesis. However, they counteracted the stimulation of glycogenolysis by dibutyryl cAMP by both allosteric inhibition and inactivation of phosphorylase. Cumulatively, the results show key differences between purine site and indole carboxamide site inhibitors in terms of (i) relative roles of dephosphorylation of phosphorylase-a as compared with allosteric inhibition, (ii) counteraction of the efficacy of the inhibitors on glycogenolysis by dibutyryl-cAMP and (iii) stimulation of glycogen synthesis.     

Cyclic AMP-mediated activation of hepatic glycogenolysis by fructose

Isolated livers from fed and fasted rats were perfused for 30 min with recirculating blood-buffer medium containing no added substrate and then switched to a flow-through perfusion using the same medium for an additional 5, 10 and 30 min. Continous infusion of fructose for the final 5, 10 or 30 min resulted in activation of glycogen phosphorylase, an increase in the activity of protein kinase, elevated levels of tissue adenosine 3′,5′-monphosphate (cylic AMP), and no consistent effect on glycogen synthase. Infusion of glucose under the same conditions resulted in activation of glycogen synthase, inactivation of glycogen phosphorylase, no change in protein kinase, and no consistent change in tissue cyclic AMP. These results demonstrate that while glucose promotes hepatic glycogen synthesis, fructose promotes activation of the enzymatic cascade responsible for glycogen breakdown.     

Cyclic AMP-mediated activation of hepatic glycogenolysis by fructose.

Isolated livers from fed and fasted rats were perfused for 30 min with recirculating blood-buffer medium containing no added substrate and then switched to a flow-through perfusion using the same medium for an additional 5, 10 and 30 min. Continuous infusion of fructose for the final 5, 10 or 30 min resulted in activation of glycogen phosphorylase, an increase in the activity of protein kinase, elevated levels of tissue adenosine 3', 5'-monophosphate (cyclic AMP), and no consistent effect on glycogen synthase. Infusion of glucose under the same conditions resulted in activation of glycogen synthase, inactivation of glycogen phosphorylase, no change in protein kinase, and no consistent change in tissue cyclic AMP. These results demonstrate that while glucose promotes hepatic glycogen synthesis, fructose promotes activation of the enzymatic cascade responsible for glycogen breakdown.     

The control of glycogen metabolism in yeast. 1. Interconversion in vivo of glycogen synthase and glycogen phosphorylase induced by glucose, a nitrogen source or uncouplers

The addition of glucose to a suspension of yeast initiated glycogen synthesis and ethanol formation. Other effects of the glucose addition were a transient rise in the concentration of cyclic AMP and a more prolonged increase in the concentration of hexose 6-monophosphate and of fructose 2,6-bisphosphate. The activity of glycogen synthase increased about 4-fold and that of glycogen phosphorylase decreased 3-5-fold. These changes could be reversed by the removal of glucose from the medium and induced again by a new addition of the sugar. These effects of glucose were also obtained with glucose derivatives known to form the corresponding 6-phosphoester. Similar changes in glycogen synthase and glycogen phosphorylase activity were induced by glucose in a thermosensitive mutant deficient in adenylate cyclase (cdc35) when incubated at the permissive temperature of 26 degrees C, but were much more pronounced at the nonpermissive temperature of 35 degrees C. Under the latter condition, glycogen synthase was nearly fully activated and glycogen phosphorylase fully inactivated. Such large effects of glucose were, however, not seen in another adenylate-cyclase-deficient mutant (cyr1), able to incorporate exogenous cyclic AMP. When a nitrogen source or uncouplers were added to the incubation medium after glucose, they had effects on glycogen metabolism and on the activity of glycogen synthase and glycogen phosphorylase which were directly opposite to those of glucose. By contrast, like glucose, these agents also caused, under most experimental conditions, a detectable rise in cyclic AMP concentration and a series of cyclic-AMP-dependent effects such as an activation of phosphofructokinase 2 and of trehalase and an increase in the concentration of fructose 2,6-bisphosphate and in the rate of glycolysis. Under all experimental conditions, the rate of glycolysis was proportional to the concentration of fructose 2,6-bisphosphate. Uncouplers, but not a nitrogen source, also induced an activation of glycogen phosphorylase and an inactivation of glycogen synthase when added to the cdc35 mutant incubated at the restrictive temperature of 35 degrees C without affecting cyclic AMP concentration.     

Role of AMP on the activation of glycogen synthase and phosphorylase by adenosine, fructose, and glutamine in rat hepatocytes

The mechanism for glycogen synthesis stimulation produced by adenosine, fructose, and glutamine has been investigated. We have analyzed the relationship between adenine nucleotides and glycogen metabolism rate-limiting enzymes upon hepatocyte incubation with these three compounds. In isolated hepatocytes, inhibition of AMP deaminase with erythro-9-(2-hydroxyl-3nonyl)adenine further increases the accumulation of AMP and the activation of glycogen synthase and phosphorylase by fructose. This ketose does not increase cyclic AMP or the activity of cyclic AMP-dependent protein kinase. Adenosine raises AMP and ATP concentration. This nucleotide also activates glycogen synthase and phosphorylase by covalent modification. The correlation coefficient between AMP and glycogen synthase activity is 0.974. Nitrobenzylthioinosine, a transport inhibitor of adenosine, blocks (by 50%) the effect of the nucleoside on AMP formation and glycogen synthase but not on phosphorylase. 2-Chloroadenosine and N6-phenylisopropyladenosine, nonmetabolizable analogues of adenosine, activate phosphorylase (6-fold) without increasing the concentration of adenine nucleotides or the activity of glycogen synthase. Cyclic AMP is not increased by adenosine in hepatocytes from starved rats but is in cells from fed animals. [Ethylenebis (oxyethylenenitrilo)]tetraacetic acid (EGTA) blocks by 60% the activation of phosphorylase by adenosine but not that of glycogen synthase. Glutamine also increases AMP concentration and glycogen synthase and phosphorylase activities, and these effects are blocked by 6-mercaptopurine, a purine synthesis inhibitor. Neither adenosine nor glutamine increases glucose 6-phosphate. It is proposed that the observed efficient glycogen synthesis from fructose, adenosine, and glutamine is due to the generation of AMP that activates glycogen synthase probably through increases in synthase phosphatase activity. It is also concluded that the activation of phosphorylase by the above-mentioned compounds can be triggered by metabolic changes.     

Regulation of the activity of rabbit skeletal muscle phosphorylase kinase by glycogen

The effects of glycogen on the non-activated and activated forms of phosphorylase kinase were studied. It was found that in the presence of glycogen the activity of non-activated kinase at pH 6.8 and 8.2 and that of the activated (in the course of phosphorylation) form are enhanced. The degree of activation depends on glycogen concentration. At saturating concentrations, this enzyme activity increases 2-3-fold; the enzyme affinity for the protein substrate, phosphorylase b, also shows an increase. The polysaccharide has no effect on the activity of phosphorylase kinase stimulated by limited proteolysis. In the presence of glycogen, the rate of autocatalytic phosphorylation of the enzyme is increased. Glycogen stabilizes the enzyme activity upon dilution. The experimental results suggest that the polysaccharide directly affects the phosphorylase kinase molecule. The maximal binding was shown to occur at the enzyme/polysaccharide ratio of 1:10 (w/w) in the presence of Ca2+ and Mg2+.     

Chronic activation of 5'-AMP-activated protein kinase increases GLUT-4, hexokinase, and glycogen in muscle.

This study was designed to determine whether chronic chemical activation of AMP-activated protein kinase (AMPK) would increase glucose transporter GLUT-4 and hexokinase in muscles similarly to periodic elevation of AMPK that accompanies endurance exercise training. The adenosine analog, 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), has previously been shown to be taken up by cells and phosphorylated to form a compound (5-aminoimidazole-4-carboxamide ribonucleotide) that mimics the effect of AMP on AMPK. A single injection of AICAR resulted in a marked increase in AMPK in epitrochlearis and gastrocnemius/plantaris muscles 60 min later. When rats were injected with AICAR (1 mg/g body wt) for 5 days in succession and were killed 1 day after the last injection, GLUT-4 was increased by 100% in epitrochlearis muscle and by 60% in gastrocnemius muscle in response to AICAR. Hexokinase was also increased approximately 2. 5-fold in the gastrocnemius/plantaris. Gastrocnemius glycogen content was twofold higher in AICAR-treated rats than in controls. Chronic chemical activation of AMPK, therefore, results in increases in GLUT-4 protein, hexokinase activity, and glycogen, similarly to those induced by endurance training.