scholarly journals The stimulation of glycogenolysis in isolated hepatocytes by opioid peptides

1986 ◽  
Vol 238 (2) ◽  
pp. 531-535 ◽  
Author(s):  
R P Leach ◽  
M A Titheradge
Keyword(s):  
Cyclic Amp ◽  
Opioid Peptides ◽  
Glycogen Phosphorylase ◽  
Glycogen Synthase ◽  
Glycogen Metabolism ◽  
Isolated Hepatocytes ◽  
Dependent Protein ◽  
Simultaneous Decrease ◽  
Glycogen Phosphorylase Activity ◽  
Stimulation Of

The opioid peptides [Leu]enkephalin and dynorphin-(1-13) were shown to enhance glycogen breakdown when added directly to hepatocytes. This was the result of a concerted effect on the enzymes of glycogen metabolism, with a stimulation of glycogen phosphorylase activity and a simultaneous decrease in glycogen synthase I activity. The latter only became significant when the enzyme was activated by incubating the cells in presence of 20 mM- or 40 mM-glucose. The effect of the opioid peptides was independent of an increase in cyclic AMP or any change in the activity ratio of the cyclic AMP-dependent protein kinase and was abolished by depleting the cells of Ca2+. Both [Leu]enkephalin and dynorphin-(1-13) produced a significant decrease in cyclic AMP formation, suggesting that in liver, as in neuronal tissue, they may act by inhibiting adenylate cyclase activity.

Protein phosphorylation and the control of glycogen metabolism in skeletal muscle

1983 ◽  
Vol 302 (1108) ◽  
pp. 13-25 ◽  
Keyword(s):  
Skeletal Muscle ◽  
Cyclic Amp ◽  
Protein Phosphatase ◽  
Glycogen Synthase ◽  
Glycogen Metabolism ◽  
Catalytic Subunit ◽  
Glycogen Synthase Kinase ◽  
Dependent Protein Kinase ◽  
Dependent Protein

Glycogen metabolism in mammalian skeletal muscle is controlled by a regulatory network in which six protein kinases, four protein phosphatases and several thermostable regulatory proteins determine the activation state of glycogen phosphorylase and glycogen synthase, the rate-limiting enzymes of this process. Thirteen phosphorylation sites are involved, twelve of which have been isolated and sequenced and shown to be phosphorylated in vivo . The effects of adrenalin and insulin on the state of phosphorylation of each site have been determined. The neural control of glycogen metabolism is mediated by calcium ions and involves phosphorylase kinase, and a specific calmodulin-dependent glycogen synthase kinase. The β-adrenergic control of the system is mediated by cyclic AMP, and involves the phosphorylation of phosphorylase kinase, glycogen synthase and inhibitor 1 by cyclic-AMP-dependent protein kinase. Inhibitor 1 is a specific inhibitor of protein phosphatase 1, the major phosphatase involved in the control of glycogen metabolism. The stimulation of glycogen synthesis by insulin results from the dephosphorylation of glycogen synthase at sites (3 a + 3 b + 3 c ), which are introduced by the enzyme glycogen synthase kinase 3. The structure, regulation and substrate specificities of the protein phosphatases involved in glycogen metabolism are reviewed. Protein phosphatase 1 can exist in an inactive form termed the Mg-ATP-dependent protein phosphatase, which consists of a complex between the catalytic subunit and a thermostable protein termed inhibitor 2. Activation of this complex is catalysed by glycogen synthase kinase 3. It involves the phosphorylation of inhibitor 2 and its dissociation from the catalytic subunit. Protein phosphatase 2A can be resolved into three forms by ion exchange chromatography. These species contain the same catalytic subunit and other subunits that may have a regulatory function. Protein phosphatase 2B is a Ca 2+ -dependent enzyme composed of two subunits, A and B. Its activity is increased tenfold by calmodulin, which interacts with the A-subunit. The B-subunit is a Ca 2+ -binding protein that is homologous with calmodulin. Its N-terminus contains the unusual myristyl blocking group, only found previously in the catalytic subunit of cyclic-AMP-dependent protein kinase. Protein phosphatase 2C is a Mg 2+ -dependent enzyme that accounts for a very small proportion of the glycogen synthase phosphatase activity in skeletal muscle. It is likely to be involved in the regulation of other metabolic processes in vivo such as cholesterol synthesis. Recent evidence suggests that many of the proteins involved in the control of glycogen metabolism have much wider roles, and that they participate in the neural and hormonal regulation of a variety of intracellular processes.

scholarly journals Effects of vasopressin and La3+ on plasma-membrane Ca2+ inflow and Ca2+ disposition in isolated hepatocytes. Evidence that vasopressin inhibits Ca2+ disposition

1986 ◽  
Vol 238 (3) ◽  
pp. 793-800 ◽  
Author(s):  
B P Hughes ◽  
S E Milton ◽  
G J Barritt
Keyword(s):  
Plasma Membrane ◽  
Glycogen Phosphorylase ◽  
Subcellular Fractionation ◽  
Isolated Hepatocytes ◽  
Ionophore A23187 ◽  
Phosphorylase Activity ◽  
45Ca Uptake ◽  
Maximal Stimulation ◽  
Glycogen Phosphorylase Activity ◽  
Stimulation Of

Vasopressin caused a 40% inhibition of 45Ca uptake after the addition of 0.1 mM-45Ca2+ to Ca2+-deprived hepatocytes. At 1.3 mM-45Ca2+, vasopressin and ionophore A23187 each caused a 10% inhibition of 45Ca2+ uptake, whereas La3+ increased the rate of 45Ca2+ uptake by Ca2+-deprived cells. Under steady-state conditions at 1.3 mM extracellular Ca2+ (Ca2+o), vasopressin and La3+ each increased the rate of 45Ca2+ exchange. The concentrations of vasopressin that gave half-maximal stimulation of 45Ca2+ exchange and glycogen phosphorylase activity were similar. At 0.1 mM-Ca2+o, La3+ increased, but vasopressin did not alter, the rate of 45Ca2+ exchange. The results of experiments performed with EGTA or A23187 or by subcellular fractionation indicate that the Ca2+ taken up by hepatocytes in the presence of La3+ is located within the cell. The addition of 1.3 mM-Ca2+o to Ca2+-deprived cells caused increases of approx. 50% in the concentration of free Ca2+ in the cytoplasm [(Ca2+]i) and in glycogen phosphorylase activity. Much larger increases in these parameters were observed in the presence of vasopressin or ionophore A23187. In contrast with vasopressin, La3+ did not cause a detectable increase in glycogen phosphorylase activity or in [Ca2+]i. It is concluded that an increase in plasma membrane Ca2+ inflow does not by itself increase [Ca2+]i, and hence that the ability of vasopressin to maintain increased [Ca2+]i over a period of time is dependent on inhibition of the intracellular removal of Ca2+.

scholarly journals The stimulation of glycogenolysis and gluconeogenesis in isolated hepatocytes by opioid peptides

1983 ◽  
Vol 216 (2) ◽  
pp. 507-510 ◽  
Author(s):  
E H Allan ◽  
I C Green ◽  
M A Titheradge
Keyword(s):  
Cyclic Amp ◽  
Opioid Peptides ◽  
Isolated Hepatocytes ◽  
Significant Inhibition ◽  
Leucine Enkephalin ◽  
Opioid Agonists ◽  
Independent Mechanism ◽  
Total Glucose ◽  
Glucose Output ◽  
Stimulation Of

The opioid agonists [leucine]enkephalin, [D-Ala2,D-Leu5]enkephalin and dynorphin-(1-13)-peptide, but not morphine, stimulated the conversion of [2-14C]pyruvate into glucose and glycogenolysis when added directly to isolated hepatocytes. Naloxone produced a small but significant inhibition of both the basal and stimulated rate of incorporation of label into glucose but had no effect on the total glucose output by the cells. The effects of the opioid peptides were mediated by a cyclic AMP-independent mechanism.

scholarly journals Diverse effects of two allosteric inhibitors on the phosphorylation state of glycogen phosphorylase in hepatocytes

2002 ◽  
Vol 368 (1) ◽  
pp. 309-316 ◽  
Author(s):  
Theodore LATSIS ◽  
Birgitte ANDERSEN ◽  
Loranne AGIUS
Keyword(s):  
Glycogen Phosphorylase ◽  
Potent Inhibitor ◽  
Glycogen Synthase ◽  
Glycogen Synthesis ◽  
Glycogen Metabolism ◽  
Small Degree ◽  
Allosteric Inhibitors ◽  
Phosphorylation State ◽  
Stimulation Of

Two distinct allosteric inhibitors of glycogen phosphorylase, 1,4-dideoxy-1,4-imino-d-arabinitol (DAB) and CP-91149 (an indole-2-carboxamide), were investigated for their effects on the phosphorylation state of the enzyme in hepatocytes in vitro. CP-91149 induced inactivation (dephosphorylation) of phosphorylase in the absence of hormones and partially counteracted the phosphorylation caused by glucagon. Inhibition of glycogenolysis by CP-91149 can be explained by dephosphorylation of phosphorylase a. This was associated with activation of glycogen synthase and stimulation of glycogen synthesis. DAB, in contrast, induced a small degree of phosphorylation of phosphorylase. This was associated with inactivation of glycogen synthase and inhibition of glycogen synthesis. Despite causing phosphorylation (activation) of phosphorylase, DAB is a very potent inhibitor of glycogenolysis in both the absence and presence of glucagon. This is explained by allosteric inhibition of phosphorylase a, which overrides the increase in activation state. In conclusion, two potent phosphorylase inhibitors exert different effects on glycogen metabolism in intact hepatocytes as a result of opposite effects on the phosphorylation state of both phosphorylase and glycogen synthase.

Regulation of the dephosphorylation of glycogen phosphorylase a and synthase b by glucose and caffeine in isolated hepatocytes

1981 ◽  
Vol 59 (6) ◽  
pp. 387-395 ◽  
Author(s):  
Peter J. Kasvinsky ◽  
Robert J. Fletterick ◽  
Neil B. Madsen
Keyword(s):  
Glycogen Phosphorylase ◽  
Glycogen Synthase ◽  
Physiological Role ◽  
Glycogen Metabolism ◽  
Isolated Hepatocytes ◽  
Effector Site ◽  
Synergistic Regulation ◽  
Hepatic Regulation

Synergistic regulation of glycogen phosphorylase a by the competitive inhibitors glucose and caffeine in vitro indicates a possible physiological role for the negative effector site which binds caffeine (nucleoside site). In intact viable hepatocytes glucose promotes the phosphorylase a to b conversion by phosphorylase phosphatase. This conversion is considered to be a necessary prelude to the activation of glycogen synthase by phosphatase and of importance in hepatic regulation of glucose homeostasis. The effects of glucose and(or) caffeine on the conversion of phosphorylase a to b and synthase b to a were studied. Assays of phosphorylase a were used which limited synergistic inhibition (in the assay) by these ligands. Such an approach is necessary to achieve an accurate measure of phosphatase activity in the viable hepatocyte when the combination of ligands is used. The data indicate that in the presence of caffeine and glucose together, the rate of loss of phosphorylase a is significantly increased (1.7-fold) over that in the presence of glucose alone. Phosphorylase phosphatase is activated. The sequential activation of glycogen synthase was also accelerated in the presence of both ligands. The results are consistent with an in vivo function for the nucleoside site, similar to that of glucose. A controlling role for phosphorylase in the regulation of glycogen metabolism by glucose is supported. Although the existence and nature of an intracellular effector is as yet unknown, crystallographic analyses of phosphorylase a crystals soaked in perchloric acid extracts of liver demonstrate that the negative effector site binds a natural metabolite.

scholarly journals Effects of C-1-substituted glucose analogue on the activation states of glycogen synthase and glycogen phosphorylase in rat hepatocytes

1995 ◽  
Vol 311 (3) ◽  
pp. 845-852 ◽  
Author(s):  
M Board ◽  
M Bollen ◽  
W Stalmans ◽  
Y Kim ◽  
G W J Fleet ◽  
...  
Keyword(s):  
Glycogen Phosphorylase ◽  
Kinase Inhibitor ◽  
Glycogen Synthase ◽  
Kinetic Studies ◽  
Rat Hepatocytes ◽  
Glycogen Metabolism ◽  
Isolated Hepatocytes ◽  
Effective Control ◽  
Subsequent Addition ◽  
Glucose Analogue

A series of glucose-analogue inhibitors of glycogen phosphorylase b (GPb) has been designed, synthesized and investigated in crystallographic binding and kinetic studies. The aim is to produce a compound that may exert more effective control over glycogen metabolism than the parent glucose molecule and which could alleviate hyperglycaemia in Type-II diabetes. N-Acetyl-beta-D-glucopyranosylamine (1-GlcNAc) has a Ki for muscle GPb in crude extracts of 30 microM, 367-fold lower than that of beta-D-glucose [Board, Hadwen and Johnson (1995) Eur. J. Biochem. 228, 753-761]. In the current work, the effects of 1-GlcNAc on the activation states of GP and glycogen synthase (GS) in cell-free preparations and in isolated hepatocytes are reported. In gel-filtered extracts of liver, which lack ATP for kinase activity, 1-GlcNAc produced a rapid and time-dependent inactivation of GP with a subsequent activation of GS. Effects of 1-GlcNAc on both enzymes were stronger than those of glucose, with 0.8 mM 1-GlcNAc being equipotent with 50 mM glucose. At 1 mM, 1-GlcNAc enhanced the dephosphorylation of exogenous GPa by liver extracts (600%) and by muscle extracts (75%). This represents an approximately 500-fold improvement on glucose for the liver activity and 40-fold for the muscle activity. In whole hepatocytes, 1-GlcNAc showed an approximately 5-fold enhancement of glucose effects for GP inactivation but failed to elicit activation of GS. Glucose-induced activation of GS in whole hepatocytes was reversed by subsequent addition of 1-GlcNAc. However, when GS activation was achieved via the adenosine analogue and kinase inhibitor, 5′-iodotubercidin (ITU), subsequent addition of 1-GlcNAc allowed continued activation of GS. Phosphorylation of 1-GlcNAc in rat hepatocytes was established using radiolabelled material. The rate of phosphorylation was 1.60 nmol/min per 10(6) cells at 20 mM 1-GlcNAc but was reduced by the presence of 50 microM ITU (0.775 nmol/min per 10(6) cells). It is suggested that the phosphorylated derivative of 1-GlcNAc formed in hepatocytes is 1-GlcNAc 6-phosphate and that the presence of this species is responsible for the failure of 1-GlcNAc to activate GS. The relative importance of the reduction in concentration of GPa versus increased glucose 6-phosphate levels for activation of GS is discussed.

scholarly journals Wine Yeast Strains Engineered for Glycogen Overproduction Display Enhanced Viability under Glucose Deprivation Conditions

2002 ◽  
Vol 68 (7) ◽  
pp. 3339-3344 ◽  
Author(s):  
R. Pérez-Torrado ◽  
J. V. Gimeno-Alcañiz ◽  
E. Matallana
Keyword(s):  
Glycogen Phosphorylase ◽  
Glycogen Synthase ◽  
Glucose Deprivation ◽  
Wine Yeast ◽  
Glycogen Metabolism ◽  
Growth Conditions ◽  
Phosphorylase Activity ◽  
Industrial Use ◽  
Glycogen Phosphorylase Activity ◽  
Glycogen Synthase Activity

ABSTRACT We used metabolic engineering to produce wine yeasts with enhanced resistance to glucose deprivation conditions. Glycogen metabolism was genetically modified to overproduce glycogen by increasing the glycogen synthase activity and eliminating glycogen phosphorylase activity. All of the modified strains had a higher glycogen content at the stationary phase, but accumulation was still regulated during growth. Strains lacking GPH1, which encodes glycogen phosphorylase, are unable to mobilize glycogen. Enhanced viability under glucose deprivation conditions occurs when glycogen accumulates in the strain that overexpresses GSY2, which encodes glycogen synthase and maintains normal glycogen phosphorylase activity. This enhanced viability is observed under laboratory growth conditions and under vinification conditions in synthetic and natural musts. Wines obtained from this modified strain and from the parental wild-type strain don't differ significantly in the analyzed enological parameters. The engineered strain might better resist some stages of nutrient depletion during industrial use.

scholarly journals Phosphorylation of β-Catenin by Cyclic AMP-Dependent Protein Kinase Stabilizes β-Catenin through Inhibition of Its Ubiquitination

2005 ◽  
Vol 25 (20) ◽  
pp. 9063-9072 ◽  
Author(s):  
Shin-ichiro Hino ◽  
Chie Tanji ◽  
Keiichi I. Nakayama ◽  
Akira Kikuchi
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Wnt Signaling ◽  
Protein Level ◽  
Glycogen Synthase ◽  
Wnt Signaling Pathway ◽  
Prostaglandin E ◽  
Dependent Protein Kinase ◽  
Intact Cells ◽  
Dependent Protein

ABSTRACT The mechanism of cross talk between the Wnt signaling and cyclic AMP (cAMP)-dependent protein kinase (protein kinase A [PKA]) pathways was studied. Prostaglandin E1 (PGE1), isoproterenol, and dibutyryl cAMP (Bt2cAMP), all of which activate PKA, increased the cytoplasmic and nuclear β-catenin protein level, and these actions were suppressed by a PKA inhibitor and RNA interference for PKA. PGE1 and Bt2cAMP also increased T-cell factor (Tcf)-dependent transcription through β-catenin. Bt2cAMP suppressed degradation of β-catenin at the protein level. Although PKA did not affect the formation of a complex between glycogen synthase kinase 3β (GSK-3β), β-catenin, and Axin, phosphorylation of β-catenin by PKA inhibited ubiquitination of β-catenin in intact cells and in vitro. Ser675 was found to be a site for phosphorylation by PKA, and substitution of this serine residue with alanine in β-catenin attenuated inhibition of the ubiquitination of β-catenin by PKA, PKA-induced stabilization of β-catenin, and PKA-dependent activation of Tcf. These results indicate that PKA inhibits the ubiquitination of β-catenin by phosphorylating β-catenin, thereby causing β-catenin to accumulate and the Wnt signaling pathway to be activated.

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