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Sample records for activate glycogen phosphorylase

  1. Activity of glycogen synthase and glycogen phosphorylase in normal and cirrhotic rat liver during glycogen synthesis from glucose or fructose.

    PubMed

    Bezborodkina, Natalia N; Chestnova, Anna Yu; Okovity, Sergey V; Kudryavtsev, Boris N

    2014-03-01

    Cirrhotic patients often demonstrate glucose intolerance, one of the possible causes being a decreased glycogen-synthesizing capacity of the liver. At the same time, information about the rates of glycogen synthesis in the cirrhotic liver is scanty and contradictory. We studied the dynamics of glycogen accumulation and the activity of glycogen synthase (GS) and glycogen phosphorylase (GP) in the course of 120min after per os administration of glucose or fructose to fasted rats with CCl4-cirrhosis or fasted normal rats. Blood serum and liver pieces were sampled for examinations. In the normal rat liver administration of glucose/fructose initiated a fast accumulation of glycogen, while in the cirrhotic liver glycogen was accumulated with a 20min delay and at a lower rate. In the normal liver GS activity rose sharply and GPa activity dropped in the beginning of glycogen synthesis, but 60min later a high synthesis rate was sustained at the background of a high GS and GPa activity. Contrariwise, in the cirrhotic liver glycogen was accumulated at the background of a decreased GS activity and a low GPa activity. Refeeding with fructose resulted in a faster increase in the GS activity in both the normal and the cirrhotic liver than refeeding with glucose. To conclude, the rate of glycogen synthesis in the cirrhotic liver is lower than in the normal one, the difference being probably associated with a low GS activity.

  2. Selective photoregulation of the activity of glycogen synthase and glycogen phosphorylase, two key enzymes in glycogen metabolism.

    PubMed

    Díaz-Lobo, Mireia; Garcia-Amorós, Jaume; Fita, Ignacio; Velasco, Dolores; Guinovart, Joan J; Ferrer, Joan C

    2015-07-14

    Glycogen is a polymer of α-1,4- and α-1,6-linked glucose units that provides a readily available source of energy in living organisms. Glycogen synthase (GS) and glycogen phosphorylase (GP) are the two enzymes that control, respectively, the synthesis and degradation of this polysaccharide and constitute adequate pharmacological targets to modulate cellular glycogen levels, by means of inhibition of their catalytic activity. Here we report on the synthesis and biological evaluation of a selective inhibitor that consists of an azobenzene moiety glycosidically linked to the anomeric carbon of a glucose molecule. In the ground state, the more stable (E)-isomer of the azobenzene glucoside had a slight inhibitory effect on rat muscle GP (RMGP, IC50 = 4.9 mM) and Escherichia coli GS (EcGS, IC50 = 1.6 mM). After irradiation and subsequent conversion to the (Z)-form, the inhibitory potency of the azobenzene glucoside did not significantly change for RMGP (IC50 = 2.4 mM), while its effect on EcGS increased 50-fold (IC50 = 32 μM). Sucrose synthase 4 from potatoes, a glycosyltransferase that does not operate on glycogen, was only slightly inhibited by the (E)-isomer (IC50 = 0.73 mM). These findings could be rationalized on the basis of kinetic and computer-aided docking analysis, which indicated that both isomers of the azobenzene glucoside mimic the EcGS acceptor substrate and exert their inhibitory effect by binding to the glycogen subsite in the active center of the enzyme. The ability to selectively photoregulate the catalytic activity of key enzymes of glycogen metabolism may represent a new approach for the treatment of glycogen metabolism disorders.

  3. Rac1 Protein Regulates Glycogen Phosphorylase Activation and Controls Interleukin (IL)-2-dependent T Cell Proliferation*

    PubMed Central

    Arrizabalaga, Onetsine; Lacerda, Hadriano M.; Zubiaga, Ana M.; Zugaza, José L.

    2012-01-01

    Small GTPases of the Rho family have been implicated in important cellular processes such as cell migration and adhesion, protein secretion, and/or gene transcription. In the lymphoid system, these GTPases participate in the signaling cascades that are activated after engagement of antigen receptors. However, little is known about the role that Rho GTPases play in IL-2-mediated responses. Here, we show that IL-2 induces Rac1 activation in Kit 225 T cells. We identified by mass spectrometry the muscle isoform of glycogen phosphorylase (PYGM) as a novel Rac1 effector molecule in IL-2-stimulated cells. The interaction between the active form of Rac1 (Rac1-GTP) and PYGM was established directly through a domain comprising amino acids 191–270 of PYGM that exhibits significant homology with the Rac binding domain of PAK1. The integrity of this region was crucial for PYGM activation. Importantly, IL-2-dependent cellular proliferation was inhibited upon blocking both the activation of Rac1 and the activity of PYGM. These results reveal a new role for Rac1 in cell signaling, showing that this GTPase triggers T cell proliferation upon IL-2 stimulation by associating with PYGM and modulating its enzymatic activity. PMID:22337875

  4. Regulation of glycogen synthase and phosphorylase during recovery from high-intensity exercise in the rat.

    PubMed Central

    Bräu, L; Ferreira, L D; Nikolovski, S; Raja, G; Palmer, T N; Fournier, P A

    1997-01-01

    The aim of this study was to determine the role of the phosphorylation state of glycogen synthase and glycogen phosphorylase in the regulation of muscle glycogen repletion in fasted animals recovering from high-intensity exercise. Groups of rats were swum to exhaustion and allowed to recover for up to 120 min without access to food. Swimming to exhaustion caused substantial glycogen breakdown and lactate accumulation in the red, white and mixed gastrocnemius muscles, whereas the glycogen content in the soleus muscle remained stable. During the first 40 min of recovery, significant repletion of glycogen occurred in all muscles examined except the soleus muscle. At the onset of recovery, the activity ratios and fractional velocities of glycogen synthase in the red, white and mixed gastrocnemius muscles were higher than basal, but returned to pre-exercise levels within 20 min after exercise. In contrast, after exercise the activity ratios of glycogen phosphorylase in the same muscles were lower than basal, and increased to pre-exercise levels within 20 min. This pattern of changes in glycogen synthase and phosphorylase activities, never reported before, suggests that the integrated regulation of the phosphorylation state of both glycogen synthase and phosphorylase might be involved in the control of glycogen deposition after high-intensity exercise. PMID:9078277

  5. Regulation of glycogen metabolism in cultured human muscles by the glycogen phosphorylase inhibitor CP-91149.

    PubMed Central

    Lerín, Carlos; Montell, Eulàlia; Nolasco, Teresa; García-Rocha, Mar; Guinovart, Joan J; Gómez-Foix, Anna M

    2004-01-01

    Pharmacological inhibition of liver GP (glycogen phosphorylase), which is currently being studied as a treatment for Type II (non-insulin-dependent) diabetes, may affect muscle glycogen metabolism. In the present study, we analysed the effects of the GP inhibitor CP-91149 on non-engineered or GP-overexpressing cultured human muscle cells. We found that CP-91149 treatment decreased muscle GP activity by (1) converting the phosphorylated AMP-independent a form into the dephosphorylated AMP-dependent b form and (2) inhibiting GP a activity and AMP-mediated GP b activation. Dephosphorylation of GP was exerted, irrespective of incubation of the cells with glucose, whereas inhibition of its activity was synergic with glucose. As expected, CP-91149 impaired the glycogenolysis induced by glucose deprivation. CP-91149 also promoted the dephosphorylation and activation of GS (glycogen synthase) in non-engineered or GP-overexpressing cultured human muscle cells, but exclusively in glucose-deprived cells. However, this inhibitor did not activate GS in glucose-deprived but glycogen-replete cells overexpressing PTG (protein targeting to glycogen), thus suggesting that glycogen inhibits the CP-91149-mediated activation of GS. Consistently, CP-91149 promoted glycogen resynthesis, but not its overaccumulation. Hence, treatment with CP-91149 impairs muscle glycogen breakdown, but enhances its recovery, which may be useful for the treatment of Type II (insulin-dependent) diabetes. PMID:14651477

  6. Insights into Brain Glycogen Metabolism: THE STRUCTURE OF HUMAN BRAIN GLYCOGEN PHOSPHORYLASE.

    PubMed

    Mathieu, Cécile; de la Sierra-Gallay, Ines Li; Duval, Romain; Xu, Ximing; Cocaign, Angélique; Léger, Thibaut; Woffendin, Gary; Camadro, Jean-Michel; Etchebest, Catherine; Haouz, Ahmed; Dupret, Jean-Marie; Rodrigues-Lima, Fernando

    2016-08-26

    Brain glycogen metabolism plays a critical role in major brain functions such as learning or memory consolidation. However, alteration of glycogen metabolism and glycogen accumulation in the brain contributes to neurodegeneration as observed in Lafora disease. Glycogen phosphorylase (GP), a key enzyme in glycogen metabolism, catalyzes the rate-limiting step of glycogen mobilization. Moreover, the allosteric regulation of the three GP isozymes (muscle, liver, and brain) by metabolites and phosphorylation, in response to hormonal signaling, fine-tunes glycogenolysis to fulfill energetic and metabolic requirements. Whereas the structures of muscle and liver GPs have been known for decades, the structure of brain GP (bGP) has remained elusive despite its critical role in brain glycogen metabolism. Here, we report the crystal structure of human bGP in complex with PEG 400 (2.5 Å) and in complex with its allosteric activator AMP (3.4 Å). These structures demonstrate that bGP has a closer structural relationship with muscle GP, which is also activated by AMP, contrary to liver GP, which is not. Importantly, despite the structural similarities between human bGP and the two other mammalian isozymes, the bGP structures reveal molecular features unique to the brain isozyme that provide a deeper understanding of the differences in the activation properties of these allosteric enzymes by the allosteric effector AMP. Overall, our study further supports that the distinct structural and regulatory properties of GP isozymes contribute to the different functions of muscle, liver, and brain glycogen. PMID:27402852

  7. Insights into Brain Glycogen Metabolism: THE STRUCTURE OF HUMAN BRAIN GLYCOGEN PHOSPHORYLASE.

    PubMed

    Mathieu, Cécile; de la Sierra-Gallay, Ines Li; Duval, Romain; Xu, Ximing; Cocaign, Angélique; Léger, Thibaut; Woffendin, Gary; Camadro, Jean-Michel; Etchebest, Catherine; Haouz, Ahmed; Dupret, Jean-Marie; Rodrigues-Lima, Fernando

    2016-08-26

    Brain glycogen metabolism plays a critical role in major brain functions such as learning or memory consolidation. However, alteration of glycogen metabolism and glycogen accumulation in the brain contributes to neurodegeneration as observed in Lafora disease. Glycogen phosphorylase (GP), a key enzyme in glycogen metabolism, catalyzes the rate-limiting step of glycogen mobilization. Moreover, the allosteric regulation of the three GP isozymes (muscle, liver, and brain) by metabolites and phosphorylation, in response to hormonal signaling, fine-tunes glycogenolysis to fulfill energetic and metabolic requirements. Whereas the structures of muscle and liver GPs have been known for decades, the structure of brain GP (bGP) has remained elusive despite its critical role in brain glycogen metabolism. Here, we report the crystal structure of human bGP in complex with PEG 400 (2.5 Å) and in complex with its allosteric activator AMP (3.4 Å). These structures demonstrate that bGP has a closer structural relationship with muscle GP, which is also activated by AMP, contrary to liver GP, which is not. Importantly, despite the structural similarities between human bGP and the two other mammalian isozymes, the bGP structures reveal molecular features unique to the brain isozyme that provide a deeper understanding of the differences in the activation properties of these allosteric enzymes by the allosteric effector AMP. Overall, our study further supports that the distinct structural and regulatory properties of GP isozymes contribute to the different functions of muscle, liver, and brain glycogen.

  8. Properties and functions of the storage sites of glycogen phosphorylase.

    PubMed

    Makino, Yasushi; Fujii, Yuta; Taniguchi, Motoi

    2015-06-01

    Glycogen phosphorylase (GP) is biologically active as a dimer of identical subunits. Each subunit has two distinct maltooligosaccharide binding sites: a storage site and a catalytic site. Our characterization of the properties of these sites suggested that GP activity consists of two activities: (i) binding to the glycogen molecule and (ii) phosphorolysis of the non-reducing-end glucose residues. Activity (i) is mainly due to the activities of the two storage sites, which depended on the ionic strength of the medium and were directly inhibited by cyclodextrins (CDs). Activity (i) is of benefit to GP because a high concentration of non-reducing-end glucose residues is localized on the surface of the glycogen molecule. Activity (ii), the total activity of the two catalytic sites, exhibited relatively little ionic strength dependence. Because the combined activity of (i) and (ii) is deduced using glycogen as an assay substrate, the sole activity of (ii) must be measured using small maltooligosyl-substrates. By using a very low concentration of pyridylaminated maltohexaose, we demonstrated that the GP catalytic sites are active even in the presence of CDs, and that the actions of the catalytic site and the storage site are independent of each other.

  9. Glycolytic potential and activity of adenosine monophosphate kinase (AMPK), glycogen phosphorylase (GP) and glycogen debranching enzyme (GDE) in steer carcasses with normal (<5.8) or high (>5.9) 24h pH determined in M. longissimus dorsi.

    PubMed

    Apaoblaza, A; Galaz, A; Strobel, P; Ramírez-Reveco, A; Jeréz-Timaure, N; Gallo, C

    2015-03-01

    Muscle glycogen concentration (MGC) and lactate (LA), activity of glycogen debranching enzyme (GDE), glycogen phosphorylase (GP) and adenosine monophosphate kinase (AMPK) were determined at 0.5h (T0) and 24h (T24) post-mortem in Longissimus dorsi samples from 38 steers that produced high pH (>5.9) and normal pH (<5.8) carcasses at 24h postmortem. MGC, LA and glycolytic potential were higher (P<0.05) in normal pH carcasses. GDE activity was similar (P>0.05) in both pH categories. GP activity increased between T0 and T24 only in normal pH carcasses. AMPK activity was four times higher in normal pH v/s high pH carcasses, without changing its activity over time. Results reinforce the idea that differences in postmortem glycogenolytic/glycolytic flow in L. dorsi of steers showing normal v/s high muscle pH at 24h, could be explained not only by the higher initial MGC in normal pH carcasses, but also by a high and sustained activity of AMPK and an increased GP activity at 24h postmortem.

  10. Glycogen phosphorylase is involved in stress endurance and biofilm formation in Azospirillum brasilense Sp7.

    PubMed

    Lerner, Anat; Castro-Sowinski, Susana; Lerner, Hadas; Okon, Yaacov; Burdman, Saul

    2009-11-01

    Here we report the identification of a glycogen phosphorylase (glgP) gene in the plant growth-promoting rhizobacterium Azospirillum brasilense, Sp7, and the characterization of a glgP marker exchange mutant of this strain. The glgP mutant showed a twofold reduction of glycogen phosphorylase activity and an increased glycogen accumulation as compared with wild-type Sp7, indicating that the identified gene indeed encodes a protein with glycogen phosphorylase activity. Interestingly, the glgP mutant had higher survival rates than the wild type after exposure to starvation, desiccation and osmotic pressure. The mutant was shown to be compromised in its biofilm formation ability. Analysis of the exopolysaccharide sugar composition of the glgP mutant revealed a decrease in the amount of glucose, accompanied by increases in rhamnose, fucose and ribose, as compared with the Sp7 exopolysaccharide. To the best of our knowledge, this is the first study that demonstrates GlgP activity in A. brasilense, and shows that glycogen accumulation may play an important role in the stress endurance of this bacterium.

  11. FR258900, a potential anti-hyperglycemic drug, binds at the allosteric site of glycogen phosphorylase

    PubMed Central

    Tiraidis, Costas; Alexacou, Kyra-Melinda; Zographos, Spyros E.; Leonidas, Demetres D.; Gimisis, Thanasis; Oikonomakos, Nikos G.

    2007-01-01

    FR258900 has been discovered as a novel inhibitor of human liver glycogen phosphorylase a and proved to suppress hepatic glycogen breakdown and reduce plasma glucose concentrations in diabetic mice models. To elucidate the mechanism of inhibition, we have determined the crystal structure of the cocrystallized rabbit muscle glycogen phosphorylase b–FR258900 complex and refined it to 2.2 Å resolution. The structure demonstrates that the inhibitor binds at the allosteric activator site, where the physiological activator AMP binds. The contacts from FR258900 to glycogen phosphorylase are dominated by nonpolar van der Waals interactions with Gln71, Gln72, Phe196, and Val45′ (from the symmetry-related subunit), and also by ionic interactions from the carboxylate groups to the three arginine residues (Arg242, Arg309, and Arg310) that form the allosteric phosphate-recognition subsite. The binding of FR258900 to the protein promotes conformational changes that stabilize an inactive T-state quaternary conformation of the enzyme. The ligand-binding mode is different from those of the potent phenoxy-phthalate and acyl urea inhibitors, previously described, illustrating the broad specificity of the allosteric site. PMID:17600143

  12. Molecular mechanisms of McArdle's disease (muscle glycogen phosphorylase deficiency). RNA and DNA analysis.

    PubMed Central

    Gautron, S; Daegelen, D; Mennecier, F; Dubocq, D; Kahn, A; Dreyfus, J C

    1987-01-01

    Lack of muscle glycogen phosphorylase activity leads to McArdle's disease, a rare metabolic myopathy. To investigate its molecular basis at the nucleic acid level, we isolated muscle phosphorylase cDNA clones from a human cDNA library in Escherichia coli plasmid pBR 322. Subcloning of one insertion of M13 bacteriophage permitted its definite identification by sequencing. Northern blot experiments revealed one specific messenger RNA of 3.4 kilobases found uniquely in tissues expressing muscle phosphorylase. We show that McArdle's disease exhibits a molecular heterogeneity at the messenger RNA level. In eight unrelated cases of McArdle's disease in which no inactive proteins had been detected, we assayed muscle biopsies for phosphorylase mRNA by Northern blotting. In five cases, no muscle phosphorylase mRNA could be detected, while in three other cases, normal length mRNA was present in lower amounts. Moreover, Southern blot analysis of DNA isolated from white blood cells in four McArdle patients revealed no major deletion or rearrangements of the phosphorylase gene as compared with controls. Images PMID:3466902

  13. The experimental type 2 diabetes therapy glycogen phosphorylase inhibition can impair aerobic muscle function during prolonged contraction.

    PubMed

    Baker, David J; Greenhaff, Paul L; MacInnes, Alan; Timmons, James A

    2006-06-01

    Glycogen phosphorylase inhibition represents a promising strategy to suppress inappropriate hepatic glucose output, while muscle glycogen is a major source of fuel during contraction. Glycogen phosphorylase inhibitors (GPi) currently being investigated for the treatment of type 2 diabetes do not demonstrate hepatic versus muscle glycogen phosphorylase isoform selectivity and may therefore impair patient aerobic exercise capabilities. Skeletal muscle energy metabolism and function are not impaired by GPi during high-intensity contraction in rat skeletal muscle; however, it is unknown whether glycogen phosphorylase inhibitors would impair function during prolonged lower-intensity contraction. Utilizing a novel red cell-perfused rodent gastrocnemius-plantaris-soleus system, muscle was pretreated for 60 min with either 3 micromol/l free drug GPi (n=8) or vehicle control (n=7). During 60 min of aerobic contraction, GPi treatment resulted in approximately 35% greater fatigue. Muscle glycogen phosphorylase a form (P<0.01) and maximal activity (P<0.01) were reduced in the GPi group, and postcontraction glycogen (121.8 +/- 16.1 vs. 168.3 +/- 8.5 mmol/kg dry muscle, P<0.05) was greater. Furthermore, lower muscle lactate efflux and glucose uptake (P<0.01), yet higher muscle Vo(2), support the conclusion that carbohydrate utilization was impaired during contraction. Our data provide new confirmation that muscle glycogen plays an essential role during submaximal contraction. Given the critical role of exercise prescription in the treatment of type 2 diabetes, it will be important to monitor endurance capacity during the clinical evaluation of nonselective GPi. Alternatively, greater effort should be devoted toward the discovery of hepatic-selective GPi, hepatic-specific drug delivery strategies, and/or alternative strategies for controlling excess hepatic glucose production in type 2 diabetes.

  14. Isoform-selective regulation of glycogen phosphorylase by energy deprivation and phosphorylation in astrocytes.

    PubMed

    Müller, Margit S; Pedersen, Sofie E; Walls, Anne B; Waagepetersen, Helle S; Bak, Lasse K

    2015-01-01

    Glycogen phosphorylase (GP) is activated to degrade glycogen in response to different stimuli, to support both the astrocyte's own metabolic demand and the metabolic needs of neurons. The regulatory mechanism allowing such a glycogenolytic response to distinct triggers remains incompletely understood. In the present study, we used siRNA-mediated differential knockdown of the two isoforms of GP expressed in astrocytes, muscle isoform (GPMM), and brain isoform (GPBB), to analyze isoform-specific regulatory characteristics in a cellular setting. Subsequently, we tested the response of each isoform to phosphorylation, triggered by incubation with norepinephrine (NE), and to AMP, increased by glucose deprivation in cells in which expression of one GP isoform had been silenced. Successful knockdown was demonstrated on the protein level by Western blot, and on a functional level by determination of glycogen content showing an increase in glycogen levels following knockdown of either GPMM or GPBB. NE triggered glycogenolysis within 15 min in control cells and after GPBB knockdown. However, astrocytes in which expression of GPMM had been silenced showed a delay in response to NE, with glycogen levels significantly reduced only after 60 min. In contrast, allosteric activation of GP by AMP, induced by glucose deprivation, seemed to mainly affect GPBB, as only knockdown of GPBB, but not of GPMM, delayed the glycogenolytic response to glucose deprivation. Our results indicate that the two GP isoforms expressed in astrocytes respond to different physiological triggers, therefore conferring distinct metabolic functions of brain glycogen.

  15. Synthesis of tartaric acid analogues of FR258900 and their evaluation as glycogen phosphorylase inhibitors.

    PubMed

    Varga, Gergely; Docsa, Tibor; Gergely, Pál; Juhász, László; Somsák, László

    2013-03-15

    Di-O-cinnamoylated, -p-coumaroylated, and -feruloylated d-, l- and meso-tartaric acids were synthesized as analogues of the natural product FR258900, a glycogen phosphorylase (GP) inhibitor with in vivo antihyperglycaemic activity. The new compounds inhibited rabbit muscle GP in the low micromolar range, and bound to the allosteric site of the enzyme. The best inhibitor was 2,3-di-O-feruloyl meso-tartaric acid and had Ki values of 2.0μM against AMP (competitive) and 3.36μM against glucose-1-phosphate (non-competitive).

  16. A glycogen phosphorylase inhibitor selectively enhances local rates of glucose utilization in brain during sensory stimulation of conscious rats: implications for glycogen turnover.

    PubMed

    Dienel, Gerald A; Ball, Kelly K; Cruz, Nancy F

    2007-07-01

    Glycogen is degraded during brain activation but its role and contribution to functional energetics in normal activated brain have not been established. In the present study, glycogen utilization in brain of normal conscious rats during sensory stimulation was assessed by three approaches, change in concentration, release of (14)C from pre-labeled glycogen and compensatory increase in utilization of blood glucose (CMR(glc)) evoked by treatment with a glycogen phosphorylase inhibitor. Glycogen level fell in cortex, (14)C release increased in three structures and inhibitor treatment caused regionally selective compensatory increases in CMR(glc) over and above the activation-induced rise in vehicle-treated rats. The compensatory rise in CMR(glc) was highest in sensory-parietal cortex where it corresponded to about half of the stimulus-induced rise in CMR(glcf) in vehicle-treated rats; this response did not correlate with metabolic rate, stimulus-induced rise in CMR(glc) or sequential station in sensory pathway. Thus, glycogen is an active fuel for specific structures in normal activated brain, not simply an emergency fuel depot and flux-generated pyruvate greatly exceeded net accumulation of lactate or net consumption of glycogen during activation. The metabolic fate of glycogen is unknown, but adding glycogen to the fuel consumed during activation would contribute to a fall in CMR(O2)/CMR(glc) ratio.

  17. Natural flavonoids as antidiabetic agents. The binding of gallic and ellagic acids to glycogen phosphorylase b.

    PubMed

    Kyriakis, Efthimios; Stravodimos, George A; Kantsadi, Anastassia L; Chatzileontiadou, Demetra S M; Skamnaki, Vassiliki T; Leonidas, Demetres D

    2015-07-01

    We present a study on the binding of gallic acid and its dimer ellagic acid to glycogen phosphorylase (GP). Ellagic acid is a potent inhibitor with Kis of 13.4 and 7.5 μM, in contrast to gallic acid which displays Kis of 1.7 and 3.9 mM for GPb and GPa, respectively. Both compounds are competitive inhibitors with respect to the substrate, glucose-1-phoshate, and non-competitive to the allosteric activator, AMP. However, only ellagic acid functions with glucose in a strongly synergistic mode. The crystal structures of the GPb-gallic acid and GPb-ellagic acid complexes were determined at high resolution, revealing that both ligands bind to the inhibitor binding site of the enzyme and highlight the structural basis for the significant difference in their inhibitory potency.

  18. KIAA1199 interacts with glycogen phosphorylase kinase β-subunit (PHKB) to promote glycogen breakdown and cancer cell survival.

    PubMed

    Terashima, Masato; Fujita, Yoshihiko; Togashi, Yosuke; Sakai, Kazuko; De Velasco, Marco A; Tomida, Shuta; Nishio, Kazuto

    2014-08-30

    The KIAA1199 gene was first discovered to be associated with non-syndromic hearing loss. Recently, several reports have shown that the up-regulation of KIAA1199 is associated with cancer cell migration or invasion and a poor prognosis. These findings indicate that KIAA1199 may be a novel target for cancer therapy. Therefore, we explored in detail the function of KIAA1199 in cancer cells. In this study, we investigated the interaction of KIAA1199 protein with intracellular proteins in cancer cells. To this end, we expressed KIAA1199-MBP fusion protein and performed a pull-down assay. In addition, KIAA1199-overexpressing cancer cell lines were constructed using a retroviral vector and were used for further experiments. A pull-down analysis showed that the glycogen phosphorylase kinase β-subunit (PHKB) interacted with the C-terminal region of KIAA1199 protein. Furthermore, we observed the interaction of KIAA1199 with glycogen phosphorylase brain form (PYGB) under serum-free conditions. The interaction promoted glycogen breakdown and cancer cell survival. Our findings indicate that KIAA1199 plays an important role in glycogen breakdown and cancer cell survival and that it may represent a novel target for cancer therapy.

  19. KIAA1199 interacts with glycogen phosphorylase kinase β-subunit (PHKB) to promote glycogen breakdown and cancer cell survival

    PubMed Central

    Terashima, Masato; Fujita, Yoshihiko; Togashi, Yosuke; Sakai, Kazuko; De Velasco, Marco A.; Tomida, Shuta; Nishio, Kazuto

    2014-01-01

    The KIAA1199 gene was first discovered to be associated with non-syndromic hearing loss. Recently, several reports have shown that the up-regulation of KIAA1199 is associated with cancer cell migration or invasion and a poor prognosis. These findings indicate that KIAA1199 may be a novel target for cancer therapy. Therefore, we explored in detail the function of KIAA1199 in cancer cells. In this study, we investigated the interaction of KIAA1199 protein with intracellular proteins in cancer cells. To this end, we expressed KIAA1199-MBP fusion protein and performed a pull-down assay. In addition, KIAA1199-overexpressing cancer cell lines were constructed using a retroviral vector and were used for further experiments. A pull-down analysis showed that the glycogen phosphorylase kinase β-subunit (PHKB) interacted with the C-terminal region of KIAA1199 protein. Furthermore, we observed the interaction of KIAA1199 with glycogen phosphorylase brain form (PYGB) under serum-free conditions. The interaction promoted glycogen breakdown and cancer cell survival. Our findings indicate that KIAA1199 plays an important role in glycogen breakdown and cancer cell survival and that it may represent a novel target for cancer therapy. PMID:25051373

  20. Glycogen phosphorylase sequences from the amitochondriate protists, Trichomonas vaginalis, Mastigamoeba balamuthi, Entamoeba histolytica and Giardia intestinalis.

    PubMed

    Wu, Gang; Müller, Miklós

    2003-01-01

    Glycogen phosphorylase genes or messages from four amitochondriate eukaryotes, Trichomonas vaginalis, Mastigamoeba balamuthi, Entamoeba histolytica (two genes) and Giardia intestinalis, have been isolated and sequenced. The sequences of the amitochondriate protist enzymes appear to share a most recent common ancestor. The clade containing these sequences is closest to that of another protist, the slime mold (Dictyostelium discoideum), and is more closely related to fungal and plant phosphorylases than to mammalian and eubacterial homologs. Structure-based amino acid alignment shows conservation of the residues and domains involved in catalysis and allosteric regulation by glucose 6-phosphate but high divergence at domains involved in phosphorylation-dependent regulation and AMP binding in fungi and animals. Protist phosphorylases, as their prokaryotic and plant counterparts, are probably not regulated by phosphorylation.

  1. The role of glycogen phosphorylase in the regulation of glycogenolysis by insulin and glucagon in isolated eel (Anguilla rostrata) hepatocytes.

    PubMed

    Foster, G D; Moon, T W

    1990-07-01

    The effects of porcine, scombroid, and salmon insulins, and bovine and anglerfish glucagons on glycogen depletion and glycogen phosphorylase (GPase) activities were examined in freshly isolated American eel (Anguilla rostrata) hepatocytes. Eel liver GPase in crude homogenates was activated (increase in % GPase a) by phosphorylating conditions and was rapidly inactivated (less than 1 h) when a phosphatase inhibitor (fluoride) was absent. Caffeine inhibits, and AMP activates, the b form of GPase consistent with their effects on rat liver GPase. Both mammalian and fish glucagons increased glucose production in eel hepatocytes, but had more ambiguous effects on glycogen levels and GPase activities. The magnitude of bovine glucagon effects were dependent on the initial glycogen content of the cells; only at glycogen concentrations less than approximately 70 μmoles.g(-1) did glucagon significantly increase % GPase a. Anglerfish glucagon significantly increased cyclic AMP (cAMP) concentrations by 90% at 10(-7) M, but had no effects at 10(-9) M and 10(-8) M. Scombroid and salmon insulins maintained hepatocyte glycogen concentrations and decreased glucose production, with these effects more pronounced at low (10(-9) to 10(-8) M) rather than high (10(-7) M) hormone concentrations. Porcine and salmon insulins decreased total GPase and % GPase a activities, and salmon insulin decreased CAMP levels, but only at 10(-8) M (by 44%).Glycogen is, therefore, depleted by glucagon and maintained by insulin in freshly isolated American eel hepatocytes, and these changes are accomplished, at least in part, by changes in the activities of GPase. Changes in cAMP do not explain all of the observed hormone effects. PMID:24220919

  2. Regulation of the Dictyostelium glycogen phosphorylase 2 gene by cyclic AMP.

    PubMed

    Sucic, J F; Selmin, O; Rutherford, C L

    1993-01-01

    A crucial developmental event in the cellular slime mold, Dictyostelium discoideum, is glycogen degradation. The enzyme that catalyzes this degradation, glycogen phosphorylase 2 (gp-2), is developmentally regulated and cAMP appears to be involved in this regulation. We have examined several aspects of the cAMP regulation of gp-2. We show that addition of exogenous cAMP to aggregation competent amoebae induced the appearance of gp-2 mRNA. The induction of gp-2 mRNA occurred within 1 and 1.5 h after the initial exposure to cAMP. Exposure to exogenous cAMP concentrations as low as 1.0 microM could induce gp-2 mRNA. We also examined the molecular mechanism through which cAMP induction of gp-2 occurs. Induction of gp-2 appears to result from a mechanism that does not require intracellular cAMP signaling, and may occur directly through a cAMP binding protein without the requirement of any intracellular signalling. We also examined the promoter region of the gp-2 gene for cis-acting elements that are involved in the cAMP regulation of gp-2. A series of deletions of the promoter were fused to a luciferase reporter gene and then analyzed for cAMP responsiveness. The results indicated that a region from -258 nucleotides to the transcriptional start site is sufficient for essentially full activity and appears to carry all necessary cis-acting sites for cAMP induction. Further deletion of 58 nucleotides from the 5' end, results in fivefold less activity in the presence of cAMP. Deletion of the next 104 nucleotides eliminates the cAMP response entirely. PMID:8222346

  3. Effects of eugenol-reduced clove extract on glycogen phosphorylase b and the development of diabetes in db/db mice.

    PubMed

    Sanae, Fujiko; Kamiyama, Ogusa; Ikeda-Obatake, Kyoko; Higashi, Yasuhiko; Asano, Naoki; Adachi, Isao; Kato, Atsushi

    2014-02-01

    We found that the 50% aqueous EtOH extract of clove (Syzygium aromaticum) had potent dose-dependent inhibitory activity toward glycogen phosphorylase b and glucagon-stimulated glucose production in primary rat hepatocytes. Among the components, eugeniin inhibited glycogen phosphorylase b and glucagon-stimulated glucose production in primary rat hepatocytes, with IC50 values of 0.14 and 4.7 μM, respectively. In sharp contrast, eugenol showed no significant inhibition toward glycogen phosphorylase b, even at a concentration of 400 μM. Eugenol-reduced clove extracts (erCE) were prepared and when fed to a db/db mouse they clearly suppressed the blood glucose and HbA1c levels. Furthermore, plasma triglyceride and non-esterified fatty acid levels in 5% and 10% erCE-fed db/db mice were significantly lowered, compared with control db/db mice without erCE supplementation. These results suggested that dietary supplementation with the erCE could beneficially modify glucose and lipid metabolism and contribute to the prevention of the progress of hyperglycemia and metabolic syndrome.

  4. Properties of a glycogen like polysaccharide produced by a mutant of Escherichia coli lacking glycogen synthase and maltodextrin phosphorylase.

    PubMed

    Kwak, Ji-Yun; Kim, Min-Gyu; Kim, Young-Wan; Ban, Hyun-Seung; Won, Mi-Sun; Park, Jong-Tae; Park, Kwan-Hwa

    2016-01-20

    Escherichia coli mutant TBP38 lacks glycogen synthase (GlgA) and maltodextrin phosphorylase (MalP). When grown on maltose in fed-batch fermentation TBP38 accumulated more than 50-fold higher glycogen-type polysaccharide than its parental strain. The polysaccharides were extracted at different growth stages and migrated as one peak in size-exclusion chromatography. TBP38 produced polysaccharides ranging 2.6 × 10(6)-4.6 × 10(6)Da. A ratio of short side-chains (DP ≦ 12) in the polysaccharides was greater than 50%, and number-average degree of polymerization varied from 9.8 to 8.4. The polysaccharides showed 70-290 times greater water-solubility than amylopectin. Km values using porcine and human pancreatic α-amylases with polysaccharides were 2- to 4-fold larger than that of amylopectin. kcat values were similar for both α-amylases. The TBP38 polysaccharides had 40-60% lower digestibility to amyloglucosidase than amylopectin. Intriguingly, the polysaccharides showed strong immunostimulating effects on mouse macrophage cell comparable to lipopolysaccharides. The lipopolysaccharide contamination levels were too low to account for this effect. PMID:26572397

  5. Liver glycogen storage diseases due to phosphorylase system deficiencies: diagnosis thanks to non invasive blood enzymatic and molecular studies.

    PubMed

    Davit-Spraul, Anne; Piraud, Monique; Dobbelaere, Dries; Valayannopoulos, Vassili; Labrune, Philippe; Habes, Dalila; Bernard, Olivier; Jacquemin, Emmanuel; Baussan, Christiane

    2011-01-01

    Glycogen storage disease (GSD) due to a deficient hepatic phosphorylase system defines a genetically heterogeneous group of disorders that mainly manifests in children. We investigated 45 unrelated children in whom a liver GSD VI or IX was suspected on the basis of clinical symptoms including hepatomegaly, increased serum transaminases, postprandial lactatemia and/or mild fasting hypoglycemia. Liver phosphorylase and phosphorylase b kinase activities studied in peripheral blood cells allowed to suspect diagnosis in 37 cases but was uninformative in 5. Sequencing of liver phosphorylase genes was useful to establish an accurate diagnosis. Causative mutations were found either in the PYGL (11 patients), PHKA2 (26 patients), PHKG2 (three patients) or in the PHKB (three patients) genes. Eleven novel disease causative mutations, five missense (p.N188K, p.D228Y, p.P382L, p.R491H, p.L500R) and six truncating mutations (c.501_502ins361pb, c.528+2T>C, c.856-29_c.1518+614del, c.1620+1G>C, p.E703del and c.2313-1G>T) were identified in the PYGL gene. Seventeen novel disease causative mutations, ten missense (p.A42P, p.Q95R, p.G131D, p.G131V, p.Q134R, p.G187R, p.G300V, p.G300A, p.C326Y, p.W820G) and seven truncating (c.537+5G>A, p.G396DfsX28, p.Q404X, p.N653X, p.L855PfsX87, and two large deletions) were identified in the PHKA2 gene. Four novel truncating mutations (p.R168X, p.Q287X, p.I268PfsX12 and c.272-1G>C) were identified in the PHKG2 gene and three (c.573_577del, p.R364X, c.2427+3A>G) in the PHKB gene. Patients with PHKG2 mutations evolved towards cirrhosis. Molecular analysis of GSD VI or IX genes allows to confirm diagnosis suspected on the basis of enzymatic analysis and to establish diagnosis and avoid liver biopsy when enzymatic studies are not informative in blood cells.

  6. EXPRESSION PATTERNS OF THE GLYCOGEN PHOSPHORYLASE GENE RELATED TO LARVAL DIAPAUSE IN Ostrinia furnacalis.

    PubMed

    Guo, Jianqing; Zhang, Honggang; Edwards, Martin; Wang, Zhenying; Bai, Shuxiong; He, Kanglai

    2016-04-01

    Glycogen phosphorylase (GP) acts in the first step in release of glucose from glycogen, a form of energy storage for most organisms. To investigate the characteristics and expression pattern of GP gene (Ofgp) in the Asian corn borer, Ostrinia furnacalis (Guenée), larvae, we cloned and analyzed tissue transcription of Ofgp. The results indicate that the open reading frame (ORF) is 2,526 bp, encoding 841 amino acid. The calculated three-dimensional structure shows 33 α-helices and 24 β-sheets. Ofgp transcription levels varied significantly during the second to fifth instars under long-day (28 °C, 16:8 L:D photoperiod, and 70-80% relative humidity (RH)) and short-day (24.5 °C, 11:13 L:D photoperiod, and 70-80% RH) conditions, remained low during the prediapause phase, and then increased after about 36 d under short-day photoperiod. In the larvae reared under long-day condition, hemolymph ranked the highest in the transcript level of Ofgp. The highest transcription was recorded in the fat body and was lower in the other tissues in larvae reared under short-day condition. We found that Ofgp transcription increased linearly from October 2012 to January 2013. The transcript level was negatively correlated with environmental temperature. We infer the higher Ofgp transcription may enhance the cold hardiness of the diapause larvae. PMID:26748939

  7. The binding of D-gluconohydroximo-1,5-lactone to glycogen phosphorylase. Kinetic, ultracentrifugation and crystallographic studies.

    PubMed Central

    Papageorgiou, A C; Oikonomakos, N G; Leonidas, D D; Bernet, B; Beer, D; Vasella, A

    1991-01-01

    Combined kinetic, ultracentrifugation and X-ray-crystallographic studies have characterized the effect of the beta-glucosidase inhibitor gluconohydroximo-1,5-lactone on the catalytic and structural properties of glycogen phosphorylase. In the direction of glycogen synthesis, gluconohydroximo-1,5-lactone was found to competitively inhibit both the b (Ki 0.92 mM) and the alpha form of the enzyme (Ki 0.76 mM) with respect to glucose 1-phosphate in synergism with caffeine. In the direction of glycogen breakdown, gluconohydroximo-1,5-lactone was found to inhibit phosphorylase b in a non-competitive mode with respect to phosphate, and no synergism with caffeine could be demonstrated. Ultracentrifugation and crystallization experiments demonstrated that gluconohydroximo-1,5-lactone was able to induce dissociation of tetrameric phosphorylase alpha and stabilization of the dimeric T-state conformation. A crystallographic binding study with 100 mM-gluconohydroximo-1,5-lactone at 0.24 nm (2.4 A) resolution showed a major peak at the catalytic site, and no significant conformational changes were observed. Analysis of the electron-density map indicated that the ligand adopts a chair conformation. The results are discussed with reference to the ability of the catalytic site of the enzyme to distinguish between two or more conformations of the glucopyranose ring. PMID:1900987

  8. Measurement of the turnover of glycogen phosphorylase by GC/MS using stable isotope derivatives of pyridoxine (vitamin B6).

    PubMed Central

    Beynon, R J; Leyland, D M; Evershed, R P; Edwards, R H; Coburn, S P

    1996-01-01

    The majority of vitamin B6 in the body is in skeletal muscle, bound as the cofactor pyridoxal 5'-phosphate to one abundant protein, glycogen phosphorylase. Previous work has established that radiolabelled vitamin B6 can be used as a turnover label for glycogen phosphorylase. In this study, a stable isotope derivative of pyridoxine ¿dideuterated pyridoxine; 3-hydroxy-4-(hydroxymethyl) -5-[hydroxymethyl-2H2]-2-methylpyridine¿ ([2H2]PN) has been used as a metabolic tracer to study the kinetics of labelling of the body pools of vitamin B6 in mice. A non-invasive method was developed in which the isotope abundance of the urinary excretory product of vitamin B6 metabolism, 4-pyridoxic acid, was analysed by GC/MS. The change in isotope abundance of urinary 4-pyridoxic acid following administration of [2H2]PN reflects the kinetics of labelling of the body pools of vitamin B6, and yields, non-invasively, the rate of degradation of glycogen phosphorylase. PMID:8713093

  9. Sequencing and characterization of glycogen synthase and glycogen phosphorylase genes from Spodoptera exigua and analysis of their function in starvation and excessive sugar intake.

    PubMed

    Tang, Bin; Xu, Qi; Zou, Qi; Fang, Qi; Wang, Shigui; Ye, Gongyin

    2012-06-01

    Glycogen and trehalose are important energy source and key regulation factors in the development of many organisms' pass through energy metabolism, including bacteria, fungi, and insects. To study glycogen metabolism pathway in Spodoptera exigua, first cDNAs for glycogen synthase (SpoexGS) and glycogen phosphorylase (SpoexGP) were cloned from S. exigua. SpoexGS cDNA contains an open reading frame of 2,010 nucleotides encoding a protein of 669 amino acids with a predicted molecular mass of 76.19 kDa and a pI of 5.84. SpoexGP contains an open reading frame of 2,946 nucleotides, which encodes a protein of 841 amino acids with a predicted molecular mass of approximately 96.63 kDa and a pI of 6.03. Second, Northern blotting revealed that SpoexGS and SpoexGP mRNAs were expressed in brain, fat body, mid-gut, Malpighian tubules, spermary, and tracheae of S. exigua. Expression patterns for SpoexGS and SpoexGP mRNAs were similar in fat body, but differed in whole body at different developmental stages. The last, under starvation conditions, SpoexGS and SpoexGP transcript expression rapidly decreased with increasing starvation time. When the starvation stress was removed, SpoexGS and SpoexGP mRNA levels were lower in the groups starved for 6 and 12 h than in the 24-h starvation and control groups. Treatment with excessive sugar intake led to higher levels of SpoexGS and SpoexGP transcripts after 12 h compared to the control group. These findings provide new data on the tissue distribution, expression patterns, and potential function of glycogen synthase and glycogen phosphorylase proteins.

  10. Contributions of Glycogen to Astrocytic Energetics during Brain Activation

    PubMed Central

    Dienel, Gerald A.; Cruz, Nancy F.

    2014-01-01

    Glycogen is the major store of glucose in brain and is mainly in astrocytes. Brain glycogen levels in unstimulated, carefully-handled rats are 10-12 mol/g, and assuming that astrocytes account for half the brain mass, astrocytic glycogen content is twice as high. Glycogen turnover is slow under basal conditions, but it is mobilized during activation. There is no net increase in incorporation of label from glucose during activation, whereas label release from pre-labeled glycogen exceeds net glycogen consumption, which increases during stronger stimuli. Because glycogen level is restored by non-oxidative metabolism, astrocytes can influence the global ratio of oxygen to glucose utilization. Compensatory increases in utilization of blood glucose during inhibition of glycogen phosphorylase are large and approximate glycogenolysis rates during sensory stimulation. In contrast, glycogenolysis rates during hypoglycemia are low due to continued glucose delivery and oxidation of endogenous substrates; rates that preserve neuronal function in the absence of glucose are also low, probably due to metabolite oxidation. Modeling studies predict that glycogenolysis maintains a high level of glucose-6-phosphate in astrocytes to maintain feedback inhibition of hexokinase, thereby diverting glucose for use by neurons. The fate of glycogen carbon in vivo is not known, but lactate efflux from brain best accounts for the major metabolic characteristics during activation of living brain. Substantial shuttling coupled with oxidation of glycogen-derived lactate is inconsistent with available evidence. Glycogen has important roles in astrocytic energetics, including glucose sparing, control of extracellular K+ level, oxidative stress management, and memory consolidation; it is a multi-functional compound. PMID:24515302

  11. Contributions of glycogen to astrocytic energetics during brain activation.

    PubMed

    Dienel, Gerald A; Cruz, Nancy F

    2015-02-01

    Glycogen is the major store of glucose in brain and is mainly in astrocytes. Brain glycogen levels in unstimulated, carefully-handled rats are 10-12 μmol/g, and assuming that astrocytes account for half the brain mass, astrocytic glycogen content is twice as high. Glycogen turnover is slow under basal conditions, but it is mobilized during activation. There is no net increase in incorporation of label from glucose during activation, whereas label release from pre-labeled glycogen exceeds net glycogen consumption, which increases during stronger stimuli. Because glycogen level is restored by non-oxidative metabolism, astrocytes can influence the global ratio of oxygen to glucose utilization. Compensatory increases in utilization of blood glucose during inhibition of glycogen phosphorylase are large and approximate glycogenolysis rates during sensory stimulation. In contrast, glycogenolysis rates during hypoglycemia are low due to continued glucose delivery and oxidation of endogenous substrates; rates that preserve neuronal function in the absence of glucose are also low, probably due to metabolite oxidation. Modeling studies predict that glycogenolysis maintains a high level of glucose-6-phosphate in astrocytes to maintain feedback inhibition of hexokinase, thereby diverting glucose for use by neurons. The fate of glycogen carbon in vivo is not known, but lactate efflux from brain best accounts for the major metabolic characteristics during activation of living brain. Substantial shuttling coupled with oxidation of glycogen-derived lactate is inconsistent with available evidence. Glycogen has important roles in astrocytic energetics, including glucose sparing, control of extracellular K(+) level, oxidative stress management, and memory consolidation; it is a multi-functional compound.

  12. Brain isoform glycogen phosphorylase as a novel hepatic progenitor cell marker.

    PubMed

    Huang, Yu-Wen; Chiu, Chien-Chang; Liang, Ja-Der; Chiou, Ling-Ling; Huang, Guan-Tarn; Yu, Ming-Jiun; Lee, Hsuan-Shu

    2015-01-01

    An appropriate liver-specific progenitor cell marker is a stepping stone in liver regenerative medicine. Here, we report brain isoform glycogen phosphorylase (GPBB) as a novel liver progenitor cell marker. GPBB was identified in a protein complex precipitated by a monoclonal antibody Ligab generated from a rat liver progenitor cell line Lig-8. Immunoblotting results show that GPBB was expressed in two liver progenitor cell lines Lig-8 and WB-F344. The levels of GPBB expression decreased in the WB-F344 cells under sodium butyrate (SB)-induced cell differentiation, consistent with roles of GPBB as a liver progenitor cell marker. Short hairpin RNA (shRNA)-mediated GPBB knockdown followed by glucose deprivation test shows that GPBB aids in liver progenitor cell survival under low glucose conditions. Furthermore, shRNA-mediated GPBB knockdown followed by SB-induced cell differentiation shows that reducing GPBB expression delayed liver progenitor cell differentiation. We conclude that GPBB is a novel liver progenitor cell marker, which facilitates liver progenitor cell survival under low glucose conditions and cell differentiation. PMID:25826279

  13. Glucose-derived spiro-isoxazolines are anti-hyperglycemic agents against type 2 diabetes through glycogen phosphorylase inhibition.

    PubMed

    Goyard, David; Kónya, Bálint; Chajistamatiou, Aikaterini S; Chrysina, Evangelia D; Leroy, Jérémy; Balzarin, Sophie; Tournier, Michel; Tousch, Didier; Petit, Pierre; Duret, Cédric; Maurel, Patrick; Somsák, László; Docsa, Tibor; Gergely, Pál; Praly, Jean-Pierre; Azay-Milhau, Jacqueline; Vidal, Sébastien

    2016-01-27

    Glycogen phosphorylase (GP) is a target for the treatment of hyperglycaemia in the context of type 2 diabetes. This enzyme is responsible for the depolymerization of glycogen into glucose thereby affecting the levels of glucose in the blood stream. Twelve new d-glucopyranosylidene-spiro-isoxazolines have been prepared from O-peracylated exo-D-glucals by regio- and stereoselective 1,3-dipolar cycloaddition of nitrile oxides generated in situ by treatment of the corresponding oximes with bleach. This mild and direct procedure appeared to be applicable to a broad range of substrates. The corresponding O-unprotected spiro-isoxazolines were evaluated as glycogen phosphorylase (GP) inhibitors and exhibited IC50 values ranging from 1 to 800 μM. Selected inhibitors were further evaluated in vitro using rat and human hepatocytes and exhibited significant inhibitory properties in the primary cell culture. Interestingly, when tested with human hepatocytes, the tetra-O-acetylated spiro-isoxazoline bearing a 2-naphthyl residue showed a much lower IC50 value (2.5 μM), compared to that of the O-unprotected analog (19.95 μM). The most promising compounds were investigated in Zucker fa/fa rat model in acute and sub-chronic assays and decreased hepatic glucose production, which is known to be elevated in type 2 diabetes. This indicates that glucose-based spiro-isoxazolines can be considered as anti-hyperglycemic agents in the context of type 2 diabetes.

  14. Function of pyridoxal 5'-phosphate in glycogen phosphorylase: a model study using 6-fluoro-5'-deoxypyridoxal- and 5'-deoxypyridoxal-reconstituted enzymes

    SciTech Connect

    Chang, Y.C.; Scott, R.D.; Graves, D.J.

    1987-01-27

    A new vitamin B/sub 6/ analogue, 6-fluoro-5'-deoxypyridoxal (6-FDPL), was synthesized and characterized. This analogue, as well as 6-fluoropyridoxal (6-FPAL), 6-fluoropyridoxal phosphate (6-FPLP), and 6-fluoropyridoxine, showed positive heteronuclear /sup 1/H-/sup 18/F nuclear Overhauser effects between the 5'-protons and the 6-fluorine. Apophosphorylase reconstituted with 6-FDLP showed 1% of the activity of the native enzyme in the presence of phosphite. The kinetic pattern, apparent pH optimum of activity, and the activity-temperature dependency of the 6-FDPL-enzyme were virtually identical with those of phosphorylase reconstituted with the parent compound, 6-FPAL except the K/sub m/ of phosphite toward the 6-FDPL-enzyme was 9 times higher than that with the 6-FPAL-enzyme and the 6-FDPL-enzyme showed a lower V/sub max/ value. Phosphorylase reconstituted with 5'-deoxypyridoxal (DPL) also showed activity in the presence of phosphite. The kinetics and the temperature-activity dependency of this reconstituted enzyme were investigated. /sup 19/F nuclear magnetic resonance studies showed that the binding of glucose 1-phosphate to a 6-FDPL-enzyme-adenosine 5'-phosphate (AMP) complex shifted the /sup 19/F signal 0.6 ppm upfield, whereas a 2.1 ppm change was observed when the 6-FPAL-enzyme-AMP formed a complex with glucose 1-phosphate. Analysis of the activation parameters, activation enthalpy and activation entropy, of the reaction of glycogen degradation catalyzed by phosphorylase containing pyridoxal phosphate, 6-FDPL, pyridoxal, or DPL showed that modifications of the coenzyme molecule affected only the activation entropy, not the activation enthalpy. Results of this study indicate that the protein structure surrounding the coenzyme molecule, as well as the coenzyme configuration, is altered upon the binding of ligands.

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

    PubMed Central

    Watson, K A; Schinzel, R; Palm, D; Johnson, L N

    1997-01-01

    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. PMID:9009262

  16. The crystal structure and activity of a putative trypanosomal nucleoside phosphorylase reveal it to be a homodimeric uridine phosphorylase

    PubMed Central

    Larson, Eric T.; Mudeppa, Devaraja G.; Gillespie, J. Robert; Mueller, Natascha; Napuli, Alberto J.; Arif, Jennifer A.; Ross, Jenni; Arakaki, Tracy L.; Lauricella, Angela; DeTitta, George; Luft, Joseph; Zucker, Frank; Verlinde, Christophe L. M. J.; Fan, Erkang; Van Voorhis, Wesley C.; Buckner, Frederick S.; Rathod, Pradipsinh K.; Hol, Wim G. J.; Merritt, Ethan A.

    2010-01-01

    Purine nucleoside phosphorylases and uridine phosphorylases are closely related enzymes involved in purine and pyrimidine salvage, respectively, which catalyze the removal of the ribosyl moiety from nucleosides so that the nucleotide base may be recycled. Parasitic protozoa generally are incapable of de novo purine biosynthesis so the purine salvage pathway is of potential therapeutic interest. Information about pyrimidine biosynthesis in these organisms is much more limited. Though all seem to carry at least a subset of enzymes from each pathway, the dependency on de novo pyrimidine synthesis versus salvage varies from organism to organism and even from one growth stage to another. We have structurally and biochemically characterized a putative nucleoside phosphorylase from the pathogenic protozoan Trypanosoma brucei and find that it is a homodimeric uridine phosphorylase. This is the first characterization of a uridine phosphorylase from a trypanosomal source despite this activity being observed decades ago. Although this gene was broadly annotated as a putative nucleoside phosphorylase, it was widely inferred to be a purine nucleoside phosphorylase. Our characterization of this trypanosomal enzyme shows that it is possible to distinguish between purine and uridine phosphorylase activity at the sequence level based on the absence or presence of a characteristic uridine phosphorylase-specificity insert. We suggest that this recognizable feature may aid in proper annotation of the substrate specificity of enzymes in the nucleoside phosphorylase family. PMID:20070944

  17. In vitro effect of fenugreek extracts on intestinal sodium-dependent glucose uptake and hepatic glycogen phosphorylase A.

    PubMed

    Al-Habori, M; Raman, A; Lawrence, M J; Skett, P

    2001-01-01

    Fenugreek (Trigonella foenum-graecum L. seed) is a food with traditional medicinal use in diabetes. Beneficial effects have been demonstrated in diabetic animals and both insulin-dependent and non-insulin-dependent diabetic subjects. Effects of a lipid extract A, crude ethanolic extract B, further sub-fractions of B (saponin-free C, saponin D and sapogenin E) and a gum fibre fraction F on intestinal sodium-dependent glucose uptake were investigated in vitro using rabbit intestinal brush border membrane vesicles. All fractions except A inhibited glucose-uptake at 0.33 and/or 3.3 mg/mL (p < 0.001). Greatest inhibition was observed with fractions D and E. Diosgenin and trigonelline (compounds reported in fenugreek) also inhibited glucose-uptake (IC50 values approximately 3 mg/ml, equivalent to 8 mM and 19 mM respectively) but did not account for the activity of the crude extracts. Fenugreek extracts had no effect on basal levels of glycogen phosphorylase a (HGPa) activity in rat hepatocyte suspensions. However fractions C and E caused a marginal but statistically significant inhibition (18.9 and 15.1% respectively, p < 0.05) of glucagon induction of this enzyme suggesting a glucagon-antagonist effect. Diosgenin (1.65 mg/ml; 4 mM) inhibited glucagon-induced HGPa activity by 20% (p < 0.05), and was more effective than trigonelline (non significant inhibition of 9.4% at 1.65 mg/ml, 10 mM). PMID:12369721

  18. Quantitative description of the absorption spectra of the coenzyme in glycogen phosphorylases based on log-normal distribution curves.

    PubMed Central

    Donoso, J; Muñoz, F; Garcia Blanco, F

    1993-01-01

    The absorption spectra of the coenzyme [pyridoxal 5'-phosphate (PLP)] in glycogen phosphorylase a (GPha), glycogen phosphorylase b (GPhb) and of the latter bound to various effectors and substrates were analysed on the basis of log-normal distribution curves. The results obtained showed that the ionization state of the PLP and GPha environment differs from that of GPhb. This divergence was interpreted in terms of tautomeric equilibria between some forms of the Schiff base of PLP and enzymic Lys-679. The ionic forms are slightly more predominant in GPha than they are in GPhb, so ionic and/or hydrogen-bonding interactions between the aromatic ring of PLP and GPha must be stronger than with GPhb. This confirms the purely structural role of the aromatic ring of the coenzyme. Binding of GPhb to AMP and Mg2+ results in the coenzyme adopting a similar state as in GPha. On the other hand, binding to IMP gives rise to no detectable changes in the tautomeric equilibrium of the coenzyme. PMID:8503849

  19. Anthranilimide-based glycogen phosphorylase inhibitors for the treatment of type 2 diabetes: 1. Identification of 1-amino-1-cycloalkyl carboxylic acid headgroups

    SciTech Connect

    Sparks, Steven M.; Banker, Pierette; Bickett, David M.; Carter, H. Luke; Clancy, Daphne C.; Dickerson, Scott H.; Dwornik, Kate A.; Garrido, Dulce M.; Golden, Pamela L.; Nolte, Robert T.; Peat, Andrew J.; Sheckler, Lauren R.; Tavares, Francis X.; Thomson, Stephen A.; Wang, Liping; Weiel, James E.

    2009-05-15

    Optimization of the amino acid residue within a series of anthranilimide-based glycogen phosphorylase inhibitors is described. These studies culminated in the identification of anthranilimides 16 and 22 which displayed potent in vitro inhibition of GPa in addition to reduced inhibition of CYP2C9 and excellent pharmacokinetic properties.

  20. Glucose-derived spiro-isoxazolines are anti-hyperglycemic agents against type 2 diabetes through glycogen phosphorylase inhibition.

    PubMed

    Goyard, David; Kónya, Bálint; Chajistamatiou, Aikaterini S; Chrysina, Evangelia D; Leroy, Jérémy; Balzarin, Sophie; Tournier, Michel; Tousch, Didier; Petit, Pierre; Duret, Cédric; Maurel, Patrick; Somsák, László; Docsa, Tibor; Gergely, Pál; Praly, Jean-Pierre; Azay-Milhau, Jacqueline; Vidal, Sébastien

    2016-01-27

    Glycogen phosphorylase (GP) is a target for the treatment of hyperglycaemia in the context of type 2 diabetes. This enzyme is responsible for the depolymerization of glycogen into glucose thereby affecting the levels of glucose in the blood stream. Twelve new d-glucopyranosylidene-spiro-isoxazolines have been prepared from O-peracylated exo-D-glucals by regio- and stereoselective 1,3-dipolar cycloaddition of nitrile oxides generated in situ by treatment of the corresponding oximes with bleach. This mild and direct procedure appeared to be applicable to a broad range of substrates. The corresponding O-unprotected spiro-isoxazolines were evaluated as glycogen phosphorylase (GP) inhibitors and exhibited IC50 values ranging from 1 to 800 μM. Selected inhibitors were further evaluated in vitro using rat and human hepatocytes and exhibited significant inhibitory properties in the primary cell culture. Interestingly, when tested with human hepatocytes, the tetra-O-acetylated spiro-isoxazoline bearing a 2-naphthyl residue showed a much lower IC50 value (2.5 μM), compared to that of the O-unprotected analog (19.95 μM). The most promising compounds were investigated in Zucker fa/fa rat model in acute and sub-chronic assays and decreased hepatic glucose production, which is known to be elevated in type 2 diabetes. This indicates that glucose-based spiro-isoxazolines can be considered as anti-hyperglycemic agents in the context of type 2 diabetes. PMID:26708111

  1. The binding of β-d-glucopyranosyl-thiosemicarbazone derivatives to glycogen phosphorylase: A new class of inhibitors.

    PubMed

    Alexacou, Kyra-Melinda; Tenchiu Deleanu, Alia-Cristina; Chrysina, Evangelia D; Charavgi, Maria-Despoina; Kostas, Ioannis D; Zographos, Spyros E; Oikonomakos, Nikos G; Leonidas, Demetres D

    2010-11-15

    Glycogen phosphorylase (GP) is a promising target for the treatment of type 2 diabetes. In the process of structure based drug design for GP, a group of 15 aromatic aldehyde 4-(β-d-glucopyranosyl)thiosemicarbazones have been synthesized and evaluated as inhibitors of rabbit muscle glycogen phosphorylase b (GPb) by kinetic studies. These compounds are competitive inhibitors of GPb with respect to α-d-glucose-1-phosphate with IC(50) values ranging from 5.7 to 524.3μM. In order to elucidate the structural basis of their inhibition, the crystal structures of these compounds in complex with GPb at 1.95-2.23Å resolution were determined. The complex structures reveal that the inhibitors are accommodated at the catalytic site with the glucopyranosyl moiety at approximately the same position as α-d-glucose and stabilize the T conformation of the 280s loop. The thiosemicarbazone part of the studied glucosyl thiosemicarbazones possess a moiety derived from substituted benzaldehydes with NO(2), F, Cl, Br, OH, OMe, CF(3), or Me at the ortho-, meta- or para-position of the aromatic ring as well as a moiety derived from 4-pyridinecarboxaldehyde. These fit tightly into the β-pocket, a side channel from the catalytic site with no access to the bulk solvent. The differences in their inhibitory potency can be interpreted in terms of variations in the interactions of the aldehyde-derived moiety with protein residues in the β-pocket. In addition, 14 out of the 15 studied inhibitors were found bound at the new allosteric site of the enzyme.

  2. Post-Exercise Muscle Glycogen Repletion in the Extreme: Effect of Food Absence and Active Recovery

    PubMed Central

    Fournier, Paul A.; Fairchild, Timothy J.; Ferreira, Luis D.; Bräu, Lambert

    2004-01-01

    Glycogen plays a major role in supporting the energy demands of skeletal muscles during high intensity exercise. Despite its importance, the amount of glycogen stored in skeletal muscles is so small that a large fraction of it can be depleted in response to a single bout of high intensity exercise. For this reason, it is generally recommended to ingest food after exercise to replenish rapidly muscle glycogen stores, otherwise one’s ability to engage in high intensity activity might be compromised. But what if food is not available? It is now well established that, even in the absence of food intake, skeletal muscles have the capacity to replenish some of their glycogen at the expense of endogenous carbon sources such as lactate. This is facilitated, in part, by the transient dephosphorylation-mediated activation of glycogen synthase and inhibition of glycogen phosphorylase. There is also evidence that muscle glycogen synthesis occurs even under conditions conducive to an increased oxidation of lactate post-exercise, such as during active recovery from high intensity exercise. Indeed, although during active recovery glycogen resynthesis is impaired in skeletal muscle as a whole because of increased lactate oxidation, muscle glycogen stores are replenished in Type IIa and IIb fibers while being broken down in Type I fibers of active muscles. This unique ability of Type II fibers to replenish their glycogen stores during exercise should not come as a surprise given the advantages in maintaining adequate muscle glycogen stores in those fibers that play a major role in fight or flight responses. Key Points Even in the absence of food intake, skeletal muscles have the capacity to replenish some of their glycogen at the expense of endogenous carbon sources such as lactate. During active recovery from exercise, skeletal muscles rich in type II fibers replenish part of their glycogen stores even in the absence of food intake. Post-exercise muscle glycogen synthesis in the

  3. Activation of Phosphorylase Kinase by Physiological Temperature.

    PubMed

    Herrera, Julio E; Thompson, Jackie A; Rimmer, Mary Ashley; Nadeau, Owen W; Carlson, Gerald M

    2015-12-29

    In the six decades since its discovery, phosphorylase kinase (PhK) from rabbit skeletal muscle has usually been studied at 30 °C; in fact, not a single study has examined functions of PhK at a rabbit's body temperature, which is nearly 10 °C greater. Thus, we have examined aspects of the activity, regulation, and structure of PhK at temperatures between 0 and 40 °C. Between 0 and 30 °C, the activity at pH 6.8 of nonphosphorylated PhK predictably increased; however, between 30 and 40 °C, there was a dramatic jump in its activity, resulting in the nonactivated enzyme having a far greater activity at body temperature than was previously realized. This anomalous change in properties between 30 and 40 °C was observed for multiple functions, and both stimulation (by ADP and phosphorylation) and inhibition (by orthophosphate) were considerably less pronounced at 40 °C than at 30 °C. In general, the allosteric control of PhK's activity is definitely more subtle at body temperature. Changes in behavior related to activity at 40 °C and its control can be explained by the near disappearance of hysteresis at physiological temperature. In important ways, the picture of PhK that has emerged from six decades of study at temperatures of ≤30 °C does not coincide with that of the enzyme studied at physiological temperature. The probable underlying mechanism for the dramatic increase in PhK's activity between 30 and 40 °C is an abrupt change in the conformations of the regulatory β and catalytic γ subunits between these two temperatures.

  4. Anthranilimide based glycogen phosphorylase inhibitors for the treatment of type 2 diabetes. Part 3: X-ray crystallographic characterization, core and urea optimization and in vivo efficacy

    SciTech Connect

    Thomson, Stephen A.; Banker, Pierette; Bickett, D. Mark; Boucheron, Joyce A.; Carter, H. Luke; Clancy, Daphne C.; Cooper, Joel P.; Dickerson, Scott H.; Garrido, Dulce M.; Nolte, Robert T.; Peat, Andrew J.; Sheckler, Lauren R.; Sparks, Steven M.; Tavares, Francis X.; Wang, Liping; Wang, Tony Y.; Weiel, James E.

    2009-05-15

    Key binding interactions of the anthranilimide based glycogen phosphorylase a (GPa) inhibitor 2 from X-ray crystallography studies are described. This series of compounds bind to the AMP site of GP. Using the binding information the core and the phenyl urea moieties were optimized. This work culminated in the identification of compounds with single nanomolar potency as well as in vivo efficacy in a diabetic model.

  5. Glycogen accumulation and degradation by the trichomonads Trichomonas vaginalis and Trichomonas tenax.

    PubMed

    Nielsen, Tyler J; Pradhan, Prajakta; Brittingham, Andrew; Wilson, Wayne A

    2012-01-01

    Several species of trichomonad have been shown to accumulate significant quantities of glycogen during growth, suggesting an important role for this compound in cell physiology. We provide the first analysis of the changes in glycogen content and glycogen phosphorylase activity that occur during in vitro growth of two trichomonad species: Trichomonas vaginalis and Trichomonas tenax. Both species accumulated glycogen following inoculation into fresh medium and utilized this compound during logarithmic growth. Glycogen phosphorylase activity also varied during growth in a species-specific manner. The expression of phosphorylase genes in T. vaginalis remained constant during growth and thus transcriptional control did not explain the observed fluctuations in phosphorylase activity. After cloning, expression, and purification, two recombinant glycogen phosphorylases from T. vaginalis and one recombinant glycogen phosphorylase from T. tenax had robust activity and, in contrast to many other eukaryotic glycogen phosphorylases, did not appear to be regulated by reversible protein phosphorylation. Furthermore, allosteric regulation, if present, was not mediated by compounds known to impact the activity of better characterized phosphorylases. PMID:22612314

  6. Synthetic, enzyme kinetic, and protein crystallographic studies of C-β-d-glucopyranosyl pyrroles and imidazoles reveal and explain low nanomolar inhibition of human liver glycogen phosphorylase.

    PubMed

    Kantsadi, Anastassia L; Bokor, Éva; Kun, Sándor; Stravodimos, George A; Chatzileontiadou, Demetra S M; Leonidas, Demetres D; Juhász-Tóth, Éva; Szakács, Andrea; Batta, Gyula; Docsa, Tibor; Gergely, Pál; Somsák, László

    2016-11-10

    C-β-d-Glucopyranosyl pyrrole derivatives were prepared in the reactions of pyrrole, 2-, and 3-aryl-pyrroles with O-peracetylated β-d-glucopyranosyl trichloroacetimidate, while 2-(β-d-glucopyranosyl) indole was obtained by a cross coupling of O-perbenzylated β-d-glucopyranosyl acetylene with N-tosyl-2-iodoaniline followed by spontaneous ring closure. An improved synthesis of O-perbenzoylated 2-(β-d-glucopyranosyl) imidazoles was achieved by reacting C-glucopyranosyl formimidates with α-aminoketones. The deprotected compounds were assayed with isoforms of glycogen phosphorylase (GP) to show no activity of the pyrroles against rabbit muscle GPb. The imidazoles proved to be the best known glucose derived inhibitors of not only the muscle enzymes (both a and b) but also of the pharmacologically relevant human liver GPa (Ki = 156 and 26 nM for the 4(5)-phenyl and -(2-naphthyl) derivatives, respectively). An X-ray crystallographic study of the rmGPb-imidazole complexes revealed structural features of the strong binding, and also allowed to explain the absence of inhibition for the pyrrole derivatives. PMID:27522507

  7. New approach to pharmacophore mapping and QSAR analysis using inductive logic programming. Application to thermolysin inhibitors and glycogen phosphorylase B inhibitors.

    PubMed

    Marchand-Geneste, Nathalie; Watson, Kimberly A; Alsberg, Bjørn K; King, Ross D

    2002-01-17

    A key problem in QSAR is the selection of appropriate descriptors to form accurate regression equations for the compounds under study. Inductive logic programming (ILP) algorithms are a class of machine-learning algorithms that have been successfully applied to a number of SAR problems. Unlike other QSAR methods, which use attributes to describe chemical structure, ILP uses relations. This gives ILP the advantages of not requiring explicit superimposition of individual compounds in a dataset, of dealing naturally with multiple conformations, and of using a language much closer to that used normally by chemists. We unify ILP and standard regression techniques to give a QSAR method that has the strength of ILP at describing steric structure with the familiarity and power of regression methods. Complex pharmacophores, correlating with activity, were identified and used as new indicator variables, along with the comparative molecular field analysis (CoMFA) prediction, to form predictive regression equations. We compared the formation of 3D-QSARs using standard CoMFA with the use of ILP on the well-studied thermolysin zinc protease inhibitor dataset and a glycogen phosphorylase inhibitor dataset. In each case the addition of ILP variables produced statistically better results (P < 0.01 for thermolysin and P < 0.05 for GP datasets) than the CoMFA analysis. Moreover, the new ILP variables were not found to increase the complexity of the final QSAR equations and gave possible insight into the binding mechanism of the ligand-protein complex under study.

  8. Cloning and expression patterns of the brine shrimp (Artemia sinica) glycogen phosphorylase (GPase) gene during development and in response to temperature stress.

    PubMed

    Zhao, Na; Hou, Ming; Wang, Ting; Chen, Yifei; Lv, Ying; Li, Zengrong; Zhang, Rui; Xin, Wenting; Zou, Xiangyang; Hou, Lin

    2014-01-01

    Glycogen serves as a metabolic reserve and is involved in macromolecular synthesis. Glycogen phosphorylase (GPase) is a key enzyme involved in intracellular glycogen catabolism, catalyzing the first step in glycogen degradation. In the diapause, GPase catalyzes glycogen into the closely related molecule, sorbitol. In this study, the full-length cDNA of the GPase gene (2,790 bp) was isolated from Artemia sinica for the first time by rapid amplification of cDNA ends technology. The GPase gene encoded a protein of 853 amino acids belonging to the Glycosyltransferase GTB type superfamily. The expression pattern and location of GPase were investigated at various stages during the embryonic development of A. sinica using real-time PCR and in situ hybridization. High GPase expression was detected at the 0 and 5 h stages. Subsequently, expression declined and was maintained at a low level during the stages from 10 to 40 h following by a small increase at day 3. Expression was downregulated at temperatures ranging from 25 to 20 °C and was subsequently upregulated in the range 15-5 °C. In situ hybridization assays showed wide distribution of the GPase gene during different developmental stages. From the results of this study, we conclude that the GPase gene expression is stress-related and might play an important role in Artemia development and metabolism. PMID:24323193

  9. Astrocyte glycogen as an emergency fuel under conditions of glucose deprivation or intense neural activity.

    PubMed

    Brown, Angus M; Ransom, Bruce R

    2015-02-01

    Energy metabolism in the brain is a complex process that is incompletely understood. Although glucose is agreed as the main energy support of the brain, the role of glucose is not clear, which has led to controversies that can be summarized as follows: the fate of glucose, once it enters the brain is unclear. It is not known the form in which glucose enters the cells (neurons and glia) within the brain, nor the degree of metabolic shuttling of glucose derived metabolites between cells, with a key limitation in our knowledge being the extent of oxidative metabolism, and how increased tissue activity alters this. Glycogen is present within the brain and is derived from glucose. Glycogen is stored in astrocytes and acts to provide short-term delivery of substrates to neural elements, although it may also contribute an important component to astrocyte metabolism. The roles played by glycogen awaits further study, but to date its most important role is in supporting neural elements during increased firing activity, where signaling molecules, proposed to be elevated interstitial K(+), indicative of elevated neural firing rates, activate glycogen phosphorylase leading to increased production of glycogen derived substrate.

  10. Targeting glycogen metabolism in bladder cancer

    PubMed Central

    Lew, Carolyn Ritterson; Guin, Sunny; Theodorescu, Dan

    2015-01-01

    Metabolism has been a heavily investigated topic in cancer research for the past decade. Although the role of aerobic glycolysis (the Warburg effect) in cancer has been extensively studied, abnormalities in other metabolic pathways are only just being understood in cancer. One such pathway is glycogen metabolism; its involvement in cancer development, particularly in urothelial malignancies, and possible ways of exploiting aberrations in this process for treatment are currently being studied. New research shows that the glycogen debranching enzyme amylo-α-1,6-glucosidase, 4-α-glucanotransferase (AGL) is a novel tumour suppressor in bladder cancer. Loss of AGL leads to rapid proliferation of bladder cancer cells. Another enzyme involved in glycogen debranching, glycogen phosphorylase, has been shown to be a tumour promoter in cancer, including in prostate cancer. Studies demonstrate that bladder cancer cells in which AGL expression is lost are more metabolically active than cells with intact AGL expression, and these cells are more sensitive to inhibition of both glycolysis and glycine synthesis—two targetable pathways. As a tumour promoter and enzyme, glycogen phosphorylase can be directly targeted, and preclinical inhibitor studies are promising. However, few of these glycogen phosphorylase inhibitors have been tested for cancer treatment in the clinical setting. Several possible limitations to the targeting of AGL and glycogen phosphorylase might also exist. PMID:26032551

  11. Targeting glycogen metabolism in bladder cancer.

    PubMed

    Ritterson Lew, Carolyn; Guin, Sunny; Theodorescu, Dan

    2015-07-01

    Metabolism has been a heavily investigated topic in cancer research for the past decade. Although the role of aerobic glycolysis (the Warburg effect) in cancer has been extensively studied, abnormalities in other metabolic pathways are only just being understood in cancer. One such pathway is glycogen metabolism; its involvement in cancer development, particularly in urothelial malignancies, and possible ways of exploiting aberrations in this process for treatment are currently being studied. New research shows that the glycogen debranching enzyme amylo-α-1,6-glucosidase, 4-α-glucanotransferase (AGL) is a novel tumour suppressor in bladder cancer. Loss of AGL leads to rapid proliferation of bladder cancer cells. Another enzyme involved in glycogen debranching, glycogen phosphorylase, has been shown to be a tumour promoter in cancer, including in prostate cancer. Studies demonstrate that bladder cancer cells in which AGL expression is lost are more metabolically active than cells with intact AGL expression, and these cells are more sensitive to inhibition of both glycolysis and glycine synthesis--two targetable pathways. As a tumour promoter and enzyme, glycogen phosphorylase can be directly targeted, and preclinical inhibitor studies are promising. However, few of these glycogen phosphorylase inhibitors have been tested for cancer treatment in the clinical setting. Several possible limitations to the targeting of AGL and glycogen phosphorylase might also exist.

  12. Increased sensitivity to glucose starvation correlates with downregulation of glycogen phosphorylase isoform PYGB in tumor cell lines resistant to 2-deoxy-d-glucose

    PubMed Central

    Philips, Katherine B.; Kurtoglu, Metin; Leung, Howard J.; Liu, Huaping; Gao, Ningguo; Lehrman, Mark A.; Murray, Timothy G.

    2015-01-01

    Background As tumors evolve, they upregulate glucose metabolism while also encountering intermittent periods of glucose deprivation. Here, we investigate mechanisms by which pancreatic cancer cells respond to therapeutic (2-deoxy-d-glucose, 2-DG) and physiologic (glucose starvation, GS) forms of glucose restriction. Methods From a tumor cell line (1420) that is unusually sensitive to 2-DG under normoxia, low (14DG2)- and high (14DG5)-dose resistant cell lines were selected and used to probe the metabolic pathways involved with their response to different forms of glucose deprivation. Results Muted induction of the unfolded protein response was found to correlate with resistance to 2-DG. Additionally, 14DG2 displayed reduced 2-DG uptake, while 14DG5 was cross-resistant to tunicamycin, suggesting it has enhanced ability to manage glycosylation defects. Conversely, 2-DG-resistant cell lines were more sensitive than their parental cell line to GS, which coincided with lowered levels of glycogen phosphorylase (PYGB) and reduced breakdown of glycogen to glucose in the 2-DG-resistant cell lines. Moreover, by inhibiting PYGB in the parental cell line, sensitivity to GS was increased. Conclusions Overall, the data demonstrate that the manner in which glucose is restricted in tumor cells, i.e., therapeutic or physiologic, leads to differential biological responses involving distinct glucose metabolic pathways. Moreover, in evolving tumors where glucose restriction occurs, the identification of PYGB as a metabolic target may have clinical application. PMID:24292700

  13. Effect of diabetes on glycogen metabolism in rat retina.

    PubMed

    Sánchez-Chávez, Gustavo; Hernández-Berrones, Jethro; Luna-Ulloa, Luis Bernardo; Coffe, Víctor; Salceda, Rocío

    2008-07-01

    Glucose is the main fuel for energy metabolism in retina. The regulatory mechanisms that maintain glucose homeostasis in retina could include hormonal action. Retinopathy is one of the chemical manifestations of long-standing diabetes mellitus. In order to better understand the effect of hyperglycemia in retina, we studied glycogen content as well as glycogen synthase and phosphorylase activities in both normal and streptozotocin-induced diabetic rat retina and compared them with other tissues. Glycogen levels in normal rat retina are low (46 +/- 4.0 nmol glucosyl residues/mg protein). However, high specific activity of glycogen synthase was found in retina, indicating a substantial capacity for glycogen synthesis. In diabetic rats, glycogen synthase activity increased between 50% and 100% in retina, brain cortex and liver of diabetic rats, but only retina exhibited an increase in glycogen content. Although, total and phosphorylated glycogen synthase levels were similar in normal and diabetic retina, activation of glycogen synthase by glucose-6-P was remarkable increased. Glycogen phosphorylase activity decreased 50% in the liver of diabetic animals; it was not modified in the other tissues examined. We conclude that the increase in glycogen levels in diabetic retina was due to alterations in glycogen synthase regulation. PMID:18274898

  14. Regulation of synthase phosphatase and phosphorylase phosphatase in rat liver.

    PubMed

    Tan, A W; Nuttall, F Q

    1976-08-12

    Using substrates purified from liver, the apparent Km values of synthase phosphatase ([UDPglucose--glycogen glucosyltransferase-D]phosphohydrolase, EC 3.1.3.42) and phosphorylase phosphatase (phosphorylase a phosphohydrolase, EC 3.1.3.17) were found to be 0.7 and 60 units/ml respectively. The maximal velocity of phosphorylase phosphatase was more than a 100 times that of synthase phosphatase. In adrenalectomized, fasted animals there was a complete loss of synthase phosphatase but only a slight decrease in phosphorylase phosphatase when activity was measured using endogenous substrates in a concentrated liver extract. When assayed under optimal conditions with purified substrates, both activities were present but had decreased to very low levels. Mixing experiments indicated that synthase D present in the extract of adrenalectomized fasted animals was altered such that it was no longer a substrate for synthase phosphatase from normal rats. Phosphorylase a substrate on the other hand was unaltered and readily converted. When glucose was given in vivo, no change in percent of synthase in the I form was seen in adrenalectomized rats but the percent of phosphorylase in the a form was reduced. Precipitation of protein from an extract of normal fed rats with ethanol produced a large activation of phosphorylase phosphatase activity with no corresponding increase in synthase phosphatase activity. Despite the low phosphorylase phosphatase present in extracts of adrenalectomized fasted animals, ethanol precipitation increased activity to the same high level as obtained in the normal fed rats. Synthase phosphatase and phosphorylase phosphatase activities were also decreased in normal fasted, diabetic fed and fasted, and adrenalectomized fed rats. Both enzymes recovered in the same manner temporally after oral glucose administration to adrenalectomized, fasted rats. These results suggest an integrated regulatory mechanism for the two phosphatase.

  15. Epinephrine-stimulated glycogen breakdown activates glycogen synthase and increases insulin-stimulated glucose uptake in epitrochlearis muscles.

    PubMed

    Kolnes, Anders J; Birk, Jesper B; Eilertsen, Einar; Stuenæs, Jorid T; Wojtaszewski, Jørgen F P; Jensen, Jørgen

    2015-02-01

    Epinephrine increases glycogen synthase (GS) phosphorylation and decreases GS activity but also stimulates glycogen breakdown, and low glycogen content normally activates GS. To test the hypothesis that glycogen content directly regulates GS phosphorylation, glycogen breakdown was stimulated in condition with decreased GS activation. Saline or epinephrine (0.02 mg/100 g rat) was injected subcutaneously in Wistar rats (∼130 g) with low (24-h-fasted), normal (normal diet), and high glycogen content (fasted-refed), and epitrochlearis muscles were removed after 3 h and incubated ex vivo, eliminating epinephrine action. Epinephrine injection reduced glycogen content in epitrochlearis muscles with high (120.7 ± 17.8 vs. 204.6 ± 14.5 mmol/kg, P < 0.01) and normal glycogen (89.5 ± 7.6 vs. 152 ± 8.1 mmol/kg, P < 0.01), but not significantly in muscles with low glycogen (90.0 ± 5.0 vs. 102.8 ± 7.8 mmol/kg, P = 0.17). In saline-injected rats, GS phosphorylation at sites 2+2a, 3a+3b, and 1b was higher and GS activity lower in muscles with high compared with low glycogen. GS sites 2+2a and 3a+3b phosphorylation decreased and GS activity increased in muscles where epinephrine decreased glycogen content; these parameters were unchanged in epitrochlearis from fasted rats where epinephrine injection did not decrease glycogen content. Incubation with insulin decreased GS site 3a+3b phosphorylation independently of glycogen content. Insulin-stimulated glucose uptake was increased in muscles where epinephrine injection decreased glycogen content. In conclusion, epinephrine stimulates glycogenolysis in epitrochlearis muscles with normal and high, but not low, glycogen content. Epinephrine-stimulated glycogenolysis decreased GS phosphorylation and increased GS activity. These data for the first time document direct regulation of GS phosphorylation by glycogen content.

  16. Allosteric inhibition of glycogen phosphorylase a by the potential antidiabetic drug 3-isopropyl 4-(2-chlorophenyl)-1,4-dihydro-1-ethyl-2-methyl-pyridine-3,5,6-tricarbo xylate.

    PubMed Central

    Oikonomakos, N. G.; Tsitsanou, K. E.; Zographos, S. E.; Skamnaki, V. T.; Goldmann, S.; Bischoff, H.

    1999-01-01

    The effect of the potential antidiabetic drug (-)(S)-3-isopropyl 4-(2-chlorophenyl)-1,4-dihydro-1-ethyl-2-methyl-pyridine-3,5,6-tricarbox ylate (W1807) on the catalytic and structural properties of glycogen phosphorylase a has been studied. Glycogen phosphorylase (GP) is an allosteric enzyme whose activity is primarily controlled by reversible phosphorylation of Ser14 of the dephosphorylated enzyme (GPb, less active, predominantly T-state) to form the phosphorylated enzyme (GPa, more active, predominantly R-state). Upon conversion of GPb to GPa, the N-terminal tail (residues 5-22), which carries the Ser14(P), changes its conformation into a distorted 3(10) helix and its contacts from intrasubunit to intersubunit. This alteration causes a series of tertiary and quaternary conformational changes that lead to activation of the enzyme through opening access to the catalytic site. As part of a screening process to identify compounds that might contribute to the regulation of glycogen metabolism in the noninsulin dependent diabetes diseased state, W1807 has been found as the most potent inhibitor of GPb (Ki = 1.6 nM) that binds at the allosteric site of T-state GPb and produces further conformational changes, characteristic of a T'-like state. Kinetics show W1807 is a potent competitive inhibitor of GPa (-AMP) (Ki = 10.8 nM) and of GPa (+1 mM AMP) (Ki = 19.4 microM) with respect to glucose 1-phosphate and acts in synergism with glucose. To elucidate the structural features that contribute to the binding, the structures of GPa in the T-state conformation in complex with glucose and in complex with both glucose and W1807 have been determined at 100 K to 2.0 A and 2.1 A resolution, and refined to crystallographic R-values of 0.179 (R(free) = 0.230) and 0.189 (R(free) = 0.263), respectively. W1807 binds tightly at the allosteric site and induces substantial conformational changes both in the vicinity of the allosteric site and the subunit interface. A disordering of the N

  17. Glycogen phosphorylase as a target for type 2 diabetes: synthetic, biochemical, structural and computational evaluation of novel N-acyl-N´-(β-D-glucopyranosyl) urea inhibitors.

    PubMed

    Kantsadi, Anastassia L; Parmenopoulou, Vanessa; Bakalov, Dimitar N; Snelgrove, Laura; Stravodimos, George A; Chatzileontiadou, Demetra S M; Manta, Stella; Panagiotopoulou, Angeliki; Hayes, Joseph M; Komiotis, Dimitri; Leonidas, Demetres D

    2015-01-01

    Glycogen phosphorylase (GP), a validated target for the development of anti-hyperglycaemic agents, has been targeted for the design of novel glycopyranosylamine inhibitors. Exploiting the two most potent inhibitors from our previous study of N-acyl-β-D-glucopyranosylamines (Parmenopoulou et al., Bioorg. Med. Chem. 2014, 22, 4810), we have extended the linking group to -NHCONHCO- between the glucose moiety and the aliphatic/aromatic substituent in the GP catalytic site β-cavity. The N-acyl-N´-(β-D-glucopyranosyl) urea inhibitors were synthesized and their efficiency assessed by biochemical methods, revealing inhibition constant values of 4.95 µM and 2.53 µM. Crystal structures of GP in complex with these inhibitors were determined and analyzed, providing data for further structure based design efforts. A novel Linear Response - Molecular Mechanics Coulomb Surface Area (LR-MM-CBSA) method has been developed which relates predicted and experimental binding free energies for a training set of N-acyl-N´-(β-D-glucopyranosyl) urea ligands with a correlation coefficient R(2) of 0.89 and leave-one-out cross-validation (LOO-cv) Q(2) statistic of 0.79. The method has significant applications to direct future lead optimization studies, where ligand entropy loss on binding is revealed as a key factor to be considered. ADMET property predictions revealed that apart from potential permeability issues, the synthesized N-acyl-N´-(β-D-glucopyranosyl) urea inhibitors have drug-like potential without any toxicity warnings.

  18. Activation of direct and indirect pathways of glycogen synthesis by hepatic overexpression of protein targeting to glycogen

    PubMed Central

    O’Doherty, Robert M.; Jensen, Per B.; Anderson, Paul; Jones, John G.; Berman, Hal K.; Kearney, Denise; Newgard, Christopher B.

    2000-01-01

    Glycogen-targeting subunits of protein phosphatase-1, such as protein targeting to glycogen (PTG), direct the phosphatase to the glycogen particle, where it stimulates glycogenesis. We have investigated the metabolic impact of overexpressing PTG in liver of normal rats. After administration of PTG cDNA in a recombinant adenovirus, animals were fasted or allowed to continue feeding for 24 hours. Liver glycogen was nearly completely depleted in fasted control animals, whereas glycogen levels in fasted or fed PTG-overexpressing animals were 70% higher than in fed controls. Nevertheless, transgenic animals regulated plasma glucose, triglycerides, FFAs, ketones, and insulin normally in the fasted and fed states. Fasted PTG-overexpressing animals receiving an oral bolus of [U-13C]glucose exhibited a large increase in hepatic glycogen content and a 70% increase in incorporation of [13C]glucose into glycogen. However, incorporation of labeled glucose accounted for only a small portion of the glycogen synthesized in PTG-overexpressing animals, consistent with our earlier finding that PTG promotes glycogen synthesis from gluconeogenic precursors. We conclude that hepatic PTG overexpression activates both direct and indirect pathways of glycogen synthesis. Because of its ability to enhance glucose storage without affecting other metabolic indicators, the glycogen-targeting subunit may prove valuable in controlling blood glucose levels in diabetes. PMID:10683377

  19. Fatal infantile cardiac glycogenosis with phosphorylase kinase deficiency and a mutation in the gamma2-subunit of AMP-activated protein kinase.

    PubMed

    Akman, Hasan O; Sampayo, James N; Ross, Fiona A; Scott, John W; Wilson, Gregory; Benson, Lee; Bruno, Claudio; Shanske, Sara; Hardie, D Grahame; Dimauro, Salvatore

    2007-10-01

    A 10-wk-old infant girl with severe hypertrophy of the septal and atrial walls by cardiac ultrasound, developed progressive ventricular wall thickening and died of aspiration pneumonia at 5 mo of age. Postmortem examination revealed ventricular hypertrophy and massive atrial wall thickening due to glycogen accumulation. A skeletal muscle biopsy showed increased free glycogen and decreased activity of phosphorylase b kinase (PHK). The report of a pathogenic mutation (R531Q) in the gene (PRKAG2) encoding the gamma2 subunit of AMP-activated protein kinase (AMPK) in three infants with congenital hypertrophic cardiomyopathy, glycogen storage, and "pseudo PHK deficiency" prompted us to screen this gene in our patient. We found a novel (R384T) heterozygous mutation in PRKAG2, affecting an arginine residue in the N-terminal AMP-binding domain. Like R531Q, this mutation reduces the binding of AMP and ATP to the isolated nucleotide-binding domains, and prevents activation of the heterotrimer by metabolic stress in intact cells. The mutation was not found in DNA from the patient's father, the only available parent, and is likely to have arisen de novo. Our studies confirm that mutations in PRKAG2 can cause fatal infantile cardiomyopathy, often associated with apparent PHK deficiency.

  20. Fetal-onset severe skeletal muscle glycogenosis associated with phosphorylase-b kinase deficiency.

    PubMed

    Bührer, C; van Landeghem, F; Brück, W; Felderhoff-Müser, U; Vorgerd, M; Obladen, M

    2000-04-01

    We report on a premature newborn girl delivered after 32 weeks of gestation by cesarean section after sparse limb movements, fetal tachycardia and late heart rate decelerations had suggested fetal distress. Following 1 day of mechanical ventilation, adequate pulmonary gas exchange was achieved by spontaneous breathing. Main symptoms were virtually complete absence of spontaneous movements, increased flexor tonus of the extremities, and hypotonia of the trunk. Inability to suck or swallow required nasogastric gavage feeding. There were no hypoglycemic episodes. Echocardiography revealed normal myocardial function. Creatine kinase was 237 U/I at 2 days of life, declining to normal values thereafter. Muscle biopsy revealed increased glycogen storage with subsarcolemmal glycogen deposits and low phosphorylase-a activity while total phosphorylase was normal after in vitro activation, suggestive of phosphorylase-b kinase deficiency. No mutation was detected in exon 1 of the myophosphorylase gene. No psychomotor development was observed, and the infant died of central apnea at 3 months of age.

  1. Increased glycogen accumulation in transgenic mice overexpressing glycogen synthase in skeletal muscle.

    PubMed Central

    Manchester, J; Skurat, A V; Roach, P; Hauschka, S D; Lawrence, J C

    1996-01-01

    To investigate the role of glycogen synthase in controlling glycogen accumulation, we generated three lines of transgenic mice in which the enzyme was overexpressed in skeletal muscle by using promoter-enhancer elements derived from the mouse muscle creatine kinase gene. In all three lines, expression was highest in muscles composed primarily of fast-twitch fibers, such as the gastrocnemius and anterior tibialis. In these muscles, glycogen synthase activity was increased by as much as 10-fold, with concomitant increases (up to 5-fold) in the glycogen content. The uridine diphosphoglucose concentrations were markedly decreased, consistent with the increase in glycogen synthase activity. Levels of glycogen phosphorylase in these muscles increased (up to 3-fold), whereas the amount of the insulin-sensitive glucose transporter 4 either remained unchanged or decreased. The observation that increasing glycogen synthase enhances glycogen accumulation supports the conclusion that the activation of glycogen synthase, as well as glucose transport, contributes to the accumulation of glycogen in response to insulin in skeletal muscle. Images Fig. 1 PMID:8855244

  2. High phosphorylase activity is correlated with increased potato minituber formation and starch content during extended clinorotation

    NASA Astrophysics Data System (ADS)

    Nedukha, O. M.; Schnyukova, E. I.; Leach, J. E.

    2003-05-01

    The major purpose of these experiments were to investigate growth of potato storage organs and starch synthesis in minitubers at slow horizontal clinorotation (2 rpm), which partly mimics microgravity, and a secondary goal was to study the activity and localization of phosphorylase (EC 2.4.1.1) in storage parenchyma under these conditions. Miniplants of Solanum tuberosum L. (cv Adreta) were grown in culture for 30 days for both the vertical control and the horizontal clinorotation. During long-term clinorotation, an acceleration of minituber formation, and an increase of amyloplast number and size in storage parenchyma cells, as well as increased starch content, was observed in the minitubers. The differences among cytochemical reaction intensity, activity of phosphorylase, and carbohydrate content in storage parenchyma cells of minitubers grown in a horizontal clinostat were established by electron-cytochemical and biochemical methods. It is shown that high phosphorylase activity is correlated with increased starch content during extended clinorotation. The results demonstrate the increase in carbohydrate metabolism and possible accelerated growth of storage organs under the influence of microgravity, as mimicked by clinorotation; therefore, clinorotation can be used as a basis for future studies on mechanisms of starch synthesis under microgravity.

  3. Uterine glycogen metabolism in mink during estrus, embryonic diapause and pregnancy.

    PubMed

    Dean, Matthew; Hunt, Jason; McDougall, Lisa; Rose, Jack

    2014-01-01

    We have determined uterine glycogen content, metabolizing enzyme expression and activity in the mink, a species that exhibits obligatory embryonic diapause, resulting in delayed implantation. Gross uterine glycogen concentrations were highest in estrus, decreased 50% by diapause and 90% in pregnancy (P ≤ 0.05). Endometrial glycogen deposits, which localized primarily to glandular and luminal epithelia, decreased 99% between estrus and diapause (P ≤ 0.05) and were nearly undetectable in pregnancy. Glycogen synthase and phosphorylase proteins were most abundant in the glandular epithelia. Glycogen phosphorylase activity (total) in uterine homogenates was higher during estrus and diapause, than pregnancy. While glycogen phosphorylase protein was detected during estrus and diapause, glycogen synthase was almost undetectable after estrus, which probably contributed to a higher glycogenolysis/glycogenesis ratio during diapause. Uterine glucose-6-phosphatase 3 gene expression was greater during diapause, when compared to estrus (P ≤ 0.05) and supports the hypothesis that glucose-6-phosphate resulting from phosphorylase activity was dephosphorylated in preparation for export into the uterine lumen. The relatively high amount of hexokinase-1 protein detected in the luminal epithelia during estrus and diapause may have contributed to glucose trapping after endometrial glycogen reserves were depleted. Collectively, our findings suggest to us that endometrial glycogen reserves may be an important source of energy, supporting uterine and conceptus metabolism up to the diapausing blastocyst stage. As a result, the size of uterine glycogen reserves accumulated prior to mating may in part, determine the number of embryos that survive to the blastocyst stage, and ultimately litter size. PMID:25225159

  4. The effects of estradiol and catecholestrogens on uterine glycogen metabolism in mink (Neovison vison)

    PubMed Central

    Rose, Jack; Hunt, Jason; Shelton, Jadd; Wyler, Steven; Mecham, Daniel

    2011-01-01

    Glycogen is a uterine histotroph nutrient synthesized by endometrial glands in response to estradiol. The effects of estradiol may be mediated, in part, through the catecholestrogens, 2-hydroxycatecholestradiol (2-OHE2) and 4-hydroxycatecholestradiol (4-OHE2), produced by hydroxylation of estradiol within the endometrium. Using ovariectomized mink, our objectives were to determine the effects of estradiol, 4-OHE2, and 2-OHE2 on uterine: 1) glycogen concentrations and tissue localization; 2) gene expression levels for glycogen synthase, glycogen phosphorylase, and glycogen synthase kinase-3B; and 3) protein expression levels for glycogen synthase kinase-3B (active) and phospho-glycogen synthase kinase-3B (inactive). Whole uterine glycogen concentrations (mean ± SEM, mg/g dry wt) were increased by estradiol (43.79 ± 5.35), 4-OHE2 (48.64 ± 4.02), and 2-OHE2 (41.36 ± 3.23) compared to controls (4.58 ± 1.16; P ≤ 0.05). Percent glycogen content of the glandular epithelia was three-fold greater than the luminal epithelia in response to estradiol and 4-OHE2 (P ≤ 0.05). Expression of glycogen synthase mRNA, the rate limiting enzyme in glycogen synthesis, was increased by 4-OHE2 and 2-OHE2 (P ≤ 0.05), but interestingly, was unaffected by estradiol. Expression of glycogen phosphorylase and glycogen synthase kinase-3B mRNAs were reduced by estradiol, 2-OHE2, and 4-OHE2 (P ≤ 0.05). Uterine phospho-glycogen synthase kinase-3B protein was barely detectable in control mink, whereas all three steroids increased phosphorylation and inactivation of the enzyme (P ≤ 0.05). We concluded that the effects of estradiol on uterine glycogen metabolism were mediated in part through catecholestrogens; perhaps the combined actions of these hormones are required for optimal uterine glycogen synthesis in mink. PMID:21196035

  5. Expression and characterization of glycogen synthase kinase-3 mutants and their effect on glycogen synthase activity in intact cells.

    PubMed Central

    Eldar-Finkelman, H; Argast, G M; Foord, O; Fischer, E H; Krebs, E G

    1996-01-01

    In these studies we expressed and characterized wild-type (WT) GSK-3 (glycogen synthase kinase-3) and its mutants, and examined their physiological effect on glycogen synthase activity. The GSK-3 mutants included mutation at serine-9 either to alanine (S9A) or glutamic acid (S9E) and an inactive mutant, K85,86MA. Expression of WT and the various mutants in a cell-free system indicated that S9A and S9E exhibit increased kinase activity as compared with WT. Subsequently, 293 cells were transiently transfected with WT GSK-3 and mutants. Cells expressing the S9A mutant exhibited higher kinase activity (2.6-fold of control cells) as compared with cells expressing WT and S9E (1.8- and 2.0-fold, respectively, of control cells). Combined, these results suggest serine-9 as a key regulatory site of GSK-3 inactivation, and indicate that glutamic acid cannot mimic the function of the phosphorylated residue. The GSK-3-expressing cell system enabled us to examine whether GSK-3 can induce changes in the endogenous glycogen synthase activity. A decrease in glycogen synthase activity (50%) was observed in cells expressing the S9A mutant. Similarly, glycogen synthase activity was suppressed in cells expressing WT and the S9E mutant (20-30%, respectively). These studies indicate that activation of GSK-3 is sufficient to inhibit glycogen synthase in intact cells, and provide evidence supporting a physiological role for GSK-3 in regulating glycogen synthase and glycogen metabolism. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8816781

  6. Glycogen shortage during fasting triggers liver–brain–adipose neurocircuitry to facilitate fat utilization

    PubMed Central

    Izumida, Yoshihiko; Yahagi, Naoya; Takeuchi, Yoshinori; Nishi, Makiko; Shikama, Akito; Takarada, Ayako; Masuda, Yukari; Kubota, Midori; Matsuzaka, Takashi; Nakagawa, Yoshimi; Iizuka, Yoko; Itaka, Keiji; Kataoka, Kazunori; Shioda, Seiji; Niijima, Akira; Yamada, Tetsuya; Katagiri, Hideki; Nagai, Ryozo; Yamada, Nobuhiro; Kadowaki, Takashi; Shimano, Hitoshi

    2013-01-01

    During fasting, animals maintain their energy balance by shifting their energy source from carbohydrates to triglycerides. However, the trigger for this switch has not yet been entirely elucidated. Here we show that a selective hepatic vagotomy slows the speed of fat consumption by attenuating sympathetic nerve-mediated lipolysis in adipose tissue. Hepatic glycogen pre-loading by the adenoviral overexpression of glycogen synthase or the transcription factor TFE3 abolished this liver–brain–adipose axis activation. Moreover, the blockade of glycolysis through the knockdown of the glycogen phosphorylase gene and the resulting elevation in the glycogen content abolished the lipolytic signal from the liver, indicating that glycogen is the key to triggering this neurocircuitry. These results demonstrate that liver glycogen shortage activates a liver–brain–adipose neural axis that has an important role in switching the fuel source from glycogen to triglycerides under prolonged fasting conditions. PMID:23939267

  7. Glycogen supercompensation in rat soleus muscle during recovery from nonweight bearing

    NASA Technical Reports Server (NTRS)

    Henriksen, Erik J.; Kirby, Christopher R.; Tischler, Marc E.

    1989-01-01

    Events leading to the normalization of the glycogen metabolism in the soleus muscle of rat, altered by 72-h three days of hind-limb suspension, were investigated during the 72-h recovery period when the animals were allowed to bear weight on all four limbs. Relative importance of the factors affecting glycogen metabolism in skeletal muscle during the recovery period was also examined. Glycogen concentration was found to decrease within 15 min and up to 2 h of recovery, while muscle glucose 6-phosphate, and the fractional activities of glycogen phosphorylase and glycogen synthase increased. From 2 to 4 h, when the glycogen synthase activity remained elevated and the phosphorylase activity declined, glycogen concentration increased, until it reached maximum values at about 24 h, after which it started to decrease, reaching control values by 72 h. At 12 and 24 h, the inverse relationship between glycogen concentration and the synthase activity ratio was lost, indicating that the reloading transiently uncoupled glycogen control of this enzyme.

  8. Myocardial glycogen dynamics: new perspectives on disease mechanisms.

    PubMed

    Chandramouli, Chanchal; Varma, Upasna; Stevens, Ellie M; Xiao, Rui-Ping; Stapleton, David I; Mellor, Kimberley M; Delbridge, Lea M D

    2015-04-01

    Cardiac glycogen regulation involves a complex interplay between multiple signalling pathways, allosteric activation of enzymes, and sequestration for autophagic degradation. Signalling pathways appear to converge on glycogen regulatory enzymes via insulin (glycogen synthase kinase 3β, protein phosphatase 1, allosteric action of glucose-6-phosphate), β-adrenergic (phosphorylase kinase protein phosphatase 1 inhibitor), and 5' adenosine monophosphate-activated protein kinase (allosteric action of glucose-6-phosphate, direct glycogen binding, insulin receptor). While cytosolic glycogen synthesis and breakdown are relatively well understood, recent findings relating to phagic glycogen degradation highlight a new area of investigation in the heart. It has been recently demonstrated that a specific glycophagy pathway is operational in the myocardium. Proteins involved in recruiting glycogen to the forming phagosome have been identified. Starch-binding domain-containing protein 1 is involved in binding glycogen and mediating membrane anchorage via interaction with a homologue of the phagosomal protein light-chain 3. Specifically, it has been shown that starch-binding domain-containing protein 1 and light-chain 3 have discrete phagosomal immunolocalization patterns in cardiomyocytes, indicating that autophagic trafficking of glycogen and protein cargo in cardiomyocytes can occur via distinct pathways. There is strong evidence from glycogen storage diseases that phagic/lysosomal glycogen breakdown is important for maintaining normal cardiac glycogen levels and does not simply constitute a redundant 'alternative' breakdown route for glycogen. Advancing understanding of glycogen handling in the heart is an important priority with relevance not only to genetic glycogen storage diseases but also to cardiac metabolic stress disorders such as diabetes and ischaemia.

  9. Glycogen metabolism in novikoff ascites-hepatoma cells

    PubMed Central

    Nigam, V. N.

    1967-01-01

    A study of the enzymes of the glycogen pathway in Novikoff ascites hepatoma shows that glycogen synthetase has the lowest activity and that the tumour contains no high-Km soluble glucokinase. However, incubation of tumour cells with metabolizable sugars in vitro, or intraperitoneal administration of glucose into the tumour-bearing rat, results in glycogen accumulation by the tumour cells. Glycogen synthesis in the tumour is supported by aerobically produced ATP but is decreased anaerobically and by uncouplers of oxidative phosphorylation. Absence of Pi from the incubation medium increases glycogen synthesis and decreases glycolysis. The optimum temperature for glycogen synthesis is 37°. The capacity of the intact tumour cell to degrade deposited glycogen is low, but is accelerated by 2,4-dinitrophenol. Tumour homogenates prepared after osmotic shock do not incorporate [14C]glucose into glycogen. The glucose moiety of glucose 1-phosphate and of UDP-glucose is incorporated into glycogen by the homogenates and the incorporation of glucose 1-phosphate is greatly enhanced by AMP. Glucose 6-phosphate is a poor precursor of glycogen in the homogenate system, probably because it inhibits activation of phosphorylase b by AMP. PMID:4291492

  10. Adsorption and enzyme activity of sucrose phosphorylase on lipid Langmuir and Langmuir-Blodgett films.

    PubMed

    Rocha, Jefferson Muniz; Caseli, Luciano

    2014-04-01

    The production of bioelectronic devices, including biosensors, can be conducted using enzymes immobilized in ultrathin solid films, for which preserving the enzymatic catalytic activity is crucial for optimal performance. In this sense, nanostructured films that allow for control over molecular architectures are of interest. In this paper, we investigate the adsorption of sucrose phosphorylase onto Langmuir monolayers of the phospholipid dimyristoylphosphatidic acid, which caused the surface pressure isotherms to expand. With polarization-modulated infrared reflection absorption spectroscopy (PM-IRRAS), the amide bands from the enzyme could be identified, with the C-N and C=O dipole moments lying parallel to the air-water interface. Structuring of the enzyme into an α-helix was noted, and this structure was preserved when the mixed enzyme-phospholipid monolayer was transferred in the form of a Langmuir-Blodgett (LB) film. The latter was demonstrated with measurements of the catalytic activity of sucrose phosphorylase, which presented the highest enzyme activity for multilayer LB film. The approach presented in this study not only allows for optimized catalytic activity toward sucrose but also permits to explain why certain film architectures exhibit superior performance.

  11. Structural basis for glucose-6-phosphate activation of glycogen synthase

    SciTech Connect

    Baskaran, Sulochanadevi; Roach, Peter J.; DePaoli-Roach, Anna A.; Hurley, Thomas D.

    2010-11-22

    Regulation of the storage of glycogen, one of the major energy reserves, is of utmost metabolic importance. In eukaryotes, this regulation is accomplished through glucose-6-phosphate levels and protein phosphorylation. Glycogen synthase homologs in bacteria and archaea lack regulation, while the eukaryotic enzymes are inhibited by protein kinase mediated phosphorylation and activated by protein phosphatases and glucose-6-phosphate binding. We determined the crystal structures corresponding to the basal activity state and glucose-6-phosphate activated state of yeast glycogen synthase-2. The enzyme is assembled into an unusual tetramer by an insertion unique to the eukaryotic enzymes, and this subunit interface is rearranged by the binding of glucose-6-phosphate, which frees the active site cleft and facilitates catalysis. Using both mutagenesis and intein-mediated phospho-peptide ligation experiments, we demonstrate that the enzyme's response to glucose-6-phosphate is controlled by Arg583 and Arg587, while four additional arginine residues present within the same regulatory helix regulate the response to phosphorylation.

  12. Glycogen function in adult central and peripheral nerves.

    PubMed

    Evans, Richard D; Brown, Angus M; Ransom, Bruce R

    2013-08-01

    We studied the roles of glycogen in axonal pathways of the central nervous system (CNS) and peripheral nervous system (PNS). By using electrophysiological recordings, in combination with biochemical glycogen assay, it was possible to determine whether glycogen was crucial to axon function under different conditions. Glycogen was present both in mouse optic nerve (MON) and in mouse sciatic nerve (MSN). Aglycemia caused loss of the compound action potential (CAP) in both pathways after a latency of 15 min (MON) and 120 min for myelinated axons (A fibers) in the MSN. With the exception of unmyelinated axons (C fibers) in the MSN, CAP decline began when usable glycogen was exhausted. Glycogen was located in astrocytes in the MON and in myelinating Schwann cells in the MSN; it was absent from the Schwann cells surrounding unmyelinated C fibers. In MON, astrocytic glycogen is metabolized to lactate and "shuttled" to axons to support metabolism. The ability of lactate to support A fiber conduction in the absence of glucose suggests a common pathway in both the CNS and the PNS. Lactate is released from MON and MSN in substantial quantities. That lactate levels fall in MSN in the presence of diaminobenzidine, which inhibits glycogen phosphorylase, strongly suggests that glycogen metabolism contributes to lactate release under resting conditions. Glycogen is a "backup" energy substrate in both the CNS and the PNS and, beyond sustaining excitability during glucose deprivation, has the capacity to subsidize the axonal energy demands during times of intense activity in the presence of glucose.

  13. Stimulating effect of phosphatidic acid on autophosphorylation of phosphorylase kinase.

    PubMed

    Negami, A I; Sasaki, H; Yamamura, H

    1985-09-16

    Autophosphorylation of phosphorylase kinase from rabbit skeletal muscle was stimulated by acidic phospholipids such as phosphatidic acid (PA), phosphatidylinositol, and phosphatidyl-serine. PA stimulated an initial velocity of autophosphorylation 3.8-fold. When fully autophosphorylated, about 11 mol of phosphate per tetramer (alpha beta gamma delta) were incorporated in the presence of PA and about 6.5 mol in the absence of PA. In the presence of PA (100 micrograms/ml), there was a concomitant enhancement of its kinase activity about 25-fold at pH 6.8. PA (100 micrograms/ml) sharply decreased an apparent Ka for Ca2+ on autophosphorylation from 4.0 X 10(-5) M to 1.0 X 10(-6) M. Available evidence indicates that the Ca2+-activated, PA-dependent autophosphorylation of phosphorylase kinase shows an ability to stimulate glycogen breakdown.

  14. Accumulation of glycogen in axotomized adult rat facial motoneurons.

    PubMed

    Takezawa, Yosuke; Baba, Otto; Kohsaka, Shinichi; Nakajima, Kazuyuki

    2015-06-01

    This study biochemically determined glycogen content in the axotomized facial nucleus of adult rats up to 35 days postinsult. The amounts of glycogen in the transected facial nucleus were significantly increased at 5 days postinsult, peaked at 7 days postinsult, and declined to the control levels at 21-35 days postinsult. Immunohistochemical analysis with antiglycogen antibody revealed that the quantity of glycogen granules in the axotomized facial nucleus was greater than that in the control nucleus at 7 days postinjury. Dual staining methods with antiglycogen antibody and a motoneuron marker clarified that the glycogen was localized mainly in motoneurons. Immunoblotting and quantification analysis revealed that the ratio of inactive glycogen synthase (GS) to total GS was significantly decreased in the injured nucleus at about 1-3 days postinsult and significantly increased from 7 to 14 days postinsult, suggesting that glycogen is actively synthesized in the early period postinjury but suppressed after 7 days postinsult. The enhanced glycogen at about 5-7 days postinsult is suggested to be responsible for the decrease in inactive GS levels, and the decrease of glycogen after 7 days postinsult is considered to be caused by increased inactive GS levels and possibly the increase in active glycogen phosphorylase.

  15. Regulation of glycogen breakdown and its consequences for skeletal muscle function after training.

    PubMed

    Katz, Abram; Westerblad, Håkan

    2014-10-01

    Repeated bouts of physical exercise, i.e., training, induce mitochondrial biogenesis and result in improved physical performance and attenuation of glycogen breakdown during submaximal exercise. It has been suggested that as a consequence of the increased mitochondrial volume, a smaller degree of metabolic stress (e.g., smaller increases in ADP and Pi) is required to maintain mitochondrial respiration in the trained state during exercise at the same absolute intensity. The lower degree of Pi accumulation is believed to account for the diminished glycogen breakdown, since Pi is a substrate for glycogen phosphorylase, the rate-limiting enzyme for glycogenolysis. However, in this review, we present an alternative explanation for the diminished glycogen breakdown. Thus, the lower degree of metabolic stress after training is also associated with smaller increases in AMP (free concentration during contraction at specific intracellular sites) and this results in less activation of phosphorylase b (the non-phosphorylated form of phosphorylase), resulting in diminished glycogen breakdown. Concomitantly, the smaller accumulation of Pi, which interferes with cross-bridge function and intracellular Ca(2+) handling, contributes to the increased fatigue resistance. The delay in glycogen depletion also contributes to enhanced performance during prolonged exercise by functioning as an energy reserve.

  16. Elevated thymidine phosphorylase activity in psoriatic lesions accounts for the apparent presence of an epidermal growth inhibitor, but is not in itself growth inhibitory

    SciTech Connect

    Hammerberg, C.; Fisher, G.J.; Voorhees, J.J.; Cooper, K.D. )

    1991-08-01

    An apparent tissue-specific growth inhibitor, or chalone, obtained from psoriatic lesions was tentatively identified in the 100-kDa fraction based upon inhibition of DNA synthesis, as measured by (3H)-thymidine uptake by a squamous cell carcinoma cell line, SCC 38. This fraction, however, failed to inhibit SCC 38 cell growth when assessed directly in a neutral red uptake assay. Characterization of the inhibitor of (3H)-thymidine uptake revealed it to have biochemical properties identical to thymidine phosphorylase: (1) molecular weight close to 100 kDa, (2) isoelectric point of 4.2, and (3) thymidine phosphorylase enzyme activity. Thus, the authors conclude that its ability to inhibit (3H)-thymidine uptake was due to thymidine catabolism rather than inhibition of DNA synthesis or growth inhibition. Examination of thymidine phosphorylase activity in keratome biopsies from psoriatic and normal skin demonstrated a twentyfold increase in activity in psoriatic lesions relative to non-lesional or normal skin. This increase in metabolism of thymidine was due to thymidine phosphorylase rather than uridine phosphorylase activity. The correlation between increased thymidine phosphorylase activity and increased keratinocyte proliferation in vitro (cultured) and in vivo (psoriasis), suggests that this enzyme may play a critical role in providing the thymidine necessary for keratinocyte proliferation.

  17. Glycogen synthesis from lactate in a chronically active muscle

    SciTech Connect

    Talmadge, R.J.; Scheide, J.I.; Silverman, H.

    1989-05-01

    In response to neural overactivity (pseudomyotonia), gastrocnemius muscle fibers from C57Bl/6Jdy2J/dy2J mice have different metabolic profiles compared with normal mice. A population of fibers in the fast-twitch superficial region of the dy2J gastrocnemius stores unusually high amounts of glycogen, leading to an increased glycogen storage in the whole muscle. The dy2J muscle also contains twice as much lactate as normal muscle. A (/sup 14/C)lactate intraperitoneal injection leads to preferential /sup 14/C incorporation into glycogen in the dy2J muscle compared with normal muscle. To determine whether skeletal muscles were incorporating lactate into glycogen without body organ (liver, kidney) input, gastrocnemius muscles were bathed in 10 mM (/sup 14/C)lactate with intact neural and arterial supply but with impeded venous return. The contralateral gastrocnemius serves as a control for body organ input. By using this in situ procedure, we demonstrate that under conditions of high lactate both normal and dy2J muscle can directly synthesize glycogen from lactate. In this case, normal whole muscle incorporates (14C) lactate into glycogen at a higher rate than dy2J whole muscle. Autoradiography, however, suggests that the high-glycogen-containing muscle fibers in the dy2J muscle incorporate lactate into glycogen at nearly four times the rate of normal or surrounding muscle fibers.

  18. Redesign of the Active Site of Sucrose Phosphorylase through a Clash-Induced Cascade of Loop Shifts.

    PubMed

    Kraus, Michael; Grimm, Clemens; Seibel, Jürgen

    2016-01-01

    Sucrose phosphorylases have been applied in the enzymatic production of glycosylated compounds for decades. However, several desirable acceptors, such as flavonoids or stilbenoids, that exhibit diverse antimicrobial, anticarcinogenic or antioxidant properties, remain poor substrates. The Q345F exchange in sucrose phosphorylase from Bifidobacterium adolescentis allows efficient glucosylation of resveratrol, (+)-catechin and (-)-epicatechin in yields of up to 97 % whereas the wild-type enzyme favours sucrose hydrolysis. Three previously undescribed products are made available. The crystal structure of the variant reveals a widened access channel with a hydrophobic aromatic surface that is likely to contribute to the improved activity towards aromatic acceptors. The generation of this channel can be explained in terms of a cascade of structural changes arising from the Q345F exchange. The observed mechanisms are likely to be relevant for the design of other tailor-made enzymes.

  19. Chronic ethanol consumption disrupts diurnal rhythms of hepatic glycogen metabolism in mice

    PubMed Central

    Udoh, Uduak S.; Swain, Telisha M.; Filiano, Ashley N.; Gamble, Karen L.; Young, Martin E.

    2015-01-01

    Chronic ethanol consumption has been shown to significantly decrease hepatic glycogen content; however, the mechanisms responsible for this adverse metabolic effect are unknown. In this study, we examined the impact chronic ethanol consumption has on time-of-day-dependent oscillations (rhythms) in glycogen metabolism processes in the liver. For this, male C57BL/6J mice were fed either a control or ethanol-containing liquid diet for 5 wk, and livers were collected every 4 h for 24 h and analyzed for changes in various genes and proteins involved in hepatic glycogen metabolism. Glycogen displayed a robust diurnal rhythm in the livers of mice fed the control diet, with the peak occurring during the active (dark) period of the day. The diurnal glycogen rhythm was significantly altered in livers of ethanol-fed mice, with the glycogen peak shifted into the inactive (light) period and the overall content of glycogen decreased compared with controls. Chronic ethanol consumption further disrupted diurnal rhythms in gene expression (glycogen synthase 1 and 2, glycogenin, glucokinase, protein targeting to glycogen, and pyruvate kinase), total and phosphorylated glycogen synthase protein, and enzyme activities of glycogen synthase and glycogen phosphorylase, the rate-limiting enzymes of glycogen metabolism. In summary, these results show for the first time that chronic ethanol consumption disrupts diurnal rhythms in hepatic glycogen metabolism at the gene and protein level. Chronic ethanol-induced disruption in these daily rhythms likely contributes to glycogen depletion and disruption of hepatic energy homeostasis, a recognized risk factor in the etiology of alcoholic liver disease. PMID:25857999

  20. ORM Promotes Skeletal Muscle Glycogen Accumulation via CCR5-Activated AMPK Pathway in Mice

    PubMed Central

    Qin, Zhen; Wan, Jing-Jing; Sun, Yang; Wang, Peng-Yuan; Su, Ding-Feng; Lei, Hong; Liu, Xia

    2016-01-01

    We found previously that acute phase protein orosomucoid reacts to fatigue and activates C-C chemokine receptor type 5 to increase muscle glycogen storage and enhance muscle endurance (Lei et al., 2016). To explore the underlying molecular mechanisms, we investigated the role of AMP-activated protein kinase, a critical fuel sensor in skeletal muscle, in C-C chemokine receptor type 5-mediated orosomucoid action. It was found orosomucoid increased skeletal muscle AMP-activated protein kinase activation in a time- and dose- dependent manner, which was largely prevented by pharmacological blocking or knockout of C-C chemokine receptor type 5. Administration of orosomucoid also significantly increased the de-phosphorylation and activity of muscle glycogen synthase, the rate-limiting enzyme for glycogen synthesis. The effect was largely absent in mice deficient in C-C chemokine receptor type 5−/− or AMP-activated protein kinase α2−/−, the predominant isoform in skeletal muscle. Moreover, deletion of AMP-activated protein kinase α2 abolished the effect of orosomucoid on fatigue and muscle glycogen. These findings indicate that orosomucoid may promote glycogen storage and enhance muscle function through C-C chemokine receptor type 5-mdiated activation of AMP-activated protein kinase, which in turn activates glycogen synthase and increases muscle glycogen. PMID:27679573

  1. ORM Promotes Skeletal Muscle Glycogen Accumulation via CCR5-Activated AMPK Pathway in Mice.

    PubMed

    Qin, Zhen; Wan, Jing-Jing; Sun, Yang; Wang, Peng-Yuan; Su, Ding-Feng; Lei, Hong; Liu, Xia

    2016-01-01

    We found previously that acute phase protein orosomucoid reacts to fatigue and activates C-C chemokine receptor type 5 to increase muscle glycogen storage and enhance muscle endurance (Lei et al., 2016). To explore the underlying molecular mechanisms, we investigated the role of AMP-activated protein kinase, a critical fuel sensor in skeletal muscle, in C-C chemokine receptor type 5-mediated orosomucoid action. It was found orosomucoid increased skeletal muscle AMP-activated protein kinase activation in a time- and dose- dependent manner, which was largely prevented by pharmacological blocking or knockout of C-C chemokine receptor type 5. Administration of orosomucoid also significantly increased the de-phosphorylation and activity of muscle glycogen synthase, the rate-limiting enzyme for glycogen synthesis. The effect was largely absent in mice deficient in C-C chemokine receptor type 5(-/-) or AMP-activated protein kinase α2(-/-), the predominant isoform in skeletal muscle. Moreover, deletion of AMP-activated protein kinase α2 abolished the effect of orosomucoid on fatigue and muscle glycogen. These findings indicate that orosomucoid may promote glycogen storage and enhance muscle function through C-C chemokine receptor type 5-mdiated activation of AMP-activated protein kinase, which in turn activates glycogen synthase and increases muscle glycogen. PMID:27679573

  2. ORM Promotes Skeletal Muscle Glycogen Accumulation via CCR5-Activated AMPK Pathway in Mice

    PubMed Central

    Qin, Zhen; Wan, Jing-Jing; Sun, Yang; Wang, Peng-Yuan; Su, Ding-Feng; Lei, Hong; Liu, Xia

    2016-01-01

    We found previously that acute phase protein orosomucoid reacts to fatigue and activates C-C chemokine receptor type 5 to increase muscle glycogen storage and enhance muscle endurance (Lei et al., 2016). To explore the underlying molecular mechanisms, we investigated the role of AMP-activated protein kinase, a critical fuel sensor in skeletal muscle, in C-C chemokine receptor type 5-mediated orosomucoid action. It was found orosomucoid increased skeletal muscle AMP-activated protein kinase activation in a time- and dose- dependent manner, which was largely prevented by pharmacological blocking or knockout of C-C chemokine receptor type 5. Administration of orosomucoid also significantly increased the de-phosphorylation and activity of muscle glycogen synthase, the rate-limiting enzyme for glycogen synthesis. The effect was largely absent in mice deficient in C-C chemokine receptor type 5−/− or AMP-activated protein kinase α2−/−, the predominant isoform in skeletal muscle. Moreover, deletion of AMP-activated protein kinase α2 abolished the effect of orosomucoid on fatigue and muscle glycogen. These findings indicate that orosomucoid may promote glycogen storage and enhance muscle function through C-C chemokine receptor type 5-mdiated activation of AMP-activated protein kinase, which in turn activates glycogen synthase and increases muscle glycogen.

  3. Putative role of glycogen as a peripheral biomarker of GSK3β activity.

    PubMed

    Frizzo, Marcos Emilio

    2013-09-01

    Glycogen synthase kinase 3-β (GSK3β) has a pivotal role in several intracellular signaling cascades that are involved in gene transcription, cytoskeletal reorganization, energy metabolism, cell cycle regulation, and apoptosis. This kinase has pleiotropic functions, and the importance of its activity has recently been shown in neurons and platelets. In addition to its regulatory function in several physiological events, changes in GSK3β activity have been associated with many psychiatric and neurodegenerative illnesses, such as Alzheimer's disease, schizophrenia and autism-spectrum disorders. Beside the reports of its involvement in several pathologies, it has become increasingly apparent that GSK3β might be a common therapeutic target for different classes of psychiatric drugs, and also that the GSK3β ratio may be a useful parameter to determine the biochemical changes that might occur during antidepressant treatment. Although GSK3β is commonly described as a key enzyme in a plethora of signaling cascades, originally it was identified as playing an important role in the regulation of glycogen synthesis, given its ability to inactivate glycogen synthase (GS) by phosphorylation. Acting as a constitutively active kinase, GSK3β phosphorylates GS, which results in a decrease of glycogen production. GSK3β phosphorylation increases glycogen synthesis and storage, while its dephosphorylation decreases glycogen synthesis. Inactivation of GSK3β leads to dephosphorylation of GS and increase in glycogen synthesis in the adipose tissue, muscle and liver. Glycogen levels are reduced by antidepressant treatment, and this effect seems to be related to an effect of drugs on GSK3β activity. Peripherally, glycogen is also abundantly found in platelets, where it is considered a major energy source, required for a variety of its functions, including the release reaction. Recently, analysis of platelets from patients with late-life major depression showed that active forms of

  4. Bacillus subtilis polynucleotide phosphorylase 3'-to-5' DNase activity is involved in DNA repair.

    PubMed

    Cardenas, Paula P; Carrasco, Begoña; Sanchez, Humberto; Deikus, Gintaras; Bechhofer, David H; Alonso, Juan C

    2009-07-01

    In the presence of Mn(2+), an activity in a preparation of purified Bacillus subtilis RecN degrades single-stranded (ss) DNA with a 3' --> 5' polarity. This activity is not associated with RecN itself, because RecN purified from cells lacking polynucleotide phosphorylase (PNPase) does not show the exonuclease activity. We show here that, in the presence of Mn(2+) and low-level inorganic phosphate (P(i)), PNPase degrades ssDNA. The limited end-processing of DNA is regulated by ATP and is inactive in the presence of Mg(2+) or high-level P(i). In contrast, the RNase activity of PNPase requires Mg(2+) and P(i), suggesting that PNPase degradation of RNA and ssDNA occur by mutually exclusive mechanisms. A null pnpA mutation (DeltapnpA) is not epistatic with Delta recA, but is epistatic with DeltarecN and Delta ku, which by themselves are non-epistatic. The addA5, Delta recO, Delta recQ (Delta recJ), Delta recU and Delta recG mutations (representative of different epistatic groups), in the context of DeltapnpA, demonstrate gain- or loss-of-function by inactivation of repair-by-recombination, depending on acute or chronic exposure to the damaging agent and the nature of the DNA lesion. Our data suggest that PNPase is involved in various nucleic acid metabolic pathways, and its limited ssDNA exonuclease activity plays an important role in RecA-dependent and RecA-independent repair pathways. PMID:19433509

  5. Alpha-glucan phosphorylase from Escherichia coli. Cloning of the gene, and purification and characterization of the protein.

    PubMed

    Yu, F; Jen, Y; Takeuchi, E; Inouye, M; Nakayama, H; Tagaya, M; Fukui, T

    1988-09-25

    By using a synthetic oligonucleotide probe identical to a part of the gene for the Escherichia coli major outer membrane lipoprotein, we have cloned a gene from E. coli chromosomal DNA. However, the cloned gene was not one of the lipoprotein genes. The amino acid sequence deduced from its nucleotide sequence shows extensive similarities instead to alpha-glucan phosphorylase (EC 2.4.1.1). The gene, glgP, is located immediately downstream from glgA, the gene for glycogen synthase. The glgP gene was inserted into pUC9 vector and expressed in the presence of the lac inducer. The gene product was purified to apparent homogeneity as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In all chromatographies, the protein was eluted accompanied by a low phosphorylase activity. The final preparation showed phosphorolytic activity to various alpha-glucans, although the specific activity was extremely low compared to other alpha-glucan phosphorylases under the standard assay conditions. Its enzymatic activity, however, increased almost linearly as the concentration of glucan increased, reaching a value comparable with those of other phosphorylases. The amino acid sequence deduced was compared with those of alpha-glucan phosphorylases from other sources. PMID:3047129

  6. Glycogen Repletion in Brown Adipose Tissue upon Refeeding Is Primarily Driven by Phosphorylation-Independent Mechanisms

    PubMed Central

    Carmean, Christopher M.; Huang, Y. Hanna; Brady, Matthew J.

    2016-01-01

    Glycogen storage in brown adipose tissue (BAT) is generally thought to take place through passive, substrate-driven activation of glycogenesis rather than programmatic shifts favoring or opposing the storage and/or retention of glycogen. This perception exists despite a growing body of evidence suggesting that BAT glycogen storage is actively regulated by covalent modification of key glycogen-metabolic enzymes, protein turnover, and endocrine hormone signaling. Members of one such class of covalent-modification regulators, glycogen-binding Phosphoprotein Phosphatase-1 (PP1)-regulatory subunits (PPP1Rs), targeting PP1 to glycogen-metabolic enzymes, were dynamically regulated in response to 24 hr of starvation and/or 24 hr of starvation followed by ad libitum refeeding. Over-expression of the PPP1R Protein Targeting to Glycogen (PTG), under the control of the aP2 promoter in mice, inactivated glycogen phosphorylase (GP) and enhanced basal- and starvation-state glycogen storage. Total interscapular BAT glycogen synthase and the constitutive activity of GS were conditionally affected. During starvation, glucose-6-phosphate (G-6-P) levels and the relative phosphorylation of Akt (p-Ser-473-Akt) were both increased in PTG-overexpressing (Tg) mice, suggesting that elevated glycogen storage during starvation modifies broader cellular metabolic pathways. During refeeding, Tg and WT mice reaccumulated glycogen similarly despite altered GS and GP activities. All observations during refeeding suggest that the phosphorylation states of GS and GP are not physiologically rate-controlling, despite there being a clear balance of endogenous kinase- and phosphatase activities. The studies presented here reveal IBAT glycogen storage to be a tightly-regulated process at all levels, with potential effects on nutrient sensing in vivo. PMID:27213961

  7. The recruitment of AMP-activated protein kinase to glycogen is regulated by autophosphorylation.

    PubMed

    Oligschlaeger, Yvonne; Miglianico, Marie; Chanda, Dipanjan; Scholz, Roland; Thali, Ramon F; Tuerk, Roland; Stapleton, David I; Gooley, Paul R; Neumann, Dietbert

    2015-05-01

    The mammalian AMP-activated protein kinase (AMPK) is an obligatory αβγ heterotrimeric complex carrying a carbohydrate-binding module (CBM) in the β-subunit (AMPKβ) capable of attaching AMPK to glycogen. Nonetheless, AMPK localizes at many different cellular compartments, implying the existence of mechanisms that prevent AMPK from glycogen binding. Cell-free carbohydrate binding assays revealed that AMPK autophosphorylation abolished its carbohydrate-binding capacity. X-ray structural data of the CBM displays the central positioning of threonine 148 within the binding pocket. Substitution of Thr-148 for a phospho-mimicking aspartate (T148D) prevents AMPK from binding to carbohydrate. Overexpression of isolated CBM or β1-containing AMPK in cellular models revealed that wild type (WT) localizes to glycogen particles, whereas T148D shows a diffuse pattern. Pharmacological AMPK activation and glycogen degradation by glucose deprivation but not forskolin enhanced cellular Thr-148 phosphorylation. Cellular glycogen content was higher if pharmacological AMPK activation was combined with overexpression of T148D mutant relative to WT AMPK. In summary, these data show that glycogen-binding capacity of AMPKβ is regulated by Thr-148 autophosphorylation with likely implications in the regulation of glycogen turnover. The findings further raise the possibility of regulated carbohydrate-binding function in a wider variety of CBM-containing proteins.

  8. The Recruitment of AMP-activated Protein Kinase to Glycogen Is Regulated by Autophosphorylation*

    PubMed Central

    Oligschlaeger, Yvonne; Miglianico, Marie; Chanda, Dipanjan; Scholz, Roland; Thali, Ramon F.; Tuerk, Roland; Stapleton, David I.; Gooley, Paul R.; Neumann, Dietbert

    2015-01-01

    The mammalian AMP-activated protein kinase (AMPK) is an obligatory αβγ heterotrimeric complex carrying a carbohydrate-binding module (CBM) in the β-subunit (AMPKβ) capable of attaching AMPK to glycogen. Nonetheless, AMPK localizes at many different cellular compartments, implying the existence of mechanisms that prevent AMPK from glycogen binding. Cell-free carbohydrate binding assays revealed that AMPK autophosphorylation abolished its carbohydrate-binding capacity. X-ray structural data of the CBM displays the central positioning of threonine 148 within the binding pocket. Substitution of Thr-148 for a phospho-mimicking aspartate (T148D) prevents AMPK from binding to carbohydrate. Overexpression of isolated CBM or β1-containing AMPK in cellular models revealed that wild type (WT) localizes to glycogen particles, whereas T148D shows a diffuse pattern. Pharmacological AMPK activation and glycogen degradation by glucose deprivation but not forskolin enhanced cellular Thr-148 phosphorylation. Cellular glycogen content was higher if pharmacological AMPK activation was combined with overexpression of T148D mutant relative to WT AMPK. In summary, these data show that glycogen-binding capacity of AMPKβ is regulated by Thr-148 autophosphorylation with likely implications in the regulation of glycogen turnover. The findings further raise the possibility of regulated carbohydrate-binding function in a wider variety of CBM-containing proteins. PMID:25792737

  9. The structure of a glycogen phosphorylase glucopyranose spirohydantoin complex at 1.8 A resolution and 100 K: the role of the water structure and its contribution to binding.

    PubMed Central

    Gregoriou, M.; Noble, M. E.; Watson, K. A.; Garman, E. F.; Krulle, T. M.; de la Fuente, C.; Fleet, G. W.; Oikonomakos, N. G.; Johnson, L. N.

    1998-01-01

    A glucopyranose spirohydantoin (a pyranose analogue of the potent herbicide, hydantocidin) has been identified as the highest affinity glucose analogue inhibitor of glycogen phosphorylase b (GPb). In order to elucidate the structural features that contribute to the binding, the structures of GPb in the native T state conformation and in complex with glucopyranose spirohydantoin have been determined at 100 K to 2.0 A and 1.8 A resolution, respectively, and refined to crystallographic R values of 0.197 (R[free] 0.248) and 0.182 (R[free] 0.229), respectively. The low temperature structure of GPb is almost identical to that of the previously determined room temperature structure, apart from a decrease in overall atomic temperature factors ((B) room temperature GPb = 34.9 A2; (B) 100 K GPb = 23.4 A2). The glucopyranose spirohydantoin inhibitor (Ki = 3.0 microM) binds at the catalytic site and induces small changes in two key regions of the protein: the 280s loop (residues 281-286) that results in a decrease in mobility of this region, and the 380s loop (residues 377-385) that undergoes more significant shifts in order to optimize contact to the ligand. The hydantoin group, that is responsible for increasing the affinity of the glucose compound by a factor of 10(3), makes only one hydrogen bond to the protein, from one of its NH groups to the main chain oxygen of His377. The other polar groups of the hydantoin group form hydrogen bonds to five water molecules. These waters are involved in extensive networks of hydrogen bonds and appear to be an integral part of the protein structure. Analysis of the water structure at the catalytic site of the native enzyme, shows that five waters are displaced by ligand binding and that there is a significant decrease in mobility of the remaining waters on formation of the GPb-hydantoin complex. The ability of the inhibitor to exploit existing waters, to displace waters and to recruit new waters appears to be important for the high

  10. Kinases and phosphatases of hepatic glycogen metabolism during fasted to refed transition in normal and streptozotocin-induced diabetic rats.

    PubMed

    Pugazhenthi, S; Khandelwal, R L

    1991-02-01

    Normal and streptozotocin-induced diabetic rats were fasted for 24 hours and refed for 4 hours. Changes in the activities of glycogen metabolizing enzymes in liver were followed during this period. In normal rats, hepatic glycogen content increased gradually after the onset of food intake. The percent of active glycogen synthase increased to a peak value at 1h which coincided with a significant (P less than 0.02) increase in synthase phosphatase activity. Phosphorylase alpha and the percent of alpha increased significantly (P less than 0.01) after the meal which correlated with similar increases in cAMP-dependent protein kinase and phosphorylase kinase activities. Activation of enzymes involved in both synthesis and degradation of glycogen during fasted to refed transition indicate a probable substrate cycling. In diabetic livers, there was marked decrease in the activities of glycogen metabolizing enzymes and their levels did not alter significantly in response to the meal indicating a poor turnover of glycogen. PMID:1652246

  11. Lck/PLCγ control migration and proliferation of interleukin (IL)-2-stimulated T cells via the Rac1 GTPase/glycogen phosphorylase pathway.

    PubMed

    Llavero, Francisco; Artaso, Alain; Lacerda, Hadriano M; Parada, Luis A; Zugaza, José L

    2016-11-01

    Recently, we have reported that the IL-2-stimulated T cells activate PKCθ in order to phosphorylate the serine residues of αPIX-RhoGEF, and to switch on the Rac1/PYGM pathway resulting in T cell migration and proliferation. However, the molecular mechanism connecting the activated IL-2-R with the PKCθ/αPIX/Rac1/PYGM pathway is still unknown. In this study, the use of a combined pharmacological and genetic approach identified Lck, a Src family member, as the tyrosine kinase phosphorylating PLCγ leading to Rac1 and PYGM activation in the IL-2-stimulated Kit 225 T cells via the PKCθ/αPIX pathway. The PLCγ tyrosine phosphorylation was required to activate first PKCθ, and then αPIX and Rac1/PYGM. The results presented here delineate a novel signalling pathway ranking equally in importance to the three major pathways controlled by the IL-2-R, i.e. PI3K, Ras/MAPK and JAK/STAT pathways. The overall evidence strongly indicates that the central biological role of the novel IL-2-R/Lck/PLCγ/PKCθ/αPIX/Rac1/PYGM signalling pathway is directly related to the control of fundamental cellular processes such as T cell migration and proliferation. PMID:27519475

  12. Pathological glycogenesis through glycogen synthase 1 and suppression of excessive AMP kinase activity in myeloid leukemia cells.

    PubMed

    Bhanot, H; Reddy, M M; Nonami, A; Weisberg, E L; Bonal, D; Kirschmeier, P T; Salgia, S; Podar, K; Galinsky, I; Chowdary, T K; Neuberg, D; Tonon, G; Stone, R M; Asara, J; Griffin, J D; Sattler, M

    2015-07-01

    The rapid proliferation of myeloid leukemia cells is highly dependent on increased glucose metabolism. Through an unbiased metabolomics analysis of leukemia cells, we found that the glycogenic precursor UDP-D-glucose is pervasively upregulated, despite low glycogen levels. Targeting the rate-limiting glycogen synthase 1 (GYS1) not only decreased glycolytic flux but also increased activation of the glycogen-responsive AMP kinase (AMPK), leading to significant growth suppression. Further, genetic and pharmacological hyper-activation of AMPK was sufficient to induce the changes observed with GYS1 targeting. Cancer genomics data also indicate that elevated levels of the glycogenic enzymes GYS1/2 or GBE1 (glycogen branching enzyme 1) are associated with poor survival in AML. These results suggest a novel mechanism whereby leukemic cells sustain aberrant proliferation by suppressing excess AMPK activity through elevated glycogenic flux and provide a therapeutic entry point for targeting leukemia cell metabolism.

  13. Myoglobinuria and Skeletal Muscle Phosphorylase Deficiency

    PubMed Central

    Nixon, J. C.; Hobbs, W. K.; Greenblatt, J.

    1966-01-01

    Investigation of a patient complaining of exercise-induced dark urine, pain, stiffness and tenderness of skeletal muscle revealed findings characteristic of McArdle's disease. The dark urine was attributable to the excretion of myoglobin, and an ischemic exercise test failed to demonstrate the usual rise and fall in blood lactate and pyruvate. Enzyme assays of skeletal muscle showed an absence of phosphorylase, a slight increase in phosphorylase b kinase and a slight decrease in phosphoglucomutase. Chemical and histochemical analyses demonstrated an increase in the skeletal muscle glycogen content and an enlargement of the muscle cells. No abnormality of liver glycogen metabolism was found. In the absence of specific therapy, an effective and practical form of treatment is reduction of exercise below the threshold of symptoms. ImagesFig. 1Fig. 2Fig. 6Fig. 7Fig. 8 PMID:4952390

  14. Glycogen content regulates peroxisome proliferator activated receptor-∂ (PPAR-∂) activity in rat skeletal muscle.

    PubMed

    Philp, Andrew; MacKenzie, Matthew G; Belew, Micah Y; Towler, Mhairi C; Corstorphine, Alan; Papalamprou, Angela; Hardie, D Grahame; Baar, Keith

    2013-01-01

    Performing exercise in a glycogen depleted state increases skeletal muscle lipid utilization and the transcription of genes regulating mitochondrial β-oxidation. Potential candidates for glycogen-mediated metabolic adaptation are the peroxisome proliferator activated receptor (PPAR) coactivator-1α (PGC-1α) and the transcription factor/nuclear receptor PPAR-∂. It was therefore the aim of the present study to examine whether acute exercise with or without glycogen manipulation affects PGC-1α and PPAR-∂ function in rodent skeletal muscle. Twenty female Wistar rats were randomly assigned to 5 experimental groups (n = 4): control [CON]; normal glycogen control [NG-C]; normal glycogen exercise [NG-E]; low glycogen control [LG-C]; and low glycogen exercise [LG-E]). Gastrocnemius (GTN) muscles were collected immediately following exercise and analyzed for glycogen content, PPAR-∂ activity via chromatin immunoprecipitation (ChIP) assays, AMPK α1/α2 kinase activity, and the localization of AMPK and PGC-1α. Exercise reduced muscle glycogen by 47 and 75% relative to CON in the NG-E and LG-E groups, respectively. Exercise that started with low glycogen (LG-E) finished with higher AMPK-α2 activity (147%, p<0.05), nuclear AMPK-α2 and PGC-1α, but no difference in AMPK-α1 activity compared to CON. In addition, PPAR-∂ binding to the CPT1 promoter was significantly increased only in the LG-E group. Finally, cell reporter studies in contracting C2C12 myotubes indicated that PPAR-∂ activity following contraction is sensitive to glucose availability, providing mechanistic insight into the association between PPAR-∂ and glycogen content/substrate availability. The present study is the first to examine PPAR-∂ activity in skeletal muscle in response to an acute bout of endurance exercise. Our data would suggest that a factor associated with muscle contraction and/or glycogen depletion activates PPAR-∂ and initiates AMPK translocation in skeletal muscle in

  15. The maximum activities of hexokinase, phosphorylase, phosphofructokinase, glycerol phosphate dehydrogenases, lactate dehydrogenase, octopine dehydrogenase, phosphoenolpyruvate carboxykinase, nucleoside diphosphatekinase, glutamate-oxaloacetate transaminase and arginine kinase in relation to carbohydrate utilization in muscles from marine invertebrates.

    PubMed Central

    Zammit, V A; Newsholme, E A

    1976-01-01

    Comparison of the activities of hexokinase, phosphorylase and phosphofructokinase in muscles from marine invertebrates indicates that they can be divided into three groups. First, the activities of the three enzymes are low in coelenterate muscles, catch muscles of molluscs and muscles of echinoderms; this indicates a low rate of carbohydrate (and energy) utilization by these muscles. Secondly, high activities of phosphorylase and phosphofructokinase relative to those of hexokinase are found in, for example, lobster abdominal and scallop snap muscles; this indicates that these muscles depend largely on anaerobic degradation of glycogen for energy production. Thirdly, high activities of hexokinase are found in the radular muscles of prosobranch molluscs and the fin muscles of squids; this indicates a high capacity for glucose utilization, which is consistent with the high activities of enzymes of the tricarboxylic acid cycle in these muscles [Alp, Newsholme & Zammit (1976) Biochem. J. 154, 689-700]. 2. The activities of lactate dehydrogenase, octopine dehydrogenase, phosphoenolpyruvate carboxykinase, cytosolic and mitochondrial glycerol 3-phosphate dehydrogenase and glutamate-oxaloacetate transaminase were measured in order to provide a qualitative indication of the importance of different processes for oxidation of glycolytically formed NADH. The muscles are divided into four groups: those that have a high activity of lactate dehydrogenase relative to the activities of phosphofructokinase (e.g. crustacean muscles); those that have high activities of octopine dehydrogenase but low activities of lactate dehydrogenase (e.g. scallop snap muscle); those that have moderate activities of both lactate dehydrogenase and octopine dehydrogenase (radular muscles of prosobranchs), and those that have low activities of both lactate dehydrogenase and octopine dehydrogenase, but which possess activities of phosphoenolpyruvate carboxykinase (oyster adductor muscles). It is

  16. Chronic corticosterone exposure reduces hippocampal glycogen level and induces depression-like behavior in mice.

    PubMed

    Zhang, Hui-yu; Zhao, Yu-nan; Wang, Zhong-li; Huang, Yu-fang

    2015-01-01

    Long-term exposure to stress or high glucocorticoid levels leads to depression-like behavior in rodents; however, the cause remains unknown. Increasing evidence shows that astrocytes, the most abundant cells in the central nervous system (CNS), are important to the nervous system. Astrocytes nourish and protect the neurons, and serve as glycogen repositories for the brain. The metabolic process of glycogen, which is closely linked to neuronal activity, can supply sufficient energy substrates for neurons. The research team probed into the effects of chronic corticosterone (CORT) exposure on the glycogen level of astrocytes in the hippocampal tissues of male C57BL/6N mice in this study. The results showed that chronic CORT injection reduced hippocampal neurofilament light protein (NF-L) and synaptophysin (SYP) levels, induced depression-like behavior in male mice, reduced hippocampal glycogen level and glycogen synthase activity, and increased glycogen phosphorylase activity. The results suggested that the reduction of the hippocampal glycogen level may be the mechanism by which chronic CORT treatment damages hippocampal neurons and induces depression-like behavior in male mice.

  17. Two Additional Phosphorylases in Developing Maize Seeds 12

    PubMed Central

    Tsai, C. Y.; Nelson, O. E.

    1969-01-01

    Two additional phosphorylases (III and IV) have been detected in developing seeds of maize. Phosphorylase IV is found only in the embryo (with scutellum). It is also present in the embryo of the germinating seed where its activity is 90-fold greater than the activity in the developing embryo 22 days after pollination. Phosphorylase IV is eluted from a DEAE-cellulose column in the same fraction as phosphorylase I of the endosperm, and the 2 enzymes are similar in many respects. Phosphorylase IV is distinguished from phosphorylase I by electrophoretic mobility, by pH optimum, and because its properties are not affected by the shrunken-4 mutation. Phosphorylase III is found both in the endosperms and embryos of developing seeds. Activity for this enzyme is not detected in crude homogenates nor eluates from a DEAE-cellulose column apparently because it complexes with a non-dialyzable, heat-labile inhibitor. High activity is found after protamine sulfate fractionation. Phosphorylase III is bound to protamine sulfate and is then removed by washing with 0.3 m phosphate buffer. Phosphorylase III activity in the endosperm is not detectable 8 days after pollination but is present 12 days after pollination. Phosphorylase III differs from phosphorylases I, II, and IV in several respects—pH optimum, pH-independent ATP inhibition, time of appearance in the endosperm, and because purine and pyrimidine nucleotides are equally inhibitory. In common with phosphorylase II, phosphorylase III apparently does not require a primer to initiate the synthesis of an amylose-like polymer. PMID:5774172

  18. Purine nucleoside phosphorylase and xanthine oxidase activities in erythrocytes and plasma from marine, semiaquatic and terrestrial mammals.

    PubMed

    López-Cruz, Roberto I; Pérez-Milicua, Myrna Barjau; Crocker, Daniel E; Gaxiola-Robles, Ramón; Bernal-Vertiz, Jaime A; de la Rosa, Alejandro; Vázquez-Medina, José P; Zenteno-Savín, Tania

    2014-05-01

    Purine nucleoside phosphorylase (PNP) and xanthine oxidase (XO) are key enzymes involved in the purine salvage pathway. PNP metabolizes purine bases to synthetize purine nucleotides whereas XO catalyzes the oxidation of purines to uric acid. In humans, PNP activity is reported to be high in erythrocytes and XO activity to be low in plasma; however, XO activity increases after ischemic events. XO activity in plasma of northern elephant seals has been reported during prolonged fasting and rest and voluntary associated apneas. The objective of this study was to analyze circulating PNP and XO activities in marine mammals adapted to tolerate repeated cycles of ischemia/reperfusion associated with diving (bottlenose dolphin, northern elephant seal) in comparison with semiaquatic (river otter) and terrestrial mammals (human, pig). PNP activities in plasma and erythrocytes, as well as XO activity in plasma, from all species were quantified by spectrophotometry. No clear relationship in circulating PNP or XO activity could be established between marine, semiaquatic and terrestrial mammals. Erythrocytes from bottlenose dolphins and humans are highly permeable to nucleosides and glucose, intraerythrocyte PNP activity may be related to a release of purine nucleotides from the liver. High-energy costs will probably mean a higher ATP degradation rate in river otters, as compared to northern elephant seals or dolphins. Lower erythrocyte PNP activity and elevated plasma XO activity in northern elephant seal could be associated with fasting and/or sleep- and dive-associated apneas.

  19. Purine nucleoside phosphorylase and xanthine oxidase activities in erythrocytes and plasma from marine, semiaquatic and terrestrial mammals.

    PubMed

    López-Cruz, Roberto I; Pérez-Milicua, Myrna Barjau; Crocker, Daniel E; Gaxiola-Robles, Ramón; Bernal-Vertiz, Jaime A; de la Rosa, Alejandro; Vázquez-Medina, José P; Zenteno-Savín, Tania

    2014-05-01

    Purine nucleoside phosphorylase (PNP) and xanthine oxidase (XO) are key enzymes involved in the purine salvage pathway. PNP metabolizes purine bases to synthetize purine nucleotides whereas XO catalyzes the oxidation of purines to uric acid. In humans, PNP activity is reported to be high in erythrocytes and XO activity to be low in plasma; however, XO activity increases after ischemic events. XO activity in plasma of northern elephant seals has been reported during prolonged fasting and rest and voluntary associated apneas. The objective of this study was to analyze circulating PNP and XO activities in marine mammals adapted to tolerate repeated cycles of ischemia/reperfusion associated with diving (bottlenose dolphin, northern elephant seal) in comparison with semiaquatic (river otter) and terrestrial mammals (human, pig). PNP activities in plasma and erythrocytes, as well as XO activity in plasma, from all species were quantified by spectrophotometry. No clear relationship in circulating PNP or XO activity could be established between marine, semiaquatic and terrestrial mammals. Erythrocytes from bottlenose dolphins and humans are highly permeable to nucleosides and glucose, intraerythrocyte PNP activity may be related to a release of purine nucleotides from the liver. High-energy costs will probably mean a higher ATP degradation rate in river otters, as compared to northern elephant seals or dolphins. Lower erythrocyte PNP activity and elevated plasma XO activity in northern elephant seal could be associated with fasting and/or sleep- and dive-associated apneas. PMID:24530799

  20. Lithium Induces Glycogen Accumulation in Salivary Glands of the Rat.

    PubMed

    Souza, D N; Mendes, F M; Nogueira, F N; Simões, A; Nicolau, J

    2016-02-01

    Lithium is administered for the treatment of mood and bipolar disorder. The aim of this study was to verify whether treatment with different concentrations of lithium may affect the glycogen metabolism in the salivary glands of the rats when compared with the liver. Mobilization of glycogen in salivary glands is important for the process of secretion. Two sets of experiments were carried out, that is, in the first, the rats received drinking water supplemented with LiCl (38,25 and 12 mM of LiCl for 15 days) and the second experiment was carried out by intraperitoneal injection of LiCl solution (12 mg/kg and 45 mg LiCl/kg body weight) for 3 days. The active form of glycogen phosphorylase was not affected by treatment with LiCl considering the two experiments. The active form of glycogen synthase presented higher activity in the submandibular glands of rats treated with 25 and 38 mM LiCl and in the liver, with 25 mM LiCl. Glycogen level was higher than that of control in the submandibular glands of rats receiving 38 and 12 mM LiCl, in the parotid of rats receiving 25 and 38 mM, and in the liver of rats receiving 12 mM LiCl. The absolute value of glycogen for the submandibular treated with 25 mM LiCl, and the liver treated with 38 mM LiCl, was higher than the control value, although not statistically significant for these tissues. No statistically significant difference was found in the submandibular and parotid salivary glands for protein concentration when comparing experimental and control groups. We concluded that LiCl administered to rats influences the metabolism of glycogen in salivary glands.

  1. Effects of diabetes, vanadium, and insulin on glycogen synthase activation in Wistar rats.

    PubMed

    Semiz, Sabina; Orvig, Chris; McNeill, John H

    2002-02-01

    In vivo effects of insulin and vanadium treatment on glycogen synthase (GS), glycogen synthase kinase-3 (GSK-3) and protein phosphatase-1 (PP1) activity were determined in Wistar rats with streptozotocin (STZ)-induced diabetes. The skeletal muscle was freeze-clamped before or following an insulin injection (5 U/kg i.v.). Diabetes, vanadium, and insulin in vivo treatment did not affect muscle GSK-3beta activity as compared to controls. Following insulin stimulation in 4-week STZ-diabetic rats muscle GS fractional activity (GSFA) was increased 3 fold (p < 0.05), while in 7-week diabetic rats it remained unchanged, suggesting development of insulin resistance in longer term diabetes. Muscle PP1 activity was increased in diabetic rats and returned to normal after vanadium treatment, while muscle GSFA remained unchanged. Therefore, it is possible that PP1 is involved in the regulation of some other cellular events of vanadium (other than regulation of glycogen synthesis). The lack of effect of vanadium treatment in stimulating glycogen synthesis in skeletal muscle suggests the involvement of other metabolic pathways in the observed glucoregulatory effect of vanadium.

  2. FLCN and AMPK Confer Resistance to Hyperosmotic Stress via Remodeling of Glycogen Stores.

    PubMed

    Possik, Elite; Ajisebutu, Andrew; Manteghi, Sanaz; Gingras, Marie-Claude; Vijayaraghavan, Tarika; Flamand, Mathieu; Coull, Barry; Schmeisser, Kathrin; Duchaine, Thomas; van Steensel, Maurice; Hall, David H; Pause, Arnim

    2015-10-01

    Mechanisms of adaptation to environmental changes in osmolarity are fundamental for cellular and organismal survival. Here we identify a novel osmotic stress resistance pathway in Caenorhabditis elegans (C. elegans), which is dependent on the metabolic master regulator 5'-AMP-activated protein kinase (AMPK) and its negative regulator Folliculin (FLCN). FLCN-1 is the nematode ortholog of the tumor suppressor FLCN, responsible for the Birt-Hogg-Dubé (BHD) tumor syndrome. We show that flcn-1 mutants exhibit increased resistance to hyperosmotic stress via constitutive AMPK-dependent accumulation of glycogen reserves. Upon hyperosmotic stress exposure, glycogen stores are rapidly degraded, leading to a significant accumulation of the organic osmolyte glycerol through transcriptional upregulation of glycerol-3-phosphate dehydrogenase enzymes (gpdh-1 and gpdh-2). Importantly, the hyperosmotic stress resistance in flcn-1 mutant and wild-type animals is strongly suppressed by loss of AMPK, glycogen synthase, glycogen phosphorylase, or simultaneous loss of gpdh-1 and gpdh-2 enzymes. Our studies show for the first time that animals normally exhibit AMPK-dependent glycogen stores, which can be utilized for rapid adaptation to either energy stress or hyperosmotic stress. Importantly, we show that glycogen accumulates in kidneys from mice lacking FLCN and in renal tumors from a BHD patient. Our findings suggest a dual role for glycogen, acting as a reservoir for energy supply and osmolyte production, and both processes might be supporting tumorigenesis.

  3. FLCN and AMPK Confer Resistance to Hyperosmotic Stress via Remodeling of Glycogen Stores

    PubMed Central

    Possik, Elite; Ajisebutu, Andrew; Manteghi, Sanaz; Gingras, Marie-Claude; Vijayaraghavan, Tarika; Flamand, Mathieu; Coull, Barry; Schmeisser, Kathrin; Duchaine, Thomas; van Steensel, Maurice; Hall, David H.; Pause, Arnim

    2015-01-01

    Mechanisms of adaptation to environmental changes in osmolarity are fundamental for cellular and organismal survival. Here we identify a novel osmotic stress resistance pathway in Caenorhabditis elegans (C. elegans), which is dependent on the metabolic master regulator 5’-AMP-activated protein kinase (AMPK) and its negative regulator Folliculin (FLCN). FLCN-1 is the nematode ortholog of the tumor suppressor FLCN, responsible for the Birt-Hogg-Dubé (BHD) tumor syndrome. We show that flcn-1 mutants exhibit increased resistance to hyperosmotic stress via constitutive AMPK-dependent accumulation of glycogen reserves. Upon hyperosmotic stress exposure, glycogen stores are rapidly degraded, leading to a significant accumulation of the organic osmolyte glycerol through transcriptional upregulation of glycerol-3-phosphate dehydrogenase enzymes (gpdh-1 and gpdh-2). Importantly, the hyperosmotic stress resistance in flcn-1 mutant and wild-type animals is strongly suppressed by loss of AMPK, glycogen synthase, glycogen phosphorylase, or simultaneous loss of gpdh-1 and gpdh-2 enzymes. Our studies show for the first time that animals normally exhibit AMPK-dependent glycogen stores, which can be utilized for rapid adaptation to either energy stress or hyperosmotic stress. Importantly, we show that glycogen accumulates in kidneys from mice lacking FLCN and in renal tumors from a BHD patient. Our findings suggest a dual role for glycogen, acting as a reservoir for energy supply and osmolyte production, and both processes might be supporting tumorigenesis. PMID:26439621

  4. Lowering Temperature is the Trigger for Glycogen Build-Up and Winter Fasting in Crucian Carp (Carassius carassius).

    PubMed

    Varis, Joonas; Haverinen, Jaakko; Vornanen, Matti

    2016-02-01

    Seasonal changes in physiology of vertebrate animals are triggered by environmental cues including temperature, day-length and oxygen availability. Crucian carp (Carassius carassius) tolerate prolonged anoxia in winter by using several physiological adaptations that are seasonally activated. This study examines which environmental cues are required to trigger physiological adjustments for winter dormancy in crucian carp. To this end, crucian carp were exposed to changing environmental factors under laboratory conditions: effects of declining water temperature, shortening day-length and reduced oxygen availability, separately and in different combinations, were examined on glycogen content and enzyme activities involved in feeding (alkaline phosphatase, AP) and glycogen metabolism (glycogen synthase, GyS; glycogen phosphorylase, GP). Lowering temperature induced a fall in activity of AP and a rise in glycogen content and rate of glycogen synthesis. Relative mass of the liver, and glycogen concentration of liver, muscle and brain increased with lowering temperature. Similarly activity of GyS in muscle and expression of GyS transcripts in brain were up-regulated by lowering temperature. Shortened day-length and oxygen availability had practically no effects on measured variables. We conclude that lowering temperature is the main trigger in preparation for winter anoxia in crucian carp. PMID:26853873

  5. Lowering Temperature is the Trigger for Glycogen Build-Up and Winter Fasting in Crucian Carp (Carassius carassius).

    PubMed

    Varis, Joonas; Haverinen, Jaakko; Vornanen, Matti

    2016-02-01

    Seasonal changes in physiology of vertebrate animals are triggered by environmental cues including temperature, day-length and oxygen availability. Crucian carp (Carassius carassius) tolerate prolonged anoxia in winter by using several physiological adaptations that are seasonally activated. This study examines which environmental cues are required to trigger physiological adjustments for winter dormancy in crucian carp. To this end, crucian carp were exposed to changing environmental factors under laboratory conditions: effects of declining water temperature, shortening day-length and reduced oxygen availability, separately and in different combinations, were examined on glycogen content and enzyme activities involved in feeding (alkaline phosphatase, AP) and glycogen metabolism (glycogen synthase, GyS; glycogen phosphorylase, GP). Lowering temperature induced a fall in activity of AP and a rise in glycogen content and rate of glycogen synthesis. Relative mass of the liver, and glycogen concentration of liver, muscle and brain increased with lowering temperature. Similarly activity of GyS in muscle and expression of GyS transcripts in brain were up-regulated by lowering temperature. Shortened day-length and oxygen availability had practically no effects on measured variables. We conclude that lowering temperature is the main trigger in preparation for winter anoxia in crucian carp.

  6. Fat and glycogen utilization in the larynx Muscles of fire-bellied toads (Bombina bombina L.) during calling activity.

    PubMed

    Eichelberg, H; Obert, H J

    1976-03-01

    Both fat and glycogen are present in abundance in the larynx muscles of anurans. To clarify their role, the Musculus dilatator laryngis of the male fire-bellied toad, Bombina bombina was studied. In untreated males, the mean fat content of this larynx muscle was 14%; the muscle contained glycogen amounting to 57% of that measured in the liver tissue of the same animal. After thirteen days of continual calling activity induced by administration of a gonadotropin, the fat content fell to 6%, the glycogen to 34%. The fat content was essentially unchanged (13%) by four hours of electrical stimulation of the muscle; the glycogen content, however, had fallen to 42% after this treatment. Neither component was altered in amount by castration, the fat content being about 13% and that of glycogen, 52%. Nor did treatment with gonadotropic hormone reduce either the fat content (13%) or the amount of glycogen (59%). From these results it was concluded that fats represent a direct source of energy for the larynx muscles, which is used up gradually over long periods of calling. The glycogen in these muscles, on the other hand, is a short-term store sufficient to supply energy for only a few hours of calling activity.

  7. Structure-Function Analysis of PPP1R3D, a Protein Phosphatase 1 Targeting Subunit, Reveals a Binding Motif for 14-3-3 Proteins which Regulates its Glycogenic Properties.

    PubMed

    Rubio-Villena, Carla; Sanz, Pascual; Garcia-Gimeno, Maria Adelaida

    2015-01-01

    Protein phosphatase 1 (PP1) is one of the major protein phosphatases in eukaryotic cells. It plays a key role in regulating glycogen synthesis, by dephosphorylating crucial enzymes involved in glycogen homeostasis such as glycogen synthase (GS) and glycogen phosphorylase (GP). To play this role, PP1 binds to specific glycogen targeting subunits that, on one hand recognize the substrates to be dephosphorylated and on the other hand recruit PP1 to glycogen particles. In this work we have analyzed the functionality of the different protein binding domains of one of these glycogen targeting subunits, namely PPP1R3D (R6) and studied how binding properties of different domains affect its glycogenic properties. We have found that the PP1 binding domain of R6 comprises a conserved RVXF motif (R102VRF) located at the N-terminus of the protein. We have also identified a region located at the C-terminus of R6 (W267DNND) that is involved in binding to the PP1 glycogenic substrates. Our results indicate that although binding to PP1 and glycogenic substrates are independent processes, impairment of any of them results in lack of glycogenic activity of R6. In addition, we have characterized a novel site of regulation in R6 that is involved in binding to 14-3-3 proteins (RARS74LP). We present evidence indicating that when binding of R6 to 14-3-3 proteins is prevented, R6 displays hyper-glycogenic activity although is rapidly degraded by the lysosomal pathway. These results define binding to 14-3-3 proteins as an additional pathway in the control of the glycogenic properties of R6.

  8. Acrylamide alters glycogen content and enzyme activities in the liver of juvenile rat.

    PubMed

    Kovac, Renata; Rajkovic, Vesna; Koledin, Ivana; Matavulj, Milica

    2015-10-01

    Acrylamide (AA) is spontaneously formed in carbohydrate-rich food during high-temperature processing. It is neurotoxic and potentially cancer causing chemical. Its harmful effects on the liver, especially in a young organism, are still to be elucidated. The study aimed to examine main liver histology, its glycogen content and enzyme activities in juvenile rats treated with 25 or 50mg/kg bw of AA for 3 weeks. Liver samples were fixed in formalin, routinely processed for paraffin embedding, sectioning and histochemical staining. Examination of haematoxylin and eosin (H&E)-stained sections showed an increase in the volume of hepatocytes, their nuclei and cytoplasm in both AA-treated groups compared to the control. In Periodic acid-Schiff (PAS)-stained sections in low-dose group was noticed glycogen reduction, while in high-dose group was present its accumulation compared to the control, respectively. Serum analysis showed increased activity of aspartate aminotransferase (AST), and decreased activity of alanine aminotransferase (ALT) in both AA-treated groups, while the activity of alkaline phosphatase (ALP) was increased in low-dose, but decreased in high-dose group compared to the control, respectively. Present results suggest a prominent hepatotoxic potential of AA which might alter the microstructural features and functional status in hepatocytes of immature liver.

  9. In vivo effects of diabetes, insulin and oleanolic acid on enzymes of glycogen metabolism in the skin of streptozotocin-induced diabetic male Sprague-Dawley rats.

    PubMed

    Mukundwa, Andrew; Langa, Silvana O; Mukaratirwa, Samson; Masola, Bubuya

    2016-03-01

    The skin is the largest organ in the body and diabetes induces pathologic changes on the skin that affect glucose homeostasis. Changes in skin glycogen and glucose levels can mirror serum glucose levels and thus the skin might contribute to whole body glucose metabolism. This study investigated the in vivo effects of diabetes, insulin and oleanolic acid (OA) on enzymes of glycogen metabolism in skin of type 1 diabetic rats. Diabetic and non-diabetic adult male Sprague-Dawley rats were treated with a single daily dose of insulin (4 IU/kg body weight), OA (80 mg/kg body weight) and a combination of OA + insulin for 14 days. Glycogen phosphorylase (GP) expression; and GP, glycogen synthase (GS) and hexokinase activities as well glycogen levels were evaluated. The results suggest that diabetes lowers hexokinase activity, GP activity and GP expression with no change in GS activity whilst the treatments increased GP expression and the activities of hexokinase, GP and GS except for the GS activity in OA treated rats. Glycogen levels were increased slightly by diabetes as well as OA treatment. In conclusion diabetes, OA and insulin can lead to changes in GS and GP activities in skin without significantly altering the glycogen content. We suggest that the skin may contribute to whole body glucose homeostasis particularly in disease states.

  10. Structural analyses reveal two distinct families of nucleoside phosphorylases.

    PubMed Central

    Pugmire, Matthew J; Ealick, Steven E

    2002-01-01

    The reversible phosphorolysis of purine and pyrimidine nucleosides is an important biochemical reaction in the salvage pathway, which provides an alternative to the de novo purine and pyrimidine biosynthetic pathways. Structural studies in our laboratory and by others have revealed that only two folds exist that catalyse the phosphorolysis of all nucleosides, and provide the basis for defining two families of nucleoside phosphorylases. The first family (nucleoside phosphorylase-I) includes enzymes that share a common single-domain subunit, with either a trimeric or a hexameric quaternary structure, and accept a range of both purine and pyrimidine nucleoside substrates. Despite differences in substrate specificity, amino acid sequence and quaternary structure, all members of this family share a characteristic subunit topology. We have also carried out a sequence motif study that identified regions of the common subunit fold that are functionally significant in differentiating the various members of the nucleoside phosphorylase-I family. Although the substrate-binding sites are arranged similarly for all members of the nucleoside phosphorylase-I family, a comparison of the active sites from the known structures of this family indicates significant differences between the trimeric and hexameric family members. Sequence comparisons also suggest structural identity between the nucleoside phosphorylase-I family and both 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase and AMP nucleosidase. Members of the second family of nucleoside phosphorylases (nucleoside phosphorylase-II) share a common two-domain subunit fold and a dimeric quaternary structure, share a significant level of sequence identity (>30%) and are specific for pyrimidine nucleosides. Members of this second family accept both thymidine and uridine substrates in lower organisms, but are specific for thymidine in mammals and other higher organisms. A possible relationship between nucleoside

  11. Expression of Genes Encoding the Enzymes for Glycogen and Trehalose Metabolism in L3 and L4 Larvae of Anisakis simplex

    PubMed Central

    Łopieńska-Biernat, E.; Zaobidna, E. A.; Dmitryjuk, M.

    2015-01-01

    Trehalose and glycogen metabolism plays an important role in supporting life processes in many nematodes, including Anisakis simplex. Nematodes, cosmopolitan helminths parasitizing sea mammals and humans, cause a disease known as anisakiasis. The aim of this study was to investigate the expression of genes encoding the enzymes involved in the metabolism of trehalose and glycogen—trehalose-6-phosphate synthase (TPS), trehalose-6-phosphate phosphatase (TPP), glycogen synthase (GS), and glycogen phosphorylase (GP)—in stage L3 and stage L4 larvae of A. simplex. The expression of mRNA all four genes, tps, tpp, gs, and gp, was examined by real-time polymerase chain reaction. The A. simplex ribosomal gene (18S) was used as a reference gene. Enzymatic activity was determined. The expression of trehalose enzyme genes was higher in L3 than in L4 larvae, but an inverse relationship was noted for the expression of gs and gp genes. PMID:26783451

  12. Changes in the activity levels of glutamine synthetase, glutaminase and glycogen synthetase in rats subjected to hypoxic stress

    NASA Astrophysics Data System (ADS)

    Vats, P.; Mukherjee, A. K.; Kumria, M. M. L.; Singh, S. N.; Patil, S. K. B.; Rangnathan, S.; Sridharan, K.

    Exposure to high altitude causes loss of body mass and alterations in metabolic processes, especially carbohydrate and protein metabolism. The present study was conducted to elucidate the role of glutamine synthetase, glutaminase and glycogen synthetase under conditions of chronic intermittent hypoxia. Four groups, each consisting of 12 male albino rats (Wistar strain), were exposed to a simulated altitude of 7620 m in a hypobaric chamber for 6 h per day for 1, 7, 14 and 21 days, respectively. Blood haemoglobin, blood glucose, protein levels in the liver, muscle and plasma, glycogen content, and glutaminase, glutamine synthetase and glycogen synthetase activities in liver and muscle were determined in all groups of exposed and in a group of unexposed animals. Food intake and changes in body mass were also monitored. There was a significant reduction in body mass (28-30%) in hypoxia-exposed groups as compared to controls, with a corresponding decrease in food intake. There was rise in blood haemoglobin and plasma protein in response to acclimatisation. Over a three-fold increase in liver glycogen content was observed following 1 day of hypoxic exposure (4.76+/-0.78 mg.g-1 wet tissue in normal unexposed rats; 15.82+/-2.30 mg.g-1 wet tissue in rats exposed to hypoxia for 1 day). This returned to normal in later stages of exposure. However, there was no change in glycogen synthetase activity except for a decrease in the 21-days hypoxia-exposed group. There was a slight increase in muscle glycogen content in the 1-day exposed group which declined significantly by 56.5, 50.6 and 42% following 7, 14, and 21 days of exposure, respectively. Muscle glycogen synthetase activity was also decreased following 21 days of exposure. There was an increase in glutaminase activity in the liver and muscle in the 7-, 14- and 21-day exposed groups. Glutamine synthetase activity was higher in the liver in 7- and 14-day exposed groups; this returned to normal following 21 days of exposure

  13. Sex-specific basal and hypoglycemic patterns of in vivo caudal dorsal vagal complex astrocyte glycogen metabolic enzyme protein expression.

    PubMed

    Tamrakar, Pratistha; Shrestha, Prem; Briski, Karen P

    2014-10-24

    Astrocytes contribute to neurometabolic stability through uptake, catabolism, and storage of glucose. These cells maintain the major brain glycogen reservoir, which is a critical fuel supply to neurons during glucose deficiency and increased brain activity. We used a combinatory approach incorporating immunocytochemistry, laser microdissection, and Western blotting to investigate the hypothesis of divergent expression of key enzymes regulating glycogen metabolism and glycolysis during in vivo normo- and/or hypoglycemia in male versus female hindbrain astrocytes. Glycogen synthase (GS) and glycogen phosphorylase (GP) levels were both enhanced in dorsal vagal complex astrocytes from vehicle-injected female versus male controls, with incremental increase in GS exceeding GP. Insulin-induced hypoglycemia (IIH) diminished GS and increased glycogen synthase kinase-3-beta (GSK3β) expression in both sexes, but decreased phosphoprotein phosphatase-1 (PP1) levels only in males. Astrocyte GP content was elevated by IIH in male, but not female rats. Data reveal sex-dependent sensitivity of these enzyme proteins to lactate as caudal hindbrain repletion of this energy substrate fully or incompletely reversed hypoglycemic inhibition of GS and prevented hypoglycemic augmentation of GSK3β and GP in females and males, respectively. Sex dimorphic patterns of glycogen branching and debranching enzyme protein expression were also observed. Levels of the rate-limiting glycolytic enzyme, phosphofructokinase, were unaffected by IIH with or without lactate repletion. Current data demonstrating sex-dependent basal and hypoglycemic patterns of hindbrain astrocyte glycogen metabolic enzyme expression imply that glycogen volume and turnover during glucose sufficiency and shortage may vary accordingly.

  14. Gain of function AMP-activated protein kinase γ3 mutation (AMPKγ3R200Q) in pig muscle increases glycogen storage regardless of AMPK activation.

    PubMed

    Scheffler, Tracy L; Park, Sungkwon; Roach, Peter J; Gerrard, David E

    2016-06-01

    Chronic activation of AMP-activated protein kinase (AMPK) increases glycogen content in skeletal muscle. Previously, we demonstrated that a mutation in the ryanodine receptor (RyR1(R615C)) blunts AMPK phosphorylation in longissimus muscle of pigs with a gain of function mutation in the AMPKγ3 subunit (AMPKγ3(R200Q)); this may decrease the glycogen storage capacity of AMPKγ3(R200Q) + RyR1(R615C) muscle. Therefore, our aim in this study was to utilize our pig model to understand how AMPKγ3(R200Q) and AMPK activation contribute to glycogen storage and metabolism in muscle. We selected and bred pigs in order to generate offspring with naturally occurring AMPKγ3(R200Q), RyR1(R615C), and AMPKγ3(R200Q) + RyR1(R615C) mutations, and also retained wild-type littermates (control). We assessed glycogen content and parameters of glycogen metabolism in longissimus muscle. Regardless of RyR1(R615C), AMPKγ3(R200Q) increased the glycogen content by approximately 70%. Activity of glycogen synthase (GS) without the allosteric activator glucose 6-phosphate (G6P) was decreased in AMPKγ3(R200Q) relative to all other genotypes, whereas both AMPKγ3(R200Q) and AMPKγ3(R200Q) + RyR1(R615C) muscle exhibited increased GS activity with G6P. Increased activity of GS with G6P was not associated with increased abundance of GS or hexokinase 2. However, AMPKγ3(R200Q) enhanced UDP-glucose pyrophosphorylase 2 (UGP2) expression approximately threefold. Although UGP2 is not generally considered a rate-limiting enzyme for glycogen synthesis, our model suggests that UGP2 plays an important role in increasing flux to glycogen synthase. Moreover, we have shown that the capacity for glycogen storage is more closely related to the AMPKγ3(R200Q) mutation than activity.

  15. PP2A inhibition results in hepatic insulin resistance despite Akt2 activation.

    PubMed

    Galbo, Thomas; Perry, Rachel J; Nishimura, Erica; Samuel, Varman T; Quistorff, Bjørn; Shulman, Gerald I

    2013-10-01

    In the liver, insulin suppresses hepatic gluconeogenesis by activating Akt, which inactivates the key gluconeogenic transcription factor FoxO1 (Forkhead Box O1). Recent studies have implicated hyperactivity of the Akt phosphatase Protein Phosphatase 2A (PP2A) and impaired Akt signaling as a molecular defect underlying insulin resistance. We therefore hypothesized that PP2A inhibition would enhance insulin-stimulated Akt activity and decrease glucose production. PP2A inhibitors increased hepatic Akt phosphorylation and inhibited FoxO1in vitro and in vivo, and suppressed gluconeogenesis in hepatocytes. Paradoxically, PP2A inhibition exacerbated insulin resistance in vivo. This was explained by phosphorylation of both hepatic glycogen synthase (GS) (inactivation) and phosphorylase (activation) resulting in impairment of glycogen storage. Our findings underline the significance of GS and Phosphorylase as hepatic PP2A substrates and importance of glycogen metabolism in acute plasma glucose regulation. PMID:24150286

  16. Rat skeletal muscle glycogen degradation pathways reveal differential association of glycogen-related proteins with glycogen granules.

    PubMed

    Xu, Hongyang; Stapleton, David; Murphy, Robyn M

    2015-06-01

    Glycogenin, glycogen-debranching enzyme (GDE) and glycogen phosphorylase (GP) are important enzymes that contribute to glycogen particle metabolism. In Long-Evans Hooded rat whole muscle homogenates prepared from extensor digitorum longus (EDL, fast-twitch) and soleus (SOL, oxidative, predominantly slow twitch), it was necessary to include α-amylase, which releases glucosyl units from glycogen, to detect glycogenin but not GDE or GP. Up to ∼12 % of intramuscular glycogen pool was broken down using either in vitro electrical stimulation or leaving muscle at room temperature >3 h (delayed, post-mortem). Electrical stimulation did not reveal glycogenin unless α-amylase was added, although in post-mortem muscle ∼50 and ∼30 % of glycogenin in EDL and SOL muscles, respectively, was detected compared to the amount detected with α-amylase treatment. Single muscle fibres were dissected from fresh or post-mortem EDL muscles, mechanically skinned to remove surface membrane and the presence of glycogenin, GDE and GP as freely diffusible proteins (i.e. cytoplasmic localization) compared by Western blotting. Diffusibility of glycogenin (∼20 %) and GP (∼60 %) was not different between muscles, although GDE increased from ∼15 % diffusible in fresh muscle to ∼60 % in post-mortem muscle. Under physiologically relevant circumstances, in rat muscle and within detection limits: (1) The total cellular pool of glycogenin is always associated with glycogen granules, (2) GDE is associated with glycogen granules with over half the total pool associated with the outer tiers of glycogen, (3) GP is only ever weakly associated with glycogen granules and (4) addition of α-amylase is necessary in order to detect glycogenin, but not GDE or GP.

  17. Glycogen synthase kinase 3β suppresses polyglutamine aggregation by inhibiting Vaccinia-related kinase 2 activity

    PubMed Central

    Lee, Eunju; Ryu, Hye Guk; Kim, Sangjune; Lee, Dohyun; Jeong, Young-Hun; Kim, Kyong-Tai

    2016-01-01

    Huntington’s disease (HD) is a neurodegenerative disorder caused by an abnormal expansion of polyglutamine repeats in the N-terminal of huntingtin. The amount of aggregate-prone protein is controlled by various mechanisms, including molecular chaperones. Vaccinia-related kinase 2 (VRK2) is known to negatively regulate chaperonin TRiC, and VRK2-facilitated degradation of TRiC increases polyQ protein aggregation, which is involved in HD. We found that VRK2 activity was negatively controlled by glycogen synthase kinase 3β (GSK3β). GSK3β directly bound to VRK2 and inhibited the catalytic activity of VRK2 in a kinase activity-independent manner. Furthermore, GSK3β increased the stability of TRiC and decreased the formation of HttQ103-GFP aggregates by inhibiting VRK2. These results indicate that GSK3β signaling may be a regulatory mechanism of HD progression and suggest targets for further therapeutic trials for HD. PMID:27377031

  18. Glycogen metabolism protects against metabolic insult to preserve carotid body function during glucose deprivation.

    PubMed

    Holmes, Andrew P; Turner, Philip J; Carter, Paul; Leadbeater, Wendy; Ray, Clare J; Hauton, David; Buckler, Keith J; Kumar, Prem

    2014-10-15

    The view that the carotid body (CB) type I cells are direct physiological sensors of hypoglycaemia is challenged by the finding that the basal sensory neuronal outflow from the whole organ is unchanged in response to low glucose. The reason for this difference in viewpoint and how the whole CB maintains its metabolic integrity when exposed to low glucose is unknown. Here we show that, in the intact superfused rat CB, basal sensory neuronal activity was sustained during glucose deprivation for 29.1 ± 1.2 min, before irreversible failure following a brief period of excitation. Graded increases in the basal discharge induced by reducing the superfusate PO2 led to proportional decreases in the time to the pre-failure excitation during glucose deprivation which was dependent on a complete run-down in glycolysis and a fall in cellular energy status. A similar ability to withstand prolonged glucose deprivation was observed in isolated type I cells. Electron micrographs and immunofluorescence staining of rat CB sections revealed the presence of glycogen granules and the glycogen conversion enzymes glycogen synthase I and glycogen phosphorylase BB, dispersed throughout the type I cell cytoplasm. Furthermore, pharmacological attenuation of glycogenolysis and functional depletion of glycogen both significantly reduced the time to glycolytic run-down by ∼33 and 65%, respectively. These findings suggest that type I cell glycogen metabolism allows for the continuation of glycolysis and the maintenance of CB sensory neuronal output in periods of restricted glucose delivery and this may act as a key protective mechanism for the organ during hypoglycaemia. The ability, or otherwise, to preserve energetic status may thus account for variation in the reported capacity of the CB to sense physiological glucose concentrations and may even underlie its function during pathological states associated with augmented CB discharge.

  19. Substrate specificity of pyrimidine nucleoside phosphorylases of NP-II family probed by X-ray crystallography and molecular modeling

    NASA Astrophysics Data System (ADS)

    Balaev, V. V.; Lashkov, A. A.; Prokofev, I. I.; Gabdulkhakov, A. G.; Seregina, T. A.; Mironov, A. S.; Betzel, C.; Mikhailov, A. M.

    2016-09-01

    Pyrimidine nucleoside phosphorylases, which are widely used in the biotechnological production of nucleosides, have different substrate specificity for pyrimidine nucleosides. An interesting feature of these enzymes is that the three-dimensional structure of thymidine-specific nucleoside phosphorylase is similar to the structure of nonspecific pyrimidine nucleoside phosphorylase. The three-dimensional structures of thymidine phosphorylase from Salmonella typhimurium and nonspecific pyrimidine nucleoside phosphorylase from Bacillus subtilis in complexes with a sulfate anion were determined for the first time by X-ray crystallography. An analysis of the structural differences between these enzymes demonstrated that Lys108, which is involved in the phosphate binding in pyrimidine nucleoside phosphorylase, corresponds to Met111 in thymidine phosphorylases. This difference results in a decrease in the charge on one of the hydroxyl oxygens of the phosphate anion in thymidine phosphorylase and facilitates the catalysis through SN2 nucleophilic substitution. Based on the results of X-ray crystallography, the virtual screening was performed for identifying a potent inhibitor (anticancer agent) of nonspecific pyrimidine nucleoside phosphorylase, which does not bind to thymidine phosphorylase. The molecular dynamics simulation revealed the stable binding of the discovered compound—2-pyrimidin-2-yl-1H-imidazole-4-carboxylic acid—to the active site of pyrimidine nucleoside phosphorylase.

  20. Activation of GABA(B) receptors inhibits protein kinase B/glycogen synthase kinase 3 signaling.

    PubMed

    Lu, Frances Fangjia; Su, Ping; Liu, Fang; Daskalakis, Zafiris J

    2012-11-28

    Accumulated evidence has suggested that potentiation of cortical GABAergic inhibitory neurotransmission may be a key mechanism in the treatment of schizophrenia. However, the downstream molecular mechanisms related to GABA potentiation remain unexplored. Recent studies have suggested that dopamine D2 receptor antagonists, which are used in the clinical treatment of schizophrenia, modulate protein kinase B (Akt)/glycogen synthase kinase (GSK)-3 signaling. Here we report that activation of GABA(B) receptors significantly inhibits Akt/GSK-3 signaling in a β-arrestin-dependent pathway. Agonist stimulation of GABA(B) receptors enhances the phosphorylation of Akt (Thr-308) and enhances the phosphorylation of GSK-3α (Ser-21)/β (Ser-9) in both HEK-293T cells expressing GABA(B) receptors and rat hippocampal slices. Furthermore, knocking down the expression of β-arrestin2 using siRNA abolishes the GABA(B) receptor-mediated modulation of GSK-3 signaling. Our data may help to identify potentially novel targets through which GABA(B) receptor agents may exert therapeutic effects in the treatment of schizophrenia.

  1. Effects of hypoxia on ionic regulation, glycogen utilization and antioxidative ability in the gills and liver of the aquatic air-breathing fish Trichogaster microlepis.

    PubMed

    Huang, Chun-Yen; Lin, Hui-Chen; Lin, Cheng-Huang

    2015-01-01

    We examined the hypothesis that Trichogaster microlepis, a fish with an accessory air-breathing organ, uses a compensatory strategy involving changes in both behavior and protein levels to enhance its gas exchange ability. This compensatory strategy enables the gill ion-regulatory metabolism to maintain homeostasis during exposure to hypoxia. The present study aimed to determine whether ionic regulation, glycogen utilization and antioxidant activity differ in terms of expression under hypoxic stresses; fish were sampled after being subjected to 3 or 12h of hypoxia and 12h of recovery under normoxia. The air-breathing behavior of the fish increased under hypoxia. No morphological modification of the gills was observed. The expression of carbonic anhydrase II did not vary among the treatments. The Na(+)/K(+)-ATPase enzyme activity did not decrease, but increases in Na(+)/K(+)-ATPase protein expression and ionocyte levels were observed. The glycogen utilization increased under hypoxia as measured by glycogen phosphorylase protein expression and blood glucose level, whereas the glycogen content decreased. The enzyme activity of several components of the antioxidant system in the gills, including catalase, glutathione peroxidase, and superoxidase dismutase, increased in enzyme activity. Based on the above data, we concluded that T. microlepis is a hypoxia-tolerant species that does not exhibit ion-regulatory suppression but uses glycogen to maintain energy utilization in the gills under hypoxic stress. Components of the antioxidant system showed increased expression under the applied experimental treatments.

  2. CARM1/PRMT4 is necessary for the glycogen gene expression programme in skeletal muscle cells.

    PubMed

    Wang, Shu-Ching Mary; Dowhan, Dennis H; Eriksson, Natalie A; Muscat, George E O

    2012-06-01

    CARM1 (co-activator-associated arginine methyltransferase 1)/PRMT4 (protein arginine methyltransferase 4), functions as a co-activator for transcription factors that are regulators of muscle fibre type and oxidative metabolism, including PGC (peroxisome-proliferator-activated receptor γ co-activator)-1α and MEF2 (myocyte enhancer factor 2). We observed significantly higher Prmt4 mRNA expression in comparison with Prmt1-Prmt6 mRNA expression in mouse muscle (in vitro and in vivo). Transfection of Prmt4 siRNA (small interfering RNA) into mouse skeletal muscle C2C12 cells attenuated PRMT4 mRNA and protein expression. We subsequently performed additional qPCR (quantitative PCR) analysis (in the context of metabolism) to examine the effect of Prmt4 siRNA expression on >200 critical genes that control (and are involved in) lipid, glucose and energy homoeostasis, and circadian rhythm. This analysis revealed a strikingly specific metabolic expression footprint, and revealed that PRMT4 is necessary for the expression of genes involved in glycogen metabolism in skeletal muscle cells. Prmt4 siRNA expression selectively suppressed the mRNAs encoding Gys1 (glycogen synthase 1), Pgam2 (muscle phosphoglycerate mutase 2) and Pygm (muscle glycogen phosphorylase). Significantly, PGAM, PYGM and GYS1 deficiency in humans causes glycogen storage diseases type X, type V/McArdle's disease and type 0 respectively. Attenuation of PRMT4 was also associated with decreased expression of the mRNAs encoding AMPK (AMP-activated protein kinase) α2/γ3 (Prkaa2 and Prkag3) and p38 MAPK (mitogen-activated protein kinase), previously implicated in Wolff-Parkinson-White syndrome and Pompe Disease (glycogen storage disease type II). Furthermore, stable transfection of two PRMT4-site-specific (methyltransferase deficient) mutants (CARM1/PRMT4 VLD and CARM1E267Q) significantly repressed the expression of Gys1, Pgam2 and AMPKγ3. Finally, in concordance, we observed increased and decreased glycogen

  3. Lactate availability is not the major factor limiting muscle glycogen repletion during recovery from an intense sprint in previously active fasted rats.

    PubMed

    Raja, Ghazala; Mills, Sally; Palmer, T Norman; Fournier, Paul A

    2004-12-01

    It is not clear whether the amount of accumulated lactate is the main factor limiting muscle glycogen accumulation during recovery from an intense sprint performed by previously active fasted laboratory rats. To address this question, groups of fasted rats swam at moderate intensity for 30 min, each animal with a lead weight equivalent to 0.5% body mass attached to its tail, followed by a 3 min high intensity swim with a 10% lead weight and a recovery period of up to 2 hours afterwards. Moderately intense exercise for 30 min caused a decrease in muscle glycogen levels in the mixed, white and red gastrocnemius and the mixed quadriceps muscles, and a further rapid fall occurred in response to the 3 min sprint effort. During recovery, glycogen increased to comparable or above pre-sprint levels across all muscles, and this occurred to a large extent at the expense of net carbon sources other than lactate, with these carbon sources accounting for at least 36-65% of the glycogen deposited. The sustained dephosphorylation-mediated activation of glycogen synthase, but not the changes in glucose 6-phosphate levels, most probably played an important role in enabling the replenishment of muscle glycogen stores. In conclusion, our findings suggest that the amount of glycogen deposited during recovery from high intensity exercise in fasted animals is not limited by the amount of accumulated lactate. PMID:15579557

  4. Ambient pH Controls Glycogen Levels by Regulating Glycogen Synthase Gene Expression in Neurospora crassa. New Insights into the pH Signaling Pathway

    PubMed Central

    Cupertino, Fernanda Barbosa; Freitas, Fernanda Zanolli; de Paula, Renato Magalhães; Bertolini, Maria Célia

    2012-01-01

    Glycogen is a polysaccharide widely distributed in microorganisms and animal cells and its metabolism is under intricate regulation. Its accumulation in a specific situation results from the balance between glycogen synthase and glycogen phosphorylase activities that control synthesis and degradation, respectively. These enzymes are highly regulated at transcriptional and post-translational levels. The existence of a DNA motif for the Aspergillus nidulans pH responsive transcription factor PacC in the promoter of the gene encoding glycogen synthase (gsn) in Neurospora crassa prompted us to investigate whether this transcription factor regulates glycogen accumulation. Transcription factors such as PacC in A. nidulans and Rim101p in Saccharomyces cerevisiae play a role in the signaling pathway that mediates adaptation to ambient pH by inducing the expression of alkaline genes and repressing acidic genes. We showed here that at pH 7.8 pacC was over-expressed and gsn was down-regulated in wild-type N. crassa coinciding with low glycogen accumulation. In the pacCKO strain the glycogen levels and gsn expression at alkaline pH were, respectively, similar to and higher than the wild-type strain at normal pH (5.8). These results characterize gsn as an acidic gene and suggest a regulatory role for PACC in gsn expression. The truncated recombinant protein, containing the DNA-binding domain specifically bound to a gsn DNA fragment containing the PacC motif. DNA-protein complexes were observed with extracts from cells grown at normal and alkaline pH and confirmed by ChIP-PCR analysis. The PACC present in these extracts showed equal molecular mass, indicating that the protein is already processed at normal pH, in contrast to A. nidulans. Together, these results show that the pH signaling pathway controls glycogen accumulation by regulating gsn expression and suggest the existence of a different mechanism for PACC activation in N. crassa. PMID:22952943

  5. One-step plasmid construction for generation of knock-out mutants in cyanobacteria: studies of glycogen metabolism in Synechococcus sp. PCC 7002.

    PubMed

    Jacobsen, Jacob H; Rosgaard, Lisa; Sakuragi, Yumiko; Frigaard, Niels-Ulrik

    2011-02-01

    Genome sequences of microorganisms typically contain hundreds of genes with vaguely defined functions. Targeted gene inactivation and phenotypic characterization of the resulting mutant strains is a powerful strategy to investigate the function of these genes. We have adapted the recently reported uracil-specific excision reagent (USER) cloning method for targeted gene inactivation in cyanobacteria and used it to inactivate genes in glycogen metabolism in Synechococcus sp. PCC 7002. Knock-out plasmid constructs were made in a single cloning step, where transformation of E. coli yielded about 90% colonies with the correct construct. The two homologous regions were chosen independently of each other and of restriction sites in the target genome. Mutagenesis of Synechococcus sp. PCC 7002 was tested with four antibiotic resistance selection markers (spectinomycin, erythromycin, kanamycin, and gentamicin), and both single-locus and double-loci mutants were prepared. We found that Synechococcus sp. PCC 7002 contains two glycogen phosphorylases (A0481/glgP and A2139/agpA) and that both need to be genetically inactivated to eliminate glycogen phosphorylase activity in the cells.

  6. The green alga Scenedesmus obliquus contains both diadenosine 5',5'''-P1,P4-tetraphosphate (asymmetrical) pyrophosphohydrolase and phosphorylase activities.

    PubMed Central

    McLennan, A G; Mayers, E; Hankin, S; Thorne, N M; Prescott, M; Powls, R

    1994-01-01

    Diadenosine 5',5'''-P1,P4-tetraphosphate (Ap4A) phosphorylase and Ap4A pyrophosphohydrolase activities have been purified from extracts of the green alga Scenedesmus obliquus. Both activities were also detected in Scenedesmus brasiliensis, Scenedesmus quadricauda and in Chlorella vulgaris. This is the first time that both types of enzyme have been detected in the same species. The Ap4A phosphorylase has a molecular mass of 46-48 kDa, a broad pH optimum between 7.5 and 9.5, and requires a divalent ion for activity (Mg2+ > Co2+ > Ca2+ = Mn2+ = Cd2+ > Zn2+). It degrades substrates with at least four phosphate groups and always produces a nucleoside 5'-diphosphate product. The Km values for Ap4A and Pi are 5.3 microM and 160 microM, respectively, and kcat. = 1.8 s-1. Arsenate, vanadate, molybdate, chromate and tungstate can substitute for phosphate. The enzyme also catalyses Ap4A synthesis (Keq. = [Ap4A] [Pi]/[ATP][ADP] = 9 x 10(-4)) and ADP arsenolysis. The Ap4A hydrolase has a molecular mass of 26-28 kDa, an alkaline pH optimum of 8.8-9.8, and prefers Zn2+ as the stimulatory ion (Zn2+ > Mg2+ > Mn2+ > Co2+ > Cd2+). It degrades substrates with at least four phosphate groups, having a slight preference for Ap5A, and always produces a nucleoside 5'-triphosphate product. The Km value for Ap4A is 6.6 microM and kcat. = 1.3 s-1. It is inhibited competitively by adenosine 5'-tetraphosphate (Ki = 0.67 microM) and non-competitively by fluoride (Ki = 150 microM). A 50-54 kDa dinucleoside 5',5'''-P1,P3-triphosphate (Ap3A) pyrophosphohydrolase was also detected in S. obliquus, S. quadricauda and C. vulgaris. The corresponding enzyme in S. brasiliensis (> 100 kDa) may be a dimer Images Figure 2 PMID:8198532

  7. [Purine nucleoside phosphorylase].

    PubMed

    Pogosian, L G; Akopian, Zh I

    2013-01-01

    Purine nucleoside phosphorylase (PNP) is one of the most important enzymes of the purine metabolism, wich promotes the recycling of purine bases. Nowadays is the actual to search for effective inhibitors of this enzyme which is necessary for creation T-cell immunodeficient status of the organism in the organs and tissues transplantation, and chemotherapy of a number pathologies as well. For their successful practical application necessary to conduct in-depth and comprehensive study of the enzyme, namely a structure, functions, and an affinity of the reaction mechanism. In the review the contemporary achievements in the study of PNP from various biological objects are presented. New data describing the structure of PNP are summarised and analysed. The physiological role of the enzyme is discussed. The enzyme basic reaction mechanisms and actions are considered. The studies on enzyme physicochemical, kinetic, and catalytic research are presented. PMID:24479338

  8. Phosphorylation of inhibitor-2 and activation of MgATP-dependent protein phosphatase by rat skeletal muscle glycogen synthase kinase

    SciTech Connect

    Hegazy, M.G.; Reimann, E.M.; Thysseril, T.J.; Schlender, K.K.

    1986-05-01

    Rat skeletal muscle contains a glycogen synthase kinase (GSK-M) which is not stimulated by Ca/sup 2 +/ or cAMP. This kinase has an apparent Mr of 62,000 and uses ATP but not GTP as a phosphoryl donor. GSK-M phosphorylated glycogen synthase at sites 2 and 3. It phosphorylated ATP-citrate lyase and activated MgATP-dependent phosphatase in the presence of ATP but not GTP. As expected, the kinase also phosphorylated phosphatase inhibitor 2 (I-2). Phosphatase incorporation reached approximately 0.3 mol/mol of I-2. Phosphopeptide maps were obtained by digesting /sup 32/P-labeled I-2 with trypsin and separating the peptides by reversed phase HPLC. Two partially separated /sup 32/P-labeled peaks were obtained when I-2 was phosphorylated with either GSK-M or glycogen synthase kinase 3 (GSK-3) and these peptides were different from those obtained when I-2 was phosphorylated with the catalytic subunit of cAMP-dependent protein kinase (CSU) or casein kinase II (CK-II). When I-2 was phosphorylated with GSK-M or GSK-3 and cleaved by CNBr, a single radioactive peak was obtained. Phosphoamino acid analysis showed that I-2 was phosphorylated by GSK-M or GSK-3 predominately in Thr whereas CSU and CK-II phosphorylated I-2 exclusively in Ser. These results indicate that GSK-M is similar to GSK-3 and to ATP-citrate lyase kinase. However, it appears to differ in Mr from ATP-citrate lyase kinase and it differs from GSK-3 in that it phosphorylates glycogen synthase at site 2 and it does not use GTP as a phosphoryl donor.

  9. Biomarker for Glycogen Storage Diseases

    ClinicalTrials.gov

    2016-08-25

    Fructose Metabolism, Inborn Errors; Glycogen Storage Disease; Glycogen Storage Disease Type I; Glycogen Storage Disease Type II; Glycogen Storage Disease Type III; Glycogen Storage Disease Type IV; Glycogen Storage Disease Type V; Glycogen Storage Disease Type VI; Glycogen Storage Disease Type VII; Glycogen Storage Disease Type VIII

  10. An evaluation of indirubin analogues as phosphorylase kinase inhibitors.

    PubMed

    Begum, Jaida; Skamnaki, Vassiliki T; Moffatt, Colin; Bischler, Nicolas; Sarrou, Josephine; Skaltsounis, Alexios-Leandros; Leonidas, Demetres D; Oikonomakos, Nikos G; Hayes, Joseph M

    2015-09-01

    Phosphorylase kinase (PhK) has been linked with a number of conditions such as glycogen storage diseases, psoriasis, type 2 diabetes and more recently, cancer (Camus et al., 2012 [6]). However, with few reported structural studies on PhK inhibitors, this hinders a structure based drug design approach. In this study, the inhibitory potential of 38 indirubin analogues have been investigated. 11 of these ligands had IC50 values in the range 0.170-0.360μM, with indirubin-3'-acetoxime (1c) the most potent. 7-Bromoindirubin-3'-oxime (13b), an antitumor compound which induces caspase-independent cell-death (Ribas et al., 2006 [20]) is revealed as a specific inhibitor of PhK (IC50=1.8μM). Binding assay experiments performed using both PhK-holo and PhK-γtrnc confirmed the inhibitory effects to arise from binding at the kinase domain (γ subunit). High level computations using QM/MM-PBSA binding free energy calculations were in good agreement with experimental binding data, as determined using statistical analysis, and support binding at the ATP-binding site. The value of a QM description for the binding of halogenated ligands exhibiting σ-hole effects is highlighted. A new statistical metric, the 'sum of the modified logarithm of ranks' (SMLR), has been defined which measures performance of a model for both the "early recognition" (ranking earlier/higher) of active compounds and their relative ordering by potency. Through a detailed structure activity relationship analysis considering other kinases (CDK2, CDK5 and GSK-3α/β), 6'(Z) and 7(L) indirubin substitutions have been identified to achieve selective PhK inhibition. The key PhK binding site residues involved can also be targeted using other ligand scaffolds in future work. PMID:26364215

  11. An evaluation of indirubin analogues as phosphorylase kinase inhibitors.

    PubMed

    Begum, Jaida; Skamnaki, Vassiliki T; Moffatt, Colin; Bischler, Nicolas; Sarrou, Josephine; Skaltsounis, Alexios-Leandros; Leonidas, Demetres D; Oikonomakos, Nikos G; Hayes, Joseph M

    2015-09-01

    Phosphorylase kinase (PhK) has been linked with a number of conditions such as glycogen storage diseases, psoriasis, type 2 diabetes and more recently, cancer (Camus et al., 2012 [6]). However, with few reported structural studies on PhK inhibitors, this hinders a structure based drug design approach. In this study, the inhibitory potential of 38 indirubin analogues have been investigated. 11 of these ligands had IC50 values in the range 0.170-0.360μM, with indirubin-3'-acetoxime (1c) the most potent. 7-Bromoindirubin-3'-oxime (13b), an antitumor compound which induces caspase-independent cell-death (Ribas et al., 2006 [20]) is revealed as a specific inhibitor of PhK (IC50=1.8μM). Binding assay experiments performed using both PhK-holo and PhK-γtrnc confirmed the inhibitory effects to arise from binding at the kinase domain (γ subunit). High level computations using QM/MM-PBSA binding free energy calculations were in good agreement with experimental binding data, as determined using statistical analysis, and support binding at the ATP-binding site. The value of a QM description for the binding of halogenated ligands exhibiting σ-hole effects is highlighted. A new statistical metric, the 'sum of the modified logarithm of ranks' (SMLR), has been defined which measures performance of a model for both the "early recognition" (ranking earlier/higher) of active compounds and their relative ordering by potency. Through a detailed structure activity relationship analysis considering other kinases (CDK2, CDK5 and GSK-3α/β), 6'(Z) and 7(L) indirubin substitutions have been identified to achieve selective PhK inhibition. The key PhK binding site residues involved can also be targeted using other ligand scaffolds in future work.

  12. Long-term effects of rapamycin treatment on insulin mediated phosphorylation of Akt/PKB and glycogen synthase activity

    SciTech Connect

    Varma, Shailly; Shrivastav, Anuraag; Changela, Sheena; Khandelwal, Ramji L.

    2008-04-01

    Protein kinase B (Akt/PKB) is a Ser/Thr kinase that is involved in the regulation of cell proliferation/survival through mammalian target of rapamycin (mTOR) and the regulation of glycogen metabolism through glycogen synthase kinase 3{beta} (GSK-3{beta}) and glycogen synthase (GS). Rapamycin is an inhibitor of mTOR. The objective of this study was to investigate the effects of rapamycin pretreatment on the insulin mediated phosphorylation of Akt/PKB phosphorylation and GS activity in parental HepG2 and HepG2 cells with overexpression of constitutively active Akt1/PKB-{alpha} (HepG2-CA-Akt/PKB). Rapamycin pretreatment resulted in a decrease (20-30%) in the insulin mediated phosphorylation of Akt1 (Ser 473) in parental HepG2 cells but showed an upregulation of phosphorylation in HepG2-CA-Akt/PKB cells. Rictor levels were decreased (20-50%) in parental HepG2 cells but were not significantly altered in the HepG2-CA-Akt/PKB cells. Furthermore, rictor knockdown decreased the phosphorylation of Akt (Ser 473) by 40-60% upon rapamycin pretreatment. GS activity followed similar trends as that of phosphorylated Akt and so with rictor levels in these cells pretreated with rapamycin; parental HepG2 cells showed a decrease in GS activity, whereas as HepG2-CA-Akt/PKB cells showed an increase in GS activity. The changes in the levels of phosphorylated Akt/PKB (Ser 473) correlated with GS and protein phoshatase-1 activity.

  13. Polymer phosphorylases: clues to the emergence of non-replicative and replicative polymers.

    PubMed

    Freire, Miguel Angel

    2011-12-01

    Polymer formation is arguably one of the essential factors that allowed the emergence, stabilisation and spread of life on Earth. Consequently, studies concerning biopolymers could shed light on the origins of life itself. Of particular interest are RNA and polysaccharide polymers, the archetypes of the contrasting proposed evolutionary scenarios and their respective polymerases. Nucleic acid polymerases were hypothesised, before their discovery, to have a functional similarity with glycogen phosphorylase. Further identification and characterisation of nucleic acid polymerases; particularly of polynucleotide phosphorylase (PNPase), provided experimental evidence for the initial premise. Once discovered, frequent similarities were found between PNPase and glycogen phosphorylase, in terms of catalytic features and biochemical properties. As a result, PNPase was seen as a model of primitive polymerase and used in laboratory precellular systems. Paradoxically, however, these similarities were not sufficient as an argument in favour of an ancestral common polymerisation mechanism prior to polysaccharides and polyribonucleotides. Here we present an overview of the common features shared by polymer phosphorylases, with new proposals for the emergence of polysaccharide and RNA polymers.

  14. 6-Methylpurine derived sugar modified nucleosides: Synthesis and in vivo antitumor activity in D54 tumor expressing M64V-Escherichia coli purine nucleoside phosphorylase.

    PubMed

    Hassan, Abdalla E A; Abou-Elkhair, Reham A I; Parker, William B; Allan, Paula W; Secrist, John A

    2016-01-27

    Impressive antitumor activity has been observed with fludarabine phosphate against tumors that express Escherichia coli purine nucleoside phosphorylase (PNP) due to the liberation of 2-fluoroadenine in the tumor tissue. 6-Methylpurine (MeP) is another cytotoxic adenine analog that does not exhibit selectivity when administered systemically, and could be very useful in a gene therapy approach to cancer treatment involving E. coli PNP. The prototype MeP releasing prodrug 9-(2-deoxy-β-d-ribofuranosyl)-6-methylpurine (1) [MeP-dR] has demonstrated good activity against tumors expressing E. coli PNP, but its antitumor activity is limited due to toxicity resulting from the generation of MeP from gut bacteria. Therefore, we have embarked on a medicinal chemistry program to identify a combination of non-toxic MeP prodrugs and non-human adenosine glycosidic bond cleaving enzymes. The two best MeP-based substrates with M64V-E coli PNP, a mutant which was engineered to tolerate modification at the 5'-position of adenosine and its analogs, were 9-(6-deoxy-α-l-talofuranosyl)-6-methylpurine (3) [methyl(talo)-MeP-R] and 9-(α-l-lyxofuranosyl)6-methylpurine (4) [lyxo-MeP-R]. The detailed synthesis methyl(talo)-MeP-R and lyxo-MeP-R, and the evaluation of their substrate activity with 4 enzymes not normally associated with cancer patients is described. In addition, we have determined the intraperitoneal pharmacokinetic (ip-PK) properties of methyl(talo)-MeP-R and have determined its in vivo bystander activity in mice bearing D54 tumors that express M64V PNP. The observed good in vivo bystander activity of [methyl(talo)-MeP-R/M64V-E coli PNP combination suggests that these agents could be useful for the treatment of cancer.

  15. The Crystal Structure of Streptococcus pyogenes Uridine Phosphorylase Reveals a Distinct Subfamily of Nucleoside Phosphorylases

    SciTech Connect

    Tran, Timothy H.; Christoffersen, S.; Allan, Paula W.; Parker, William B.; Piskur, Jure; Serra, I.; Terreni, M.; Ealick, Steven E.

    2011-09-20

    Uridine phosphorylase (UP), a key enzyme in the pyrimidine salvage pathway, catalyzes the reversible phosphorolysis of uridine or 2'-deoxyuridine to uracil and ribose 1-phosphate or 2'-deoxyribose 1-phosphate. This enzyme belongs to the nucleoside phosphorylase I superfamily whose members show diverse specificity for nucleoside substrates. Phylogenetic analysis shows Streptococcus pyogenes uridine phosphorylase (SpUP) is found in a distinct branch of the pyrimidine subfamily of nucleoside phosphorylases. To further characterize SpUP, we determined the crystal structure in complex with the products, ribose 1-phosphate and uracil, at 1.8 {angstrom} resolution. Like Escherichia coli UP (EcUP), the biological unit of SpUP is a hexamer with an ?/? monomeric fold. A novel feature of the active site is the presence of His169, which structurally aligns with Arg168 of the EcUP structure. A second active site residue, Lys162, is not present in previously determined UP structures and interacts with O2 of uracil. Biochemical studies of wild-type SpUP showed that its substrate specificity is similar to that of EcUP, while EcUP is {approx}7-fold more efficient than SpUP. Biochemical studies of SpUP mutants showed that mutations of His169 reduced activity, while mutation of Lys162 abolished all activity, suggesting that the negative charge in the transition state resides mostly on uracil O2. This is in contrast to EcUP for which transition state stabilization occurs mostly at O4.

  16. The phosphate site of trehalose phosphorylase from Schizophyllum commune probed by site-directed mutagenesis and chemical rescue studies.

    PubMed

    Goedl, Christiane; Nidetzky, Bernd

    2008-03-01

    Schizophyllum communealpha,alpha-trehalose phosphorylase utilizes a glycosyltransferase-like catalytic mechanism to convert its disaccharide substrate into alpha-d-glucose 1-phosphate and alpha-d-glucose. Recruitment of phosphate by the free enzyme induces alpha,alpha-trehalose binding recognition and promotes the catalytic steps. Like the structurally related glycogen phosphorylase and other retaining glycosyltransferases of fold family GT-B, the trehalose phosphorylase contains an Arg507-XXXX-Lys512 consensus motif (where X is any amino acid) comprising key residues of its putative phosphate-binding sub-site. Loss of wild-type catalytic efficiency for reaction with phosphate (kcat/Km=21,000 m(-1).s(-1)) was dramatic (>or=10(7)-fold) in purified Arg507-->Ala (R507A) and Lys512-->Ala (K512A) enzymes, reflecting a corresponding change of comparable magnitude in kcat (Arg507) and Km (Lys512). External amine and guanidine derivatives selectively enhanced the activity of the K512A mutant and the R507A mutant respectively. Analysis of the pH dependence of chemical rescue of the K512A mutant by propargylamine suggested that unprotonated amine in combination with H2PO4-, the protonic form of phosphate presumably utilized in enzymatic catalysis, caused restoration of activity. Transition state-like inhibition of the wild-type enzyme A by vanadate in combination with alpha,alpha-trehalose (Ki=0.4 microm) was completely disrupted in the R507A mutant but only weakened in the K512A mutant (Ki=300 microm). Phosphate (50 mm) enhanced the basal hydrolase activity of the K512A mutant toward alpha,alpha-trehalose by 60% but caused its total suppression in wild-type and R507A enzymes. The results portray differential roles for the side chains of Lys512 and Arg507 in trehalose phosphorylase catalysis, reactant state binding of phosphate and selective stabilization of the transition state respectively. PMID:18205830

  17. Modified 5-fluorouracil: Uridine phosphorylase inhibitor

    NASA Astrophysics Data System (ADS)

    Lashkov, A. A.; Shchekotikhin, A. A.; Shtil, A. A.; Sotnichenko, S. E.; Mikhailov, A. M.

    2016-09-01

    5-Fluorouracil (5-FU) is a medication widely used in chemotherapy to treat various types of cancer. Being a substrate for the reverse reaction catalyzed by uridine phosphorylase (UPase), 5-FU serves as a promising prototype molecule (molecular scaffold) for the design of a selective UPase inhibitor that enhances the antitumor activity of 5-FU and exhibits intrinsic cytostatic effects on cancer cells. The chemical formula of the new compound, which binds to the uracil-binding site and, in the presence of a phosphate anion, to the phosphate-binding site of UPase, is proposed and investigated by molecular simulation methods.

  18. Identification of the maize amyloplast stromal 112-kD protein as a plastidic starch phosphorylase.

    PubMed

    Yu, Y; Mu, H H; Wasserman, B P; Carman, G M

    2001-01-01

    Amyloplast is the site of starch synthesis in the storage tissue of maize (Zea mays). The amyloplast stroma contains an enriched group of proteins when compared with the whole endosperm. Proteins with molecular masses of 76 and 85 kD have been identified as starch synthase I and starch branching enzyme IIb, respectively. A 112-kD protein was isolated from the stromal fraction by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subjected to tryptic digestion and amino acid sequence analysis. Three peptide sequences showed high identity to plastidic forms of starch phosphorylase (SP) from sweet potato, potato, and spinach. SP activity was identified in the amyloplast stromal fraction and was enriched 4-fold when compared with the activity in the whole endosperm fraction. Native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses showed that SP activity was associated with the amyloplast stromal 112-kD protein. In addition, antibodies raised against the potato plastidic SP recognized the amyloplast stromal 112-kD protein. The amyloplast stromal 112-kD SP was expressed in whole endosperm isolated from maize harvested 9 to 24 d after pollination. Results of affinity electrophoresis and enzyme kinetic analyses showed that the amyloplast stromal 112-kD SP preferred amylopectin over glycogen as a substrate in the synthetic reaction. The maize shrunken-4 mutant had reduced SP activity due to a decrease of the amyloplast stromal 112-kD enzyme.

  19. [Use of properties and regulation peculiarities of enzymes of glycogenolysis in fish skeletal muscle depending on peculiarities of motor activity of species].

    PubMed

    Serebrenikova, T P; Nesterov, V P

    2008-01-01

    Levels of activity, properties, and peculiarities of activation of glycogen phosphorylase (GP; EC 2.4.1.1) and glycogen phosphorylase kinase (GPK; EC 2.7.1.38) were studied in the white skeletal muscle of fish differing in motor behavior. No differences in the GP and GPK activity levels were revealed in laskir Diplodus annularis (L.), horse mackerel Trachurus mediterraneus ponticus, salmon Salmo trutta morphario, scorpena Scorpaena porcus, Scophtalnus 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. In fish capable for the spurt type of swimming (scorpena, salmon) the more rapid ion regulation plays the predominant role. In other species, the glycogenolysis hormonal regulation leading to a change of the GPK activity index has been found.

  20. O-GlcNAc Protein Modification in Cancer Cells Increases in Response to Glucose Deprivation through Glycogen Degradation*

    PubMed Central

    Kang, Jeong Gu; Park, Sang Yoon; Ji, Suena; Jang, Insook; Park, Sujin; Kim, Hyun Sil; Kim, Sung-Min; Yook, Jong In; Park, Yong-Il; Roth, Jürgen; Cho, Jin Won

    2009-01-01

    When cellular glucose concentrations fall below normal levels, in general the extent of protein O-GlcNAc modification (O-GlcNAcylation) decreases. However, recent reports demonstrated increased O-GlcNAcylation by glucose deprivation in HepG2 and Neuro-2a cells. Here, we report increased O-GlcNAcylation in non-small cell lung carcinoma A549 cells and various other cells in response to glucose deprivation. Although the level of O-GlcNAc transferase was unchanged, the enzyme contained less O-GlcNAc, and its activity was increased. Moreover, O-GlcNAcase activity was reduced. The studied cells contain glycogen, and we show that its degradation in response to glucose deprivation provides a source for UDP-GlcNAc required for increased O-GlcNAcylation under this condition. This required active glycogen phosphorylase and resulted in increased glutamine:fructose-6-phosphate amidotransferase, the first and rate-limiting enzyme in the hexosamine biosynthetic pathway. Interestingly, glucose deprivation reduced the amount of phosphofructokinase 1, a regulatory glycolytic enzyme, and blocked ATP synthesis. These findings suggest that glycogen is the source for increased O-GlcNAcylation but not for generating ATP in response to glucose deprivation and that this may be useful for cancer cells to survive. PMID:19833729

  1. Glycogen catabolism, but not its biosynthesis, affects virulence of Fusarium oxysporum on the plant host.

    PubMed

    Corral-Ramos, Cristina; Roncero, M Isabel G

    2015-04-01

    The role of glycogen metabolism was investigated in the fungal pathogen Fusarium oxysporum. Targeted inactivation was performed of genes responsible for glycogen biosynthesis: gnn1 encoding glycogenin, gls1 encoding glycogen synthase, and gbe1 encoding glycogen branching enzyme. Moreover genes involved in glycogen catabolism were deleted: gph1 encoding glycogen phosphorylase and gdb1 encoding glycogen de-branching enzyme. Glycogen reserves increased steadily during growth of the wild type strain in axenic cultures, to reach up to 1500μg glucose equivalents mg(-1) protein after 14 days. Glycogen accumulation was abolished in mutants lacking biosynthesis genes, whereas it increased by 20-40% or 80%, respectively, in the single and double mutants affected in catabolic genes. Transcript levels of glycogen metabolism genes during tomato plant infection peaked at four days post inoculation, similar to the results observed during axenic culture. Significant differences were observed between gdb mutants and the wild type strain for vegetative hyphal fusion ability. The single mutants defective in glycogen metabolism showed similar levels of virulence in the invertebrate animal model Galleria mellonella. Interestingly, the deletion of gdb1 reduced virulence on the plant host up to 40% compared to the wild type in single and in double mutant backgrounds, whereas the other mutants showed the virulence at the wild-type level.

  2. Brain glycogen in health and disease.

    PubMed

    Duran, Jordi; Guinovart, Joan J

    2015-12-01

    Glycogen is present in the brain at much lower concentrations than in muscle or liver. However, by characterizing an animal depleted of brain glycogen, we have shown that the polysaccharide plays a key role in learning capacity and in activity-dependent changes in hippocampal synapse strength. Since glycogen is essentially found in astrocytes, the diverse roles proposed for this polysaccharide in the brain have been attributed exclusively to these cells. However, we have demonstrated that neurons have an active glycogen metabolism that contributes to tolerance to hypoxia. However, these cells can store only minute amounts of glycogen, since the progressive accumulation of this molecule leads to neuronal loss. Loss-of-function mutations in laforin and malin cause Lafora disease. This condition is characterized by the presence of high numbers of insoluble polyglucosan bodies, known as Lafora bodies, in neuronal cells. Our findings reveal that the accumulation of this aberrant glycogen accounts for the neurodegeneration and functional consequences, as well as the impaired autophagy, observed in models of this disease. Similarly glycogen synthase is responsible for the accumulation of corpora amylacea, which are polysaccharide-based aggregates present in the neurons of aged human brains. Our findings change the current view of the role of glycogen in the brain and reveal that endogenous neuronal glycogen metabolism is important under stress conditions and that neuronal glycogen accumulation contributes to neurodegenerative diseases and to aging-related corpora amylacea formation.

  3. Microwave-assisted synthesis of C-8 aryl and heteroaryl inosines and determination of their inhibitory activities against Plasmodium falciparum purine nucleoside phosphorylase.

    PubMed

    Gigante, Alba; Priego, Eva-María; Sánchez-Carrasco, Paula; Ruiz-Pérez, Luis Miguel; Vande Voorde, Johan; Camarasa, María-José; Balzarini, Jan; González-Pacanowska, Dolores; Pérez-Pérez, María-Jesús

    2014-07-23

    8-Arylinosines have been scarcely studied for therapeutic purposes, probably due to difficulties in their synthesis. The recently described direct arylation reaction at position 8 of purine nucleosides has been employed to synthesize a series of 8-aryl and 8-pyridylinosines. These compounds have been studied for hydrolytic stability and subjected to biological evaluation. Three compounds have shown a pronounced specific inhibition of Plasmodium falciparum-encoded purine nucleoside phosphorylase, an important target for antimalarial chemotherapy. PMID:24929343

  4. Multiple Glycogen-binding Sites in Eukaryotic Glycogen Synthase Are Required for High Catalytic Efficiency toward Glycogen

    SciTech Connect

    Baskaran, Sulochanadevi; Chikwana, Vimbai M.; Contreras, Christopher J.; Davis, Keri D.; Wilson, Wayne A.; DePaoli-Roach, Anna A.; Roach, Peter J.; Hurley, Thomas D.

    2012-12-10

    Glycogen synthase is a rate-limiting enzyme in the biosynthesis of glycogen and has an essential role in glucose homeostasis. The three-dimensional structures of yeast glycogen synthase (Gsy2p) complexed with maltooctaose identified four conserved maltodextrin-binding sites distributed across the surface of the enzyme. Site-1 is positioned on the N-terminal domain, site-2 and site-3 are present on the C-terminal domain, and site-4 is located in an interdomain cleft adjacent to the active site. Mutation of these surface sites decreased glycogen binding and catalytic efficiency toward glycogen. Mutations within site-1 and site-2 reduced the V{sub max}/S{sub 0.5} for glycogen by 40- and 70-fold, respectively. Combined mutation of site-1 and site-2 decreased the V{sub max}/S{sub 0.5} for glycogen by >3000-fold. Consistent with the in vitro data, glycogen accumulation in glycogen synthase-deficient yeast cells ({Delta}gsy1-gsy2) transformed with the site-1, site-2, combined site-1/site-2, or site-4 mutant form of Gsy2p was decreased by up to 40-fold. In contrast to the glycogen results, the ability to utilize maltooctaose as an in vitro substrate was unaffected in the site-2 mutant, moderately affected in the site-1 mutant, and almost completely abolished in the site-4 mutant. These data show that the ability to utilize maltooctaose as a substrate can be independent of the ability to utilize glycogen. Our data support the hypothesis that site-1 and site-2 provide a 'toehold mechanism,' keeping glycogen synthase tightly associated with the glycogen particle, whereas site-4 is more closely associated with positioning of the nonreducing end during catalysis.

  5. Synthesis of benzimidazole based thiadiazole and carbohydrazide conjugates as glycogen synthase kinase-3β inhibitors with anti-depressant activity.

    PubMed

    Khan, Imran; Tantray, Mushtaq A; Hamid, Hinna; Alam, Mohammad Sarwar; Kalam, Abul; Dhulap, Abhijeet

    2016-08-15

    A series of benzimidazole based thiadiazole and carbohydrazide conjugates have been synthesized and evaluated for inhibition of glycogen synthase kinase-3β and anti-depressant effect. Compounds 4f, 4j, 5b, 5g and 5i were found to be the most potent inhibitors of GSK-3β in vitro amongst the twenty-five benzimidazole based thiadiazole and carbohydrazide conjugates synthesized. Compound 5i was also found to exhibit significant antidepressant activity in vivo at 50mg/kg, when compared to fluoxetine, a known antidepressant drug. The molecular docking studies revealed multiple hydrogen bond interactions by the synthesized compounds with various amino acid residues, viz, ASP-133, LYS-183, PRO-136, VAL-135, TYR-134, or LYS-60 at the GSK-3β receptor site. PMID:27406796

  6. Glycogen metabolism in cancer.

    PubMed

    Zois, Christos E; Favaro, Elena; Harris, Adrian L

    2014-11-01

    Since its identification more than 150 years ago, there has been an extensive characterisation of glycogen metabolism and its regulatory pathways in the two main glycogen storage organs of the body, i.e. liver and muscle. In recent years, glycogen metabolism has also been demonstrated to be upregulated in many tumour types, suggesting it is an important aspect of cancer cell pathophysiology. Here, we provide an overview of glycogen metabolism and its regulation, with a focus on its role in metabolic reprogramming of cancer cells. The various methods to detect glycogen in tumours in vivo are also reviewed. Finally, we discuss the targeting of glycogen metabolism as a strategy for cancer treatment.

  7. Pathway-level acceleration of glycogen catabolism by a response regulator in the cyanobacterium Synechocystis species PCC 6803.

    PubMed

    Osanai, Takashi; Oikawa, Akira; Numata, Keiji; Kuwahara, Ayuko; Iijima, Hiroko; Doi, Yoshiharu; Saito, Kazuki; Hirai, Masami Yokota

    2014-04-01

    Response regulators of two-component systems play pivotal roles in the transcriptional regulation of responses to environmental signals in bacteria. Rre37, an OmpR-type response regulator, is induced by nitrogen depletion in the unicellular cyanobacterium Synechocystis species PCC 6803. Microarray and quantitative real-time polymerase chain reaction analyses revealed that genes related to sugar catabolism and nitrogen metabolism were up-regulated by rre37 overexpression. Protein levels of GlgP(slr1367), one of the two glycogen phosphorylases, in the rre37-overexpressing strain were higher than those of the parental wild-type strain under both nitrogen-replete and nitrogen-depleted conditions. Glycogen amounts decreased to less than one-tenth by rre37 overexpression under nitrogen-replete conditions. Metabolome analysis revealed that metabolites of the sugar catabolic pathway and amino acids were altered in the rre37-overexpressing strain after nitrogen depletion. These results demonstrate that Rre37 is a pathway-level regulator that activates the metabolic flow from glycogen to polyhydroxybutyrate and the hybrid tricarboxylic acid and ornithine cycle, unraveling the mechanism of the transcriptional regulation of primary metabolism in this unicellular cyanobacterium.

  8. Phosphorylation and activation of calcineurin by glycogen synthase (casein) kinase-1 and cyclic AMP-dependent protein kinase

    SciTech Connect

    Singh, T.J.; Wang, J.H.

    1986-05-01

    Calcineurin is a phosphoprotein phosphatase that is activated by divalent cations and further stimulated by calmodulin. In this study calcineurin is shown to be a substrate for both glycogen synthase (casein) kinase-1 (CK-1) and cyclic AMP-dependent protein kinase (A-kinase). Either kinase can catalyze the incorporation of 1.0-1.4 mol /sup 32/P/mol calcineurin. Analysis by SDS-PAGE revealed that only the ..cap alpha.. subunit is phosphorylated. Phosphorylation of calcineurin by either kinase leads to its activation. Using p-nitrophenyl phosphate as a substrate the authors observed a 2-3 fold activation of calcineurin by either Mn/sup 2 +/ or Ni/sup 2 +/ (in the presence or absence of calmodulin) after phosphorylation of calcineurin by either CK-1 or A-kinase. In the absence of Mn/sup 2 +/ or Ni/sup 2 +/ phosphorylated calcineurin, like the nonphosphorylated enzyme, showed very little activity. Ni/sup 2 +/ was a more potent activator of phosphorylated calcineurin compared to Mn/sup 2 +/. Higher levels of activation (5-8 fold) of calcineurin by calmodulin was observed when phosphorylated calcineurin was pretreated with Ni/sup 2 +/ before measurement of phosphatase activity. These results indicate that phosphorylation may be an important mechanism by which calcineurin activity is regulated by Ca/sup 2 +/.

  9. far4, far5, and far6 define three genes required for efficient activation of MAPKs Fus3 and Kss1 and accumulation of glycogen.

    PubMed

    Cherkasova, V; Elion, E A

    2001-08-01

    In Saccharomyces cerevisiae, mating pheromones induce G1 arrest through the activation of two MAP kinases, Fus3 and Kss1. Here we report the isolation of three mutants, far4, far5, and far6, that have the novel phenotype of regulating both the activity of Fus3 and Kss1 and the accumulation of glycogen. A far4 mutation constitutively activates Fus3 and Kss1, reduces glycogen, and blocks G1 arrest in the presence of alpha factor. In contrast, far5 and far6 mutations increase glycogen and reduce activation of Fus3 and Kss1 by pheromone. far4, far5, and far6 are recessive and not allelic to FAR1, FAR3, or 14 genes known to regulate the pheromone response. Non-allelic noncomplementation occurs between far6 and both far4 and far5, suggesting that FAR6 functionally interacts with FAR4 and FAR5. Additional observations suggest that FAR4 has functional overlap with FAR3, which we also find to regulate glycogen accumulation. Our results suggest that the activation of the mating MAPK cascade and subsequent G1 arrest is influenced by a signal transduction pathway that regulates glycogen. In support of this possibility, we find that Fus3 is activated to a greater extent in a "wimp" strain with defective protein kinase A. Finally, BIM1 and BIK1 have been identified as CEN suppressors of far5, suggesting that the microtubule apparatus may regulate the ability of the pheromone response pathway to promote G1 arrest. PMID:11570512

  10. Development of a quantitative 96-well method to image glycogen storage in primary rat hepatocytes.

    PubMed

    Pilling, James; Garside, Helen; Ainscow, Edward

    2010-08-01

    Within the liver, hormonal control of glycogen metabolism allows for rapid release and uptake of glucose from the circulation, providing a reserve of glucose that can be utilised by other organs. Traditionally, cellular glycogen storage has been detected using Periodic acid Schiff (PAS) staining of histopathology samples or a biochemical assay. Colorimetric measurement of glycogen content using PAS staining is hard to quantify whilst biochemical techniques give limited information about events such as cytotoxicity or allow analysis of hepatic heterogeneity. Here, we describe the development of an imaging based method to quantify glycogen storage in 96-well cultures of primary rat hepatocytes using the inherent fluorescence properties of the Schiff reagent. PAS-stained hepatocytes were imaged using an automated fluorescent microscope, with the amount of glycogen present in each cell being quantified. Using this technique, we found an increase in glycogen storage in response to insulin (EC50 = 0.31 nM) that was in agreement with that determined using biochemical quantification (EC50 = 0.32 nM). Furthermore, a dose dependent increase in glycogen storage was also seen in response to glycogen synthase kinase inhibitors and glycogen phosphorylase inhibitors. This technique allows rapid assessment of cellular glycogen storage in response to hormones and small molecule inhibitors.

  11. Effect of increased free fatty acid supply on glucose metabolism and skeletal muscle glycogen synthase activity in normal man.

    PubMed

    Johnson, A B; Argyraki, M; Thow, J C; Cooper, B G; Fulcher, G; Taylor, R

    1992-02-01

    1. Experimental elevation of plasma non-esterified fatty acid concentrations has been postulated to decrease insulin-stimulated glucose oxidation and storage rates. Possible mechanisms were examined by measuring skeletal muscle glycogen synthase activity and muscle glycogen content before and during hyperinsulinaemia while fasting plasma non-esterified fatty acid levels were maintained. 2. Fasting plasma non-esterified fatty acid levels were maintained in seven healthy male subjects by infusion of 20% (w/v) Intralipid (1 ml/min) for 120 min before and during a 240 min hyperinsulinaemic euglycaemic clamp (100 m-units h-1 kg-1) combined with indirect calorimetry. On the control day, 0.154 mol/l NaCl was infused. Vastus lateralis muscle biopsy was performed before and at the end of the insulin infusion. 3. On the Intralipid study day serum triacylglycerol (2.24 +/- 0.20 versus 0.67 +/- 0.10 mmol/l), plasma nonesterified fatty acid (395 +/- 13 versus 51 +/- 1 mumol/l), blood glycerol (152 +/- 2 versus 11 +/- 1 mumol/l) and blood 3-hydroxybutyrate clamp levels [mean (95% confidence interval)] [81 (64-104) versus 4 (3-5) mumol/l] were all significantly higher (all P less than 0.001) than on the control study day. Lipid oxidation rates were also elevated (1.07 +/- 0.07 versus 0.27 +/- 0.08 mg min-1 kg-1, P less than 0.001). During the clamp with Intralipid infusion, insulin-stimulated whole-body glucose disposal decreased by 28% (from 8.53 +/- 0.77 to 6.17 +/- 0.71 mg min-1 kg-1, P less than 0.005).(ABSTRACT TRUNCATED AT 250 WORDS)

  12. Experimental evaluation of decrease in the activities of polyphosphate/glycogen-accumulating organisms due to cell death and activity decay in activated sludge.

    PubMed

    Hao, Xiaodi; Wang, Qilin; Cao, Yali; van Loosdrecht, Mark C M

    2010-06-15

    Decrease in bacterial activity (biomass decay) in activated sludge can result from cell death (reduction in the amount of active bacteria) and activity decay (reduction in the specific activity of active bacteria). The goal of this study was to experimentally differentiate between cell death and activity decay as the cause of decrease in bacterial activity. By means of measuring maximal anaerobic phosphate release rates, verifying membrane integrity by live/dead staining and verifying presence of 16S rRNA with fluorescence in situ hybridization (FISH), the decay rates and death rates of polyphosphate-accumulating organisms (PAOs) in a biological nutrient removal (BNR) system and a laboratory phosphate removing sequencing batch reactor (SBR) system were determined, respectively, under famine conditions. In addition, the decay rate and death rate of glycogen-accumulating organisms (GAOs) in a SBR system with an enrichment culture of GAOs were also measured under famine conditions. Hereto the maximal anaerobic volatile fatty acid uptake rates, live/dead staining, and FISH were used. The experiments revealed that in the BNR and enriched PAO-SBR systems, activity decay contributed 58% and 80% to the decreased activities of PAOs, and that cell death was responsible for 42% and 20% of decreases in their respective activities. In the enriched GAOs system, activity decay constituted a proportion of 74% of the decreased activity of GAOs, and cell death only accounted for 26% of the decrease of their activity.

  13. SIRT3 Blocks Aging-Associated Tissue Fibrosis in Mice by Deacetylating and Activating Glycogen Synthase Kinase 3β

    PubMed Central

    Sundaresan, Nagalingam R.; Bindu, Samik; Pillai, Vinodkumar B.; Samant, Sadhana; Pan, Yong; Huang, Jing-Yi; Gupta, Madhu; Nagalingam, Raghu S.; Wolfgeher, Donald

    2015-01-01

    Tissue fibrosis is a major cause of organ dysfunction during chronic diseases and aging. A critical step in this process is transforming growth factor β1 (TGF-β1)-mediated transformation of fibroblasts into myofibroblasts, cells capable of synthesizing extracellular matrix. Here, we show that SIRT3 controls transformation of fibroblasts into myofibroblasts via suppressing the profibrotic TGF-β1 signaling. We found that Sirt3 knockout (KO) mice with age develop tissue fibrosis of multiple organs, including heart, liver, kidney, and lungs but not whole-body SIRT3-overexpressing mice. SIRT3 deficiency caused induction of TGF-β1 expression and hyperacetylation of glycogen synthase kinase 3β (GSK3β) at residue K15, which negatively regulated GSK3β activity to phosphorylate the substrates Smad3 and β-catenin. Reduced phosphorylation led to stabilization and activation of these transcription factors regulating expression of the profibrotic genes. SIRT3 deacetylated and activated GSK3β and thereby blocked TGF-β1 signaling and tissue fibrosis. These data reveal a new role of SIRT3 to negatively regulate aging-associated tissue fibrosis and discloses a novel phosphorylation-independent mechanism controlling the catalytic activity of GSK3β. PMID:26667039

  14. PfIRR Interacts with HrIGF-I and Activates the MAP-kinase and PI3-kinase Signaling Pathways to Regulate Glycogen Metabolism in Pinctada fucata

    PubMed Central

    Shi, Yu; He, Mao-xian

    2016-01-01

    The insulin-induced mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) pathways are major intracellular signaling modules and conserved among eukaryotes that are known to regulate diverse cellular processes. However, they have not been investigated in the mollusk species Pinctada fucata. Here, we demonstrate that insulin-related peptide receptor of P. fucata (pfIRR) interacts with human recombinant insulin-like growth factor I (hrIGF-I), and stimulates the MAPK and PI3K signaling pathways in P. fucata oocytes. We also show that inhibition of pfIRR by the inhibitor PQ401 significantly attenuates the basal and hrIGF-I-induced phosphorylation of MAPK and PI3K/Akt at amino acid residues threonine 308 and serine 473. Furthermore, our experiments show that there is cross-talk between the MAPK and PI3K/Akt pathways, in which MAPK kinase positively regulates the PI3K pathway, and PI3K positively regulates the MAPK cascade. Intramuscular injection of hrIGF-I stimulates the PI3K and MAPK pathways to increase the expression of pfirr, protein phosphatase 1, glucokinase, and the phosphorylation of glycogen synthase, decreases the mRNA expression of glycogen synthase kinase-3 beta, decreases glucose levels in hemocytes, and increases glycogen levels in digestive glands. These results suggest that the MAPK and PI3K pathways in P. fucata transmit the hrIGF-I signal to regulate glycogen metabolism. PMID:26911653

  15. PfIRR Interacts with HrIGF-I and Activates the MAP-kinase and PI3-kinase Signaling Pathways to Regulate Glycogen Metabolism in Pinctada fucata.

    PubMed

    Shi, Yu; He, Mao-xian

    2016-01-01

    The insulin-induced mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) pathways are major intracellular signaling modules and conserved among eukaryotes that are known to regulate diverse cellular processes. However, they have not been investigated in the mollusk species Pinctada fucata. Here, we demonstrate that insulin-related peptide receptor of P. fucata (pfIRR) interacts with human recombinant insulin-like growth factor I (hrIGF-I), and stimulates the MAPK and PI3K signaling pathways in P. fucata oocytes. We also show that inhibition of pfIRR by the inhibitor PQ401 significantly attenuates the basal and hrIGF-I-induced phosphorylation of MAPK and PI3K/Akt at amino acid residues threonine 308 and serine 473. Furthermore, our experiments show that there is cross-talk between the MAPK and PI3K/Akt pathways, in which MAPK kinase positively regulates the PI3K pathway, and PI3K positively regulates the MAPK cascade. Intramuscular injection of hrIGF-I stimulates the PI3K and MAPK pathways to increase the expression of pfirr, protein phosphatase 1, glucokinase, and the phosphorylation of glycogen synthase, decreases the mRNA expression of glycogen synthase kinase-3 beta, decreases glucose levels in hemocytes, and increases glycogen levels in digestive glands. These results suggest that the MAPK and PI3K pathways in P. fucata transmit the hrIGF-I signal to regulate glycogen metabolism.

  16. Fructose effect to enhance liver glycogen deposition is due to inhibition of glycogenolysis

    SciTech Connect

    Youn, J.; Kaslow, H.; Bergman, R.

    1987-05-01

    The effect of fructose on glycogen degradation was examined by measuring flux of (/sup 14/C) from prelabeled glycogen in perfused rat livers. During 2 h refeeding of fasted rats hepatic glycogen was labeled by injection of (U /sup 14/C) galactose (0.1 mg and 0.02 ..mu..Ci/g of body weight). Refed livers were perfused for 30 min with glucose only (10 mM) and for 60 min with glucose (10 mM) without (n=5) or with fructose (1, 2, 10 mM; n=5 for each). With fructose, label production immediately declined and remained suppressed through the end of perfusion (P < 0.05). Suppression was dose-dependent: steady state label production was suppressed 45, 64, and 72% by 1, 2, and 10 mM fructose (P < 0.0001), without significant changes in glycogen synthase or phosphorylase. These results suggest the existence of allosteric inhibition of phosphorylase in the presence of fructose. Fructose 1-phosphate (F1P) accumulated in proportion to fructose (0.11 +/- 0.01 without fructose, 0.86 +/- 0.03, 1.81 +/- 0.18, and 8.23 +/- 0.6 ..mu..moles/g of liver with 1, 2, and 10 mM fructose. Maximum inhibition of phosphorylase was 82%; FIP concentration for half inhibition was 0.57 ..mu..moles/g of liver, well within the concentration of F1P attained in refeeding. Fructose enhances net glycogen synthesis in liver by suppressing glycogenolysis and the suppression is presumably caused by allosteric inhibition of phosphorylase by F1P.

  17. NK cell function triggered by multiple activating receptors is negatively regulated by glycogen synthase kinase-3β.

    PubMed

    Kwon, Hyung-Joon; Kwon, Soon Jae; Lee, Heejae; Park, Hye-Ran; Choi, Go-Eun; Kang, Sang-Wook; Kwon, Seog Woon; Kim, Nacksung; Lee, Soo Young; Ryu, Sangryeol; Kim, Sun Chang; Kim, Hun Sik

    2015-09-01

    Activation of NK cells is triggered by combined signals from multiple activating receptors that belong to different families. Several NK cell activating receptors have been identified, but their role in the regulation of effector functions is primarily understood in the context of their individual engagement. Therefore, little is known about the signaling pathways broadly implicated by the multiple NK cell activation cues. Here we provide evidence pointing to glycogen synthase kinase (GSK)-3β as a negative regulator of multiple NK cell activating signals. Using an activation model that combines NKG2D and 2B4 and tests different signaling molecules, we found that GSK-3 undergoes inhibitory phosphorylation at regulatory serine residues by the engagement of NKG2D and 2B4, either individually or in combination. The extent of such phosphorylation was closely correlated with the degree of NK cell activation. NK cell functions, such as cytokine production and cytotoxicity, were consistently enhanced by the knockdown of GSK-3β or its inhibition with different pharmacological inhibitors, whereas inhibition of the GSK-3α isoform had no effect. In addition, NK cell function was augmented by the overexpression of a catalytically inactive form of GSK-3β. Importantly, the regulation of NK cell function by GSK-3β was common to diverse activating receptors that signal through both ITAM and non-ITAM pathways. Thus, our results suggest that GSK-3β negatively regulates NK cell activation and that modulation of GSK-3β function could be used to enhance NK cell activation.

  18. Prodigiosin induces the proapoptotic gene NAG-1 via glycogen synthase kinase-3beta activity in human breast cancer cells.

    PubMed

    Soto-Cerrato, Vanessa; Viñals, Francesc; Lambert, James R; Kelly, Julie A; Pérez-Tomás, Ricardo

    2007-01-01

    Prodigiosin (2-methyl-3-pentyl-6-methoxyprodigiosene) is a bacterial metabolite that has anticancer and antimetastatic properties. However, the molecular mechanisms responsible for these abilities are not fully understood. Gene expression profiling of the human breast cancer cell line MCF-7 treated with prodigiosin was analyzed by cDNA array technology. The majority of the significantly modified genes were related to apoptosis, cell cycle, cellular adhesion, or transcription regulation. The dramatic increase of the nonsteroidal anti-inflammatory drug-activated gene 1 (NAG-1) made this gene an interesting candidate regarding the possible mechanism by which prodigiosin induces cytotoxicity in MCF-7 cells. Our results show that prodigiosin triggers accumulation of the DNA-damage response tumor-suppressor protein p53 but that NAG-1 induction was independent of p53 accumulation. Moreover, prodigiosin caused AKT dephosphorylation and glycogen synthase kinase-3beta (GSK-3beta) activation, which correlated with NAG-1 expression. Prodigiosin-induced apoptosis was recovered by inhibiting GSK-3beta, which might be due, at least in part, to the blockade of the GSK-3beta-dependent up-regulation of death receptors 4 and 5 expression. These findings suggest that prodigiosin-mediated GSK-3beta activation is a key event in regulating the molecular pathways that trigger the apoptosis induced by this anticancer agent.

  19. Dual activity of certain HIT-proteins: A. thaliana Hint4 and C. elegans DcpS act on adenosine 5'-phosphosulfate as hydrolases (forming AMP) and as phosphorylases (forming ADP).

    PubMed

    Guranowski, Andrzej; Wojdyła, Anna Maria; Zimny, Jarosław; Wypijewska, Anna; Kowalska, Joanna; Jemielity, Jacek; Davis, Richard E; Bieganowski, Paweł

    2010-01-01

    Histidine triad (HIT)-family proteins interact with different mono- and dinucleotides and catalyze their hydrolysis. During a study of the substrate specificity of seven HIT-family proteins, we have shown that each can act as a sulfohydrolase, catalyzing the liberation of AMP from adenosine 5'-phosphosulfate (APS or SO(4)-pA). However, in the presence of orthophosphate, Arabidopsis thaliana Hint4 and Caenorhabditis elegans DcpS also behaved as APS phosphorylases, forming ADP. Low pH promoted the phosphorolytic and high pH the hydrolytic activities. These proteins, and in particular Hint4, also catalyzed hydrolysis or phosphorolysis of some other adenylyl-derivatives but at lower rates than those for APS cleavage. A mechanism for these activities is proposed and the possible role of some HIT-proteins in APS metabolism is discussed. PMID:19896942

  20. Inhibition of glycogen synthase kinase 3β activity with lithium prevents and attenuates paclitaxel-induced neuropathic pain.

    PubMed

    Gao, M; Yan, X; Weng, H-R

    2013-12-19

    Paclitaxel (taxol) is a first-line chemotherapy-drug used to treat many types of cancers. Neuropathic pain and sensory dysfunction are the major toxicities, which are dose-limiting and significantly reduce the quality of life in patients. Two known critical spinal mechanisms underlying taxol-induced neuropathic pain are an increased production of pro-inflammatory cytokines including interleukin-1β (IL-1β) and suppressed glial glutamate transporter activities. In this study, we uncovered that increased activation of glycogen synthase kinase 3beta (GSK3β) in the spinal dorsal horn was concurrently associated with increased protein expressions of GFAP, IL-1β and a decreased protein expression of glial glutamate transporter 1 (GLT-1), as well as the development and maintenance of taxol-induced neuropathic pain. The enhanced GSK3β activities were supported by the concurrently decreased AKT and mTOR activities. The changes of all these biomarkers were basically prevented when animals received pre-emptive lithium (a GSK3β inhibitor) treatment, which also prevented the development of taxol-induced neuropathic pain. Further, chronic lithium treatment, which began on day 11 after the first taxol injection, reversed the existing mechanical and thermal allodynia induced by taxol. The taxol-induced increased GSK3β activities and decreased AKT and mTOR activities in the spinal dorsal horn were also reversed by lithium. Meanwhile, protein expressions of GLT-1, GFAP and IL-1β in the spinal dorsal horn were improved. Hence, suppression of spinal GSK3β activities is a key mechanism used by lithium to reduce taxol-induced neuropathic pain, and targeting spinal GSK3β is an effective approach to ameliorate GLT-1 expression and suppress the activation of astrocytes and IL-1β over-production in the spinal dorsal horn.

  1. Micropruina glycogenica gen. nov., sp. nov., a new Gram-positive glycogen-accumulating bacterium isolated from activated sludge.

    PubMed

    Shintani, T; Liu, W T; Hanada, S; Kamagata, Y; Miyaoka, S; Suzuki, T; Nakamura, K

    2000-01-01

    A new Gram-positive non-spore-forming bacterium, strain Lg2T, was isolated from an activated sludge reactor showing enhanced biological phosphorus removal activity. The new isolate was a slowly growing organism and was capable of accumulating large amounts of intracellular glycogen from substrate taken up. Both oxidase and catalase were produced. The new isolate contained meso-diaminopimelic acid (DAP) in the cell wall. Complex fatty acid patterns with iso-C14:0, anteiso-C15:0, C16:0, iso-C16:0 and four other minor saturated or unsaturated straight-chain fatty acids were detected. The isolate contained a high genomic G+C content (70.5 mol%). Phylogenetic analysis based on the 16S rRNA gene sequence placed the isolate in the high G+C Gram-positive group with Microlunatus phosphovorus and Friedmanniella antarctica as the closest relatives (sequence similarities are 93 and 92 %, respectively). These three organisms shared common features in morphology, but strain Lg2T could be differentiated from the other species by its peptidoglycan type (meso-DAP), fatty acid composition, carbon source utilization profile and G+C content. On the basis of these findings, it is proposed that a new genus and species, Micropruina glycogenica, should be created for the new isolate; the type strain is strain Lg2T (= JCM 10248T).

  2. Evidence for the absence of cerebral glucose-6-phosphatase activity in glycogen storage disease type I (Von Gierke's disease)

    SciTech Connect

    Phelps, M.E.; Mazziotta, J.C.; Hawkins, R.A.; Philippart, M.

    1981-01-01

    Glycogen storage disease type I (GSD-I) is characterized by a functional deficit in glucose-6-phosphatase that normally hydrolyzes glucose-6-PO/sub 4/ to glucose. This enzyme is primarily found in liver, kidney, and muscle but it is also present in brain, where it appears to participate in the regulation of cerebral tissue glucose. Since most neurological symptoms in GSD-I patients involve systemic hypoglycemia, previous reports have not examined possible deficiencies in phosphatase activity in the brain. Positron computed tomography, F-18-labeled 2-fluorodeoxyglucose (FDG) and a tracer kinetic model for FDG were used to measure the cortical plasma/tissue forward and reverse transport, phosphorylation and dephosphorylation rate constants, tissue/plasma concentration gradient, tissue concentration turnover rate for this competitive analog of glucose, and the cortical metabolic rates for glucose. Studies were carried out in age-matched normals (N = 13) and a single GSD-I patient. The dephosphorylation rate constant in the GSD-I patient was about one tenth the normal value indicating a low level of cerebral phosphatase activity. The other measured parameters were within normal limits except for the rate of glucose phosphorylation which reflected a cortical glucose metabolic rate one half the normal value. Since glucose transport and tissue glucose concentration was normal, the reduced cortical glucose metabolism probably results from the use of alternative substrates (..beta..-hydroxybutyrate and acetoacetate) which are consistently elevated in the plasma of GSD-I patients.

  3. Identification of the Maize Amyloplast Stromal 112-kD Protein as a Plastidic Starch Phosphorylase12

    PubMed Central

    Yu, Ying; Mu, Helen He; Wasserman, Bruce P.; Carman, George M.

    2001-01-01

    Amyloplast is the site of starch synthesis in the storage tissue of maize (Zea mays). The amyloplast stroma contains an enriched group of proteins when compared with the whole endosperm. Proteins with molecular masses of 76 and 85 kD have been identified as starch synthase I and starch branching enzyme IIb, respectively. A 112-kD protein was isolated from the stromal fraction by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subjected to tryptic digestion and amino acid sequence analysis. Three peptide sequences showed high identity to plastidic forms of starch phosphorylase (SP) from sweet potato, potato, and spinach. SP activity was identified in the amyloplast stromal fraction and was enriched 4-fold when compared with the activity in the whole endosperm fraction. Native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses showed that SP activity was associated with the amyloplast stromal 112-kD protein. In addition, antibodies raised against the potato plastidic SP recognized the amyloplast stromal 112-kD protein. The amyloplast stromal 112-kD SP was expressed in whole endosperm isolated from maize harvested 9 to 24 d after pollination. Results of affinity electrophoresis and enzyme kinetic analyses showed that the amyloplast stromal 112-kD SP preferred amylopectin over glycogen as a substrate in the synthetic reaction. The maize shrunken-4 mutant had reduced SP activity due to a decrease of the amyloplast stromal 112-kD enzyme. PMID:11154342

  4. The role of glycogen, glucose and lactate in neuronal activity during hypoxia in the hooded seal (Cystophora cristata) brain.

    PubMed

    Czech-Damal, N U; Geiseler, S J; Hoff, M L M; Schliep, R; Ramirez, J-M; Folkow, L P; Burmester, T

    2014-09-01

    The brains of diving mammals are repeatedly exposed to hypoxic conditions during diving. Brain neurons of the hooded seal (Cystophora cristata) have been shown to be more hypoxia tolerant than those of mice, but the underlying mechanisms are not clear. Here we investigated the roles of different metabolic substrates for maintenance of neuronal activity and integrity, by comparing the in vitro spontaneous neuronal activity of brain slices from layer V of the visual cortex of hooded seals with those in mice (Mus musculus). Studies were conducted by manipulating the composition of the artificial cerebrospinal fluid (aCSF), containing either 10 mM glucose, or 20 mM lactate, or no external carbohydrate supply (aglycemia). Normoxic, hypoxic and ischemic conditions were applied. The lack of glucose or the application of lactate in the aCSF containing no glucose had little effect on the neuronal activity of seal neurons in either normoxia or hypoxia, while neurons from mice survived in hypoxia only few minutes regardless of the composition of the aCSF. We propose that seal neurons have higher intrinsic energy stores. Indeed, we found about three times higher glycogen stores in the seal brain (∼4.1 ng per μg total protein in the seal cerebrum) than in the mouse brain. Notably, in aCSF containing no glucose, seal neurons can tolerate 20 mM lactate while in mouse neuronal activity vanished after few minutes even in normoxia. This can be considered as an adaptation to long dives, during which lactate accumulates in the blood. PMID:24959743

  5. The role of glycogen, glucose and lactate in neuronal activity during hypoxia in the hooded seal (Cystophora cristata) brain.

    PubMed

    Czech-Damal, N U; Geiseler, S J; Hoff, M L M; Schliep, R; Ramirez, J-M; Folkow, L P; Burmester, T

    2014-09-01

    The brains of diving mammals are repeatedly exposed to hypoxic conditions during diving. Brain neurons of the hooded seal (Cystophora cristata) have been shown to be more hypoxia tolerant than those of mice, but the underlying mechanisms are not clear. Here we investigated the roles of different metabolic substrates for maintenance of neuronal activity and integrity, by comparing the in vitro spontaneous neuronal activity of brain slices from layer V of the visual cortex of hooded seals with those in mice (Mus musculus). Studies were conducted by manipulating the composition of the artificial cerebrospinal fluid (aCSF), containing either 10 mM glucose, or 20 mM lactate, or no external carbohydrate supply (aglycemia). Normoxic, hypoxic and ischemic conditions were applied. The lack of glucose or the application of lactate in the aCSF containing no glucose had little effect on the neuronal activity of seal neurons in either normoxia or hypoxia, while neurons from mice survived in hypoxia only few minutes regardless of the composition of the aCSF. We propose that seal neurons have higher intrinsic energy stores. Indeed, we found about three times higher glycogen stores in the seal brain (∼4.1 ng per μg total protein in the seal cerebrum) than in the mouse brain. Notably, in aCSF containing no glucose, seal neurons can tolerate 20 mM lactate while in mouse neuronal activity vanished after few minutes even in normoxia. This can be considered as an adaptation to long dives, during which lactate accumulates in the blood.

  6. Crystallization of the glycogen-binding domain of the AMP-activated protein kinase β subunit and preliminary X-ray analysis

    SciTech Connect

    Polekhina, Galina Feil, Susanne C.; Gupta, Abhilasha; O’Donnell, Paul; Stapleton, David; Parker, Michael W.

    2005-01-01

    The glycogen-binding domain of the AMP-activated kinase β subunit has been crystallized in the presence of β-cyclodextrin. The structure has been determined by single isomorphous replacement and threefold averaging using in-house X-ray data collected from selenomethionine-substituted protein. AMP-activated protein kinase (AMPK) is an intracellular energy sensor that regulates metabolism in response to energy demand and supply by adjusting the ATP-generating and ATP-consuming pathways. AMPK potentially plays a critical role in diabetes and obesity as it is known to be activated by metforin and rosiglitazone, drugs used for the treatment of type II diabetes. AMPK is a heterotrimer composed of a catalytic α subunit and two regulatory subunits, β and γ. Mutations in the γ subunit are known to cause glycogen accumulation, leading to cardiac arrhythmias. Recently, a functional glycogen-binding domain (GBD) has been identified in the β subunit. Here, the crystallization of GBD in the presence of β-cyclodextrin is reported together with preliminary X-ray data analysis allowing the determination of the structure by single isomorphous replacement and threefold averaging using in-house X-ray data collected from a selenomethionine-substituted protein.

  7. Regulation of phosphorylase kinase by low concentrations of Ca ions upon muscle contraction: the connection between metabolism and muscle contraction and the connection between muscle physiology and Ca-dependent signal transduction

    PubMed Central

    OZAWA, Eijiro

    2011-01-01

    It had long been one of the crucial questions in muscle physiology how glycogenolysis is regulated in connection with muscle contraction, when we found the answer to this question in the last half of the 1960s. By that time, the two principal currents of muscle physiology, namely, the metabolic flow starting from glycogen and the mechanisms of muscle contraction, had already been clarified at the molecular level thanks to our senior researchers. Thus, the final question we had to answer was how to connect these two currents. We found that low concentrations of Ca ions (10−7–10−4 M) released from the sarcoplasmic reticulum for the regulation of muscle contraction simultaneously reversibly activate phosphorylase kinase, the enzyme regulating glycogenolysis. Moreover, we found that adenosine 3′,5′-monophosphate (cyclic AMP), which is already known to activate muscle phosphorylase kinase, is not effective in the absence of such concentrations of Ca ions. Thus, cyclic AMP is not effective by itself alone and only modifies the activation process in the presence of Ca ions (at that time, cyclic AMP-dependent protein kinase had not yet been identified). After a while, it turned out that our works have not only provided the solution to the above problem on muscle physiology, but have also been considered as the first report of Ca-dependent protein phosphorylation, which is one of the central problems in current cell biology. Phosphorylase kinase is the first protein kinase to phosphorylate a protein resulting in the change in the function of the phosphorylated protein, as shown by Krebs and Fischer. Our works further showed that this protein kinase is regulated in a Ca-dependent manner. Accordingly, our works introduced the concept of low concentrations of Ca ions, which were first identified as the regulatory substance of muscle contraction, to the vast field of Ca biology including signal transduction. PMID:21986313

  8. A practical method for quantification of phosphorus- and glycogen-accumulating organism populations in activated sludge systems.

    PubMed

    López-Vázquez, Carlos M; Hooijmans, Christine M; Brdjanovic, Damir; Gijzen, Huub J; van Loosdrecht, Mark C M

    2007-12-01

    Enhanced biological phosphorus removal (EBPR) from wastewater relies on the enrichment of activated sludge with phosphorus-accumulating organisms (PAOs). The presence and proliferation of glycogen-accumulating organisms (GAOs), which compete for substrate with PAOs, may be detrimental for EBPR systems, leading to deterioration and, in extreme cases, failure of the process. Therefore, from both process evaluation and modeling perspectives, the estimation of PAO and GAO populations in activated sludge systems is a relevant issue. A simple method for the quantification of PAO and GAO population fractions in activated sludge systems is presented in this paper. To develop such a method, the activity observed in anaerobic batch tests executed with different PAO/GAO ratios, by mixing highly enriched PAO and GAO cultures, was studied. Strong correlations between PAO/GAO population ratios and biomass activity were observed (R2 > 0.97). This served as a basis for the proposal of a simple and practical method to quantify the PAO and GAO populations in activated sludge systems, based on commonly measured and reliable analytical parameters (i.e., mixed liquor suspended solids, acetate, and orthophosphate) without requiring molecular techniques. This method relies on the estimation of the total active biomass population under anaerobic conditions (PAO plus GAO populations), by measuring the maximum acetate uptake rate in the presence of excess acetate. Later, the PAO and GAO populations present in the activated sludge system can be estimated, by taking into account the PAO/GAO ratio calculated on the basis of the anaerobic phosphorus release-to-acetate consumed ratio. The proposed method was evaluated using activated sludge from municipal wastewater treatment plants. The results from the quantification performed following the proposed method were compared with direct population estimations carried out with fluorescence in situ hybridization analysis (determining Candidatus

  9. A modification to the Activated Sludge Model No. 2 based on the competition between phosphorus-accumulating organisms and glycogen-accumulating organisms.

    PubMed

    Manga, J; Ferrer, J; Garcia-Usach, F; Seco, A

    2001-01-01

    A modification to the ASM2 is proposed which permits representation of the competition between phosphorus accumulating organisms (PAOs) and glycogen accumulating organisms (GAOs) in a nutrient removal activated sludge system. Some important aspects, which are not considered in ASM2, are discussed. The proposed modification includes denitrification by PAOs, PAO glycogen storage capability and GAO metabolism model. It is shown that the proposed modification is capable of describing pilot plant data using a single set of stoichiometric and kinetic parameters over three different sludge ages (16, 14 and 12 days). The modified ASM2 may be applicable to a wide range of situations where PAOs and GAOs can compete. This modification may well provide a better understanding about GAO behaviour.

  10. Anti-malarial Activities of Two Soil Actinomycete Isolates from Sabah via Inhibition of Glycogen Synthase Kinase 3β

    PubMed Central

    Dahari, Dhiana Efani; Salleh, Raifana Mohamad; Mahmud, Fauze; Chin, Lee Ping; Embi, Noor; Sidek, Hasidah Mohd

    2016-01-01

    Exploiting natural resources for bioactive compounds is an attractive drug discovery strategy in search for new anti-malarial drugs with novel modes of action. Initial screening efforts in our laboratory revealed two preparations of soil-derived actinomycetes (H11809 and FH025) with potent anti-malarial activities. Both crude extracts showed glycogen synthase kinase 3β (GSK3β)-inhibitory activities in a yeast-based kinase assay. We have previously shown that the GSK3 inhibitor, lithium chloride (LiCl), was able to suppress parasitaemia development in a rodent model of malarial infection. The present study aims to evaluate whether anti-malarial activities of H11809 and FH025 involve the inhibition of GSK3β. The acetone crude extracts of H11809 and FH025 each exerted strong inhibition on the growth of Plasmodium falciparum 3D7 in vitro with 50% inhibitory concentration (IC50) values of 0.57 ± 0.09 and 1.28 ± 0.11 µg/mL, respectively. The tested extracts exhibited Selectivity Index (SI) values exceeding 10 for the 3D7 strain. Both H11809 and FH025 showed dosage-dependent chemo-suppressive activities in vivo and improved animal survivability compared to non-treated infected mice. Western analysis revealed increased phosphorylation of serine (Ser 9) GSK3β (by 6.79 to 6.83-fold) in liver samples from infected mice treated with H11809 or FH025 compared to samples from non-infected or non-treated infected mice. A compound already identified in H11809 (data not shown), dibutyl phthalate (DBP) showed active anti-plasmodial activity against 3D7 (IC50 4.87 ± 1.26 µg/mL which is equivalent to 17.50 µM) and good chemo-suppressive activity in vivo (60.80% chemo-suppression at 300 mg/kg body weight [bw] dosage). DBP administration also resulted in increased phosphorylation of Ser 9 GSK3β compared to controls. Findings from the present study demonstrate that the potent anti-malarial activities of H11809 and FH025 were mediated via inhibition of host GSK3β. In addition

  11. Anti-malarial Activities of Two Soil Actinomycete Isolates from Sabah via Inhibition of Glycogen Synthase Kinase 3β.

    PubMed

    Dahari, Dhiana Efani; Salleh, Raifana Mohamad; Mahmud, Fauze; Chin, Lee Ping; Embi, Noor; Sidek, Hasidah Mohd

    2016-08-01

    Exploiting natural resources for bioactive compounds is an attractive drug discovery strategy in search for new anti-malarial drugs with novel modes of action. Initial screening efforts in our laboratory revealed two preparations of soil-derived actinomycetes (H11809 and FH025) with potent anti-malarial activities. Both crude extracts showed glycogen synthase kinase 3β (GSK3β)-inhibitory activities in a yeast-based kinase assay. We have previously shown that the GSK3 inhibitor, lithium chloride (LiCl), was able to suppress parasitaemia development in a rodent model of malarial infection. The present study aims to evaluate whether anti-malarial activities of H11809 and FH025 involve the inhibition of GSK3β. The acetone crude extracts of H11809 and FH025 each exerted strong inhibition on the growth of Plasmodium falciparum 3D7 in vitro with 50% inhibitory concentration (IC50) values of 0.57 ± 0.09 and 1.28 ± 0.11 µg/mL, respectively. The tested extracts exhibited Selectivity Index (SI) values exceeding 10 for the 3D7 strain. Both H11809 and FH025 showed dosage-dependent chemo-suppressive activities in vivo and improved animal survivability compared to non-treated infected mice. Western analysis revealed increased phosphorylation of serine (Ser 9) GSK3β (by 6.79 to 6.83-fold) in liver samples from infected mice treated with H11809 or FH025 compared to samples from non-infected or non-treated infected mice. A compound already identified in H11809 (data not shown), dibutyl phthalate (DBP) showed active anti-plasmodial activity against 3D7 (IC50 4.87 ± 1.26 µg/mL which is equivalent to 17.50 µM) and good chemo-suppressive activity in vivo (60.80% chemo-suppression at 300 mg/kg body weight [bw] dosage). DBP administration also resulted in increased phosphorylation of Ser 9 GSK3β compared to controls. Findings from the present study demonstrate that the potent anti-malarial activities of H11809 and FH025 were mediated via inhibition of host GSK3β. In addition

  12. Anti-malarial Activities of Two Soil Actinomycete Isolates from Sabah via Inhibition of Glycogen Synthase Kinase 3β

    PubMed Central

    Dahari, Dhiana Efani; Salleh, Raifana Mohamad; Mahmud, Fauze; Chin, Lee Ping; Embi, Noor; Sidek, Hasidah Mohd

    2016-01-01

    Exploiting natural resources for bioactive compounds is an attractive drug discovery strategy in search for new anti-malarial drugs with novel modes of action. Initial screening efforts in our laboratory revealed two preparations of soil-derived actinomycetes (H11809 and FH025) with potent anti-malarial activities. Both crude extracts showed glycogen synthase kinase 3β (GSK3β)-inhibitory activities in a yeast-based kinase assay. We have previously shown that the GSK3 inhibitor, lithium chloride (LiCl), was able to suppress parasitaemia development in a rodent model of malarial infection. The present study aims to evaluate whether anti-malarial activities of H11809 and FH025 involve the inhibition of GSK3β. The acetone crude extracts of H11809 and FH025 each exerted strong inhibition on the growth of Plasmodium falciparum 3D7 in vitro with 50% inhibitory concentration (IC50) values of 0.57 ± 0.09 and 1.28 ± 0.11 µg/mL, respectively. The tested extracts exhibited Selectivity Index (SI) values exceeding 10 for the 3D7 strain. Both H11809 and FH025 showed dosage-dependent chemo-suppressive activities in vivo and improved animal survivability compared to non-treated infected mice. Western analysis revealed increased phosphorylation of serine (Ser 9) GSK3β (by 6.79 to 6.83-fold) in liver samples from infected mice treated with H11809 or FH025 compared to samples from non-infected or non-treated infected mice. A compound already identified in H11809 (data not shown), dibutyl phthalate (DBP) showed active anti-plasmodial activity against 3D7 (IC50 4.87 ± 1.26 µg/mL which is equivalent to 17.50 µM) and good chemo-suppressive activity in vivo (60.80% chemo-suppression at 300 mg/kg body weight [bw] dosage). DBP administration also resulted in increased phosphorylation of Ser 9 GSK3β compared to controls. Findings from the present study demonstrate that the potent anti-malarial activities of H11809 and FH025 were mediated via inhibition of host GSK3β. In addition

  13. Candidate Gene Polymorphisms and their Association with Glycogen Content in the Pacific Oyster Crassostrea gigas

    PubMed Central

    She, Zhicai; Li, Li; Qi, Haigang; Song, Kai; Que, Huayong; Zhang, Guofan

    2015-01-01

    Background The Pacific oyster Crassostrea gigas is an important cultivated shellfish that is rich in nutrients. It contains high levels of glycogen, which is of high nutritional value. To investigate the genetic basis of this high glycogen content and its variation, we conducted a candidate gene association analysis using a wild population, and confirmed our results using an independent population, via targeted gene resequencing and mRNA expression analysis. Results We validated 295 SNPs in the 90 candidate genes surveyed for association with glycogen content, 86 of were ultimately genotyped in all 144 experimental individuals from Jiaonan (JN). In addition, 732 SNPs were genotyped via targeted gene resequencing. Two SNPs (Cg_SNP_TY202 and Cg_SNP_3021) in Cg_GD1 (glycogen debranching enzyme) and one SNP (Cg_SNP_4) in Cg_GP1 (glycogen phosphorylase) were identified as being associated with glycogen content. The glycogen content of individuals with genotypes TT and TC in Cg_SNP_TY202 was higher than that of individuals with genotype CC. The transcript abundance of both glycogen-associated genes was differentially expressed in high glycogen content and low glycogen content individuals. Conclusions This study identified three polymorphisms in two genes associated with oyster glycogen content, via candidate gene association analysis. The transcript abundance differences in Cg_GD1 and Cg_GP1 between low- and the high-glycogen content individuals suggests that it is possible that transcript regulation is mediated by variations of Cg_SNP_TY202, Cg_SNP_3021, and Cg_SNP_4. These findings will not only provide insights into the genetic basis of oyster quality, but also promote research into the molecular breeding of oysters. PMID:25951187

  14. Lithium Enhances Axonal Regeneration in Peripheral Nerve by Inhibiting Glycogen Synthase Kinase 3β Activation

    PubMed Central

    Su, Huanxing; Yuan, Qiuju; Qin, Dajiang; Yang, Xiaoying; So, Kwok-Fai; Wu, Wutian

    2014-01-01

    Brachial plexus injury often involves traumatic root avulsion resulting in permanent paralysis of the innervated muscles. The lack of sufficient regeneration from spinal motoneurons to the peripheral nerve (PN) is considered to be one of the major causes of the unsatisfactory outcome of various surgical interventions for repair of the devastating injury. The present study was undertaken to investigate potential inhibitory signals which influence axonal regeneration after root avulsion injury. The results of the study showed that root avulsion triggered GSK-3β activation in the injured motoneurons and remaining axons in the ventral funiculus. Systemic application of a clinical dose of lithium suppressed activated GSK-3β in the lesioned spinal cord to the normal level and induced extensive axonal regeneration into replanted ventral roots. Our study suggests that GSK-3β activity is involved in negative regulation for axonal elongation and regeneration and lithium, the specific GSK-3β inhibitor, enhances motoneuron regeneration from CNS to PNS. PMID:24967390

  15. Processivity and Subcellular Localization of Glycogen Synthase Depend on a Non-catalytic High Affinity Glycogen-binding Site*

    PubMed Central

    Díaz, Adelaida; Martínez-Pons, Carlos; Fita, Ignacio; Ferrer, Juan C.; Guinovart, Joan J.

    2011-01-01

    Glycogen synthase, a central enzyme in glucose metabolism, catalyzes the successive addition of α-1,4-linked glucose residues to the non-reducing end of a growing glycogen molecule. A non-catalytic glycogen-binding site, identified by x-ray crystallography on the surface of the glycogen synthase from the archaeon Pyrococcus abyssi, has been found to be functionally conserved in the eukaryotic enzymes. The disruption of this binding site in both the archaeal and the human muscle glycogen synthases has a large impact when glycogen is the acceptor substrate. Instead, the catalytic efficiency remains essentially unchanged when small oligosaccharides are used as substrates. Mutants of the human muscle enzyme with reduced affinity for glycogen also show an altered intracellular distribution and a marked decrease in their capacity to drive glycogen accumulation in vivo. The presence of a high affinity glycogen-binding site away from the active center explains not only the long-recognized strong binding of glycogen synthase to glycogen but also the processivity and the intracellular localization of the enzyme. These observations demonstrate that the glycogen-binding site is a critical regulatory element responsible for the in vivo catalytic efficiency of GS. PMID:21464127

  16. Exercise training increases glycogen synthase activity and GLUT4 expression but not insulin signaling in overweight nondiabetic and type 2 diabetic subjects.

    PubMed

    Christ-Roberts, Christine Y; Pratipanawatr, Thongchai; Pratipanawatr, Wilailak; Berria, Rachele; Belfort, Renata; Kashyap, Sangeeta; Mandarino, Lawrence J

    2004-09-01

    Exercise training improves insulin sensitivity in subjects with and without type 2 diabetes. However, the mechanism by which this occurs is unclear. The present study was undertaken to determine how improved insulin signaling, GLUT4 expression, and glycogen synthase activity contribute to this improvement. Euglycemic clamps with indirect calorimetry and muscle biopsies were performed before and after 8 weeks of exercise training in 16 insulin-resistant nondiabetic subjects and 6 type 2 diabetic patients. Training increased peak aerobic capacity (Vo(2peak)) in both nondiabetic (from 34 +/- 2 to 39 +/- 2 mL O(2)/kg fat-free mass [FFM]/min, 14% +/- 2%, P <.001) and diabetic (from 26 +/- 3 to 34 +/- 3 mL O(2)/kg FFM/min, 32% +/- 4%) subjects. Training also increased insulin-stimulated glucose disposal in nondiabetic (from 6.2 +/- 0.5 to 7.1 +/- 0.7 mg/kg FFM/min) and diabetic subjects (from 4.3 +/- 0.6 to 5.5 +/- 0.6 mg/kg FFM/min). Total glycogen synthase activity was increased by 46% +/- 17% and 45% +/- 12% in nondiabetic and diabetic subjects, respectively, in response to training (P <.01 v before training). Moreover, after training, glycogen synthase fractional velocity was correlated with insulin-stimulated glucose storage (r = 0.53, P <.05) and the training-induced improvement in glucose disposal was accounted for primarily by increased insulin-stimulated glucose storage. Training also increased GLUT4 protein by 38% +/- 8% and 22% +/- 10% in nondiabetic and diabetic subjects, respectively (P <.05 v. before training). Akt protein expression, which was decreased by 29% +/- 3% (P <.05) in the diabetic subjects before training (compared to the nondiabetics), increased significantly in both groups (P <.001). In contrast, exercise training did not enhance the ability of insulin to stimulate insulin receptor substrate-1 (IRS-1)-associated phosphatidylinositol 3 (PI 3)-kinase activity. The present data are consistent with a working model whereby 8 weeks of exercise

  17. Dihydromyricetin protects neurons in an MPTP-induced model of Parkinson's disease by suppressing glycogen synthase kinase-3 beta activity

    PubMed Central

    Ren, Zhao-xiang; Zhao, Ya-fei; Cao, Ting; Zhen, Xue-chu

    2016-01-01

    Aim: It is general believed that mitochondrial dysfunction and oxidative stress play critical roles in the pathology of Parkinson's disease (PD). Dihydromyricetin (DHM), a natural flavonoid extracted from Ampelopsis grossedentata, has recently been found to elicit potent anti-oxidative effects. In the present study, we explored the role of DHM in protecting dopaminergic neurons. Methods: Male C57BL/6 mice were intraperitoneally injected with 1-methyl4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) for 7 d to induce PD. Additionally, mice were treated with either 5 or 10 mg/kg DHM for a total of 13 d (3 d before the start of MPTP, during MPTP administration (7 d) and 3 d after the end of MPTP). For the saline or DHM alone treatment groups, mice were injected with saline or DHM for 13 d. On d 14, behavioral tests (locomotor activity, the rotarod test and the pole test) were administered. After the behavioral tests, the mice were sacrificed, and brain tissue was collected for immunofluorescence staining and Western blotting. In addition, MES23.5 cells were treated with MPP+ and DHM, and evaluated using cell viability assays, reactive oxygen species (ROS) measurements, apoptosis analysis and Western blotting. Results: DHM significantly attenuated MPTP-induced mouse behavioral impairments and dopaminergic neuron loss. In the MES23.5 cells, DHM attenuated MPP+-induced cell injury and ROS production in a dose-dependent manner. In addition, DHM increased glycogen synthase kinase-3 beta phosphorylation in a dose- and time-dependent manner, which may be associated with DHM-induced dopaminergic neuronal protection. Conclusion: The present study demonstrated that DHM is a potent neuroprotective agent for DA neurons by modulating the Akt/GSK-3β pathway, which suggests that DHM may be a promising therapeutic candidate for PD. PMID:27374489

  18. Discovery of Isonicotinamides as Highly Selective, Brain Penetrable, and Orally Active Glycogen Synthase Kinase-3 Inhibitors.

    PubMed

    Luo, Guanglin; Chen, Ling; Burton, Catherine R; Xiao, Hong; Sivaprakasam, Prasanna; Krause, Carol M; Cao, Yang; Liu, Nengyin; Lippy, Jonathan; Clarke, Wendy J; Snow, Kimberly; Raybon, Joseph; Arora, Vinod; Pokross, Matt; Kish, Kevin; Lewis, Hal A; Langley, David R; Macor, John E; Dubowchik, Gene M

    2016-02-11

    GSK-3 is a serine/threonine kinase that has numerous substrates. Many of these proteins are involved in the regulation of diverse cellular functions, including metabolism, differentiation, proliferation, and apoptosis. Inhibition of GSK-3 may be useful in treating a number of diseases including Alzheimer's disease (AD), type II diabetes, mood disorders, and some cancers, but the approach poses significant challenges. Here, we present a class of isonicotinamides that are potent, highly kinase-selective GSK-3 inhibitors, the members of which demonstrated oral activity in a triple-transgenic mouse model of AD. The remarkably high kinase selectivity and straightforward synthesis of these compounds bode well for their further exploration as tool compounds and therapeutics.

  19. Discovery of Isonicotinamides as Highly Selective, Brain Penetrable, and Orally Active Glycogen Synthase Kinase-3 Inhibitors.

    PubMed

    Luo, Guanglin; Chen, Ling; Burton, Catherine R; Xiao, Hong; Sivaprakasam, Prasanna; Krause, Carol M; Cao, Yang; Liu, Nengyin; Lippy, Jonathan; Clarke, Wendy J; Snow, Kimberly; Raybon, Joseph; Arora, Vinod; Pokross, Matt; Kish, Kevin; Lewis, Hal A; Langley, David R; Macor, John E; Dubowchik, Gene M

    2016-02-11

    GSK-3 is a serine/threonine kinase that has numerous substrates. Many of these proteins are involved in the regulation of diverse cellular functions, including metabolism, differentiation, proliferation, and apoptosis. Inhibition of GSK-3 may be useful in treating a number of diseases including Alzheimer's disease (AD), type II diabetes, mood disorders, and some cancers, but the approach poses significant challenges. Here, we present a class of isonicotinamides that are potent, highly kinase-selective GSK-3 inhibitors, the members of which demonstrated oral activity in a triple-transgenic mouse model of AD. The remarkably high kinase selectivity and straightforward synthesis of these compounds bode well for their further exploration as tool compounds and therapeutics. PMID:26751161

  20. Valproic acid potentiates the anticancer activity of capecitabine in vitro and in vivo in breast cancer models via induction of thymidine phosphorylase expression

    PubMed Central

    Terranova-Barberio, Manuela; Roca, Maria Serena; Zotti, Andrea Ilaria; Leone, Alessandra; Bruzzese, Francesca; Vitagliano, Carlo; Scogliamiglio, Giosuè; Russo, Domenico; D'Angelo, Giovanni; Franco, Renato; Budillon, Alfredo; Di Gennaro, Elena

    2016-01-01

    The prognosis of patients with metastatic breast cancer remains poor, and thus novel therapeutic approaches are needed. Capecitabine, which is commonly used for metastatic breast cancer in different settings, is an inactive prodrug that takes advantage of elevated levels of thymidine phosphorylase (TP), a key enzyme that is required for its conversion to 5-fluororacil, in tumors. We demonstrated that histone deacetylase inhibitors (HDACi), including low anticonvulsant dosage of VPA, induced the dose- and time-dependent up-regulation of TP transcript and protein expression in breast cancer cells, but not in the non-tumorigenic breast MCF-10A cell line. Through the use of siRNA or isoform-specific HDACi, we demonstrated that HDAC3 is the main isoform whose inhibition is involved in the modulation of TP. The combined treatment with capecitabine and HDACi, including valproic acid (VPA), resulted in synergistic/additive antiproliferative and pro-apoptotic effects in breast cancer cells but not in TP-knockout cells, both in vitro and in vivo, highlighting the crucial role of TP in the synergism observed. Overall, this study suggests that the combination of HDACi (e.g., VPA) and capecitabine is an innovative antitumor strategy that warrants further clinical evaluation for the treatment of metastatic breast cancer. PMID:26735339

  1. Glycogen metabolism in the rat retina.

    PubMed

    Coffe, Víctor; Carbajal, Raymundo C; Salceda, Rocío

    2004-02-01

    It has been reported that glycogen levels in retina vary with retinal vascularization. However, the electrical activity of isolated retina depends on glucose supply, suggesting that it does not contain energetic reserves. We determined glycogen levels and pyruvate and lactate production under various conditions in isolated retina. Ex vivo retinas from light- and dark-adapted rats showed values of 44 +/- 0.3 and 19.5 +/- 0.4 nmol glucosyl residues/mg protein, respectively. The glycogen content of retinas from light-adapted animals was reduced by 50% when they were transferred to darkness. Glycogen levels were low in retinas incubated in glucose-free media and increased in the presence of glucose. The highest glycogen values were found in media containing 20 mm of glucose. A rapid increase in lactate production was observed in the presence of glucose. Surprisingly, glycogen levels were the lowest and lactate production was also very low in the presence of 30 mm glucose. Our results suggest that glycogen can be used as an immediate accessible energy reserve in retina. We speculate on the possibility that gluconeogenesis may play a protective role by removal of lactic acid. PMID:14756809

  2. High liver glycogen in hereditary fructose intolerance

    PubMed Central

    Cain, A. R. R.; Ryman, Brenda E.

    1971-01-01

    A case of hereditary fructose intolerance is reported in a girl aged 2 years at the time of her death. She had apparently progressed normally until the age of 14 months. At 19 months she was admitted to hospital with failure to thrive, hepatomegaly, and superficial infections. Investigations revealed hypoglycaemia, persistent acidosis, aminoaciduria, and a high liver glycogen level which suggested that she had glycogen storage disease. There was also some evidence of malabsorption. At necropsy the liver enzyme estimations showed that fructose 1-phosphate aldolase activity was absent and that fructose 1,6-diphosphate aldolase activity was reduced. Hereditary fructose intolerance and glycogen storage disease have been confused in the past on clinical grounds, but a high liver glycogen level has not previously been reported in hereditary fructose intolerance. PMID:5289293

  3. Glycal Formation in Crystals of Uridine Phosphorylase

    SciTech Connect

    Paul, Debamita; O’Leary, Sen E.; Rajashankar, Kanagalaghatta; Bu, Weiming; Toms, Angela; Settembre, Ethan C.; Sanders, Jennie M.; Begley, Tadhg P.; Ealick, Steven E.

    2010-06-22

    Uridine phosphorylase is a key enzyme in the pyrimidine salvage pathway. This enzyme catalyzes the reversible phosphorolysis of uridine to uracil and ribose 1-phosphate (or 2{prime}-deoxyuridine to 2{prime}-deoxyribose 1-phosphate). Here we report the structure of hexameric Escherichia coli uridine phosphorylase treated with 5-fluorouridine and sulfate and dimeric bovine uridine phosphorylase treated with 5-fluoro-2{prime}-deoxyuridine or uridine, plus sulfate. In each case the electron density shows three separate species corresponding to the pyrimidine base, sulfate, and a ribosyl species, which can be modeled as a glycal. In the structures of the glycal complexes, the fluorouracil O2 atom is appropriately positioned to act as the base required for glycal formation via deprotonation at C2{prime}. Crystals of bovine uridine phosphorylase treated with 2{prime}-deoxyuridine and sulfate show intact nucleoside. NMR time course studies demonstrate that uridine phosphorylase can catalyze the hydrolysis of the fluorinated nucleosides in the absence of phosphate or sulfate, without the release of intermediates or enzyme inactivation. These results add a previously unencountered mechanistic motif to the body of information on glycal formation by enzymes catalyzing the cleavage of glycosyl bonds.

  4. Optimization of molecular design in the evolution of metabolism: the glycogen molecule.

    PubMed Central

    Meléndez-Hevia, E; Waddell, T G; Shelton, E D

    1993-01-01

    The animal glycogen molecule has to be designed in accordance with its metabolic function as a very effective fuel store allowing quick release of large amounts of glucose. In addition, the design should account for a high capacity of glucose storage in the least possible space. We have studied the optimization of these variables by means of a mathematical model of the glycogen molecule. Our results demonstrate that the structure is optimized to maximize (a) the total glucose stored in the smallest possible volume, (b) the proportion of it that can be directly released by phosphorylase before any debranching occurs, and (c) the number of non-reducing ends (points of attack for phosphorylase), which maximizes the speed of fuel release. The optimization of these four variables is achieved with appropriate values for two key parameters in glycogen design: the degree of branching and the length of the chains. The optimal values of these two parameters are precisely those found in cellular glycogen. PMID:8240246

  5. Study of the hydrolysis and ionization constants of Schiff base from pyridoxal 5'-phosphate and n-hexylamine in partially aqueous solvents. An application to phosphorylase b.

    PubMed Central

    Donoso, J; Muñoz, F; García Del Vado, A; Echevarría, G; García Blanco, F

    1986-01-01

    Formation and hydrolysis rate constants as well as equilibrium constants of the Schiff base derived from pyridoxal 5'-phosphate and n-hexylamine were determined between pH 3.5 and 7.5 in ethanol/water mixtures (3:17, v/v, and 49:1, v/v). The results indicate that solvent polarity scarcely alters the values of these constants but that they are dependent on the pH. Spectrophotometric titration of this Schiff base was also carried out. We found that a pKa value of 6.1, attributed in high-polarity media to protonation of the pyridine nitrogen atom, is independent of solvent polarity, whereas the pKa of the monoprotonated form of the imine falls from 12.5 in ethanol/water (3:17) to 11.3 in ethanol/water (49:1). Fitting of the experimental results for the hydrolysis to a theoretical model indicates the existence of a group with a pKa value of 6.1 that is crucial in the variation of kinetic constant of hydrolysis with pH. Studies of the reactivity of the coenzyme (pyridoxal 5'-phosphate) of glycogen phosphorylase b with hydroxylamine show that this reaction only occurs when the pH value of solution is below 6.5 and the hydrolysis of imine bond has started. We propose that the decrease in activity of phosphorylase b when the pH value is less than 6.2 must be caused by the cleavage of enzyme-coenzyme binding and that this may be related with protonation of the pyridine nitrogen atom of pyridoxal 5'-phosphate. PMID:3099764

  6. Characterization and crystal structure determination of β-1,2-mannobiose phosphorylase from Listeria innocua.

    PubMed

    Tsuda, Tomohiro; Nihira, Takanori; Chiku, Kazuhiro; Suzuki, Erika; Arakawa, Takatoshi; Nishimoto, Mamoru; Kitaoka, Motomitsu; Nakai, Hiroyuki; Fushinobu, Shinya

    2015-12-21

    Glycoside hydrolase family 130 consists of phosphorylases and hydrolases for β-mannosides. Here, we characterized β-1,2-mannobiose phosphorylase from Listeria innocua (Lin0857) and determined its crystal structures complexed with β-1,2-linked mannooligosaccharides. β-1,2-Mannotriose was bound in a U-shape, interacting with a phosphate analog at both ends. Lin0857 has a unique dimer structure connected by a loop, and a significant open-close loop displacement was observed for substrate entry. A long loop, which is exclusively present in Lin0857, covers the active site to limit the pocket size. A structural basis for substrate recognition and phosphorolysis was provided.

  7. Non-invasive quantification of brain glycogen absolute concentration

    PubMed Central

    van Heeswijk, Ruud B.; Xin, Lijing; Laus, Sabrina; Frenkel, Hanne; Lei, Hongxia; Gruetter, Rolf

    2009-01-01

    The only currently available method to measure brain glycogen in vivo is 13C NMR spectroscopy. Incorporation of 13C-labeled glucose (Glc) is necessary to allow glycogen measurement, but might be affected by turnover changes. Our aim was to measure glycogen absolute concentration in the rat brain by eliminating label turnover as variable. The approach is based on establishing an increased, constant 13C isotopic enrichment (IE). 13C-Glc infusion is then performed at the IE of brain glycogen. As glycogen IE cannot be assessed in vivo, we validated that it can be inferred from that of N-acetyl-aspartate IE in vivo: After [1-13C]-Glc ingestion, glycogen IE was 2.2 ± 0.1 fold that of N-acetyl-aspartate (n = 11, R2 = 0.77). After subsequent Glc infusion, glycogen IE equaled brain Glc IE (n = 6, paired t-test, p = 0.37), implying isotopic steady-state achievement and complete turnover of the glycogen molecule. Glycogen concentration measured in vivo by 13C NMR (mean ± SD: 5.8 ± 0.7 μmol/g) was in excellent agreement with that in vitro (6.4 ± 0.6 μmol/g, n = 5). When insulin was administered, the stability of glycogen concentration was analogous to previous biochemical measurements implying that glycogen turnover is activated by insulin. We conclude that the entire glycogen molecule is turned over and that insulin activates glycogen turnover. PMID:19013831

  8. Nrf2-Mediated Regulation of Skeletal Muscle Glycogen Metabolism.

    PubMed

    Uruno, Akira; Yagishita, Yoko; Katsuoka, Fumiki; Kitajima, Yasuo; Nunomiya, Aki; Nagatomi, Ryoichi; Pi, Jingbo; Biswal, Shyam S; Yamamoto, Masayuki

    2016-06-01

    Nrf2 (NF-E2-related factor 2) contributes to the maintenance of glucose homeostasis in vivo Nrf2 suppresses blood glucose levels by protecting pancreatic β cells from oxidative stress and improving peripheral tissue glucose utilization. To elucidate the molecular mechanisms by which Nrf2 contributes to the maintenance of glucose homeostasis, we generated skeletal muscle (SkM)-specific Keap1 knockout (Keap1MuKO) mice that express abundant Nrf2 in their SkM and then examined Nrf2 target gene expression in that tissue. In Keap1MuKO mice, blood glucose levels were significantly downregulated and the levels of the glycogen branching enzyme (Gbe1) and muscle-type PhKα subunit (Phka1) mRNAs, along with those of the glycogen branching enzyme (GBE) and the phosphorylase b kinase α subunit (PhKα) protein, were significantly upregulated in mouse SkM. Consistent with this result, chemical Nrf2 inducers promoted Gbe1 and Phka1 mRNA expression in both mouse SkM and C2C12 myotubes. Chromatin immunoprecipitation analysis demonstrated that Nrf2 binds the Gbe1 and Phka1 upstream promoter regions. In Keap1MuKO mice, muscle glycogen content was strongly reduced and forced GBE expression in C2C12 myotubes promoted glucose uptake. Therefore, our results demonstrate that Nrf2 induction in SkM increases GBE and PhKα expression and reduces muscle glycogen content, resulting in improved glucose tolerance. Our results also indicate that Nrf2 differentially regulates glycogen metabolism in SkM and the liver. PMID:27044864

  9. Novel benzothiazinones (BTOs) as allosteric modulator or substrate competitive inhibitor of glycogen synthase kinase 3β (GSK-3β) with cellular activity of promoting glucose uptake.

    PubMed

    Zhang, Peng; Li, Shufen; Gao, Yang; Lu, Wenbo; Huang, Ke; Ye, Deyong; Li, Xi; Chu, Yong

    2014-12-15

    Glycogen synthase kinase 3β (GSK-3β) plays a key role in insulin metabolizing pathway and therefore inhibition of the enzyme might provide an important therapeutic approach for treatment of insulin resistance and type 2 diabetes. Recently, discovery of ATP noncompetitive inhibitors is gaining importance not only due to their generally increased selectivity but also for the potentially subtle modulation of the target. These kinds of compounds include allosteric modulators and substrate competitive inhibitors. Here we reported two benzothiazinone compounds (BTO), named BTO-5h (IC50=8 μM) and BTO-5s (IC50=10 μM) as novel allosteric modulator and substrate competitive inhibitor of GSK-3β, respectively. Their different action modes were proved by kinetic experiments. Furthermore, BTO-5s was selected to check the kinases profile and showed little or even no activity to a panel of ten protein kinases at 100 μM, indicating it has good selectivity. Docking studies were performed to give suggesting binding modes which can well explain their impacts on the enzyme. Moreover, cell experiments displayed both compounds reduced the phosphorylation level of glycogen synthase in an intact cell, and greatly enhanced the glucose uptake in both HpG2 and 3T3-L1 cells. All of these results suggested BTO-5s and BTO-5h maybe have potentially therapeutic value for anti-diabetes. The results also offer a new scaffold for designing and developing selective inhibitors with novel mechanisms of action.

  10. Role of phosphorylase in the mechanism of potato minituber storage cell changes during clinorotation

    NASA Astrophysics Data System (ADS)

    Nedukha, O.; Shnyukova, E.

    The differences between the cytochemical reaction intensity and activity of phosphorylase (EC 2.4.1.1) and carbohydrate content in storage parenchyma cells of Solanum tuberosum L. (cv Adreta) minitubers grown for 30 days in the horizontal clinostate (2 rev/min) and in the control have been studied by electroncytochemical and biochemical methods. It is established an acceleration of minitubers formation and storage parenchyma cell differentiation at clinorotation. Electroncytochemical investigation of phosphorylase activity localization in the storage parenchyma cells of minitubers grown in control and at clinorotation showed the product of the reaction as electron-dense precipitate was marked plastids. Intensity and density of precipitate was increase in stroma of plastids and on starch grain surface during of intensive growth of starch in amyloplast (on 10- and 20-days of the minituber formation) of clinorotated minitubers in comparison with that in the control. The precipitate amount was decreased in the plastids on 30 day of growth in both variants. Using biochemical methods it is found that activity of phosphorylase and content of mono- and disaccharide and also starch content changed in minitubers formed during clinorotation and in the control. Data obtained are discussed regarding the possible mechanism of phosphorylase activity change and the role of mono- and disaccharide in acceleration of storage organ formation during clinorotation.

  11. Minimal hepatic glucose-6-phosphatase-α activity required to sustain survival and prevent hepatocellular adenoma formation in murine glycogen storage disease type Ia.

    PubMed

    Lee, Young Mok; Kim, Goo-Young; Pan, Chi-Jiunn; Mansfield, Brian C; Chou, Janice Y

    2015-06-01

    Glycogen storage disease type Ia (GSD-Ia), characterized by impaired glucose homeostasis and chronic risk of hepatocellular adenoma (HCA), is caused by a deficiency in glucose-6-phosphatase-α (G6Pase-α or G6PC) activity. In a previous 70-90 week-study, we showed that a recombinant adeno-associated virus (rAAV) vector-mediated gene transfer that restores more than 3% of wild-type hepatic G6Pase-α activity in G6pc (-/-) mice corrects hepatic G6Pase-α deficiency with no evidence of HCA. We now examine the minimal hepatic G6Pase-α activity required to confer therapeutic efficacy. We show that rAAV-treated G6pc (-/-) mice expressing 0.2% of wild-type hepatic G6Pase-α activity suffered from frequent hypoglycemic seizures at age 63-65 weeks but mice expressing 0.5-1.3% of wild-type hepatic G6Pase-α activity (AAV-LL mice) sustain 4-6 h of fast and grow normally to age 75-90 weeks. Despite marked increases in hepatic glycogen accumulation, the AAV-LL mice display no evidence of hepatic abnormalities, hepatic steatosis, or HCA. Interprandial glucose homeostasis is maintained by the G6Pase-α/glucose-6-phosphate transporter (G6PT) complex, and G6PT-mediated microsomal G6P uptake is the rate-limiting step in endogenous glucose production. We show that hepatic G6PT activity is increased in AAV-LL mice. These findings are encouraging for clinical studies of G6Pase-α gene-based therapy for GSD-Ia.

  12. Glycogen Synthase Kinase 3 Protein Kinase Activity Is Frequently Elevated in Human Non-Small Cell Lung Carcinoma and Supports Tumour Cell Proliferation

    PubMed Central

    O′Flaherty, Linda; Pardo, Olivier E.; Dzien, Piotr; Phillips, Lois; Morgan, Carys; Pawade, Joya; May, Margaret T.; Sohail, Muhammad; Hetzel, Martin R.; Seckl, Michael J.; Tavaré, Jeremy M.

    2014-01-01

    Background Glycogen synthase kinase 3 (GSK3) is a central regulator of cellular metabolism, development and growth. GSK3 activity was thought to oppose tumourigenesis, yet recent studies indicate that it may support tumour growth in some cancer types including in non-small cell lung carcinoma (NSCLC). We examined the undefined role of GSK3 protein kinase activity in tissue from human NSCLC. Methods The expression and protein kinase activity of GSK3 was determined in 29 fresh frozen samples of human NSCLC and patient-matched normal lung tissue by quantitative immunoassay and western blotting for the phosphorylation of three distinct GSK3 substrates in situ (glycogen synthase, RelA and CRMP-2). The proliferation and sensitivity to the small-molecule GSK3 inhibitor; CHIR99021, of NSCLC cell lines (Hcc193, H1975, PC9 and A549) and non-neoplastic type II pneumocytes was further assessed in adherent culture. Results Expression and protein kinase activity of GSK3 was elevated in 41% of human NSCLC samples when compared to patient-matched control tissue. Phosphorylation of GSK3α/β at the inhibitory S21/9 residue was a poor biomarker for activity in tumour samples. The GSK3 inhibitor, CHIR99021 dose-dependently reduced the proliferation of three NSCLC cell lines yet was ineffective against type II pneumocytes. Conclusion NSCLC tumours with elevated GSK3 protein kinase activity may have evolved dependence on the kinase for sustained growth. Our results provide further important rationale for exploring the use of GSK3 inhibitors in treating NSCLC. PMID:25486534

  13. Precursors to glycogen in ovine fetuses

    SciTech Connect

    Levitsky, L.L.; Paton, J.B.; Fisher, D.E. )

    1988-11-01

    Postprandial hepatic glycogenesis in the adult animal is now felt to proceed largely through gluconeogenic pathways rather than directly from glucose. The ovine fetus, like the mature sheep, lacks specific hepatic glucokinase. Therefore, the authors examined the role of lactate as a fetal glycogenic precursor in seven chronically catheterized 125-day sheep fetuses. Fetuses were infused with L-(U-{sup 14}C)lactate and D-(3-{sup 3}H)glucose, while maternal glucose was maintained at 50 mg/dl. Mean fetal hepatic glycogen specific activity ({mu}Ci/mg {times} 10{sup 3}) was 0.82 {plus minus} 0.08 for {sup 14}C and 2.6 {plus minus} 0.4 for {sup 3}H, whereas fetal renal glycogen specific activity was 0.46 {plus minus} 0.22 for {sup 14}C and 0.78 {plus minus} 0.16 for {sup 3}H. In contrast, ({sup 14}C)glucose specific activity was undetectable in blood and mean ({sup 3}H)glucose specific activity was 8.9 {plus minus} 1.3 {mu}Ci/mg {times} 10{sup 3}. The least detectable specific activity of ({sup 14}C)glucose did not differ significantly from the ({sup 14}C)glycogen enrichment in liver, whereas ({sup 3}H)glucose specific activity was significantly greater than ({sup 3}H)glycogen enrichment. The authors conclude that glycogenesis from glucose is partly through the indirect gluconeogenic route and that lactate may be a glycogenic precursor in the ovine fetus.

  14. Glycogen contains phosphodiester groups that can be introduced by UDPglucose: glycogen glucose 1-phosphotransferase.

    PubMed

    Lomako, J; Lomako, W M; Whelan, W J; Marchase, R B

    1993-08-30

    Rabbit-muscle glycogen contains covalently bound phosphorus, equivalent to 1 phosphate group per 208 glucose residues. This often disputed, minor component was previously thought to represent a phosphomonoester group at C-6 of a glucose residue. Here we show that more than half the phosphorus is present as a phosphodiester, the remainder being monoester. A novel enzyme activity has been found in muscle that can account for the presence of the phosphodiester in glycogen. This is a UDPglucose: glycogen glucose 1-phosphotransferase that positions glucose 1-phosphate on C-6 of glucose residues in glycogen, forming a diester. The phosphomonoester groups present may arise by removal of the glucose residue originally transferred as glucose 1-phosphate. PMID:8396041

  15. Isolation, crystallization and preliminary crystallographic analysis of Salmonella typhimurium uridine phosphorylase crystallized with 2,2′-anhydrouridine

    SciTech Connect

    Timofeev, Vladimir I.; Lashkov, Alexander A.; Gabdoulkhakov, Azat G.; Pavlyuk, Bogdan Ph.; Kachalova, Galina S.; Betzel, Christian

    2007-10-01

    S. typhimurium uridine phosphorylase has been isolated and crystallized in the presence of ligand. Uridine phosphorylase (UPh; EC 2.4.2.3) is a member of the pyrimidine nucleoside phosphorylase family of enzymes which catalyzes the phosphorolytic cleavage of the C—N glycoside bond of uridine, with the formation of ribose 1-phosphate and uracil. This enzyme has been shown to be important in the activation and catabolism of fluoropyrimidines. Modulation of its enzymatic activity may affect the therapeutic efficacy of chemotherapeutic agents. The structural investigation of the bacterial uridine phosphorylases, both unliganded and complexed with substrate/product analogues and inhibitors, may help in understanding the catalytic mechanism of the phosphorolytic cleavage of uridine. Salmonella typhimurium uridine phosphorylase has been crystallized with 2,2′-anhydrouridine. X-ray diffraction data were collected to 2.15 Å. Preliminary analysis of the diffraction data indicates that the crystal belongs to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 88.52, b = 123.98, c = 133.52 Å. The solvent content is 45.51%, assuming the presence of one hexamer molecule per asymmetric unit.

  16. The activity of carbohydrate-degrading enzymes in the development of brood and newly emerged workers and drones of the Carniolan honeybee, Apis mellifera carnica.

    PubMed

    Żółtowska, Krystyna; Lipiński, Zbigniew; Łopieńska-Biernat, Elżbieta; Farjan, Marek; Dmitryjuk, Małgorzata

    2012-01-01

    The activity of glycogen Phosphorylase and carbohydrate hydrolyzing enzymes α-amylase, glucoamylase, trehalase, and sucrase was studied in the development of the Carniolan honey bee, Apis mellifera carnica Pollman (Hymenoptera: Apidae), from newly hatched larva to freshly emerged imago of worker and drone. Phosphorolytic degradation of glycogen was significantly stronger than hydrolytic degradation in all developmental stages. Developmental profiles of hydrolase activity were similar in both sexes of brood; high activity was found in unsealed larvae, the lowest in prepupae followed by an increase in enzymatic activity. Especially intensive increases in activity occurred in the last stage of pupae and newly emerged imago. Besides α-amylase, the activities of other enzymes were higher in drone than in worker broods. Among drones, activity of glucoamylase was particularly high, ranging from around three times higher in the youngest larvae to 13 times higher in the oldest pupae. This confirms earlier suggestions about higher rates of metabolism in drone broods than in worker broods.

  17. Glycogen phosphorylation and Lafora disease.

    PubMed

    Roach, Peter J

    2015-12-01

    Covalent phosphorylation of glycogen, first described 35 years ago, was put on firm ground through the work of the Whelan laboratory in the 1990s. But glycogen phosphorylation lay fallow until interest was rekindled in the mid 2000s by the finding that it could be removed by a glycogen-binding phosphatase, laforin, and that mutations in laforin cause a fatal teenage-onset epilepsy, called Lafora disease. Glycogen phosphorylation is due to phosphomonoesters at C2, C3 and C6 of glucose residues. Phosphate is rare, ranging from 1:500 to 1:5000 phosphates/glucose depending on the glycogen source. The mechanisms of glycogen phosphorylation remain under investigation but one hypothesis to explain C2 and perhaps C3 phosphate is that it results from a rare side reaction of the normal synthetic enzyme glycogen synthase. Lafora disease is likely caused by over-accumulation of abnormal glycogen in insoluble deposits termed Lafora bodies in neurons. The abnormality in the glycogen correlates with elevated phosphorylation (at C2, C3 and C6), reduced branching, insolubility and an enhanced tendency to aggregate and become insoluble. Hyperphosphorylation of glycogen is emerging as an important feature of this deadly childhood disease.

  18. Constitutive glycogen synthase kinase-3α/β activity protects against chronic β-adrenergic remodelling of the heart

    PubMed Central

    Webb, Ian G.; Nishino, Yasuhiro; Clark, James E.; Murdoch, Colin; Walker, Simon J.; Makowski, Marcus R.; Botnar, Rene M.; Redwood, Simon R.; Shah, Ajay M.; Marber, Michael S.

    2010-01-01

    Aims Glycogen synthase kinase 3 (GSK-3) signalling is implicated in the growth of the heart during development and in response to stress. However, its precise role remains unclear. We set out to characterize developmental growth and response to chronic isoproterenol (ISO) stress in knockin (KI) mice lacking the critical N-terminal serines, 21 of GSK-3α and 9 of GSK-3β respectively, required for inactivation by upstream kinases. Methods and results Between 5 and 15 weeks, KI mice grew more rapidly, but normalized heart weight and contractile performance were similar to wild-type (WT) mice. Isolated hearts of both genotypes responded comparably to acute ISO infusion with increases in heart rate and contractility. In WT mice, chronic subcutaneous ISO infusion over 14 days resulted in cardiac hypertrophy, interstitial fibrosis, and impaired contractility, accompanied by foetal gene reactivation. These effects were all significantly attenuated in KI mice. Indeed, ISO-treated KI hearts demonstrated reversible physiological remodelling traits with increased stroke volume and a preserved contractile response to acute adrenergic stimulation. Furthermore, simultaneous pharmacological inhibition of GSK-3 in KI mice treated with chronic subcutaneous ISO recapitulated the adverse remodelling phenotype seen in WT hearts. Conclusion Expression of inactivation-resistant GSK-3α/β does not affect eutrophic myocardial growth but protects against pathological hypertrophy induced by chronic adrenergic stimulation, maintaining cardiac function and attenuating interstitial fibrosis. Accordingly, strategies to prevent phosphorylation of Ser-21/9, and consequent inactivation of GSK-3α/β, may enable a sustained cardiac response to chronic β-agonist stimulation while preventing pathological remodelling. PMID:20299330

  19. Kaposi's sarcoma-associated herpesvirus LANA protein downregulates nuclear glycogen synthase kinase 3 activity and consequently blocks differentiation.

    PubMed

    Liu, Jianyong; Martin, Heather; Shamay, Meir; Woodard, Crystal; Tang, Qi-Qun; Hayward, S Diane

    2007-05-01

    The Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen (LANA) protein interacts with glycogen synthase kinase 3 (GSK-3) and relocalizes GSK-3 in a manner that leads to stabilization of beta-catenin and upregulation of beta-catenin-responsive cell genes. The LANA-GSK-3 interaction was further examined to determine whether there were additional downstream consequences. In the present study, the nuclear GSK-3 bound to LANA in transfected cells and in BCBL1 primary effusion lymphoma cells was found to be enriched for the inactive serine 9-phosphorylated form of GSK-3. The mechanism of inactivation of nuclear GSK-3 involved LANA recruitment of the extracellular signal-regulated kinases 1 and 2 (ERK1/2) and the ribosomal S6 kinase 1 (RSK1). ERK1/2 and RSK1 coprecipitated with LANA, and LANA was a substrate for ERK1 in vitro. A model is proposed for the overall inactivation of nuclear GSK-3 that incorporates the previously described GSK-3 phosphorylation of LANA itself. Functional inactivation of nuclear GSK-3 was demonstrated by the ability of LANA to limit phosphorylation of the known GSK-3 substrates C/EBPbeta and C/EBPalpha. The effect of LANA-mediated ablation of C/EBP phosphorylation on differentiation was modeled in the well-characterized 3T3L1 adipogenesis system. LANA-expressing 3T3L1 cells were impaired in their ability to undergo differentiation and adipogenesis. C/EBPbeta induction followed the same time course as that seen in vector-transduced cells, but there was delayed and reduced induction of C/EBPbeta transcriptional targets in LANA-expressing cells. We conclude that LANA inactivates nuclear GSK-3 and modifies the function of proteins that are GSK-3 substrates. In the case of C/EBPs, this translates into LANA-mediated inhibition of differentiation. PMID:17314169

  20. Nicotinamide riboside and nicotinic acid riboside salvage in fungi and mammals. Quantitative basis for Urh1 and purine nucleoside phosphorylase function in NAD+ metabolism.

    PubMed

    Belenky, Peter; Christensen, Kathryn C; Gazzaniga, Francesca; Pletnev, Alexandre A; Brenner, Charles

    2009-01-01

    NAD+ is a co-enzyme for hydride transfer enzymes and an essential substrate of ADP-ribose transfer enzymes and sirtuins, the type III protein lysine deacetylases related to yeast Sir2. Supplementation of yeast cells with nicotinamide riboside extends replicative lifespan and increases Sir2-dependent gene silencing by virtue of increasing net NAD+ synthesis. Nicotinamide riboside elevates NAD+ levels via the nicotinamide riboside kinase pathway and by a pathway initiated by splitting the nucleoside into a nicotinamide base followed by nicotinamide salvage. Genetic evidence has established that uridine hydrolase, purine nucleoside phosphorylase, and methylthioadenosine phosphorylase are required for Nrk-independent utilization of nicotinamide riboside in yeast. Here we show that mammalian purine nucleoside phosphorylase but not methylthioadenosine phosphorylase is responsible for mammalian nicotinamide riboside kinase-independent nicotinamide riboside utilization. We demonstrate that so-called uridine hydrolase is 100-fold more active as a nicotinamide riboside hydrolase than as a uridine hydrolase and that uridine hydrolase and mammalian purine nucleoside phosphorylase cleave nicotinic acid riboside, whereas the yeast phosphorylase has little activity on nicotinic acid riboside. Finally, we show that yeast nicotinic acid riboside utilization largely depends on uridine hydrolase and nicotinamide riboside kinase and that nicotinic acid riboside bioavailability is increased by ester modification. PMID:19001417

  1. Further characterization of the polynucleotide phosphorylase of Micrococcus luteus

    PubMed Central

    Letendre, Carol H.; Singer, Maxine F.

    1975-01-01

    The purification of polynucleotide phosphorylase from Micrococcus luteus by chromatography on phosphocellulose columns is described. This procedure offers several advantages over previous procedures. Previously determined molecular weights for Form-I enzyme and Form-T enzyme derived from Form-I by limited tryptic hydrolysis were confirmed as 2.7 and 2.3 × 105, respectively. Form-I appears homogeneous in the ultracentrifuge, but multiple active protein species are separable by polyacrylamide gel electrophoresis. The multiple species are probably the result of proteolysis. On polyacrylamide gel electrophoresis under denaturing conditions, Form-T yielded a single size of subunit of 71,000 daltons, and Form-I yielded several bands of different molecular sizes. These results differ from earlier determinations. The amino acid compositions of Form-I and Form-T are reported. Form-I contains only between 8 and 10 cysteine residues per molecule and Form-T half that many. Images PMID:1121422

  2. Exercise training-induced adaptations associated with increases in skeletal muscle glycogen content.

    PubMed

    Manabe, Yasuko; Gollisch, Katja S C; Holton, Laura; Kim, Young-Bum; Brandauer, Josef; Fujii, Nobuharu L; Hirshman, Michael F; Goodyear, Laurie J

    2013-02-01

    Chronic exercise training results in numerous skeletal muscle adaptations, including increases in insulin sensitivity and glycogen content. To understand the mechanism leading to increased muscle glycogen, we studied the effects of exercise training on glycogen regulatory proteins in rat skeletal muscle. Female Sprague Dawley rats performed voluntary wheel running for 1, 4 or 7 weeks. After 7 weeks of training, insulin-stimulated glucose uptake was increased in epitrochlearis muscle. As compared with sedentary control rats, muscle glycogen did not change after 1 week of training, but increased significantly after 4 and 7 weeks. The increases in muscle glycogen were accompanied by elevated glycogen synthase activity and protein expression. To assess the regulation of glycogen synthase, we examined its major activator, protein phosphatase 1 (PP1), and its major deactivator, glycogen synthase kinase (GSK)-3. Consistent with glycogen synthase activity, PP1 activity was unchanged after 1 week of training but significantly increased after 4 and 7 weeks of training. Protein expression of R(GL)(G(M)), another regulatory PP1 subunit, significantly decreased after 4 and 7 weeks of training. Unlike PP1 activity, GSK-3 phosphorylation did not follow the pattern of glycogen synthase activity. The ~ 40% decrease in GSK-3α phosphorylation after 1 week of exercise training persisted until 7 weeks, and may function as a negative feedback mechanism in response to elevated glycogen. Our findings suggest that exercise training-induced increases in muscle glycogen content could be regulated by multiple mechanisms, including enhanced insulin sensitivity, glycogen synthase expression, allosteric activation of glycogen synthase, and PP1 activity.

  3. Dorsomedial hindbrain catecholamine regulation of hypothalamic astrocyte glycogen metabolic enzyme protein expression: Impact of estradiol.

    PubMed

    Tamrakar, P; Shrestha, P K; Briski, K P

    2015-04-30

    The brain astrocyte glycogen reservoir is a vital energy reserve and, in the cerebral cortex, subject among other factors to noradrenergic control. The ovarian steroid estradiol potently stimulates nerve cell aerobic respiration, but its role in glial glycogen metabolism during energy homeostasis or mismatched substrate supply/demand is unclear. This study examined the premise that estradiol regulates hypothalamic astrocyte glycogen metabolic enzyme protein expression during normo- and hypoglycemia in vivo through dorsomedial hindbrain catecholamine (CA)-dependent mechanisms. Individual astrocytes identified in situ by glial fibrillary acidic protein immunolabeling were laser-microdissected from the ventromedial hypothalamic (VMH), arcuate hypothalamic (ARH), and paraventricular hypothalamic (PVH) nuclei and the lateral hypothalamic area (LHA) of estradiol (E)- or oil (O)-implanted ovariectomized (OVX) rats after insulin or vehicle injection, and pooled within each site. Stimulation [VMH, LHA] or suppression [PVH, ARH] of basal glycogen synthase (GS) protein expression by E was reversed in the former three sites by caudal fourth ventricular pretreatment with the CA neurotoxin 6-hydroxydopamine (6-OHDA). E diminished glycogen phosphorylase (GP) protein profiles by CA-dependent [VMH, PVH] or -independent mechanisms [LHA]. Insulin-induced hypoglycemia (IIH) increased GS expression in the PVH in OVX+E, but reduced this protein in the PVH, ARH, and LHA in OVX+O. Moreover, IIH augmented GP expression in the VMH, LHA, and ARH in OVX+E and in the ARH in OVX+O, responses that normalized by 6-OHDA. Results demonstrate site-specific effects of E on astrocyte glycogen metabolic enzyme expression in the female rat hypothalamus, and identify locations where dorsomedial hindbrain CA input is required for such action. Evidence that E correspondingly increases and reduces basal GS and GP in the VMH and LHA, but augments the latter protein during IIH suggests that E regulates

  4. Insights into glycogen metabolism in Lactobacillus acidophilus: impact on carbohydrate metabolism, stress tolerance and gut retention.

    PubMed

    Goh, Yong Jun; Klaenhammer, Todd R

    2014-11-20

    In prokaryotic species equipped with glycogen metabolism machinery, the co-regulation of glycogen biosynthesis and degradation has been associated with the synthesis of energy storage compounds and various crucial physiological functions, including global cellular processes such as carbon and nitrogen metabolism, energy sensing and production, stress response and cell-cell communication. In addition, the glycogen metabolic pathway was proposed to serve as a carbon capacitor that regulates downstream carbon fluxes, and in some microorganisms the ability to synthesize intracellular glycogen has been implicated in host persistence. Among lactobacilli, complete glycogen metabolic pathway genes are present only in select species predominantly associated with mammalian hosts or natural environments. This observation highlights the potential involvement of glycogen biosynthesis in probiotic activities and persistence of intestinal lactobacilli in the human gastrointestinal tract. In this review, we summarize recent findings on (i) the presence and potential ecological distribution of glycogen metabolic pathways among lactobacilli, (ii) influence of carbon substrates and growth phases on glycogen metabolic gene expression and glycogen accumulation in L. acidophilus, and (iii) the involvement of glycogen metabolism on growth, sugar utilization and bile tolerance. Our present in vivo studies established the significance of glycogen biosynthesis on the competitive retention of L. acidophilus in the mouse intestinal tract, demonstrating for the first time that the ability to synthesize intracellular glycogen contributes to gut fitness and retention among probiotic microorganisms.

  5. Altered energy state reversibly controls smooth muscle contractile function in human saphenous vein during acute hypoxia-reoxygenation: Role of glycogen, AMP-activated protein kinase, and insulin-independent glucose uptake.

    PubMed

    Pyla, Rajkumar; Pichavaram, Prahalathan; Fairaq, Arwa; Park, Mary Anne; Kozak, Mark; Kamath, Vinayak; Patel, Vijay S; Segar, Lakshman

    2015-09-01

    Hypoxia is known to promote vasodilation of coronary vessels through several mediators including cardiac-derived adenosine and endothelium-derived prostanoids and nitric oxide. To date, the impact of endogenous glycogen depletion in vascular smooth muscle and the resultant alterations in cellular energy state (e.g., AMP-activated protein kinase, AMPK) on the contractile response to G protein-coupled receptor agonists (e.g., serotonin, 5-HT) has not yet been studied. In the present study, ex vivo exposure of endothelium-denuded human saphenous vein rings to hypoxic and glucose-deprived conditions during KCl-induced contractions for 30 min resulted in a marked depletion of endogenous glycogen by ∼80% (from ∼1.78 μmol/g under normoxia to ∼0.36 μmol/g under hypoxia). Importantly, glycogen-depleted HSV rings, which were maintained under hypoxia/reoxygenation and glucose-deprived conditions, exhibited significant increases in basal AMPK phosphorylation (∼6-fold ↑) and 5-HT-induced AMPK phosphorylation (∼19-fold ↑) with an accompanying suppression of 5-HT-induced maximal contractile response (∼68% ↓), compared with respective controls. Exposure of glycogen-depleted HSV rings to exogenous D-glucose, but not the inactive glucose analogs, prevented the exaggerated increase in 5-HT-induced AMPK phosphorylation and restored 5-HT-induced maximal contractile response. In addition, the ability of exogenous D-glucose to rescue cellular stress and impaired contractile function occurred through GLUT1-mediated but insulin/GLUT4-independent mechanisms. Together, the present findings from clinically-relevant human saphenous vein suggest that the loss of endogenous glycogen in vascular smooth muscle and the resultant accentuation of AMPK phosphorylation by GPCR agonists may constitute a yet another mechanism of metabolic vasodilation of coronary vessels in ischemic heart disease.

  6. Altered energy state reversibly controls smooth muscle contractile function in human saphenous vein during acute hypoxia-reoxygenation: Role of glycogen, AMP-activated protein kinase, and insulin-independent glucose uptake

    PubMed Central

    Pyla, Rajkumar; Pichavaram, Prahalathan; Fairaq, Arwa; Park, Mary Anne; Kozak, Mark; Kamath, Vinayak; Patel, Vijay S.; Segar, Lakshman

    2015-01-01

    Hypoxia is known to promote vasodilation of coronary vessels through several mediators including cardiac-derived adenosine and endothelium-derived prostanoids and nitric oxide. To date, the impact of endogenous glycogen depletion in vascular smooth muscle and the resultant alterations in cellular energy state (e.g., AMP-activated protein kinase, AMPK) on the contractile response to G protein-coupled receptor agonists (e.g., serotonin, 5-HT) has not yet been studied. In the present study, ex vivo exposure of endothelium-denuded human saphenous vein rings to hypoxic and glucose-deprived conditions during KCl-induced contractions for 30 min resulted in a marked depletion of endogenous glycogen by ~80% (from ~1.78 μmol/g under normoxia to ~0.36 μmol/g under hypoxia). Importantly, glycogen-depleted HSV rings, which were maintained under hypoxia/reoxygenation and glucose-deprived conditions, exhibited significant increases in basal AMPK phosphorylation (~6-fold ↑) and 5-HT-induced AMPK phosphorylation (~19-fold ↑) with an accompanying suppression of 5-HT-induced maximal contractile response (~68% ↓), compared with respective controls. Exposure of glycogen-depleted HSV rings to exogenous D-glucose, but not the inactive glucose analogs, prevented the exaggerated increase in 5-HT-induced AMPK phosphorylation and restored 5-HT-induced maximal contractile response. In addition, the ability of exogenous D-glucose to rescue cellular stress and impaired contractile function occurred through GLUT1-mediated but insulin/GLUT4-independent mechanisms. Together, the present findings from clinically-relevant human saphenous vein suggest that the loss of endogenous glycogen in vascular smooth muscle and the resultant accentuation of AMPK phosphorylation by GPCR agonists may constitute a yet another mechanism of metabolic vasodilation of coronary vessels in ischemic heart disease. PMID:26212549

  7. Impairment of Liver Glycogen Storage in the db/db Animal Model of Type 2 Diabetes: A Potential Target for Future Therapeutics?

    PubMed

    Sullivan, Mitchell A; Harcourt, Brooke E; Xu, Ping; Forbes, Josephine M; Gilbert, Robert G

    2015-01-01

    After the discovery of the db gene in 1966, it was determined that a blood-borne satiety factor was produced excessively, but was not responded to, in db/db mice. This model for type 2 diabetes is widely used since it phenocopies human disease and its co-morbidities including obesity, progressive deterioration in glucose tolerance, hypertension and hyperlipidaemia. Db/db mice, unlike their non-diabetic controls, have consistently elevated levels of liver glycogen, most likely due to hyperphagia. In transmission electron micrographs, liver glycogen usually shows a composite cauliflower-like morphology of large "α particles" (with a wide range of sizes) made up of smaller "β particles" bound together. New studies have explored the size distribution of liver glycogen molecules and found that α particles in db/db mice are more chemically fragile than those in healthy mice, and can readily break apart to smaller β particles. There is evidence that smaller glycogen particles have a higher association with glycogen phosphorylase, a key enzyme involved in glycogen degradation, as well as being degraded more rapidly in vitro; therefore the inability to form stable large glycogen α particles is predicted to result in a faster, less controlled degradation into glucose. The implications of this for glycaemic control remain to be fully elucidated. However, "rescuing" the more fragile diabetic glycogen to decrease hepatic glucose output in type 2 diabetes, may provide a potential therapeutic target which is the subject of this review.

  8. Nimbolide, a neem limonoid inhibits Phosphatidyl Inositol-3 Kinase to activate Glycogen Synthase Kinase-3β in a hamster model of oral oncogenesis

    PubMed Central

    Sophia, Josephraj; Kiran Kishore T., Kranthi; Kowshik, Jaganathan; Mishra, Rajakishore; Nagini, Siddavaram

    2016-01-01

    Glycogen synthase kinase-3β (GSK-3β), a serine/threonine kinase is frequently inactivated by the oncogenic signalling kinases PI3K/Akt and MAPK/ERK in diverse malignancies. The present study was designed to investigate GSK-3β signalling circuits in the 7,12-dimethylbenz[a]anthracene (DMBA)-induced hamster buccal pouch (HBP) carcinogenesis model and the therapeutic potential of the neem limonoid nimbolide. Inactivation of GSK-3β by phosphorylation at serine 9 and activation of PI3K/Akt, MAPK/ERK and β-catenin was associated with increased cell proliferation and apoptosis evasion during stepwise evolution of HBP carcinomas. Administration of nimbolide inhibited PI3K/Akt signalling with consequent activation of GSK-3β thereby inducing trafficking of β-catenin away from the nucleus and enhancing the expression of miR-126 and let-7. Molecular docking studies confirmed interaction of nimbolide with PI3K, Akt, ERK and GSK-3β. Furthermore, nimbolide attenuated cell proliferation and induced apoptosis as evidenced by increased p-cyclin D1Thr286 and pro-apoptotic proteins. The present study has unravelled aberrant phosphorylation as a key determinant for oncogenic signalling and acquisition of cancer hallmarks in the HBP model. The study has also provided mechanistic insights into the chemotherapeutic potential of nimbolide that may be a useful addition to the armamentarium of natural compounds targeting PI3K for oral cancer treatment. PMID:26902162

  9. Nimbolide, a neem limonoid inhibits Phosphatidyl Inositol-3 Kinase to activate Glycogen Synthase Kinase-3β in a hamster model of oral oncogenesis.

    PubMed

    Sophia, Josephraj; Kiran Kishore T, Kranthi; Kowshik, Jaganathan; Mishra, Rajakishore; Nagini, Siddavaram

    2016-02-23

    Glycogen synthase kinase-3β (GSK-3β), a serine/threonine kinase is frequently inactivated by the oncogenic signalling kinases PI3K/Akt and MAPK/ERK in diverse malignancies. The present study was designed to investigate GSK-3β signalling circuits in the 7,12-dimethylbenz[a]anthracene (DMBA)-induced hamster buccal pouch (HBP) carcinogenesis model and the therapeutic potential of the neem limonoid nimbolide. Inactivation of GSK-3β by phosphorylation at serine 9 and activation of PI3K/Akt, MAPK/ERK and β-catenin was associated with increased cell proliferation and apoptosis evasion during stepwise evolution of HBP carcinomas. Administration of nimbolide inhibited PI3K/Akt signalling with consequent activation of GSK-3β thereby inducing trafficking of β-catenin away from the nucleus and enhancing the expression of miR-126 and let-7. Molecular docking studies confirmed interaction of nimbolide with PI3K, Akt, ERK and GSK-3β. Furthermore, nimbolide attenuated cell proliferation and induced apoptosis as evidenced by increased p-cyclin D1(Thr286) and pro-apoptotic proteins. The present study has unravelled aberrant phosphorylation as a key determinant for oncogenic signalling and acquisition of cancer hallmarks in the HBP model. The study has also provided mechanistic insights into the chemotherapeutic potential of nimbolide that may be a useful addition to the armamentarium of natural compounds targeting PI3K for oral cancer treatment.

  10. Nimbolide, a neem limonoid inhibits Phosphatidyl Inositol-3 Kinase to activate Glycogen Synthase Kinase-3β in a hamster model of oral oncogenesis.

    PubMed

    Sophia, Josephraj; Kiran Kishore T, Kranthi; Kowshik, Jaganathan; Mishra, Rajakishore; Nagini, Siddavaram

    2016-01-01

    Glycogen synthase kinase-3β (GSK-3β), a serine/threonine kinase is frequently inactivated by the oncogenic signalling kinases PI3K/Akt and MAPK/ERK in diverse malignancies. The present study was designed to investigate GSK-3β signalling circuits in the 7,12-dimethylbenz[a]anthracene (DMBA)-induced hamster buccal pouch (HBP) carcinogenesis model and the therapeutic potential of the neem limonoid nimbolide. Inactivation of GSK-3β by phosphorylation at serine 9 and activation of PI3K/Akt, MAPK/ERK and β-catenin was associated with increased cell proliferation and apoptosis evasion during stepwise evolution of HBP carcinomas. Administration of nimbolide inhibited PI3K/Akt signalling with consequent activation of GSK-3β thereby inducing trafficking of β-catenin away from the nucleus and enhancing the expression of miR-126 and let-7. Molecular docking studies confirmed interaction of nimbolide with PI3K, Akt, ERK and GSK-3β. Furthermore, nimbolide attenuated cell proliferation and induced apoptosis as evidenced by increased p-cyclin D1(Thr286) and pro-apoptotic proteins. The present study has unravelled aberrant phosphorylation as a key determinant for oncogenic signalling and acquisition of cancer hallmarks in the HBP model. The study has also provided mechanistic insights into the chemotherapeutic potential of nimbolide that may be a useful addition to the armamentarium of natural compounds targeting PI3K for oral cancer treatment. PMID:26902162

  11. Decreased Activity in Neuropathic Pain Form and Gene Expression of Cyclin-Dependent Kinase5 and Glycogen Synthase Kinase-3 Beta in Soleus Muscle of Wistar Male Rats

    PubMed Central

    Rahmati, Masoud; Taherabadi, Seyed Jalal; Mehrabi, Mahmoud

    2015-01-01

    Background: The relationship between decreased activity/neuropathic pain and gene expression alterations in soleus muscle has remained elusive. Objectives: In this experimental study, we investigated the effects of decreased activity in neuropathic pain form on Cyclin-Dependent Kinase 5 (CDK5) and Glycogen Synthase Kinase-3 β (GSK-3β) gene expression in soleus muscle of rats. Materials and Methods: Twelve male Wistar rats were randomly divided into three groups: (1) tight ligation of the L5 spinal nerve (SNL: n = 4); (2) sham surgery (Sham: n = 4), and (3) control (C: n = 4). The threshold to produce a withdrawal response to a mechanical and thermal stimulus was measured using von Frey filaments and radiation heat apparatus, respectively. Following 4 weeks after surgery, the left soleus muscle was removed and mRNA levels were determined by real-time Polymerase Chain Reaction (PCR). Results: Compared to control animals, L5 ligated animals developed mechanical and heat hypersensitivity during total period of study. Soleus muscle weight as well as CDK5 mRNA levels (less than ~ 0.4 fold) was decreased and GSK-3β mRNA levels (up to ~ 7 folds) increased in L5 ligated animals. Conclusions: These results showed enhanced muscle atrophy processes following peripheral nerve damage and might provide a useful approach to study underlying muscle mechanisms associated with clinical neuropathic pain syndromes. PMID:26290750

  12. Quantitative Structure-Activity Relationship Analysis and a Combined Ligand-Based/Structure-Based Virtual Screening Study for Glycogen Synthase Kinase-3.

    PubMed

    Fu, Gang; Liu, Sheng; Nan, Xiaofei; Dale, Olivia R; Zhao, Zhendong; Chen, Yixin; Wilkins, Dawn E; Manly, Susan P; Cutler, Stephen J; Doerksen, Robert J

    2014-09-01

    Glycogen synthase kinase-3 (GSK-3) is a multifunctional serine/threonine protein kinase which regulates a wide range of cellular processes, involving various signalling pathways. GSK-3β has emerged as an important therapeutic target for diabetes and Alzheimer's disease. To identify structurally novel GSK-3β inhibitors, we performed virtual screening by implementing a combined ligand-based/structure-based approach, which included quantitative structure-activity relationship (QSAR) analysis and docking prediction. To integrate and analyze complex data sets from multiple experimental sources, we drafted and validated a hierarchical QSAR method, which adopts a two-level structure to take data heterogeneity into account. A collection of 728 GSK-3 inhibitors with diverse structural scaffolds was obtained from published papers that used different experimental assay protocols. Support vector machines and random forests were implemented with wrapper-based feature selection algorithms to construct predictive learning models. The best models for each single group of compounds were then used to build the final hierarchical QSAR model, with an overall R(2) of 0.752 for the 141 compounds in the test set. The compounds obtained from the virtual screening experiment were tested for GSK-3β inhibition. The bioassay results confirmed that 2 hit compounds are indeed GSK-3β inhibitors exhibiting sub-micromolar inhibitory activity, and therefore validated our combined ligand-based/structure-based approach as effective for virtual screening experiments. PMID:27486081

  13. Glycogen metabolism and the homeostatic regulation of sleep.

    PubMed

    Petit, Jean-Marie; Burlet-Godinot, Sophie; Magistretti, Pierre J; Allaman, Igor

    2015-02-01

    In 1995 Benington and Heller formulated an energy hypothesis of sleep centered on a key role of glycogen. It was postulated that a major function of sleep is to replenish glycogen stores in the brain that have been depleted during wakefulness which is associated to an increased energy demand. Astrocytic glycogen depletion participates to an increase of extracellular adenosine release which influences sleep homeostasis. Here, we will review some evidence obtained by studies addressing the question of a key role played by glycogen metabolism in sleep regulation as proposed by this hypothesis or by an alternative hypothesis named "glycogenetic" hypothesis as well as the importance of the confounding effect of glucocorticoïds. Even though actual collected data argue in favor of a role of sleep in brain energy balance-homeostasis, they do not support a critical and direct involvement of glycogen metabolism on sleep regulation. For instance, glycogen levels during the sleep-wake cycle are driven by different physiological signals and therefore appear more as a marker-integrator of brain energy status than a direct regulator of sleep homeostasis. In support of this we provide evidence that blockade of glycogen mobilization does not induce more sleep episodes during the active period while locomotor activity is reduced. These observations do not invalidate the energy hypothesis of sleep but indicate that underlying cellular mechanisms are more complex than postulated by Benington and Heller.

  14. Endoplasmic reticulum stress-activated glycogen synthase kinase 3β aggravates liver inflammation and hepatotoxicity in mice with acute liver failure.

    PubMed

    Ren, Feng; Zhou, Li; Zhang, Xiangying; Wen, Tao; Shi, Hongbo; Xie, Bangxiang; Li, Zhuo; Chen, Dexi; Wang, Zheling; Duan, Zhongping

    2015-01-01

    Endoplasmic reticulum stress (ER stress) has been increasingly recognized as an important mechanism in various liver diseases. However, its intrinsic physiological role in acute liver failure (ALF) remains largely undetermined. This study aimed to examine how ER stress orchestrates glycogen synthase kinase 3β (GSK3β) and inflammation to affect ALF. In a murine ALF model induced by D-galactosamine (D-GalN) and lipopolysaccharide (LPS), 4-phenylbutyric acid (4-PBA) is to be administered to relieve ER stress. The lethality rate, liver damage, cytokine expression, and the activity of GSK3β were evaluated. How to regulate LPS-induced inflammation and TNF-α-induced hepatocyte apoptosis by ER stress was investigated in vitro. In vivo, ER stress was triggered in the liver with the progression of mice ALF model. ER stress was essential for the development of ALF because ER stress inhibition by 4-PBA ameliorated the liver damage through decreasing liver inflammation and hepatocyte apoptosis. 4-PBA also decreased GSK3β activity in the livers of ALF mice. In vitro, ER stress induced by tunicamycin synergistically increased LPS-triggered pro-inflammatory cytokine induction and promoted the activation of nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) pathway in bone marrow-derived macrophages; moreover, tunicamycin also cooperated with TNF-α to increase hepatocyte apoptosis. ER stress promoted LPS-triggered inflammation depending on GSK3β activation because inhibition of GSK3β by SB216763, the specific inhibitor of GSK3β, resulted in downregulation of pro-inflammatory genes. ER stress contributes to liver inflammation and hepatotoxicity in ALF, particularly by regulating GSK3β, and is therefore a potential therapeutic target for ALF.

  15. Glycogen synthase kinase 3{beta} regulation of nuclear factor of activated T-cells isoform c1 in the vascular smooth muscle cell response to injury

    SciTech Connect

    Chow Winsion; Hou Guangpei; Bendeck, Michelle P.

    2008-10-01

    The migration and proliferation of vascular smooth muscle cells (vSMCs) are critical events in neointima formation during atherosclerosis and restenosis. The transcription factor nuclear factor of activated T-cells-isoform c1 (NFATc1) is regulated by atherogenic cytokines, and has been implicated in the migratory and proliferative responses of vSMCs through the regulation of gene expression. In T-cells, calcineurin de-phosphorylates NFATc1, leading to its nuclear import, while glycogen synthase kinase 3 {beta} (GSK3{beta}) phosphorylates NFATc1 and promotes its nuclear export. However, the relationship between NFATc1 and GSK3{beta} has not been studied during SMC migration and proliferation. We investigated this by scrape wounding vSMCs in vitro, and studying wound repair. NFATc1 protein was transiently increased, reaching a peak at 8 h after wounding. Cell fractionation and immunocytochemistry revealed that NFATc1 accumulation in the nucleus was maximal at 4 h after injury, and this was coincident with a significant 9 fold increase in transcriptional activity. Silencing NFATc1 expression with siRNA or inhibition of NFAT with cyclosporin A (CsA) attenuated wound closure by vSMCs. Phospho-GSK3{beta} (inactive) increased to a peak at 30 min after injury, preceding the nuclear accumulation of NFATc1. Overexpression of a constitutively active mutant of GSK3{beta} delayed the nuclear accumulation of NFATc1, caused a 50% decrease in NFAT transcriptional activity, and attenuated vSMC wound repair. We conclude that NFATc1 promotes the vSMC response to injury, and that inhibition of GSK3{beta} is required for the activation of NFAT during wound repair.

  16. Glycogen synthase kinase 3β dictates podocyte motility and focal adhesion turnover by modulating paxillin activity: implications for the protective effect of low-dose lithium in podocytopathy.

    PubMed

    Xu, Weiwei; Ge, Yan; Liu, Zhihong; Gong, Rujun

    2014-10-01

    Aberrant focal adhesion turnover is centrally involved in podocyte actin cytoskeleton disorganization and foot process effacement. The structural and dynamic integrity of focal adhesions is orchestrated by multiple cell signaling molecules, including glycogen synthase kinase 3β (GSK3β), a multitasking kinase lately identified as a mediator of kidney injury. However, the role of GSK3β in podocytopathy remains obscure. In doxorubicin (Adriamycin)-injured podocytes, lithium, a GSK3β inhibitor and neuroprotective mood stabilizer, obliterated the accelerated focal adhesion turnover, rectified podocyte hypermotility, and restored actin cytoskeleton integrity. Mechanistically, lithium counteracted the doxorubicin-elicited GSK3β overactivity and the hyperphosphorylation and overactivation of paxillin, a focal adhesion-associated adaptor protein. Moreover, forced expression of a dominant negative kinase dead mutant of GSK3β highly mimicked, whereas ectopic expression of a constitutively active GSK3β mutant abolished, the effect of lithium in doxorubicin-injured podocytes, suggesting that the effect of lithium is mediated, at least in part, through inhibition of GSK3β. Furthermore, paxillin interacted with GSK3β and served as its substrate. In mice with doxorubicin nephropathy, a single low dose of lithium ameliorated proteinuria and glomerulosclerosis. Consistently, lithium therapy abrogated GSK3β overactivity, blunted paxillin hyperphosphorylation, and reinstated actin cytoskeleton integrity in glomeruli associated with an early attenuation of podocyte foot process effacement. Thus, GSK3β-modulated focal adhesion dynamics might serve as a novel therapeutic target for podocytopathy.

  17. Maintained activity of glycogen synthase kinase-3{beta} despite of its phosphorylation at serine-9 in okadaic acid-induced neurodegenerative model

    SciTech Connect

    Lim, Yong-Whan; Yoon, Seung-Yong; Choi, Jung-Eun; Kim, Sang-Min; Lee, Hui-Sun; Choe, Han; Lee, Seung-Chul; Kim, Dong-Hou

    2010-04-30

    Glycogen synthase kinase-3{beta} (GSK3{beta}) is recognized as one of major kinases to phosphorylate tau in Alzheimer's disease (AD), thus lots of AD drug discoveries target GSK3{beta}. However, the inactive form of GSK3{beta} which is phosphorylated at serine-9 is increased in AD brains. This is also inconsistent with phosphorylation status of other GSK3{beta} substrates, such as {beta}-catenin and collapsin response mediator protein-2 (CRMP2) since their phosphorylation is all increased in AD brains. Thus, we addressed this paradoxical condition of AD in rat neurons treated with okadaic acid (OA) which inhibits protein phosphatase-2A (PP2A) and induces tau hyperphosphorylation and cell death. Interestingly, OA also induces phosphorylation of GSK3{beta} at serine-9 and other substrates including tau, {beta}-catenin and CRMP2 like in AD brains. In this context, we observed that GSK3{beta} inhibitors such as lithium chloride and 6-bromoindirubin-3'-monoxime (6-BIO) reversed those phosphorylation events and protected neurons. These data suggest that GSK3{beta} may still have its kinase activity despite increase of its phosphorylation at serine-9 in AD brains at least in PP2A-compromised conditions and that GSK3{beta} inhibitors could be a valuable drug candidate in AD.

  18. The Role of Skeletal Muscle Glycogen Breakdown for Regulation of Insulin Sensitivity by Exercise

    PubMed Central

    Jensen, Jørgen; Rustad, Per Inge; Kolnes, Anders Jensen; Lai, Yu-Chiang

    2011-01-01

    Glycogen is the storage form of carbohydrates in mammals. In humans the majority of glycogen is stored in skeletal muscles (∼500 g) and the liver (∼100 g). Food is supplied in larger meals, but the blood glucose concentration has to be kept within narrow limits to survive and stay healthy. Therefore, the body has to cope with periods of excess carbohydrates and periods without supplementation. Healthy persons remove blood glucose rapidly when glucose is in excess, but insulin-stimulated glucose disposal is reduced in insulin resistant and type 2 diabetic subjects. During a hyperinsulinemic euglycemic clamp, 70–90% of glucose disposal will be stored as muscle glycogen in healthy subjects. The glycogen stores in skeletal muscles are limited because an efficient feedback-mediated inhibition of glycogen synthase prevents accumulation. De novo lipid synthesis can contribute to glucose disposal when glycogen stores are filled. Exercise physiologists normally consider glycogen’s main function as energy substrate. Glycogen is the main energy substrate during exercise intensity above 70% of maximal oxygen uptake (Vo2max⁡) and fatigue develops when the glycogen stores are depleted in the active muscles. After exercise, the rate of glycogen synthesis is increased to replete glycogen stores, and blood glucose is the substrate. Indeed insulin-stimulated glucose uptake and glycogen synthesis is elevated after exercise, which, from an evolutional point of view, will favor glycogen repletion and preparation for new “fight or flight” events. In the modern society, the reduced glycogen stores in skeletal muscles after exercise allows carbohydrates to be stored as muscle glycogen and prevents that glucose is channeled to de novo lipid synthesis, which over time will causes ectopic fat accumulation and insulin resistance. The reduction of skeletal muscle glycogen after exercise allows a healthy storage of carbohydrates after meals and prevents development of type 2

  19. Impaired activation of skeletal muscle glycogen synthase in non-insulin-dependent diabetes mellitus is unrelated to the degree of obesity.

    PubMed

    Johnson, A B; Argyraki, M; Thow, J C; Jones, I R; Broughton, D; Miller, M; Taylor, R

    1991-03-01

    Twenty-five newly presenting, untreated, white, non-insulin-dependent diabetic (NIDDM) subjects were studied within 72 hours of diagnosis. They were allocated to three groups according to their body mass index [BMI] (lean BMI less than 25.0, n = 9; overweight BMI 25.0 to 30.0, n = 6; obese BMI greater than .30.0 kg/m2, n = 10). All three groups exhibited equivalent hyperglycemia. Eleven normal control subjects were also studied. The degree of activation of skeletal muscle glycogen synthase (GS) was used as an intracellular marker of insulin action, before and during a 240-minute insulin infusion (100 mU/kg/h). Fractional GS activity did not increase in the lean (change, -0.9 +/- 3.3%), the overweight (-1.9 +/- 2.7%), or the obese (+2.2 +/- 1.6%) NIDDM subjects during the insulin infusion and was markedly decreased compared with the control subjects (change, +14.6 +/- 2.4%, all P less than .001). Glucose requirement was also significantly decreased in all three NIDDM groups (103 +/- 23 v 81 +/- 14 v 53 +/- 14 mg/m2/min, respectively) compared with the control subjects (319 +/- 18 mg/m2/min, all P less than .001). There was a significant negative correlation with BMI (r = -.51, P less than .01), but the difference in glucose requirement between the lean and obese NIDDM groups was not significant. Muscle GS activity at the end of the euglycemic clamp correlated with glucose requirement (r = .53, P less than .001), and a similar correlation was observed between the insulin-induced change in muscle GS activity from basal and glucose requirement (r = .47, P less than .005).(ABSTRACT TRUNCATED AT 250 WORDS)

  20. Regulation of glycogen metabolism in yeast and bacteria

    PubMed Central

    Wilson, Wayne A.; Roach, Peter J.; Montero, Manuel; Baroja-Fernández, Edurne; Muñoz, Francisco José; Eydallin, Gustavo; Viale, Alejandro M.; Pozueta-Romero, Javier

    2010-01-01

    Microorganisms have the capacity to utilize a variety of nutrients and adapt to continuously changing environmental conditions. Many microorganisms, including yeast and bacteria, accumulate carbon and energy reserves to cope with starvation conditions temporarily present in the environment. Glycogen biosynthesis is a main strategy for such metabolic storage and a variety of sensing and signaling mechanisms have evolved in evolutionarily distant species to guarantee the production of this homopolysaccharide. At the most fundamental level, the processes of glycogen synthesis and degradation in yeast and bacteria share certain broad similarities. However, the regulation of these processes is sometimes quite distinct, indicating that they have evolved separately to respond optimally to the habitat conditions of each species. This review aims to highlight the mechanisms, both at the transcriptional and post-transcriptional levels, which regulate glycogen metabolism in yeast and bacteria, focusing on selected areas where the greatest increase in knowledge has occurred during the last few years. In the yeast system, we focus particularly on the various signaling pathways that control the activity of the enzymes of glycogen storage. We also discuss our recent understanding of the important role played by the vacuole in glycogen metabolism. In the case of bacterial glycogen, especial emphasis is given to aspects related with genetic regulation of glycogen metabolism and its connection with other biological processes. PMID:20412306

  1. Purification, crystallization, and preliminary X-ray diffraction study of purine nucleoside phosphorylase from E. coli

    SciTech Connect

    Abramchik, Yu. A. Timofeev, V. I. Zhukhlistova, N. E.; Muravieva, T. I.; Esipov, R. S.; Kuranova, I. P.

    2015-07-15

    Crystals of E. coli purine nucleoside phosphorylase were grown in microgravity by the capillary counter-diffusion method through a gel layer. The X-ray diffraction data set suitable for the determination of the three-dimensional structure at atomic resolution was collected from one crystal at the Spring-8 synchrotron facility to 0.99 Å resolution. The crystals belong to sp. gr. P2{sub 1} and have the following unit-cell parameters: a = 74.1 Å, b = 110.2 Å, c = 88.2 Å, α = γ = 90°, β = 111.08°. The crystal contains six subunits of the enzyme comprising a hexamer per asymmetric unit. The hexamer is the biological active form of E. coli. purine nucleoside phosphorylase.

  2. Crystal Structure of Full-length Mycobacterium tuberculosis H37Rv Glycogen Branching Enzyme; Insights of N-Terminal [beta]-Sandwich in Sustrate Specifity and Enzymatic Activity

    SciTech Connect

    Pal, Kuntal; Kumar, Shiva; Sharma, Shikha; Garg, Saurabh Kumar; Alam, Mohammad Suhail; Xu, H. Eric; Agrawal, Pushpa; Swaminathan, Kunchithapadam

    2010-07-13

    The open reading frame Rv1326c of Mycobacterium tuberculosis (Mtb) H37Rv encodes for an {alpha}-1,4-glucan branching enzyme (MtbGlgB, EC 2.4.1.18, Uniprot entry Q10625). This enzyme belongs to glycoside hydrolase (GH) family 13 and catalyzes the branching of a linear glucose chain during glycogenesis by cleaving a 1 {yields} 4 bond and making a new 1 {yields} 6 bond. Here, we show the crystal structure of full-length MtbGlgB (MtbGlgBWT) at 2.33-{angstrom} resolution. MtbGlgBWT contains four domains: N1 {beta}-sandwich, N2 {beta}-sandwich, a central ({beta}/{alpha}){sub 8} domain that houses the catalytic site, and a C-terminal {beta}-sandwich. We have assayed the amylase activity with amylose and starch as substrates and the glycogen branching activity using amylose as a substrate for MtbGlgBWT and the N1 domain-deleted (the first 108 residues deleted) Mtb{Delta}108GlgB protein. The N1 {beta}-sandwich, which is formed by the first 105 amino acids and superimposes well with the N2 {beta}-sandwich, is shown to have an influence in substrate binding in the amylase assay. Also, we have checked and shown that several GH13 family inhibitors are ineffective against MtbGlgBWT and Mtb{Delta}108GlgB. We propose a two-step reaction mechanism, for the amylase activity (1 {yields} 4 bond breakage) and isomerization (1 {yields} 6 bond formation), which occurs in the same catalytic pocket. The structural and functional properties of MtbGlgB and Mtb{Delta}108GlgB are compared with those of the N-terminal 112-amino acid-deleted Escherichia coli GlgB (EC{Delta}112GlgB).

  3. Muscle glycogen stores and fatigue.

    PubMed

    Ørtenblad, Niels; Westerblad, Håkan; Nielsen, Joachim

    2013-09-15

      Studies performed at the beginning of the last century revealed the importance of carbohydrate as a fuel during exercise, and the importance of muscle glycogen on performance has subsequently been confirmed in numerous studies. However, the link between glycogen depletion and impaired muscle function during fatigue is not well understood and a direct cause-and-effect relationship between glycogen and muscle function remains to be established. The use of electron microscopy has revealed that glycogen is not homogeneously distributed in skeletal muscle fibres, but rather localized in distinct pools. Furthermore, each glycogen granule has its own metabolic machinery with glycolytic enzymes and regulating proteins. One pool of such glycogenolytic complexes is localized within the myofibrils in close contact with key proteins involved in the excitation-contraction coupling and Ca2+ release from the sarcoplasmic reticulum (SR). We and others have provided experimental evidence in favour of a direct role of decreased glycogen, localized within the myofibrils, for the reduction in SR Ca2+ release during fatigue. This is consistent with compartmentalized energy turnover and distinctly localized glycogen pools being of key importance for SR Ca2+ release and thereby affecting muscle contractility and fatigability.

  4. Muscle glycogen stores and fatigue

    PubMed Central

    Ørtenblad, Niels; Westerblad, Håkan; Nielsen, Joachim

    2013-01-01

    Studies performed at the beginning of the last century revealed the importance of carbohydrate as a fuel during exercise, and the importance of muscle glycogen on performance has subsequently been confirmed in numerous studies. However, the link between glycogen depletion and impaired muscle function during fatigue is not well understood and a direct cause-and-effect relationship between glycogen and muscle function remains to be established. The use of electron microscopy has revealed that glycogen is not homogeneously distributed in skeletal muscle fibres, but rather localized in distinct pools. Furthermore, each glycogen granule has its own metabolic machinery with glycolytic enzymes and regulating proteins. One pool of such glycogenolytic complexes is localized within the myofibrils in close contact with key proteins involved in the excitation–contraction coupling and Ca2+ release from the sarcoplasmic reticulum (SR). We and others have provided experimental evidence in favour of a direct role of decreased glycogen, localized within the myofibrils, for the reduction in SR Ca2+ release during fatigue. This is consistent with compartmentalized energy turnover and distinctly localized glycogen pools being of key importance for SR Ca2+ release and thereby affecting muscle contractility and fatigability. PMID:23652590

  5. Structure analysis of archaeal AMP phosphorylase reveals two unique modes of dimerization.

    PubMed

    Nishitani, Yuichi; Aono, Riku; Nakamura, Akira; Sato, Takaaki; Atomi, Haruyuki; Imanaka, Tadayuki; Miki, Kunio

    2013-08-01

    AMP phosphorylase (AMPpase) catalyzes the initial reaction in a novel AMP metabolic pathway recently found in archaea, converting AMP and phosphate into adenine and ribose 1,5-bisphosphate. Gel-filtration chromatography revealed that AMPpase from Thermococcus kodakarensis (Tk-AMPpase) forms an exceptionally large macromolecular structure (>40-mers) in solution. To investigate its unique multimerization feature, we determined the first crystal structures of Tk-AMPpase, in the apo-form and in complex with substrates. Structures of two truncated forms of Tk-AMPpase (Tk-AMPpaseΔN84 and Tk-AMPpaseΔC10) clarified that this multimerization is achieved by two dimer interfaces within a single molecule: one by the central domain and the other by the C-terminal domain, which consists of an unexpected domain-swapping interaction. The N-terminal domain, characteristic of archaeal enzymes, is essential for enzymatic activity, participating in multimerization as well as domain closure of the active site upon substrate binding. Moreover, biochemical analysis demonstrated that the macromolecular assembly of Tk-AMPpase contributes to its high thermostability, essential for an enzyme from a hyperthermophile. Our findings unveil a unique archaeal nucleotide phosphorylase that is distinct in both function and structure from previously known members of the nucleoside phosphorylase II family.

  6. Glucose uptake and glycogen synthesis in muscles from immobilized limbs

    NASA Technical Reports Server (NTRS)

    Nicholson, W. F.; Watson, P. A.; Booth, F. W.

    1984-01-01

    Defects in glucose metabolism in muscles of immobilized limbs of mice were related to alterations in insulin binding, insulin responsiveness, glucose supply, and insulin activation of glycogen synthase. These were tested by in vitro methodology. A significant lessening in the insulin-induced maximal response of 2-deoxyglucose uptake into the mouse soleus muscle occurred between the 3rd and 8th h of limb immobilization, suggesting a decreased insulin responsiveness. Lack of change in the specific binding of insulin to muscles of 24-h immobilized limbs indicates that a change in insulin receptor number did not play a role in the failure of insulin to stimulate glucose metabolism. Its inability to stimulate glycogen synthesis in muscle from immobilized limbs is due, in part, to a lack of glucose supply to glycogen synthesis and also to the ineffectiveness of insulin to increase the percentage of glycogen synthase in its active form in muscles from 24-h immobilized limbs.

  7. Immobilized phosphorylase for synthesis of polysaccharides from glucose

    NASA Technical Reports Server (NTRS)

    Marshall, D. L.

    1972-01-01

    Continuous processes for enzymatic production of carbohydrates from glucose are discussed. Key reactant in process is identified as phosphorylase which catalyzes reversible formation or degradation of polysaccharide. Chemical compounds and reactions to synthesize polysaccharides are analyzed.

  8. Effects of intensive dietary treatment on insulin-stimulated skeletal muscle glycogen synthase activation and insulin secretion in newly presenting type 2 diabetic patients.

    PubMed

    Johnson, A B; Argyraki, M; Thow, J C; Broughton, D; Jones, I R; Taylor, R

    1990-06-01

    Ten newly presenting, untreated, Europid Type 2 diabetic patients were studied before and after 8 weeks treatment with intensive diet alone. Nine normal control subjects were also studied. The degree of activation of skeletal muscle glycogen synthase (GS) was used as an intracellular marker of insulin action, prior to and during a 240-min insulin infusion (100 mU kg-1 h-1). Fasting blood glucose decreased from 12.1 +/- 0.9 (+/- SE) to 9.2 +/- 0.8 mmol l-1 (p less than 0.01), but there was no change in fasting insulin concentrations, 9.9 +/- 2.3 vs 9.3 +/- 2.1 mU l-1. Fractional GS activity did not increase in the Type 2 diabetic patients during the insulin infusion either at presentation (change -1.5 +/- 1.9%) or after treatment (change +0.9 +/- 1.8%), and was markedly decreased compared with the control subjects (change +14.5 +/- 2.8%, both p less than 0.001). Glucose requirement during the clamp was decreased in the Type 2 diabetic patients at presentation (2.2 +/- 0.7 vs 7.3 +/- 0.6 mg kg-1 min-1, p less than 0.001), and despite improvement following dietary treatment to 3.3 +/- 0.6 mg kg-1 min-1 (p less than 0.01) remained lower than in the control subjects (p less than 0.001). Fasting plasma non-esterified fatty acid (NEFA) concentrations were elevated at presentation (p less than 0.05), and failed to suppress normally during the insulin infusion. After treatment fasting NEFA concentrations decreased (p less than 0.05) and suppressed normally (p less than 0.05). Insulin secretion was assessed following an intravenous bolus of glucose (0.5 g kg-1) at euglycaemia before and after treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

  9. The impact of metformin therapy on hepatic glucose production and skeletal muscle glycogen synthase activity in overweight type II diabetic patients.

    PubMed

    Johnson, A B; Webster, J M; Sum, C F; Heseltine, L; Argyraki, M; Cooper, B G; Taylor, R

    1993-09-01

    The effect of metformin therapy on glucose metabolism was examined in eight overweight newly presenting untreated type II diabetic patients (five males, three females). Patients were treated for 12 weeks with either metformin (850 mg x 3) or matching placebo using a double-blind crossover study design; patients were studied at presentation and at the end of each treatment period. Insulin action was assessed by measuring activation of skeletal muscle glycogen synthase (GS) before and during a 4-hour hyperinsulinemic euglycemic clamp (100 mU.kg-1 x h-1). Metformin therapy was associated with a significant decrease in fasting blood glucose (6.8 +/- 0.6 v 8.3 +/- 0.9 mmol.L-1, P < .01) and glycosylated hemoglobin ([HbA1] 7.7% +/- 0.4% v 8.5% +/- 0.5%, P < .01) levels. Fasting hepatic glucose production (HGP) was also significantly decreased following metformin therapy (1.98 +/- 0.13 v 2.41 +/- 0.20 mg.kg-1 x min-1, P < .02), whereas fasting insulin and C-peptide concentrations remained unaltered. The decrease in basal HGP correlated closely with the decrease in fasting blood glucose concentration (r = .92, P < .001). Insulin-stimulated glucose uptake was assessed using the hyperinsulinemic euglycemic clamp technique and was increased post-metformin (3.8 +/- 0.6 v 3.1 +/- 0.7 mg.kg-1 x min-1, P < .05). This was primarily the result of increased nonoxidative glucose metabolism (1.1 +/- 0.6 v 0.4 +/- 0.6 mg.kg-1 x min-1, P < .05); oxidative glucose metabolism did not change. Metformin had no measurable effect on insulin activation of skeletal muscle GS, the rate-limiting enzyme controlling muscle glucose storage.(ABSTRACT TRUNCATED AT 250 WORDS)

  10. Requirement for active glycogen synthase kinase-3β in TGF-β1 upregulation of connective tissue growth factor (CCN2/CTGF) levels in human gingival fibroblasts

    PubMed Central

    Bahammam, Maha; Black, Samuel A.; Sume, Siddika Selva; Assaggaf, Mohammad A.; Faibish, Michael

    2013-01-01

    Connective tissue growth factor (CCN2/CTGF) mediates transforming growth factor-β (TGF-β)-induced fibrosis. Drug-induced gingival overgrowth is tissue specific. Here the role of the phosphoinositol 3-kinase (PI3K) pathway in mediating TGF-β1-stimulated CCN2/CTGF expression in primary human adult gingival fibroblasts and human adult lung fibroblasts was compared. Data indicate that PI3K inhibitors attenuate upregulation of TGF-β1-induced CCN2/CTGF expression in human gingival fibroblasts independent of reducing JNK MAP kinase activation. Pharmacologic inhibitors and small interfering (si)RNA-mediated knockdown studies indicate that calcium-dependent isoforms and an atypical isoform of protein kinase C (PKC-δ) do not mediate TGF-β1-stimulated CCN2/CTGF expression in gingival fibroblasts. As glycogen synthase kinase-3β (GSK-3β) can undergo phosphorylation by the PI3K/pathway, the effects of GSK-3β inhibitor kenpaullone and siRNA knockdown were investigated. Data in gingival fibroblasts indicate that kenpaullone attenuates TGF-β1-mediated CCN2/CTGF expression. Activation of the Wnt canonical pathways with Wnt3a, which inhibits GSK-3β, similarly inhibits TGF-β1-stimulated CCN2/CTGF expression. In contrast, inhibition of GSK-3β by Wnt3a does not inhibit, but modestly stimulates, CCN2/CTGF levels in primary human adult lung fibroblasts and is β-catenin dependent, consistent with previous studies performed in other cell models. These data identify a novel pathway in gingival fibroblasts in which inhibition of GSK-3β attenuates CCN2/CTGF expression. In adult lung fibroblasts inhibition of GSK-3β modestly stimulates TGF-β1-regulated CCN2/CTGF expression. These studies have potential clinical relevance to the tissue specificity of drug-induced gingival overgrowth. PMID:23824844

  11. Glycogen synthase kinase 3 β activity is required for hBora/Aurora A-mediated mitotic entry.

    PubMed

    Lee, Yu-Cheng; Liao, Po-Chi; Liou, Yih-Cherng; Hsiao, Michael; Huang, Chi-Ying; Lu, Pei-Jung

    2013-03-15

    The synthesis and degradation of hBora is important for the regulation of mitotic entry and exist. In G 2 phase, hBora can complex with Aurora A to activate Plk1 and control mitotic entry. However, whether the post-translational modification of hBora is relevant to the mitotic entry still unclear. Here, we used the LC-MS/MS phosphopeptide mapping assay to identify 13 in vivo hBora phosphorylation sites and characterized that GSK3β can interact with hBora and phosphorylate hBora at Ser274 and Ser278. Pharmacological inhibitors of GSK3β reduced the retarded migrating band of hBora in cells and diminished the phosphorylation of hBora by in vitro kinase assay. Moreover, as well as in GSK3β activity-inhibited cells, specific knockdown of GSK3β by shRNA and S274A/S278 hBora mutant-expressing cells also exhibited the reduced Plk1 activation and a delay in mitotic entry. It suggests that GSK3β activity is required for hBora-mediated mitotic entry through Ser274 and Ser278 phosphorylation.

  12. Crystallization and X-ray diffraction studies of cellobiose phosphorylase from Cellulomonas uda.

    PubMed

    Van Hoorebeke, Annelies; Stout, Jan; Kyndt, John; De Groeve, Manu; Dix, Ina; Desmet, Tom; Soetaert, Wim; Van Beeumen, Jozef; Savvides, Savvas N

    2010-03-01

    Disaccharide phosphorylases are able to catalyze both the synthesis and the breakdown of disaccharides and have thus emerged as attractive platforms for tailor-made sugar synthesis. Cellobiose phosphorylase from Cellulomonas uda (CPCuda) is an enzyme that belongs to glycoside hydrolase family 94 and catalyzes the reversible breakdown of cellobiose [beta-D-glucopyranosyl-(1,4)-D-glucopyranose] to alpha-D-glucose-1-phosphate and D-glucose. Crystals of ligand-free recombinant CPCuda and of its complexes with substrates and reaction products yielded complete X-ray diffraction data sets to high resolution using synchrotron radiation but suffered from significant variability in diffraction quality. In at least one case an intriguing space-group transition from a primitive monoclinic to a primitive orthorhombic lattice was observed during data collection. The structure of CPCuda was determined by maximum-likelihood molecular replacement, thus establishing a starting point for an investigation of the structural and mechanistic determinants of disaccharide phosphorylase activity.

  13. [Properties of sucrose phosphorylase from recombinant Escherichia coli and enzymatic synthesis of alpha-arbutin].

    PubMed

    Wan, Yuejia; Ma, Jiangfeng; Xu, Rong; He, Aiyong; Jiang, Min; Chen, Kequan; Jiang, Yin

    2012-12-01

    Sucrose phosphorylase (EC 2.4.1.7, Sucrose phosphorylase, SPase) can be produced by recombinant strain Escherichia coli Rosetta(DE3)/Pet-SPase. Crude enzyme was obtained from the cells by the high pressure disruption and centrifugation. Sucrose phosphorylase was purified by Ni-NTA affinity column chromatography and desalted by ultrafiltration. The specific enzyme activity was 1.1-fold higher than that of the crude enzyme, and recovery rate was 82.7%. The purified recombinant SPase had a band of 59 kDa on SDS-PAGE. Thermostability of the enzyme was shown at temperatures up to 37 degrees C, and pH stability between pH 6.0 and 6.7. The optimum temperature and pH were 37 degrees C and 6.7, respectively. The K(m) of SPase for sucrose was 7.3 mmol/L, and Vmax was 0.2 micromol/(min x mg). Besides, alpha-arbutin was synthesized from sucrose and hydroquinone by transglucosylation with recombinant SPase. The optimal conditions for synthesis of alpha-arbutin were 200 U/mL of recombinant SPase, 20% of sucrose, and 1.6% hydroquinone at pH 6-6.5 and 25 degrees C for 21 h. Under these conditions, alpha-arbutin was obtained with a 78.3% molar yield with respect to hydroquinone, and the concentration of alpha-arbutin was about 31 g/L.

  14. Structure determination of glycogen synthase kinase-3 from Leishmania major and comparative inhibitor structure-activity relationships with Trypanosoma brucei GSK-3

    SciTech Connect

    Ojo, Kayode K; Arakaki, Tracy L; Napuli, Alberto J; Inampudi, Krishna K; Keyloun, Katelyn R; Zhang, Li; Hol, Wim G.J.; Verlind, Christophe L.M.J.; Merritt, Ethan A; Van Voorhis, Wesley C

    2012-04-24

    Glycogen synthase kinase-3 (GSK-3) is a drug target under intense investigation in pharmaceutical companies and constitutes an attractive piggyback target for eukaryotic pathogens. Two different GSKs are found in trypanosomatids, one about 150 residues shorter than the other. GSK-3 short (GeneDB: Tb927.10.13780) has previously been validated genetically as a drug target in Trypanosoma brucei by RNAi induced growth retardation; and chemically by correlation between enzyme and in vitro growth inhibition. Here, we report investigation of the equivalent GSK-3 short enzymes of L. major (LmjF18.0270) and L. infantum (LinJ18_V3.0270, identical in amino acid sequences to LdonGSK-3 short) and a crystal structure of LmajGSK-3 short at 2 Å resolution. The inhibitor structure-activity relationships (SARs) of L. major and L. infantum are virtually identical, suggesting that inhibitors could be useful for both cutaneous and visceral leishmaniasis. Leishmania spp. GSK-3 short has different inhibitor SARs than TbruGSK-3 short, which can be explained mostly by two variant residues in the ATP-binding pocket. Indeed, mutating these residues in the ATP-binding site of LmajGSK-3 short to the TbruGSK-3 short equivalents results in a mutant LmajGSK-3 short enzyme with SAR more similar to that of TbruGSK-3 short. The differences between human GSK-3β (HsGSK-3β) and LmajGSK-3 short SAR suggest that compounds which selectively inhibit LmajGSK-3 short may be found.

  15. Activation of the Saccharomyces cerevisiae filamentation/invasion pathway by osmotic stress in high-osmolarity glycogen pathway mutants.

    PubMed

    Davenport, K D; Williams, K E; Ullmann, B D; Gustin, M C

    1999-11-01

    Mitogen-activated protein kinase (MAPK) cascades are frequently used signal transduction mechanisms in eukaryotes. Of the five MAPK cascades in Saccharomyces cerevisiae, the high-osmolarity glycerol response (HOG) pathway functions to sense and respond to hypertonic stress. We utilized a partial loss-of-function mutant in the HOG pathway, pbs2-3, in a high-copy suppressor screen to identify proteins that modulate growth on high-osmolarity media. Three high-copy suppressors of pbs2-3 osmosensitivity were identified: MSG5, CAK1, and TRX1. Msg5p is a dual-specificity phosphatase that was previously demonstrated to dephosphorylate MAPKs in yeast. Deletions of the putative MAPK targets of Msg5p revealed that kss1delta could suppress the osmosensitivity of pbs2-3. Kss1p is phosphorylated in response to hyperosmotic shock in a pbs2-3 strain, but not in a wild-type strain nor in a pbs2-3 strain overexpressing MSG5. Both TEC1 and FRE::lacZ expressions are activated in strains lacking a functional HOG pathway during osmotic stress in a filamentation/invasion-pathway-dependent manner. Additionally, the cellular projections formed by a pbs2-3 mutant on high osmolarity are absent in strains lacking KSS1 or STE7. These data suggest that the loss of filamentation/invasion pathway repression contributes to the HOG mutant phenotype.

  16. Activation of the Saccharomyces cerevisiae filamentation/invasion pathway by osmotic stress in high-osmolarity glycogen pathway mutants

    NASA Technical Reports Server (NTRS)

    Davenport, K. D.; Williams, K. E.; Ullmann, B. D.; Gustin, M. C.; McIntire, L. V. (Principal Investigator)

    1999-01-01

    Mitogen-activated protein kinase (MAPK) cascades are frequently used signal transduction mechanisms in eukaryotes. Of the five MAPK cascades in Saccharomyces cerevisiae, the high-osmolarity glycerol response (HOG) pathway functions to sense and respond to hypertonic stress. We utilized a partial loss-of-function mutant in the HOG pathway, pbs2-3, in a high-copy suppressor screen to identify proteins that modulate growth on high-osmolarity media. Three high-copy suppressors of pbs2-3 osmosensitivity were identified: MSG5, CAK1, and TRX1. Msg5p is a dual-specificity phosphatase that was previously demonstrated to dephosphorylate MAPKs in yeast. Deletions of the putative MAPK targets of Msg5p revealed that kss1delta could suppress the osmosensitivity of pbs2-3. Kss1p is phosphorylated in response to hyperosmotic shock in a pbs2-3 strain, but not in a wild-type strain nor in a pbs2-3 strain overexpressing MSG5. Both TEC1 and FRE::lacZ expressions are activated in strains lacking a functional HOG pathway during osmotic stress in a filamentation/invasion-pathway-dependent manner. Additionally, the cellular projections formed by a pbs2-3 mutant on high osmolarity are absent in strains lacking KSS1 or STE7. These data suggest that the loss of filamentation/invasion pathway repression contributes to the HOG mutant phenotype.

  17. Physiological aggregation of maltodextrin phosphorylase from Pyrococcus furiosus and its application in a process of batch starch degradation to alpha-D-glucose-1-phosphate.

    PubMed

    Nahálka, Jozef

    2008-04-01

    Maltodextrin phosphorylase from Pyrococcus furiosus (PF1535) was fused with the cellulose-binding domain of Clostridium cellulovorans serving as an aggregation module. After molecular cloning of the corresponding gene fusion construct and controlled expression in Escherichia coli BL21, 83% of total maltodextrin phosphorylase activity (0.24 U/mg of dry cell weight) was displayed in active inclusion bodies. These active inclusion bodies were easily isolated by nonionic detergent treatment and directly used for maltodextrin conversion to alpha-D-glucose-1-phosphate in a repetitive batch mode. Only 10% of enzyme activity was lost after ten conversion cycles at optimum conditions.

  18. The effect of sulphonylurea therapy on skeletal muscle glycogen synthase activity and insulin secretion in newly presenting type 2 (non-insulin-dependent) diabetic patients.

    PubMed

    Johnson, A B; Argyraki, M; Thow, J C; Jones, I R; Broughton, D; Miller, M; Taylor, R

    1991-04-01

    Ten newly presenting, Type 2 (non-insulin-dependent), Caucasian diabetic patients were studied before and after 8 weeks treatment with the sulphonylurea gliclazide, and in parallel 13 similar patients were studied before and after 8 weeks treatment with diet alone. Eight non-diabetic subjects were also studied. Insulin action was assessed by measuring activation of skeletal muscle glycogen synthase (GS) prior to and during a 4-h hyperinsulinaemic euglycaemic clamp (100 mU kg-1 h-1). Fasting plasma glucose (+/- SE) and glycosylated haemoglobin decreased to a greater extent in the gliclazide treated patients (fall of 6.2 +/- 0.7 vs 2.1 +/- 0.5 mmol l-1, p less than 0.005 and 4.7 +/- 0.5 vs 2.1 +/- 0.5%, p less than 0.005). This was accompanied by an increase in fasting serum insulin concentrations in the gliclazide treated patients (7.0 +/- 1.3 to 10.1 +/- 1.1 mU l-1, p less than 0.005), but no change in the diet treated patients. Fractional GS activity did not increase during the clamp at presentation in either treatment group (change +2.9 +/- 1.8 and -1.5 +/- 1.9%, respectively) whereas it increased markedly in the control subjects (+16.4 +/- 3.4%, both p less than 0.001). After 8-week treatment there was a significant increase in GS activity during the clamp in the patients receiving gliclazide (+6.9 +/- 2.7%, p less than 0.05), but no change in GS activity in the patients on diet alone (+0.5 +/- 1.4%). The difference in post-treatment muscle insulin action was significant (p less than 0.05). There was no correlation between the degree of improvement in metabolic control and the improvement in response of GS to insulin in the gliclazide treated patients (r = -0.06), suggesting a possible direct drug effect on skeletal muscle. Glucose requirement during the clamp at presentation was markedly lower in both treatment groups than in the non-diabetic subjects (gliclazide 2.1 +/- 0.3, diet 2.0 +/- 0.6 vs 7.8 +/- 0.4 mg kg-1 min-1, both p less than 0.001), and despite a

  19. Glycogen Synthase Kinase-3 is involved in glycogen metabolism control and embryogenesis of Rhodnius prolixus.

    PubMed

    Mury, Flávia B; Lugon, Magda D; DA Fonseca, Rodrigo Nunes; Silva, Jose R; Berni, Mateus; Araujo, Helena M; Fontenele, Marcio Ribeiro; Abreu, Leonardo Araujo DE; Dansa, Marílvia; Braz, Glória; Masuda, Hatisaburo; Logullo, Carlos

    2016-10-01

    Rhodnius prolixus is a blood-feeding insect that transmits Trypanosoma cruzi and Trypanosoma rangeli to vertebrate hosts. Rhodnius prolixus is also a classical model in insect physiology, and the recent availability of R. prolixus genome has opened new avenues on triatomine research. Glycogen synthase kinase 3 (GSK-3) is classically described as a key enzyme involved in glycogen metabolism, also acting as a downstream component of the Wnt pathway during embryogenesis. GSK-3 has been shown to be highly conserved among several organisms, mainly in the catalytic domain region. Meanwhile, the role of GSK-3 during R. prolixus embryogenesis or glycogen metabolism has not been investigated. Here we show that chemical inhibition of GSK-3 by alsterpaullone, an ATP-competitive inhibitor of GSK3, does not affect adult survival rate, though it alters oviposition and egg hatching. Specific GSK-3 gene silencing by dsRNA injection in adult females showed a similar phenotype. Furthermore, bright field and 4'-6-diamidino-2-phenylindole (DAPI) staining analysis revealed that ovaries and eggs from dsGSK-3 injected females exhibited specific morphological defects. We also demonstrate that glycogen content was inversely related to activity and transcription levels of GSK-3 during embryogenesis. Lastly, after GSK-3 knockdown, we observed changes in the expression of the Wingless (Wnt) downstream target β-catenin as well as in members of other pathways such as the receptor Notch. Taken together, our results show that GSK-3 regulation is essential for R. prolixus oogenesis and embryogenesis.

  20. Glycogen Synthase Kinase-3 is involved in glycogen metabolism control and embryogenesis of Rhodnius prolixus.

    PubMed

    Mury, Flávia B; Lugon, Magda D; DA Fonseca, Rodrigo Nunes; Silva, Jose R; Berni, Mateus; Araujo, Helena M; Fontenele, Marcio Ribeiro; Abreu, Leonardo Araujo DE; Dansa, Marílvia; Braz, Glória; Masuda, Hatisaburo; Logullo, Carlos

    2016-10-01

    Rhodnius prolixus is a blood-feeding insect that transmits Trypanosoma cruzi and Trypanosoma rangeli to vertebrate hosts. Rhodnius prolixus is also a classical model in insect physiology, and the recent availability of R. prolixus genome has opened new avenues on triatomine research. Glycogen synthase kinase 3 (GSK-3) is classically described as a key enzyme involved in glycogen metabolism, also acting as a downstream component of the Wnt pathway during embryogenesis. GSK-3 has been shown to be highly conserved among several organisms, mainly in the catalytic domain region. Meanwhile, the role of GSK-3 during R. prolixus embryogenesis or glycogen metabolism has not been investigated. Here we show that chemical inhibition of GSK-3 by alsterpaullone, an ATP-competitive inhibitor of GSK3, does not affect adult survival rate, though it alters oviposition and egg hatching. Specific GSK-3 gene silencing by dsRNA injection in adult females showed a similar phenotype. Furthermore, bright field and 4'-6-diamidino-2-phenylindole (DAPI) staining analysis revealed that ovaries and eggs from dsGSK-3 injected females exhibited specific morphological defects. We also demonstrate that glycogen content was inversely related to activity and transcription levels of GSK-3 during embryogenesis. Lastly, after GSK-3 knockdown, we observed changes in the expression of the Wingless (Wnt) downstream target β-catenin as well as in members of other pathways such as the receptor Notch. Taken together, our results show that GSK-3 regulation is essential for R. prolixus oogenesis and embryogenesis. PMID:27574112

  1. Vorinostat synergises with capecitabine through upregulation of thymidine phosphorylase

    PubMed Central

    Di Gennaro, E; Piro, G; Chianese, M I; Franco, R; Cintio, A Di; Moccia, T; Luciano, A; de Ruggiero, I; Bruzzese, F; Avallone, A; Arra, C; Budillon, A

    2010-01-01

    Background: Potentiation of anticancer activity of capecitabine is required to improve its therapeutic index. In colorectal cancer (CRC) cells, we evaluated whether the histone deacetylase-inhibitor vorinostat may induce synergistic antitumour effects in combination with capecitabine by modulating the expression of thymidine phosphorylase (TP), a key enzyme in the conversion of capecitabine to 5-florouracil (5-FU), and thymidylate synthase (TS), the target of 5-FU. Methods: Expression of TP and TS was measured by real-time PCR, western blotting and immunohistochemistry. Knockdown of TP was performed by specific small interfering RNA. Antitumour activity of vorinostat was assessed in vitro in combination with the capecitabine active metabolite deoxy-5-fluorouridine (5′-DFUR) according to the Chou and Talay method and by evaluating apoptosis as well as in xenografts-bearing nude mice in combination with capecitabine. Results: Vorinostat induced both in vitro and in vivo upregulation of TP as well as downregulation of TS in cancer cells, but not in ex vivo treated peripheral blood lymphocytes. Combined treatment with vorinostat and 5′-DFUR resulted in a synergistic antiproliferative effect and increased apoptotic cell death in vitro. This latter effect was impaired in cells where TP was knocked. In vivo, vorinostat plus capecitabine potently inhibited tumour growth, increased apoptosis and prolonged survival compared with control or single-agent treatments. Conclusions: Overall, this study suggests that the combination of vorinostat and capecitabine is an innovative antitumour strategy and warrants further clinical evaluation for the treatment of CRC. PMID:21045833

  2. Crystal structure of glycogen debranching enzyme and insights into its catalysis and disease-causing mutations

    PubMed Central

    Zhai, Liting; Feng, Lingling; Xia, Lin; Yin, Huiyong; Xiang, Song

    2016-01-01

    Glycogen is a branched glucose polymer and serves as an important energy store. Its debranching is a critical step in its mobilization. In animals and fungi, the 170 kDa glycogen debranching enzyme (GDE) catalyses this reaction. GDE deficiencies in humans are associated with severe diseases collectively termed glycogen storage disease type III (GSDIII). We report crystal structures of GDE and its complex with oligosaccharides, and structure-guided mutagenesis and biochemical studies to assess the structural observations. These studies reveal that distinct domains in GDE catalyse sequential reactions in glycogen debranching, the mechanism of their catalysis and highly specific substrate recognition. The unique tertiary structure of GDE provides additional contacts to glycogen besides its active sites, and our biochemical experiments indicate that they mediate its recruitment to glycogen and regulate its activity. Combining the understanding of the GDE catalysis and functional characterizations of its disease-causing mutations provides molecular insights into GSDIII. PMID:27088557

  3. Crystal structure of glycogen debranching enzyme and insights into its catalysis and disease-causing mutations.

    PubMed

    Zhai, Liting; Feng, Lingling; Xia, Lin; Yin, Huiyong; Xiang, Song

    2016-04-18

    Glycogen is a branched glucose polymer and serves as an important energy store. Its debranching is a critical step in its mobilization. In animals and fungi, the 170 kDa glycogen debranching enzyme (GDE) catalyses this reaction. GDE deficiencies in humans are associated with severe diseases collectively termed glycogen storage disease type III (GSDIII). We report crystal structures of GDE and its complex with oligosaccharides, and structure-guided mutagenesis and biochemical studies to assess the structural observations. These studies reveal that distinct domains in GDE catalyse sequential reactions in glycogen debranching, the mechanism of their catalysis and highly specific substrate recognition. The unique tertiary structure of GDE provides additional contacts to glycogen besides its active sites, and our biochemical experiments indicate that they mediate its recruitment to glycogen and regulate its activity. Combining the understanding of the GDE catalysis and functional characterizations of its disease-causing mutations provides molecular insights into GSDIII.

  4. Glycogen depletion and resynthesis during 14 days of chronic low-frequency stimulation of rabbit muscle.

    PubMed

    Prats, C; Bernal, C; Cadefau, J A; Frias, J; Tibolla, M; Cussó, R

    2002-10-10

    Electro-stimulation alters muscle metabolism and the extent of this change depends on application intensity and duration. The effect of 14 days of chronic electro-stimulation on glycogen turnover and on the regulation of glycogen synthase in fast-twitch muscle was studied. The results showed that macro- and proglycogen degrade simultaneously during the first hour of stimulation. After 3 h, the muscle showed net synthesis, with an increase in the proglycogen fraction. The glycogen content peaked after 4 days of stimulation, macroglycogen being the predominant fraction at that time. Glycogen synthase was determined during electro-stimulation. The activity of this enzyme was measured at low UDPG concentration with either high or low Glu-6-P content. Western blots were performed against glycogen synthase over a range of stimulation periods. Activation of this enzyme was maximum before the net synthesis of glycogen, partial during net synthesis, and low during late synthesis. These observations suggest that the more active, dephosphorylated and very low phosphorylated forms of glycogen synthase may participate in the first steps of glycogen resynthesis before net synthesis is observed, while partially phosphorylated forms are most active during glycogen elongation.

  5. Thymidine phosphorylase, 2-deoxy-D-ribose and angiogenesis.

    PubMed Central

    Brown, N S; Bicknell, R

    1998-01-01

    Angiogenesis is the term used to describe the formation of new blood vessels from the existing vasculature. In order to attract new vessels, a tissue must release an endothelial-cell chemoattractant. 2-Deoxy-D-ribose is produced in vivo by the catalytic action of thymidine phosphorylase (TP) on thymidine and has recently been identified as an endothelial-cell chemoattractant and angiogenesis-inducing factor. TP, previously known only for its role in nucleotide salvage, is now known to be angiogenic. TP expression is elevated in many solid tumours and in chronically inflamed tissues, both known areas of active angiogenesis. There is evidence that TP is also involved in physiological angiogenesis such as endometrial angiogenesis during the menstrual cycle. The majority of known endothelial-cell chemoattractants are polypeptides that bind to endothelial-cell-surface receptors. In contrast, 2-deoxy-D-ribose appears to lack a cell-surface receptor. Glucose is another sugar that acts as an endothelial-cell chemoattractant. The migratory activity of glucose is blocked by ouabain. It is possible that 2-deoxy-D-ribose and glucose stimulate endothelial-cell migration via a similar mechanistic pathway. PMID:9693094

  6. Digestion of glycogen by a glucosidase released by Trichomonas vaginalis.

    PubMed

    Huffman, Ryan D; Nawrocki, Lauren D; Wilson, Wayne A; Brittingham, Andrew

    2015-12-01

    Trichomonas vaginalis is a protozoan parasite that is the causative agent of trichomoniasis, a widespread sexually transmitted disease. In vitro culture of T. vaginalis typically employs a medium supplemented with either maltose or glucose and carbohydrates are considered essential for growth. Although the nature of the carbohydrates utilized by T. vaginalis in vivo is undefined, the vaginal epithelium is rich in glycogen, which appears to provide a source of carbon for the vaginal microbiota. Here, we show that T. vaginalis grows equally well in growth media supplemented with simple sugars or with glycogen. Analysis of conditioned growth medium by thin layer chromatography indicates that growth on glycogen is accompanied by glycogen breakdown to a mixture of products including maltose, glucose, and oligosaccharides. Enzymatic assays with conditioned growth medium show that glycogen breakdown is accomplished via the release of a glucosidase activity having the properties of an α-amylase into the growth medium. Furthermore, we find that released glucosidase activity increases upon removal of carbohydrate from the growth medium, indicating regulation of synthesis and/or secretion in response to environmental cues. Lastly, we show that addition of T. vaginalis glucosidase activity to a growth medium containing glycogen generates sufficient simple sugar to support the growth of lactobacilli which, themselves, are unable to degrade glycogen. Thus, not only does the glucosidase activity likely play an important role in allowing T. vaginalis to secure simple sugars for its own use, it has the potential to impact the growth of other members of the vaginal microbiome.

  7. Axonal and dendritic localization of mRNAs for glycogen-metabolizing enzymes in cultured rodent neurons

    PubMed Central

    2014-01-01

    Background Localization of mRNAs encoding cytoskeletal or signaling proteins to neuronal processes is known to contribute to axon growth, synaptic differentiation and plasticity. In addition, a still increasing spectrum of mRNAs has been demonstrated to be localized under different conditions and developing stages thus reflecting a highly regulated mechanism and a role of mRNA localization in a broad range of cellular processes. Results Applying fluorescence in-situ-hybridization with specific riboprobes on cultured neurons and nervous tissue sections, we investigated whether the mRNAs for two metabolic enzymes, namely glycogen synthase (GS) and glycogen phosphorylase (GP), the key enzymes of glycogen metabolism, may also be targeted to neuronal processes. If it were so, this might contribute to clarify the so far enigmatic role of neuronal glycogen. We found that the mRNAs for both enzymes are localized to axonal and dendritic processes in cultured lumbar spinal motoneurons, but not in cultured trigeminal neurons. In cultured cortical neurons which do not store glycogen but nevertheless express glycogen synthase, the GS mRNA is also subject to axonal and dendritic localization. In spinal motoneurons and trigeminal neurons in situ, however, the mRNAs could only be demonstrated in the neuronal somata but not in the nerves. Conclusions We could demonstrate that the mRNAs for major enzymes of neural energy metabolism can be localized to neuronal processes. The heterogeneous pattern of mRNA localization in different culture types and developmental stages stresses that mRNA localization is a versatile mechanism for the fine-tuning of cellular events. Our findings suggest that mRNA localization for enzymes of glycogen metabolism could allow adaptation to spatial and temporal energy demands in neuronal events like growth, repair and synaptic transmission. PMID:24898526

  8. Substrate-induced Nuclear Export and Peripheral Compartmentalization of Hepatic Glucokinase Correlates with Glycogen Deposition

    PubMed Central

    Shiota, Masa; Knobel, Susan M.; Piston, David W.; Cherrington, Alan D.; Magnuson, Mark A.

    2001-01-01

    Hepatic glucokinase (GK) is acutely regulated by binding to its nuclear-anchored regulatory protein (GKRP). Although GK release by GKRP is tightly coupled to the rate of glycogen synthesis, the nature of this association is obscure. To gain insight into this coupling mechanism under physiological stimulating conditions in primary rat hepatocytes, we analyzed the subcellular distribution of GK and GKRP with immunofluorescence, and glycogen deposition with glycogen cytochemical fluorescence, using confocal microscopyand quantitative image analysis. Following stimulation, a fraction of the GK signal translocated from the nucleus to the cytoplasm. The reduction in the nuclear to cytoplasmic ratio of GK, an index of nuclear export, correlated with a >50% increase in glycogen cytochemical fluorescence over a 60min stimulation period. Furthermore, glycogen accumulation was initially deposited in a peripheral pattern in hepatocytes similar to that of GK. These data suggest that a compartmentalization exists of both active GK and the initial sites of glycogen deposition at the hepatocyte surface. PMID:12369705

  9. Thymidine phosphorylase expression in Kaposi sarcoma.

    PubMed Central

    Dada, M A; Boshoff, C H; Comley, M A; Turley, H; Schneider, J W; Chetty, R; Gatter, K C

    1996-01-01

    AIMS: To examine the immunohistochemical distribution of thymidine phosphorylase (TP) in all clinicopathological subtypes of Kaposi sarcoma. METHODS: Thirty two biopsy specimens of Kaposi sarcoma (29 patients) were studied. Six of these patients represented classic, six endemic, eight HIV associated, seven post-immunosuppression/transplant related, and two unclassified variants of Kaposi sarcoma. The average age was 49 years (range 22-83 years) and the male: female ratio 24:5. Four samples of angiosarcoma and one of spindle cell haemangio-endothelioma were stained in parallel. All specimens were fixed in formalin, embedded in paraffin wax and processed routinely. Immunohistochemistry was carried out using an antibody directed against CD31 (JC70) and the monoclonal antibody P-GF.44C against TP. RESULTS: All biopsy specimens showed immunoexpression for TP. The spindle cell component stained more strongly than newly formed endothelium lined vessels and normal, resident vessels at a distance from the lesions. CONCLUSIONS: The strong immunoexpression of TP suggests up-regulation of TP and a role for TP in angiogensis in Kaposi sarcoma. The mechanism for the up-regulation of TP remains unknown, but viral infections may trigger it. The differential staining of the various cell components of Kaposi sarcoma also suggest that TP either plays a role in the differentiation and maturation of Kaposi sarcoma or is a reflection of such changes. Images PMID:8707955

  10. Phosphorylase re-expression, increase in the force of contraction and decreased fatigue following notexin-induced muscle damage and regeneration in the ovine model of McArdle disease.

    PubMed

    Howell, J McC; Walker, K R; Creed, K E; Dunton, E; Davies, L; Quinlivan, R; Karpati, G

    2014-02-01

    McArdle disease is caused by a deficiency of myophosphorylase and currently a satisfactory treatment is not available. The injection of notexin into, or the layering of notexin onto, the muscles of affected sheep resulted in necrosis followed by regeneration of muscle fibres with the expression of both non-muscle isoforms of phosphorylase within the fibres and a reduction of the amount of glycogen in the muscle with an increase in the strength of contraction and a decrease in fatiguability in the muscle fibres. The sustained re-expression of both the brain and liver isoforms of phosphorylase within the muscle fibres provides further emphasis that strategies to enhance the re-expression of these isoforms should be investigated as a possible treatment for McArdle disease.

  11. A novel GDP-D-glucose phosphorylase involved in quality control of the nucleoside diphosphate sugar pool in Caenorhabditis elegans and mammals.

    PubMed

    Adler, Lital N; Gomez, Tara A; Clarke, Steven G; Linster, Carole L

    2011-06-17

    The plant VTC2 gene encodes GDP-L-galactose phosphorylase, a rate-limiting enzyme in plant vitamin C biosynthesis. Genes encoding apparent orthologs of VTC2 exist in both mammals, which produce vitamin C by a distinct metabolic pathway, and in the nematode worm Caenorhabditis elegans where vitamin C biosynthesis has not been demonstrated. We have now expressed cDNAs of the human and worm VTC2 homolog genes (C15orf58 and C10F3.4, respectively) and found that the purified proteins also display GDP-hexose phosphorylase activity. However, as opposed to the plant enzyme, the major reaction catalyzed by these enzymes is the phosphorolysis of GDP-D-glucose to GDP and D-glucose 1-phosphate. We detected activities with similar substrate specificity in worm and mouse tissue extracts. The highest expression of GDP-D-glucose phosphorylase was found in the nervous and male reproductive systems. A C. elegans C10F3.4 deletion strain was found to totally lack GDP-D-glucose phosphorylase activity; this activity was also found to be decreased in human HEK293T cells transfected with siRNAs against the human C15orf58 gene. These observations confirm the identification of the worm C10F3.4 and the human C15orf58 gene expression products as the GDP-D-glucose phosphorylases of these organisms. Significantly, we found an accumulation of GDP-D-glucose in the C10F3.4 mutant worms, suggesting that the GDP-D-glucose phosphorylase may function to remove GDP-D-glucose formed by GDP-D-mannose pyrophosphorylase, an enzyme that has previously been shown to lack specificity for its physiological D-mannose 1-phosphate substrate. We propose that such removal may prevent the misincorporation of glucosyl residues for mannosyl residues into the glycoconjugates of worms and mammals.

  12. Purine nucleoside phosphorylase polymorphism in the genus Littorina (Prosobranchia: Mollusca).

    PubMed

    Knight, A J; Ward, R D

    1986-06-01

    Examination of eight Atlantic species of the genus Littorina by starch gel electrophoresis of purine nucleoside phosphorylase revealed extensive polymorphism within the L. saxatilis complex. In this group, four alleles have been identified. Heterozygotes are four banded, and thus, as in vertebrates, the enzyme is likely to be a trimer. Breeding experiments confirmed the genetic interpretation of the phenotype patterns. Where species of the saxatilis complex [L. saxatilis (=L. rudis), L. arcana, L. nigrolineata, L. neglecta] are sympatric, there are sometimes significant allele frequency differences between them. A fifth allele was present at a high frequency in L. obtusata and L. mariae, and L. littorea and L. neritoides each possessed unique alleles. A total of eight alleles was identified. Densitometric scanning of heterozygote patterns pointed to activity differences between alleles and also showed that, while the heterotrimeric bands were never less intense than the homotrimeric bands, the heterotrimeric bands were sometimes less intense than expected. It is not clear whether this represents nonrandom association of subunits, decreased stability of heterotrimers, or simply an artifact of the staining and quantifying process. PMID:3091000

  13. Genetics Home Reference: glycogen storage disease type IV

    MedlinePlus

    ... gene provides instructions for making the glycogen branching enzyme. This enzyme is involved in the production of glycogen , which ... to a shortage (deficiency) of the glycogen branching enzyme. As a result, glycogen is not formed properly. ...

  14. Pho85p, a cyclin-dependent protein kinase, and the Snf1p protein kinase act antagonistically to control glycogen accumulation in Saccharomyces cerevisiae.

    PubMed Central

    Huang, D; Farkas, I; Roach, P J

    1996-01-01

    In Saccharomyces cerevisiae, nutrient levels control multiple cellular processes. Cells lacking the SNF1 gene cannot express glucose-repressible genes and do not accumulate the storage polysaccharide glycogen. The impaired glycogen synthesis is due to maintenance of glycogen synthase in a hyperphosphorylated, inactive state. In a screen for second site suppressors of the glycogen storage defect of snf1 cells, we identified a mutant gene that restored glycogen accumulation and which was allelic with PHO85, which encodes a member of the cyclin-dependent kinase family. In cells with disrupted PHO85 genes, we observed hyperaccumulation of glycogen, activation of glycogen synthase, and impaired glycogen synthase kinase activity. In snf1 cells, glycogen synthase kinase activity was elevated. Partial purification of glycogen synthase kinase activity from yeast extracts resulted in the separation of two fractions by phenyl-Sepharose chromatography, both of which phosphorylated and inactivated glycogen synthase. The activity of one of these, GPK2, was inhibited by olomoucine, which potently inhibits cyclin-dependent protein kinases, and contained an approximately 36-kDa species that reacted with antibodies to Pho85p. Analysis of Ser-to-Ala mutations at the three potential Gsy2p phosphorylation sites in pho85 cells implicated Ser-654 and/or Thr-667 in PHO85 control of glycogen synthase. We propose that Pho85p is a physiological glycogen synthase kinase, possibly acting downstream of Snf1p. PMID:8754836

  15. Surface Induced Dissociation Yields Quaternary Substructure of Refractory Noncovalent Phosphorylase B and Glutamate Dehydrogenase Complexes

    NASA Astrophysics Data System (ADS)

    Ma, Xin; Zhou, Mowei; Wysocki, Vicki H.

    2014-03-01

    Ion mobility (IM) and tandem mass spectrometry (MS/MS) coupled with native MS are useful for studying noncovalent protein complexes. Collision induced dissociation (CID) is the most common MS/MS dissociation method. However, some protein complexes, including glycogen phosphorylase B kinase (PHB) and L-glutamate dehydrogenase (GDH) examined in this study, are resistant to dissociation by CID at the maximum collision energy available in the instrument. Surface induced dissociation (SID) was applied to dissociate the two refractory protein complexes. Different charge state precursor ions of the two complexes were examined by CID and SID. The PHB dimer was successfully dissociated to monomers and the GDH hexamer formed trimeric subcomplexes that are informative of its quaternary structure. The unfolding of the precursor and the percentages of the distinct products suggest that the dissociation pathways vary for different charge states. The precursors at lower charge states (+21 for PHB dimer and +27 for GDH hexamer) produce a higher percentage of folded fragments and dissociate more symmetrically than the precusors at higher charge states (+29 for PHB dimer and +39 for GDH hexamer). The precursors at lower charge state may be more native-like than the higher charge state because a higher percentage of folded fragments and a lower percentage of highly charged unfolded fragments are detected. The combination of SID and charge reduction is shown to be a powerful tool for quaternary structure analysis of refractory noncovalent protein complexes, as illustrated by the data for PHB dimer and GDH hexamer.

  16. A purine nucleoside phosphorylase in Solanum tuberosum L. (potato) with specificity for cytokinins contributes to the duration of tuber endodormancy.

    PubMed

    Bromley, Jennifer R; Warnes, Barbara J; Newell, Christine A; Thomson, Jamie C P; James, Celia M; Turnbull, Colin G N; Hanke, David E

    2014-03-01

    StCKP1 (Solanum tuberosum cytokinin riboside phosphorylase) catalyses the interconversion of the N9-riboside form of the plant hormone CK (cytokinin), a subset of purines, with its most active free base form. StCKP1 prefers CK to unsubstituted aminopurines. The protein was discovered as a CK-binding activity in extracts of tuberizing potato stolon tips, from which it was isolated by affinity chromatography. The N-terminal amino acid sequence matched the translation product of a set of ESTs, enabling a complete mRNA sequence to be obtained by RACE-PCR. The predicted polypeptide includes a cleavable signal peptide and motifs for purine nucleoside phosphorylase activity. The expressed protein was assayed for purine nucleoside phosphorylase activity against CKs and adenine/adenosine. Isopentenyladenine, trans-zeatin, dihydrozeatin and adenine were converted into ribosides in the presence of ribose 1-phosphate. In the opposite direction, isopentenyladenosine, trans-zeatin riboside, dihydrozeatin riboside and adenosine were converted into their free bases in the presence of Pi. StCKP1 had no detectable ribohydrolase activity. Evidence is presented that StCKP1 is active in tubers as a negative regulator of CKs, prolonging endodormancy by a chill-reversible mechanism.

  17. Redox regulation of glycogen biosynthesis in the cyanobacterium Synechocystis sp. PCC 6803: analysis of the AGP and glycogen synthases.

    PubMed

    Díaz-Troya, Sandra; López-Maury, Luis; Sánchez-Riego, Ana María; Roldán, Miguel; Florencio, Francisco J

    2014-01-01

    Glycogen constitutes the major carbon storage source in cyanobacteria, as starch in algae and higher plants. Glycogen and starch synthesis is linked to active photosynthesis and both of them are degraded to glucose in the dark to maintain cell metabolism. Control of glycogen biosynthesis in cyanobacteria could be mediated by the regulation of the enzymes involved in this process, ADP-glucose pyrophosphorylase (AGP) and glycogen synthase, which were identified as putative thioredoxin targets. We have analyzed whether both enzymes were subjected to redox modification using purified recombinant enzymes or cell extracts in the model cyanobacterium Synechocystis sp. PCC 6803. Our results indicate that both AGP and glycogen synthases are sensitive to copper oxidation. However, only AGP exhibits a decrease in its enzymatic activity, which is recovered after reduction by DTT or reduced thioredoxin (TrxA), suggesting a redox control of AGP. In order to elucidate the role in redox control of the cysteine residues present on the AGP sequence (C45, C185, C320, and C337), they were replaced with serine. All AGP mutant proteins remained active when expressed in Synechocystis, although they showed different electrophoretic mobility profiles after copper oxidation, reflecting a complex pattern of cysteines interaction.

  18. Preparation, proteolysis and reversible oxidation of highly purified Azotobacter vinelandii polynucleotide phosphorylase

    PubMed Central

    Gajda, A. T.; de Behrens, G. Zaror; Fitt, P. S.

    1970-01-01

    1. A new method has been developed for the preparation in good yield of highly purified Azotobacter vinelandii polynucleotide phosphorylase in its reduced form. 2. Aging or digestion with trypsin causes the enzyme to develop a primer requirement that is not eliminated by β-mercaptoethanol. 3. The development of a primer requirement is accompanied by marked changes of the electrophoretic mobility of the enzyme in polyacrylamide gels. 4. The enzyme is inactivated by aerial oxidation or thiol-specific reagents. The lost activity is restored by β-mercaptoethanol, but not by oligonucleotide primers. PMID:5495150

  19. Assignment of the gene encoding glycogen synthase (GYS) to human chromosome 19, band q13,3

    SciTech Connect

    Lehto, M. Helsinki Univ. ); Stoffel, M.; Espinosa, R. III; Beau, M.M. le; Bell, G.I. ); Groop, L. )

    1993-02-01

    The enzyme glycogen synthase (UDP glocose:glycogen 4-[alpha]-D-glucosyltransferase, EC 2.4.1.11) catalyzes the formation of glycogen from uridine diphosphate glucose (UPDG). Impaired activation of muscle glycogen synthase by insulin has been noted in patients with genetic risk of developing non-insulin-dependent diabets mellitus (NIDDM) and this may represent an early defect in the pathogenesis of this disorder. As such, glycogen synthase represents a candidate gene for contributing to genetic susceptibility. As a first step in studying the role of glycogen synthase in the genetics of NIDDM, we have isolated a cosmid encoding the human glycogen synthase gene (gene symbol GYS) and determined its chromosomal localization by fluorescence in situ hybridization. 4 refs., 1 fig.

  20. Enzymatic Properties and Substrate Specificity of the Trehalose Phosphorylase from Caldanaerobacter subterraneus▿

    PubMed Central

    Van der Borght, Jef; Chen, Chao; Hoflack, Lieve; Van Renterghem, Lucas; Desmet, Tom; Soetaert, Wim

    2011-01-01

    A putative glycoside phosphorylase from Caldanaerobacter subterraneus subsp. pacificus was recombinantly expressed in Escherichia coli, after codon optimization and chemical synthesis of the encoding gene. The enzyme was purified by His tag chromatography and was found to be specifically active toward trehalose, with an optimal temperature of 80°C. In addition, no loss of activity could be detected after 1 h of incubation at 65°C, which means that it is the most stable trehalose phosphorylase reported so far. The substrate specificity was investigated in detail by measuring the relative activity on a range of alternative acceptors, applied in the reverse synthetic reaction, and determining the kinetic parameters for the best acceptors. These results were rationalized based on the enzyme-substrate interactions observed in a homology model with a docked ligand. The specificity for the orientation of the acceptor's hydroxyl groups was found to decrease in the following order: C-3 > C-2 > C-4. This results in a particularly high activity on the monosaccharides d-fucose, d-xylose, l-arabinose, and d-galactose, as well as on l-fucose. However, determination of the kinetic parameters revealed that these acceptors bind less tightly in the active site than the natural acceptor d-glucose, resulting in drastically increased Km values. Nevertheless, the enzyme's high thermostability and broad acceptor specificity make it a valuable candidate for industrial disaccharide synthesis. PMID:21803886

  1. Structural determinants of the 5'-methylthioinosine specificity of Plasmodium purine nucleoside phosphorylase.

    PubMed

    Donaldson, Teraya M; Ting, Li-Min; Zhan, Chenyang; Shi, Wuxian; Zheng, Renjian; Almo, Steven C; Kim, Kami

    2014-01-01

    Plasmodium parasites rely upon purine salvage for survival. Plasmodium purine nucleoside phosphorylase is part of the streamlined Plasmodium purine salvage pathway that leads to the phosphorylysis of both purines and 5'-methylthiopurines, byproducts of polyamine synthesis. We have explored structural features in Plasmodium falciparum purine nucleoside phosphorylase (PfPNP) that affect efficiency of catalysis as well as those that make it suitable for dual specificity. We used site directed mutagenesis to identify residues critical for PfPNP catalytic activity as well as critical residues within a hydrophobic pocket required for accommodation of the 5'-methylthio group. Kinetic analysis data shows that several mutants had disrupted binding of the 5'-methylthio group while retaining activity for inosine. A triple PfPNP mutant that mimics Toxoplasma gondii PNP had significant loss of 5'-methylthio activity with retention of inosine activity. Crystallographic investigation of the triple mutant PfPNP with Tyr160Phe, Val66Ile, andVal73Ile in complex with the transition state inhibitor immucillin H reveals fewer hydrogen bond interactions for the inhibitor in the hydrophobic pocket. PMID:24416224

  2. Structural determinants of the 5'-methylthioinosine specificity of Plasmodium purine nucleoside phosphorylase.

    PubMed

    Donaldson, Teraya M; Ting, Li-Min; Zhan, Chenyang; Shi, Wuxian; Zheng, Renjian; Almo, Steven C; Kim, Kami

    2014-01-01

    Plasmodium parasites rely upon purine salvage for survival. Plasmodium purine nucleoside phosphorylase is part of the streamlined Plasmodium purine salvage pathway that leads to the phosphorylysis of both purines and 5'-methylthiopurines, byproducts of polyamine synthesis. We have explored structural features in Plasmodium falciparum purine nucleoside phosphorylase (PfPNP) that affect efficiency of catalysis as well as those that make it suitable for dual specificity. We used site directed mutagenesis to identify residues critical for PfPNP catalytic activity as well as critical residues within a hydrophobic pocket required for accommodation of the 5'-methylthio group. Kinetic analysis data shows that several mutants had disrupted binding of the 5'-methylthio group while retaining activity for inosine. A triple PfPNP mutant that mimics Toxoplasma gondii PNP had significant loss of 5'-methylthio activity with retention of inosine activity. Crystallographic investigation of the triple mutant PfPNP with Tyr160Phe, Val66Ile, andVal73Ile in complex with the transition state inhibitor immucillin H reveals fewer hydrogen bond interactions for the inhibitor in the hydrophobic pocket.

  3. Structural basis of AMPK regulation by adenine nucleotides and glycogen

    SciTech Connect

    Li, Xiaodan; Wang, Lili; Zhou, X. Edward; Ke, Jiyuan; de Waal, Parker W.; Gu, Xin; Tan, M. H. Eileen; Wang, Dongye; Wu, Donghai; Xu, H. Eric; Melcher, Karsten

    2014-11-21

    AMP-activated protein kinase (AMPK) is a central cellular energy sensor and regulator of energy homeostasis, and a promising drug target for the treatment of diabetes, obesity, and cancer. Here we present low-resolution crystal structures of the human α1β2γ1 holo-AMPK complex bound to its allosteric modulators AMP and the glycogen-mimic cyclodextrin, both in the phosphorylated (4.05 Å) and non-phosphorylated (4.60 Å) state. In addition, we have solved a 2.95 Å structure of the human kinase domain (KD) bound to the adjacent autoinhibitory domain (AID) and have performed extensive biochemical and mutational studies. Altogether, these studies illustrate an underlying mechanism of allosteric AMPK modulation by AMP and glycogen, whose binding changes the equilibria between alternate AID (AMP) and carbohydrate-binding module (glycogen) interactions.

  4. Glucagon-like peptide 1: a potent glycogenic hormone.

    PubMed

    Valverde, I; Morales, M; Clemente, F; López-Delgado, M I; Delgado, E; Perea, A; Villanueva-Peñacarrillo, M L

    1994-08-01

    GLP-1(7-36)amide is an insulinotropic peptide derived from the intestinal post-translational proglucagon process, the release of which is increased mainly after a carbohydrate meal; also, its anti-diabetogenic effect in normal and diabetic states has been reported. In this study, GLP-1(7-36)amide stimulates the formation of glycogen from glucose in isolated rat hepatocytes, such a glycogenic effect being achieved with physiological concentrations of the peptide. The GLP-1(7-36)amide-induced glycogenesis is abolished by glucagon, and it is accompanied by stimulation of the glycogen synthase alpha activity and by a decrease in the basal and glucagon-stimulated cyclic AMP content. These findings could explain, at least in part, the GLP-1(7-36)amide insulin-independent plasma glucose lowering effect.

  5. Structural basis of AMPK regulation by adenine nucleotides and glycogen

    DOE PAGES

    Li, Xiaodan; Wang, Lili; Zhou, X. Edward; Ke, Jiyuan; de Waal, Parker W.; Gu, Xin; Tan, M. H. Eileen; Wang, Dongye; Wu, Donghai; Xu, H. Eric; et al

    2014-11-21

    AMP-activated protein kinase (AMPK) is a central cellular energy sensor and regulator of energy homeostasis, and a promising drug target for the treatment of diabetes, obesity, and cancer. Here we present low-resolution crystal structures of the human α1β2γ1 holo-AMPK complex bound to its allosteric modulators AMP and the glycogen-mimic cyclodextrin, both in the phosphorylated (4.05 Å) and non-phosphorylated (4.60 Å) state. In addition, we have solved a 2.95 Å structure of the human kinase domain (KD) bound to the adjacent autoinhibitory domain (AID) and have performed extensive biochemical and mutational studies. Altogether, these studies illustrate an underlying mechanism of allostericmore » AMPK modulation by AMP and glycogen, whose binding changes the equilibria between alternate AID (AMP) and carbohydrate-binding module (glycogen) interactions.« less

  6. Expression of a cDNA for the catalytic subunit of skeletal-muscle phosphorylase kinase in transfected 3T3 cells.

    PubMed Central

    Cawley, K C; Akita, C G; Walsh, D A

    1989-01-01

    Phosphorylase kinase is a multimeric enzyme of composition (alpha, beta, gamma, delta)4 whose catalytic activity resides in the gamma-subunit. As an approach to understand further its regulation, a cDNA for the gamma-subunit of phosphorylase kinase (gamma PhK) has been cloned into a mammalian expression vector behind the mouse metallothionein-1 promoter. NIH 3T3 cells were co-transfected with this construct (pEV gamma PhK) and pSV2neo, G418-resistant clones were selected, and several were found to have stably incorporated the gamma-subunit cDNA into their genomic DNA. Phosphorylase kinase activity was clearly present in extracts from cultures of pEV gamma PhK-transformed cells and increased several-fold after 24 h of incubation with Zn2+, whereas it was undetectable in the parent 3T3 cells. A significant, but variable, proportion (15-70%) of the activity was Ca2+-dependent. We conclude that the phosphorylase kinase activity expressed by the cells transformed with pEV gamma PhK is due to free gamma-subunit and gamma-subunit associated with cellular calmodulin, which replaces the delta-subunit normally associated with the gamma-subunit in the holoenzyme. Images Fig. 1. Fig. 2. Fig. 3. PMID:2481439

  7. Brain glycogen-new perspectives on its metabolic function and regulation at the subcellular level.

    PubMed

    Obel, Linea F; Müller, Margit S; Walls, Anne B; Sickmann, Helle M; Bak, Lasse K; Waagepetersen, Helle S; Schousboe, Arne

    2012-01-01

    Glycogen is a complex glucose polymer found in a variety of tissues, including brain, where it is localized primarily in astrocytes. The small quantity found in brain compared to e.g., liver has led to the understanding that brain glycogen is merely used during hypoglycemia or ischemia. In this review evidence is brought forward highlighting what has been an emerging understanding in brain energy metabolism: that glycogen is more than just a convenient way to store energy for use in emergencies-it is a highly dynamic molecule with versatile implications in brain function, i.e., synaptic activity and memory formation. In line with the great spatiotemporal complexity of the brain and thereof derived focus on the basis for ensuring the availability of the right amount of energy at the right time and place, we here encourage a closer look into the molecular and subcellular mechanisms underlying glycogen metabolism. Based on (1) the compartmentation of the interconnected second messenger pathways controlling glycogen metabolism (calcium and cAMP), (2) alterations in the subcellular location of glycogen-associated enzymes and proteins induced by the metabolic status and (3) a sequential component in the intermolecular mechanisms of glycogen metabolism, we suggest that glycogen metabolism in astrocytes is compartmentalized at the subcellular level. As a consequence, the meaning and importance of conventional terms used to describe glycogen metabolism (e.g., turnover) is challenged. Overall, this review represents an overview of contemporary knowledge about brain glycogen and its metabolism and function. However, it also has a sharp focus on what we do not know, which is perhaps even more important for the future quest of uncovering the roles of glycogen in brain physiology and pathology.

  8. Revisiting Glycogen Content in the Human Brain.

    PubMed

    Öz, Gülin; DiNuzzo, Mauro; Kumar, Anjali; Moheet, Amir; Seaquist, Elizabeth R

    2015-12-01

    Glycogen provides an important glucose reservoir in the brain since the concentration of glucosyl units stored in glycogen is several fold higher than free glucose available in brain tissue. We have previously reported 3-4 µmol/g brain glycogen content using in vivo (13)C magnetic resonance spectroscopy (MRS) in conjunction with [1-(13)C]glucose administration in healthy humans, while higher levels were reported in the rodent brain. Due to the slow turnover of bulk brain glycogen in humans, complete turnover of the glycogen pool, estimated to take 3-5 days, was not observed in these prior studies. In an attempt to reach complete turnover and thereby steady state (13)C labeling in glycogen, here we administered [1-(13)C]glucose to healthy volunteers for 80 h. To eliminate any net glycogen synthesis during this period and thereby achieve an accurate estimate of glycogen concentration, volunteers were maintained at euglycemic blood glucose levels during [1-(13)C]glucose administration and (13)C-glycogen levels in the occipital lobe were measured by (13)C MRS approximately every 12 h. Finally, we fitted the data with a biophysical model that was recently developed to take into account the tiered structure of the glycogen molecule and additionally incorporated blood glucose levels and isotopic enrichments as input function in the model. We obtained excellent fits of the model to the (13)C-glycogen data, and glycogen content in the healthy human brain tissue was found to be 7.8 ± 0.3 µmol/g, a value substantially higher than previous estimates of glycogen content in the human brain.

  9. Nerve growth factor (NGF) regulates activity of nuclear factor of activated T-cells (NFAT) in neurons via the phosphatidylinositol 3-kinase (PI3K)-Akt-glycogen synthase kinase 3β (GSK3β) pathway.

    PubMed

    Kim, Man-Su; Shutov, Leonid P; Gnanasekaran, Aswini; Lin, Zhihong; Rysted, Jacob E; Ulrich, Jason D; Usachev, Yuriy M

    2014-11-01

    The Ca(2+)/calcineurin-dependent transcription factor nuclear factor of activated T-cells (NFAT) plays an important role in regulating many neuronal functions, including excitability, axonal growth, synaptogenesis, and neuronal survival. NFAT can be activated by action potential firing or depolarization that leads to Ca(2+)/calcineurin-dependent dephosphorylation of NFAT and its translocation to the nucleus. Recent data suggest that NFAT and NFAT-dependent functions in neurons can also be potently regulated by NGF and other neurotrophins. However, the mechanisms of NFAT regulation by neurotrophins are not well understood. Here, we show that in dorsal root ganglion sensory neurons, NGF markedly facilitates NFAT-mediated gene expression induced by mild depolarization. The effects of NGF were not associated with changes in [Ca(2+)]i and were independent of phospholipase C activity. Instead, the facilitatory effect of NGF depended on activation of the PI3K/Akt pathway downstream of the TrkA receptor and on inhibition of glycogen synthase kinase 3β (GSK3β), a protein kinase known to phosphorylate NFAT and promote its nuclear export. Knockdown or knockout of NFATc3 eliminated this facilitatory effect. Simultaneous monitoring of EGFP-NFATc3 nuclear translocation and [Ca(2+)]i changes in dorsal root ganglion neurons indicated that NGF slowed the rate of NFATc3 nuclear export but did not affect its nuclear import rate. Collectively, our data suggest that NGF facilitates depolarization-induced NFAT activation by stimulating PI3K/Akt signaling, inactivating GSK3β, and thereby slowing NFATc3 export from the nucleus. We propose that NFAT serves as an integrator of neurotrophin action and depolarization-driven calcium signaling to regulate neuronal gene expression.

  10. Structural basis for the mechanism of inhibition of uridine phosphorylase from Salmonella typhimurium

    SciTech Connect

    Lashkov, A. A.; Zhukhlistova, N. E.; Sotnichenko, S. E.; Gabdulkhakov, A. G.; Mikhailov, A. M.

    2010-01-15

    The three-dimensional structures of three complexes of Salmonella typhimurium uridine phosphorylase with the inhibitor 2,2'-anhydrouridine, the substrate PO{sub 4}, and with both the inhibitor 2,2'-anhydrouridine and the substrate PO{sub 4} (a binary complex) were studied in detail by X-ray diffraction. The structures of the complexes were refined at 2.38, 1.5, and 1.75 A resolution, respectively. Changes in the three-dimensional structure of the subunits in different crystal structures are considered depending on the presence or absence of the inhibitor molecule and (or) the phosphate ion in the active site of the enzyme. The presence of the phosphate ion in the phosphate-binding site was found to substantially change the orientations of the side chains of the amino-acid residues Arg30, Arg91, and Arg48 coordinated to this ion. A comparison showed that the highly flexible loop L9 is unstable. The atomic coordinates of the refined structures of the complexes and the corresponding structure factors were deposited in the Protein Data Bank (their PDB ID codes are 3DD0 and 3C74). The experimental data on the spatial reorganization of the active site caused by changes in its functional state from the unligated to the completely inhibited state suggest the structural basis for the mechanism of inhibition of Salmonella typhimurium uridine phosphorylase.

  11. Brain Glycogen Decreases During Intense Exercise Without Hypoglycemia: The Possible Involvement of Serotonin.

    PubMed

    Matsui, Takashi; Soya, Shingo; Kawanaka, Kentaro; Soya, Hideaki

    2015-07-01

    Brain glycogen stored in astrocytes, a source of lactate as a neuronal energy source, decreases during prolonged exercise with hypoglycemia. However, brain glycogen dynamics during exercise without hypoglycemia remain unknown. Since intense exercise increases brain noradrenaline and serotonin as known inducers for brain glycogenolysis, we hypothesized that brain glycogen decreases with intense exercise not accompanied by hypoglycemia. To test this hypothesis, we employed a well-established acute intense exercise model of swimming in rats. Rats swam for fourteen 20 s bouts with a weight equal to 8 % of their body mass and were sacrificed using high-power (10 kW) microwave irradiation to inactivate brain enzymes for accurate detection of brain glycogen and monoamines. Intense exercise did not alter blood glucose, but did increase blood lactate levels. Immediately after exercise, brain glycogen decreased and brain lactate increased in the hippocampus, cerebellum, cortex, and brainstem. Simultaneously, serotonin turnover in the hippocampus and brainstem mutually increased and were associated with decreased brain glycogen. Intense swimming exercise that does not induce hypoglycemia decreases brain glycogen associated with increased brain lactate, implying an importance of glycogen in brain energetics during intense exercise even without hypoglycemia. Activated serotonergic regulation is a possible underlying mechanism for intense exercise-induced glycogenolysis at least in the hippocampus and brainstem.

  12. QSAR modeling of the inhibition of glycogen synthase kinase-3.

    PubMed

    Katritzky, Alan R; Pacureanu, Liliana M; Dobchev, Dimitar A; Fara, Dan C; Duchowicz, Pablo R; Karelson, Mati

    2006-07-15

    Quantitative structure-activity relationship (QSAR) models of the biological activity (pIC50) of 277 inhibitors of Glycogen Synthase Kinase-3 (GSK-3) are developed using geometrical, topological, quantum mechanical, and electronic descriptors calculated by CODESSA PRO. The linear (multilinear regression) and nonlinear (artificial neural network) models obtained link the structures to their reported activity pIC50. The results are discussed in the light of the main factors that influence the inhibitory activity of the GSK-3 enzyme.

  13. Glycogen Synthase in Sertoli Cells: More Than Glycogenesis?

    PubMed

    Maldonado, Rodrigo; Mancilla, Héctor; Villarroel-Espíndola, Franz; Slebe, Felipe; Slebe, Juan Carlos; Méndez, Raúl; Guinovart, Joan J; Concha, Ilona I

    2016-11-01

    Sertoli cell metabolism actively maintains the nutritional needs of germ cells. It has been described that after glucose incorporation in Sertoli cells, less than 1% is converted to glycogen suggesting low levels of glycogen synthase activity. Phosphorylation of muscle glycogen synthase (MGS) at serine 640 (pS640MGS) decreases its activity, and this form of the enzyme was discovered as a non-ribosomal protein that modulates the translation of a subset of transcripts in HeLa cells. The aim of our study was to functionally characterize MGS in cultured Sertoli cells, as well as to explore this new feature related to RNA molecules. We detected MGS in the cytoplasm of Sertoli cells as well as in the nuclei. The activity rates of the enzyme were extremely low indicating that MGS is expressed but almost inactive. Protein targeting to glycogen (PTG) overexpression was performed to activate MGS by dephosphorylation. PTG induced glycogen synthesis massively, confirming that this enzyme is present but inactive. This finding correlates with high levels of pS640MGS, which were assayed by phosphatase treatment. To explore a putative new function for MGS in Sertoli cells, we performed RNA immunoprecipitation coupled to microarray studies. The results revealed that MGS co-immunoprecipitated with the several mRNAs and also rRNAs. These findings indicate that MGS is expressed Sertoli cells but in an inactive form, and also support a possibly novel feature of this metabolic enzyme associated with RNA-related molecules. J. Cell. Biochem. 117: 2597-2607, 2016. © 2016 Wiley Periodicals, Inc. PMID:27017955

  14. Glycogen Synthase in Sertoli Cells: More Than Glycogenesis?

    PubMed

    Maldonado, Rodrigo; Mancilla, Héctor; Villarroel-Espíndola, Franz; Slebe, Felipe; Slebe, Juan Carlos; Méndez, Raúl; Guinovart, Joan J; Concha, Ilona I

    2016-11-01

    Sertoli cell metabolism actively maintains the nutritional needs of germ cells. It has been described that after glucose incorporation in Sertoli cells, less than 1% is converted to glycogen suggesting low levels of glycogen synthase activity. Phosphorylation of muscle glycogen synthase (MGS) at serine 640 (pS640MGS) decreases its activity, and this form of the enzyme was discovered as a non-ribosomal protein that modulates the translation of a subset of transcripts in HeLa cells. The aim of our study was to functionally characterize MGS in cultured Sertoli cells, as well as to explore this new feature related to RNA molecules. We detected MGS in the cytoplasm of Sertoli cells as well as in the nuclei. The activity rates of the enzyme were extremely low indicating that MGS is expressed but almost inactive. Protein targeting to glycogen (PTG) overexpression was performed to activate MGS by dephosphorylation. PTG induced glycogen synthesis massively, confirming that this enzyme is present but inactive. This finding correlates with high levels of pS640MGS, which were assayed by phosphatase treatment. To explore a putative new function for MGS in Sertoli cells, we performed RNA immunoprecipitation coupled to microarray studies. The results revealed that MGS co-immunoprecipitated with the several mRNAs and also rRNAs. These findings indicate that MGS is expressed Sertoli cells but in an inactive form, and also support a possibly novel feature of this metabolic enzyme associated with RNA-related molecules. J. Cell. Biochem. 117: 2597-2607, 2016. © 2016 Wiley Periodicals, Inc.

  15. Purification and characterization of purine nucleoside phosphorylase from developing embryos of Hyalomma dromedarii.

    PubMed

    Kamel, M Y; Fahmy, A S; Ghazy, A H; Mohamed, M A

    1991-04-01

    Purine nucleoside phosphorylase from Hyalomma dromedarii, the camel tick, was purified to apparent homogeneity. A molecular weight of 56,000 - 58,000 was estimated for both the native and denatured enzyme, suggesting that the enzyme is monomeric. Unlike purine nucleoside phosphorylase preparations from other tissues, the H. dromedarii enzyme was unstable in the presence of beta-mercaptoethanol. The enzyme had a sharp pH optimum at pH 6.5. It catalyzed the phosphorolysis and arsenolysis of ribo- and deoxyribo-nucleosides of hypoxanthine and guanine, but not of adenine or pyrimidine nucleosides. The Km values of the enzyme at the optimal pH for inosine, deoxyinosine, guanosine, and deoxyguanosine were 0.31, 0.67, 0.55, and 0.33 mM, respectively. Inactivation and kinetic studies suggested that histidine and cysteine residues were essential for activity. The pKa values determined for catalytic ionizable groups were 6-7 and 8-9. The enzyme was completely inactivated by thiol reagents and reactivated by excess beta-mercaptoethanol. The enzyme was also susceptible to pH-dependent photooxidation in the presence of methylene blue, implicating histidine. Initial velocity studies showed an intersecting pattern of double-reciprocal plots of the data, consistent with a sequential mechanism. PMID:1905141

  16. MicroRNA-31 targets FIH-1 to positively regulate corneal epithelial glycogen metabolism.

    PubMed

    Peng, Han; Hamanaka, Robert B; Katsnelson, Julia; Hao, Liang-Liang; Yang, Wending; Chandel, Navdeep S; Lavker, Robert M

    2012-08-01

    Corneal epithelium relies on abundant glycogen stores as its primary energy source. MicroRNA-31 (miR-31), a corneal epithelial-preferred miRNA, negatively regulates factor inhibiting hypoxia-inducible factor-1 (FIH-1). Since HIF-1α is involved in anaerobic energy production, we investigated the role that miR-31 and FIH-1 play in regulating corneal epithelial glycogen. We used antagomirs (antago) to reduce the level of miR-31 in primary human corneal epithelial keratinocytes (HCEKs), and a miR-31-resistant FIH-1 to increase FIH-1 levels. Antago-31 raised FIH-1 levels and significantly reduced glycogen stores in HCEKs compared to irrelevant-antago treatment. Similarly, HCEKs retrovirally transduced with a miR-31-resistant FIH-1 had markedly reduced glycogen levels compared with empty vector controls. In addition, we observed no change in a HIF-1α reporter or known genes downstream of HIF-1α indicating that the action of FIH-1 and miR-31 on glycogen is HIF-1α-independent. An enzyme-dead FIH-1 mutation failed to restore glycogen stores, indicating that FIH-1 negatively regulates glycogen in a hydroxylase-independent manner. FIH-1 overexpression in HCEKs decreased AKT signaling, activated GSK-3β, and inactivated glycogen synthase. Treatment of FIH-1-transduced HCEKs with either a myristolated Akt or a GSK-3β inhibitor restored glycogen stores, confirming the direct involvement of Akt/GSK-3β signaling. Silencing FIH-1 in HCEKs reversed the observed changes in Akt-signaling. Glycogen regulation in a HIF-1α-independent manner is a novel function for FIH-1 and provides new insight into how the corneal epithelium regulates its energy requirements.

  17. Mutations in PHKA2 are responsible for X-linked liver glycogen storage disease

    SciTech Connect

    Hendrickx, J.; Coucke, P.; Dams, E.

    1994-09-01

    X-linked liver glycogenosis type I (XLG I) is due to a deficiency of phosphorylase kinase (PHK), a key enzyme in the control of glycogen breakdown. XLG I is the most common glycogen storage disease. Patients show hepatomegaly, growth retardation and elevation of liver enzymes as their main clinical symptoms. We assigned the XLG I gene to the chromosomal region Xp22 by linkage analysis in six XLG I families. As the liver {alpha}-subunit of PHK (PHKA2) was also localized to Xp22, PHKA2 was considered a candidate gene for XLG I. In this study, we searched for mutations in 6 exons of the PHDA2 gene of 9 unrelated XLG I patients by SSCP analysis. This revealed three point mutations present in three different patients. Two of these mutations introduce a premature stop codon leading to a truncated protein. The third mutation abolishes a 5{prime} splice site consensus sequence leading to exon skipping. All three mutations therefore result in a PHKA2 protein that lacks several amino acids, what most probably affects enzyme function or stability. These findings indicate that PHKA2 is the XLG I gene.

  18. Genetics Home Reference: glycogen storage disease type VII

    MedlinePlus

    ... Health Conditions glycogen storage disease type VII glycogen storage disease type VII Enable Javascript to view the ... Download PDF Open All Close All Description Glycogen storage disease type VII (GSDVII) is an inherited disorder ...

  19. Pluralistic roles for glycogen in the central and peripheral nervous systems.

    PubMed

    Fryer, Kirsty L; Brown, Angus M

    2015-02-01

    Glycogen is present in the mammalian nervous system, but at concentrations of up to one hundred times lower than those found in liver and skeletal muscle. This relatively low concentration has resulted in neglect of assigning a role(s) for brain glycogen, but in the last 15 years enormous progress has been made in revealing the multifaceted roles that glycogen plays in the mammalian nervous system. Initial studies highlighted a role for glycogen in supporting neural elements (neurons and axons) during aglycemia, where glycogen supplied supplementary energy substrate in the form of lactate to fuel neural oxidative metabolism. The appropriate enzymes and membrane bound transporters have been localized to cellular locations consistent with astrocyte to neuron energy substrate shuttling. A role for glycogen in supporting the induction of long term potential (LTP) in the hippocampus has recently been described, where glycogen is metabolized to lactate and shuttled to neurons via the extracellular space by monocarboxylate transporters, where it plays an integral role in the induction process of LTP. This is the first time that glycogen has been assigned a role in a distinct, complex physiological brain function, where the lack of glycogen, in the presence of normoglycemia, results in disturbance of the function. The signalling pathway that alerts astrocytes to increased neuronal activity has been recently described, highlighting a pivotal role for increased extracellular potassium ([K(+)]o) that routinely accompanies increased neural activity. An astrocyte membrane bound bicarbonate transporter is activated by the [K(+)]o, the resulting increase in intracellular bicarbonate alkalizing the cell's interior and activating soluble adenyl cyclase (sAC). The sAC promotes glycogenolysis via increases in cyclic AMP, ultimately producing lactate, which is shuttled out of the astrocyte and presumably taken up by neurons from the extracellular space.

  20. Natural Progression of Canine Glycogen Storage Disease Type IIIa

    PubMed Central

    Brooks, Elizabeth D; Yi, Haiqing; Austin, Stephanie L; Thurberg, Beth L; Young, Sarah P; Fyfe, John C; Kishnani, Priya S; Sun, Baodong

    2016-01-01

    Glycogen storage disease type IIIa (GSD IIIa) is caused by a deficiency of glycogen debranching enzyme activity. Hepatomegaly, muscle degeneration, and hypoglycemia occur in human patients at an early age. Long-term complications include liver cirrhosis, hepatic adenomas, and generalized myopathy. A naturally occurring canine model of GSD IIIa that mimics the human disease has been described, with progressive liver disease and skeletal muscle damage likely due to excess glycogen deposition. In the current study, long-term follow-up of previously described GSD IIIa dogs until 32 mo of age (n = 4) and of family-owned GSD IIIa dogs until 11 to 12 y of age (n = 2) revealed that elevated concentrations of liver and muscle enzyme (AST, ALT, ALP, and creatine phosphokinase) decreased over time, consistent with hepatic cirrhosis and muscle fibrosis. Glycogen deposition in many skeletal muscles; the tongue, diaphragm, and heart; and the phrenic and sciatic nerves occurred also. Furthermore, the urinary biomarker Glc4, which has been described in many types of GSD, was first elevated and then decreased later in life. This urinary biomarker demonstrated a similar trend as AST and ALT in GSD IIIa dogs, indicating that Glc4 might be a less invasive biomarker of hepatocellular disease. Finally, the current study further demonstrates that the canine GSD IIIa model adheres to the clinical course in human patients with this disorder and is an appropriate model for developing novel therapies. PMID:26884409

  1. Brain glycogen supercompensation following exhaustive exercise.

    PubMed

    Matsui, Takashi; Ishikawa, Taro; Ito, Hitoshi; Okamoto, Masahiro; Inoue, Koshiro; Lee, Min-Chul; Fujikawa, Takahiko; Ichitani, Yukio; Kawanaka, Kentaro; Soya, Hideaki

    2012-02-01

    Brain glycogen localized in astrocytes, a critical energy source for neurons, decreases during prolonged exhaustive exercise with hypoglycaemia. However, it is uncertain whether exhaustive exercise induces glycogen supercompensation in the brain as in skeletal muscle. To explore this question, we exercised adult male rats to exhaustion at moderate intensity (20 m min(-1)) by treadmill, and quantified glycogen levels in several brain loci and skeletal muscles using a high-power (10 kW) microwave irradiation method as a gold standard. Skeletal muscle glycogen was depleted by 82-90% with exhaustive exercise, and supercompensated by 43-46% at 24 h after exercise. Brain glycogen levels decreased by 50-64% with exhaustive exercise, and supercompensated by 29-63% (whole brain 46%, cortex 60%, hippocampus 33%, hypothalamus 29%, cerebellum 63% and brainstem 49%) at 6 h after exercise. The brain glycogen supercompensation rates after exercise positively correlated with their decrease rates during exercise. We also observed that cortical and hippocampal glycogen supercompensation were sustained until 24 h after exercise (long-lasting supercompensation), and their basal glycogen levels increased with 4 weeks of exercise training (60 min day(-1) at 20 m min(-1)). These results support the hypothesis that, like the effect in skeletal muscles, glycogen supercompensation also occurs in the brain following exhaustive exercise, and the extent of supercompensation is dependent on that of glycogen decrease during exercise across brain regions. However, supercompensation in the brain preceded that of skeletal muscles. Further, the long-lasting supercompensation of the cortex and hippocampus is probably a prerequisite for their training adaptation (increased basal levels), probably to meet the increased energy demands of the brain in exercising animals.

  2. [The effect of light on glycogen turnover in the retina of the honeybee drone (author's transl)].

    PubMed

    Tsacopoulos, M; Evèquoz, V

    1980-04-01

    The retina of the compound eye of the drone (Apis mellifera) comprises two distinct classes of cells: the photoreceptors and the glial cells. The photoreceptors contain mitochondriae and the photopigment (rhodopsin). The glial cells do not contain mitochondriae, but large amounts of glycogen granules. Light stimulation causes intraglial glycogen metabolism to accelerate. Since glial cells are not directly excitable by light, it is deduced that the absorption of light by rhodopsin must somehow cause a signal to pass the glial cells to activate the glycogen metabolism.

  3. Enzymatic Glycosylation of Phenolic Antioxidants: Phosphorylase-Mediated Synthesis and Characterization.

    PubMed

    De Winter, Karel; Dewitte, Griet; Dirks-Hofmeister, Mareike E; De Laet, Sylvie; Pelantová, Helena; Křen, Vladimír; Desmet, Tom

    2015-11-25

    Although numerous biologically active molecules exist as glycosides in nature, information on the activity, stability, and solubility of glycosylated antioxidants is rather limited to date. In this work, a wide variety of antioxidants were glycosylated using different phosphorylase enzymes. The resulting antioxidant library, containing α/β-glucosides, different regioisomers, cellobiosides, and cellotriosides, was then characterized. Glycosylation was found to significantly increase the solubility and stability of all evaluated compounds. Despite decreased radical-scavenging abilities, most glycosides were identified to be potent antioxidants, outperforming the commonly used 2,6-bis(1,1-dimethylethyl)-4-methylphenol (BHT). Moreover, the point of attachment, the anomeric configuration, and the glycosidic chain length were found to influence the properties of these phenolic glycosides.

  4. Enzymatic Glycosylation of Phenolic Antioxidants: Phosphorylase-Mediated Synthesis and Characterization.

    PubMed

    De Winter, Karel; Dewitte, Griet; Dirks-Hofmeister, Mareike E; De Laet, Sylvie; Pelantová, Helena; Křen, Vladimír; Desmet, Tom

    2015-11-25

    Although numerous biologically active molecules exist as glycosides in nature, information on the activity, stability, and solubility of glycosylated antioxidants is rather limited to date. In this work, a wide variety of antioxidants were glycosylated using different phosphorylase enzymes. The resulting antioxidant library, containing α/β-glucosides, different regioisomers, cellobiosides, and cellotriosides, was then characterized. Glycosylation was found to significantly increase the solubility and stability of all evaluated compounds. Despite decreased radical-scavenging abilities, most glycosides were identified to be potent antioxidants, outperforming the commonly used 2,6-bis(1,1-dimethylethyl)-4-methylphenol (BHT). Moreover, the point of attachment, the anomeric configuration, and the glycosidic chain length were found to influence the properties of these phenolic glycosides. PMID:26540621

  5. Prolyl isomerase Pin1 promotes amyloid precursor protein (APP) turnover by inhibiting glycogen synthase kinase-3β (GSK3β) activity: novel mechanism for Pin1 to protect against Alzheimer disease.

    PubMed

    Ma, Suk Ling; Pastorino, Lucia; Zhou, Xiao Zhen; Lu, Kun Ping

    2012-03-01

    Alzheimer disease (AD) is characterized by the presence of senile plaques of amyloid-β (Aβ) peptides derived from amyloid precursor protein (APP) and neurofibrillary tangles made of hyperphosphorylated Tau. Increasing APP gene dosage or expression has been shown to cause familial early-onset AD. However, whether and how protein stability of APP is regulated is unclear. The prolyl isomerase Pin1 and glycogen synthase kinase-3β (GSK3β) have been shown to have the opposite effects on APP processing and Tau hyperphosphorylation, relevant to the pathogenesis of AD. However, nothing is known about their relationship. In this study, we found that Pin1 binds to the pT330-P motif in GSK3β to inhibit its kinase activity. Furthermore, Pin1 promotes protein turnover of APP by inhibiting GSK3β activity. A point mutation either at Thr-330, the Pin1-binding site in GSK3β, or at Thr-668, the GSK3β phosphorylation site in APP, abolished the regulation of GSK3β activity, Thr-668 phosphorylation, and APP stability by Pin1, resulting in reduced non-amyloidogenic APP processing and increased APP levels. These results uncover a novel role of Pin1 in inhibiting GSK3β kinase activity to reduce APP protein levels, providing a previously unrecognized mechanism by which Pin1 protects against Alzheimer disease.

  6. Glycogen in honeybee queens, workers and drones (Apis mellifera carnica Pollm.).

    PubMed

    Crailsheim, K; Panzenböck, U

    1997-02-21

    Honey bees (Apis mellifera carnica Pollm.) have low glycogen reserves in summer. Upon emergence drones have significantly larger amounts per unit weight when emerging, than workers; perhaps as adaption to the risk of not being fed as intensely as young workers. Maximum content was 0.23mg for workers (28d), and 0.59mg for drones (after emergence). Workers have relatively constant glycogen contents during their life, and very young drones have more glycogen than older ones. Young queens are similar to workers. In workers and queens in summer the greatest amounts of glycogen are found in the thorax. When the bees start flying (6th-8th day of life), drones have the highest amounts in the head (probably to supply their eyes), and upon maturity, drones have the least glycogen in the abdomen.Workers in winter show different glycogen values depending on whether they are active bees from the core area (0.23mg) or inactive ones from the outer surface of the winter cluster (0.37mg). They use glycogen from the thorax and the abdomen for their ongoing energy need.

  7. Human α-amylase present in lower-genital-tract mucosal fluid processes glycogen to support vaginal colonization by Lactobacillus.

    PubMed

    Spear, Gregory T; French, Audrey L; Gilbert, Douglas; Zariffard, M Reza; Mirmonsef, Paria; Sullivan, Thomas H; Spear, William W; Landay, Alan; Micci, Sandra; Lee, Byung-Hoo; Hamaker, Bruce R

    2014-10-01

    Lactobacillus colonization of the lower female genital tract provides protection from the acquisition of sexually transmitted diseases, including human immunodeficiency virus, and from adverse pregnancy outcomes. While glycogen in vaginal epithelium is thought to support Lactobacillus colonization in vivo, many Lactobacillus isolates cannot utilize glycogen in vitro. This study investigated how glycogen could be utilized by vaginal lactobacilli in the genital tract. Several Lactobacillus isolates were confirmed to not grow in glycogen, but did grow in glycogen-breakdown products, including maltose, maltotriose, maltopentaose, maltodextrins, and glycogen treated with salivary α-amylase. A temperature-dependent glycogen-degrading activity was detected in genital fluids that correlated with levels of α-amylase. Treatment of glycogen with genital fluids resulted in production of maltose, maltotriose, and maltotetraose, the major products of α-amylase digestion. These studies show that human α-amylase is present in the female lower genital tract and elucidates how epithelial glycogen can support Lactobacillus colonization in the genital tract.

  8. Biochemical titration of glycogen in vitro.

    PubMed

    Pelletier, Joffrey; Bellot, Grégory; Pouysségur, Jacques; Mazure, Nathalie M

    2013-01-01

    Glycogen is the main energetic polymer of glucose in vertebrate animals and plays a crucial role in whole body metabolism as well as in cellular metabolism. Many methods to detect glycogen already exist but only a few are quantitative. We describe here a method using the Abcam Glycogen assay kit, which is based on specific degradation of glycogen to glucose by glucoamylase. Glucose is then specifically oxidized to a product that reacts with the OxiRed probe to produce fluorescence. Titration is accurate, sensitive and can be achieved on cell extracts or tissue sections. However, in contrast to other techniques, it does not give information about the distribution of glycogen in the cell. As an example of this technique, we describe here the titration of glycogen in two cell lines, Chinese hamster lung fibroblast CCL39 and human colon carcinoma LS174, incubated in normoxia (21% O2) versus hypoxia (1% O2). We hypothesized that hypoxia is a signal that prepares cells to synthesize and store glycogen in order to survive(1).

  9. Biochemical Titration of Glycogen In vitro

    PubMed Central

    Pelletier, Joffrey; Bellot, Grégory; Pouysségur, Jacques; Mazure, Nathalie M.

    2013-01-01

    Glycogen is the main energetic polymer of glucose in vertebrate animals and plays a crucial role in whole body metabolism as well as in cellular metabolism. Many methods to detect glycogen already exist but only a few are quantitative. We describe here a method using the Abcam Glycogen assay kit, which is based on specific degradation of glycogen to glucose by glucoamylase. Glucose is then specifically oxidized to a product that reacts with the OxiRed probe to produce fluorescence. Titration is accurate, sensitive and can be achieved on cell extracts or tissue sections. However, in contrast to other techniques, it does not give information about the distribution of glycogen in the cell. As an example of this technique, we describe here the titration of glycogen in two cell lines, Chinese hamster lung fibroblast CCL39 and human colon carcinoma LS174, incubated in normoxia (21% O2) versus hypoxia (1% O2). We hypothesized that hypoxia is a signal that prepares cells to synthesize and store glycogen in order to survive1. PMID:24300406

  10. Evaluation of capillary chromatographic supports for immobilized human purine nucleoside phosphorylase in frontal affinity chromatography studies.

    PubMed

    de Moraes, Marcela Cristina; Temporini, Caterina; Calleri, Enrica; Bruni, Giovanna; Ducati, Rodrigo Gay; Santos, Diógenes Santiago; Cardoso, Carmen Lucia; Cass, Quezia Bezerra; Massolini, Gabriella

    2014-04-18

    The aim of this work was to optimize the preparation of a capillary human purine nucleoside phosphorylase (HsPNP) immobilized enzyme reactor (IMER) for characterization and affinity screening studies of new inhibitors by frontal affinity chromatography coupled to mass spectrometry (FAC-MS). For this purpose two monolithic supports, a Chromolith Speed Rod (0.1mm I.D.×5cm) and a methacrylate-based monolithic epoxy polymeric capillary column (0.25mm I.D.×5cm) with epoxy reactive groups were considered and compared to an IMER previously developed using an open fused silica capillary. Each HsPNP-IMER was characterized in terms of catalytic activity using Inosine as standard substrate. Furthermore, they were also explored for affinity ranking experiments. Kd determination was carried out with the based fused silica HsPNP-IMER and the results are herein discussed.

  11. Hit Optimization of 5-Substituted-N-(piperidin-4-ylmethyl)-1H-indazole-3-carboxamides: Potent Glycogen Synthase Kinase-3 (GSK-3) Inhibitors with in Vivo Activity in Model of Mood Disorders.

    PubMed

    Furlotti, Guido; Alisi, Maria Alessandra; Cazzolla, Nicola; Dragone, Patrizia; Durando, Lucia; Magarò, Gabriele; Mancini, Francesca; Mangano, Giorgina; Ombrato, Rosella; Vitiello, Marco; Armirotti, Andrea; Capurro, Valeria; Lanfranco, Massimiliano; Ottonello, Giuliana; Summa, Maria; Reggiani, Angelo

    2015-11-25

    Novel treatments for bipolar disorder with improved efficacy and broader spectrum of activity are urgently needed. Glycogen synthase kinase 3β (GSK-3β) has been suggested to be a key player in the pathophysiology of bipolar disorder. A series of novel GSK-3β inhibitors having the common N-[(1-alkylpiperidin-4-yl)methyl]-1H-indazole-3-carboxamide scaffold were prepared taking advantage of an X-ray cocrystal structure of compound 5 with GSK-3β. We probed different substitutions at the indazole 5-position and at the piperidine-nitrogen to obtain potent ATP-competitive GSK-3β inhibitors with good cell activity. Among the compounds assessed in the in vivo PK experiments, 14i showed, after i.p. dosing, encouraging plasma PK profile and brain exposure, as well as efficacy in a mouse model of mania. Compound 14i was selected for further in vitro/in vivo pharmacological evaluation, in order to elucidate the use of ATP-competitive GSK-3β inhibitors as new tools in the development of new treatments for mood disorders.

  12. Effect of pH on Cleavage of Glycogen by Vaginal Enzymes.

    PubMed

    Spear, Greg T; McKenna, Mary; Landay, Alan L; Makinde, Hadijat; Hamaker, Bruce; French, Audrey L; Lee, Byung-Hoo

    2015-01-01

    Glycogen expressed by the lower genital tract epithelium is believed to support Lactobacillus growth in vivo, although most genital isolates of Lactobacillus are not able to use glycogen as an energy source in vitro. We recently reported that α-amylase is present in the genital fluid of women and that it breaks down glycogen into small carbohydrates that support growth of lactobacilli. Since the pH of the lower genital tract can be very low, we determined how low pH affects glycogen processing by α-amylase. α-amylase in saliva degraded glycogen similarly at pH 6 and 7, but activity was reduced by 52% at pH 4. The glycogen degrading activity in nine genital samples from seven women showed a similar profile with an average reduction of more than 50% at pH 4. However, two samples collected from one woman at different times had a strikingly different pH profile with increased glycogen degradation at pH 4, 5 and 6 compared to pH 7. This second pH profile did not correlate with levels of human α-acid glucosidase or human intestinal maltase glucoamylase. High-performance anion-exchange chromatography showed that mostly maltose was produced from glycogen by samples with the second pH profile in contrast to genital α-amylase that yielded maltose, maltotriose and maltotetraose. These studies show that at low pH, α-amylase activity is reduced to low but detectable levels, which we speculate helps maintain Lactobacillus growth at a limited but sustained rate. Additionally, some women have a genital enzyme distinct from α-amylase with higher activity at low pH. Further studies are needed to determine the identity and distribution of this second enzyme, and whether its presence influences the makeup of genital microbiota.

  13. Effect of pH on Cleavage of Glycogen by Vaginal Enzymes.

    PubMed

    Spear, Greg T; McKenna, Mary; Landay, Alan L; Makinde, Hadijat; Hamaker, Bruce; French, Audrey L; Lee, Byung-Hoo

    2015-01-01

    Glycogen expressed by the lower genital tract epithelium is believed to support Lactobacillus growth in vivo, although most genital isolates of Lactobacillus are not able to use glycogen as an energy source in vitro. We recently reported that α-amylase is present in the genital fluid of women and that it breaks down glycogen into small carbohydrates that support growth of lactobacilli. Since the pH of the lower genital tract can be very low, we determined how low pH affects glycogen processing by α-amylase. α-amylase in saliva degraded glycogen similarly at pH 6 and 7, but activity was reduced by 52% at pH 4. The glycogen degrading activity in nine genital samples from seven women showed a similar profile with an average reduction of more than 50% at pH 4. However, two samples collected from one woman at different times had a strikingly different pH profile with increased glycogen degradation at pH 4, 5 and 6 compared to pH 7. This second pH profile did not correlate with levels of human α-acid glucosidase or human intestinal maltase glucoamylase. High-performance anion-exchange chromatography showed that mostly maltose was produced from glycogen by samples with the second pH profile in contrast to genital α-amylase that yielded maltose, maltotriose and maltotetraose. These studies show that at low pH, α-amylase activity is reduced to low but detectable levels, which we speculate helps maintain Lactobacillus growth at a limited but sustained rate. Additionally, some women have a genital enzyme distinct from α-amylase with higher activity at low pH. Further studies are needed to determine the identity and distribution of this second enzyme, and whether its presence influences the makeup of genital microbiota. PMID:26171967

  14. Computer-generated Model of Purine Nucleoside Phosphorylase (PNP)

    NASA Technical Reports Server (NTRS)

    1987-01-01

    Purine Nucleoside Phosphorylase (PNP) is an important target enzyme for the design of anti-cancer and immunosuppressive drugs. Bacterial PNP, which is slightly different from the human enzyme, is used to synthesize chemotherapuautic agents. Knowledge of the three-dimensional structure of the bacterial PNP molecule is useful in efforts to engineer different types of PNP enzymes, that can be used to produce new chemotherapeutic agents. This picture shows a computer model of bacterial PNP, which looks a lot like a display of colorful ribbons. Principal Investigator was Charles Bugg.

  15. AMPK and autophagy in glucose/glycogen metabolism.

    PubMed

    Ha, Joohun; Guan, Kun-Liang; Kim, Joungmok

    2015-12-01

    Glucose/glycogen metabolism is a primary metabolic pathway acting on a variety of cellular needs, such as proliferation, growth, and survival against stresses. The multiple regulatory mechanisms underlying a specific metabolic fate have been documented and explained the molecular basis of various pathophysiological conditions, including metabolic disorders and cancers. AMP-activated protein kinase (AMPK) has been appreciated for many years as a central metabolic regulator to inhibit energy-consuming pathways as well as to activate the compensating energy-producing programs. In fact, glucose starvation is a potent physiological AMPK activating condition, in which AMPK triggers various subsequent metabolic events depending on cells or tissues. Of note, the recent studies show bidirectional interplay between AMPK and glycogen. A growing number of studies have proposed additional level of metabolic regulation by a lysosome-dependent catabolic program, autophagy. Autophagy is a critical degradative pathway not only for maintenance of cellular homeostasis to remove potentially dangerous constituents, such as protein aggregates and dysfunctional subcellular organelles, but also for adaptive responses to metabolic stress, such as nutrient starvation. Importantly, many lines of evidence indicate that autophagy is closely connected with nutrient signaling modules, including AMPK, to fine-tune the metabolic pathways in response to many different cellular cues. In this review, we introduce the studies demonstrating the role of AMPK and autophagy in glucose/glycogen metabolism. Also, we describe the recent advances on their contributions to the metabolic disorders.

  16. Carbohydrates, Muscle Glycogen, and Improved Performance.

    ERIC Educational Resources Information Center

    Sherman, W. Mike

    1987-01-01

    One way to improve athletic performance without harming the athlete's health is diet manipulation. This article explores the relationship between muscular endurance and muscle glycogen and discusses a diet and training approach to competition. (Author/MT)

  17. ¹³C MRS reveals a small diurnal variation in the glycogen content of human thigh muscle.

    PubMed

    Takahashi, Hideyuki; Kamei, Akiko; Osawa, Takuya; Kawahara, Takashi; Takizawa, Osamu; Maruyama, Katsuya

    2015-06-01

    There is marked diurnal variation in the glycogen content of skeletal muscles of animals, but few studies have addressed such variations in human muscles. (13)C MRS can be used to noninvasively measure the glycogen content of human skeletal muscle, but no study has explored the diurnal variations in this parameter. This study aimed to investigate whether a diurnal variation in glycogen content occurs in human muscles and, if so, to what extent it can be identified using (13)C MRS. Six male volunteers were instructed to maintain their normal diet and not to perform strenuous exercise for at least 3 days before and during the experiment. Muscle glycogen and blood glucose concentrations were measured six times in 24 h under normal conditions in these subjects. The glycogen content in the thigh muscle was determined noninvasively by natural abundance (13)C MRS using a clinical MR system at 3 T. Nutritional analysis revealed that the subjects' mean carbohydrate intake was 463 ± 137 g, being approximately 6.8 ± 2.4 g/kg body weight. The average sleeping time was 5.9 ± 1.0 h. The glycogen content in the thigh muscle at the starting point was 64.8 ± 20.6 mM. Although absolute and relative individual variations in muscle glycogen content were 7.0 ± 2.1 mM and 11.3 ± 4.6%, respectively, no significant difference in glycogen content was observed among the different time points. This study demonstrates that normal food intake (not fat and/or carbohydrate rich), sleep and other daily activities have a negligible influence on thigh muscle glycogen content, and that the diurnal variation of the glycogen content in human muscles is markedly smaller than that in animal muscles. Moreover, the present results also support the reproducibility and availability of (13)C MRS for the evaluation of the glycogen content in human muscles.

  18. Oligosaccharide Binding in Escherichia coli Glycogen Synthase

    SciTech Connect

    Sheng, Fang; Yep, Alejandra; Feng, Lei; Preiss, Jack; Geiger, James H.

    2010-11-17

    Glycogen/starch synthase elongates glucan chains and is the key enzyme in the synthesis of glycogen in bacteria and starch in plants. Cocrystallization of Escherichia coli wild-type glycogen synthase (GS) with substrate ADPGlc and the glucan acceptor mimic HEPPSO produced a closed form of GS and suggests that domain-domain closure accompanies glycogen synthesis. Cocrystallization of the inactive GS mutant E377A with substrate ADPGlc and oligosaccharide results in the first oligosaccharide-bound glycogen synthase structure. Four bound oligosaccharides are observed, one in the interdomain cleft (G6a) and three on the N-terminal domain surface (G6b, G6c, and G6d). Extending from the center of the enzyme to the interdomain cleft opening, G6a mostly interacts with the highly conserved N-terminal domain residues lining the cleft of GS. The surface-bound oligosaccharides G6c and G6d have less interaction with enzyme and exhibit a more curled, helixlike structural arrangement. The observation that oligosaccharides bind only to the N-terminal domain of GS suggests that glycogen in vivo probably binds to only one side of the enzyme to ensure unencumbered interdomain movement, which is required for efficient, continuous glucan-chain synthesis.

  19. Structural bases for N-glycan processing by mannoside phosphorylase.

    PubMed

    Ladevèze, Simon; Cioci, Gianluca; Roblin, Pierre; Mourey, Lionel; Tranier, Samuel; Potocki-Véronèse, Gabrielle

    2015-06-01

    The first crystal structure of Uhgb_MP, a β-1,4-mannopyranosyl-chitobiose phosphorylase belonging to the GH130 family which is involved in N-glycan degradation by human gut bacteria, was solved at 1.85 Å resolution in the apo form and in complex with mannose and N-acetylglucosamine. SAXS and crystal structure analysis revealed a hexameric structure, a specific feature of GH130 enzymes among other glycoside phosphorylases. Mapping of the -1 and +1 subsites in the presence of phosphate confirmed the conserved Asp104 as the general acid/base catalytic residue, which is in agreement with a single-step reaction mechanism involving Man O3 assistance for proton transfer. Analysis of this structure, the first to be solved for a member of the GH130_2 subfamily, revealed Met67, Phe203 and the Gly121-Pro125 loop as the main determinants of the specificity of Uhgb_MP and its homologues towards the N-glycan core oligosaccharides and mannan, and the molecular bases of the key role played by GH130 enzymes in the catabolism of dietary fibre and host glycans.

  20. Structural bases for N-glycan processing by mannoside phosphorylase

    PubMed Central

    Ladevèze, Simon; Cioci, Gianluca; Roblin, Pierre; Mourey, Lionel; Tranier, Samuel; Potocki-Véronèse, Gabrielle

    2015-01-01

    The first crystal structure of Uhgb_MP, a β-1,4-mannopyranosyl-chitobiose phosphorylase belonging to the GH130 family which is involved in N-glycan degradation by human gut bacteria, was solved at 1.85 Å resolution in the apo form and in complex with mannose and N-acetylglucosamine. SAXS and crystal structure analysis revealed a hexameric structure, a specific feature of GH130 enzymes among other glycoside phosphorylases. Mapping of the −1 and +1 subsites in the presence of phosphate confirmed the conserved Asp104 as the general acid/base catalytic residue, which is in agreement with a single-step reaction mechanism involving Man O3 assistance for proton transfer. Analysis of this structure, the first to be solved for a member of the GH130_2 subfamily, revealed Met67, Phe203 and the Gly121–Pro125 loop as the main determinants of the specificity of Uhgb_MP and its homologues towards the N-glycan core oligosaccharides and mannan, and the molecular bases of the key role played by GH130 enzymes in the catabolism of dietary fibre and host glycans. PMID:26057673

  1. The Saccharomyces cerevisiae YPR184w gene encodes the glycogen debranching enzyme.

    PubMed

    Teste, M A; Enjalbert, B; Parrou, J L; François, J M

    2000-12-01

    The YPR184w gene encodes a 1536-amino acid protein that is 34-39% identical to the mammal, Drosophila melanogaster and Caenorhabditis elegans glycogen debranching enzyme. The N-terminal part of the protein possesses the four conserved sequences of the alpha-amylase superfamily, while the C-terminal part displays 50% similarity with the C-terminal of other eukaryotic glycogen debranching enzymes. Reliable measurement of alpha-1,4-glucanotransferase and alpha-1, 6-glucosidase activity of the yeast debranching enzyme was determined in strains overexpressing YPR184w. The alpha-1, 4-glucanotransferase activity of a partially purified preparation of debranching enzyme preferentially transferred maltosyl units than maltotriosyl. Deletion of YPR184w prevents glycogen degradation, whereas overexpression had no effect on the rate of glycogen breakdown. In response to stress and growth conditions, the transcriptional control of YPR184w gene, renamed GDB1 (for Glycogen DeBranching gene), is strictly identical to that of other genes involved in glycogen metabolism.

  2. Direct vs. indirect pathway of hepatic glycogen synthesis as a function of glucose infusion rate

    SciTech Connect

    Bagby, G.J.; Lang, C.H.; Johnson, J.L.; Blakesly, H.L.; Spitzer, J.J.

    1986-03-05

    This study was initiated to determine the influence of the rate of exogenous glucose administration on liver glycogen synthesis by the direct (glucose uptake and incorporation into glycogen) vs the indirect pathway (glucose degradation to 3-carbon intermediates, e.g., lactate, prior to incorporation into glycogen). Catheterized rats were fasted 2 days prior to receiving a 3 hr infusion of glucose at rates of 0 to 230 ..mu..mol/min/kg containing tracer (6-/sup 3/H)- and (U-/sup 14/C)-glucose. Plasma glucose (r = 0.80), insulin (r = 0.90) and lactate (r = 0.84) were correlated with glucose infusion rate. The rate of liver glycogen deposition (0.46 +/- 0.03 ..mu..mol/min/g) did not differ between a glucose infusion rate of 20 and 230 ..mu..mol/min/kg. At the lowest and highest glucose infusion rates hepatic glycogenesis accounted for 87 +/- 6 and 9 +/- 1% of the total glucose load, respectively. The percent contribution of the direct pathways to glycogen deposition ((/sup 3/H) specific activity in hepatic glycogen/(/sup 3/H) specific activity in plasma glucose) increased from 16 +/- 3 to 83 +/- 5% from lowest to highest glucose infusion rates (prevailing plasma glucose concentrations: 9 +/- 1 and 21 +/- 2 mM, respectively). The results indicate that the relative contribution of the direct and indirect pathways of glucogen synthesis are dependent upon the glucose load or plasma glucose concentration.

  3. Characterization of the highly branched glycogen from the thermoacidophilic red microalga Galdieria sulphuraria and comparison with other glycogens.

    PubMed

    Martinez-Garcia, Marta; Stuart, Marc C A; van der Maarel, Marc J E C

    2016-08-01

    The thermoacidophilic red microalga Galdieria sulphuraria synthesizes glycogen when growing under heterotrophic conditions. Structural characterization revealed that G. sulphuraria glycogen is the most highly branched glycogen described to date, with 18% of α-(1→6) linkages. Moreover, it differs from other glycogens because it is composed of short chains only and has a substantially smaller molecular weight and particle size. The physiological role of this highly branched glycogen in G. sulphuraria is discussed. PMID:27107958

  4. Identification of mutations in Type IV glycogen storage disease

    SciTech Connect

    Bao, Y.; Kishnani, P.; Chen, Y.T.

    1994-09-01

    Type IV glycogen storage disease (GSD IV, Andersen disease) is caused by a deficiency of glycogen branching enzyme (GBE) activity, which results in the accumulation of glycogen with unbranched, long, outer chains in the tissues. The molecular basis of the disease is not known. We studied four patients with the disease; three with typical presentation of progressive liver cirrhosis and failure, and one with severe and fatal neonatal hypotonia and cardiomyopathy. Southern blot analysis with EcoRI or MspI did not detect gross DNA rearrangement, deletion or duplication in patients` glycogen branching enzyme genes. Northern analysis with total cellular RNAs isolated from skin fibroblast MI strains of three patients with typical clinical presentation showed a normal level and size (2.95 kb) of GBE mRNA hybridization band in two and absent mRNA hybridization band in the remaining one. The patient with atypical severe neonatal hypotonia demonstrated a less intense and smaller size (2.75 kb) of mRNA hybridization band. A 210 hp deletion from nucleotide sequence 873 to 1082 which causes 70 amino acids missing from amino acid sequence 262 to 331 was detected in all 17 clones sequenced from the fatal hypotonia patient. This deletion is located in the region which is highly conserved between prokaryotic, yeast and human GBE polypeptide sequences, and also includes the first of the four regions which constitute the catalytic active sites of most of amylolytic enzymes. A point mutation C-T (1633) which changes the amino acid from Arginine to Cystine was found in 19 of 20 cDNA clones from a patient with classical clinical presentation. This point mutation was unique to this patient and was not observed in three other patients or normal controls. This is the first report on the molecular basis of GSD IV and our data indicated the presence of extensive genetic heterogeneity in the disease.

  5. [The regulation of glucose-6-phosphate dehydrogenase and glycogen synthase activities by insulin superfamily peptides in myometrium of pregnant women and its impairments under different types of diabetes mellitus].

    PubMed

    Kuznetsova, L A; Chistiakova, O V

    2009-01-01

    The regulatory effects of insulin, insulin-like growth factor 1 (IGF-1), and relaxin on glucose-6-phosphate dehydrogenase (G6PDH) and glycogen synthase (GS) activities have been studied in myometrium of pregnant women of control group and with diabetes mellitus of different etiology. In patients with type 1 diabetes G6PDH activity did not differ from the control group, but the enzyme activity was sharply decreased in pregnant women with type 2 diabetes and gestational diabetes. In the control group maximal stimulation of G6PDH activity was observed at 10(-9) M of peptides and their stimulating effect decreased in the following order: insulin > relaxin > IGF-1. In pregnant women with types 1 diabetes insulin effect on the enzyme activity was lower than in the control, and the effects of IGF-1 and relaxin were absent. In the group of pregnant women with type 2 diabetes and gestational diabetes the effects of insulin and IGF-1 were decreased, but the effect of relaxin was somewhat higher thus giving the following order in their efficiency relaxin > IGF-1 = insulin. At 10(-9) M peptides exhibited similar stimulating effects on the active form of GS-I, but had no influence on the total enzyme activity in the control group of pregnant women. In patients with type 1 diabetes GS activity remained unchanged (versus control), and peptides did not stimulate the enzyme activity. In patients with type 2 diabetes a significant decrease in GS activity was accompanied by the decrease in the effect of peptides, giving the following order of their efficiency: insulin = IGF-1 > relaxin. In myometrium of pregnant women with gestational (treated and untreated) diabetes GS activity decreased, the effect of insulin was weaker, whereas the effects of relaxin and IGF-1 increased thus giving the following order of their efficiency: relaxin > IGF-1 > insulin. Insulin therapy of type 1 diabetes incompletely restored sensitivity of the enzymes to the peptide actions. At the same time, in women

  6. Palmitate action to inhibit glycogen synthase and stimulate protein phosphatase 2A increases with risk factors for type 2 diabetes

    PubMed Central

    Mott, David M.; Stone, Karen; Gessel, Mary C.; Bunt, Joy C.; Bogardus, Clifton

    2008-01-01

    Recent studies have suggested that abnormal regulation of protein phosphatase 2A (PP2A) is associated with Type 2 diabetes in rodent and human tissues. Results with cultured mouse myotubes support a mechanism for palmitate activation of PP2A, leading to activation of glycogen synthase kinase 3. Phosphorylation and inactivation of glycogen synthase by glycogen synthase kinase 3 could be the mechanism for long-chain fatty acid inhibition of insulin-mediated carbohydrate storage in insulin-resistant subjects. Here, we test the effects of palmitic acid on cultured muscle glycogen synthase and PP2A activities. Palmitate inhibition of glycogen synthase fractional activity is increased in subjects with high body mass index compared with subjects with lower body mass index (r = −0.43, P = 0.03). Palmitate action on PP2A varies from inhibition in subjects with decreased 2-h plasma glucose concentration to activation in subjects with increased 2-h plasma glucose concentration (r = 0.45, P < 0.03) during oral glucose tolerance tests. The results do not show an association between palmitate effects on PP2A and glycogen synthase fractional activity. We conclude that subjects at risk for Type 2 diabetes have intrinsic differences in palmitate regulation of at least two enzymes (PP2A and glycogen synthase), contributing to abnormal insulin regulation of glucose metabolism. PMID:18056794

  7. Palmitate action to inhibit glycogen synthase and stimulate protein phosphatase 2A increases with risk factors for type 2 diabetes.

    PubMed

    Mott, David M; Stone, Karen; Gessel, Mary C; Bunt, Joy C; Bogardus, Clifton

    2008-02-01

    Recent studies have suggested that abnormal regulation of protein phosphatase 2A (PP2A) is associated with Type 2 diabetes in rodent and human tissues. Results with cultured mouse myotubes support a mechanism for palmitate activation of PP2A, leading to activation of glycogen synthase kinase 3. Phosphorylation and inactivation of glycogen synthase by glycogen synthase kinase 3 could be the mechanism for long-chain fatty acid inhibition of insulin-mediated carbohydrate storage in insulin-resistant subjects. Here, we test the effects of palmitic acid on cultured muscle glycogen synthase and PP2A activities. Palmitate inhibition of glycogen synthase fractional activity is increased in subjects with high body mass index compared with subjects with lower body mass index (r = -0.43, P = 0.03). Palmitate action on PP2A varies from inhibition in subjects with decreased 2-h plasma glucose concentration to activation in subjects with increased 2-h plasma glucose concentration (r = 0.45, P < 0.03) during oral glucose tolerance tests. The results do not show an association between palmitate effects on PP2A and glycogen synthase fractional activity. We conclude that subjects at risk for Type 2 diabetes have intrinsic differences in palmitate regulation of at least two enzymes (PP2A and glycogen synthase), contributing to abnormal insulin regulation of glucose metabolism.

  8. Energy Metabolism of the Brain, Including the Cooperation between Astrocytes and Neurons, Especially in the Context of Glycogen Metabolism

    PubMed Central

    Falkowska, Anna; Gutowska, Izabela; Goschorska, Marta; Nowacki, Przemysław; Chlubek, Dariusz; Baranowska-Bosiacka, Irena

    2015-01-01

    Glycogen metabolism has important implications for the functioning of the brain, especially the cooperation between astrocytes and neurons. According to various research data, in a glycogen deficiency (for example during hypoglycemia) glycogen supplies are used to generate lactate, which is then transported to neighboring neurons. Likewise, during periods of intense activity of the nervous system, when the energy demand exceeds supply, astrocyte glycogen is immediately converted to lactate, some of which is transported to the neurons. Thus, glycogen from astrocytes functions as a kind of protection against hypoglycemia, ensuring preservation of neuronal function. The neuroprotective effect of lactate during hypoglycemia or cerebral ischemia has been reported in literature. This review goes on to emphasize that while neurons and astrocytes differ in metabolic profile, they interact to form a common metabolic cooperation. PMID:26528968

  9. Energy Metabolism of the Brain, Including the Cooperation between Astrocytes and Neurons, Especially in the Context of Glycogen Metabolism.

    PubMed

    Falkowska, Anna; Gutowska, Izabela; Goschorska, Marta; Nowacki, Przemysław; Chlubek, Dariusz; Baranowska-Bosiacka, Irena

    2015-10-29

    Glycogen metabolism has important implications for the functioning of the brain, especially the cooperation between astrocytes and neurons. According to various research data, in a glycogen deficiency (for example during hypoglycemia) glycogen supplies are used to generate lactate, which is then transported to neighboring neurons. Likewise, during periods of intense activity of the nervous system, when the energy demand exceeds supply, astrocyte glycogen is immediately converted to lactate, some of which is transported to the neurons. Thus, glycogen from astrocytes functions as a kind of protection against hypoglycemia, ensuring preservation of neuronal function. The neuroprotective effect of lactate during hypoglycemia or cerebral ischemia has been reported in literature. This review goes on to emphasize that while neurons and astrocytes differ in metabolic profile, they interact to form a common metabolic cooperation.

  10. Studies on glycogen synthesis in pigeon liver homogenates. Incorporation of hexose into glycogen

    PubMed Central

    Nigam, V. N.; Fridland, A.

    1967-01-01

    Liver homogenates of avian species, but not of mammals, form glycogen from glucose, mannose, fructose and galactose. Incorporation of labelled glucose, fructose and mannose, but not of labelled galactose, into glycogen is diluted isotopically by unlabelled glucose. Except for fructose, glycogen formation from other substrates by pigeon liver homogenates compares favourably with that from the same substrates in pigeon liver slices. Optimum conditions for glycogen synthesis from glucose by pigeon liver homogenate are: medium of incubation, 0·175m-sucrose–45mm-potassium chloride–15mm-glycylglycine buffer, pH7·5; concentration of substrate, 15mm; concentration of tissue, less than 120mg./ml.; temperature of incubation, 37–43°; atmosphere, oxygen. Uncouplers of oxidative phosphorylation, Ca2+, EDTA, PPi, 2-deoxyglucose 6-phosphate and microsomal fraction of rat liver are inhibitory to glycogen synthesis from glucose. Starvation of pigeons for 24 and 48hr. leads to a slight stimulation of glycogen synthesis in their liver homogenates as compared with fed controls. Pigeon liver homogenates can be separated into subcellular fractions that on reconstitution can synthesize glycogen. All the enzymes of the glycogen pathway except soluble high-Km glucokinase are present in pigeon liver. PMID:5583993

  11. Investigation of potential glycogen synthase kinase 3 inhibitors using pharmacophore mapping and virtual screening.

    PubMed

    Dessalew, Nigus; Bharatam, Prasad V

    2006-09-01

    Glycogen synthase kinase-3 is a serine/threonine kinase that has attracted significant drug discovery attention in recent years. To investigate the identification of new potential glycogen synthase kinase-3 inhibitors, a pharmacophore mapping study was carried out using a set of 21 structurally diverse glycogen synthase kinase-3 inhibitors. A hypothesis containing four features: two hydrophobic, one hydrogen bond donor and another hydrogen bond acceptor was found to be the best from the 10 common feature hypotheses produced by HipHop module of Catalyst. The best hypothesis has a high cost of 156.592 and higher best fit values were obtained for the 21 inhibitors using this best hypothesis than the other HipHop hypotheses. The best hypothesis was then used to screen electronically the NCI2000 database. The hits obtained were docked into glycogen synthase kinase-3beta active site. A total of five novel potential leads were proposed after: (i) visual examination of how well they dock into the glycogen synthase kinase-3beta-binding site, (ii) comparative analysis of their FlexX, G-Score, PMF-Score, ChemScore and D-Scores values, (iii) comparison of their best fit value with the known inhibitors and (iv) examination of the how the hits retain interactions with the important amino acid residues of glycogen synthase kinase-3beta-binding site. PMID:17062013

  12. Investigation of potential glycogen synthase kinase 3 inhibitors using pharmacophore mapping and virtual screening.

    PubMed

    Dessalew, Nigus; Bharatam, Prasad V

    2006-09-01

    Glycogen synthase kinase-3 is a serine/threonine kinase that has attracted significant drug discovery attention in recent years. To investigate the identification of new potential glycogen synthase kinase-3 inhibitors, a pharmacophore mapping study was carried out using a set of 21 structurally diverse glycogen synthase kinase-3 inhibitors. A hypothesis containing four features: two hydrophobic, one hydrogen bond donor and another hydrogen bond acceptor was found to be the best from the 10 common feature hypotheses produced by HipHop module of Catalyst. The best hypothesis has a high cost of 156.592 and higher best fit values were obtained for the 21 inhibitors using this best hypothesis than the other HipHop hypotheses. The best hypothesis was then used to screen electronically the NCI2000 database. The hits obtained were docked into glycogen synthase kinase-3beta active site. A total of five novel potential leads were proposed after: (i) visual examination of how well they dock into the glycogen synthase kinase-3beta-binding site, (ii) comparative analysis of their FlexX, G-Score, PMF-Score, ChemScore and D-Scores values, (iii) comparison of their best fit value with the known inhibitors and (iv) examination of the how the hits retain interactions with the important amino acid residues of glycogen synthase kinase-3beta-binding site.

  13. Neuronal glycogen synthesis contributes to physiological aging

    PubMed Central

    Sinadinos, Christopher; Valles-Ortega, Jordi; Boulan, Laura; Solsona, Estel; Tevy, Maria F; Marquez, Mercedes; Duran, Jordi; Lopez-Iglesias, Carmen; Calbó, Joaquim; Blasco, Ester; Pumarola, Marti; Milán, Marco; Guinovart, Joan J

    2014-01-01

    Glycogen is a branched polymer of glucose and the carbohydrate energy store for animal cells. In the brain, it is essentially found in glial cells, although it is also present in minute amounts in neurons. In humans, loss-of-function mutations in laforin and malin, proteins involved in suppressing glycogen synthesis, induce the presence of high numbers of insoluble polyglucosan bodies in neuronal cells. Known as Lafora bodies (LBs), these deposits result in the aggressive neurodegeneration seen in Lafora’s disease. Polysaccharide-based aggregates, called corpora amylacea (CA), are also present in the neurons of aged human brains. Despite the similarity of CA to LBs, the mechanisms and functional consequences of CA formation are yet unknown. Here, we show that wild-type laboratory mice also accumulate glycogen-based aggregates in the brain as they age. These structures are immunopositive for an array of metabolic and stress-response proteins, some of which were previously shown to aggregate in correlation with age in the human brain and are also present in LBs. Remarkably, these structures and their associated protein aggregates are not present in the aged mouse brain upon genetic ablation of glycogen synthase. Similar genetic intervention in Drosophila prevents the accumulation of glycogen clusters in the neuronal processes of aged flies. Most interestingly, targeted reduction of Drosophila glycogen synthase in neurons improves neurological function with age and extends lifespan. These results demonstrate that neuronal glycogen accumulation contributes to physiological aging and may therefore constitute a key factor regulating age-related neurological decline in humans. PMID:25059425

  14. Neuronal glycogen synthesis contributes to physiological aging.

    PubMed

    Sinadinos, Christopher; Valles-Ortega, Jordi; Boulan, Laura; Solsona, Estel; Tevy, Maria F; Marquez, Mercedes; Duran, Jordi; Lopez-Iglesias, Carmen; Calbó, Joaquim; Blasco, Ester; Pumarola, Marti; Milán, Marco; Guinovart, Joan J

    2014-10-01

    Glycogen is a branched polymer of glucose and the carbohydrate energy store for animal cells. In the brain, it is essentially found in glial cells, although it is also present in minute amounts in neurons. In humans, loss-of-function mutations in laforin and malin, proteins involved in suppressing glycogen synthesis, induce the presence of high numbers of insoluble polyglucosan bodies in neuronal cells. Known as Lafora bodies (LBs), these deposits result in the aggressive neurodegeneration seen in Lafora's disease. Polysaccharide-based aggregates, called corpora amylacea (CA), are also present in the neurons of aged human brains. Despite the similarity of CA to LBs, the mechanisms and functional consequences of CA formation are yet unknown. Here, we show that wild-type laboratory mice also accumulate glycogen-based aggregates in the brain as they age. These structures are immunopositive for an array of metabolic and stress-response proteins, some of which were previously shown to aggregate in correlation with age in the human brain and are also present in LBs. Remarkably, these structures and their associated protein aggregates are not present in the aged mouse brain upon genetic ablation of glycogen synthase. Similar genetic intervention in Drosophila prevents the accumulation of glycogen clusters in the neuronal processes of aged flies. Most interestingly, targeted reduction of Drosophila glycogen synthase in neurons improves neurological function with age and extends lifespan. These results demonstrate that neuronal glycogen accumulation contributes to physiological aging and may therefore constitute a key factor regulating age-related neurological decline in humans.

  15. A glycogene mutation map for discovery of diseases of glycosylation

    PubMed Central

    Hansen, Lars; Lind-Thomsen, Allan; Joshi, Hiren J; Pedersen, Nis Borbye; Have, Christian Theil; Kong, Yun; Wang, Shengjun; Sparso, Thomas; Grarup, Niels; Vester-Christensen, Malene Bech; Schjoldager, Katrine; Freeze, Hudson H; Hansen, Torben; Pedersen, Oluf; Henrissat, Bernard; Mandel, Ulla; Clausen, Henrik; Wandall, Hans H; Bennett, Eric P

    2015-01-01

    Glycosylation of proteins and lipids involves over 200 known glycosyltransferases (GTs), and deleterious defects in many of the genes encoding these enzymes cause disorders collectively classified as congenital disorders of glycosylation (CDGs). Most known CDGs are caused by defects in glycogenes that affect glycosylation globally. Many GTs are members of homologous isoenzyme families and deficiencies in individual isoenzymes may not affect glycosylation globally. In line with this, there appears to be an underrepresentation of disease-causing glycogenes among these larger isoenzyme homologous families. However, genome-wide association studies have identified such isoenzyme genes as candidates for different diseases, but validation is not straightforward without biomarkers. Large-scale whole-exome sequencing (WES) provides access to mutations in, for example, GT genes in populations, which can be used to predict and/or analyze functional deleterious mutations. Here, we constructed a draft of a functional mutational map of glycogenes, GlyMAP, from WES of a rather homogenous population of 2000 Danes. We cataloged all missense mutations and used prediction algorithms, manual inspection and in case of carbohydrate-active enzymes family GT27 experimental analysis of mutations to map deleterious mutations. GlyMAP (http://glymap.glycomics.ku.dk) provides a first global view of the genetic stability of the glycogenome and should serve as a tool for discovery of novel CDGs. PMID:25267602

  16. The functional impact of Pgm amino acid polymorphism on glycogen content in Drosophila melanogaster.

    PubMed Central

    Verrelli, B C; Eanes, W F

    2001-01-01

    Earlier studies of the common PGM allozymes in Drosophila melanogaster reported no in vitro activity differences. However, our study of nucleotide variation observed that PGM allozymes are a heterogeneous mixture of amino acid polymorphisms. In this study, we analyze 10 PGM protein haplotypes with respect to PGM activity, thermostability, and adult glycogen content. We find a twofold difference in activity among PGM protein haplotypes that is associated with a threefold difference in glycogen content. The latitudinal clines for several Pgm amino acid polymorphisms show that high PGM activity, and apparently higher flux to glycogen synthesis, parallel the low activity clines at G6PD for reduced pentose shunt flux in northern latitudes. This suggests that amino acid polymorphism is under selection at this branch point and may be favored for increased metabolic storage associated with stress resistance and adaptation to temperate regions. PMID:11560897

  17. The Saccharomyces cerevisiae fermentation stress response protein Igd1p/Yfr017p regulates glycogen levels by inhibiting the glycogen debranching enzyme.

    PubMed

    Walkey, Christopher J; Luo, Zongli; Borchers, Christoph H; Measday, Vivien; van Vuuren, Hennie J J

    2011-09-01

    Wine fermentation imposes a number of stresses on Saccharomyces cerevisiae, and wine yeasts respond to this harsh environment by altering their transcriptional profile (Marks et al., 2008). We have labeled this change in gene expression patterns the fermentation stress response (FSR). An important component of the FSR is the increased expression of 62 genes for which no function has been identified for their protein products. We hypothesize that a function for these proteins may only be revealed late in grape must fermentation, when the yeast cells are facing conditions much more extreme than those normally encountered in laboratory media. We used affinity copurification to identify interaction partners for the FSR protein Yfr017p, and found that it interacts specifically with the glycogen debranching enzyme (Gdb1p). The expression of both of these proteins is strongly induced during wine fermentation. Therefore, we investigated the role of Yfr017p in glycogen metabolism by constructing wine yeast strains that lack this protein. These YFR017C null cells displayed a significant reduction in their ability to accumulate glycogen during aerobic growth and fermentation. Moreover, Yfr017p inhibits Gdb1p activity in vitro. These results suggest that Yfr017p functions as an inhibitor of Gdb1p, enhancing the ability of yeast cells to store glucose as glycogen. Therefore, we propose IGD1 (for inhibitor of glycogen debranching) as a gene name for the YFR017C ORF.

  18. Exercise in muscle glycogen storage diseases.

    PubMed

    Preisler, Nicolai; Haller, Ronald G; Vissing, John

    2015-05-01

    Glycogen storage diseases (GSD) are inborn errors of glycogen or glucose metabolism. In the GSDs that affect muscle, the consequence of a block in skeletal muscle glycogen breakdown or glucose use, is an impairment of muscular performance and exercise intolerance, owing to 1) an increase in glycogen storage that disrupts contractile function and/or 2) a reduced substrate turnover below the block, which inhibits skeletal muscle ATP production. Immobility is associated with metabolic alterations in muscle leading to an increased dependence on glycogen use and a reduced capacity for fatty acid oxidation. Such changes may be detrimental for persons with GSD from a metabolic perspective. However, exercise may alter skeletal muscle substrate metabolism in ways that are beneficial for patients with GSD, such as improving exercise tolerance and increasing fatty acid oxidation. In addition, a regular exercise program has the potential to improve general health and fitness and improve quality of life, if executed properly. In this review, we describe skeletal muscle substrate use during exercise in GSDs, and how blocks in metabolic pathways affect exercise tolerance in GSDs. We review the studies that have examined the effect of regular exercise training in different types of GSD. Finally, we consider how oral substrate supplementation can improve exercise tolerance and we discuss the precautions that apply to persons with GSD that engage in exercise.

  19. Impaired glucose tolerance and predisposition to the fasted state in liver glycogen synthase knock-out mice.

    PubMed

    Irimia, Jose M; Meyer, Catalina M; Peper, Caron L; Zhai, Lanmin; Bock, Cheryl B; Previs, Stephen F; McGuinness, Owen P; DePaoli-Roach, Anna; Roach, Peter J

    2010-04-23

    Conversion to glycogen is a major fate of ingested glucose in the body. A rate-limiting enzyme in the synthesis of glycogen is glycogen synthase encoded by two genes, GYS1, expressed in muscle and other tissues, and GYS2, primarily expressed in liver (liver glycogen synthase). Defects in GYS2 cause the inherited monogenic disease glycogen storage disease 0. We have generated mice with a liver-specific disruption of the Gys2 gene (liver glycogen synthase knock-out (LGSKO) mice), using Lox-P/Cre technology. Conditional mice carrying floxed Gys2 were crossed with mice expressing Cre recombinase under the albumin promoter. The resulting LGSKO mice are viable, develop liver glycogen synthase deficiency, and have a 95% reduction in fed liver glycogen content. They have mild hypoglycemia but dispose glucose less well in a glucose tolerance test. Fed, LGSKO mice also have a reduced capacity for exhaustive exercise compared with mice carrying floxed alleles, but the difference disappears after an overnight fast. Upon fasting, LGSKO mice reach within 4 h decreased blood glucose levels attained by control floxed mice only after 24 h of food deprivation. The LGSKO mice maintain this low blood glucose for at least 24 h. Basal gluconeogenesis is increased in LGSKO mice, and insulin suppression of endogenous glucose production is impaired as assessed by euglycemic-hyperinsulinemic clamp. This observation correlates with an increase in the liver gluconeogenic enzyme phosphoenolpyruvate carboxykinase expression and activity. This mouse model mimics the pathophysiology of glycogen storage disease 0 patients and highlights the importance of liver glycogen stores in whole body glucose homeostasis.

  20. Three-dimensional structures of unligated uridine phosphorylase from Yersinia pseudotuberculosis at 1.4 Å resolution and its complex with an antibacterial drug

    NASA Astrophysics Data System (ADS)

    Balaev, V. V.; Lashkov, A. A.; Gabdulkhakov, A. G.; Dontsova, M. V.; Mironov, A. S.; Betzel, C.; Mikhailov, A. M.

    2015-07-01

    Uridine phosphorylases play an essential role in the cellular metabolism of some antibacterial agents. Acute infectious diseases (bubonic plague, yersiniosis, pseudotuberculosis, etc., caused by bacteria of the genus Yersinia) are treated using both sulfanilamide medicines and antibiotics, including trimethoprim. The action of an antibiotic on a bacterial cell is determined primarily by the character of its interactions with cellular components, including those which are not targets (for example, with pyrimidine phosphorylases). This type of interaction should be taken into account in designing drugs. The three-dimensional structure of uridine phosphorylase from the bacterium Yersinia pseudotuberculosis ( YptUPh) with the free active site was determined for the first time by X-ray crystallography and refined at 1.40 Å resolution (DPI = 0.062 Å; ID PDB: 4OF4). The structure of the complex of YptUPh with the bacteriostatic drug trimethoprim was studied by molecular docking and molecular dynamics methods. The trimethoprim molecule was shown to be buffered by the enzyme YptUPh, resulting in a decrease in the efficiency of the treatment of infectious diseases caused by bacteria of the genus Yersinia with trimethoprim.

  1. Reaction of phosphorylase-a with α-D-glucose 1-phosphate and maltodextrin acceptors to give products with degree of polymerization 6-89.

    PubMed

    Kazłowski, Bartosz; Ko, Yuan-Tih

    2014-06-15

    A series of linear glucan saccharides (GS) with defined quantity and degree of polymerization (DP) were synthesized from α-d-glucose 1-phosphate (α-d-Glc 1-P) by phosphorylase-a. The GS product fractions with average DP 11, 22, 38, 52, 60, 70, and 79 were measured by HPSEC-ELSD system. Then the same seven fractions were resolved into individual peaks with DP: 6-14, 10-32, 27-55, 37-67, 44-75, 49-83 and 53-89 by HPAEC-PAD system. Results showed that measurement of α-d-Glc 1-P amount consuming during GS synthesis by both systems enable calculation of reaction yield. The reaction yield for the 24h biosynthesis of the GS product was 25.3% (measured by HPSEC-ELSD) or 29.1% (measured by HPAEC-PAD). The HPSEC-ELSD and HPAEC-PAD systems were also successfully used for phosphorylase-a activity measurement in order to perform its kinetic characterization. This study established feasible systems for preparation of various sizes of the GS with defined DP and quantity as well as characterization of phosphorylase-a kinetics.

  2. The Activity of Carbohydrate-Degrading Enzymes in the Development of Brood and Newly Emerged workers and Drones of the Carniolan Honeybee, Apis mellifera carnica

    PubMed Central

    Żółtowska, Krystyna; Lipiński, Zbigniew; Łopieńska-Biernat, Elżbieta; Farjan, Marek; Dmitryjuk, Małgorzata

    2012-01-01

    The activity of glycogen Phosphorylase and carbohydrate hydrolyzing enzymes α-amylase, glucoamylase, trehalase, and sucrase was studied in the development of the Carniolan honey bee, Apis mellifera carnica Pollman (Hymenoptera: Apidae), from newly hatched larva to freshly emerged imago of worker and drone. Phosphorolytic degradation of glycogen was significantly stronger than hydrolytic degradation in all developmental stages. Developmental profiles of hydrolase activity were similar in both sexes of brood; high activity was found in unsealed larvae, the lowest in prepupae followed by an increase in enzymatic activity. Especially intensive increases in activity occurred in the last stage of pupae and newly emerged imago. Besides α-amylase, the activities of other enzymes were higher in drone than in worker broods. Among drones, activity of glucoamylase was particularly high, ranging from around three times higher in the youngest larvae to 13 times higher in the oldest pupae. This confirms earlier suggestions about higher rates of metabolism in drone broods than in worker broods. PMID:22943407

  3. Quantitative comparison of pathways of hepatic glycogen repletion in fed and fasted humans

    SciTech Connect

    Shulman, G.I.; Cline, G.; Schumann, W.C.; Chandramouli, V.; Kumaran, K.; Landau, B.R. )

    1990-09-01

    The effect of fasting vs. refeeding on hepatic glycogen repletion by the direct pathway, i.e., glucose----glucose 6-phosphate (G-6-P)----glycogen, was determined. Acetaminophen was administered during an infusion of glucose labeled with (1-13C)- and (6-14C)glucose into four healthy volunteers after an overnight fast and into the same subjects 4 h after breakfast. 13C enrichments in C-1 and C-6 of glucose formed from urinary acetaminophen glucuronide compared with enrichments in C-1 and C-6 of plasma glucose provided an estimate of glycogen formation by the direct pathway. The specific activity of glucose from the glucuronide compared with the specific activity of the plasma glucose, along with the percentages of 14C in C-1 and C-6 of the glucose from the glucuronide, also provided an estimate of the amount of glycogen formed by the direct pathway. The estimates were similar. Those from (6-14C)glucose would have been higher than from (1-13C)glucose if the pentose cycle contribution to overall glucose utilization had been significant. After an overnight fast, during the last hour of infusion, 49 +/- 3% of the glycogen formed was formed via the direct pathway. After breakfast, at similar plasma glucose and insulin concentrations, the percentage increased to 69 +/- 7% (P less than 0.02). Thus the contributions of the pathways to hepatic glycogen formation depend on the dietary state of the individual. For a dietary regimen in which individuals consume multiple meals per day containing at least a moderate amount of carbohydrates most glycogen synthesis occurs by the direct pathway.

  4. Chromatin binding and polymerization of the endogenous Xenopus egg lamins: the opposing effects of glycogen and ATP.

    PubMed

    Lourim, D; Krohne, G

    1998-12-18

    We have previously identified and quantitated three B-type lamin isoforms present in the nuclei of mature Xenopus laevis oocytes, and in cell-free egg extracts. As Xenopus egg extracts are frequently used to analyze nuclear envelope assembly and lamina functions, we felt it was imperative that the polymerization and chromatin-binding properties of the endogenous B-type egg lamins be investigated. While we have demonstrated that soluble B-type lamins bind to chromatin, we have also observed that the polymerization of egg lamins does not require membranes or chromatin. Lamin assembly is enhanced by the addition of glycogen/glucose, or by the depletion of ATP from the extract. Moreover, the polymerization of egg cytosol lamins and their binding to demembranated sperm or chromatin assembled from naked lambda-DNA is inhibited by an ATP regeneration system. These ATP-dependent inhibitory activities can be overcome by the coaddition of glycogen to egg cytosol. We have observed that glycogen does not alter ATP levels during cytosol incubation, but rather, as glycogen-enhanced lamin polymerization is inhibited by okadaic acid, we conclude that glycogen activates protein phosphatases. Because protein phosphatase 1 (PP1) is the only phosphatase known to be specifically regulated by glycogen our data indicate that PP1 is involved in lamin polymerization. Our results show that ATP and glycogen effect lamin polymerization and chromatin binding by separate and opposing mechanisms. PMID:9819358

  5. Muscle glycogen remodeling and glycogen phosphate metabolism following exhaustive exercise of wild type and laforin knockout mice.

    PubMed

    Irimia, Jose M; Tagliabracci, Vincent S; Meyer, Catalina M; Segvich, Dyann M; DePaoli-Roach, Anna A; Roach, Peter J

    2015-09-11

    Glycogen, the repository of glucose in many cell types, contains small amounts of covalent phosphate, of uncertain function and poorly understood metabolism. Loss-of-function mutations in the laforin gene cause the fatal neurodegenerative disorder, Lafora disease, characterized by increased glycogen phosphorylation and the formation of abnormal deposits of glycogen-like material called Lafora bodies. It is generally accepted that the phosphate is removed by the laforin phosphatase. To study the dynamics of skeletal muscle glycogen phosphorylation in vivo under physiological conditions, mice were subjected to glycogen-depleting exercise and then monitored while they resynthesized glycogen. Depletion of glycogen by exercise was associated with a substantial reduction in total glycogen phosphate and the newly resynthesized glycogen was less branched and less phosphorylated. Branching returned to normal on a time frame of days, whereas phosphorylation remained suppressed over a longer period of time. We observed no change in markers of autophagy. Exercise of 3-month-old laforin knock-out mice caused a similar depletion of glycogen but no loss of glycogen phosphate. Furthermore, remodeling of glycogen to restore the basal branching pattern was delayed in the knock-out animals. From these results, we infer that 1) laforin is responsible for glycogen dephosphorylation during exercise and acts during the cytosolic degradation of glycogen, 2) excess glycogen phosphorylation in the absence of laforin delays the normal remodeling of the branching structure, and 3) the accumulation of glycogen phosphate is a relatively slow process involving multiple cycles of glycogen synthesis-degradation, consistent with the slow onset of the symptoms of Lafora disease.

  6. Genetics Home Reference: glycogen storage disease type I

    MedlinePlus

    ... Orphanet: Glycogen storage disease due to glucose-6-phosphatase deficiency Patient Support and Advocacy Resources (7 links) ... JY, Mansfield BC. Mutations in the glucose-6-phosphatase-alpha (G6PC) gene that cause type Ia glycogen ...

  7. Identification of differentially expressed genes in chickens differing in muscle glycogen content and meat quality

    PubMed Central

    2011-01-01

    Background The processing ability of poultry meat is highly related to its ultimate pH, the latter being mainly determined by the amount of glycogen in the muscle at death. The genetic determinism of glycogen and related meat quality traits has been established in the chicken but the molecular mechanisms involved in variations in these traits remain to be fully described. In this study, Chicken Genome Arrays (20 K) were used to compare muscle gene expression profiles of chickens from Fat (F) and Lean (L) lines that exhibited high and low muscle glycogen content, respectively, and of individuals exhibiting extremely high (G+) or low (G-) muscle glycogen content originating from the F2 cross between the Fat and Lean lines. Real-time RT-PCR was subsequently performed to validate the differential expression of genes either selected from the microarray analysis or whose function in regulating glycogen metabolism was well known. Results Among the genes found to be expressed in chicken P. major muscle, 197 and 254 transcripts appeared to be differentially expressed on microarrays for the F vs. L and the G+ vs. G- comparisons, respectively. Some involved particularly in lipid and carbohydrate metabolism were selected for further validation studies by real-time RT-PCR. We confirmed that, as in mammals, the down-regulation of CEBPB and RGS2 coincides with a decrease in peripheral adiposity in the chicken, but these genes are also suggested to affect muscle glycogen turnover through their role in the cAMP-dependent signalling pathway. Several other genes were suggested to have roles in the regulation of glycogen storage in chicken muscle. PDK4 may act as a glycogen sensor in muscle, UGDH may compete for glycogen synthesis by using UDP-glucose for glucoronidation, and PRKAB1, PRKAG2, and PHKD may impact on glycogen turnover in muscle, through AMP-activated signalling pathways. Conclusions This study is the first stage in the understanding of molecular mechanisms underlying

  8. Genetics Home Reference: glycogen storage disease type VI

    MedlinePlus

    ... a result, liver cells cannot use glycogen for energy. Since glycogen cannot be broken down, it accumulates within liver cells, causing these cells to become enlarged and dysfunctional. Learn more about the gene associated with glycogen storage disease type VI PYGL Related Information What is ...

  9. Genetics Home Reference: glycogen storage disease type IX

    MedlinePlus

    ... cellular energy is a simple sugar called glucose. Glucose is stored in muscle and liver cells in a form called glycogen. Glycogen can ... result, glycogen accumulates in and damages cells, and glucose is not available for ... in the liver leads to hepatomegaly, and the liver's inability to ...

  10. Variation in glycogen concentrations within mantle and foot tissue in Amblema plicata plicata: Implications for tissue biopsy sampling

    USGS Publications Warehouse

    Naimo, T.J.; Monroe, E.M.

    1999-01-01

    With the development of techniques to non-lethally biopsy tissue from unionids, a new method is available to measure changes in biochemical, contaminant, and genetic constituents in this imperiled faunal group. However, before its widespread application, information on the variability of biochemical components within and among tissues needs to be evaluated. We measured glycogen concentrations in foot and mantle tissue in Amblema plicata plicata (Say, 1817) to determine if glycogen was evenly distributed within and between tissues and to determine which tissue might be more responsive to the stress associated with relocating mussels. Glycogen was measured in two groups of mussels: those sampled from their native environment (undisturbed mussels) and quickly frozen for analysis and those relocated into an artificial pond (relocated mussels) for 24 months before analysis. In both undisturbed and relocated mussels, glycogen concentrations were evenly distributed within foot, but not within mantle tissue. In mantle tissue, concentrations of glycogen varied about 2-fold among sections. In addition, glycogen varied significantly between tissues in undisturbed mussels, but not in relocated mussels. Twenty-four months after relocation, glycogen concentrations had declined by 80% in mantle tissue and by 56% in foot tissue relative to the undisturbed mussels. These data indicate that representative biopsy samples can be obtained from foot tissue, but not mantle tissue. We hypothesize that mantle tissue could be more responsive to the stress of relocation due to its high metabolic activity associated with shell formation.

  11. In silico analysis of the three-dimensional structures of the homodimer of uridine phosphorylase from Yersinia Pseudotuberculosis in the ligand-free state and in a complex with 5-fluorouracil

    SciTech Connect

    Lashkov, A. A. Sotnichenko, S. E.; Mikhailov, A. M.

    2013-03-15

    Pseudotuberculosis is an acute infectious disease characterized by a lesion of the gastrointestinal tract. A positive therapeutic effect can be achieved by selectively suppressing the activity of uridine phosphorylase from the causative agent of the disease Yersinia pseudotuberculosis. The synergistic effect of a combination of the chemotherapeutic agent 5-fluorouracil and antimicrobial drugs, which block the synthesis of pyrimidine bases, on the cells of pathogenic protozoa and bacteria is described in the literature. The three-dimensional structures of uridine phosphorylase from Yersinia pseudotuberculosis (YptUPh) both in the ligand-free state and in complexes with pharmacological agents are unknown, which hinders the search for and design of selective inhibitors of YptUPh. The three-dimensional structure of the ligand-free homodimer of YptUPh was determined by homology-based molecular modeling. The three-dimensional structure of the subunit of the YptUPh molecule belongs to {alpha}/{beta} proteins, and its topology is a three-layer {alpha}/{beta}/{alpha} sandwich. The subunit monomer of the YptUPh molecule consists of 38% helices and 24% {beta} strands. A model of the homodimer structure of YptUPh in a complex with 5-FU was obtained by the molecular docking. The position of 5-FU in the active site of the molecule is very consistent with the known data on the X-ray diffraction structures of other bacterial uridine phosphorylases (the complex of uridine phosphorylase from Salmonella typhimurium (StUPh) with 5-FU, ID PDB: 4E1V and the complex of uridine phosphorylase from Escherichia coli (EcUPh) with 5-FU and ribose 1-phosphate, ID PDB: 1RXC).

  12. In silico analysis of the three-dimensional structures of the homodimer of uridine phosphorylase from Yersinia Pseudotuberculosis in the ligand-free state and in a complex with 5-fluorouracil

    NASA Astrophysics Data System (ADS)

    Lashkov, A. A.; Sotnichenko, S. E.; Mikhailov, A. M.

    2013-03-01

    Pseudotuberculosis is an acute infectious disease characterized by a lesion of the gastrointestinal tract. A positive therapeutic effect can be achieved by selectively suppressing the activity of uridine phosphorylase from the causative agent of the disease Yersinia pseudotuberculosis. The synergistic effect of a combination of the chemotherapeutic agent 5-fluorouracil and antimicrobial drugs, which block the synthesis of pyrimidine bases, on the cells of pathogenic protozoa and bacteria is described in the literature. The three-dimensional structures of uridine phosphorylase from Yersinia pseudotuberculosis ( YptUPh) both in the ligand-free state and in complexes with pharmacological agents are unknown, which hinders the search for and design of selective inhibitors of YptUPh. The three-dimensional structure of the ligand-free homodimer of YptUPh was determined by homology-based molecular modeling. The three-dimensional structure of the subunit of the YptUPh molecule belongs to α/β proteins, and its topology is a three-layer α/β/α sandwich. The subunit monomer of the YptUPh molecule consists of 38% helices and 24% β strands. A model of the homodimer structure of YptUPh in a complex with 5-FU was obtained by the molecular docking. The position of 5-FU in the active site of the molecule is very consistent with the known data on the X-ray diffraction structures of other bacterial uridine phosphorylases (the complex of uridine phosphorylase from Salmonella typhimurium ( StUPh) with 5-FU, ID PDB: 4E1V and the complex of uridine phosphorylase from Escherichia coli ( EcUPh) with 5-FU and ribose 1-phosphate, ID PDB: 1RXC).

  13. Molecular Structure of Human-Liver Glycogen

    PubMed Central

    Deng, Bin; Sullivan, Mitchell A.; Chen, Cheng; Li, Jialun; Powell, Prudence O.; Hu, Zhenxia; Gilbert, Robert G.

    2016-01-01

    Glycogen is a highly branched glucose polymer which is involved in maintaining blood-sugar homeostasis. Liver glycogen contains large composite α particles made up of linked β particles. Previous studies have shown that the binding which links β particles into α particles is impaired in diabetic mice. The present study reports the first molecular structural characterization of human-liver glycogen from non-diabetic patients, using transmission electron microscopy for morphology and size-exclusion chromatography for the molecular size distribution; the latter is also studied as a function of time during acid hydrolysis in vitro, which is sensitive to certain structural features, particularly glycosidic vs. proteinaceous linkages. The results are compared with those seen in mice and pigs. The molecular structural change during acid hydrolysis is similar in each case, and indicates that the linkage of β into α particles is not glycosidic. This result, and the similar morphology in each case, together imply that human liver glycogen has similar molecular structure to those of mice and pigs. This knowledge will be useful for future diabetes drug targets. PMID:26934359

  14. Crystal structure and molecular dynamics studies of purine nucleoside phosphorylase from Mycobacterium tuberculosis associated with acyclovir.

    PubMed

    Caceres, Rafael A; Timmers, Luís F S M; Ducati, Rodrigo G; da Silva, Diego O N; Basso, Luiz A; de Azevedo, Walter F; Santos, Diógenes S

    2012-01-01

    Consumption has been a scourge of mankind since ancient times. This illness has charged a high price to human lives. Many efforts have been made to defeat Mycobacterium tuberculosis (Mt). The M. tuberculosis purine nucleoside phosphorylase (MtPNP) is considered an interesting target to pursuit new potential inhibitors, inasmuch it belongs to the purine salvage pathway and its activity might be involved in the mycobacterial latency process. Here we present the MtPNP crystallographic structure associated with acyclovir and phosphate (MtPNP:ACY:PO(4)) at 2.10 Å resolution. Molecular dynamics simulations were carried out in order to dissect MtPNP:ACY:PO(4) structural features, and the influence of the ligand in the binding pocket stability. Our results revealed that the ligand leads to active site lost of stability, in agreement with experimental results, which demonstrate a considerable inhibitory activity against MtPNP (K(i) = 150 nM). Furthermore, we observed that some residues which are important in the proper ligand's anchor into the human homologous enzyme do not present the same importance to MtPNP. Therewithal, these findings contribute to the search of new specific inhibitors for MtPNP, since peculiarities between the mycobacterial and human enzyme binding sites have been identified, making a structural-based drug design feasible.

  15. The 3T3-L1 adipocyte glycogen proteome

    PubMed Central

    2013-01-01

    Background Glycogen is a branched polysaccharide of glucose residues, consisting of α-1-4 glycosidic linkages with α-1-6 branches that together form multi-layered particles ranging in size from 30 nm to 300 nm. Glycogen spatial conformation and intracellular organization are highly regulated processes. Glycogen particles interact with their metabolizing enzymes and are associated with a variety of proteins that intervene in its biology, controlling its structure, particle size and sub-cellular distribution. The function of glycogen in adipose tissue is not well understood but appears to have a pivotal role as a regulatory mechanism informing the cells on substrate availability for triacylglycerol synthesis. To provide new molecular insights into the role of adipocyte glycogen we analyzed the glycogen-associated proteome from differentiated 3T3-L1-adipocytes. Results Glycogen particles from 3T3-L1-adipocytes were purified using a series of centrifugation steps followed by specific elution of glycogen bound proteins using α-1,4 glucose oligosaccharides, or maltodextrins, and tandem mass spectrometry. We identified regulatory proteins, 14-3-3 proteins, RACK1 and protein phosphatase 1 glycogen targeting subunit 3D. Evidence was also obtained for a regulated subcellular distribution of the glycogen particle: metabolic and mitochondrial proteins were abundant. Unlike the recently analyzed hepatic glycogen proteome, no endoplasmic proteins were detected, along with the recently described starch-binding domain protein 1. Other regulatory proteins which have previously been described as glycogen-associated proteins were not detected, including laforin, the AMPK beta-subunit and protein targeting to glycogen (PTG). Conclusions These data provide new molecular insights into the regulation of glycogen-bound proteins that are associated with the maintenance, organization and localization of the adipocyte glycogen particle. PMID:23521774

  16. Glycogen with short average chain length enhances bacterial durability

    NASA Astrophysics Data System (ADS)

    Wang, Liang; Wise, Michael J.

    2011-09-01

    Glycogen is conventionally viewed as an energy reserve that can be rapidly mobilized for ATP production in higher organisms. However, several studies have noted that glycogen with short average chain length in some bacteria is degraded very slowly. In addition, slow utilization of glycogen is correlated with bacterial viability, that is, the slower the glycogen breakdown rate, the longer the bacterial survival time in the external environment under starvation conditions. We call that a durable energy storage mechanism (DESM). In this review, evidence from microbiology, biochemistry, and molecular biology will be assembled to support the hypothesis of glycogen as a durable energy storage compound. One method for testing the DESM hypothesis is proposed.

  17. Enzymatic characterization of AMP phosphorylase and ribose-1,5-bisphosphate isomerase functioning in an archaeal AMP metabolic pathway.

    PubMed

    Aono, Riku; Sato, Takaaki; Yano, Ayumu; Yoshida, Shosuke; Nishitani, Yuichi; Miki, Kunio; Imanaka, Tadayuki; Atomi, Haruyuki

    2012-12-01

    AMP phosphorylase (AMPpase), ribose-1,5-bisphosphate (R15P) isomerase, and type III ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) have been proposed to constitute a novel pathway involved in AMP metabolism in the Archaea. Here we performed a biochemical examination of AMPpase and R15P isomerase from Thermococcus kodakarensis. R15P isomerase was specific for the α-anomer of R15P and did not recognize other sugar compounds. We observed that activity was extremely low with the substrate R15P alone but was dramatically activated in the presence of AMP. Using AMP-activated R15P isomerase, we reevaluated the substrate specificity of AMPpase. AMPpase exhibited phosphorylase activity toward CMP and UMP in addition to AMP. The [S]-v plot (plot of velocity versus substrate concentration) of the enzyme toward AMP was sigmoidal, with an increase in activity observed at concentrations higher than approximately 3 mM. The behavior of the two enzymes toward AMP indicates that the pathway is intrinsically designed to prevent excess degradation of intracellular AMP. We further examined the formation of 3-phosphoglycerate from AMP, CMP, and UMP in T. kodakarensis cell extracts. 3-Phosphoglycerate generation was observed from AMP alone, and from CMP or UMP in the presence of dAMP, which also activates R15P isomerase. 3-Phosphoglycerate was not formed when 2-carboxyarabinitol 1,5-bisphosphate, a Rubisco inhibitor, was added. The results strongly suggest that these enzymes are actually involved in the conversion of nucleoside monophosphates to 3-phosphoglycerate in T. kodakarensis.

  18. Defects in polynucleotide phosphorylase impairs virulence in Escherichia coli O157:H7.

    PubMed

    Hu, Jia; Zhu, Mei-Jun

    2015-01-01

    Polynucleotide phosphorylase (PNPase) is reported to regulate virulence in Salmonella, Yersinia sp. and Campylobacter jejuni, yet its role in Escherichia coli O157:H7 has not been investigated. To gain insights into its roles in E. coli O157:H7 virulence, pnp deletion mutants were generated and the major virulence factors were compared to their parental wild type strains. Deletion of pnp in E. coli O157:H7 dramatically decreased stx2 mRNA expression and Stx2 protein production, and impaired lambdoid prophage activation in E. coli O157:H7. Quantitative PCR further confirmed that the Stx2 phage lytic growth was repressed by pnp deletion. Consistent with reduced Stx2 production and Stx2 phage activation, the transcriptional levels of genes involved in phage lysis and replication were down-regulated. In addition, disruption of pnp in E. coli O157:H7 decreased its adhesion to intestinal epithelial cells as well as cattle colonic explant tissues. On the other hand, PNPase inactivation in E. coli O157:H7 enhanced Tir protein content and the transcription of type three secretion system components, including genes encoding intimin, Tir, and EspB as well as locus of enterocyte and effacement positive regulator, Ler. Collectively, data indicate that PNPase has pleiotropic effects on the virulence of E. coli O157:H7. PMID:26347717

  19. The kinetic mechanism of Human Thymidine Phosphorylase - a molecular target for cancer drug development.

    PubMed

    Deves, Candida; Rostirolla, Diana Carolina; Martinelli, Leonardo Kras Borges; Bizarro, Cristiano Valim; Santos, Diogenes Santiago; Basso, Luiz Augusto

    2014-03-01

    Human Thymidine Phosphorylase (HTP), also known as the platelet-derived endothelial cell growth factor (PD-ECGF) or gliostatin, catalyzes the reversible phosphorolysis of thymidine (dThd) to thymine and 2-deoxy-α-d-ribose-1-phosphate (2dR1P). HTP is a key enzyme in the pyrimidine salvage pathway involved in dThd homeostasis in cells. HTP is a target for anticancer drug development as its enzymatic activity promotes angiogenesis. Here, we describe cloning, expression, and purification to homogeneity of recombinant TYMP-encoded HTP. Peptide fingerprinting and the molecular mass value of the homogenous protein confirmed its identity as HTP assessed by mass spectrometry. Size exclusion chromatography showed that HTP is a dimer in solution. Kinetic studies revealed that HTP displayed substrate inhibition for dThd. Initial velocity and isothermal titration calorimetry (ITC) studies suggest that HTP catalysis follows a rapid-equilibrium random bi-bi kinetic mechanism. ITC measurements also showed that dThd and Pi binding are favorable processes. The pH-rate profiles indicated that maximal enzyme activity was achieved at low pH values. Functional groups with apparent pK values of 5.2 and 9.0 are involved in dThd binding and groups with pK values of 6.1 and 7.8 are involved in phosphate binding. PMID:24407036

  20. Thymidine phosphorylase exerts complex effects on bone resorption and formation in myeloma.

    PubMed

    Liu, Huan; Liu, Zhiqiang; Du, Juan; He, Jin; Lin, Pei; Amini, Behrang; Starbuck, Michael W; Novane, Nora; Shah, Jatin J; Davis, Richard E; Hou, Jian; Gagel, Robert F; Yang, Jing

    2016-08-24

    Myelomatous bone disease is characterized by the development of lytic bone lesions and a concomitant reduction in bone formation, leading to chronic bone pain and fractures. To understand the underlying mechanism, we investigated the contribution of myeloma-expressed thymidine phosphorylase (TP) to bone lesions. In osteoblast progenitors, TP up-regulated the methylation of RUNX2 and osterix, leading to decreased bone formation. In osteoclast progenitors, TP up-regulated the methylation of IRF8 and thereby enhanced expression of NFATc1 (nuclear factor of activated T cells, cytoplasmic 1 protein), leading to increased bone resorption. TP reversibly catalyzes thymidine into thymine and 2-deoxy-d-ribose (2DDR). Myeloma-secreted 2DDR bound to integrin αVβ3/α5β1 in the progenitors, activated PI3K (phosphoinositide 3-kinase)/Akt signaling, and increased DNMT3A (DNA methyltransferase 3A) expression, resulting in hypermethylation of RUNX2, osterix, and IRF8 This study elucidates an important mechanism for myeloma-induced bone lesions, suggesting that targeting TP may be a viable approach to healing resorbed bone in patients. Because TP overexpression is common in bone-metastatic tumors, our findings could have additional mechanistic implications. PMID:27559096

  1. Production and application of a rare disaccharide using sucrose phosphorylase from Leuconostoc mesenteroides.

    PubMed

    Morimoto, Kenji; Yoshihara, Akihide; Furumoto, Toshio; Takata, Goro

    2015-06-01

    Sucrose phosphorylase (SPase) from Leuconostoc mesenteroides exhibited activity towards eight ketohexoses, which behaved as D-glucosyl acceptors, and α-D-glucose-1-phosphate (G1P), which behaved as a donor. All eight of these ketohexoses were subsequently transformed into the corresponding d-glucosyl-ketohexoses. Of the eight ketohexoses evaluated in the current study, d-allulose behaved as the best substrate for SPase, and the resulting d-glucosyl-d-alluloside product was found to be a non-reducing sugar with a specific optical rotation of [α]D(20) + 74.36°. D-Glucosyl-D-alluloside was identified as α-D-glucopyranosyl-(1→2)-β-D-allulofuranoside by NMR analysis. D-Glucosyl-D-alluloside exhibited an inhibitory activity towards an invertase from yeast with a Km value of 50 mM, where it behaved as a competitive inhibitor with a Ki value of 9.2 mM. D-Glucosyl-D-alluloside was also successfully produced from sucrose using SPase and D-tagatose 3-epimerase. This process also allowed for the production of G1P from sucrose and d-allulose from D-fructose, which suggested that this method could be used to prepare d-glucosyl-d-alluloside without the need for expensive reagents such as G1P and d-allulose. PMID:25499751

  2. Translocation of glycogen synthase kinase-3β (GSK-3β), a trigger of permeability transition, is kinase activity-dependent and mediated by interaction with voltage-dependent anion channel 2 (VDAC2).

    PubMed

    Tanno, Masaya; Kuno, Atsushi; Ishikawa, Satoko; Miki, Takayuki; Kouzu, Hidemichi; Yano, Toshiyuki; Murase, Hiromichi; Tobisawa, Toshiyuki; Ogasawara, Makoto; Horio, Yoshiyuki; Miura, Tetsuji

    2014-10-17

    Glycogen synthase kinase-3β (GSK-3β) is a major positive regulator of the mitochondrial permeability transition pore (mPTP), a principle trigger of cell death, under the condition of oxidative stress. However, the mechanism by which cytosolic GSK-3β translocates to mitochondria, promoting mPTP opening, remains unclear. Here we addressed this issue by analyses of the effect of site-directed mutations in GSK-3β on mitochondrial translocation and protein/protein interactions upon oxidative stress. H9c2 cardiomyoblasts were transfected with GFP-tagged GSK-3β (WT), a mutant GSK-3β insensitive to inhibitory phosphorylation (S9A), or kinase-deficient GSK-3β (K85R). Time lapse observation revealed that WT and S9A translocated from the cytosol to the mitochondria more promptly than did K85R after exposure to oxidative stress. H2O2 increased the density of nine spots on two-dimensional gel electrophoresis of anti-GSK-3β-immunoprecipitates by more than 3-fold. MALDI-TOF/MS analysis revealed that one of the spots contained voltage-dependent anion channel 2 (VDAC2). Knockdown of VDAC2, but not VDAC1 or VDAC3, by siRNA attenuated both the mitochondrial translocation of GSK-3β and mPTP opening under stress conditions. The mitochondrial translocation of GSK-3β was attenuated also when Lys-15, but not Arg-4 or Arg-6, in the N-terminal domain of GSK-3β was replaced with alanine. The oxidative stress-induced mitochondrial translocation of GSK-3β was associated with an increase in cell death, which was suppressed by lithium chloride (LiCl), a GSK-3β inhibitor. These results demonstrate that GSK-3β translocates from the cytosol to mitochondria in a kinase activity- and VDAC2-dependent manner in which an N-terminal domain of GSK-3β may function as a mitochondrial targeting sequence.

  3. Translocation of Glycogen Synthase Kinase-3β (GSK-3β), a Trigger of Permeability Transition, Is Kinase Activity-dependent and Mediated by Interaction with Voltage-dependent Anion Channel 2 (VDAC2)*

    PubMed Central

    Tanno, Masaya; Kuno, Atsushi; Ishikawa, Satoko; Miki, Takayuki; Kouzu, Hidemichi; Yano, Toshiyuki; Murase, Hiromichi; Tobisawa, Toshiyuki; Ogasawara, Makoto; Horio, Yoshiyuki; Miura, Tetsuji

    2014-01-01

    Glycogen synthase kinase-3β (GSK-3β) is a major positive regulator of the mitochondrial permeability transition pore (mPTP), a principle trigger of cell death, under the condition of oxidative stress. However, the mechanism by which cytosolic GSK-3β translocates to mitochondria, promoting mPTP opening, remains unclear. Here we addressed this issue by analyses of the effect of site-directed mutations in GSK-3β on mitochondrial translocation and protein/protein interactions upon oxidative stress. H9c2 cardiomyoblasts were transfected with GFP-tagged GSK-3β (WT), a mutant GSK-3β insensitive to inhibitory phosphorylation (S9A), or kinase-deficient GSK-3β (K85R). Time lapse observation revealed that WT and S9A translocated from the cytosol to the mitochondria more promptly than did K85R after exposure to oxidative stress. H2O2 increased the density of nine spots on two-dimensional gel electrophoresis of anti-GSK-3β-immunoprecipitates by more than 3-fold. MALDI-TOF/MS analysis revealed that one of the spots contained voltage-dependent anion channel 2 (VDAC2). Knockdown of VDAC2, but not VDAC1 or VDAC3, by siRNA attenuated both the mitochondrial translocation of GSK-3β and mPTP opening under stress conditions. The mitochondrial translocation of GSK-3β was attenuated also when Lys-15, but not Arg-4 or Arg-6, in the N-terminal domain of GSK-3β was replaced with alanine. The oxidative stress-induced mitochondrial translocation of GSK-3β was associated with an increase in cell death, which was suppressed by lithium chloride (LiCl), a GSK-3β inhibitor. These results demonstrate that GSK-3β translocates from the cytosol to mitochondria in a kinase activity- and VDAC2-dependent manner in which an N-terminal domain of GSK-3β may function as a mitochondrial targeting sequence. PMID:25187518

  4. [Studies on the initiation of glycogen metabolism in Escherichia coli (author's transl)].

    PubMed

    Barengo, R; Krisman, C R

    1976-01-01

    Glycogen biosynthesis was studied in Escherichia coli. An enzyme complex composed of UDP-glucose; protein glucosyltransferase, ADP-glucose: protein glucosyltransferase and ADP-glucose: alpha-1,4 glucan alpha-4-glucosyltransferase was found. Further results revealed that while glycogen concentration remained unchanged, the specific activity of the glucosyltransferase complex increased during the growth phase of the culture. The detergents Lubrol and Brij provoked a decrease of 80% and 20% in the glucose transfer to protein from ADP-glucose and UDP-glucose, respectively. These detergents did not inhibit the glucose incorporation into glycogen by ADP-glucose: alpha-1,4-glucosyltransferase. We postulated that the biosynthesis of glycogen in Escherichia coli could be initiated by two different enzymes which catalyze the transfer of glucose from UDP-glucose or ADP-glucose to an acceptor protien. In a second step, the glucan protein formed is used as primer by the ADP-glucose: alpha-1,4 glucan alpha-1-glucosyltransferase for glycogen formation.

  5. Patterns of glycogen turnover in liver characterized by computer modeling

    SciTech Connect

    Youn, J.H.; Bergman, R.N. )

    1987-10-01

    The authors used a computer model of liver glycogen turnover to reexamine the data of Devos and Hers, who reported the time course of accumulation in and loss from glycogen of label originating in (1-{sup 14}C)galactose injected at different times after the start of refeeding of 40-h fasted mice or rats. In the present study computer representation of individual glycogen molecules was utilized to account for growth and degradation of glycogen according to specific hypothetical patterns. Using this model they could predict the accumulation and localization within glycogen of labeled glucose residues and compare the predictions with the previously published data. They considered three specific hypotheses of glycogen accumulation during refeeding: (1) simultaneous, (2) sequential, and (3) accelerating growth. Hypothetical patterns of glycogen degradation were (1) ordered and (2) random degradation. The pattern of glycogen synthesis consistent with experimental data was a steadily increasing number of growing glycogen molecules, whereas during degradation glycogen molecules are exposed to degrading enzymes randomly, rather than in a specific reverse order of synthesis. These patterns predict the existence of a specific mechanism for the steadily increasing seeding of new glycogen molecules during synthesis.

  6. Glycogen and its metabolism: some new developments and old themes

    PubMed Central

    Roach, Peter J.; Depaoli-Roach, Anna A.; Hurley, Thomas D.; Tagliabracci, Vincent S.

    2016-01-01

    Glycogen is a branched polymer of glucose that acts as a store of energy in times of nutritional sufficiency for utilization in times of need. Its metabolism has been the subject of extensive investigation and much is known about its regulation by hormones such as insulin, glucagon and adrenaline (epinephrine). There has been debate over the relative importance of allosteric compared with covalent control of the key biosynthetic enzyme, glycogen synthase, as well as the relative importance of glucose entry into cells compared with glycogen synthase regulation in determining glycogen accumulation. Significant new developments in eukaryotic glycogen metabolism over the last decade or so include: (i) three-dimensional structures of the biosynthetic enzymes glycogenin and glycogen synthase, with associated implications for mechanism and control; (ii) analyses of several genetically engineered mice with altered glycogen metabolism that shed light on the mechanism of control; (iii) greater appreciation of the spatial aspects of glycogen metabolism, including more focus on the lysosomal degradation of glycogen; and (iv) glycogen phosphorylation and advances in the study of Lafora disease, which is emerging as a glycogen storage disease. PMID:22248338

  7. Glycogen synthase kinase 3β inhibition enhanced proliferation, migration and functional re-endothelialization of endothelial progenitor cells in hypercholesterolemia microenvironment

    PubMed Central

    Cui, Bin; Jin, Jun; Ding, Xiaohan; Deng, Mengyang; Yu, Shiyong; Song, MingBao; Yu, Yang; Zhao, Xiaohui; Chen, Jianfei

    2015-01-01

    Hypercholesterolemia impairs the quantity and function of endothelial progenitor cell. We hypothesized that glycogen synthase kinase 3β activity is involved in regulating biological function of endothelial progenitor cells in hypercholesterolemia microenvironment. For study, endothelial progenitor cells derived from apolipoprotein E-deficient mice fed with high-fat diet were used. Glycogen synthase kinase 3β activity was interfered with glycogen synthase kinase 3β inhibitor lithium chloride or transduced with replication defective adenovirus vector expressing catalytically inactive glycogen synthase kinase 3β (GSK3β-KM). Functions of endothelial progenitor cells, proliferation, migration, secretion and network formation of endothelial progenitor cells were assessed in vitro. The expression of phospho-glycogen synthase kinase 3β, β-catenin and cyclinD1 in endothelial progenitor cells was detected by Western blot. The in vivo function re-endothelialization and vasodilation were also analyzed by artery injury model transplanted with glycogen synthase kinase 3β-inhibited endothelial progenitor cells. We demonstrated that while the proliferation, migration, network formation as well as VEGF and NO secretion were impaired in apolipoprotein E-deficient endothelial progenitor cells, glycogen synthase kinase 3β inhibition significantly improved all these functions. Apolipoprotein E-deficient endothelial progenitor cells showed decreased phospho-glycogen synthase kinase 3β, β-catenin and cyclinD1 expression, whereas these signals were enhanced by glycogen synthase kinase 3β inhibition and accompanied with β-catenin nuclear translocation. Our in vivo model showed that glycogen synthase kinase 3β inhibition remarkably increased re-endothelial and vasodilation. Taken together, our data suggest that inhibition of glycogen synthase kinase 3β is associated with endothelial progenitor cell biological functions both in vitro and in vivo. It might be an important

  8. Ordered synthesis and mobilization of glycogen in the perfused heart

    SciTech Connect

    Brainard, J.R.; Hutson, J.Y. ); Hoekenga, D.E.; Lenhoff, R. )

    1989-12-12

    The molecular order of synthesis and mobilization of glycogen in the perfused heart was studied by {sup 13}C NMR. By varying the glucose isotopomer ((1-{sup 13}C)glucose or (2-{sup 13}C)glucose) supplied to the heart, glycogen synthesized at different times during the perfusion was labeled at different carbon sites. Subsequently, the in situ mobilization of glycogen during ischemia was observed by detection of labeled lactate derived from glycolysis of the glucosyl monomers. When (1-{sup 13}C)glucose was given initially in the perfusion and (2-{sup 13}C)glucose was given second, (2-{sup 13}C)lactate was detected first during ischemia and (3-{sup 13}C)lactate second. This result, and the equivalent result when the glucose labels were given in the reverse order, demonstrates that glycogen synthesis and mobilization are ordered in the heart, where glycogen is found morphologically only as {beta} particles. Previous studies of glycogen synthesis and mobilization in liver and adipocytes have suggested that the organization of {beta} particles into {alpha} particles was partially responsible for ordered synthesis and mobilization. The observations reported here for cardiac glycogen suggest that another mechanism is responsible. In addition to examine the ordered synthesis and mobilization of cardiac glycogen, the authors have selectively monitored the NMR properties of {sup 13}C-labeled glycogen synthesized early in the perfusion during further glycogen synthesis from a second, differently labeled substrate. During synthesis from the second labeled glucose monomer, the glycogen resonance from the first label decreased in integrated intensity and increased in line width. These results suggest either that there is significant isotopic exchange of glucosyl monomers in glycogen during net synthesis or that glucosyl residues incorporated into glycogen undergo motional restrictions as further glycogen synthesis occurs.

  9. The essential role of methylthioadenosine phosphorylase in prostate cancer

    PubMed Central

    Foster, Barbara A.; Karasik, Ellen; Gillard, Bryan; Morrison, Carl; Mohler, James; Phillips, James G.; Smiraglia, Dominic J.

    2016-01-01

    Prostatic epithelial cells secrete high levels of acetylated polyamines into the prostatic lumen. This distinctive characteristic places added strain on the connected pathways, which are forced to increase metabolite production to maintain pools. The methionine salvage pathway recycles the one-carbon unit lost to polyamine biosynthesis back to the methionine cycle, allowing for replenishment of SAM pools providing a mechanism to help mitigate metabolic stress associated with high flux through these pathways. The rate-limiting enzyme involved in this process is methylthioadenosine phosphorylase (MTAP), which, although commonly deleted in many cancers, is protected in prostate cancer. We report near universal retention of MTAP expression in a panel of human prostate cancer cell lines as well as patient samples. Upon metabolic perturbation, prostate cancer cell lines upregulate MTAP and this correlates with recovery of SAM levels. Furthermore, in a mouse model of prostate cancer we find that both normal prostate and diseased prostate maintain higher SAM levels than other tissues, even under increased metabolic stress. Finally, we show that knockdown of MTAP, both genetically and pharmacologically, blocks androgen sensitive prostate cancer growth in vivo. Our findings strongly suggest that the methionine salvage pathway is a major player in homeostatic regulation of metabolite pools in prostate cancer due to their high level of flux through the polyamine biosynthetic pathway. Therefore, this pathway, and specifically the MTAP enzyme, is an attractive therapeutic target for prostate cancer. PMID:26910893

  10. Polynucleotide Phosphorylase Protects Escherichia coli against Oxidative Stress†

    PubMed Central

    Wu, Jinhua; Jiang, Zhe; Liu, Min; Gong, Xin; Wu, Shaohui; Burns, Christopher M.; Li, Zhongwei

    2009-01-01

    Escherichia coli polynucleotide phosphorylase (PNPase) primarily functions in RNA degradation. It is an exoribonuclease and integral component of the multienzyme RNA degradosome complex [Carpousis et al. (1994) Cell 76, 889]. PNPase was previously shown to specifically bind a synthetic RNA containing the oxidative lesion 8-hydroxyguanine (8-oxoG) [Hayakawa et al. (2001) Biochemistry 40, 9977], suggesting a possible role in removing oxidatively damaged RNA. Here we show that PNPase binds to RNA molecules of natural sequence that were oxidatively damaged by treatment with hydrogen peroxide (H2O2) postsynthetically. PNPase bound oxidized RNA with higher affinity than untreated RNA of the same sequence, raising the possibility that it may act against a wide variety of lesions. The importance of such a protective role is illustrated by the observation that, under conditions known to cause oxidative damage to cytoplasmic components, PNPase-deficient cells are less viable than wild-type cells. Further, when challenged with H2O2, PNPase-deficient cells accumulate 8-oxoG in cellular RNA to a greater extent than wild-type cells, suggesting that this RNase functions in minimizing oxidized RNA in vivo. Introducing the pnp gene encoding PNPase rescues defects in growth and RNA quality of the pnp mutant cells. Our results also suggest that protection against oxidative stress is an intrinsic function of PNPase because association with the RNA degradosome or with RNA helicase B (RhlB) is not required. PMID:19219992

  11. Inhibition and Structure of Toxoplasma gondii Purine Nucleoside Phosphorylase

    PubMed Central

    Donaldson, Teraya M.; Cassera, María B.; Ho, Meng-Chiao; Zhan, Chenyang; Merino, Emilio F.; Evans, Gary B.; Tyler, Peter C.; Almo, Steven C.; Schramm, Vern L.

    2014-01-01

    The intracellular pathogen Toxoplasma gondii is a purine auxotroph that relies on purine salvage for proliferation. We have optimized T. gondii purine nucleoside phosphorylase (TgPNP) stability and crystallized TgPNP with phosphate and immucillin-H, a transition-state analogue that has high affinity for the enzyme. Immucillin-H bound to TgPNP with a dissociation constant of 370 pM, the highest affinity of 11 immucillins selected to probe the catalytic site. The specificity for transition-state analogues indicated an early dissociative transition state for TgPNP. Compared to Plasmodium falciparum PNP, large substituents surrounding the 5′-hydroxyl group of inhibitors demonstrate reduced capacity for TgPNP inhibition. Catalytic discrimination against large 5′ groups is consistent with the inability of TgPNP to catalyze the phosphorolysis of 5′-methylthioinosine to hypoxanthine. In contrast to mammalian PNP, the 2′-hydroxyl group is crucial for inhibitor binding in the catalytic site of TgPNP. This first crystal structure of TgPNP describes the basis for discrimination against 5′-methylthioinosine and similarly 5′-hydroxy-substituted immucillins; structural differences reflect the unique adaptations of purine salvage pathways of Apicomplexa. PMID:24585883

  12. Neisseria meningitidis Polynucleotide Phosphorylase Affects Aggregation, Adhesion, and Virulence

    PubMed Central

    Engman, Jakob; Negrea, Aurel; Sigurlásdóttir, Sara; Geörg, Miriam; Eriksson, Jens; Eriksson, Olaspers Sara; Kuwae, Asaomi; Sjölinder, Hong

    2016-01-01

    Neisseria meningitidis autoaggregation is an important step during attachment to human cells. Aggregation is mediated by type IV pili and can be modulated by accessory pilus proteins, such as PilX, and posttranslational modifications of the major pilus subunit PilE. The mechanisms underlying the regulation of aggregation remain poorly characterized. Polynucleotide phosphorylase (PNPase) is a 3′–5′ exonuclease that is involved in RNA turnover and the regulation of small RNAs. In this study, we biochemically confirm that NMC0710 is the N. meningitidis PNPase, and we characterize its role in N. meningitidis pathogenesis. We show that deletion of the gene encoding PNPase leads to hyperaggregation and increased adhesion to epithelial cells. The aggregation induced was found to be dependent on pili and to be mediated by excessive pilus bundling. PNPase expression was induced following bacterial attachment to human cells. Deletion of PNPase led to global transcriptional changes and the differential regulation of 469 genes. We also demonstrate that PNPase is required for full virulence in an in vivo model of N. meningitidis infection. The present study shows that PNPase negatively affects aggregation, adhesion, and virulence in N. meningitidis. PMID:26930706

  13. Early alterations in soleus GLUT-4, glucose transport, and glycogen in voluntary running rats

    NASA Technical Reports Server (NTRS)

    Henriksen, Erik J.; Halseth, Amy E.

    1994-01-01

    Voluntary wheel running (WR) by juvenile female rats was used as a noninterventional model of soleus muscle functional overload to study the regulation of insulin-stimulated glucose transport activity by the glucose transporter (GLUT-4 isoform) protein level and glycogen concentration. Soleus total protein content was significantly greater (+18%;P greater than 0.05) than in age-matched controls after 1 wk of WR, and this hypertrophic response continued in weeks 2-4 (+24-32%). GLUT-4 protein was 39% greater than in controls in 1-wk WR soleus, and this adaptation was accompanied by a similar increase in in vitro insulin-stimulated glucose transport activity(+29%). After 2 and 4 wk of WR, however, insulin-stimulated glucose transport activity had returned to control levels, despite a continued elevation (+25-28%) of GLUT-4 protein. At these two time points, glycogen concentration was significantly enhanced in WR soleus (+21-42%), which coincided with significant reductions in glycogen synthase activity ratios (-23 to-41%). These results indicate that, in this model of soleus muscle functional overload, the GLUT-4 protein level may initially regulate insulin-stimulated glucose transport activity in the absence of changes in other modifying factors. However,this regulation of glucose transport activity by GLUT-4 protein may be subsequently overridden by elevated glycogen concentration.

  14. Multiple cellobiohydrolases and cellobiose phosphorylases cooperate in the ruminal bacterium Ruminococcus albus 8 to degrade cellooligosaccharides

    PubMed Central

    Devendran, Saravanan; Abdel-Hamid, Ahmed M.; Evans, Anton F.; Iakiviak, Michael; Kwon, In Hyuk; Mackie, Roderick I.; Cann, Isaac

    2016-01-01

    Digestion of plant cell wall polysaccharides is important in energy capture in the gastrointestinal tract of many herbivorous and omnivorous mammals, including humans and ruminants. The members of the genus Ruminococcus are found in both the ruminant and human gastrointestinal tract, where they show versatility in degrading both hemicellulose and cellulose. The available genome sequence of Ruminococcus albus 8, a common inhabitant of the cow rumen, alludes to a bacterium well-endowed with genes that target degradation of various plant cell wall components. The mechanisms by which R. albus 8 employs to degrade these recalcitrant materials are, however, not clearly understood. In this report, we demonstrate that R. albus 8 elaborates multiple cellobiohydrolases with multi-modular architectures that overall enhance the catalytic activity and versatility of the enzymes. Furthermore, our analyses show that two cellobiose phosphorylases encoded by R. albus 8 can function synergistically with a cognate cellobiohydrolase and endoglucanase to completely release, from a cellulosic substrate, glucose which can then be fermented by the bacterium for production of energy and cellular building blocks. We further use transcriptomic analysis to confirm the over-expression of the biochemically characterized enzymes during growth of the bacterium on cellulosic substrates compared to cellobiose. PMID:27748409

  15. A conserved loop in polynucleotide phosphorylase (PNPase) essential for both RNA and ADP/phosphate binding.

    PubMed

    Carzaniga, Thomas; Mazzantini, Elisa; Nardini, Marco; Regonesi, Maria Elena; Greco, Claudio; Briani, Federica; De Gioia, Luca; Dehò, Gianni; Tortora, Paolo

    2014-02-01

    Polynucleotide phosphorylase (PNPase) reversibly catalyzes RNA phosphorolysis and polymerization of nucleoside diphosphates. Its homotrimeric structure forms a central channel where RNA is accommodated. Each protomer core is formed by two paralogous RNase PH domains: PNPase1, whose function is largely unknown, hosts a conserved FFRR loop interacting with RNA, whereas PNPase2 bears the putative catalytic site, ∼20 Å away from the FFRR loop. To date, little is known regarding PNPase catalytic mechanism. We analyzed the kinetic properties of two Escherichia coli PNPase mutants in the FFRR loop (R79A and R80A), which exhibited a dramatic increase in Km for ADP/Pi binding, but not for poly(A), suggesting that the two residues may be essential for binding ADP and Pi. However, both mutants were severely impaired in shifting RNA electrophoretic mobility, implying that the two arginines contribute also to RNA binding. Additional interactions between RNA and other PNPase domains (such as KH and S1) may preserve the enzymatic activity in R79A and R80A mutants. Inspection of enzyme structure showed that PNPase has evolved a long-range acting hydrogen bonding network that connects the FFRR loop with the catalytic site via the F380 residue. This hypothesis was supported by mutation analysis. Phylogenetic analysis of PNPase domains and RNase PH suggests that such network is a unique feature of PNPase1 domain, which coevolved with the paralogous PNPase2 domain.

  16. Multiple cellobiohydrolases and cellobiose phosphorylases cooperate in the ruminal bacterium Ruminococcus albus 8 to degrade cellooligosaccharides

    NASA Astrophysics Data System (ADS)

    Devendran, Saravanan; Abdel-Hamid, Ahmed M.; Evans, Anton F.; Iakiviak, Michael; Kwon, In Hyuk; Mackie, Roderick I.; Cann, Isaac

    2016-10-01

    Digestion of plant cell wall polysaccharides is important in energy capture in the gastrointestinal tract of many herbivorous and omnivorous mammals, including humans and ruminants. The members of the genus Ruminococcus are found in both the ruminant and human gastrointestinal tract, where they show versatility in degrading both hemicellulose and cellulose. The available genome sequence of Ruminococcus albus 8, a common inhabitant of the cow rumen, alludes to a bacterium well-endowed with genes that target degradation of various plant cell wall components. The mechanisms by which R. albus 8 employs to degrade these recalcitrant materials are, however, not clearly understood. In this report, we demonstrate that R. albus 8 elaborates multiple cellobiohydrolases with multi-modular architectures that overall enhance the catalytic activity and versatility of the enzymes. Furthermore, our analyses show that two cellobiose phosphorylases encoded by R. albus 8 can function synergistically with a cognate cellobiohydrolase and endoglucanase to completely release, from a cellulosic substrate, glucose which can then be fermented by the bacterium for production of energy and cellular building blocks. We further use transcriptomic analysis to confirm the over-expression of the biochemically characterized enzymes during growth of the bacterium on cellulosic substrates compared to cellobiose.

  17. Multiple disulfide bridges modulate conformational stability and flexibility in hyperthermophilic archaeal purine nucleoside phosphorylase.

    PubMed

    Bagarolo, Maria Libera; Porcelli, Marina; Martino, Elisa; Feller, Georges; Cacciapuoti, Giovanna

    2015-10-01

    5'-Deoxy-5'-methylthioadenosine phosphorylase from Sulfolobus solfataricus is a hexameric hyperthermophilic protein containing in each subunit two pairs of disulfide bridges, a CXC motif, and one free cysteine. The contribution of each disulfide bridge to the protein conformational stability and flexibility has been assessed by comparing the thermal unfolding and the limited proteolysis of the wild-type enzyme and its variants obtained by site-directed mutagenesis of the seven cysteine residues. All variants catalyzed efficiently MTA cleavage with specific activity similar to the wild-type enzyme. The elimination of all cysteine residues caused a substantial decrease of ΔHcal (850 kcal/mol) and Tmax (39°C) with respect to the wild-type indicating that all cysteine pairs and especially the CXC motif significantly contribute to the enzyme thermal stability. Disulfide bond Cys200-Cys262 and the CXC motif weakly affected protein flexibility while the elimination of the disulfide bond Cys138-Cys205 lead to an increased protease susceptibility. Experimental evidence from limited proteolysis, differential scanning calorimetry, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing and nonreducing conditions also allowed to propose a stabilizing role for the free Cys164.

  18. Crystal structure and molecular dynamics studies of human purine nucleoside phosphorylase complexed with 7-deazaguanine.

    PubMed

    Caceres, Rafael Andrade; Timmers, Luis Fernando Saraiva Macedo; Pauli, Ivani; Gava, Lisandra Marques; Ducati, Rodrigo Gay; Basso, Luiz Augusto; Santos, Diógenes Santiago; de Azevedo, Walter Filgueira

    2010-03-01

    In humans, purine nucleoside phosphorylase (HsPNP) is responsible for degradation of deoxyguanosine, and genetic deficiency of this enzyme leads to profound T-cell mediated immunosuppression. HsPNP is a target for inhibitor development aiming at T-cell immune response modulation. Here we report the crystal structure of HsPNP in complex with 7-deazaguanine (HsPNP:7DG) at 2.75 A. Molecular dynamics simulations were employed to assess the structural features of HsPNP in both free form and in complex with 7DG. Our results show that some regions, responsible for entrance and exit of substrate, present a conformational variability, which is dissected by dynamics simulation analysis. Enzymatic assays were also carried out and revealed that 7-deazaguanine presents a lower inhibitory activity against HsPNP (K(i)=200 microM). The present structure may be employed in both structure-based design of PNP inhibitors and in development of specific empirical scoring functions.

  19. Capillary bioreactors based on human purine nucleoside phosphorylase: a new approach for ligands identification and characterization.

    PubMed

    de Moraes, Marcela Cristina; Ducati, Rodrigo Gay; Donato, Augusto José; Basso, Luiz Augusto; Santos, Diógenes Santiago; Cardoso, Carmen Lucia; Cass, Quezia Bezerra

    2012-04-01

    The enzyme purine nucleoside phosphorylase (PNP) is a target for the discovery of new lead compounds employed on the treatment severe T-cell mediated disorders. Within this context, the development of new, direct, and reliable methods for ligands screening is an important task. This paper describes the preparation of fused silica capillaries human PNP (HsPNP) immobilized enzyme reactor (IMER). The activity of the obtained IMER is monitored on line in a multidimensional liquid chromatography system, by the quantification of the product formed throughout the enzymatic reaction. The K(M) value for the immobilized enzyme was about twofold higher than that measured for the enzyme in solution (255 ± 29.2 μM and 133 ± 14.9 μM, respectively). A new fourth-generation immucillin derivative (DI4G; IC(50)=40.6 ± 0.36 nM), previously identified and characterized in HsPNP free enzyme assays, was used to validate the IMER as a screening method for HsPNP ligands. The validated method was also used for mechanistic studies with this inhibitor. This new approach is a valuable tool to PNP ligand screening, since it directly measures the hypoxanthine released by inosine phosphorolysis, thus furnishing more reliable results than those one used in a coupled enzymatic spectrophotometric assay.

  20. A mutation in polynucleotide phosphorylase from Escherichia coli impairing RNA binding and degradosome stability

    PubMed Central

    Regonesi, Maria Elena; Briani, Federica; Ghetta, Andrea; Zangrossi, Sandro; Ghisotti, Daniela; Tortora, Paolo; Dehò, Gianni

    2004-01-01

    Polynucleotide phosphorylase (PNPase), a 3′ to 5′ exonuclease encoded by pnp, plays a key role in Escherichia coli RNA decay. The enzyme, made of three identical 711 amino acid subunits, may also be assembled in the RNA degradosome, a heteromultimeric complex involved in RNA degradation. PNPase autogenously regulates its expression by promoting the decay of pnp mRNA, supposedly by binding at the 5′-untranslated leader region of an RNase III-processed form of this transcript. The KH and S1 RNA-binding domains at the C-terminus of the protein (amino acids 552–711) are thought to be involved in pnp mRNA recognition. Here we show that a G454D substitution in E.coli PNPase impairs autogenous regulation whereas it does not affect the catalytic activities of the enzyme. Although the mutation maps outside of the KH and S1 RNA-binding domains, analysis of the mutant protein revealed a defective RNA binding, thus suggesting that other determinants may be involved in PNPase–RNA interactions. The mutation also caused a looser association with the degradosome and an abnormal electrophoretic mobility in native gels. The latter feature suggests an altered structural conformation of PNPase, which may account for the properties of the mutant protein. PMID:14963263

  1. Diffuse reticuloendothelial system involvement in type IV glycogen storage disease with a novel GBE1 mutation: a case report and review.

    PubMed

    Magoulas, Pilar L; El-Hattab, Ayman W; Roy, Angshumoy; Bali, Deeksha S; Finegold, Milton J; Craigen, William J

    2012-06-01

    Glycogen storage disease type IV is a rare autosomal recessive disorder of glycogen metabolism caused by mutations in the GBE1 gene that encodes the 1,4-alpha-glucan-branching enzyme 1. Its clinical presentation is variable, with the most common form presenting in early childhood with primary hepatic involvement. Histologic manifestations in glycogen storage disease type IV typically consist of intracytoplasmic non-membrane-bound inclusions containing abnormally branched glycogen (polyglucosan bodies) within hepatocytes and myocytes. We report a female infant with classic hepatic form of glycogen storage disease type IV who demonstrated diffuse reticuloendothelial system involvement with the spleen, bone marrow, and lymph nodes infiltrated by foamy histiocytes with intracytoplasmic polyglucosan deposits. Sequence analysis of the GBE1 gene revealed compound heterozygosity for a previously described frameshift mutation (c.1239delT) and a novel missense mutation (c.1279G>A) that is predicted to alter a conserved glycine residue. GBE enzyme analysis revealed no detectable activity. A review of the literature for glycogen storage disease type IV patients with characterized molecular defects and deficient enzyme activity reveals most GBE1 mutations to be missense mutations clustering in the catalytic enzyme domain. Individuals with the classic hepatic form of glycogen storage disease type IV tend to be compound heterozygotes for null and missense mutations. Although the extensive reticuloendothelial system involvement that was observed in our patient is not typical of glycogen storage disease type IV, it may be associated with severe enzymatic deficiency and a poor outcome.

  2. Structure of purine nucleoside phosphorylase (DeoD) from Bacillus anthracis

    SciTech Connect

    Grenha, Rosa; Levdikov, Vladimir M.; Fogg, Mark J.; Blagova, Elena V.; Brannigan, James A. Wilkinson, Anthony J.; Wilson, Keith S.

    2005-05-01

    The crystal structure of purine nucleoside phosphorylase (DeoD) from B. anthracis was solved by X-ray crystallography using molecular replacement and refined at a resolution of 2.24 Å. Protein structures from the causative agent of anthrax (Bacillus anthracis) are being determined as part of a structural genomics programme. Amongst initial candidates for crystallographic analysis are enzymes involved in nucleotide biosynthesis, since these are recognized as potential targets in antibacterial therapy. Purine nucleoside phosphorylase is a key enzyme in the purine-salvage pathway. The crystal structure of purine nucleoside phosphorylase (DeoD) from B. anthracis has been solved by molecular replacement at 2.24 Å resolution and refined to an R factor of 18.4%. This is the first report of a DeoD structure from a Gram-positive bacterium.

  3. Effect of eccentric exercise with reduced muscle glycogen on plasma interleukin-6 and neuromuscular responses of musculus quadriceps femoris.

    PubMed

    Gavin, James P; Myers, Stephen D; Willems, Mark E T

    2016-07-01

    Eccentric exercise can result in muscle damage and interleukin-6 (IL-6) secretion. Glycogen availability is a potent stimulator of IL-6 secretion. We examined effects of eccentric exercise in a low-glycogen state on neuromuscular function and plasma IL-6 secretion. Twelve active men (23 ± 4 yr, 179 ± 5 cm, 77 ± 10 kg, means ± SD) completed two downhill treadmill runs (gradient, -12%, 5 × 8 min; speed, 12.1 ± 1.1 km/h) with normal (NG) and reduced muscle glycogen (RG) in randomized order and at least 6 wk apart. Muscle glycogen was reduced using an established cycling protocol until exhaustion and dietary manipulation the evening before the morning run. Physiological responses were measured up to 48 h after the downhill runs. During recovery, force deficits of musculus quadriceps femoris by maximal isometric contractions were similar. Changes in low-frequency fatigue were larger with RG. Voluntary activation and plasma IL-6 levels were similar in recovery between conditions. It is concluded that unaccustomed, damaging eccentric exercise with low muscle glycogen of the m. quadriceps femoris 1) exacerbated low-frequency fatigue but 2) had no additional effect on IL-6 secretion. Neuromuscular impairment after eccentric exercise with low muscle glycogen appears to have a greater peripheral component in early recovery. PMID:27150832

  4. Effect of eccentric exercise with reduced muscle glycogen on plasma interleukin-6 and neuromuscular responses of musculus quadriceps femoris.

    PubMed

    Gavin, James P; Myers, Stephen D; Willems, Mark E T

    2016-07-01

    Eccentric exercise can result in muscle damage and interleukin-6 (IL-6) secretion. Glycogen availability is a potent stimulator of IL-6 secretion. We examined effects of eccentric exercise in a low-glycogen state on neuromuscular function and plasma IL-6 secretion. Twelve active men (23 ± 4 yr, 179 ± 5 cm, 77 ± 10 kg, means ± SD) completed two downhill treadmill runs (gradient, -12%, 5 × 8 min; speed, 12.1 ± 1.1 km/h) with normal (NG) and reduced muscle glycogen (RG) in randomized order and at least 6 wk apart. Muscle glycogen was reduced using an established cycling protocol until exhaustion and dietary manipulation the evening before the morning run. Physiological responses were measured up to 48 h after the downhill runs. During recovery, force deficits of musculus quadriceps femoris by maximal isometric contractions were similar. Changes in low-frequency fatigue were larger with RG. Voluntary activation and plasma IL-6 levels were similar in recovery between conditions. It is concluded that unaccustomed, damaging eccentric exercise with low muscle glycogen of the m. quadriceps femoris 1) exacerbated low-frequency fatigue but 2) had no additional effect on IL-6 secretion. Neuromuscular impairment after eccentric exercise with low muscle glycogen appears to have a greater peripheral component in early recovery.

  5. Postexercise Glycogen Recovery and Exercise Performance is Not Significantly Different Between Fast Food and Sport Supplements.

    PubMed

    Cramer, Michael J; Dumke, Charles L; Hailes, Walter S; Cuddy, John S; Ruby, Brent C

    2015-10-01

    A variety of dietary choices are marketed to enhance glycogen recovery after physical activity. Past research informs recommendations regarding the timing, dose, and nutrient compositions to facilitate glycogen recovery. This study examined the effects of isoenergetic sport supplements (SS) vs. fast food (FF) on glycogen recovery and exercise performance. Eleven males completed two experimental trials in a randomized, counterbalanced order. Each trial included a 90-min glycogen depletion ride followed by a 4-hr recovery period. Absolute amounts of macronutrients (1.54 ± 0.27 g·kg-1 carbohydrate, 0.24 ± 0.04 g·kg fat-1, and 0.18 ±0.03g·kg protein-1) as either SS or FF were provided at 0 and 2 hr. Muscle biopsies were collected from the vastus lateralis at 0 and 4 hr post exercise. Blood samples were analyzed at 0, 30, 60, 120, 150, 180, and 240 min post exercise for insulin and glucose, with blood lipids analyzed at 0 and 240 min. A 20k time-trial (TT) was completed following the final muscle biopsy. There were no differences in the blood glucose and insulin responses. Similarly, rates of glycogen recovery were not different across the diets (6.9 ± 1.7 and 7.9 ± 2.4 mmol·kg wet weight- 1·hr-1 for SS and FF, respectively). There was also no difference across the diets for TT performance (34.1 ± 1.8 and 34.3 ± 1.7 min for SS and FF, respectively. These data indicate that short-term food options to initiate glycogen resynthesis can include dietary options not typically marketed as sports nutrition products such as fast food menu items.

  6. Investigation and management of the hepatic glycogen storage diseases

    PubMed Central

    2015-01-01

    The glycogen storage diseases (GSD) comprise a group of disorders that involve the disruption of metabolism of glycogen. Glycogen is stored in various organs including skeletal muscle, the kidneys and liver. The liver stores glycogen to supply the rest of the body with glucose when required. Therefore, disruption of this process can lead to hypoglycaemia. If glycogen is not broken down effectively, this can lead to hepatomegaly. Glycogen synthase deficiency leads to impaired glycogen synthesis and consequently the liver is small. Glycogen brancher deficiency can lead to abnormal glycogen being stored in the liver leading to a quite different disorder of progressive liver dysfunction. Understanding the physiology of GSD I, III, VI and IX guides dietary treatments and the provision of appropriate amounts and types of carbohydrates. There has been recent re-emergence in the literature of the use of ketones in therapy, either in the form of the salt D,L-3-hydroxybutyrate or medium chain triglyceride (MCT). High protein diets have also been advocated. Alternative waxy maize based starches seem to show promising early data of efficacy. There are many complications of each of these disorders and they need to be prospectively surveyed and managed. Liver and kidney transplantation is still indicated in severe refractory disease. PMID:26835382

  7. Investigation and management of the hepatic glycogen storage diseases.

    PubMed

    Bhattacharya, Kaustuv

    2015-07-01

    The glycogen storage diseases (GSD) comprise a group of disorders that involve the disruption of metabolism of glycogen. Glycogen is stored in various organs including skeletal muscle, the kidneys and liver. The liver stores glycogen to supply the rest of the body with glucose when required. Therefore, disruption of this process can lead to hypoglycaemia. If glycogen is not broken down effectively, this can lead to hepatomegaly. Glycogen synthase deficiency leads to impaired glycogen synthesis and consequently the liver is small. Glycogen brancher deficiency can lead to abnormal glycogen being stored in the liver leading to a quite different disorder of progressive liver dysfunction. Understanding the physiology of GSD I, III, VI and IX guides dietary treatments and the provision of appropriate amounts and types of carbohydrates. There has been recent re-emergence in the literature of the use of ketones in therapy, either in the form of the salt D,L-3-hydroxybutyrate or medium chain triglyceride (MCT). High protein diets have also been advocated. Alternative waxy maize based starches seem to show promising early data of efficacy. There are many complications of each of these disorders and they need to be prospectively surveyed and managed. Liver and kidney transplantation is still indicated in severe refractory disease. PMID:26835382

  8. Crystal Structure and Substrate Recognition of Cellobionic Acid Phosphorylase, Which Plays a Key Role in Oxidative Cellulose Degradation by Microbes*

    PubMed Central

    Nam, Young-Woo; Nihira, Takanori; Arakawa, Takatoshi; Saito, Yuka; Kitaoka, Motomitsu; Nakai, Hiroyuki; Fushinobu, Shinya

    2015-01-01

    The microbial oxidative cellulose degradation system is attracting significant research attention after the recent discovery of lytic polysaccharide mono-oxygenases. A primary product of the oxidative and hydrolytic cellulose degradation system is cellobionic acid (CbA), the aldonic acid form of cellobiose. We previously demonstrated that the intracellular enzyme belonging to glycoside hydrolase family 94 from cellulolytic fungus and bacterium is cellobionic acid phosphorylase (CBAP), which catalyzes reversible phosphorolysis of CbA into glucose 1-phosphate and gluconic acid (GlcA). In this report, we describe the biochemical characterization and the three-dimensional structure of CBAP from the marine cellulolytic bacterium Saccharophagus degradans. Structures of ligand-free and complex forms with CbA, GlcA, and a synthetic disaccharide product from glucuronic acid were determined at resolutions of up to 1.6 Å. The active site is located near the dimer interface. At subsite +1, the carboxylate group of GlcA and CbA is recognized by Arg-609 and Lys-613. Additionally, one residue from the neighboring protomer (Gln-190) is involved in the carboxylate recognition of GlcA. A mutational analysis indicated that these residues are critical for the binding and catalysis of the aldonic and uronic acid acceptors GlcA and glucuronic acid. Structural and sequence comparisons with other glycoside hydrolase family 94 phosphorylases revealed that CBAPs have a unique subsite +1 with a distinct amino acid residue conservation pattern at this site. This study provides molecular insight into the energetically efficient metabolic pathway of oxidized sugars that links the oxidative cellulolytic pathway to the glycolytic and pentose phosphate pathways in cellulolytic microbes. PMID:26041776

  9. Thymidine phosphorylase in cancer cells stimulates human endothelial cell migration and invasion by the secretion of angiogenic factors

    PubMed Central

    Bijnsdorp, I V; Capriotti, F; Kruyt, F A E; Losekoot, N; Fukushima, M; Griffioen, A W; Thijssen, V L; Peters, G J

    2011-01-01

    Background: Thymidine phosphorylase (TP) is often overexpressed in tumours and has a role in tumour aggressiveness and angiogenesis. Here, we determined whether TP increased tumour invasion and whether TP-expressing cancer cells stimulated angiogenesis. Methods: Angiogenesis was studied by exposing endothelial cells (HUVECs) to conditioned medium (CM) derived from cancer cells with high (Colo320TP1=CT-CM, RT112/TP=RT-CM) and no TP expression after which migration (wound-healing-assay) and invasion (transwell-assay) were determined. The involvement of several angiogenic factors were examined by RT–PCR, ELISA and blocking antibodies. Results: Tumour invasion was not dependent on intrinsic TP expression. The CT-CM and RT-CM stimulated HUVEC-migration and invasion by about 15 and 40%, respectively. Inhibition by 10 μ TPI and 100 μ L-dR, blocked migration and reduced the invasion by 50–70%. Thymidine phosphorylase activity in HUVECs was increased by CT-CM. Reverse transcription-polymerase chain reaction revealed a higher mRNA expression of bFGF (Colo320TP1), IL-8 (RT112/TP) and TNF-α, but not VEGF. Blocking antibodies targeting these factors decreased the migration and invasion that was induced by the CT-CM and RT-CM, except for IL-8 in CT-CM and bFGF in RT-CM. Conclusion: In our cell line panels, TP did not increase the tumour invasion, but stimulated the migration and invasion of HUVECs by two different mechanisms. Hence, TP targeting seems to provide a potential additional strategy in the field of anti-angiogenic therapy. PMID:21386840

  10. Volume I. Glycogen: A historical overview, an adjunct to thesis. Volume II. Non-glucose components of glycogen

    SciTech Connect

    Kirkman, B.R.

    1988-01-01

    Investigations have been carried out on three non-glucose components of native glycogen: protein, glucosamine, and phosphate. The protein, glycogenin, appears to serve as the primer upon which new molecules of glycogen are synthesized. When cell extracts are incubated with ({sup 14}C)UDPG, ({sup 14}C)glucose becomes transferred onto pre-existing chains of alpha-1,4 linked glucose associated with free glycogenin. The transferase and glycogenin remain associated during various purification steps. Liver glycogen appears to contain less than 0.02% protein which may correspond to the presence of one molecule of glycogenin (37 kDa) per alpha particle of liver glycogen. The core beta particle within each alpha particle may be synthesized upon glycogenin, while the remaining 20-40 beta particles may arise from each other. The author has demonstrated the natural occurrence of glucosamine in liver glycogen (but not muscle glycogen) from various species in an amount of about one molecule per molecule of glycogen. The glucosamine is underivatized, appears to be randomly scattered in the glycogen, and may be derived from dietary galactosamine. Similar to Fontana (1980), the author observed that native liver glycogen could be fractionated on DEAE-cellulose apparently on the basis of phosphate content. The more strongly bound glycogen possessed a greater molecular weight and content of glucosamine and phosphate. Possible explanations for these subfractions are considered. The phosphate appears to be concentrated near the center of the glycogen molecules. About 30% appears to be associated with glucose-6P and the remainder with an unidentified phosphodiester. The phosphate may stimulate glycogen synthesis. How the phosphate becomes incorporated is unknown.

  11. Methylthioinosine Phosphorylase from Pseudomonas aeruginosa. Structure and Annotation of a Novel Enzyme in Quorum Sensing†

    PubMed Central

    Guan, Rong; Ho, Meng-Chiao; Almo, Steven C.; Schramm, Vern L.

    2011-01-01

    The PA3004 gene of Pseudomonas aeruginosa PAO1 was originally annotated as a 5’-methylthioadenosine phosphorylase (MTAP). However, the PA3004 encoded protein uses 5’-methylthioinosine (MTI) as a preferred substrate and represents the only known example of a specific MTI phosphorylase (MTIP). MTIP does not utilize 5’-methylthioadenosine (MTA). Inosine is a weak substrate with a kcat/Km value 290-fold less than MTI and is the second best substrate identified. The crystal structure of P. aeruginosa MTIP (PaMTIP) in complex with hypoxanthine was determined to 2.8 Å resolution and revealed a three-fold symmetric homotrimer. The methylthioribose and phosphate binding regions of PaMTIP are similar to MTAPs, and the purine binding region is similar to that of purine nucleoside phosphorylases (PNPs). The catabolism of MTA in P. aeruginosa involves deamination to MTI and phosphorolysis to hypoxanthine (MTA → MTI → hypoxanthine). This pathway also exists in Plasmodium falciparum, where the purine nucleoside phosphorylase (PfPNP) acts on both inosine and MTI. Three tight-binding transition state analogue inhibitors of PaMTIP are identified with dissociation constants in the picomolar range. Inhibitor specificity suggests an early dissociative transition state for PaMTIP. Quorum sensing molecules are associated with MTA metabolism in bacterial pathogens suggesting PaMTIP as a potential therapeutic target. PMID:21197954

  12. The GH130 Family of Mannoside Phosphorylases Contains Glycoside Hydrolases That Target β-1,2-Mannosidic Linkages in Candida Mannan*

    PubMed Central

    Cuskin, Fiona; Baslé, Arnaud; Ladevèze, Simon; Day, Alison M.; Gilbert, Harry J.; Davies, Gideon J.; Potocki-Véronèse, Gabrielle; Lowe, Elisabeth C.

    2015-01-01

    The depolymerization of complex glycans is an important biological process that is of considerable interest to environmentally relevant industries. β-Mannose is a major component of plant structural polysaccharides and eukaryotic N-glycans. These linkages are primarily cleaved by glycoside hydrolases, although recently, a family of glycoside phosphorylases, GH130, have also been shown to target β-1,2- and β-1,4-mannosidic linkages. In these phosphorylases, bond cleavage was mediated by a single displacement reaction in which phosphate functions as the catalytic nucleophile. A cohort of GH130 enzymes, however, lack the conserved basic residues that bind the phosphate nucleophile, and it was proposed that these enzymes function as glycoside hydrolases. Here we show that two Bacteroides enzymes, BT3780 and BACOVA_03624, which lack the phosphate binding residues, are indeed β-mannosidases that hydrolyze β-1,2-mannosidic linkages through an inverting mechanism. Because the genes encoding these enzymes are located in genetic loci that orchestrate the depolymerization of yeast α-mannans, it is likely that the two enzymes target the β-1,2-mannose residues that cap the glycan produced by Candida albicans. The crystal structure of BT3780 in complex with mannose bound in the −1 and +1 subsites showed that a pair of glutamates, Glu227 and Glu268, hydrogen bond to O1 of α-mannose, and either of these residues may function as the catalytic base. The candidate catalytic acid and the other residues that interact with the active site mannose are conserved in both GH130 mannoside phosphorylases and β-1,2-mannosidases. Functional phylogeny identified a conserved lysine, Lys199 in BT3780, as a key specificity determinant for β-1,2-mannosidic linkages. PMID:26286752

  13. Scintigraphic abnormalities in glycogen storage disease.

    PubMed

    Miller, J H; Gates, G F; Landing, B H; Kogut, M D; Roe, T F

    1978-04-01

    Fifteen patients with glycogen-storage disease type 1 (von Gierke's disease) were evaluated by serial scintigraphy, with a clearly recognizable pattern of an enlarged liver with diminished radionuclide accumulation, splenomegaly with considerably increased uptake and renomegaly. In seven of these patients with GSD-1 scintigraphy demonstrated focal defects of varying size. Small or stable defects suggest benign hepatic adenomata, whereas malignant change occurred in growing large lesions. The potential malignant end-point of hepatic-cell carcinoma in GSD-1 warrants careful serial liver scintigraphy with scintiangiography on a routine basis. PMID:204758

  14. Liver as a source for thymidine phosphorylase replacement in mitochondrial neurogastrointestinal encephalomyopathy.

    PubMed

    Boschetti, Elisa; D'Alessandro, Roberto; Bianco, Francesca; Carelli, Valerio; Cenacchi, Giovanna; Pinna, Antonio D; Del Gaudio, Massimo; Rinaldi, Rita; Stanghellini, Vincenzo; Pironi, Loris; Rhoden, Kerry; Tugnoli, Vitaliano; Casali, Carlo; De Giorgio, Roberto

    2014-01-01

    Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a rare autosomal recessive mitochondrial disease associated with mutations in the nuclear TYMP gene. As a result, the thymidine phosphorylase (TP) enzyme activity is markedly reduced leading to toxic accumulation of thymidine and therefore altered mitochondrial DNA. MNGIE is characterized by severe gastrointestinal dysmotility, neurological impairment, reduced life expectancy and poor quality of life. There are limited therapeutic options for MNGIE. In the attempt to restore TP activity, allogenic hematopoietic stem cell transplantation has been used as cellular source of TP. The results of this approach on ∼ 20 MNGIE patients showed gastrointestinal and neurological improvement, although the 5-year mortality rate is about 70%. In this study we tested whether the liver may serve as an alternative source of TP. We investigated 11 patients (7M; 35-55 years) who underwent hepatic resection for focal disorders. Margins of normal liver tissue were processed to identify, quantify and localize the TP protein by Western Blot, ELISA, and immunohistochemistry, and to evaluate TYMP mRNA expression by qPCR. Western Blot identified TP in liver with a TP/GAPDH ratio of 0.9 ± 0.5. ELISA estimated TP content as 0.5 ± 0.07 ng/μg of total protein. TP was identified in both nuclei and cytoplasm of hepatocytes and sinusoidal lining cells. Finally, TYMP mRNA was expressed in the liver. Overall, our study demonstrates that the liver is an important source of TP. Orthotopic liver transplantation may be considered as a therapeutic alternative for MNGIE patients.

  15. Effects of short and long term exercise on intracellular glycogen and fat in pigeon pectoralis.

    PubMed

    Parker, G H; George, J C

    1975-01-01

    In an attempt to determine the functional role of the component broad white and narrow red fibre types during activity, the pectroalis muscle of the adult pigeon (Columba livia) was electrically stimulated in situ after anaesthetizing the birds with sodium pentobarbital. For examination of the effect of short term exercise, the pectoral muscle of one side was stimulated through the innervating brachial plexus, using an electronic stimulator for 1 hr, and a biopsy sample was taken from the stimulated muscle. The stimulation was resumed for 17 hr more with additional doses of anaesthesia, and a muscle sample was taken after the decapitation of the birds in the long term experiment. Muscle samples from non-stimulated pigeons under identical conditions served as control. Fixed and frozen sections were made from the samples and were stained with Periodic Acid-Schiff's reagent for the demonstration of glycogen, while additional frozen muscle sections were stained with Fettrot 7B according to Pearse, A.G.E. (1960) for the demonstration of neutral lipids in the specimen. Thus histochemical examinations were made to study effects of short and long term exercises on energy stores in skeletal muscles. Short-term stimulation resulted in a selective depletion of intracellular glycogen stores from white fibre populations whereas prolonged long term stimulation resulted in the resynthesis of glycogen within these fibres and a concomitant reduction in the intracellular fat and glycogen reserves in most red fibres. It is postulated that during flight the white fibres indulge only in brief bursts of intense phasic activity such as in quick take-off, rapid accelerations or sudden manoeuvres and utilize glycogen as fuel whereas the red fibres perform sustained activity as in cruising flight and metabolize chiefly fat.

  16. Anopheles gambiae Purine Nucleoside Phosphorylase: Catalysis, Structure, and Inhibition

    SciTech Connect

    Taylor,E.; Rinaldo-Matthis, A.; Li, L.; Ghanem, M.; Hazleton, K.; Cassera, M.; Almo, S.; Schramm, V.

    2007-01-01

    The purine salvage pathway of Anopheles gambiae, a mosquito that transmits malaria, has been identified in genome searches on the basis of sequence homology with characterized enzymes. Purine nucleoside phosphorylase (PNP) is a target for the development of therapeutic agents in humans and purine auxotrophs, including malarial parasites. The PNP from Anopheles gambiae (AgPNP) was expressed in Escherichia coli and compared to the PNPs from Homo sapiens (HsPNP) and Plasmodium falciparum (PfPNP). AgPNP has kcat values of 54 and 41 s-1 for 2'-deoxyinosine and inosine, its preferred substrates, and 1.0 s-1 for guanosine. However, the chemical step is fast for AgPNP at 226 s-1 for guanosine in pre-steady-state studies. 5'-Deaza-1'-aza-2'-deoxy-1'-(9-methylene)-Immucillin-H (DADMe-ImmH) is a transition-state mimic for a 2'-deoxyinosine ribocation with a fully dissociated N-ribosidic bond and is a slow-onset, tight-binding inhibitor with a dissociation constant of 3.5 pM. This is the tightest-binding inhibitor known for any PNP, with a remarkable Km/Ki* of 5.4 x 107, and is consistent with enzymatic transition state predictions of enhanced transition-state analogue binding in enzymes with enhanced catalytic efficiency. Deoxyguanosine is a weaker substrate than deoxyinosine, and DADMe-Immucillin-G is less tightly bound than DADMe-ImmH, with a dissociation constant of 23 pM for AgPNP as compared to 7 pM for HsPNP. The crystal structure of AgPNP was determined in complex with DADMe-ImmH and phosphate to a resolution of 2.2 Angstroms to reveal the differences in substrate and inhibitor specificity. The distance from the N1' cation to the phosphate O4 anion is shorter in the AgPNP{center_dot}DADMe-ImmH{center_dot}PO4 complex than in HsPNP{center_dot}DADMe-ImmH{center_dot}SO4, offering one explanation for the stronger inhibitory effect of DADMe-ImmH for AgPNP.

  17. The human liver glycogen synthase isozyme gene is located on the short arm of chromosome 12

    SciTech Connect

    Nuttall, F.Q.; Gannon, M.C. ); Kubic, V.L.; Hoyt, K.J. )

    1994-01-15

    Glycogen synthase catalyzes the rate-limiting step in glycogen synthesis. Its activity is regulated by a complex phosphorylation-dephosphorylation mechanism and by allosteric stimulators and inhibitors. Two isozymes of synthase, a skeletal muscle type and liver type, have been identified in rabbit and rat tissues using specific polyclonal antibodies. The skeletal muscle type isozyme is present in several organs in addition to skeletal muscle; the liver isozyme has been identified only in liver. Recently, we have purified and characterized the human liver synthase isozyme. We also have cloned and sequenced the gene from a human liver cDNA library. Using the entire cDNA coding sequence as a probe, we report here the localization of the liver synthase isozyme gene to the short arm of chromosome 12. These studies revealed a centromeric signal on chromosome 12 together with signal to glycogen synthase on the short arm of this chromosome in the p11.2-p12.2 region. Measurements of the relative distance from the midpoint of the centromere to the signal corresponding to glycogen synthase suggests that the locus is in the p12.2 band rather than in the more centromeric location.

  18. Glycogen Synthase Kinase-3 (GSK-3)-Targeted Therapy and Imaging.

    PubMed

    Pandey, Mukesh K; DeGrado, Timothy R

    2016-01-01

    Glycogen synthase kinase-3 (GSK-3) is associated with various key biological processes, including glucose regulation, apoptosis, protein synthesis, cell signaling, cellular transport, gene transcription, proliferation, and intracellular communication. Accordingly, GSK-3 has been implicated in a wide variety of diseases and specifically targeted for both therapeutic and imaging applications by a large number of academic laboratories and pharmaceutical companies. Here, we review the structure, function, expression levels, and ligand-binding properties of GSK-3 and its connection to various diseases. A selected list of highly potent GSK-3 inhibitors, with IC50 <20 nM for adenosine triphosphate (ATP)-competitive inhibitors and IC50 <5 μM for non-ATP-competitive inhibitors, were analyzed for structure activity relationships. Furthermore, ubiquitous expression of GSK-3 and its possible impact on therapy and imaging are also highlighted. Finally, a rational perspective and possible route to selective and effective GSK-3 inhibitors is discussed. PMID:26941849

  19. Glycogen Synthase Kinase-3 (GSK-3)-Targeted Therapy and Imaging

    PubMed Central

    Pandey, Mukesh K.; DeGrado, Timothy R.

    2016-01-01

    Glycogen synthase kinase-3 (GSK-3) is associated with various key biological processes, including glucose regulation, apoptosis, protein synthesis, cell signaling, cellular transport, gene transcription, proliferation, and intracellular communication. Accordingly, GSK-3 has been implicated in a wide variety of diseases and specifically targeted for both therapeutic and imaging applications by a large number of academic laboratories and pharmaceutical companies. Here, we review the structure, function, expression levels, and ligand-binding properties of GSK-3 and its connection to various diseases. A selected list of highly potent GSK-3 inhibitors, with IC50 <20 nM for adenosine triphosphate (ATP)-competitive inhibitors and IC50 <5 μM for non-ATP-competitive inhibitors, were analyzed for structure activity relationships. Furthermore, ubiquitous expression of GSK-3 and its possible impact on therapy and imaging are also highlighted. Finally, a rational perspective and possible route to selective and effective GSK-3 inhibitors is discussed. PMID:26941849

  20. Hepatocellular carcinoma in glycogen storage disease type IV

    PubMed Central

    de Moor, R A; Schweizer, J; van Hoek, B; Wasser, M; Vink, R; Maaswinkel-Mooy, P

    2000-01-01

    A 13 year old patient with juvenile type IV glycogen storage disease died of the complications of hepatocellular carcinoma. To our knowledge this is the first reported case of hepatocellular carcinoma in association with type IV glycogen storage disease.

 PMID:10833181

  1. Muscle glycogen and cell function--Location, location, location.

    PubMed

    Ørtenblad, N; Nielsen, J

    2015-12-01

    The importance of glycogen, as a fuel during exercise, is a fundamental concept in exercise physiology. The use of electron microscopy has revealed that glycogen is not evenly distributed in skeletal muscle fibers, but rather localized in distinct pools. In this review, we present the available evidence regarding the subcellular localization of glycogen in skeletal muscle and discuss this from the perspective of skeletal muscle fiber function. The distribution of glycogen in the defined pools within the skeletal muscle varies depending on exercise intensity, fiber phenotype, training status, and immobilization. Furthermore, these defined pools may serve specific functions in the cell. Specifically, reduced levels of these pools of glycogen are associated with reduced SR Ca(2+) release, muscle relaxation rate, and membrane excitability. Collectively, the available literature strongly demonstrates that the subcellular localization of glycogen has to be considered to fully understand the role of glycogen metabolism and signaling in skeletal muscle function. Here, we propose that the effect of low muscle glycogen on excitation-contraction coupling may serve as a built-in mechanism, which links the energetic state of the muscle fiber to energy utilization.

  2. Genetics Home Reference: glycogen storage disease type 0

    MedlinePlus

    ... the expand/collapse boxes. Download PDF Open All Close All Description Glycogen storage disease type 0 (also known as GSD 0) is a condition caused by the body's inability to form a complex sugar called glycogen , which is a major source of stored energy in the body. GSD 0 ...

  3. Purine nucleoside phosphorylase and the enzymatic antioxidant defense system in breast milk from women with different levels of arsenic exposure.

    PubMed

    Gaxiola-Robles, Ramón; Labrada-Martagón, Vanessa; Bitzer-Quintero, Oscar Kurt; Zenteno-Savín, Tania; Méndez-Rodríguez, Lía Celina

    2015-05-01

    Purine nucleoside phosphorylase (PNP) is an ubiquitous enzyme which plays an important role in arsenic (As) detoxification. As is a toxic metalloid present in air, soil and water; is abundant in the environment and is readily transferred along the trophic chain, being found even in human breast milk. Milk is the main nutrient source for the growth and development of neonates. Information on breast milk synthesis and its potential defense mechanism against As toxicity is scarce. In this study, PNP and antioxidant enzymes activities, as well as glutathione (GSH) and total arsenic (TAs) concentrations, were quantified in breast milk samples. PNP, superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST), glutathione peroxidase (GPx), glutathione reductase (GR) activities and GSH concentration were determined spectrophotometrically; TAs concentration ([TAs]) was measured by atomic absorption spectrometry. Data suggest an increase in PNP activity (median = 0.034 U mg protein-1) in the presence of TAs (median = 1.16 g L(-1)). To explain the possible association of PNP activity in breast milk with the activity of the antioxidant enzymes as well as with GSH and TAs concentrations, generalized linear models were built. In the adjusted model, GPx and GR activities showed a statistically significant (p<0.01) association with PNP activity. These results may suggest that PNP activity increases in the presence of TAs as part of the detoxification mechanism in breast milk.

  4. Four Generations of Transition State Analogues for Human Purine Nucleoside Phosphorylase

    SciTech Connect

    Ho, M.; Shi, W; Rinaldo-Mathis, A; Tyler, P; Evans, G; Almo, S; Schramm, V

    2010-01-01

    Inhibition of human purine nucleoside phosphorylase (PNP) stops growth of activated T-cells and the formation of 6-oxypurine bases, making it a target for leukemia, autoimmune disorders, and gout. Four generations of ribocation transition-state mimics bound to PNP are structurally characterized. Immucillin-H (K*{sub i} = 58 pM, first-generation) contains an iminoribitol cation with four asymmetric carbons. DADMe-Immucillin-H (K*{sub i} = 9 pM, second-generation), uses a methylene-bridged dihydroxypyrrolidine cation with two asymmetric centers. DATMe-Immucillin-H (K*{sub i} = 9 pM, third-generation) contains an open-chain amino alcohol cation with two asymmetric carbons. SerMe-ImmH (K*{sub i} = 5 pM, fourth-generation) uses achiral dihydroxyaminoalcohol seramide as the ribocation mimic. Crystal structures of PNPs establish features of tight binding to be; (1) ion-pair formation between bound phosphate (or its mimic) and inhibitor cation, (2) leaving-group interactions to N1, O6, and N7 of 9-deazahypoxanthine, (3) interaction between phosphate and inhibitor hydroxyl groups, and (4) His257 interacting with the 5{prime}-hydroxyl group. The first generation analogue is an imperfect fit to the catalytic site with a long ion pair distance between the iminoribitol and bound phosphate and weaker interactions to the leaving group. Increasing the ribocation to leaving-group distance in the second- to fourth-generation analogues provides powerful binding interactions and a facile synthetic route to powerful inhibitors. Despite chemical diversity in the four generations of transition-state analogues, the catalytic site geometry is almost the same for all analogues. Multiple solutions in transition-state analogue design are available to convert the energy of catalytic rate enhancement to binding energy in human PNP.

  5. Conformational States of Human Purine Nucleoside Phosphorylase at Rest, at Work and with Transition State Analogues†

    PubMed Central

    Edwards, Achelle A.; Tipton, Jeremiah D.; Brenowitz, Michael D.; Emmett, Mark R.; Marshall, Alan G.; Evans, Gary B.; Tyler, Peter C.; Schramm, Vern L.

    2010-01-01

    Human purine nucleoside phosphorylase (PNP) is a homotrimer binding tightly to the transition state analogues Immucillin-H (ImmH, Kd = 56 pM) and DATMe-ImmH-Immucillin-H (DATMe-ImmH, Kd = 8.6 pM). ImmH binds with a larger entropic penalty than DATMe-ImmH, a chemically more flexible inhibitor. The testable hypothesis is that PNP conformational states are more relaxed (dynamic) with DATMe-ImmH, despite tighter binding than with ImmH. PNP conformations are probed by peptide amide deuterium exchange (HDX) using liquid chromatography high-resolution Fourier transform ion cyclotron resonance mass spectrometry and by sedimentation rates. Catalytically equilibrating Michaelis complexes (PNP•PO4•Inosine ↔ PNP•Hx•R-1-P) and inhibited complexes (PNP•PO4•DATMe-ImmH and PNP•PO4•ImmH) show protection from HDX at 9, 13 and 15 sites per subunit relative to resting PNP (PNP•PO4) in extended incubations. The PNP•PO4•ImmH complex is more compact (by sedimentation rate) than the other complexes. HDX kinetic analysis of ligand-protected sites corresponds to peptides near the catalytic sites. HDX and sedimentation results establish that PNP protein conformation (dynamic motion) correlates more closely to entropy of binding than to affinity. Catalytically active turnover with saturated substrate sites causes less change in HDX and sedimentation rates than binding of transition state analogues. DATMe-ImmH more closely mimics the transition of human PNP than does ImmH, and achieves strong binding interactions at the catalytic site while causing relatively modest alterations of the protein dynamic motion. Transition state analogues causing the most rigid, closed protein conformation are therefore not necessarily the most tightly bound. Close mimics of the transition state are hypothesized to retain enzymatic dynamic motions related to transition state formation. PMID:20108972

  6. Transition Path Sampling Study of the Reaction Catalyzed by Purine Nucleoside Phosphorylase

    PubMed Central

    Saen-oon, Suwipa; Schramm, Vern L.; Schwartz, Steven D.

    2010-01-01

    The Transition Path Sampling (TPS) method is a powerful technique for studying rare events in complex systems, that allows description of reactive events in atomic detail without prior knowledge of reaction coordinates and transition states. We have applied TPS in combination with a hybrid Quantum Mechanical/Molecular Mechanical (QM/MM) method to study the enzyme human purine nucleoside phosphorylase (hPNP). This enzyme catalyzes the reversible phosphorolysis of 6-oxypurine (deoxy)nucleosides to generate the corresponding purine base and (deoxy)ribose 1-phosphate. Hundreds of reactive trajectories were generated. Analysis of this transition path ensembles provides insight into the detailed mechanistic dynamics of reaction in the enzyme. Our studies have indicated a reaction mechanism involving the cleavage of the N-ribosidic bond to form transition states with substantial ribooxacarbenium ion character, that is then followed by conformational changes in the enzyme and the ribosyl group leading to migration of the anomeric carbon of the ribosyl group toward phosphate to form the product ribose 1-phosphate. This latter process is crucial in PNP, because several strong H-bonds form between active site residues in order to capture and align the phosphate nucleophile. Calculations of the commitment probability along reactive paths demonstrated the presence of a broad energy barrier at the transition state. Analysis of these transition state structures showed that bond-breaking and bond-forming distances are not a good choice for the reaction coordinate, but that the pseudorotational phase of the ribose ring is also a significant variable. PMID:20664707

  7. Cryoelectron microscopy reveals new features in the three-dimensional structure of phosphorylase kinase.

    PubMed

    Nadeau, Owen W; Gogol, Edward P; Carlson, Gerald M

    2005-04-01

    Phosphorylase kinase (PhK), a regulatory enzyme in the cascade activation of glycogenolysis, is a 1.3-MDa hexadecameric complex, (alphabetagammadelta)(4). PhK comprises two arched octameric (alphabetagammadelta)(2) lobes that are oriented back-to-back with overall D(2) symmetry and connected by small bridges. These interlobal bridges, arguably the most questionable structural component of PhK, are one of several structural features that potentially are artifactually generated or altered by conventional sample preparation techniques for electron microscopy (EM). To minimize such artifacts, we have solved by cryoEM the first three-dimensional (3D) structure of nonactivated PhK from images of frozen hydrated molecules of the kinase. Minimal dose electron micrographs of PhK in vitreous ice revealed particles in a multitude of orientations. A simple model was used to orient the individual images for 3D reconstruction, followed by multiple rounds of refinement. Three-dimensional reconstruction of nonactivated PhK from approximately 5000 particles revealed a bridged, bilobal molecule with a resolution estimated by Fourier shell correlation analysis at 25 A. This new structure suggests that several prominent features observed in the structure of PhK derived from negatively stained particles arise as artifacts of specimen preparation. In comparison to the structure from negative staining, the cryoEM structure shows three important differences: (1) a dihedral angle between the two lobes of approximately 90 degrees instead of 68 degrees, (2) a compact rather than extended structure for the lobes, and (3) the presence of four, rather than two, connecting bridges, which provides the first direct evidence for these components as authentic elements of the kinase solution structure. PMID:15741332

  8. Role of the direct and indirect pathways for glycogen synthesis in rat liver in the postprandial state

    SciTech Connect

    Huang, M.T.; Veech, R.L.

    1988-03-01

    The pathway for hepatic glycogen synthesis in the postprandial state was studied in meal-fed rats chronically cannulated in the portal vein. Plasma glucose concentration in the portal vein was found to be 4.50 +/- 1.01 mM (mean +/- SE; n = 3) before a meal and 11.54 +/- 0.70 mM (mean +/- SE; n = 4) after a meal in rats meal-fed a diet consisting of 100% commercial rat chow for 7 d. The hepatic-portal difference of plasma glucose concentration showed that liver released glucose in the fasted state and either extracted or released glucose after feeding depending on plasma glucose concentration in the portal vein. The concentration of portal vein glucose at which liver changes from glucose releasing to glucose uptake was 8 mM, the Km of glucokinase. The rate of glycogen synthesis in liver during meal-feeding was found to be approximately 1 mumol glucosyl U/g wet wt/min in rats meal-fed a 50% glucose supplemented chow diet. The relative importance of the direct vs. indirect pathway for the replenishment of hepatic glycogen was determined by the incorporation of (3-/sup 3/H,U-/sup 14/C)glucose into liver glycogen. Labeled glucose was injected into the portal vein at the end of meal-feeding. The ratio of /sup 3/H//sup 14/C in the glucosyl units of glycogen was found to be 83-92% of the ratio in liver free glucose six minutes after the injection, indicating that the majority of exogenous glucose incorporated into glycogen did not go through glycolysis. The percent contribution of the direct versus indirect pathway was quantitated from the difference in the relative specific activity (RSA) of (/sup 3/H) and (/sup 14/C)-glycogen in rats infused with (3-/sup 3/H,U-/sup 14/C)glucose. No significant difference was found between the RSA of (/sup 3/H)glycogen and (/sup 14/C)glycogen, indicating further that the pathway for glycogen synthesis in liver from exogenous glucose is from the direct pathway.

  9. Pyruvate Incubation Enhances Glycogen Stores and Sustains Neuronal Function during Subsequent Glucose Deprivation

    PubMed Central

    Shetty, Pavan K; Sadgrove, Matthew P; Galeffi, Francesca; Turner, Dennis A.

    2011-01-01

    The use of energy substrates, such as lactate and pyruvate, has been shown to improve synaptic function when administered during glucose deprivation. In the present study, we investigatedwhether prolonged incubation with monocarboxylate (pyruvate or lactate) prior rather than duringglucose deprivation can also sustainsynaptic and metabolic function.Pyruvate pre-incubation(3-4 hr) significantly prolonged (>25 min) the tolerance of rat hippocampal slices to delayed glucose deprivation compared to control and lactate pre-incubated slices, as revealed by field excitatory post synaptic potentials (fEPSPs); pre-incubation with pyruvate alsoreduced the marked decrease in NAD(P)H fluorescence resulting from glucose deprivation. Moreover, pyruvate exposure led to enhancement of glycogen stores with time, compared to glucose alone(12 μmol/g tissue at 4 hr vs. 3.5 μmol/g tissue). Prolonged resistance to glucose deprivation following exogenous pyruvate incubation was prevented by glycogenolysis inhibitors, suggesting that enhanced glycogen mediates the delay in synaptic activity failure.The application of an adenosine A1 receptor antagonist enhanced glycogen utilization and prolonged the time to synaptic failure, further confirming this hypothesis of the importance of glycogen.Moreover, tissue levels of ATP werealso significantly maintained duringglucose deprivation in pyruvate pre-treated slices compared to control and lactate. In summary, these experiments indicate that pyruvate exposure prior to glucose deprivation significantly increased the energy buffering capacity of hippocampal slices, particularly by enhancing internal glycogen stores, delaying synaptic failure during glucose deprivation by maintaining ATP levels, and minimizing the decreasein the levels of NAD(P)H. PMID:21854850

  10. Pyruvate incubation enhances glycogen stores and sustains neuronal function during subsequent glucose deprivation.

    PubMed

    Shetty, Pavan K; Sadgrove, Matthew P; Galeffi, Francesca; Turner, Dennis A

    2012-01-01

    The use of energy substrates, such as lactate and pyruvate, has been shown to improve synaptic function when administered during glucose deprivation. In the present study, we investigated whether prolonged incubation with monocarboxylate (pyruvate or lactate) prior rather than during glucose deprivation can also sustain synaptic and metabolic function. Pyruvate pre-incubation(3-4h) significantly prolonged (>25 min) the tolerance of rat hippocampal slices to delayed glucose deprivation compared to control and lactate pre-incubated slices, as revealed by field excitatory post synaptic potentials (fEPSPs); pre-incubation with pyruvate also reduced the marked decrease in NAD(P)H fluorescence resulting from glucose deprivation. Moreover, pyruvate exposure led to the enhancement of glycogen stores with time, compared to glucose alone (12 μmol/g tissue at 4h vs. 3.5 μmol/g tissue). Prolonged resistance to glucose deprivation following exogenous pyruvate incubation was prevented by glycogenolysis inhibitors, suggesting that enhanced glycogen mediates the delay in synaptic activity failure. The application of an adenosine A1 receptor antagonist enhanced glycogen utilization and prolonged the time to synaptic failure, further confirming this hypothesis of the importance of glycogen. Moreover, tissue levels of ATP were also significantly maintained during glucose deprivation in pyruvate pretreated slices compared to control and lactate. In summary, these experiments indicate that pyruvate exposure prior to glucose deprivation significantly increased the energy buffering capacity of hippocampal slices, particularly by enhancing internal glycogen stores, delaying synaptic failure during glucose deprivation by maintaining ATP levels, and minimizing the decrease in the levels of NAD(P)H.

  11. An intermittent exhaustion of the pool of glycogen in the human organism as a simple universal health promoting mechanism.

    PubMed

    Cherkas, Andriy; Golota, Sergii

    2014-03-01

    Glycogen storage in human organism is providing reserve source of glucose which is critical for normal functioning of the nervous system during periods between meals and is also important for many other tissues. Overwhelming excessive consumption of carbohydrates and decreasing physical activity among the world population lead to dramatic increase in incidence and mortality related to cardiovascular diseases, metabolic syndrome and diabetes mellitus type 2. There is an observation that many interventions with proved clinical efficiency like physical activity, intermittent fasting, caloric restriction and some pharmacological treatments have in common the ability to decrease content of glycogen in the liver and skeletal muscles. This effect leads to increased ability of these organs to uptake the next dose of glucose and store it in the form of glycogen. Moreover these interventions lead to significant life span extension, provide better body fitness and prevent development of multiple age-related diseases. In contrast excessive glucose load and saturation of tissues with glycogen provide a metabolic shift toward synthesis of fatty acids by liver. In advanced stages decreased glucose tolerance, insulin resistance, hyperinsulinemia, fatty liver disease, impairment of liver function and derangements of cholesterol metabolism are observed. It is suggested that noninvasive measurement of glycogen content in tissues could serve as important diagnostic and follow-up parameter for clinical practice and healthy lifestyle in wide population groups.

  12. An intermittent exhaustion of the pool of glycogen in the human organism as a simple universal health promoting mechanism.

    PubMed

    Cherkas, Andriy; Golota, Sergii

    2014-03-01

    Glycogen storage in human organism is providing reserve source of glucose which is critical for normal functioning of the nervous system during periods between meals and is also important for many other tissues. Overwhelming excessive consumption of carbohydrates and decreasing physical activity among the world population lead to dramatic increase in incidence and mortality related to cardiovascular diseases, metabolic syndrome and diabetes mellitus type 2. There is an observation that many interventions with proved clinical efficiency like physical activity, intermittent fasting, caloric restriction and some pharmacological treatments have in common the ability to decrease content of glycogen in the liver and skeletal muscles. This effect leads to increased ability of these organs to uptake the next dose of glucose and store it in the form of glycogen. Moreover these interventions lead to significant life span extension, provide better body fitness and prevent development of multiple age-related diseases. In contrast excessive glucose load and saturation of tissues with glycogen provide a metabolic shift toward synthesis of fatty acids by liver. In advanced stages decreased glucose tolerance, insulin resistance, hyperinsulinemia, fatty liver disease, impairment of liver function and derangements of cholesterol metabolism are observed. It is suggested that noninvasive measurement of glycogen content in tissues could serve as important diagnostic and follow-up parameter for clinical practice and healthy lifestyle in wide population groups. PMID:24495563

  13. Melatonin ameliorates high fat diet-induced diabetes and stimulates glycogen synthesis via a PKCzeta-Akt-GSK3beta pathway in hepatic cells.

    PubMed

    Shieh, Jiunn-Min; Wu, Hung-Tsung; Cheng, Kai-Chun; Cheng, Juei-Tang

    2009-11-01

    Low levels of melatonin in circulation had been reported to be related to the development of diabetes. Melatonin administration in animals increases hepatic glycogen content to lower blood glucose. However, the signaling pathway for these effects is still unclear. The present study shows that intraperitoneal injection of 10 mg/kg melatonin ameliorated glucose utilization and insulin sensitivity in high fat diet-induced diabetic mice with an increase in hepatic glycogen and improvement in liver steatosis. We used HepG2 cells to investigate the signaling pathways for the melatonin-stimulated hepatic glycogen increment. Treatment of HepG2 cells with 1 nm melatonin markedly increased glycogen synthesis which was blocked by the melatonin receptor antagonist luzindole. In addition, melatonin increased the phosphorylation of subcellular signals at the level of protein kinase C zeta (PKCzeta), Akt, and glycogen synthase kinase 3beta (GSK3beta) while the increase in glycogen synthesis induced by melatonin was inhibited by PKCzeta pseudo-peptide. However, 3',5'-cyclic adenosine monophosphate-activated protein kinase (AMPK) was not influenced by melatonin treatment. Taken together, melatonin improves glucose intolerance and insulin resistance in high fat diet-induced diabetic mice and stimulates glycogen synthesis via a PKCzeta-Akt-GSK3beta pathway in HepG2 cells.

  14. Concerted action of two subunits of the functional dimer of Shewanella oneidensis MR-1 uridine phosphorylase derived from a comparison of the C212S mutant and the wild-type enzyme.

    PubMed

    Safonova, T N; Mordkovich, N N; Veiko, V P; Okorokova, N A; Manuvera, V A; Dorovatovskii, P V; Popov, V O; Polyakov, K M

    2016-02-01

    Uridine phosphorylase (UP; EC 2.4.2.3), a key enzyme in the pyrimidine-salvage pathway, catalyzes the reversible phosphorolysis of uridine to uracil and ribose 1-phosphate. The structure of the C212S mutant of uridine phosphorylase from the facultatively aerobic Gram-negative γ-proteobacterium Shewanella oneidensis MR-1 (SoUP) was determined at 1.68 Å resolution. A comparison of the structures of the mutant and the wild-type enzyme showed that one dimer in the mutant hexamer differs from all other dimers in the mutant and wild-type SoUP (both in the free form and in complex with uridine). The key difference is the `maximum open' state of one of the subunits comprising this dimer, which has not been observed previously for uridine phosphorylases. Some conformational features of the SoUP dimer that provide access of the substrate into the active site are revealed. The binding of the substrate was shown to require the concerted action of two subunits of the dimer. The changes in the three-dimensional structure induced by the C212S mutation account for the lower affinity of the mutant for inorganic phosphate, while the affinity for uridine remains unchanged.

  15. Mutation of the Plastidial α-Glucan Phosphorylase Gene in Rice Affects the Synthesis and Structure of Starch in the Endosperm[W

    PubMed Central

    Satoh, Hikaru; Shibahara, Kensuke; Tokunaga, Takashi; Nishi, Aiko; Tasaki, Mikako; Hwang, Seon-Kap; Okita, Thomas W.; Kaneko, Nanae; Fujita, Naoko; Yoshida, Mayumi; Hosaka, Yuko; Sato, Aya; Utsumi, Yoshinori; Ohdan, Takashi; Nakamura, Yasunori

    2008-01-01

    Plastidial phosphorylase (Pho1) accounts for ∼96% of the total phosphorylase activity in developing rice (Oryza sativa) seeds. From mutant stocks induced by N-methyl-N-nitrosourea treatment, we identified plants with mutations in the Pho1 gene that are deficient in Pho1. Strikingly, the size of mature seeds and the starch content in these mutants showed considerable variation, ranging from shrunken to pseudonormal. The loss of Pho1 caused smaller starch granules to accumulate and modified the amylopectin structure. Variation in the morphological and biochemical phenotype of individual seeds was common to all 15 pho1-independent homozygous mutant lines studied, indicating that this phenotype was caused solely by the genetic defect. The phenotype of the pho1 mutation was temperature dependent. While the mutant plants grown at 30°C produced mainly plump seeds at maturity, most of the seeds from plants grown at 20°C were shrunken, with a significant proportion showing severe reduction in starch accumulation. These results strongly suggest that Pho1 plays a crucial role in starch biosynthesis in rice endosperm at low temperatures and that one or more other factors can complement the function of Pho1 at high temperatures. PMID:18621947

  16. Impact of Oxidative Stress on Ascorbate Biosynthesis in Chlamydomonas via Regulation of the VTC2 Gene Encoding a GDP-l-galactose Phosphorylase*

    PubMed Central

    Urzica, Eugen I.; Adler, Lital N.; Page, M. Dudley; Linster, Carole L.; Arbing, Mark A.; Casero, David; Pellegrini, Matteo; Merchant, Sabeeha S.; Clarke, Steven G.

    2012-01-01

    The l-galactose (Smirnoff-Wheeler) pathway represents the major route to l-ascorbic acid (vitamin C) biosynthesis in higher plants. Arabidopsis thaliana VTC2 and its paralogue VTC5 function as GDP-l-galactose phosphorylases converting GDP-l-galactose to l-galactose-1-P, thus catalyzing the first committed step in the biosynthesis of l-ascorbate. Here we report that the l-galactose pathway of ascorbate biosynthesis described in higher plants is conserved in green algae. The Chlamydomonas reinhardtii genome encodes all the enzymes required for vitamin C biosynthesis via the l-galactose pathway. We have characterized recombinant C. reinhardtii VTC2 as an active GDP-l-galactose phosphorylase. C. reinhardtii cells exposed to oxidative stress show increased VTC2 mRNA and l-ascorbate levels. Genes encoding enzymatic components of the ascorbate-glutathione system (e.g. ascorbate peroxidase, manganese superoxide dismutase, and dehydroascorbate reductase) are also up-regulated in response to increased oxidative stress. These results indicate that C. reinhardtii VTC2, like its plant homologs, is a highly regulated enzyme in ascorbate biosynthesis in green algae and that, together with the ascorbate recycling system, the l-galactose pathway represents the major route for providing protective levels of ascorbate in oxidatively stressed algal cells. PMID:22393048

  17. Long-Term Administration of Dehydroepiandrosterone Accelerates Glucose Catabolism via Activation of PI3K/Akt-PFK-2 Signaling Pathway in Rats Fed a High-Fat Diet.

    PubMed

    Kang, Jian; Ge, Chongyang; Yu, Lei; Li, Longlong; Ma, Haitian

    2016-01-01

    Dehydroepiandrosterone (DHEA) has a fat-reducing effect, while little information is available on whether DHEA regulates glucose metabolism, which would in turn affect fat deposition. To investigate the effects of DHEA on glucose metabolism, rats were administered a high-fat diet containing either 0 (HCG), 25 (HLG), 50 (HMG), or 100 (HHG) mg·kg-1 DHEA per day via gavage for 8 weeks. Results showed that long-term administration of DHEA inhibited body weight gain in rats on a high-fat diet. No statistical differences in serum glucose levels were observed, whereas hepatic glycogen content in HMG and HHG groups and muscle glycogen content in HLG and HMG groups were higher than those in HCG group. Glucokinase, malate dehydrogenase and phosphofructokinase-2 activities in HMG and HHG groups, pyruvate kinase and succinate dehydrogenase activities in HMG group, and pyruvate dehydrogenase activity in all DHEA treatment groups were increased compared with those in HCG group. Phosphoenolpyruvate carboxykinase and glycogen phosphorylase mRNA levels were decreased in HMG and HHG groups, whereas glycogen synthase-2 mRNA level was increased in HMG group compared with those in HCG. The abundance of Glut2 mRNA in HMG and HHG groups and Glut4 mRNA in HMG group was higher than that in HCG group. DHEA treatment increased serum leptin content in HMG and HHG groups compared with that in HCG group. Serum insulin content and insulin receptor mRNA level in HMG group and insulin receptor substrate-2 mRNA level in HMG and HHG group were increased compared with those in HCG group. Furthermore, Pi3k mRNA level in HMG and Akt mRNA level in HMG and HHG groups were significantly increased than those in HCG group. These data showed that DHEA treatment could enhance glycogen storage and accelerate glucose catabolism in rats fed a high-fat diet, and this effect may be associated with the activation of PI3K/Akt-PFK-2 signaling pathway. PMID:27410429

  18. Long-Term Administration of Dehydroepiandrosterone Accelerates Glucose Catabolism via Activation of PI3K/Akt-PFK-2 Signaling Pathway in Rats Fed a High-Fat Diet

    PubMed Central

    Kang, Jian; Ge, Chongyang; Yu, Lei; Li, Longlong; Ma, Haitian

    2016-01-01

    Dehydroepiandrosterone (DHEA) has a fat-reducing effect, while little information is available on whether DHEA regulates glucose metabolism, which would in turn affect fat deposition. To investigate the effects of DHEA on glucose metabolism, rats were administered a high-fat diet containing either 0 (HCG), 25 (HLG), 50 (HMG), or 100 (HHG) mg·kg-1 DHEA per day via gavage for 8 weeks. Results showed that long-term administration of DHEA inhibited body weight gain in rats on a high-fat diet. No statistical differences in serum glucose levels were observed, whereas hepatic glycogen content in HMG and HHG groups and muscle glycogen content in HLG and HMG groups were higher than those in HCG group. Glucokinase, malate dehydrogenase and phosphofructokinase-2 activities in HMG and HHG groups, pyruvate kinase and succinate dehydrogenase activities in HMG group, and pyruvate dehydrogenase activity in all DHEA treatment groups were increased compared with those in HCG group. Phosphoenolpyruvate carboxykinase and glycogen phosphorylase mRNA levels were decreased in HMG and HHG groups, whereas glycogen synthase-2 mRNA level was increased in HMG group compared with those in HCG. The abundance of Glut2 mRNA in HMG and HHG groups and Glut4 mRNA in HMG group was higher than that in HCG group. DHEA treatment increased serum leptin content in HMG and HHG groups compared with that in HCG group. Serum insulin content and insulin receptor mRNA level in HMG group and insulin receptor substrate-2 mRNA level in HMG and HHG group were increased compared with those in HCG group. Furthermore, Pi3k mRNA level in HMG and Akt mRNA level in HMG and HHG groups were significantly increased than those in HCG group. These data showed that DHEA treatment could enhance glycogen storage and accelerate glucose catabolism in rats fed a high-fat diet, and this effect may be associated with the activation of PI3K/Akt-PFK-2 signaling pathway. PMID:27410429

  19. Improving size-exclusion chromatography separation for glycogen.

    PubMed

    Sullivan, Mitchell A; Powell, Prudence O; Witt, Torsten; Vilaplana, Francisco; Roura, Eugeni; Gilbert, Robert G

    2014-03-01

    Glycogen is a hyperbranched glucose polymer comprised of glycogen β particles, which can also form much larger composite α particles. The recent discovery using size-exclusion chromatography (SEC) that fewer, smaller, α particles are found in diabetic-mouse liver compared to healthy mice highlights the need to achieve greater accuracy in the size separation methods used to analyze α and β particles. While past studies have used dimethyl sulfoxide as the SEC eluent to analyze the molecular size and structure of native glycogen, an aqueous eluent has not been rigorously tested and compared with dimethyl sulfoxide. The conditions for SEC of pig-liver glycogen, phytoglycogen and oyster glycogen were optimized by comparing two different eluents, aqueous 50 mM NH₄NO₃/0.02% NaN₃ and dimethyl sulfoxide/0.5% LiBr, run through different column materials and pore sizes at various flow rates. The aqueous system gave distinct size separation of α- and β-particle peaks, allowing for a more detailed and quantitative analysis and comparison between liver glycogen samples. This greater resolution has also revealed key differences between the structure of liver glycogen and phytoglycogen.

  20. Glycogen and glucose metabolism are essential for early embryonic development of the red flour beetle Tribolium castaneum.

    PubMed

    Fraga, Amanda; Ribeiro, Lupis; Lobato, Mariana; Santos, Vitória; Silva, José Roberto; Gomes, Helga; da Cunha Moraes, Jorge Luiz; de Souza Menezes, Jackson; de Oliveira, Carlos Jorge Logullo; Campos, Eldo; da Fonseca, Rodrigo Nunes

    2013-01-01

    Control of energy metabolism is an essential process for life. In insects, egg formation (oogenesis) and embryogenesis is dependent on stored molecules deposited by the mother or transcribed later by the zygote. In oviparous insects the egg becomes an isolated system after egg laying with all energy conversion taking place during embryogenesis. Previous studies in a few vector species showed a strong correlation of key morphogenetic events and changes in glucose metabolism. Here, we investigate glycogen and glucose metabolism in the red flour beetle Tribolium castaneum, an insect amenable to functional genomic studies. To examine the role of the key enzymes on glycogen and glucose regulation we cloned and analyzed the function of glycogen synthase kinase 3 (GSK-3) and hexokinase (HexA) genes during T. castaneum embryogenesis. Expression analysis via in situ hybridization shows that both genes are expressed only in the embryonic tissue, suggesting that embryonic and extra-embryonic cells display different metabolic activities. dsRNA adult female injection (parental RNAi) of both genes lead a reduction in egg laying and to embryonic lethality. Morphological analysis via DAPI stainings indicates that early development is impaired in Tc-GSK-3 and Tc-HexA1 RNAi embryos. Importantly, glycogen levels are upregulated after Tc-GSK-3 RNAi and glucose levels are upregulated after Tc-HexA1 RNAi, indicating that both genes control metabolism during embryogenesis and oogenesis, respectively. Altogether our results show that T. castaneum embryogenesis depends on the proper control of glucose and glycogen.

  1. The modulation of the symbiont/host interaction between Wolbachia pipientis and Aedes fluviatilis embryos by glycogen metabolism.

    PubMed

    da Rocha Fernandes, Mariana; Martins, Renato; Pessoa Costa, Evenilton; Pacidônio, Etiene Casagrande; Araujo de Abreu, Leonardo; da Silva Vaz, Itabajara; Moreira, Luciano A; da Fonseca, Rodrigo Nunes; Logullo, Carlos

    2014-01-01

    Wolbachia pipientis, a maternally transmitted bacterium that colonizes arthropods, may affect the general aspects of insect physiology, particularly reproduction. Wolbachia is a natural endosymbiont of Aedes fluviatilis, whose effects in embryogenesis and reproduction have not been addressed so far. In this context, we investigated the correlation between glucose metabolism and morphological alterations during A. fluviatilis embryo development in Wolbachia-positive (W+) and Wolbachia-negative (W-) mosquito strains. While both strains do not display significant morphological and larval hatching differences, larger differences were observed in hexokinase activity and glycogen contents during early and mid-stages of embryogenesis, respectively. To investigate if glycogen would be required for parasite-host interaction, we reduced Glycogen Synthase Kinase-3 (GSK-3) levels in adult females and their eggs by RNAi. GSK-3 knock-down leads to embryonic lethality, lower levels of glycogen and total protein and Wolbachia reduction. Therefore, our results suggest that the relationship between A. fluviatilis and Wolbachia may be modulated by glycogen metabolism.

  2. The Modulation of the Symbiont/Host Interaction between Wolbachia pipientis and Aedes fluviatilis Embryos by Glycogen Metabolism

    PubMed Central

    da Rocha Fernandes, Mariana; Martins, Renato; Pessoa Costa, Evenilton; Casagrande Pacidônio, Etiene; Araujo de Abreu, Leonardo; da Silva Vaz, Itabajara; Moreira, Luciano A.; da Fonseca, Rodrigo Nunes; Logullo, Carlos

    2014-01-01

    Wolbachia pipientis, a maternally transmitted bacterium that colonizes arthropods, may affect the general aspects of insect physiology, particularly reproduction. Wolbachia is a natural endosymbiont of Aedes fluviatilis, whose effects in embryogenesis and reproduction have not been addressed so far. In this context, we investigated the correlation between glucose metabolism and morphological alterations during A. fluviatilis embryo development in Wolbachia-positive (W+) and Wolbachia-negative (W−) mosquito strains. While both strains do not display significant morphological and larval hatching differences, larger differences were observed in hexokinase activity and glycogen contents during early and mid-stages of embryogenesis, respectively. To investigate if glycogen would be required for parasite-host interaction, we reduced Glycogen Synthase Kinase-3 (GSK-3) levels in adult females and their eggs by RNAi. GSK-3 knock-down leads to embryonic lethality, lower levels of glycogen and total protein and Wolbachia reduction. Therefore, our results suggest that the relationship between A. fluviatilis and Wolbachia may be modulated by glycogen metabolism. PMID:24926801

  3. Three-dimensional structure of E. Coli purine nucleoside phosphorylase at 0.99 Å resolution

    NASA Astrophysics Data System (ADS)

    Timofeev, V. I.; Abramchik, Yu. A.; Zhukhlistova, N. E.; Muravieva, T. I.; Esipov, R. S.; Kuranova, I. P.

    2016-03-01

    Purine nucleoside phosphorylases (PNPs) catalyze the reversible phosphorolysis of nucleosides and are key enzymes involved in nucleotide metabolism. They are essential for normal cell function and can catalyze the transglycosylation. Crystals of E. coli PNP were grown in microgravity by the capillary counterdiffusion method through a gel layer. The three-dimensional structure of the enzyme was determined by the molecular-replacement method at 0.99 Å resolution. The structural features are considered, and the structure of E. coli PNP is compared with the structures of the free enzyme and its complexes with purine base derivatives established earlier. A comparison of the environment of the purine base in the complex of PNP with formycin A and of the pyrimidine base in the complex of uridine phosphorylase with thymidine revealed the main structural features of the base-binding sites. Coordinates of the atomic model determined with high accuracy were deposited in the Protein Data Bank (PDB_ID: 4RJ2).

  4. Diastereoselective Synthesis of Glycosyl Phosphates by Using a Phosphorylase-Phosphatase Combination Catalyst.

    PubMed

    Wildberger, Patricia; Pfeiffer, Martin; Brecker, Lothar; Nidetzky, Bernd

    2015-12-21

    Sugar phosphates play an important role in metabolism and signaling, but also as constituents of macromolecular structures. Selective phosphorylation of sugars is chemically difficult, particularly at the anomeric center. We report phosphatase-catalyzed diastereoselective "anomeric" phosphorylation of various aldose substrates with α-D-glucose 1-phosphate, derived from phosphorylase-catalyzed conversion of sucrose and inorganic phosphate, as the phosphoryl donor. Simultaneous and sequential two-step transformations by the phosphorylase-phosphatase combination catalyst yielded glycosyl phosphates of defined anomeric configuration in yields of up to 70 % based on the phosphate applied to the reaction. An efficient enzyme-assisted purification of the glycosyl phosphate products from reaction mixtures was established.

  5. Development of a capillary electrophoresis method for analyzing adenosine deaminase and purine nucleoside phosphorylase and its application in inhibitor screening.

    PubMed

    Qi, Yanfei; Li, Youxin; Bao, James J

    2016-08-01

    A novel capillary electrophoresis (CE) method was developed for simultaneous analysis of adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) in red blood cells (RBCs). The developed method considered and took advantage of the natural conversion from the ADA product, inosine to hypoxanthine. The transformation ratio was introduced for ADA and PNP analysis to obtain more reliable results. After optimizing the enzymatic incubation and electrophoresis separation conditions, the determined activities of ADA and PNP in 12 human RBCs were 0.237-0.833 U/ml and 9.013-10.453 U/ml packed cells, respectively. The analysis of ADA in mice RBCs indicated that there was an apparent activity difference between healthy and hepatoma mice. In addition, the proposed method was also successfully applied in the inhibitor screening from nine traditional Chinese medicines, and data showed that ADA activities were strongly inhibited by Rhizoma Chuanxiong and Angelica sinensis. The inhibition effect of Angelica sinensis on ADA is first reported here and could also inhibit PNP activity. PMID:27173606

  6. L-Enantiomers of Transition State Analogue Inhibitors Bound to Human Purine Nucleoside Phosphorylase

    SciTech Connect

    Rinaldo-Matthis,A.; Murkin, A.; Ramagopal, U.; Clinch, K.; Mee, S.; Evans, G.; Tyler, P.; Furneaux, R.; Almo, S.; Schramm, v.

    2008-01-01

    Human purine nucleoside phosphorylase (PNP) was crystallized with transition-state analogue inhibitors Immucillin-H and DADMe-Immucillin-H synthesized with ribosyl mimics of l-stereochemistry. The inhibitors demonstrate that major driving forces for tight binding of these analogues are the leaving group interaction and the cationic mimicry of the transition state, even though large geometric changes occur with d-Immucillins and l-Immucillins bound to human PNP.

  7. GlgS, described previously as a glycogen synthesis control protein, negatively regulates motility and biofilm formation in Escherichia coli.

    PubMed

    Rahimpour, Mehdi; Montero, Manuel; Almagro, Goizeder; Viale, Alejandro M; Sevilla, Ángel; Cánovas, Manuel; Muñoz, Francisco J; Baroja-Fernández, Edurne; Bahaji, Abdellatif; Eydallin, Gustavo; Dose, Hitomi; Takeuchi, Rikiya; Mori, Hirotada; Pozueta-Romero, Javier

    2013-06-15

    Escherichia coli glycogen metabolism involves the regulation of glgBXCAP operon expression and allosteric control of the GlgC [ADPG (ADP-glucose) pyrophosphorylase]-mediated catalysis of ATP and G1P (glucose-1-phosphate) to ADPG linked to glycogen biosynthesis. E. coli glycogen metabolism is also affected by glgS. Though the precise function of the protein it encodes is unknown, its deficiency causes both reduced glycogen content and enhanced levels of the GlgC-negative allosteric regulator AMP. The transcriptomic analyses carried out in the present study revealed that, compared with their isogenic BW25113 wild-type strain, glgS-null (ΔglgS) mutants have increased expression of the operons involved in the synthesis of type 1 fimbriae adhesins, flagella and nucleotides. In agreement, ΔglgS cells were hyperflagellated and hyperfimbriated, and displayed elevated swarming motility; these phenotypes all reverted to the wild-type by ectopic glgS expression. Also, ΔglgS cells accumulated high colanic acid content and displayed increased ability to form biofilms on polystyrene surfaces. F-driven conjugation based on large-scale interaction studies of glgS with all the non-essential genes of E. coli showed that deletion of purine biosynthesis genes complement the glycogen-deficient, high motility and high biofilm content phenotypes of ΔglgS cells. Overall the results of the present study indicate that glycogen deficiency in ΔglgS cells can be ascribed to high flagellar propulsion and high exopolysaccharide and purine nucleotides biosynthetic activities competing with GlgC for the same ATP and G1P pools. Supporting this proposal, glycogen-less ΔglgC cells displayed an elevated swarming motility, and accumulated high levels of colanic acid and biofilm. Furthermore, glgC overexpression reverted the glycogen-deficient, high swarming motility, high colanic acid and high biofilm content phenotypes of ΔglgS cells to the wild-type. As on the basis of the present study Glg

  8. Structural Basis of Glycogen Biosynthesis Regulation in Bacteria.

    PubMed

    Cifuente, Javier O; Comino, Natalia; Madariaga-Marcos, Julene; López-Fernández, Sonia; García-Alija, Mikel; Agirre, Jon; Albesa-Jové, David; Guerin, Marcelo E

    2016-09-01

    ADP-glucose pyrophosphorylase (AGPase) catalyzes the rate-limiting step of bacterial glycogen and plant starch biosynthesis, the most common carbon storage polysaccharides in nature. A major challenge is to understand how AGPase activity is regulated by metabolites in the energetic flux within the cell. Here we report crystal structures of the homotetrameric AGPase from Escherichia coli in complex with its physiological positive and negative allosteric regulators, fructose-1,6-bisphosphate (FBP) and AMP, and sucrose in the active site. FBP and AMP bind to partially overlapping sites located in a deep cleft between glycosyltransferase A-like and left-handed β helix domains of neighboring protomers, accounting for the fact that sensitivity to inhibition by AMP is modulated by the concentration of the activator FBP. We propose a model in which the energy reporters regulate EcAGPase catalytic activity by intra-protomer interactions and inter-protomer crosstalk, with a sensory motif and two regulatory loops playing a prominent role.

  9. Structural Basis of Glycogen Biosynthesis Regulation in Bacteria.

    PubMed

    Cifuente, Javier O; Comino, Natalia; Madariaga-Marcos, Julene; López-Fernández, Sonia; García-Alija, Mikel; Agirre, Jon; Albesa-Jové, David; Guerin, Marcelo E

    2016-09-01

    ADP-glucose pyrophosphorylase (AGPase) catalyzes the rate-limiting step of bacterial glycogen and plant starch biosynthesis, the most common carbon storage polysaccharides in nature. A major challenge is to understand how AGPase activity is regulated by metabolites in the energetic flux within the cell. Here we report crystal structures of the homotetrameric AGPase from Escherichia coli in complex with its physiological positive and negative allosteric regulators, fructose-1,6-bisphosphate (FBP) and AMP, and sucrose in the active site. FBP and AMP bind to partially overlapping sites located in a deep cleft between glycosyltransferase A-like and left-handed β helix domains of neighboring protomers, accounting for the fact that sensitivity to inhibition by AMP is modulated by the concentration of the activator FBP. We propose a model in which the energy reporters regulate EcAGPase catalytic activity by intra-protomer interactions and inter-protomer crosstalk, with a sensory motif and two regulatory loops playing a prominent role. PMID:27545622

  10. Hepatic glycogen synthesis in the fetal mouse: An ultrastructural, morphometric, and autoradiographic investigation of the relationship between the smooth endoplasmic reticulum and glycogen

    SciTech Connect

    Breslin, J.S.

    1989-01-01

    Fetal rodent hepatocytes undergo a rapid and significant accumulation of glycogen prior to birth. The distinct association of the smooth endoplasmic reticulum (SER) with glycogen during glycogen synthesis documented in the adult hepatocyte has not been clearly demonstrated in the fetus. The experiments described in this dissertation tested the hypothesis that SER is present and functions in the synthesis of fetal hepatic glycogen. Biochemical analysis, light microscopic (LM) histochemistry and electron microscope (EM) morphometry demonstrated that fetal hepatic glycogen synthesis began on day 15, with maximum accumulation occurring between days 17-19. Glycogen accumulation began in a small population of cells. Both the number of cells containing glycogen and the quantity of glycogen per cell increased as glycogen accumulated. Smooth endoplasmic reticulum (SER) was observed on day 14 of gestation and throughout fetal hepatic glycogen synthesis, primarily as dilated ribosome-free terminal extensions of rough endoplasmic reticulum (RER), frequently associated with glycogen. SER was in close proximity to isolated particles of glycogen and at the periphery of large compact glycogen deposits. Morphometry demonstrated that the membrane surface of SER in the average fetal hepatocyte increased as glycogen accumulated through day 18 and dropped significantly as glycogen levels peaked on day 19. Parallel alterations in RER membrane surface, indicated overall increases in ER membrane surface. Autoradiography following administration of {sup 3}H-galactose demonstrated that newly synthesized glycogen was deposited near profiles of SER at day 16 and at day 18; however, at day 18 the majority of label was uniformly distributed over glycogen remote from profiles of SER.

  11. Crystal structure of Caulobacter crescentus polynucleotide phosphorylase reveals a mechanism of RNA substrate channelling and RNA degradosome assembly.

    PubMed

    Hardwick, Steven W; Gubbey, Tobias; Hug, Isabelle; Jenal, Urs; Luisi, Ben F

    2012-04-01

    Polynucleotide phosphorylase (PNPase) is an exoribonuclease that cleaves single-stranded RNA substrates with 3'-5' directionality and processive behaviour. Its ring-like, trimeric architecture creates a central channel where phosphorolytic active sites reside. One face of the ring is decorated with RNA-binding K-homology (KH) and S1 domains, but exactly how these domains help to direct the 3' end of single-stranded RNA substrates towards the active sites is an unsolved puzzle. Insight into this process is provided by our crystal structures of RNA-bound and apo Caulobacter crescentus PNPase. In the RNA-free form, the S1 domains adopt a 'splayed' conformation that may facilitate capture of RNA substrates. In the RNA-bound structure, the three KH domains collectively close upon the RNA and direct the 3' end towards a constricted aperture at the entrance of the central channel. The KH domains make non-equivalent interactions with the RNA, and there is a marked asymmetry within the catalytic core of the enzyme. On the basis of these data, we propose that structural non-equivalence, induced upon RNA binding, helps to channel substrate to the active sites through mechanical ratcheting. Structural and biochemical analyses also reveal the basis for PNPase association with RNase E in the multi-enzyme RNA degradosome assembly of the α-proteobacteria.

  12. A Ca(2+)-dependent global conformational change in the 3D structure of phosphorylase kinase obtained from electron microscopy.

    PubMed

    Nadeau, Owen W; Carlson, Gerald M; Gogol, Edward P

    2002-01-01

    Phosphorylase kinase (PhK), a Ca(2+)-dependent regulatory enzyme of the glycogenolytic cascade in skeletal muscle, is a 1.3 MDa hexadecameric oligomer comprising four copies of four distinct subunits, termed alpha, beta, gamma, and delta, the last being endogenous calmodulin. The structures of both nonactivated and Ca(2+)-activated PhK were determined to elucidate Ca(2+)-induced structural changes associated with PhK's activation. Reconstructions of both conformers of the kinase, each including over 11,000 particles, yielded bridged, bilobal structures with resolutions estimated by Fourier shell correlation at 24 A using a 0.5 correlation cutoff, or at 18 A by the 3sigma (corrected for D(2) symmetry) threshold curve. Extensive Ca(2+)-induced structural changes were observed in regions encompassing both the lobes and bridges, consistent with changes in subunit interactions upon activation. The relative placement of the alpha, beta, gamma, and delta subunits in the nonactivated three-dimensional structure, relying upon previous two-dimensional localizations, is in agreement with the known effects of Ca(2+) on subunit conformations and interactions in the PhK complex. PMID:11796107

  13. Comparative genomic and phylogenetic analyses of Gammaproteobacterial glg genes traced the origin of the Escherichia coli glycogen glgBXCAP operon to the last common ancestor of the sister orders Enterobacteriales and Pasteurellales.

    PubMed

    Almagro, Goizeder; Viale, Alejandro M; Montero, Manuel; Rahimpour, Mehdi; Muñoz, Francisco José; Baroja-Fernández, Edurne; Bahaji, Abdellatif; Zúñiga, Manuel; González-Candelas, Fernando; Pozueta-Romero, Javier

    2015-01-01

    Production of branched α-glucan, glycogen-like polymers is widely spread in the Bacteria domain. The glycogen pathway of synthesis and degradation has been fairly well characterized in the model enterobacterial species Escherichia coli (order Enterobacteriales, class Gammaproteobacteria), in which the cognate genes (branching enzyme glgB, debranching enzyme glgX, ADP-glucose pyrophosphorylase glgC, glycogen synthase glgA, and glycogen phosphorylase glgP) are clustered in a glgBXCAP operon arrangement. However, the evolutionary origin of this particular arrangement and of its constituent genes is unknown. Here, by using 265 complete gammaproteobacterial genomes we have carried out a comparative analysis of the presence, copy number and arrangement of glg genes in all lineages of the Gammaproteobacteria. These analyses revealed large variations in glg gene presence, copy number and arrangements among different gammaproteobacterial lineages. However, the glgBXCAP arrangement was remarkably conserved in all glg-possessing species of the orders Enterobacteriales and Pasteurellales (the E/P group). Subsequent phylogenetic analyses of glg genes present in the Gammaproteobacteria and in other main bacterial groups indicated that glg genes have undergone a complex evolutionary history in which horizontal gene transfer may have played an important role. These analyses also revealed that the E/P glgBXCAP genes (a) share a common evolutionary origin, (b) were vertically transmitted within the E/P group, and (c) are closely related to glg genes of some phylogenetically distant betaproteobacterial species. The overall data allowed tracing the origin of the E. coli glgBXCAP operon to the last common ancestor of the E/P group, and also to uncover a likely glgBXCAP transfer event from the E/P group to particular lineages of the Betaproteobacteria.

  14. Comparative Genomic and Phylogenetic Analyses of Gammaproteobacterial glg Genes Traced the Origin of the Escherichia coli Glycogen glgBXCAP Operon to the Last Common Ancestor of the Sister Orders Enterobacteriales and Pasteurellales

    PubMed Central

    Almagro, Goizeder; Viale, Alejandro M.; Montero, Manuel; Rahimpour, Mehdi; Muñoz, Francisco José; Baroja-Fernández, Edurne; Bahaji, Abdellatif; Zúñiga, Manuel; González-Candelas, Fernando; Pozueta-Romero, Javier

    2015-01-01

    Production of branched α-glucan, glycogen-like polymers is widely spread in the Bacteria domain. The glycogen pathway of synthesis and degradation has been fairly well characterized in the model enterobacterial species Escherichia coli (order Enterobacteriales, class Gammaproteobacteria), in which the cognate genes (branching enzyme glgB, debranching enzyme glgX, ADP-glucose pyrophosphorylase glgC, glycogen synthase glgA, and glycogen phosphorylase glgP) are clustered in a glgBXCAP operon arrangement. However, the evolutionary origin of this particular arrangement and of its constituent genes is unknown. Here, by using 265 complete gammaproteobacterial genomes we have carried out a comparative analysis of the presence, copy number and arrangement of glg genes in all lineages of the Gammaproteobacteria. These analyses revealed large variations in glg gene presence, copy number and arrangements among different gammaproteobacterial lineages. However, the glgBXCAP arrangement was remarkably conserved in all glg-possessing species of the orders Enterobacteriales and Pasteurellales (the E/P group). Subsequent phylogenetic analyses of glg genes present in the Gammaproteobacteria and in other main bacterial groups indicated that glg genes have undergone a complex evolutionary history in which horizontal gene transfer may have played an important role. These analyses also revealed that the E/P glgBXCAP genes (a) share a common evolutionary origin, (b) were vertically transmitted within the E/P group, and (c) are closely related to glg genes of some phylogenetically distant betaproteobacterial species. The overall data allowed tracing the origin of the E. coli glgBXCAP operon to the last common ancestor of the E/P group, and also to uncover a likely glgBXCAP transfer event from the E/P group to particular lineages of the Betaproteobacteria. PMID:25607991

  15. Genetics Home Reference: glycogen storage disease type III

    MedlinePlus

    ... blood (hyperlipidemia), and elevated blood levels of liver enzymes. As they get older, children with this condition ... gene provides instructions for making the glycogen debranching enzyme. This enzyme is involved in the breakdown of ...

  16. Expression of a dominant-negative Ras mutant does not affect stimulation of glucose uptake and glycogen synthesis by insulin.

    PubMed

    Dorrestijn, J; Ouwens, D M; Van den Berghe, N; Bos, J L; Maassen, J A

    1996-05-01

    It has previously been shown that insulin-induced stimulation of glucose uptake and glycogen synthesis requires activation of phosphatidylinositol-3-kinase (PI3kinase). Insulin also induces formation of RasGTP in cells and various studies have yielded inconsistent data with respect to the contribution of signalling pathways activated by RasGTP, to insulin-stimulated glucose uptake and glycogen synthesis. We have examined the requirement of RasGTP-mediated signalling for these insulin responses by expression of a dominant negative mutant of Ras (RasN17) in cells by vaccinia virus mediated gene transfer. This Ras-mutant abrogates the signalling pathways mediated by endogenous RasGTP. Subsequently, the ability of insulin to stimulate 2-deoxyglucose uptake and glycogen was examined. We observed that expression of RasN17 in 3T3L1 adipocytes did not affect the stimulation of hexose uptake by insulin. Similarly, expression of RasN17 in A14 cells, an NIH 3T3-derived cell line with high expression of insulin receptors, did not affect insulin-induced stimulation of glycogen synthesis. In both cell lines, insulin-induced phosphorylation of Mapkinase (Erk1,2) was abrogated after expression of RasN17, demonstrating the functional interference by RasN17 with signalling mediated by endogenous RasGTP. Wortmannin, an inhibitor of PI3kinase, abolished dose-dependently the insulin-induced stimulation of hexose uptake and glycogen synthesis without an effect on RasGTP levels in both cell types. We conclude that stimulation of glucose transport and glycogen synthesis by insulin occurs independently of RasGTP-mediated signalling.

  17. The interaction between AMPKβ2 and the PP1-targeting subunit R6 is dynamically regulated by intracellular glycogen content.

    PubMed

    Oligschlaeger, Yvonne; Miglianico, Marie; Dahlmans, Vivian; Rubio-Villena, Carla; Chanda, Dipanjan; Garcia-Gimeno, Maria Adelaida; Coumans, Will A; Liu, Yilin; Voncken, J Willem; Luiken, Joost J F P; Glatz, Jan F C; Sanz, Pascual; Neumann, Dietbert

    2016-04-01

    AMP-activated protein kinase (AMPK) is a metabolic stress-sensing kinase. We previously showed that glucose deprivation induces autophosphorylation of AMPKβ at Thr-148, which prevents the binding of AMPK to glycogen. Furthermore, in MIN6 cells, AMPKβ1 binds to R6 (PPP1R3D), a glycogen-targeting subunit of protein phosphatase type 1 (PP1), thereby regulating the glucose-induced inactivation of AMPK. In the present study, we further investigated the interaction of R6 with AMPKβ and the possible dependency on Thr-148 phosphorylation status. Yeast two-hybrid (Y2H) analyses and co-immunoprecipitation (IP) of the overexpressed proteins in human embryonic kidney (HEK) 293T) cells revealed that both AMPKβ1 and AMPK-β2 wild-type (WT) isoforms bind to R6. The AMPKβ-R6 interaction was stronger with the muscle-specific AMPKβ2-WT and required association with the substrate-binding motif of R6. When HEK293T cells or C2C12 myotubes were cultured in high-glucose medium, AMPKβ2-WT and R6 weakly interacted. In contrast, glycogen depletion significantly enhanced this protein interaction. Mutation of AMPKβ2 Thr-148 prevented the interaction with R6 irrespective of the intracellular glycogen content. Treatment with the AMPK activator oligomycin enhanced the AMPKβ2-R6 interaction in conjunction with increased Thr-148 phosphorylation in cells grown in low-glucose medium. These data are in accordance with R6 binding directly to AMPKβ2 when both proteins detach from the diminishing glycogen particle, which is simultaneous with increased AMPKβ2 Thr-148 autophosphorylation. Such a model points to a possible control of AMPK by PP1-R6 upon glycogen depletion in muscle.

  18. MiR-19a regulates PTEN expression to mediate glycogen synthesis in hepatocytes.

    PubMed

    Dou, Lin; Meng, Xiangyu; Sui, Xiaofang; Wang, Shuyue; Shen, Tao; Huang, Xiuqing; Guo, Jun; Fang, Weiwei; Man, Yong; Xi, Jianzhong; Li, Jian

    2015-06-26

    MiR-19a, a member of mir-17-92 microRNA clusters, has been demonstrated to promote cell proliferation and angiogenesis via regulating the PI3K/AKT pathway, the major insulin signaling pathway. However, whether miR-19a plays an important role in glycogen synthesis in hepatocytes remains unknown. Here, we define the impact of miR-19a on glycogen synthesis and IL-6-induced reduced glycogenesis in hepatocytes and its underlying mechanisms. Our studies indicate that miR-19a was down-regulated in the livers of db/db mice and mice injected with IL-6, as well as mouse NCTC 1469 hepatocytes and HEP 1-6 hepatocytes treated by IL-6. We found that over-expression of miR-19a in NCTC 1469 cells and HEP 1-6 cells led to increased activation of the AKT/GSK pathway and synthesis of glycogen, whereas down-regulation of miR-19a impaired AKT/GSK phosphorylation and glycogenesis. Over-expression of miR-19a ameliorated IL-6-induced reduced glycogen synthesis in hepatocytes. Moreover, we identified PTEN as the target of miR-19a by a luciferase assay. Down-regulation of PTEN rescued the effects of miR-19a suppression on the activation of the AKT/GSK pathway and improved glycogenesis in NTC 1469 cells. These findings show for the first time that miR-19a might activate the AKT/GSK pathway and glycogenesis via down-regulation of PTEN expression.

  19. Differences in glycogen, lipids, and enzymes in livers from rats flown on COSMOS 2044.

    PubMed

    Merrill, A H; Wang, E; LaRocque, R; Mullins, R E; Morgan, E T; Hargrove, J L; Bonkovsky, H L; Popova, I A

    1992-08-01

    Livers from rats flown aboard COSMOS 2044 were analyzed for protein, carbohydrate (glycogen), and lipids as well as the activities of a number of key enzymes involved in metabolism of these compounds and xenobiotics. The major differences between the flight group and the synchronous control were elevations in microsomal protein, liver glycogen content, tyrosine aminotransferase, and tryptophan oxygenase and reductions in sphingolipids and the rate-limiting enzyme of heme biosynthesis, delta-aminolevulinic acid synthase. These results provide further evidence that spaceflight has pronounced and diverse effects on liver function; however, some of the results with samples from COSMOS 2044 differed notably from those from previous spaceflights. This may be due to conditions of spaceflight and/or the postflight recovery period for COSMOS 2044.

  20. Differences in glycogen, lipids, and enzymes in livers from rats flown on Cosmos 2044

    NASA Technical Reports Server (NTRS)

    Merrill, Alfred H., Jr.; Wang, Elaine; Laroque, Regina; Mullins, Richard E.; Morgan, Edward T.; Hargrove, James L.; Bonkovsky, Herbert L.; Popova, Irina A.

    1992-01-01

    Livers from rats flown aboard Cosmos 2044 were analyzed for protein, carbohydrate (glycogen), and lipids as well as the activities of a number of key enzymes involved in metabolism of these compounds and xenobiotics. The major differences between the flight group and the synchronous control were elevations in microsomal protein, liver glycogen content, tyrosine aminotransferase, and tryptophan oxygenase and reductions in sphingolipids and the rate-limiting enzyme of heme biosynthesis delta-aminolevulinic acid synthase. These results provide further evidence that spaceflight has pronounced and diverse effects on liver function; however, some of the results with samples from Cosmos 2044 differed notably from those from previous spaceflights. This may be due to conditions of spaceflight and/or the postflight recovery period for Cosmos 2044.

  1. Transition state analogue inhibitors of human methylthioadenosine phosphorylase and bacterial methylthioadenosine/S-adenosylhomocysteine nucleosidase incorporating acyclic ribooxacarbenium ion mimics

    PubMed Central

    Clinch, Keith; Evans, Gary B.; Fröhlich, Richard F. G.; Gulab, Shivali A.; Gutierrez, Jemy A.; Mason, Jennifer M.; Schramm, Vern L.; Tyler, Peter C.; Woolhouse, Anthony D.

    2012-01-01

    Several acyclic hydroxy-methylthio-amines with 3 to 5 carbon atoms were prepared and coupled via a methylene link to 9-deazaadenine. The products were tested for inhibition against human MTAP and E. coli and N. meningitidis MTANs and gave Ki values as low as 0.23 nM. These results were compared to those obtained with 1st and 2nd generation inhibitors (1S)-1-(9-deazaadenin-9-yl)-1,4-dideoxy-1,4-imino-5-methylthio-d-ribitol (MT-Immucillin-A, 3) and (3R,4S)-1-[9-deazaadenin-9-yl)methyl]3-hydroxy-4-methylthiomethylpyrrolidine (MT-DADMe-Immucillin-A, 4). The best inhibitors were found to exhibit binding affinities of approximately 2- to 4-fold those of 3 but were significantly weaker than 4. Cleavage of the 2,3 carbon–carbon bond in MT-Immucillin-A (3) gave an acyclic product (79) with a 21,500 fold loss of activity against E. coli MTAN. In another case, N-methylation of a side chain secondary amine resulted in a 250-fold loss of activity against the same enzyme [(±)-65 vs (±)-68]. The inhibition results were also contrasted with those acyclic derivatives previously prepared as inhibitors for a related enzyme, purine nucleoside phosphorylase (PNP), where some inhibitors in the latter case were found to be more potent than their cyclic counterparts. PMID:22854195

  2. Design of vectors for efficient expression of human purine nucleoside phosphorylase in skin fibroblasts from enzyme-deficient humans

    SciTech Connect

    Osborne, W.R.A.; Miller, A.D.

    1988-09-01

    Purine nucleoside phosphorylase deficiency is an inherited disorder associated with a severe immune defect that is fatal. Enzyme replacement therapy is an attractive approach to treatment of this disease. To this aim the authors constructed retroviral vectors containing a human PNP cDNA and a selectable gene encoding neomycin phosphotransferase. PNP expression was controlled by either the early promoter from simian virus 40, the immediate early promoter from human cytomegalovirus, or the retroviral promoter. Cultured skin fibroblasts from two unrelated PNP-deficient patients that were infected with these vectors expressed mean PNP activities of 0.03, 0.74, and 5.9 /mu/mol/hr per mg of protein, respectively. The latter infectants had PNP activities eight times the level of 0.74 /mu/mol/hr per mg of protein observed in normal skin fibroblasts, enabling rapid metabolism of exogenous deoxyguanosine, the cytotoxic metabolite that accumulates in the plasma of PNP-deficient patients. These experiments indicate that viral long terminal repeat was the strongest promoter for expression of PNP and suggest the potential of human skin fibroblasts as vehicles for therapeutic gene expression.

  3. Characterization of the Methylthioadenosine Phosphorylase Polymorphism rs7023954 - Incidence and Effects on Enzymatic Function in Malignant Melanoma

    PubMed Central

    Limm, Katharina; Dettmer, Katja; Reinders, Jörg; Oefner, Peter J.; Bosserhoff, Anja-Katrin

    2016-01-01

    Deficiency of methylthioadenosine phosphorylase (MTAP) supports melanoma development and progression through accumulation of its substrate 5’-methylthioadenosine (MTA), which leads amongst others to a constitutive inhibition of protein arginine methyltransferases (PRMTs) and activation of the transcription factor AP-1 via the receptor ADORA2B. Genetic association studies have also suggested that genetic polymorphism in MTAP may modulate the risk of melanoma. Here, we investigated the only globally common non-synonymous single nucleotide polymorphism (SNP) reported to date for MTAP. The SNP rs7023954 is located in exon 3 (c.166G>A), and leads to the conservative substitution of one branched-chain amino acid residue (valine) for another (isoleucine) at position 56 (p.Val56Ile). Whereas genotype frequencies in normal and primary melanoma tissues or cell lines were in Hardy-Weinberg equilibrium based on cDNA amplicon sequencing, a marked (P = 0.00019) deviation was observed in metastatic melanoma tissues and cell lines due to a deficit of heterozygotes. Enzyme assays conducted on the co-dominantly expressed alleles revealed no difference in the conversion rate of MTA to adenine and 5-methylthioribose-1-phosphate, indicating that this known enzymatic activity does not modulate the tumor suppressive function of MTAP. PMID:27479139

  4. Increasing free-energy (ATP) conservation in maltose-grown Saccharomyces cerevisiae by expression of a heterologous maltose phosphorylase.

    PubMed

    de Kok, Stefan; Yilmaz, Duygu; Suir, Erwin; Pronk, Jack T; Daran, Jean-Marc; van Maris, Antonius J A

    2011-09-01

    Increasing free-energy conservation from the conversion of substrate into product is crucial for further development of many biotechnological processes. In theory, replacing the hydrolysis of disaccharides by a phosphorolytic cleavage reaction provides an opportunity to increase the ATP yield on the disaccharide. To test this concept, we first deleted the native maltose metabolism genes in Saccharomyces cerevisiae. The knockout strain showed no maltose-transport activity and a very low residual maltase activity (0.03 μmol mg protein(-1)min(-1)). Expression of a maltose phosphorylase gene from Lactobacillus sanfranciscensis and the MAL11 maltose-transporter gene resulted in relatively slow growth (μ(aerobic) 0.09 ± 0.03 h(-1)). Co-expression of Lactococcus lactis β-phosphoglucomutase accelerated maltose utilization via this route (μ(aerobic) 0.21 ± 0.01 h(-1), μ(anaerobic) 0.10 ± 0.00 h(-1)). Replacing maltose hydrolysis with phosphorolysis increased the anaerobic biomass yield on maltose in anaerobic maltose-limited chemostat cultures by 26%, thus demonstrating the potential of phosphorolysis to improve the free-energy conservation of disaccharide metabolism in industrial microorganisms.

  5. Determination of pathways of glycogen synthesis and the dilution of the three-carbon pool with (U- sup 13 C)glucose

    SciTech Connect

    Katz, J.; Wals, P.A. ); Lee, W.N.P. )

    1991-03-15

    Rats were infused with glucose at 30 mg/min, containing 18% enriched (U-{sup 13}C)glucose and (1-{sup 14}C)- and (3-{sup 3}H)glucose and liver glycogen were determined by gas chromatography/mass spectroscopy. The contribution of the direct pathway to glycogen was calculated from the three tracers, and the values by all three were nearly identical, about 50%. The {sup 14}C specific activity in carbon 6 of glycogen glucose was about 6% that of carbon 1. The ({sup 3}H)glucose/(1-{sup 14}C)glucose ratio in glycogen was 80-90% that is blood glucose. The enrichment of {sup 13}C and the specific activity of {sup 14}C in glycogen formed by the indirect path were 20-25% of glycogen formed directly from glucose. The dilution is of two kinds: (1) an exchange of labeled carbon with unlabeled carbon in the tricarboxylic acid cycle and (2) dilution by unlabeled nonglucose carbon. Methods to calculate the two types of dilution are presented. In rate preinjected with glucagon, the dilution through the tricarboxylic acid cycle was unaffected but that by nonglucose carbon was decreased.

  6. Structure and Mechanism of an ADP-Glucose Phosphorylase from Arabidopsis thaliana†,‡

    PubMed Central

    McCoy, Jason G.; Arabshahi, Abolfazl; Bitto, Eduard; Bingman, Craig A.; Ruzicka, Frank J.; Frey, Perry A.; Phillips, George N.

    2008-01-01

    The X-ray crystal structure of the At5g18200.1 protein has been solved to a nominal resolution of 2.30 Å. The structure has a histidine triad (HIT)-like fold containing two distinct HIT-like motifs. The sequence of At5g18200.1 indicates a distant family relationship to the Escherichia coli galactose-1-P uridylyltransferase (GalT): the determined structure of the At5g18200.1 protein confirms this relationship. The At5g18200.1 protein does not demonstrate GalT activity but instead catalyzes adenylyltransfer in the reaction of ADP-glucose with various phosphates. The best acceptor among those evaluated is phosphate itself, thus the At5g18200.1 enzyme appears to be an ADP-glucose phosphorylase. The enzyme catalyzes the exchange of 14C between ADP-[14C]glucose and glucose-1-P in the absence of phosphate. The steady state kinetics of exchange follows the ping pong bi bi kinetic mechanism, with kcat = 4.1 s−1 and Km-values of 1.4 µM and 83 µM for ADP-[14C]glucose and glucose-1-P, respectively, at pH 8.5 and 25 °C. The overall reaction of ADP-glucose with phosphate to produce ADP and glucose-1-P follows ping pong bi bi steady state kinetics, with kcat = 2.7 s−1 and Km-values of 6.9 µM and 90 µM for ADP-glucose and phosphate, respectively, at pH 8.5 and 25 °C. The kinetics are consistent with a double displacement mechanism that involves a covalent adenylyl-enzyme intermediate. The X-ray crystal structure of this intermediate was solved to 1.83 Å resolution, and shows the AMP-group bonded to His186. The value of Keq in the direction of ADP and glucose-1-P formation is 5.0 at pH 7.0 and 25 °C in the absence of a divalent metal ion, and it is 40 in the presence of 1 mM MgCl2. PMID:16519510

  7. Inhibition of Glycogen Synthase Kinase-3ß Enhances Cognitive Recovery after Stroke: The Role of TAK1

    ERIC Educational Resources Information Center

    Venna, Venugopal Reddy; Benashski, Sharon E.; Chauhan, Anjali; McCullough, Louise D.

    2015-01-01

    Memory deficits are common among stroke survivors. Identifying neuroprotective agents that can prevent memory impairment or improve memory recovery is a vital area of research. Glycogen synthase kinase-3ß (GSK-3ß) is involved in several essential intracellular signaling pathways. Unlike many other kinases, GSK-3ß is active only when…

  8. Acid Hydrolysis and Molecular Density of Phytoglycogen and Liver Glycogen Helps Understand the Bonding in Glycogen α (Composite) Particles

    PubMed Central

    Powell, Prudence O.; Sullivan, Mitchell A.; Sheehy, Joshua J.; Schulz, Benjamin L.; Warren, Frederick J.; Gilbert, Robert G.

    2015-01-01

    Phytoglycogen (from certain mutant plants) and animal glycogen are highly branched glucose polymers with similarities in structural features and molecular size range. Both appear to form composite α particles from smaller β particles. The molecular size distribution of liver glycogen is bimodal, with distinct α and β components, while that of phytoglycogen is monomodal. This study aims to enhance our understanding of the nature of the link between liver-glycogen β particles resulting in the formation of large α particles. It examines the time evolution of the size distribution of these molecules during acid hydrolysis, and the size dependence of the molecular density of both glucans. The monomodal distribution of phytoglycogen decreases uniformly in time with hydrolysis, while with glycogen, the large particles degrade significantly more quickly. The size dependence of the molecular density shows qualitatively different shapes for these two types of molecules. The data, combined with a quantitative model for the evolution of the distribution during degradation, suggest that the bonding between β into α particles is different between phytoglycogen and liver glycogen, with the formation of a glycosidic linkage for phytoglycogen and a covalent or strong non-covalent linkage, most probably involving a protein, for glycogen as most likely. This finding is of importance for diabetes, where α-particle structure is impaired. PMID:25799321

  9. Acid hydrolysis and molecular density of phytoglycogen and liver glycogen helps understand the bonding in glycogen α (composite) particles.

    PubMed

    Powell, Prudence O; Sullivan, Mitchell A; Sheehy, Joshua J; Schulz, Benjamin L; Warren, Frederick J; Gilbert, Robert G

    2015-01-01

    Phytoglycogen (from certain mutant plants) and animal glycogen are highly branched glucose polymers with similarities in structural features and molecular size range. Both appear to form composite α particles from smaller β particles. The molecular size distribution of liver glycogen is bimodal, with distinct α and β components, while that of phytoglycogen is monomodal. This study aims to enhance our understanding of the nature of the link between liver-glycogen β particles resulting in the formation of large α particles. It examines the time evolution of the size distribution of these molecules during acid hydrolysis, and the size dependence of the molecular density of both glucans. The monomodal distribution of phytoglycogen decreases uniformly in time with hydrolysis, while with glycogen, the large particles degrade significantly more quickly. The size dependence of the molecular density shows qualitatively different shapes for these two types of molecules. The data, combined with a quantitative model for the evolution of the distribution during degradation, suggest that the bonding between β into α particles is different between phytoglycogen and liver glycogen, with the formation of a glycosidic linkage for phytoglycogen and a covalent or strong non-covalent linkage, most probably involving a protein, for glycogen as most likely. This finding is of importance for diabetes, where α-particle structure is impaired.

  10. Body Morphology, Energy Stores, and Muscle Enzyme Activity Explain Cricket Acoustic Mate Attraction Signaling Variation

    PubMed Central

    Thomson, Ian R.; Darveau, Charles-A.; Bertram, Susan M.

    2014-01-01

    High mating success in animals is often dependent on males signalling attractively with high effort. Since males should be selected to maximize their reproductive success, female preferences for these traits should result in minimal signal variation persisting in the population. However, extensive signal variation persists. The genic capture hypothesis proposes genetic variation persists because fitness-conferring traits depend on an individual's basic processes, including underlying physiological, morphological, and biochemical traits, which are themselves genetically variable. To explore the traits underlying signal variation, we quantified among-male differences in signalling, morphology, energy stores, and the activities of key enzymes associated with signalling muscle metabolism in two species of crickets, Gryllus assimilis (chirper: <20 pulses/chirp) and G. texensis (triller: >20 pulses/chirp). Chirping G. assimilis primarily fuelled signalling with carbohydrate metabolism: smaller individuals and individuals with increased thoracic glycogen stores signalled for mates with greater effort; individuals with greater glycogen phosphorylase activity produced more attractive mating signals. Conversely, the more energetic trilling G. texensis fuelled signalling with both lipid and carbohydrate metabolism: individuals with increased β-hydroxyacyl-CoA dehydrogenase activity and increased thoracic free carbohydrate content signalled for mates with greater effort; individuals with higher thoracic and abdominal carbohydrate content and higher abdominal lipid stores produced more attractive signals. Our findings suggest variation in male reproductive success may be driven by hidden physiological trade-offs that affect the ability to uptake, retain, and use essential nutrients, although the results remain correlational in nature. Our findings indicate that a physiological perspective may help us to understand some of the causes of variation in behaviour. PMID:24608102

  11. Regulation of glycogen metabolism by the CRE-1, RCO-1 and RCM-1 proteins in Neurospora crassa. The role of CRE-1 as the central transcriptional regulator.

    PubMed

    Cupertino, Fernanda Barbosa; Virgilio, Stela; Freitas, Fernanda Zanolli; Candido, Thiago de Souza; Bertolini, Maria Célia

    2015-04-01

    The transcription factor CreA/Mig1/CRE-1 is a repressor protein that regulates the use of alternative carbon sources via a mechanism known as Carbon Catabolite Repression (CCR). In Saccharomyces cerevisiae, Mig1 recruits the complex Ssn6-Tup1, the Neurospora crassa RCM-1 and RCO-1 orthologous proteins, respectively, to bind to promoters of glucose-repressible genes. We have been studying the regulation of glycogen metabolism in N. crassa and the identification of the RCO-1 corepressor as a regulator led us to investigate the regulatory role of CRE-1 in this process. Glycogen content is misregulated in the rco-1(KO), rcm-1(RIP) and cre-1(KO) strains, and the glycogen synthase phosphorylation is decreased in all strains, showing that CRE-1, RCO-1 and RCM-1 proteins are involved in glycogen accumulation and in the regulation of GSN activity by phosphorylation. We also confirmed the regulatory role of CRE-1 in CCR and its nuclear localization under repressing condition in N. crassa. The expression of all glycogenic genes is misregulated in the cre-1(KO) strain, suggesting that CRE-1 also controls glycogen metabolism by regulating gene expression. The existence of a high number of the Aspergillus nidulans CreA motif (5'-SYGGRG-3') in the glycogenic gene promoters led us to analyze the binding of CRE-1 to some DNA motifs both in vitro by DNA gel shift and in vivo by ChIP-qPCR analysis. CRE-1 bound in vivo to all motifs analyzed demonstrating that it down-regulates glycogen metabolism by controlling gene expression and GSN phosphorylation.

  12. Computer Simulations Reveal Substrate Specificity of Glycosidic Bond Cleavage in Native and Mutant Human Purine Nucleoside Phosphorylase.

    PubMed

    Isaksen, Geir Villy; Hopmann, Kathrin Helen; Åqvist, Johan; Brandsdal, Bjørn Olav

    2016-04-12

    Purine nucleoside phosphorylase (PNP) catalyzes the reversible phosphorolysis of purine ribonucleosides and 2'-deoxyribonucleosides, yielding the purine base and (2'-deoxy)ribose 1-phosphate as products. While this enzyme has been extensively studied, several questions with respect to the catalytic mechanism have remained largely unanswered. The role of the phosphate and key amino acid residues in the catalytic reaction as well as the purine ring protonation state is elucidated using density functional theory calculations and extensive empirical valence bond (EVB) simulations. Free energy surfaces for adenosine, inosine, and guanosine are fitted to ab initio data and yield quantitative agreement with experimental data when the surfaces are used to model the corresponding enzymatic reactions. The cognate substrates 6-aminopurines (inosine and guanosine) interact with PNP through extensive hydrogen bonding, but the substrate specificity is found to be a direct result of the electrostatic preorganization energy along the reaction coordinate. Asn243 has previously been identified as a key residue providing substrate specificity. Mutation of Asn243 to Asp has dramatic effects on the substrate specificity, making 6-amino- and 6-oxopurines equally good as substrates. The principal effect of this particular mutation is the change in the electrostatic preorganization energy between the native enzyme and the Asn243Asp mutant, clearly favoring adenosine over inosine and guanosine. Thus, the EVB simulations show that this particular mutation affects the electrostatic preorganization of the active site, which in turn can explain the substrate specificity. PMID:26985580

  13. Site-Selective Ribosylation of Fluorescent Nucleobase Analogs Using Purine-Nucleoside Phosphorylase as a Catalyst: Effects of Point Mutations.

    PubMed

    Stachelska-Wierzchowska, Alicja; Wierzchowski, Jacek; Bzowska, Agnieszka; Wielgus-Kutrowska, Beata

    2015-12-28

    Enzymatic ribosylation of fluorescent 8-azapurine derivatives, like 8-azaguanine and 2,6-diamino-8-azapurine, with purine-nucleoside phosphorylase (PNP) as a catalyst, leads to N9, N8, and N7-ribosides. The final proportion of the products may be modulated by point mutations in the enzyme active site. As an example, ribosylation of the latter substrate by wild-type calf PNP gives N7- and N8-ribosides, while the N243D mutant directs the ribosyl substitution at N9- and N7-positions. The same mutant allows synthesis of the fluorescent N7-β-d-ribosyl-8-azaguanine. The mutated form of the E. coli PNP, D204N, can be utilized to obtain non-typical ribosides of 8-azaadenine and 2,6-diamino-8-azapurine as well. The N7- and N8-ribosides of the 8-azapurines can be analytically useful, as illustrated by N7-β-d-ribosyl-2,6-diamino-8-azapurine, which is a good fluorogenic substrate for mammalian forms of PNP, including human blood PNP, while the N8-riboside is selective to the E. coli enzyme.

  14. The metabolic zonation of glycogen synthesis in rat liver after fasting and refeeding.

    PubMed

    Kudryavtseva, M V; Sakuta, G A; Stein, G I; Kudryavtsev, B N

    1992-01-01

    The quantitative analysis of total glycogen and two fractions of the glycogen content was made by means of cytophotometry in hepatocytes with respect to the portal and central zones of the liver lobule after 48 hr starvation and 15, 30, 60, 120 min after refeeding using the Magiscan image analyzer. It was shown that glycogen content was minimal after 48 hr starvation, although a few cells of the central zone contained a noticeable glycogen quantity. Glycogen synthesis initiation was observed after 15 min refeeding. Glycogen synthesis has been characterized by an increasing glycogen content in the portal zone of the liver lobule compared to the pericentral zone, and this difference increased with time. The distinctive morphological changes were observed in the total glycogen content as well as fractions with different optical density in the process of glycogen synthesis after starvation of rats.

  15. The Multiple Functions of Common Microbial Carbon Polymers, Glycogen and PHB, during Stress Responses in the Non-Diazotrophic Cyanobacterium Synechocystis sp. PCC 6803

    PubMed Central

    Damrow, Ramon; Maldener, Iris; Zilliges, Yvonne

    2016-01-01

    Classical microbial carbon polymers such as glycogen and polyhydroxybutyrate (PHB) have a crucial impact as both a sink and a reserve under macronutrient stress conditions. Most microbial species exclusively synthesize and degrade either glycogen or PHB. A few bacteria such as the phototrophic model organism Synechocystis sp. PCC 6803 surprisingly produce both physico-chemically different polymers under conditions of high C to N ratios. For the first time, the function and interrelation of both carbon polymers in non-diazotrophic cyanobacteria are analyzed in a comparative physiological study of single- and double-knockout mutants (ΔglgC; ΔphaC; ΔglgC/ΔphaC), respectively. Most of the observed phenotypes are explicitly related to the knockout of glycogen synthesis, highlighting the metabolic, energetic, and structural impact of this process whenever cells switch from an active, photosynthetic ‘protein status’ to a dormant ‘glycogen status’. The carbon flux regulation into glycogen granules is apparently crucial for both phycobilisome degradation and thylakoid layer disassembly in the presence of light. In contrast, PHB synthesis is definitely not involved in this primary acclimation response. Moreover, the very weak interrelations between the two carbon-polymer syntheses indicate that the regulation and role of PHB synthesis in Synechocystis sp. PCC 6803 is different from glycogen synthesis. PMID:27446007

  16. The Multiple Functions of Common Microbial Carbon Polymers, Glycogen and PHB, during Stress Responses in the Non-Diazotrophic Cyanobacterium Synechocystis sp. PCC 6803.

    PubMed

    Damrow, Ramon; Maldener, Iris; Zilliges, Yvonne

    2016-01-01

    Classical microbial carbon polymers such as glycogen and polyhydroxybutyrate (PHB) have a crucial impact as both a sink and a reserve under macronutrient stress conditions. Most microbial species exclusively synthesize and degrade either glycogen or PHB. A few bacteria such as the phototrophic model organism Synechocystis sp. PCC 6803 surprisingly produce both physico-chemically different polymers under conditions of high C to N ratios. For the first time, the function and interrelation of both carbon polymers in non-diazotrophic cyanobacteria are analyzed in a comparative physiological study of single- and double-knockout mutants (ΔglgC; ΔphaC; ΔglgC/ΔphaC), respectively. Most of the observed phenotypes are explicitly related to the knockout of glycogen synthesis, highlighting the metabolic, energetic, and structural impact of this process whenever cells switch from an active, photosynthetic 'protein status' to a dormant 'glycogen status'. The carbon flux regulation into glycogen granules is apparently crucial for both phycobilisome degradation and thylakoid layer disassembly in the presence of light. In contrast, PHB synthesis is definitely not involved in this primary acclimation response. Moreover, the very weak interrelations between the two carbon-polymer syntheses indicate that the regulation and role of PHB synthesis in Synechocystis sp. PCC 6803 is different from glycogen synthesis. PMID:27446007

  17. Quantitation of the pathways of hepatic glycogen formation on ingesting a glucose load

    SciTech Connect

    Magnusson, I.; Chandramouli, V.; Schumann, W.C.; Kumaran, K.; Wahren, J.; Landau, B.R.

    1987-12-01

    Diflunisal, 5-(2',4'-difluorophenyl)salicylic acid, excreted in urine as its glucuronide, was given to normal humans (n = 6) along with a glucose load specifically labeled with /sup 14/C. Glucuronide excreted by each subject was reduced to its glucoside and glucose from it degraded to yield the distribution of 14 C in its six carbons. Randomization of the /sup 14/C from the specifically labeled glucose was taken as a measure of the extent to which glucose was deposited indirectly (i.e., glucose----lactate----glucose----6-P----glycogen), rather than directly (i.e., glucose----glucose-6-P----glycogen). The maximum contribution to glycogen formation by the direct pathway was estimated to be 65 +/- 1%, on the assumption that glucuronide and glycogen are derived from the same hepatic pool of glucose-6-P in liver. Evidence that supports that assumption was obtained by comparing the randomization of /sup 14/C in the urinary glucuronide with that in glucose in blood from the hepatic vein of four of the subjects before and after they were given glucagon. Other evidence supporting the assumption was obtained by comparing in two subjects /sup 3/H//sup 14/C ratios in glucose from hepatic vein blood before and after glucagon administration with that in urinary glucuronide, having labeled the uridine diphosphate (UDP)-glucose in their livers with /sup 14/C by giving them 1-(/sup 14/C)galactose and their circulating glucose with /sup 3/H by giving a 5-(/sup 3/H)glucose-labeled load. It is concluded that glucuronide formation in humans can be used to trace glucose metabolism in the liver, and that in humans the indirect pathway of glucose metabolism is active.

  18. Diurnal variation in skeletal muscle and liver glycogen in humans with normal health and Type 2 diabetes.

    PubMed

    Macauley, Mavin; Smith, Fiona E; Thelwall, Peter E; Hollingsworth, Kieren G; Taylor, Roy

    2015-05-01

    In health, food carbohydrate is stored as glycogen in muscle and liver, preventing a deleterious rise in osmotically active plasma glucose after eating. Glycogen concentrations increase sequentially after each meal to peak in the evening, and fall to fasting levels thereafter. Skeletal muscle accounts for the larger part of this diurnal buffering capacity with liver also contributing. The effectiveness of this diurnal mechanism has not been previously studied in Type 2 diabetes. We have quantified the changes in muscle and liver glycogen concentration with 13C magnetic resonance spectroscopy at 3.0 T before and after three meals consumed at 4 h intervals. We studied 40 (25 males; 15 females) well-controlled Type 2 diabetes subjects on metformin only (HbA1c (glycated haemoglobin) 6.4±0.07% or 47±0.8 mmol/mol) and 14 (8 males; 6 females) glucose-tolerant controls matched for age, weight and body mass index (BMI). Muscle glycogen concentration increased by 17% after day-long eating in the control group (68.1±4.8 to 79.7±4.2 mmol/l; P=0.006), and this change inversely correlated with homoeostatic model assessment of insulin resistance [HOMA-IR] (r=-0.56; P=0.02). There was no change in muscle glycogen in the Type 2 diabetes group after day-long eating (68.3±2.6 to 67.1±2.0 mmol/mol; P=0.62). Liver glycogen rose similarly in normal control (325.9±25.0 to 388.1±30.3 mmol/l; P=0.005) and Type 2 diabetes groups (296.1±16.0 to 350.5±6.7 mmol/l; P<0.0001). In early Type 2 diabetes, the major physiological mechanism for skeletal muscle postprandial glycogen storage is completely inactive. This is directly related to insulin resistance, although liver glycogen storage is normal.

  19. Late form of Pompe disease with glycogen storage in peripheral nerves axons.

    PubMed

    Fidziańska, Anna; Ługowska, Agnieszka; Tylki-Szymańska, Anna

    2011-02-15

    Pompe disease is caused by the deficiency of acid α-glucosidase (GAA), which degrades glycogen into glucose. Its manifestation is characterized by a broad and continuous spectrum of clinical severity ranging from severe infantile to relatively benign adult form. We describe a 12-year-old girl diagnosed at a presymptomatic stage of late form Pompe disease due to fortuitous detection of an elevated level of serum creatine kinase (CK) at the age of 4. Biopsies were taken from the quadriceps muscle and studied with histological and histochemical techniques, as well as in electron microscope. Sporadic muscle cells showed the accumulation of lysosomal glycogen, suggesting Pompe disease. Interestingly, we found lysosomal bound glycogen, located in the axons of intramuscular nerves. The diagnosis was confirmed by deficient GAA activity in leukocytes. Mutation analysis revealed changes IVS1-13T>G and p.C103G in the GAA gene. The patient was able to obtain enzyme replacement therapy in the early asymptomatic stage of the disease.

  20. Effects of diabetes on brain metabolism--is brain glycogen a significant player?

    PubMed

    Sickmann, Helle M; Waagepetersen, Helle S

    2015-02-01

    Brain glycogen, being an intracellular glucose reservoir, contributes to maintain energy and neurotransmitter homeostasis under physiological as well as pathological conditions. Under conditions with a disturbance in systemic glucose metabolism such as in diabetes, the supply of glucose to the brain may be affected and have important impacts on brain metabolism and neurotransmission. This also implies that brain glycogen may serve an essential role in the diabetic state to sustain appropriate brain function. There are two main types of diabetes; type 1 and type 2 diabetes and both types may be associated with brain impairments e.g. cognitive decline and dementia. It is however, not clear how these impairments on brain function are linked to alterations in brain energy and neurotransmitter metabolism. In this review, we will illuminate how rodent diabetes models have contributed to a better understanding of how brain energy and neurotransmitter metabolism is affected in diabetes. There will be a particular focus on the role of brain glycogen to support glycolytic and TCA cycle activity as well as glutamate-glutamine cycle in type 1 and type 2 diabetes.

  1. Neuromuscular responses to mild-muscle damaging eccentric exercise in a low glycogen state.

    PubMed

    Gavin, James P; Myers, Stephen D; Willems, Mark E T

    2015-02-01

    The aim of this study was to examine the effect of low muscle glycogen on the neuromuscular responses to maximal eccentric contractions. Fourteen healthy men (22 ± 3 years) performed single-leg cycling (20 min at ~75% maximal oxygen uptake (V̇O2 max); eight 90 s sprints at a 1:1 work-to-rest ratio (5% decrements from 90% to 55% V̇O2 max until exhaustion) the evening before 100 eccentric (1.57 rads(-1)) with reduced (RED) and normal glycogen (NORM). Neuromuscular responses were measured during and up to 48 h after with maximal voluntary and involuntary (twitch, 20 Hz and 50 Hz) isometric contractions. During eccentric contractions, peak torque decreased (RED: -16.1 ± 2.5%; NORM: -6.2 ± 5.1%) and EMG frequency increased according to muscle length. EMG activity decreased for RED only. After eccentric contractions, maximal isometric force was reduced up to 24h for NORM (-13.5 ± 5.8%) and 48 h for RED (-7.4 ± 10.9%). Twelve hours after eccentric contractions, twitch force and the 20:50 Hz ratio were decreased for RED but not for NORM. Immediate involuntary with prolonged voluntary force loss suggests that reduced glycogen is associated with increased susceptibility to mild muscle-damaging eccentric exercise with contributions of peripheral and central mechanisms to be different during recovery.

  2. Molecular and metabolomic effects of voluntary running wheel activity on skeletal muscle in late middle-aged rats

    PubMed Central

    Garvey, Sean M; Russ, David W; Skelding, Mary B; Dugle, Janis E; Edens, Neile K

    2015-01-01

    We examined the molecular and metabolomic effects of voluntary running wheel activity in late middle-aged male Sprague Dawley rats (16–17 months). Rats were assigned either continuous voluntary running wheel access for 8 weeks (RW+) or cage-matched without running wheel access (RW−). The 9 RW+ rats averaged 83 m/day (range: 8–163 m), yet exhibited both 84% reduced individual body weight gain (4.3 g vs. 26.3 g, P = 0.02) and 6.5% reduced individual average daily food intake (20.6 g vs. 22.0 g, P = 0.09) over the 8 weeks. Hindlimb muscles were harvested following an overnight fast. Muscle weights and myofiber cross-sectional area showed no difference between groups. Western blots of gastrocnemius muscle lysates with a panel of antibodies suggest that running wheel activity improved oxidative metabolism (53% increase in PGC1α, P = 0.03), increased autophagy (36% increase in LC3B-II/-I ratio, P = 0.03), and modulated growth signaling (26% increase in myostatin, P = 0.04). RW+ muscle also showed 43% increased glycogen phosphorylase expression (P = 0.04) and 45% increased glycogen content (P = 0.04). Metabolomic profiling of plantaris and soleus muscles indicated that even low-volume voluntary running wheel activity is associated with decreases in many long-chain fatty acids (e.g., palmitoleate, myristoleate, and eicosatrienoate) relative to RW− rats. Relative increases in acylcarnitines and acyl glycerophospholipids were also observed in RW+ plantaris. These data establish that even modest amounts of physical activity during late middle-age promote extensive metabolic remodeling of skeletal muscle. PMID:25716928

  3. Molecular and metabolomic effects of voluntary running wheel activity on skeletal muscle in late middle-aged rats.

    PubMed

    Garvey, Sean M; Russ, David W; Skelding, Mary B; Dugle, Janis E; Edens, Neile K

    2015-02-01

    We examined the molecular and metabolomic effects of voluntary running wheel activity in late middle-aged male Sprague Dawley rats (16-17 months). Rats were assigned either continuous voluntary running wheel access for 8 weeks (RW+) or cage-matched without running wheel access (RW-). The 9 RW+ rats averaged 83 m/day (range: 8-163 m), yet exhibited both 84% reduced individual body weight gain (4.3 g vs. 26.3 g, P = 0.02) and 6.5% reduced individual average daily food intake (20.6 g vs. 22.0 g, P = 0.09) over the 8 weeks. Hindlimb muscles were harvested following an overnight fast. Muscle weights and myofiber cross-sectional area showed no difference between groups. Western blots of gastrocnemius muscle lysates with a panel of antibodies suggest that running wheel activity improved oxidative metabolism (53% increase in PGC1α, P = 0.03), increased autophagy (36% increase in LC3B-II/-I ratio, P = 0.03), and modulated growth signaling (26% increase in myostatin, P = 0.04). RW+ muscle also showed 43% increased glycogen phosphorylase expression (P = 0.04) and 45% increased glycogen content (P = 0.04). Metabolomic profiling of plantaris and soleus muscles indicated that even low-volume voluntary running wheel activity is associated with decreases in many long-chain fatty acids (e.g., palmitoleate, myristoleate, and eicosatrienoate) relative to RW- rats. Relative increases in acylcarnitines and acyl glycerophospholipids were also observed in RW+ plantaris. These data establish that even modest amounts of physical activity during late middle-age promote extensive metabolic remodeling of skeletal muscle.

  4. Dietary management of Type I glycogen storage disease.

    PubMed

    Folk, C C; Greene, H L

    1984-03-01

    The most commonly recognized type of glycogen storage disease (von Gierke's disease) results from deficient glucose-6-phosphatase activity. This enzyme is the last step in the release of free glucose from the liver into the circulation. Thus, the most prominent and life-threatening complication in the illness is severe and often prolonged hypoglycemia, which occurs after the dietary glucose is normally removed from the circulation. With an optimal dietary intake spaced at 2 1/2- to 3 1/2-hour intervals, the blood glucose can be maintained in the normal range during the daytime, but hypoglycemia may occur during overnight fasting. Recent advances in the understanding of the pathophysiology of the illness have led to the use of frequent high-starch feedings during the day and nocturnal intragastric infusions of liquid formulas containing glucose polymers. The liquid formula is infused through either a nasogastric or a gastrostomy tube continuously at night while the patient sleeps. The success of this treatment not only has improved the survival rate but also has corrected the abnormal blood chemistries and generated a more normal rate of growth and development. Because patients with this disease are reaching adulthood in greater numbers, it is necessary for dietitians caring for adults as well as for children to become familiar with the prescribed methods of treatment. PMID:6583274

  5. A new non-degradative method to purify glycogen.

    PubMed

    Tan, Xinle; Sullivan, Mitchell A; Gao, Fei; Li, Shihan; Schulz, Benjamin L; Gilbert, Robert G

    2016-08-20

    Liver glycogen, a complex branched glucose polymer containing a small amount of protein, is important for maintaining glucose homeostasis (blood-sugar control) in humans. It has recently been found that glycogen molecular structure is impaired in diabetes. Isolating the carbohydrate polymer and any intrinsically-attached protein(s) is an essential prerequisite for studying this structural impairment. This requires an effective, non-degradative and efficient purification method to exclude the many other proteins present in liver. Proteins and glycogen have different ranges of molecular sizes. Despite the plethora of proteins that might still be present in significant abundance after other isolation techniques, SEC (size exclusion chromatography, also known as GPC), which separates by molecular size, should separate those extraneous to glycogen from glycogen with any intrinsically associated protein(s). A novel purification method is developed for this, based on preparative SEC following sucrose gradient centrifugation. Proteomics is used to show that the new method compares favourably with current methods in the literature. PMID:27178921

  6. On artifacts appearing in the histochemical fixation of glycogen.

    PubMed

    TAKAHASHI, K; IWASE, S

    1955-09-25

    1. Fixation artifacts associated with glycogen translocation are prevalent in tissues of parenchymatous type and scarce or non-existent in tissues of loose type. 2. Liver tissue treated with M/3 NaOH solution before fixation did not show an uneven distribution of glycogen. This was interpreted as indicating that the liver, a tissue of parenchymatous type, was changed, so to speak, into a loose type of tissue by alkali treatment. 3. The so called Alkohol-flucht of glycogen was produced in Yoshida's ascites tumor cells by a procedure which changed a loose type of tissue into a parenchymatous one, that is, by packing the tumor cells tightly. 4. The translocation of glycogen in cells appeared to occur when the fixatives penetrated the cells rapidly from a single direction, but failed to occur when the cells were attacked by the fixative from all directions. 5. In dried smears of Yoshida's ascites tumor cells and bone marrow cells, the glycogen particles are translocated to the peripheral regions of the cells, and coalesce there. The production of these artifacts is related in some way to the physicochemical properties of the protoplasm and plasma membrane of the cells.

  7. Crystallographic and docking studies of purine nucleoside phosphorylase from Mycobacterium tuberculosis.

    PubMed

    Ducati, Rodrigo G; Basso, Luiz A; Santos, Diógenes S; de Azevedo, Walter F

    2010-07-01

    This work describes for the first time the structure of purine nucleoside phosphorylase from Mycobacterium tuberculosis (MtPNP) in complex with sulfate and its natural substrate, 2'-deoxyguanosine, and its application to virtual screening. We report docking studies of a set of molecules against this structure. Application of polynomial empirical scoring function was able to rank docking solutions with good predicting power which opens the possibility to apply this new criterion to analyze docking solutions and screen small-molecule databases for new chemical entities to inhibit MtPNP.

  8. Compositions and methods involving methyladenosine phosphorylase in the diagnosis and treatment of proliferative disorders

    DOEpatents

    Olopade, Olufunmilayo I.

    2007-03-20

    Disclosed are novel nucleic acid and peptide compositions comprising methylthioadenosine phosphorylase (MTAP) and methods of use for MTAP amino acid sequences and DNA segments comprising MTAP in the diagnosis of human cancers and development of MTAP-specific antibodies. Also disclosed are methods for the diagnosis and treatment of tumors and other proliferative cell disorders, and identification of tumor suppressor genes and gene products from the human 9p21-p22 chromosome region. Such methods are useful in the diagnosis of multiple tumor types such as bladder cancer, lung cancer, breast cancer, pancreatic cancer, brain tumors, lymphomas, gliomas, melanomas, and leukemias.

  9. Methylthioadenosine phosphorylase compositions and methods of use in the diagnosis and treatment of proliferative disorders

    DOEpatents

    Olopade, Olufunmilayo I.

    2005-03-22

    Disclosed are novel nucleic acid and peptide compositions comprising methythlioadenosine phosphorylase (MTAP) and methods of use for MTAP amino acid sequences and DNA segments comprising MTAP in the diagnosis of human cancers and development of MTAP-specific antibodies. Also disclosed are methods for the diagnosis and treatment of tumors and other proliferative cell disorders, and idenification tumor suppressor genes and gene products from the human 9p21-p22 chromosome region. Such methods are useful in the diagnosis of multiple tumor types such as bladder cancer, lung cancer, breast cancer, pancreatic cancer, brain tumors, lymphomas, gliomas, melanomas, and leukemias.

  10. Role in Tumor Growth of a Glycogen Debranching Enzyme Lost in Glycogen Storage Disease

    PubMed Central

    Guin, Sunny; Pollard, Courtney; Ru, Yuanbin; Ritterson Lew, Carolyn; Duex, Jason E.; Dancik, Garrett; Owens, Charles; Spencer, Andrea; Knight, Scott; Holemon, Heather; Gupta, Sounak; Hansel, Donna; Hellerstein, Marc; Lorkiewicz, Pawel; Lane, Andrew N.; Fan, Teresa W.-M.

    2014-01-01

    Background Bladder cancer is the most common malignancy of the urinary system, yet our molecular understanding of this disease is incomplete, hampering therapeutic advances. Methods Here we used a genome-wide functional short-hairpin RNA (shRNA) screen to identify suppressors of in vivo bladder tumor xenograft growth (n = 50) using bladder cancer UMUC3 cells. Next-generation sequencing was used to identify the most frequently occurring shRNAs in tumors. Genes so identified were studied in 561 patients with bladder cancer for their association with stratification of clinical outcome by Kaplan-Meier analysis. The best prognostic marker was studied to determine its mechanism in tumor suppression using anchorage-dependent and -independent growth, xenograft (n = 20), and metabolomic assays. Statistical significance was determined using two-sided Student t test and repeated-measures statistical analysis. Results We identified the glycogen debranching enzyme AGL as a prognostic indicator of patient survival (P = .04) and as a novel regulator of bladder cancer anchorage-dependent (P < .001), anchorage-independent (mean ± standard deviation, 180 ± 23.1 colonies vs 20±9.5 in control, P < .001), and xenograft growth (P < .001). Rescue experiments using catalytically dead AGL variants revealed that this effect is independent of AGL enzymatic functions. We demonstrated that reduced AGL enhances tumor growth by increasing glycine synthesis through increased expression of serine hydroxymethyltransferase 2. Conclusions Using an in vivo RNA interference screen, we discovered that AGL, a glycogen debranching enzyme, has a biologically and statistically significant role in suppressing human cancer growth. PMID:24700805

  11. Familial nephropathy associated with hepatic type of glycogen storage disease.

    PubMed

    Sonobe, H; Ogawa, K; Takahashi, I

    1976-11-01

    The female patient was diagnosed as having Von Gierke's disease at 14 years of age, based on clinical manifestations, laboratory examination and liver biopsy. At 19 years of age she had uremia and died from its deterioration at 24 years of age. The parents were consanguineous, and a 27-year-old sister is presently hospitalized for renal insufficiency with hepatomegaly. On autopsy, the patient's kidneys were highly contracted and contained a number of small cysts, mainly in the medulla. Histological examination indicated periglomerular fibrosis, glomerular hyalinization, tubular atrophy or cystic dilatation and intersitial fibrosis with round cell infiltration. These findings correspond to Fanconi's familial juvenile nephronophthisis, except for age. The liver was markedly enlarged and indicated severe, glycogen deposits, but the kidney did not contain glycogen deposits. It can, therefore, be presumed that the renal lesions were not a secondary consequence of long-term glycogen deposits but that renal and hepatic lesions were associated with each other. PMID:1070908

  12. Modified glycogen as construction material for functional biomimetic microfibers.

    PubMed

    Rabyk, Mariia; Hruby, Martin; Vetrik, Miroslav; Kucka, Jan; Proks, Vladimir; Parizek, Martin; Konefal, Rafal; Krist, Pavel; Chvatil, David; Bacakova, Lucie; Slouf, Miroslav; Stepanek, Petr

    2016-11-01

    We describe a conceptually new, microfibrous, biodegradable functional material prepared from a modified storage polysaccharide also present in humans (glycogen) showing strong potential as direct-contact dressing/interface material for wound healing. Double bonds were introduced into glycogen via allylation and were further exploited for crosslinking of the microfibers. Triple bonds were introduced by propargylation and served for further click functionalization of the microfibers with bioactive peptide. A simple solvent-free method allowing the preparation of thick layers was used to produce microfibers (diameter ca 2μm) from allylated and/or propargylated glycogen. Crosslinking of the samples was performed by microtron beta-irradiation, and the irradiation dose was optimized to 2kGy. The results from biological testing showed that these highly porous, hydrophilic, readily functionalizable materials were completely nontoxic to cells growing in their presence. The fibers were gradually degraded in the presence of cells. PMID:27516273

  13. Ursolic acid and luteolin-7-glucoside improve lipid profiles and increase liver glycogen content through glycogen synthase kinase-3.

    PubMed

    Azevedo, Marisa F; Camsari, Cagri; Sá, Carla M; Lima, Cristovao F; Fernandes-Ferreira, Manuel; Pereira-Wilson, Cristina

    2010-06-01

    In the present study, two phytochemicals - ursolic acid (UA) and luteolin-7-glucoside (L7G) - were assessed in vivo in healthy rats regarding effects on plasma glucose and lipid profile (total cholesterol, HDL and LDL), as well as liver glycogen content, in view of their importance in the aetiology of diabetes and associated complications. Both UA and L7G significantly decreased plasma glucose concentration. UA also significantly increased liver glycogen levels accompanied by phosphorylation of glycogen synthase kinase-3 (GSK3). The increase in glycogen deposition induced by UA (mediated by GSK3) could have contributed to the lower plasma glucose levels observed. Both compounds significantly lowered total plasma cholesterol and low-density lipoprotein levels, and, in addition, UA increased plasma high-density lipoprotein levels. Our results show that UA particularly may be useful in preventable strategies for people at risk of developing diabetes and associated cardiovascular complications by imp