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Sample records for akt glycogen synthase

  1. 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.

  2. Repulsive axon guidance by Draxin is mediated by protein Kinase B (Akt), glycogen synthase kinase-3β (GSK-3β) and microtubule-associated protein 1B.

    PubMed

    Meli, Rajeshwari; Weisová, Petronela; Propst, Friedrich

    2015-01-01

    Draxin is an important axon guidance cue necessary for the formation of forebrain commissures including the corpus callosum, but the molecular details of draxin signaling are unknown. To unravel how draxin signals are propagated we used murine cortical neurons and genetic and pharmacological approaches. We found that draxin-induced growth cone collapse critically depends on draxin receptors (deleted in colorectal cancer, DCC), inhibition of protein kinase B/Akt, activation of GSK-3β (glycogen synthase kinase-3β) and the presence of microtubule-associated protein MAP1B. This study, for the first time elucidates molecular events in draxin repulsion, links draxin and DCC to MAP1B and identifies a novel MAP1B-depenent GSK-3β pathway essential for chemo-repulsive axon guidance cue signaling.

  3. 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.

  4. 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.

  5. 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

  6. 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.

  7. 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

  8. 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.

  9. 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.

  10. 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

  11. 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.

  12. 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

  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.

  14. 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.

  15. 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.

  16. 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

  17. 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.

  18. 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.

  19. Irisin inhibits hepatic gluconeogenesis and increases glycogen synthesis via the PI3K/Akt pathway in type 2 diabetic mice and hepatocytes.

    PubMed

    Liu, Tong-Yan; Shi, Chang-Xiang; Gao, Run; Sun, Hai-Jian; Xiong, Xiao-Qing; Ding, Lei; Chen, Qi; Li, Yue-Hua; Wang, Jue-Jin; Kang, Yu-Ming; Zhu, Guo-Qing

    2015-11-01

    Increased glucose production and reduced hepatic glycogen storage contribute to metabolic abnormalities in diabetes. Irisin, a newly identified myokine, induces the browning of white adipose tissue, but its effects on gluconeogenesis and glycogenesis are unknown. In the present study, we investigated the effects and underlying mechanisms of irisin on gluconeogenesis and glycogenesis in hepatocytes with insulin resistance, and its therapeutic role in type 2 diabetic mice. Insulin resistance was induced by glucosamine (GlcN) or palmitate in human hepatocellular carcinoma (HepG2) cells and mouse primary hepatocytes. Type 2 diabetes was induced by streptozotocin/high-fat diet (STZ/HFD) in mice. In HepG2 cells, irisin ameliorated the GlcN-induced increases in glucose production, phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) expression, and glycogen synthase (GS) phosphorylation; it prevented GlcN-induced decreases in glycogen content and the phosphoinositide 3-kinase (PI3K) p110α subunit level, and the phosphorylation of Akt/protein kinase B, forkhead box transcription factor O1 (FOXO1) and glycogen synthase kinase-3 (GSK3). These effects of irisin were abolished by the inhibition of PI3K or Akt. The effects of irisin were confirmed in mouse primary hepatocytes with GlcN-induced insulin resistance and in human HepG2 cells with palmitate-induced insulin resistance. In diabetic mice, persistent subcutaneous perfusion of irisin improved the insulin sensitivity, reduced fasting blood glucose, increased GSK3 and Akt phosphorylation, glycogen content and irisin level, and suppressed GS phosphorylation and PEPCK and G6Pase expression in the liver. Irisin improves glucose homoeostasis by reducing gluconeogenesis via PI3K/Akt/FOXO1-mediated PEPCK and G6Pase down-regulation and increasing glycogenesis via PI3K/Akt/GSK3-mediated GS activation. Irisin may be regarded as a novel therapeutic strategy for insulin resistance and type 2 diabetes.

  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. 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

  2. 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

  3. 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

  4. 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

  5. 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.

  6. Mechanism of estrogen-mediated improvement in cardiac function after trauma-hemorrhage: p38-dependent normalization of cardiac Akt phosphorylation and glycogen levels.

    PubMed

    Hsu, Jun-Te; Kan, Wen-Hong; Hsieh, Ya-Ching; Choudhry, Mashkoor A; Schwacha, Martin G; Bland, Kirby I; Chaudry, Irshad H

    2008-10-01

    Both p38 mitogen-activated protein kinase (p38) activation and protein kinase B (Akt) activation have been reported to regulate glucose transport during myocardial I/R. An increase in cardiac glycogen levels prevents myocardial injury in the ischemic or stressed heart. Although studies have shown that 17"-estradiol (E2)-mediated improvement in cardiac function after trauma-hemorrhage is via p38 activation, it remains unknown whether p38/Akt plays any role in regulation of cardiac glycogen levels under these conditions. To study this, male rats underwent trauma-hemorrhage(mean blood pressure, x40 mmHg for 90 min) followed by fluid resuscitation. At the onset of resuscitation, rats (n=6 per group) were treated with vehicle, E2 (1 mg/kg body weight), the p38 inhibitor SB203580 (2 mg/kg body weight), or E2 and SB203580. Various parameters were measured at 2 h after resuscitation. One-way ANOVA and Tukey test were used for statistical analysis, and differences were considered significant at P<0.05. The depressed cardiac function after trauma-hemorrhage was restored by E2 treatment (P<0.05). Administration of E2 after trauma-hemorrhage also normalized the p38/Akt phosphorylation, which was associated with restoration of cardiac glycogen, glycogen synthase kinase 3"activation, glucose transporter 4 translocation, and increased hexokinase II levels (all parameters, P<0.05). Inhibition of the p38 pathway abolished the E2-induced restoration in above parameters after trauma-hemorrhage. These results suggest that p38-dependent normalization of cardiac Akt phosphorylation and glycogen levels plays an important role in E2-mediated restoration of cardiac function after trauma-hemorrhage.

  7. Glycogen synthase kinase-3 (GSK3): regulation, actions, and diseases

    PubMed Central

    Beurel, Eleonore; Grieco, Steven F.; Jope, Richard S.

    2014-01-01

    Glycogen synthase kinase-3 (GSK3) may be the busiest kinase in most cells, with over 100 known substrates to deal with. How does GSK3 maintain control to selectively phosphorylate each substrate, and why was it evolutionarily favorable for GSK3 to assume such a large responsibility? GSK3 must be particularly adaptable for incorporating new substrates into its repertoire, and we discuss the distinct properties of GSK3 that may contribute to its capacity to fulfill its roles in multiple signaling pathways. The mechanisms regulating GSK3 (predominantly post-translational modifications, substrate priming, cellular trafficking, protein complexes) have been reviewed previously, so here we focus on newly identified complexities in these mechanisms, how each of these regulatory mechanism contributes to the ability of GSK3 to select which substrates to phosphorylate, and how these mechanisms may have contributed to its adaptability as new substrates evolved. The current understanding of the mechanisms regulating GSK3 is reviewed, as are emerging topics in the actions of GSK3, particularly its interactions with receptors and receptor-coupled signal transduction events, and differential actions and regulation of the two GSK3 isoforms, GSK3α and GSK3β. Another remarkable characteristic of GSK3 is its involvement in many prevalent disorders, including psychiatric and neurological diseases, inflammatory diseases, cancer, and others. We address the feasibility of targeting GSK3 therapeutically, and provide an update of its involvement in the etiology and treatment of several disorders. PMID:25435019

  8. Glycogen synthase kinase-3 inhibitors: Rescuers of cognitive impairments

    PubMed Central

    King, Margaret K.; Pardo, Marta; Cheng, Yuyan; Downey, Kimberlee; Jope, Richard S.; Beurel, Eléonore

    2013-01-01

    Impairment of cognitive processes is a devastating outcome of many diseases, injuries, and drugs affecting the central nervous system (CNS). Most often, very little can be done by available therapeutic interventions to improve cognitive functions. Here we review evidence that inhibition of glycogen synthase kinase-3 (GSK3) ameliorates cognitive deficits in a wide variety of animal models of CNS diseases, including Alzheimer's disease, Fragile X syndrome, Down syndrome, Parkinson's disease, spinocerebellar ataxia type 1, traumatic brain injury, and others. GSK3 inhibitors also improve cognition following impairments caused by therapeutic interventions, such as cranial irradiation for brain tumors. These findings demonstrate that GSK3 inhibitors are able to ameliorate cognitive impairments caused by a diverse array of diseases, injury, and treatments. The improvements in impaired cognition instilled by administration of GSK3 inhibitors appear to involve a variety of different mechanisms, such as supporting long-term potentiation and diminishing long-term depression, promotion of neurogenesis, reduction of inflammation, and increasing a number of neuroprotective mechanisms. The potential for GSK3 inhibitors to repair cognitive deficits associated with many conditions warrants further investigation of their potential for therapeutic interventions, particularly considering the current dearth of treatments available to reduce loss of cognitive functions. PMID:23916593

  9. 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.

  10. 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.

  11. Glycogen synthase kinase 3β inhibition promotes human iTreg differentiation and suppressive function.

    PubMed

    Xia, Yongxiang; Zhuo, Han; Lu, Yunjie; Deng, Lei; Jiang, Runqiu; Zhang, Long; Zhu, Qin; Pu, Liyong; Wang, Xuehao; Lu, Ling

    2015-05-01

    Induced regulatory T cells (iTregs) are essential to maintain immunological tolerance, immune homeostasis and prevention of autoimmunity. Some studies suggest that glycogen synthase kinase 3β (GSK3β) is involved in the mouse iTreg differentiation; however, whether GSK3β inhibits or enhances iTreg differentiation is still a matter of controversy. To address this issue, we have utilized human naïve CD4(+) T cells and investigated whether GSK3 activity changes during iTreg differentiation and whether altering GSK3 activity influences the development of iTregs and its suppressive function. As a constitutively activated kinase, during iTreg differentiation GSK3β became quickly deactivated (phosphorylated at serine 9), which is dependent on MAPK pathway rather than PI3-kinase/Akt pathway. Our results indicated that inhibition of GSK3β by specific inhibitors, SB216763 or TDZD-8, promoted the differentiation of iTreg and increased their suppressive activity. In contrast, overexpression of GSK3β significantly inhibited iTreg differentiation. Furthermore, GSK3β inhibition enhanced iTreg differentiation through the TGF-β/Smad3 pathway. Taken together, this study demonstrates that inhibition of GSK3β enhances human iTreg differentiation and its suppressive activity, and provides a rationale to target GSK3β as a novel immunotherapeutic strategy.

  12. 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 improving plasma glucose levels and lipid profile, as well as by promoting liver glycogen deposition.

  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 improving plasma glucose levels and lipid profile, as well as by promoting liver glycogen deposition. PMID:20127879

  14. 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.

  15. Glycogen synthase kinase-3β modulation of glucocorticoid responsiveness in COPD

    PubMed Central

    Hoffmann, Roland F.; Durham, Andrew L.; Marwick, John A.; Brandenburg, Simone M.; de Bruin, Harold G.; Jonker, Marnix R.; Rossios, Christos; Tsitsiou, Eleni; Caramori, Gaetano; Contoli, Marco; Casolari, Paolo; Monaco, Francesco; Andò, Filippo; Speciale, Giuseppe; Kilty, Iain; Chung, Kian F.; Papi, Alberto; Lindsay, Mark A.; ten Hacken, Nick H. T.; van den Berge, Maarten; Timens, Wim; Barnes, Peter J.; van Oosterhout, Antoon J.; Kirkham, Paul A.; Heijink, Irene H.

    2015-01-01

    In chronic obstructive pulmonary disease (COPD), oxidative stress regulates the inflammatory response of bronchial epithelium and monocytes/macrophages through kinase modulation and has been linked to glucocorticoid unresponsiveness. Glycogen synthase-3β (GSK3β) inactivation plays a key role in mediating signaling processes upon reactive oxygen species (ROS) exposure. We hypothesized that GSK3β is involved in oxidative stress-induced glucocorticoid insensitivity in COPD. We studied levels of phospho-GSK3β-Ser9, a marker of GSK3β inactivation, in lung sections and cultured monocytes and bronchial epithelial cells of COPD patients, control smokers, and nonsmokers. We observed increased levels of phospho-GSK3β-Ser9 in monocytes, alveolar macrophages, and bronchial epithelial cells from COPD patients and control smokers compared with nonsmokers. Pharmacological inactivation of GSK3β did not affect CXCL8 or granulocyte-macrophage colony-stimulating factor (GM-CSF) expression but resulted in glucocorticoid insensitivity in vitro in both inflammatory and structural cells. Further mechanistic studies in monocyte and bronchial epithelial cell lines showed that GSK3β inactivation is a common effector of oxidative stress-induced activation of the MEK/ERK-1/2 and phosphatidylinositol 3-kinase/Akt signaling pathways leading to glucocorticoid unresponsiveness. In primary monocytes, the mechanism involved modulation of histone deacetylase 2 (HDAC2) activity in response to GSK3β inactivation. In conclusion, we demonstrate for the first time that ROS-induced glucocorticoid unresponsiveness in COPD is mediated through GSK3β, acting as a ROS-sensitive hub. PMID:26320152

  16. Glycogen synthase kinase-3β modulation of glucocorticoid responsiveness in COPD.

    PubMed

    Ngkelo, Anta; Hoffmann, Roland F; Durham, Andrew L; Marwick, John A; Brandenburg, Simone M; de Bruin, Harold G; Jonker, Marnix R; Rossios, Christos; Tsitsiou, Eleni; Caramori, Gaetano; Contoli, Marco; Casolari, Paolo; Monaco, Francesco; Andò, Filippo; Speciale, Giuseppe; Kilty, Iain; Chung, Kian F; Papi, Alberto; Lindsay, Mark A; Ten Hacken, Nick H T; van den Berge, Maarten; Timens, Wim; Barnes, Peter J; van Oosterhout, Antoon J; Adcock, Ian M; Kirkham, Paul A; Heijink, Irene H

    2015-11-15

    In chronic obstructive pulmonary disease (COPD), oxidative stress regulates the inflammatory response of bronchial epithelium and monocytes/macrophages through kinase modulation and has been linked to glucocorticoid unresponsiveness. Glycogen synthase-3β (GSK3β) inactivation plays a key role in mediating signaling processes upon reactive oxygen species (ROS) exposure. We hypothesized that GSK3β is involved in oxidative stress-induced glucocorticoid insensitivity in COPD. We studied levels of phospho-GSK3β-Ser9, a marker of GSK3β inactivation, in lung sections and cultured monocytes and bronchial epithelial cells of COPD patients, control smokers, and nonsmokers. We observed increased levels of phospho-GSK3β-Ser9 in monocytes, alveolar macrophages, and bronchial epithelial cells from COPD patients and control smokers compared with nonsmokers. Pharmacological inactivation of GSK3β did not affect CXCL8 or granulocyte-macrophage colony-stimulating factor (GM-CSF) expression but resulted in glucocorticoid insensitivity in vitro in both inflammatory and structural cells. Further mechanistic studies in monocyte and bronchial epithelial cell lines showed that GSK3β inactivation is a common effector of oxidative stress-induced activation of the MEK/ERK-1/2 and phosphatidylinositol 3-kinase/Akt signaling pathways leading to glucocorticoid unresponsiveness. In primary monocytes, the mechanism involved modulation of histone deacetylase 2 (HDAC2) activity in response to GSK3β inactivation. In conclusion, we demonstrate for the first time that ROS-induced glucocorticoid unresponsiveness in COPD is mediated through GSK3β, acting as a ROS-sensitive hub. PMID:26320152

  17. Glycogen synthase kinase 3 promotes p53 mRNA translation via phosphorylation of RNPC1

    PubMed Central

    Zhang, Min; Zhang, Jin; Chen, Xiangling; Cho, Seong-Jun; Chen, Xinbin

    2013-01-01

    The RNPC1 RNA-binding protein, also called Rbm38, is a target of p53 and a repressor of p53 mRNA translation. Thus, the p53–RNPC1 loop is critical for modulating p53 tumor suppression, but it is not clear how the loop is regulated. Here, we showed that RNPC1 is phosphorylated at Ser195 by glycogen synthase kinase 3 (GSK3). We also showed that GSK3 promotes p53 mRNA translation through phosphorylation of RNPC1. Interestingly, we found that the phosphor-mimetic mutant S195D and the deletion mutant Δ189–204, which lacks the GSK3 phosphorylation site, are unable to repress p53 mRNA translation due to loss of interaction with eukaryotic translation factor eIF4E on p53 mRNA. Additionally, we found that phosphorylated RNPC1, RNPC1-S195D, and RNPC1(Δ189–204) promote p53 mRNA translation through interaction with eukaryotic translation factor eIF4G, which then facilitates the assembly of the eIF4F complex on p53 mRNA. Furthermore, we showed that upon inhibition of the phosphatidylinositol 3-kinase (PI3K)–Akt pathway, GSK3 is activated, leading to increased RNPC1 phosphorylation and increased p53 expression in a RNPC1-dependent manner. Together, we postulate that the p53–RNPC1 loop can be explored to increase or decrease p53 activity for cancer therapy. PMID:24142875

  18. 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

  19. 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.

  20. A novel mutation in the glycogen synthase 2 gene in a child with glycogen storage disease type 0

    PubMed Central

    2010-01-01

    Background Glycogen storage disease type 0 is an autosomal recessive disease presenting in infancy or early childhood and characterized by ketotic hypoglycemia after prolonged fasting and postprandial hyperglycemia and hyperlactatemia. Sixteen different mutations have been identified to date in the gene which encodes hepatic glycogen synthase, resulting in reduction of glycogen storage in the liver. Case Presentation Biochemical evaluation as well as direct sequencing of exons and exon-intron boundary regions of the GYS2 gene were performed in a patient presenting fasting hypoglycemia and postprandial hyperglycemia and her parents. The patient was found to be compound heterozygous for one previously reported nonsense mutation (c.736 C>T; R243X) and a novel frameshift mutation (966_967delGA/insC) which introduces a stop codon 21 aminoacids downstream from the site of the mutation that presumably leads to loss of 51% of the COOH-terminal part of the protein. The glycemia and lactatemia of the parents after an oral glucose tolerance test were evaluated to investigate a possible impact of the carrier status on the metabolic profile. The mother, who presented a positive family history of type 2 diabetes, was classified as glucose intolerant and the father, who did not exhibit metabolic changes after the glucose overload, had an antecedent history of hypoglycemia after moderate alcohol ingestion. Conclusion The current results expand the spectrum of known mutations in GYS2 and suggest that haploinsufficiency could explain metabolic abnormalities in heterozygous carriers in presence of predisposing conditions. PMID:20051115

  1. NPM-ALK signals through glycogen synthase kinase 3β to promote oncogenesis.

    PubMed

    McDonnell, S R P; Hwang, S R; Basrur, V; Conlon, K P; Fermin, D; Wey, E; Murga-Zamalloa, C; Zeng, Z; Zu, Y; Elenitoba-Johnson, K S J; Lim, M S

    2012-08-01

    Anaplastic large cell lymphoma (ALCL) is the most common type of pediatric peripheral T-cell lymphoma. In 70-80% of cases, the chromosomal aberration t(2;5)(p23;q35) results in the juxtaposition of anaplastic lymphoma kinase (ALK) with nucleophosmin (NPM) and the subsequent expression of the NPM-ALK fusion protein. NPM-ALK is a chimeric tyrosine kinase, which induces numerous signaling pathways that drive proliferation and abrogate apoptosis. However, the mechanisms that lead to activation of downstream growth regulatory molecules have not been completely elucidated. Using a mass spectrometry-based phosphoproteomic screen, we identified GSK3β as a signaling mediator of NPM-ALK. Using a selective inhibitor of ALK, we demonstrated that the tyrosine kinase activity of ALK regulates the serine-9 phosphorylation of GSK3β. Expression of NPM-ALK in 293T cells led to an increase of pS(9)-GSK3β (glycogen synthase kinase 3 beta) compared with kinase-defective K210R mutant NPM-ALK, but did not affect total GSK3β levels. Phosphorylation of pS(9)-GSK3β by NPM-ALK was mediated by the PI3K/AKT signaling pathway. ALK inhibition resulted in degradation of GSK3β substrates Mcl-1 and CDC25A, which was recovered upon chemical inhibition of the proteasome (MG132). Furthermore, the degradation of Mcl-1 was recoverable with inhibition of GSK3β. ALK inhibition also resulted in decreased cell viability, which was rescued by GSK3β inhibition. Furthermore, stable knockdown of GSK3β conferred resistance to the growth inhibitory effects of ALK inhibition using viability and colony formation assays. pS(9)-GSK3β and CDC25A were selectively expressed in neoplastic cells of ALK+ALCL tissue biopsies, and showed a significant correlation (P<0.001). Conversely, ALK-ALCL tissue biopsies did not show significant correlation of pS(9)-GSK3β and CDC25A expression (P<0.2). Our results demonstrate that NPM-ALK regulates the phosphorylation of S(9)-GSK3β by PI3K/AKT. The subsequent inhibition of

  2. Molecular and functional characterization of glycogen synthase in the porcine satellite cells under insulin treatment.

    PubMed

    Wang, Linjie; Xiong, Yuanzhu; Zuo, Bo; Lei, Minggang; Ren, Zhuqing; Xu, Dequan

    2012-01-01

    Glycogen synthase (GS) catalyzes the key step of glycogen synthesis and plays an important role in glycogen metabolism in liver and muscle. In this study, we cloned the cDNA and promoter sequences of porcine glycogen synthesis genes (GYS1 and GYS2). Expression analysis revealed that porcine GYS1 was highly expressed in the skeletal muscle and heart. GYS2 was expressed specifically in liver and subcutaneous adipose tissue. The expression level of GYS1 was up-regulated from proliferation to differentiation in the porcine satellite cells, and insulin did not significantly affect the transcription of GYS1. Insulin stimulated 72-h-differentiated satellite cells as indicated by decrease in phosphorylation of GS, but did not affect GYS1 transcription and total GS protein level, suggesting that the effect of insulin is primarily mediated via posttranscriptional control rather than regulated at the transcriptional level. Four single-nucleotide polymorphisms (SNPs) were detected in the promoter and cDNA sequences of porcine GYS1. Association analyses revealed that the GYS1 Hin6I and MvaI polymorphisms both had significant associations (P < 0.05) with pH of M. longissimus dorsi (pHLD), M. biceps femoris (pHBF) and M. semipinalis capitis (pHSC) at 45 min postmortem. These results provide useful information for further investigation on the function of glycogen synthase in porcine skeletal muscle.

  3. Insulin Receptor Substrate 2-mediated Phosphatidylinositol 3-kinase Signaling Selectively Inhibits Glycogen Synthase Kinase 3β to Regulate Aerobic Glycolysis*

    PubMed Central

    Landis, Justine; Shaw, Leslie M.

    2014-01-01

    Insulin receptor substrate 1 (IRS-1) and IRS-2 are cytoplasmic adaptor proteins that mediate the activation of signaling pathways in response to ligand stimulation of upstream cell surface receptors. Despite sharing a high level of homology and the ability to activate PI3K, only Irs-2 positively regulates aerobic glycolysis in mammary tumor cells. To determine the contribution of Irs-2-dependent PI3K signaling to this selective regulation, we generated an Irs-2 mutant deficient in the recruitment of PI3K. We identified four tyrosine residues (Tyr-649, Tyr-671, Tyr-734, and Tyr-814) that are essential for the association of PI3K with Irs-2 and demonstrate that combined mutation of these tyrosines inhibits glucose uptake and lactate production, two measures of aerobic glycolysis. Irs-2-dependent activation of PI3K regulates the phosphorylation of specific Akt substrates, most notably glycogen synthase kinase 3β (Gsk-3β). Inhibition of Gsk-3β by Irs-2-dependent PI3K signaling promotes glucose uptake and aerobic glycolysis. The regulation of unique subsets of Akt substrates by Irs-1 and Irs-2 may explain their non-redundant roles in mammary tumor biology. Taken together, our study reveals a novel mechanism by which Irs-2 signaling preferentially regulates tumor cell metabolism and adds to our understanding of how this adaptor protein contributes to breast cancer progression. PMID:24811175

  4. 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.

  5. Characterization of Function of the GlgA2 Glycogen/Starch Synthase in Cyanobacterium sp. Clg1 Highlights Convergent Evolution of Glycogen Metabolism into Starch Granule Aggregation.

    PubMed

    Kadouche, Derifa; Ducatez, Mathieu; Cenci, Ugo; Tirtiaux, Catherine; Suzuki, Eiji; Nakamura, Yasunori; Putaux, Jean-Luc; Terrasson, Amandine Durand; Diaz-Troya, Sandra; Florencio, Francisco Javier; Arias, Maria Cecilia; Striebeck, Alexander; Palcic, Monica; Ball, Steven G; Colleoni, Christophe

    2016-07-01

    At variance with the starch-accumulating plants and most of the glycogen-accumulating cyanobacteria, Cyanobacterium sp. CLg1 synthesizes both glycogen and starch. We now report the selection of a starchless mutant of this cyanobacterium that retains wild-type amounts of glycogen. Unlike other mutants of this type found in plants and cyanobacteria, this mutant proved to be selectively defective for one of the two types of glycogen/starch synthase: GlgA2. This enzyme is phylogenetically related to the previously reported SSIII/SSIV starch synthase that is thought to be involved in starch granule seeding in plants. This suggests that, in addition to the selective polysaccharide debranching demonstrated to be responsible for starch rather than glycogen synthesis, the nature and properties of the elongation enzyme define a novel determinant of starch versus glycogen accumulation. We show that the phylogenies of GlgA2 and of 16S ribosomal RNA display significant congruence. This suggests that this enzyme evolved together with cyanobacteria when they diversified over 2 billion years ago. However, cyanobacteria can be ruled out as direct progenitors of the SSIII/SSIV ancestral gene found in Archaeplastida. Hence, both cyanobacteria and plants recruited similar enzymes independently to perform analogous tasks, further emphasizing the importance of convergent evolution in the appearance of starch from a preexisting glycogen metabolism network.

  6. Characterization of Function of the GlgA2 Glycogen/Starch Synthase in Cyanobacterium sp. Clg1 Highlights Convergent Evolution of Glycogen Metabolism into Starch Granule Aggregation.

    PubMed

    Kadouche, Derifa; Ducatez, Mathieu; Cenci, Ugo; Tirtiaux, Catherine; Suzuki, Eiji; Nakamura, Yasunori; Putaux, Jean-Luc; Terrasson, Amandine Durand; Diaz-Troya, Sandra; Florencio, Francisco Javier; Arias, Maria Cecilia; Striebeck, Alexander; Palcic, Monica; Ball, Steven G; Colleoni, Christophe

    2016-07-01

    At variance with the starch-accumulating plants and most of the glycogen-accumulating cyanobacteria, Cyanobacterium sp. CLg1 synthesizes both glycogen and starch. We now report the selection of a starchless mutant of this cyanobacterium that retains wild-type amounts of glycogen. Unlike other mutants of this type found in plants and cyanobacteria, this mutant proved to be selectively defective for one of the two types of glycogen/starch synthase: GlgA2. This enzyme is phylogenetically related to the previously reported SSIII/SSIV starch synthase that is thought to be involved in starch granule seeding in plants. This suggests that, in addition to the selective polysaccharide debranching demonstrated to be responsible for starch rather than glycogen synthesis, the nature and properties of the elongation enzyme define a novel determinant of starch versus glycogen accumulation. We show that the phylogenies of GlgA2 and of 16S ribosomal RNA display significant congruence. This suggests that this enzyme evolved together with cyanobacteria when they diversified over 2 billion years ago. However, cyanobacteria can be ruled out as direct progenitors of the SSIII/SSIV ancestral gene found in Archaeplastida. Hence, both cyanobacteria and plants recruited similar enzymes independently to perform analogous tasks, further emphasizing the importance of convergent evolution in the appearance of starch from a preexisting glycogen metabolism network. PMID:27208262

  7. Enantioselective synthesis of the novel chiral sulfoxide derivative as a glycogen synthase kinase 3beta inhibitor.

    PubMed

    Saitoh, Morihisa; Kunitomo, Jun; Kimura, Eiji; Yamano, Toru; Itoh, Fumio; Kori, Masakuni

    2010-09-01

    Glycogen synthase kinase 3beta (GSK-3beta) inhibitors are expected to be attractive therapeutic agents for the treatment of Alzheimer's disease (AD). Recently we discovered sulfoxides (S)-1 as a novel GSK-3beta inhibitor having in vivo efficacy. We investigated practical asymmetric preparation methods for the scale-up synthesis of (S)-1. The highly enantioselective synthesis of (S)-1 (94% ee) was achieved by titanium-mediated oxidation with D-(-)-diethyl tartrate on gram scale.

  8. 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.

  9. 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

  10. 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.

  11. Glycogen synthase kinase 3 regulates IL-1β mediated iNOS expression in hepatocytes by down-regulating c-Jun.

    PubMed

    Lakshmanan, Jaganathan; Zhang, Baochun; Nweze, Ikenna C; Du, Yibo; Harbrecht, Brian G

    2015-01-01

    Excessive nitric oxide from the inducible nitric oxide synthase (iNOS) increases shock-induced hepatic injury, hepatic dysfunction, inflammation, and mortality in animal models. Cytokines increase the expression of iNOS in hepatocytes, but the signaling mechanisms involved are not completely understood. We have previously demonstrated that Akt mediates the inhibitory effect of cAMP and insulin on cytokine-induced hepatocyte iNOS expression. We hypothesized that glycogen synthase kinase 3 (GSK3), a target of Akt phosphorylation, would regulate hepatocyte iNOS expression. In cultured rat hepatocytes, GSK3 inhibitors decreased IL-1β mediated nitric oxide (NO) production and iNOS protein expression, while the phosphatidylinositol 3-kinase (PI3K)/Akt pathway inhibitor LY294002 increased the cytokine-mediated NO production and iNOS expression. Over-expression of the constitutively active form of GSK3β enhanced IL-1β-mediated iNOS expression. GSK3 catalyzes the phosphorylation of c-Jun at the c-terminal Thr239 that facilitates c-Jun degradation. Inhibition of GSK3 with SB216763 and lithium chloride significantly reduced, whereas blocking PI3K/Akt increased phosphorylation of c-Jun at Thr239. The levels of total-c-Jun and c-Jun phosphorylated at Ser63 inversely correlated with c-Jun phosphorylated at Thr239, GSK3 activation and iNOS expression. Over-expression of a dominant negative c-Jun not only caused an increase in IL-1β-mediated iNOS promoter activity and iNOS protein expression but was also able to reverse the SB216763-mediated suppression of iNOS. These results demonstrate that GSK3, a downstream target of Akt, regulates IL-1β-stimulated iNOS expression in hepatocytes by directly phosphorylating c-Jun in an inhibitory manner. PMID:25160751

  12. 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

  13. 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.

  14. 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

  15. 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

  16. 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.

  17. 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.

  18. Glycogen Synthase Kinase 3: A Point of Integration in Alzheimer's Disease and a Therapeutic Target?

    PubMed Central

    Mondragón-Rodríguez, Siddhartha; Perry, George; Zhu, Xiongwei; Moreira, Paula I.; Williams, Sylvain

    2012-01-01

    Glycogen synthase kinase 3 (GSK3) has been implicated in neurological disorders; therefore, it is not surprising that there has been an increased focus towards developing therapies directed to this kinase. Unfortunately, these current therapies have not taken into consideration the physiological role of GSK3 in crucial events like synaptic plasticity. With this in mind we will discuss the relationship of synaptic plasticity with GSK3 and tau protein and their role as potential targets for the development of therapeutic strategies. Finally, we will provide perspectives in developing a cocktail therapy for Alzheimer's treatment. PMID:22779025

  19. 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

  20. Neurodegeneration and functional impairments associated with glycogen synthase accumulation in a mouse model of Lafora disease

    PubMed Central

    Valles-Ortega, Jordi; Duran, Jordi; Garcia-Rocha, Mar; Bosch, Carles; Saez, Isabel; Pujadas, Lluís; Serafin, Anna; Cañas, Xavier; Soriano, Eduardo; Delgado-García, José M; Gruart, Agnès; Guinovart, Joan J

    2011-01-01

    Lafora disease (LD) is caused by mutations in either the laforin or malin gene. The hallmark of the disease is the accumulation of polyglucosan inclusions called Lafora Bodies (LBs). Malin knockout (KO) mice present polyglucosan accumulations in several brain areas, as do patients of LD. These structures are abundant in the cerebellum and hippocampus. Here, we report a large increase in glycogen synthase (GS) in these mice, in which the enzyme accumulates in LBs. Our study focused on the hippocampus where, under physiological conditions, astrocytes and parvalbumin-positive (PV+) interneurons expressed GS and malin. Although LBs have been described only in neurons, we found this polyglucosan accumulation in the astrocytes of the KO mice. They also had LBs in the soma and some processes of PV+ interneurons. This phenomenon was accompanied by the progressive loss of these neuronal cells and, importantly, neurophysiological alterations potentially related to impairment of hippocampal function. Our results emphasize the relevance of the laforin–malin complex in the control of glycogen metabolism and highlight altered glycogen accumulation as a key contributor to neurodegeneration in LD. PMID:21882344

  1. Evidence of positive selection for a glycogen synthase (GYS1) mutation in domestic horse populations.

    PubMed

    McCoy, Annette M; Schaefer, Robert; Petersen, Jessica L; Morrell, Peter L; Slamka, Megan A; Mickelson, James R; Valberg, Stephanie J; McCue, Molly E

    2014-01-01

    A dominantly inherited gain-of-function mutation in the glycogen synthase (GYS1) gene, resulting in excess skeletal muscle glycogen, has been identified in more than 30 horse breeds. This mutation is associated with the disease Equine Polysaccharide Storage Myopathy Type 1, yet persists at high frequency in some breeds. Under historical conditions of daily work and limited feed, excess muscle glycogen may have been advantageous, driving the increase in frequency of this allele. Fine-scale DNA sequencing in 80 horses and genotype assays in 279 horses revealed a paucity of haplotypes carrying the mutant allele when compared with the wild-type allele. Additionally, we found increased linkage disequilibrium, measured by relative extended haplotype homozygosity, in haplotypes carrying the mutation compared with haplotypes carrying the wild-type allele. Coalescent simulations of Belgian horse populations demonstrated that the high frequency and extended haplotype associated with the GYS1 mutation were unlikely to have arisen under neutrality or due to population demography. In contrast, in Quarter Horses, elevated relative extended haplotype homozygosity was associated with multiple haplotypes and may be the result of recent population expansion or a popular sire effect. These data suggest that the GYS1 mutation underwent historical selection in the Belgian, but not in the Quarter Horse.

  2. The Crystal Structures of the Open and Catalytically Competent Closed Conformation of Escherichia coli Glycogen Synthase

    SciTech Connect

    Sheng, Fang; Jia, Xiaofei; Yep, Alejandra; Preiss, Jack; Geiger, James H.

    2009-07-06

    Escherichia coli glycogen synthase (EcGS, EC 2.4.1.21) is a retaining glycosyltransferase (GT) that transfers glucose from adenosine diphosphate glucose to a glucan chain acceptor with retention of configuration at the anomeric carbon. EcGS belongs to the GT-B structural superfamily. Here we report several EcGS x-ray structures that together shed considerable light on the structure and function of these enzymes. The structure of the wild-type enzyme bound to ADP and glucose revealed a 15.2 degrees overall domain-domain closure and provided for the first time the structure of the catalytically active, closed conformation of a glycogen synthase. The main chain carbonyl group of His-161, Arg-300, and Lys-305 are suggested by the structure to act as critical catalytic residues in the transglycosylation. Glu-377, previously thought to be catalytic is found on the alpha-face of the glucose and plays an electrostatic role in the active site and as a glucose ring locator. This is also consistent with the structure of the EcGS(E377A)-ADP-HEPPSO complex where the glucose moiety is either absent or disordered in the active site

  3. 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.

  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. 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…

  6. Critical role of glycogen synthase kinase-3ß in regulating the avian heterophil response to Salmonella enterica serovar Enteritidis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A microarray-assisted gene expression screen of chicken heterophils revealed glycogen synthase kinase-3ß (GSK-3ß), a multifunctional Ser/Thr kinase, to be consistently up-regulated 30-180 min following stimulation with Salmonella enterica serovar Enteritidis (S. Enteritidis). The present study was ...

  7. In silico deconstruction of ATP-competitive inhibitors of glycogen synthase kinase-3β.

    PubMed

    Bisignano, Paola; Lambruschini, Chiara; Bicego, Manuele; Murino, Vittorio; Favia, Angelo D; Cavalli, Andrea

    2012-12-21

    Fragment-based methods have emerged in the last two decades as alternatives to traditional high throughput screenings for the identification of chemical starting points in drug discovery. One arguable yet popular assumption about fragment-based design is that the fragment binding mode remains conserved upon chemical expansion. For instance, the question of the binding conservation upon fragmentation of a molecule is still unclear. A number of papers have challenged this hypothesis by means of experimental techniques, with controversial results, "underlining" the idea that a simple generalization, maybe, is not possible. From a computational standpoint, the issue has been rarely addressed and mostly to test novel protocols on limited data sets. To fill this gap, we here report on a computational retrospective study concerned with the in silico deconstruction of leadlike compounds, active on the pharmaceutically relevant enzyme glycogen synthase kinase-3β.

  8. Evaluation of Improved Glycogen Synthase Kinase-3α Inhibitors in Models of Acute Myeloid Leukemia

    PubMed Central

    Neumann, Theresa; Benajiba, Lina; Göring, Stefan; Stegmaier, Kimberly; Schmidt, Boris

    2016-01-01

    The challenge for Glycogen Synthase Kinase-3 (GSK-3) inhibitor design lies in achieving high selectivity for one isoform over the other. The therapy of certain diseases, such as acute myeloid leukemia (AML) may require α-isoform specific targeting. The scorpion shaped GSK-3 inhibitors developed by our group achieved the highest GSK-3α selectivity reported so far, but suffered from insufficient aqueous solubility. This work presents the solubility-driven optimization of our isoform-selective inhibitors using a scorpion shaped lead. Among 15 novel compounds, compound 27 showed high activity against GSK-3α/β with the highest GSK-3α selectivity reported to date. Compound 27 was profiled for bioavailability and toxicity in a zebrafish embryo phenotype assay. Selective GSK-3α targeting in AML cell lines was achieved with compound 27, resulting in a strong differentiation phenotype and colony formation impairment, confirming the potential of GSK-3α inhibition in AML therapy. PMID:26496242

  9. Glycogen Synthase Kinase 3 Inhibitors in the Next Horizon for Alzheimer's Disease Treatment

    PubMed Central

    Martinez, Ana; Gil, Carmen; Perez, Daniel I.

    2011-01-01

    Glycogen synthase kinase 3 (GSK-3), a proline/serine protein kinase ubiquitously expressed and involved in many cellular signaling pathways, plays a key role in the pathogenesis of Alzheimer's disease (AD) being probably the link between β-amyloid and tau pathology. A great effort has recently been done in the discovery and development of different new molecules, of synthetic and natural origin, able to inhibit this enzyme, and several kinetics mechanisms of binding have been described. The small molecule called tideglusib belonging to the thiadiazolidindione family is currently on phase IIb clinical trials for AD. The potential risks and benefits of this new kind of disease modifying drugs for the future therapy of AD are discussed in this paper. PMID:21760986

  10. Glycogen synthase kinase 3 inhibitors in the next horizon for Alzheimer's disease treatment.

    PubMed

    Martinez, Ana; Gil, Carmen; Perez, Daniel I

    2011-01-01

    Glycogen synthase kinase 3 (GSK-3), a proline/serine protein kinase ubiquitously expressed and involved in many cellular signaling pathways, plays a key role in the pathogenesis of Alzheimer's disease (AD) being probably the link between β-amyloid and tau pathology. A great effort has recently been done in the discovery and development of different new molecules, of synthetic and natural origin, able to inhibit this enzyme, and several kinetics mechanisms of binding have been described. The small molecule called tideglusib belonging to the thiadiazolidindione family is currently on phase IIb clinical trials for AD. The potential risks and benefits of this new kind of disease modifying drugs for the future therapy of AD are discussed in this paper.

  11. Glycogen synthase kinase 3 phosphorylates kinesin light chains and negatively regulates kinesin-based motility

    NASA Technical Reports Server (NTRS)

    Morfini, Gerardo; Szebenyi, Gyorgyi; Elluru, Ravindhra; Ratner, Nancy; Brady, Scott T.

    2002-01-01

    Membrane-bounded organelles (MBOs) are delivered to different domains in neurons by fast axonal transport. The importance of kinesin for fast antero grade transport is well established, but mechanisms for regulating kinesin-based motility are largely unknown. In this report, we provide biochemical and in vivo evidence that kinesin light chains (KLCs) interact with and are in vivo substrates for glycogen synthase kinase 3 (GSK3). Active GSK3 inhibited anterograde, but not retrograde, transport in squid axoplasm and reduced the amount of kinesin bound to MBOs. Kinesin microtubule binding and microtubule-stimulated ATPase activities were unaffected by GSK3 phosphorylation of KLCs. Active GSK3 was also localized preferentially to regions known to be sites of membrane delivery. These data suggest that GSK3 can regulate fast anterograde axonal transport and targeting of cargos to specific subcellular domains in neurons.

  12. 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

  13. Calcineurin B homologous protein 3 negatively regulates cardiomyocyte hypertrophy via inhibition of glycogen synthase kinase 3 phosphorylation.

    PubMed

    Kobayashi, Soushi; Nakamura, Tomoe Y; Wakabayashi, Shigeo

    2015-07-01

    Cardiac hypertrophy is a leading cause of serious heart diseases. Although many signaling molecules are involved in hypertrophy, the functions of some proteins in this process are still unknown. Calcineurin B homologous protein 3 (CHP3)/tescalcin is an EF-hand Ca(2+)-binding protein that is abundantly expressed in the heart; however, the function of CHP3 is unclear. Here, we aimed to identify the cardiac functions of CHP3. CHP3 was expressed in hearts at a wide range of developmental stages and was specifically detected in neonatal rat ventricular myocytes (NRVMs) but not in cardiac fibroblasts in culture. Moreover, knockdown of CHP3 expression using adenoviral-based RNA interference in NRVMs resulted in enlargement of cardiomyocyte size, concomitant with increased expression of a pathological hypertrophy marker ANP. This same treatment elevated glycogen synthase kinase (GSK3α/β) phosphorylation, which is known to inhibit GSK3 function. In contrast, CHP3 overexpression blocked the insulin-induced phosphorylation of GSK3α/β without affecting the phosphorylation of Akt, which is an upstream kinase of GSK3α/β, in HEK293 cells, and it inhibited both IGF-1-induced phosphorylation of GSK3β and cardiomyocyte hypertrophy in NRVMs. Co-immunoprecipitation experiments revealed that GSK3β interacted with CHP3. However, a Ca(2+)-binding-defective mutation of CHP3 (CHP3-D123A) also interacted with GSK3β and had the same inhibitory effect on GSK3α/β phosphorylation, suggesting that the action of CHP3 was independent of Ca(2+). These findings suggest that CHP3 functions as a novel negative regulator of cardiomyocyte hypertrophy via inhibition of GSK3α/β phosphorylation and subsequent enzymatic activation of GSK3α/β. PMID:25935310

  14. Calcineurin B homologous protein 3 negatively regulates cardiomyocyte hypertrophy via inhibition of glycogen synthase kinase 3 phosphorylation.

    PubMed

    Kobayashi, Soushi; Nakamura, Tomoe Y; Wakabayashi, Shigeo

    2015-07-01

    Cardiac hypertrophy is a leading cause of serious heart diseases. Although many signaling molecules are involved in hypertrophy, the functions of some proteins in this process are still unknown. Calcineurin B homologous protein 3 (CHP3)/tescalcin is an EF-hand Ca(2+)-binding protein that is abundantly expressed in the heart; however, the function of CHP3 is unclear. Here, we aimed to identify the cardiac functions of CHP3. CHP3 was expressed in hearts at a wide range of developmental stages and was specifically detected in neonatal rat ventricular myocytes (NRVMs) but not in cardiac fibroblasts in culture. Moreover, knockdown of CHP3 expression using adenoviral-based RNA interference in NRVMs resulted in enlargement of cardiomyocyte size, concomitant with increased expression of a pathological hypertrophy marker ANP. This same treatment elevated glycogen synthase kinase (GSK3α/β) phosphorylation, which is known to inhibit GSK3 function. In contrast, CHP3 overexpression blocked the insulin-induced phosphorylation of GSK3α/β without affecting the phosphorylation of Akt, which is an upstream kinase of GSK3α/β, in HEK293 cells, and it inhibited both IGF-1-induced phosphorylation of GSK3β and cardiomyocyte hypertrophy in NRVMs. Co-immunoprecipitation experiments revealed that GSK3β interacted with CHP3. However, a Ca(2+)-binding-defective mutation of CHP3 (CHP3-D123A) also interacted with GSK3β and had the same inhibitory effect on GSK3α/β phosphorylation, suggesting that the action of CHP3 was independent of Ca(2+). These findings suggest that CHP3 functions as a novel negative regulator of cardiomyocyte hypertrophy via inhibition of GSK3α/β phosphorylation and subsequent enzymatic activation of GSK3α/β.

  15. Inactivation of Glycogen Synthase Kinase-3β Is Required for Osteoclast Differentiation*

    PubMed Central

    Jang, Hyun Duk; Shin, Ji Hye; Park, Doo Ri; Hong, Jin Hee; Yoon, Kwiyeom; Ko, Ryeojin; Ko, Chang-Yong; Kim, Han-Sung; Jeong, Daewon; Kim, Nacksung; Lee, Soo Young

    2011-01-01

    Glycogen synthase kinase-3β (GSK-3β) is a serine/threonine kinase originally identified as a regulator of glycogen deposition. Although the role of GSK-3β in osteoblasts is well characterized as a negative regulator of β-catenin, its effect on osteoclast formation remains largely unidentified. Here, we show that the GSK-3β inactivation upon receptor activator of NF-κB ligand (RANKL) stimulation is crucial for osteoclast differentiation. Regulation of GSK-3β activity in bone marrow macrophages by retroviral expression of the constitutively active GSK-3β (GSK3β-S9A) mutant inhibits RANKL-induced osteoclastogenesis, whereas expression of the catalytically inactive GSK-3β (GSK3β-K85R) or small interfering RNA (siRNA)-mediated GSK-3β silencing enhances osteoclast formation. Pharmacological inhibition of GSK-3β further confirmed the negative role of GSK-3β in osteoclast formation. We also show that overexpression of the GSK3β-S9A mutant in bone marrow macrophages inhibits RANKL-mediated NFATc1 induction and Ca2+ oscillations. Remarkably, transgenic mice expressing the GSK3β-S9A mutant show an osteopetrotic phenotype due to impaired osteoclast differentiation. Further, osteoclast precursor cells from the transgenic mice show defects in expression and nuclear localization of NFATc1. These findings demonstrate a novel role for GSK-3β in the regulation of bone remodeling through modulation of NFATc1 in RANKL signaling. PMID:21949120

  16. Glycogen Synthase Kinase 3 Inhibition Promotes Adult Hippocampal Neurogenesis in Vitro and in Vivo

    PubMed Central

    2012-01-01

    Glycogen synthase kinase-3 (GSK-3) is a serine/threonine kinase originally identified as a regulator of glycogen metabolism but it also plays a pivotal role in numerous cellular functions, including differentiation, cell cycle regulation, and proliferation. The dentate gyrus of the hippocampus, together with the subventricular zone of the lateral ventricles, is one of the regions in which neurogenesis takes place in the adult brain. Here, using a chemical genetic approach that involves the use of several diverse inhibitors of GSK-3 as pharmacological tools, we show that inhibition of GSK-3 induces proliferation, migration, and differentiation of neural stem cells toward a neuronal phenotype in in vitro studies. Also, we demonstrate that inhibition of GSK-3 with the small molecule NP03112, called tideglusib, induces neurogenesis in the dentate gyrus of the hippocampus of adult rats. Taken together, our results suggest that GSK-3 should be considered as a new target molecule for modulating the production and integration of new neurons in the hippocampus as a treatment for neurodegenerative diseases or brain injury and, consequently, its inhibitors may represent new potential therapeutic drugs in neuroregenerative medicine. PMID:23173075

  17. Glycogen synthase kinase 3 inhibition promotes adult hippocampal neurogenesis in vitro and in vivo.

    PubMed

    Morales-Garcia, Jose A; Luna-Medina, Rosario; Alonso-Gil, Sandra; Sanz-Sancristobal, Marina; Palomo, Valle; Gil, Carmen; Santos, Angel; Martinez, Ana; Perez-Castillo, Ana

    2012-11-21

    Glycogen synthase kinase-3 (GSK-3) is a serine/threonine kinase originally identified as a regulator of glycogen metabolism but it also plays a pivotal role in numerous cellular functions, including differentiation, cell cycle regulation, and proliferation. The dentate gyrus of the hippocampus, together with the subventricular zone of the lateral ventricles, is one of the regions in which neurogenesis takes place in the adult brain. Here, using a chemical genetic approach that involves the use of several diverse inhibitors of GSK-3 as pharmacological tools, we show that inhibition of GSK-3 induces proliferation, migration, and differentiation of neural stem cells toward a neuronal phenotype in in vitro studies. Also, we demonstrate that inhibition of GSK-3 with the small molecule NP03112, called tideglusib, induces neurogenesis in the dentate gyrus of the hippocampus of adult rats. Taken together, our results suggest that GSK-3 should be considered as a new target molecule for modulating the production and integration of new neurons in the hippocampus as a treatment for neurodegenerative diseases or brain injury and, consequently, its inhibitors may represent new potential therapeutic drugs in neuroregenerative medicine.

  18. Expression and purification of functional human glycogen synthase-1:glycogenin-1 complex in insect cells.

    PubMed

    Hunter, Roger W; Zeqiraj, Elton; Morrice, Nicholas; Sicheri, Frank; Sakamoto, Kei

    2015-04-01

    We report the successful expression and purification of functional human muscle glycogen synthase (GYS1) in complex with human glycogenin-1 (GN1). Stoichiometric GYS1:GN1 complex was produced by co-expression of GYS1 and GN1 using a bicistronic pFastBac™-Dual expression vector, followed by affinity purification and subsequent size-exclusion chromatography. Mass spectrometry analysis identified that GYS1 is phosphorylated at several well-characterised and uncharacterised Ser/Thr residues. Biochemical analysis, including activity ratio (in the absence relative to that in the presence of glucose-6-phosphate) measurement, covalently attached phosphate estimation as well as phosphatase treatment, revealed that recombinant GYS1 is substantially more heavily phosphorylated than would be observed in intact human or rodent muscle tissues. A large quantity of highly-pure stoichiometric GYS1:GN1 complex will be useful to study its structural and biochemical properties in the future, which would reveal mechanistic insights into its functional role in glycogen biosynthesis.

  19. Glycogen synthase kinase-3 (GSK3) inhibition induces prosurvival autophagic signals in human pancreatic cancer cells.

    PubMed

    Marchand, Benoît; Arsenault, Dominique; Raymond-Fleury, Alexandre; Boisvert, François-Michel; Boucher, Marie-Josée

    2015-02-27

    Glycogen synthase kinase-3 (GSK3) are ubiquitously expressed serine-threonine kinases involved in a plethora of functions ranging from the control of glycogen metabolism to transcriptional regulation. We recently demonstrated that GSK3 inhibition triggers JNK-cJUN-dependent apoptosis in human pancreatic cancer cells. However, the comprehensive picture of downstream GSK3-regulated pathways/functions remains elusive. Herein, counterbalancing the death signals, we show that GSK3 inhibition induces prosurvival signals through increased activity of the autophagy/lysosomal network. Our data also reveal a contribution of GSK3 in the regulation of the master transcriptional regulator of autophagy and lysosomal biogenesis, transcription factor EB (TFEB) in pancreatic cancer cells. Similarly to mammalian target of rapamycin (mTOR) inhibition, GSK3 inhibitors promote TFEB nuclear localization and leads to TFEB dephosphorylation through endogenous serine/threonine phosphatase action. However, GSK3 and mTOR inhibition impinge differently and independently on TFEB phosphorylation suggesting that TFEB is regulated by a panel of kinases and/or phosphatases. Despite their differential impact on TFEB phosphorylation, both GSK3 and mTOR inhibitors promote 14-3-3 dissociation and TFEB nuclear localization. Quantitative mass spectrometry analyses further reveal an increased association of TFEB with nuclear proteins upon GSK3 and mTOR inhibition suggesting a positive impact on TFEB transcriptional function. Finally, a predominant nuclear localization of TFEB is unveiled in fully fed pancreatic cancer cells, whereas a reduction in TFEB expression significantly impairs their capacity for growth in an anchorage-independent manner. In addition, TFEB-restricted cells are more sensitive to apoptosis upon GSK3 inhibition. Altogether, our data uncover new functions under the control of GSK3 in pancreatic cancer cells in addition to providing key insight into TFEB regulation.

  20. 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.

  1. Glycogen synthase kinase-3 facilitates con a-induced IFN-γ-- mediated immune hepatic injury.

    PubMed

    Tsai, Cheng-Chieh; Huang, Wei-Ching; Chen, Chia-Ling; Hsieh, Chia-Yuan; Lin, Yee-Shin; Chen, Shun-Hua; Yang, Kao-Chi; Lin, Chiou-Feng

    2011-10-01

    Immune hepatic injury induced by Con A results primarily from IFN-γ-mediated inflammation, followed by hepatic cell death. Glycogen synthase kinase (GSK)-3, which acts proapoptotically and is proinflammatory, is also important for facilitating IFN-γ signaling. We hypothesized a pathogenic role for GSK-3 in Con A hepatic injury. Con A stimulation caused GSK-3 activation in the livers of C57BL/6 mice. Inhibiting GSK-3 reduced Con A hepatic injury, including hepatic necrosis and apoptosis, inflammation, infiltration of T cells and granulocytes, and deregulated expression of adhesion molecule CD54. Con A induced hepatic injury in an IFN-γ receptor 1-dependent manner. Con A/IFN-γ induced activation and expression of STAT1 in a GSK-3-dependent manner. GSK-3 facilitated IFN-γ-induced inducible NO synthase, but had limited effects on CD95 upregulation and CD95-mediated hepatocyte apoptosis in vitro. Notably, inhibiting GSK-3 decreased Con A-induced IFN-γ production in both wild-type and IFN-γ receptor 1-deficient C57BL/6 mice. In Con A-activated NKT cells, GSK-3 was also activated and was required for nuclear translocation of T-box transcription factor Tbx21, a transcription factor of IFN-γ, but it was not required for CD95 ligand expression or activation-induced cell death. These results demonstrate the dual and indispensable role of GSK-3 in Con A hepatic injury by facilitating IFN-γ-induced hepatopathy.

  2. Impaired glucose metabolism and exercise capacity with muscle-specific glycogen synthase 1 (gys1) deletion in adult mice

    PubMed Central

    Xirouchaki, Chrysovalantou E.; Mangiafico, Salvatore P.; Bate, Katherine; Ruan, Zheng; Huang, Amy M.; Tedjosiswoyo, Bing Wilari; Lamont, Benjamin; Pong, Wynne; Favaloro, Jenny; Blair, Amy R.; Zajac, Jeffrey D.; Proietto, Joseph; Andrikopoulos, Sofianos

    2016-01-01

    Objective Muscle glucose storage and muscle glycogen synthase (gys1) defects have been associated with insulin resistance. As there are multiple mechanisms for insulin resistance, the specific role of glucose storage defects is not clear. The aim of this study was to examine the effects of muscle-specific gys1 deletion on glucose metabolism and exercise capacity. Methods Tamoxifen inducible and muscle specific gys-1 KO mice were generated using the Cre/loxP system. Mice were subjected to glucose tolerance tests, euglycemic/hyperinsulinemic clamps and exercise tests. Results gys1-KO mice showed ≥85% reduction in muscle gys1 mRNA and protein concentrations, 70% reduction in muscle glycogen levels, postprandial hyperglycaemia and hyperinsulinaemia and impaired glucose tolerance. Under insulin-stimulated conditions, gys1-KO mice displayed reduced glucose turnover and muscle glucose uptake, indicative of peripheral insulin resistance, as well as increased plasma and muscle lactate levels and reductions in muscle hexokinase II levels. gys1-KO mice also exhibited markedly reduced exercise and endurance capacity. Conclusions Thus, muscle-specific gys1 deletion in adult mice results in glucose intolerance due to insulin resistance and reduced muscle glucose uptake as well as impaired exercise and endurance capacity. In brief This study demonstrates why the body prioritises muscle glycogen storage over liver glycogen storage despite the critical role of the liver in supplying glucose to the brain in the fasting state and shows that glycogen deficiency results in impaired glucose metabolism and reduced exercise capacity. PMID:26977394

  3. 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

  4. Effect of glycogen synthase kinase-3 inactivation on mouse mammary gland development and oncogenesis

    PubMed Central

    Dembowy, J; Adissu, H A; Liu, J C; Zacksenhaus, E; Woodgett, J R

    2015-01-01

    Many components of the Wnt/β-catenin signaling pathway have critical functions in mammary gland development and tumor formation, yet the contribution of glycogen synthase kinase-3 (GSK-3α and GSK-3β) to mammopoiesis and oncogenesis is unclear. Here, we report that WAP-Cre-mediated deletion of GSK-3 in the mammary epithelium results in activation of Wnt/β-catenin signaling and induces mammary intraepithelial neoplasia that progresses to squamous transdifferentiation and development of adenosquamous carcinomas at 6 months. To uncover possible β-catenin-independent activities of GSK-3, we generated mammary-specific knockouts of GSK-3 and β-catenin. Squamous transdifferentiation of the mammary epithelium was largely attenuated, however, mammary epithelial cells lost the ability to form mammospheres suggesting perturbation of stem cell properties unrelated to loss of β-catenin alone. At 10 months, adenocarcinomas that developed in glands lacking GSK-3 and β-catenin displayed elevated levels of γ-catenin/plakoglobin as well as activation of the Hedgehog and Notch pathways. Collectively, these results establish the two isoforms of GSK-3 as essential integrators of multiple developmental signals that act to maintain normal mammary gland function and suppress tumorigenesis. PMID:25195860

  5. Glycogen Synthase Kinase-3β Is a Negative Regulator of Cardiomyocyte Hypertrophy

    PubMed Central

    Haq, Syed; Choukroun, Gabriel; Kang, Zhao Bin; Ranu, Hardeep; Matsui, Takashi; Rosenzweig, Anthony; Molkentin, Jeffrey D.; Alessandrini, Alessandro; Woodgett, James; Hajjar, Roger; Michael, Ashour; Force, Thomas

    2000-01-01

    Hypertrophy is a basic cellular response to a variety of stressors and growth factors, and has been best characterized in myocytes. Pathologic hypertrophy of cardiac myocytes leads to heart failure, a major cause of death and disability in the developed world. Several cytosolic signaling pathways have been identified that transduce prohypertrophic signals, but to date, little work has focused on signaling pathways that might negatively regulate hypertrophy. Herein, we report that glycogen synthase kinase-3β (GSK-3β), a protein kinase previously implicated in processes as diverse as development and tumorigenesis, is inactivated by hypertrophic stimuli via a phosphoinositide 3-kinase–dependent protein kinase that phosphorylates GSK-3β on ser 9. Using adenovirus-mediated gene transfer of GSK-3β containing a ser 9 to alanine mutation, which prevents inactivation by hypertrophic stimuli, we demonstrate that inactivation of GSK-3β is required for cardiomyocytes to undergo hypertrophy. Furthermore, our data suggest that GSK-3β regulates the hypertrophic response, at least in part, by modulating the nuclear/cytoplasmic partitioning of a member of the nuclear factor of activated T cells family of transcription factors. The identification of GSK-3β as a transducer of antihypertrophic signals suggests that novel therapeutic strategies to treat hypertrophic diseases of the heart could be designed that target components of the GSK-3 pathway. PMID:11018058

  6. Glycogen Synthase Kinase-3 Inhibitors as Potent Therapeutic Agents for the Treatment of Parkinson Disease.

    PubMed Central

    2012-01-01

    Parkinson's disease (PD) is a devastating neurodegenerative disorder characterized by degeneration of the nigrostriatal dopaminergic pathway. Because the current therapies only lead to temporary, limited improvement and have severe side effects, new approaches to treat PD need to be developed. To discover new targets for potential therapeutic intervention, a chemical genetic approach involving the use of small molecules as pharmacological tools has been implemented. First, a screening of an in-house chemical library on a well-established cellular model of PD was done followed by a detailed pharmacological analysis of the hits. Here, we report the results found for the small heterocyclic derivative called SC001, which after different enzymatic assays was revealed to be a new glycogen synthase kinase-3 (GSK-3) inhibitor with IC50 = 3.38 ± 0.08 μM. To confirm that GSK-3 could be a good target for PD, the evaluation of a set of structurally diverse GSK-3 inhibitors as neuroprotective agents for PD was performed. Results show that inhibitors of GSK-3 have neuroprotective effects in vitro representing a new pharmacological option for the disease-modifying treatment of PD. Furthermore, we show that SC001 is able to cross the blood–brain barrier, protects dopaminergic neurons, and reduces microglia activation in in vivo models of Parkinson disease, being a good candidate for further drug development. PMID:23421686

  7. Crosstalk between Phosphodiesterase 7 and Glycogen Synthase Kinase-3: Two Relevant Therapeutic Targets for Neurological Disorders

    PubMed Central

    2014-01-01

    Chronic neuroinflammation has been increasingly recognized as a primary mechanism underlying acute brain injury and neurodegenerative diseases. Enhanced expression of diverse pro-inflammatory agents in glial cells has been shown to contribute to the cell death that takes place in these disorders. Previous data from our group have shown that different inhibitors of the cyclic adenosine monophosphate (cAMP) specific phosphodiesterase 7 (PDE7) and glycogen synthase kinase-3 (GSK-3) enzymes are potent anti-inflammatory agents in different models of brain injury. In this study, we investigated cross-talk between PDE7 and GSK-3, two relevant therapeutic targets for neurological disorders, using a chemical approach. To this end, we compared specific inhibitors of GSK-3 and PDE7 with dual inhibitors of both enzymes with regard to anti-inflammatory effects in primary cultures of glial cells treated with lipopolysaccharide. Our results show that the GSK-3 inhibitors act exclusively by inhibition of this enzyme. By contrast, PDE7 inhibitors exert their effects via inhibition of PDE7 to increase intracellular cAMP levels but also through indirect inhibition of GSK-3. Activation of protein kinase A by cAMP results in phosphorylation of Ser9 of GSK-3 and subsequent inhibition. Our results indicate that the indirect inhibition of GSK-3 by PDE7 inhibitors is an important mechanism that should be considered in the future development of pharmacological treatments. PMID:24437940

  8. Glycogen Synthase Kinase-3: A Potential Preventive Target for Prostate Cancer Management

    PubMed Central

    Li, Benyi; Thrasher, J. Brantley; Terranova, Paul

    2016-01-01

    Prostate cancers are the frequently diagnosed cancers in men and patients with metastatic disease only have 28% chance for 5-year survival. Patients with low risk tumors are subjected to active surveillance while high risk cases are actively treated. Unfortunately, there is no cure for late-stage patients. Glycogen synthase kinase-3 (GSK-3, α and β) is a protein serine/threonine kinase and has diverse cellular functions and numerous substrates. Accumulating evidence indicates that GSK-3α is mainly expressed in low-risk prostate cancers and is related to hormone-dependent androgen receptor (AR)-mediated gene expression, while GSK-3β is mainly expressed in high-risk prostate cancers and is related to hormone-independent AR-mediated gene expression. GSK-3 has been demonstrated as a positive regulator in AR transactivation and prostate cancer growth independent of the Wnt/β-catenin pathway. Different types of GSK-3 inhibitors including lithium show promising results in suppressing tumor growth in different animal models of prostate cancer. Importantly, clinical use of lithium is associated with reduced cancer incidence in psychiatric patients. Taken together, GSK-3 inhibition might be implicated in prostate cancer management as a preventive treatment. PMID:26051358

  9. Glycogen Synthase Kinase-3: A Promising Therapeutic Target for Fragile X Syndrome

    PubMed Central

    Mines, Marjelo A.; Jope, Richard S.

    2011-01-01

    Recent advances in understanding the pathophysiological mechanisms contributing to fragile X syndrome (FXS) have increased optimism that drug interventions can provide significant therapeutic benefits. FXS results from inadequate expression of functional fragile X mental retardation protein (FMRP). FMRP may have several functions, but it is most well-established as an RNA binding protein that regulates translation, and it is thought that by this mechanism FMRP is capable of affecting numerous cellular processes by selectively regulating protein levels. The multiple cellular functions regulated by FMRP suggest that multiple interventions may be required for reversing the effects of deficient FMRP. Evidence that inhibitors of glycogen synthase kinase-3 (GSK3) may contribute to the therapeutic treatment of FXS is reviewed here. Lithium, a GSK3 inhibitor, improved function in the Drosophila model of FXS. In mice lacking FMRP expression (FX mice), GSK3 is hyperactive in several brain regions. Significant improvements in several FX-related phenotypes have been obtained in FX mice following the administration of lithium, and in some case other GSK3 inhibitors. These responses include normalization of heightened audiogenic seizure susceptibility and of hyperactive locomotor behavior, enhancement of passive avoidance learning retention and of sociability behaviors, and corrections of macroorchidism, neuronal spine density, and neural plasticity measured electrophysiologically as long term depression. A pilot clinical trial of lithium in patients with FXS also found improvements in several measures of behavior. Taken together, these findings indicate that lithium and other inhibitors of GSK3 are promising candidate therapeutic agents for treating FXS. PMID:22053151

  10. The Role of Glycogen Synthase Kinase 3 Beta in Neuroinflammation and Pain

    PubMed Central

    Maixner, Dylan Warren; Weng, Han-Rong

    2013-01-01

    Neuroinflammation is a crucial mechanism related to many neurological diseases. Extensive studies in recent years have indicated that dysregulation of Glycogen Synthase Kinase 3 Beta (GSK3β) contributes to the development and progression of these disorders through regulating the neuroinflammation processes. Inhibitors of GSK3β have been shown to be beneficial in many neuroinflammatory disease models including Alzheimer's disease, multiple sclerosis and AIDS dem entia complex. Glial activation and elevated pro-inflammation cytokines (signs of neuroinflammation) in the spinal cord have been widely recognized as a pivotal mechanism underlying the development and maintenance of many types of pathological pain. The role of GSK3β in the pathogenesis of pain has recently emerged. In this review, we will first review the GSK3β structure, regulation, and mechanisms by which GSK3βregulates inflammation. We will then describe neuroinflammationin general and in specific types of neurological diseases and the potential beneficial effects induced by inhibiting GSK3β. Finally, we will provide new evidence linking aberrant levels of GSK3β in the development of pathological pain. PMID:25309941

  11. Glycogen Synthase Kinase-3 is an Intermediate Modulator of Serotonin Neurotransmission

    PubMed Central

    Polter, Abigail M.; Li, Xiaohua

    2011-01-01

    Serotonin is a neurotransmitter with broad functions in brain development, neuronal activity, and behaviors; and serotonin is the prominent drug target in several major neuropsychiatric diseases. The multiple actions of serotonin are mediated by diverse serotonin receptor subtypes and associated signaling pathways. However, the key signaling components that mediate specific function of serotonin neurotransmission have not been fully identified. This review will provide evidence from biochemical, pharmacological, and animal behavioral studies showing that serotonin regulates the activation states of brain glycogen synthase kinase-3 (GSK3) via type 1 and type 2 serotonin receptors. In return, GSK3 directly interacts with serotonin receptors in a highly selective manner, with a prominent effect on modulating serotonin 1B receptor activity. Therefore, GSK3 acts as an intermediate modulator in the serotonin neurotransmission system, and balanced GSK3 activity is essential for serotonin-regulated brain function and behaviors. Particularly important, several classes of serotonin-modulating drugs, such as antidepressants and atypical antipsychotics, regulate GSK3 by inhibiting its activity in brain, which reinforces the importance of GSK3 as a potential therapeutic target in neuropsychiatric diseases associated with abnormal serotonin function. PMID:22028682

  12. Phosphorylation of insulin receptor substrate 1 by glycogen synthase kinase 3 impairs insulin action

    PubMed Central

    Eldar-Finkelman, Hagit; Krebs, Edwin G.

    1997-01-01

    The phosphorylation of insulin receptor substrate 1 (IRS-1) on tyrosine residues by the insulin receptor (IR) tyrosine kinase is involved in most of the biological responses of insulin. IRS-1 mediates insulin signaling by recruiting SH2 proteins through its multiple tyrosine phosphorylation sites. The phosphorylation of IRS-1 on serine/threonine residues also occurs in cells; however, the particular protein kinase(s) promoting this type of phosphorylation are unknown. Here we report that glycogen synthase kinase 3 (GSK-3) is capable of phosphorylating IRS-1 and that this modification converts IRS-1 into an inhibitor of IR tyrosine kinase activity in vitro. Expression of wild-type GSK-3 or an “unregulated” mutant of the kinase (S9A) in CHO cells overexpressing IRS-1 and IR, resulted in increased serine phosphorylation levels of IRS-1, suggesting that IRS-1 is a cellular target of GSK-3. Furthermore, insulin-induced tyrosine phosphorylation of IRS-1 and IR was markedly suppressed in cells expressing wild-type or the S9A mutant, indicating that expression of GSK-3 impairs IR tyrosine kinase activity. Taken together, our studies suggest a new role for GSK-3 in attenuating insulin signaling via its phosphorylation of IRS-1 and may provide new insight into mechanisms important in insulin resistance. PMID:9275179

  13. Brain derived neurotrophic factor is involved in the regulation of glycogen synthase kinase 3β (GSK3β) signalling

    SciTech Connect

    Gupta, Vivek; Chitranshi, Nitin; You, Yuyi; Gupta, Veer; Klistorner, Alexander; Graham, Stuart

    2014-11-21

    Highlights: • BDNF knockdown leads to activation of GSK3β in the neuronal cells. • BDNF knockdown can induce GSK3β activation beyond TrkB mediated effects. • BDNF impairment in vivo leads to age dependent activation of GSK3β in the retina. • Systemic treatment with TrkB agonist induces inhibition of retinal GSK3β. - Abstract: Glycogen synthase kinase 3β (GSK3β) is involved in several biochemical processes in neurons regulating cellular survival, gene expression, cell fate determination, metabolism and proliferation. GSK3β activity is inhibited through the phosphorylation of its Ser-9 residue. In this study we sought to investigate the role of BDNF/TrkB signalling in the modulation of GSK3β activity. BDNF/TrkB signalling regulates the GSK3β activity both in vivo in the retinal tissue as well as in the neuronal cells under culture conditions. We report here for the first time that BDNF can also regulate GSK3β activity independent of its effects through the TrkB receptor signalling. Knockdown of BDNF lead to a decline in GSK3β phosphorylation without having a detectable effect on the TrkB activity or its downstream effectors Akt and Erk1/2. Treatment with TrkB receptor agonist had a stimulating effect on the GSK3β phosphorylation, but the effect was significantly less pronounced in the cells in which BDNF was knocked down. The use of TrkB receptor antagonist similarly, manifested itself in the form of downregulation of GSK3β phosphorylation, but a combined TrkB inhibition and BDNF knockdown exhibited a much stronger negative effect. In vivo, we observed reduced levels of GSK3β phosphorylation in the retinal tissues of the BDNF{sup +/−} animals implicating critical role of BDNF in the regulation of the GSK3β activity. Concluding, BDNF/TrkB axis strongly regulates the GSK3β activity and BDNF also exhibits GSK3β regulatory effect independent of its actions through the TrkB receptor signalling.

  14. Parallel evolution of the glycogen synthase 1 (muscle) gene Gys1 between Old World and New World fruit bats (Order: Chiroptera).

    PubMed

    Fang, Lu; Shen, Bin; Irwin, David M; Zhang, Shuyi

    2014-10-01

    Glycogen synthase, which catalyzes the synthesis of glycogen, is especially important for Old World (Pteropodidae) and New World (Phyllostomidae) fruit bats that ingest high-carbohydrate diets. Glycogen synthase 1, encoded by the Gys1 gene, is the glycogen synthase isozyme that functions in muscles. To determine whether Gys1 has undergone adaptive evolution in bats with carbohydrate-rich diets, in comparison to insect-eating sister bat taxa, we sequenced the coding region of the Gys1 gene from 10 species of bats, including two Old World fruit bats (Pteropodidae) and a New World fruit bat (Phyllostomidae). Our results show no evidence for positive selection in the Gys1 coding sequence on the ancestral Old World and the New World Artibeus lituratus branches. Tests for convergent evolution indicated convergence of the sequences and one parallel amino acid substitution (T395A) was detected on these branches, which was likely driven by natural selection.

  15. Evidence for a role of glycogen synthase kinase-3 beta in rodent spermatogenesis.

    PubMed

    Guo, Taylor B; Chan, Kam C; Hakovirta, Harri; Xiao, Yang; Toppari, Jorma; Mitchell, Aaron P; Salameh, Wael A

    2003-01-01

    Glycogen synthase kinase-3 beta (GSK-3 beta) regulates cell metabolism, cell cycle, and cell fate through the phosphorylation of a diverse array of substrates. Herein, we provide evidence that supports a role for GSK-3 in mammalian meiosis and spermatogenesis. Immunostaining of testis sections showed that while GSK-3 alpha was ubiquitous in the seminiferous tubules, GSK-3 beta was expressed in premeiotic type B spermatogonia, in both meiotic preleptotene and leptotene spermatocytes, as well as in Sertoli cells in both the mouse and rat. Thus, GSK-3 beta is expressed in germ cells entering meiosis. In addition, intense immunoreactivity was detected in rat step 6 though 11 spermatids. In situ hybridization (ISH) in rat testis confirmed the immunostaining pattern in leptotene and spermatids and showed a GSK-3 beta messenger RNA (mRNA) signal in some pachytene spermatocytes. The restricted pattern of expression suggests cell-specific regulation of Gsk-3 beta mRNA. To determine whether GSK-3 is required for meiosis entry, rat stage VIIa seminiferous tubule segments were cultured with selective small-molecule GSK-3 inhibitors. These compounds markedly and dose-dependently suppressed meiotic synthesis (S)-phase DNA. Since a yeast GSK-3 homolog, Rim11p (regulator of inducer of meiosis), is pivotal to meiosis entry, we tested whether GSK-3 beta complements Rim11p function in meiosis. Rim11p phosphorylates transcription factors Ume6p (unscheduled meiotic gene expression) and Ime1p (inducer of meiosis) to induce meiosis entry. Overexpression of murine GSK-3 beta in a rim11 mutant yeast failed to rescue the sporulation defect. Our finding that GSK-3 beta interacted only with Ume6p but not with IME1 in a yeast 2-hybrid assay suggests that noncomplementation reflects partial divergence in substrate specificity. This work provides the basis for future studies of GSK-3 beta signaling in mammalian meiosis and spermatogenesis.

  16. Glycogen synthase kinase-3beta phosphorylates Bax and promotes its mitochondrial localization during neuronal apoptosis.

    PubMed

    Linseman, Daniel A; Butts, Brent D; Precht, Thomas A; Phelps, Reid A; Le, Shoshona S; Laessig, Tracey A; Bouchard, Ron J; Florez-McClure, Maria L; Heidenreich, Kim A

    2004-11-01

    Glycogen synthase kinase-3beta (GSK-3beta) is a critical activator of neuronal apoptosis induced by a diverse array of neurotoxic insults. However, the downstream substrates of GSK-3beta that ultimately induce neuronal death are unknown. Here, we show that GSK-3beta phosphorylates and regulates the activity of Bax, a pro-apoptotic Bcl-2 family member that stimulates the intrinsic (mitochondrial) death pathway by eliciting cytochrome c release from mitochondria. In cerebellar granule neurons undergoing apoptosis, inhibition of GSK-3beta suppressed both the mitochondrial translocation of an expressed green fluorescent protein (GFP)-Bax(alpha) fusion protein and the conformational activation of endogenous Bax. GSK-3beta directly phosphorylated Bax(alpha) on Ser163, a residue found within a species-conserved, putative GSK-3beta phosphorylation motif. Coexpression of GFP-Bax(alpha) with a constitutively active mutant of GSK-3beta, GSK-3beta(Ser9Ala), enhanced the in vivo phosphorylation of wild-type Bax(alpha), but not a Ser163Ala mutant of Bax(alpha), in transfected human embryonic kidney 293 (HEK293) cells. Moreover, cotransfection with constitutively active GSK-3beta promoted the localization of Bax(alpha) to mitochondria and induced apoptosis in both transfected HEK293 cells and cerebellar granule neurons. In contrast, neither a Ser163Ala point mutant of Bax(alpha) nor a naturally occurring splice variant that lacks 13 amino acids encompassing Ser163 (Bax(sigma)) were driven to mitochondria in HEK293 cells coexpressing constitutively active GSK-3beta. In a similar manner, either mutation or deletion of the identified GSK-3beta phosphorylation motif prevented the localization of Bax to mitochondria in cerebellar granule neurons undergoing apoptosis. Our results indicate that GSK-3beta exerts some of its pro-apoptotic effects in neurons by regulating the mitochondrial localization of Bax, a key component of the intrinsic apoptotic cascade.

  17. Inhibition of glycogen synthase kinase-3β prevents sympathetic hyperinnervation in infarcted rats

    PubMed Central

    Lee, Tsung-Ming; Lin, Shinn-Zong

    2015-01-01

    We have demonstrated that nerve growth factor (NGF) expression in the myocardium is selectively increased during chronic stage of myocardial infarction, resulting in sympathetic hyperinnervation. Glycogen synthase kinase-3 (GSK-3) signal has been shown to play key roles in the regulation of cytoskeletal assembly during axon regeneration. We assessed whether lithium, a GSK-3 inhibitor, attenuates cardiac sympathetic reinnervation after myocardial infarction through attenuated NGF expression and Tau expression. Twenty-four hours after ligation of the anterior descending artery, male Wistar rats were randomized to either LiCl or SB216763, chemically unrelated inhibitors of GSK-3β, a combination of LiCl and SB216763, or vehicle for four weeks. Myocardial norepinephrine levels revealed a significant elevation in vehicle-treated rats compared with sham-operated rats, consistent with excessive sympathetic reinnervation after infarction. Immunohistochemical analysis for sympathetic nerve also confirmed the change of myocardial norepinephrine. This was paralleled by a significant upregulation of NGF protein and mRNA in the vehicle-treated rats, which was reduced after administering either LiCl, SB216763, or combination. Arrhythmic scores during programmed stimulation in the vehicle-treated rats were significantly higher than those treated with GSK-3 inhibitors. Addition of SB216763 did not have additional beneficial effects compared with those seen in rats treated with LiCl alone. Furthermore, lithium treatment increased Tau1 and decreased AT8 and AT180 levels. Chronic use of lithium after infarction, resulting in attenuated sympathetic reinnervation by GSK-3 inhibition, may modify the arrhythmogenic response to programmed electrical stimulation. PMID:25576342

  18. Progranulin enhances neural progenitor cell proliferation through glycogen synthase kinase 3β phosphorylation.

    PubMed

    Nedachi, T; Kawai, T; Matsuwaki, T; Yamanouchi, K; Nishihara, M

    2011-06-30

    Progranulin (PGRN) is an estrogen-inducible growth factor thought to affect multiple processes in the CNS, including brain sexual differentiation, adult neurogenesis in the hippocampus, and development of neurodegenerative diseases. However, the precise physiological functions of PGRN in individual nerve cells are not fully understood. The aim of the present study was to enhance the understanding of PGRN function in the CNS by investigating the effects of PGRN on neural progenitor cells (NPCs). We found that significant amounts of endogenous PGRN were secreted from isolated NPCs in cultures. To assess the bioactivities of endogenous and exogenous PGRN, we studied NPCs derived from wild-type mice (WT-NPCs) and PGRN-deficient mice (KO-NPCs). We found that proliferation of KO-NPCs was significantly enhanced by PGRN treatment; however, PGRN treatment apparently did not affect proliferation of WT-NPCs perhaps because of the high levels of endogenous PGRN expression. NPC death and asymmetric cellular division of KO-NPCs and WT-NPCs, which results in production of neural stem cells, astrocytes, or oligodendrocytes, were not affected by PGRN treatment. We also investigated the signaling mechanism(s) that mediate PGRN-induced NPC proliferation and found that phosphorylation of serine 9 (S9) of glycogen synthase kinase 3-beta (GSK3β), which was dependent on phosphatidylinositol 3-kinase (PI3K) activity, was induced by PGRN treatment. In addition, a GSK3β-specific inhibitor enhanced NPC proliferation. Taken together, our observations indicate that PGRN enhanced NPC proliferation, at least in part, via inducing GSK3β phosphorylation. PMID:21540081

  19. Glycogen Synthase Kinase-3 in the Etiology and Treatment of Mood Disorders

    PubMed Central

    Jope, Richard Scott

    2011-01-01

    The mood disorders major depressive disorder and bipolar disorder are prevalent, are inadequately treated, and little is known about their etiologies. A better understanding of the causes of mood disorders would benefit from improved animal models of mood disorders, which now rely on behavioral measurements. This review considers the limitations in relating measures of rodent behaviors to mood disorders, and the evidence from behavioral assessments indicating that glycogen synthase kinase-3 (GSK3) dysregulation promotes mood disorders and is a potential target for treating mood disorders. The classical mood stabilizer lithium was identified by studying animal behaviors and later was discovered to be an inhibitor of GSK3. Several mood-relevant behavioral effects of lithium in rodents have been identified, and most have now been shown to be due to its inhibition of GSK3. An extensive variety of pharmacological and molecular approaches for manipulating GSK3 are discussed, the results of which strongly support the proposal that inhibition of GSK3 reduces both depression-like and manic-like behaviors. Studies in human postmortem brain and peripheral cells also have identified correlations between alterations in GSK3 and mood disorders. Evidence is reviewed that depression may be associated with impaired inhibitory control of GSK3, and mania by hyper-stimulation of GSK3. Taken together, these studies provide substantial support for the hypothesis that inhibition of GSK3 activity is therapeutic for mood disorders. Future research should identify the causes of dysregulated GSK3 in mood disorders and the actions of GSK3 that contribute to these diseases. PMID:21886606

  20. Regulation of Th1 cells and experimental autoimmune encephalomyelitis (EAE) by glycogen synthase kinase-3

    PubMed Central

    Beurel, Eléonore; Kaidanovich-Beilin, Oksana; Yeh, Wen-I; Song, Ling; Palomo, Valle; Michalek, Suzanne M.; Woodgett, James R.; Harrington, Laurie E.; Eldar-Finkelman, Hagit; Martinez, Ana; Jope, Richard S.

    2013-01-01

    Experimental autoimmune encephalomyelitis (EAE) is a rodent model of multiple sclerosis (MS), a debilitating autoimmune disease of the central nervous system, for which only limited therapeutic interventions are available. Since MS is mediated in part by autoreactive T cells, particularly Th17 and Th1 cells, in the present study, we tested if inhibitors of glycogen synthase kinase-3 (GSK3), previously reported to reduce Th17 cell generation, also alter Th1 cell production or ameliorate EAE. GSK3 inhibitors were found to impede the production of Th1 cells by reducing STAT1 activation. Molecularly reducing the expression of either of the two GSK3 isoforms demonstrated that Th17 cell production was sensitive to reduced levels of GSK3β, and Th1 cell production was inhibited in GSK3α-deficient cells. Administration of the selective GSK3 inhibitors TDZD-8, VP2.51, VP0.7, or L803-mts, significantly reduced the clinical symptoms of MOG35-55-induced EAE in mice, nearly eliminating the chronic progressive phase, and reduced the number of Th17 and Th1 cells in the spinal cord. Administration of TDZD-8 or L803-mts after the initial disease episode ameliorated clinical symptoms in a relapsing/remitting model of PLP139-151-induced EAE. Furthermore, deletion of GSK3β specifically in T cells was sufficient to ameliorate MOG35-55-induced EAE. These results demonstrate isoform-selective effects of GSK3 on T cell generation, therapeutic effects of GSK3 inhibitors in EAE, and that GSK3 inhibition in T cells is sufficient to reduce the severity of EAE, suggesting that GSK3 may be a feasible target for developing new therapeutic interventions for MS. PMID:23606540

  1. Identification of a Glycogen Synthase Kinase-3[beta] Inhibitor that Attenuates Hyperactivity in CLOCK Mutant Mice

    SciTech Connect

    Kozikowski, Alan P.; Gunosewoyo, Hendra; Guo, Songpo; Gaisina, Irina N.; Walter, Richard L.; Ketcherside, Ariel; McClung, Colleen A.; Mesecar, Andrew D.; Caldarone, Barbara

    2012-05-02

    Bipolar disorder is characterized by a cycle of mania and depression, which affects approximately 5 million people in the United States. Current treatment regimes include the so-called 'mood-stabilizing drugs', such as lithium and valproate that are relatively dated drugs with various known side effects. Glycogen synthase kinase-3{beta} (GSK-3{beta}) plays a central role in regulating circadian rhythms, and lithium is known to be a direct inhibitor of GSK-3{beta}. We designed a series of second generation benzofuran-3-yl-(indol-3-yl)maleimides containing a piperidine ring that possess IC{sub 50} values in the range of 4 to 680 nM against human GSK-3{beta}. One of these compounds exhibits reasonable kinase selectivity and promising preliminary absorption, distribution, metabolism, and excretion (ADME) data. The administration of this compound at doses of 10 to 25 mg kg{sup -1} resulted in the attenuation of hyperactivity in amphetamine/chlordiazepoxide-induced manic-like mice together with enhancement of prepulse inhibition, similar to the effects found for valproate (400 mg kg{sup -1}) and the antipsychotic haloperidol (1 mg kg{sup -1}). We also tested this compound in mice carrying a mutation in the central transcriptional activator of molecular rhythms, the CLOCK gene, and found that the same compound attenuates locomotor hyperactivity in response to novelty. This study further demonstrates the use of inhibitors of GSK-3{beta} in the treatment of manic episodes of bipolar/mood disorders, thus further validating GSK-3{beta} as a relevant therapeutic target in the identification of new therapies for bipolar patients.

  2. Inhibition of glycogen synthase kinase-3β attenuates organ injury and dysfunction associated with liver ischemia-reperfusion and thermal injury in the rat.

    PubMed

    Rocha, Joao; Figueira, Maria-Eduardo; Barateiro, Andreia; Fernandes, Adelaide; Brites, Dora; Pinto, Rui; Freitas, Marisa; Fernandes, Eduarda; Mota-Filipe, Helder; Sepodes, Bruno

    2015-04-01

    Glycogen synthase kinase 3 (GSK-3) is a serine-threonine kinase discovered decades ago to have an important role in glycogen metabolism. Today, we know that this kinase is involved in the regulation of many cell functions, including insulin signaling, specification of cell fate during embryonic development, and the control of cell division and apoptosis. Insulin and TDZD-8 (4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione) are inhibitors of GSK-3β that have been shown to possess organ-protective effects in inflammatory-mediated organ injury models. We aimed to evaluate the cytoprotective effect of GSK-3β inhibition on rat models of liver ischemia-reperfusion and thermal injury. In the liver ischemia-reperfusion model, TDZD-8 and insulin were administered at 5 mg/kg (i.v.) and 1.4 IU/kg (i.v.), respectively, 30 min before induction of ischemia and led to the significant reduction of the serum concentration of aspartate aminotransferase, alanine aminotransferase, γ-glutamyltransferase, and lactate dehydrogenase. Beneficial effects were found to be independent from blood glucose levels. In the thermal injury model, TDZD-8 was administered at 5 mg/kg (i.v.) 5 min before induction of injury and significantly reduced multiple organ dysfunction markers (liver, neuromuscular, and lung). In the lung, TDZD-8 reduced the histological signs of tissue injury, inflammatory markers (cytokines), and neutrophil chemotaxis/infiltration; reduced GSK-3β, nuclear factor-κB, and Akt activation; reduced caspase-3 and metalloproteinase-9 activation. Our study provides a new insight on the beneficial effects of GSK-3β inhibition on systemic inflammation and further elucidates the mechanism and pathway crosstalks by which TDZD-8 reduces the multiple organ injury elicited by thermal injury.

  3. The Glycogen Synthase Kinase 3α and β Isoforms Differentially Regulates Interleukin-12p40 Expression in Endothelial Cells Stimulated with Peptidoglycan from Staphylococcus aureus

    PubMed Central

    Huante-Mendoza, Alejandro; Bravo-Patiño, Alejandro; Valdez-Alarcón, Juan J.; Finlay, B. Brett; Baizabal-Aguirre, Víctor M.

    2015-01-01

    Glycogen synthase kinase 3 (GSK3) is a constitutively active regulatory enzyme that is important in cancer, diabetes, and cardiovascular, neurodegenerative, and psychiatric diseases. While GSK3α is usually important in neurodegenerative and psychiatric diseases GSK3β is fundamental in the inflammatory response caused by bacterial components. Peptidoglycan (PGN), one of the most abundant cell-wall structures of Gram-positive bacteria, is an important inducer of inflammation. To evaluate whether inhibition of GSK3α and GSK3β activity in bovine endothelial cells (BEC) regulates the expression of the pro-inflammatory cytokine IL-12p40, we treated BEC with SDS-purified PGN from Staphylococcus aureus. We found that PGN triggered a TLR2/PI3K/Akt-dependent phosphorylation of GSK3α at Ser21, GSK3β at Ser9, and NF-κB p65 subunit (p65) at Ser536, and the phosphorylation of GSK3α was consistently higher than that of GSK3β. The expression of IL-12p40 was inhibited in BEC stimulated with PGN and pre-treated with a specific neutralizing anti-TLR2 antibody that targets the extracellular domain of TLR2 or by the addition of Akt-i IV (an Akt inhibitor). Inhibition of GSK3α and GSK3β with LiCl or SB216763 induced an increase in IL-12p40 mRNA and protein. The effect of each isoform on IL-12p40 expression was evaluated by siRNA-gene expression silencing of GSK3α and GSK3β. GSK3α gene silencing resulted in a marked increase in IL-12p40 mRNA and protein while GSK3β gene silencing had the opposite effect on IL-12p40 expression. These results indicate that the TLR2/PI3K/Akt-dependent inhibition of GSK3α activity also plays an important role in the inflammatory response caused by stimulation of BEC with PGN from S. aureus. PMID:26200352

  4. Inhibition of Glycogen Synthase Kinase-3β Is Sufficient for Airway Smooth Muscle Hypertrophy*

    PubMed Central

    Deng, Huan; Dokshin, Gregoriy A.; Lei, Jing; Goldsmith, Adam M.; Bitar, Khalil N.; Fingar, Diane C.; Hershenson, Marc B.; Bentley, J. Kelley

    2008-01-01

    We examined the role of glycogen synthase kinase-3β (GSK-3β) inhibition in airway smooth muscle hypertrophy, a structural change found in patients with severe asthma. LiCl, SB216763, and specific small interfering RNA (siRNA) against GSK-3β, each of which inhibit GSK-3β activity or expression, increased human bronchial smooth muscle cell size, protein synthesis, and expression of the contractile proteins α-smooth muscle actin, myosin light chain kinase, smooth muscle myosin heavy chain, and SM22. Similar results were obtained following treatment of cells with cardiotrophin (CT)-1, a member of the interleukin-6 superfamily, and transforming growth factor (TGF)-β, a proasthmatic cytokine. GSK-3β inhibition increased mRNA expression of α-actin and transactivation of nuclear factors of activated T cells and serum response factor. siRNA against eukaryotic translation initiation factor 2Bε (eIF2Bε) attenuated LiCl- and SB216763-induced protein synthesis and expression of α-actin and SM22, indicating that eIF2B is required for GSK-3β-mediated airway smooth muscle hypertrophy. eIF2Bε siRNA also blocked CT-1- but not TGF-β-induced protein synthesis. Infection of human bronchial smooth muscle cells with pMSCV GSK-3β-A9, a retroviral vector encoding a constitutively active, nonphosphorylatable GSK-3β, blocked protein synthesis and α-actin expression induced by LiCl, SB216763, and CT-1 but not TGF-β. Finally, lungs from ovalbumin-sensitized and -challenged mice demonstrated increased α-actin and CT-1 mRNA expression, and airway myocytes isolated from ovalbumin-treated mice showed increased cell size and GSK-3β phosphorylation. These data suggest that inhibition of the GSK-3β/eIF2Bε translational control pathway contributes to airway smooth muscle hypertrophy in vitro and in vivo. On the other hand, TGF-β-induced hypertrophy does not depend on GSK-3β/eIF2B signaling. PMID:18252708

  5. Glycogen Synthase Kinase 3beta Contributes to Proliferation of Arterial Smooth Muscle Cells in Pulmonary Hypertension

    PubMed Central

    Tian, Xia; Ghofrani, Hossein Ardeschir; Weissmann, Norbert; Sedding, Daniel; Kashour, Tarek; Seeger, Werner; Grimminger, Friedrich; Pullamsetti, Soni Savai

    2011-01-01

    Rationale Pulmonary arterial hypertension (PAH) is a rare progressive pulmonary vascular disorder associated with vascular remodeling and right heart failure. Vascular remodeling involves numerous signaling cascades governing pulmonary arterial smooth muscle cell (PASMC) proliferation, migration and differentiation. Glycogen synthase kinase 3beta (GSK3ß) is a serine/threonine kinase and can act as a downstream regulatory switch for numerous signaling pathways. Hence, we hypothesized that GSK3ß plays a crucial role in pulmonary vascular remodeling. Methods All experiments were done with lung tissue or isolated PASMCs in a well-established monocrotaline (MCT)-induced PAH rat model. The mRNA expression of Wnt ligands (Wnt1, Wnt3a, Wnt5a), upstream Wnt signaling regulator genes (Frizzled Receptors 1, 2 and secreted Frizzled related protein sFRP-1) and canonical Wnt intracellular effectors (GSK3ß, Axin1) were assessed by real-time polymerase chain reaction and protein levels of GSK3ß, phospho-GSK3ß (ser 9) by western blotting and localization by immunohistochemistry. The role of GSK3ß in PASMCs proliferation was assessed by overexpression of wild-type GSK3ß (WT) and constitutively active GSK3ß S9A by [3H]-thymidine incorporation assay. Results Increased levels of total and phosphorylated GSK3ß (inhibitory phosphorylation) were observed in lungs and PASMCs isolated from MCT-induced PAH rats compared to controls. Further, stimulation of MCT-PASMCs with growth factors induced GSK3ß inactivation. Most importantly, treatment with the PDGFR inhibitor, Imatinib, attenuated PDGF-BB and FCS induced GSK3ß phosphorylation. Increased expression of GSK3ß observed in lungs and PASMC isolated from MCT-induced PAH rats was confirmed to be clinically relevant as the same observation was identified in human iPAH lung explants. Overexpression of GSK3ß significantly increased MCT-PASMCs proliferation by regulating ERK phosphorylation. Constitutive activation of GSK3ß (GSK3

  6. The Effects of Glycogen Synthase Kinase-3beta in Serotonin Neurons

    PubMed Central

    Zhou, Wenjun; Chen, Ligong; Paul, Jodi; Yang, Sufen; Li, Fuzeng; Sampson, Karen; Woodgett, Jim R.; Beaulieu, Jean Martin; Gamble, Karen L.; Li, Xiaohua

    2012-01-01

    Glycogen synthase kinase-3 (GSK3) is a constitutively active protein kinase in brain. Increasing evidence has shown that GSK3 acts as a modulator in the serotonin neurotransmission system, including direct interaction with serotonin 1B (5-HT1B) receptors in a highly selective manner and prominent modulating effect on 5-HT1B receptor activity. In this study, we utilized the serotonin neuron-selective GSK3β knockout (snGSK3β-KO) mice to test if GSK3β in serotonin neurons selectively modulates 5-HT1B autoreceptor activity and function. The snGSK3β-KO mice were generated by crossbreeding GSK3β-floxed mice and ePet1-Cre mice. These mice had normal growth and physiological characteristics, similar numbers of tryptophan hydroxylase-2 (TpH2)-expressing serotonin neurons, and the same brain serotonin content as in littermate wild type mice. However, the expression of GSK3β in snGSK3β-KO mice was diminished in TpH2-expressing serotonin neurons. Compared to littermate wild type mice, snGSK3β-KO mice had a reduced response to the 5-HT1B receptor agonist anpirtoline in the regulation of serotonergic neuron firing, cAMP production, and serotonin release, whereas these animals displayed a normal response to the 5-HT1A receptor agonist 8-OH-DPAT. The effect of anpirtoline on the horizontal, center, and vertical activities in the open field test was differentially affected by GSK3β depletion in serotonin neurons, wherein vertical activity, but not horizontal activity, was significantly altered in snGSK3β-KO mice. In addition, there was an enhanced anti-immobility response to anpirtoline in the tail suspension test in snGSK3β-KO mice. Therefore, results of this study demonstrated a serotonin neuron-targeting function of GSK3β by regulating 5-HT1B autoreceptors, which impacts serotonergic neuron firing, serotonin release, and serotonin-regulated behaviors. PMID:22912839

  7. Glycogen Synthase Kinase-3 Inhibitor Protects Against Microvascular Hyperpermeability Following Hemorrhagic Shock

    PubMed Central

    Sawant, Devendra A.; Tharakan, Binu; Hunter, Felicia A.; Childs, Ed W.

    2015-01-01

    Background Hemorrhagic shock (HS)-induce microvascular hyperpermeability involves disruption of endothelial cell adherens junctions leading to increase in paracellular permeability. β-Catenin, an integral component of the adherens junctional complex and Wnt pathway, and caspase-3 via its apoptotic signaling regulate endothelial cell barrier integrity. We have hypothesized that inhibiting phosphorylation of β-catenin and caspase-3 activity using glycogen synthase kinase-3 (GSK-3) specific inhibitor SB216763, would attenuate microvascular hyperpermeability following HS. Methods In Sprague-Dawley rats, HS was induced by withdrawing blood to reduce mean arterial pressure to 40 mmHg for 60 minutes followed by resuscitation. Rats were given SB216763 (600 μg/kg) intravenously 10 minutes prior to shock. To study microvascular permeability, the rats were injected intravenously with FITC-albumin (50 mg/kg) and its flux across the mesenteric post-capillary venules was determined using intravital microscopy. In cell-culture studies, rat lung microvascular endothelial cell (RLMEC) monolayers grown on Transwell plates were pre-treated with SB216763 (5 μM) followed by BAK (5 μg/mL) and caspase-3 (5 μg/mL) protein transfection. FITC-albumin (5 mg/mL) flux across cell monolayers indicates change in monolayer permeability. Activity of canonical Wnt pathway was determined by luciferase assay. Caspase-3 enzyme activity was assayed fluorometrically. Results The HS group showed significant increase in FITC-albumin extravasation (p<0.05) compared with sham. SB216763 significantly decrease HS-induced FITC-albumin extravasation (p<0.05). Pre-treatment with SB216763, protected against a BAK-induced increase in RLMEC monolayer permeability and caspase-3 activity, but failed to show similar results with a caspase-3-induced increase in monolayer permeability. Wnt3a treatment showed an increase in β-catenin dependent TCF-mediated transcription. Conclusion Inhibiting phosphorylation of

  8. MDS1, a dosage suppressor of an mck1 mutant, encodes a putative yeast homolog of glycogen synthase kinase 3.

    PubMed Central

    Puziss, J W; Hardy, T A; Johnson, R B; Roach, P J; Hieter, P

    1994-01-01

    The yeast gene MCK1 encodes a serine/threonine protein kinase that is thought to function in regulating kinetochore activity and entry into meiosis. Disruption of MCK1 confers a cold-sensitive phenotype, a temperature-sensitive phenotype, and sensitivity to the microtubule-destabilizing drug benomyl and leads to loss of chromosomes during growth on benomyl. A dosage suppression selection was used to identify genes that, when present at high copy number, could suppress the cold-sensitive phenotype of mck1::HIS3 mutant cells. Several unique classes of clones were identified, and one of these, designated MDS1, has been characterized in some detail. Nucleotide sequence data reveal that MDS1 encodes a serine/threonine protein kinase that is highly homologous to the shaggy/zw3 kinase in Drosophila melanogaster and its functional homolog, glycogen synthase kinase 3, in rats. The presence of MDS1 in high copy number rescues both the cold-sensitive and the temperature-sensitive phenotypes, but not the benomyl-sensitive phenotype, associated with the disruption of MCK1. Analysis of strains harboring an mds1 null mutation demonstrates that MDS1 is not essential during normal vegetative growth but appears to be required for meiosis. Finally, in vitro experiments indicate that the proteins encoded by both MCK1 and MDS1 possess protein kinase activity with substrate specificity similar to that of mammalian glycogen synthase kinase 3. Images PMID:8264650

  9. c-Src regulates cell cycle proteins expression through protein kinase B/glycogen synthase kinase 3 beta and extracellular signal-regulated kinases 1/2 pathways in MCF-7 cells.

    PubMed

    Liu, Xiang; Du, Liying; Feng, Renqing

    2013-07-01

    We have demonstrated that c-Src suppression inhibited the epithelial to mesenchymal transition in human breast cancer cells. Here, we investigated the role of c-Src on the cell cycle progression using siRNAs and small molecule inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2). Western blot analysis demonstrated the down-regulation of cyclin D1 and cyclin E and up-regulation of p27 Kip1 after c-Src suppression by PP2. Incubation of cells in the presence of PP2 significantly blocked the phosphorylation of extracellular signal-regulated kinases 1/2 (ERK1/2), protein kinase B (AKT), and glycogen synthase kinase 3 beta (GSK3β). Specific pharmacological inhibitors of MEK1/2/ERK1/2 and phosphatidylinositide 3-kinase/AKT pathways were used to demonstrate the relationship between the signal cascade and cell cycle proteins expression. The expression of cyclin D1 and cyclin E were decreased after inhibition of ERK1/2 or AKT activity, whereas the p27 Kip1 expression was increased. In addition, knockdown of c-Src by siRNAs reduced cell proliferation and phosphorylation of ERK1/2, AKT, and GSK3β. After c-Src depletion by siRNAs, we observed significant down-regulation of cyclin D1 and cyclin E, and up-regulation of p27 Kip1. These results suggest that c-Src suppression by PP2 or siRNAs may regulate the progression of cell cycle through AKT/GSK3β and ERK1/2 pathways.

  10. 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

  11. Exogenous zinc protects cardiac cells from reperfusion injury by targeting mitochondrial permeability transition pore through inactivation of glycogen synthase kinase-3β

    PubMed Central

    Chanoit, Guillaume; Lee, SungRyul; Xi, Jinkun; Zhu, Min; McIntosh, Rachel A.; Mueller, Robert A.; Norfleet, Edward A.; Xu, Zhelong

    2008-01-01

    The purpose of this study was to determine whether exogenous zinc prevents cardiac reperfusion injury by targeting the mitochondrial permeability transition pore (mPTP) via glycogen synthase kinase-3β (GSK-3β). The treatment of cardiac H9c2 cells with ZnCl2 (10 μM) in the presence of zinc ionophore pyrithione for 20 min significantly enhanced GSK-3β phosphorylation at Ser9, indicating that exogenous zinc can inactivate GSK-3β in H9c2 cells. The effect of zinc on GSK-3β activity was blocked by the phosphatidylinositol 3-kinase (PI3K) inhibitor LY-294002 but not by the mammalian target of rapamycin (mTOR) inhibitor rapamycin or the PKC inhibitor chelerythrine, implying that PI3K but not mTOR or PKC accounts for the action of zinc. In support of this interpretation, zinc induced a significant increase in Akt but not mTOR phosphorylation. Further experiments found that zinc also increased mitochondrial GSK-3β phosphorylation. This may indicate an involvement of the mitochondria in the action of zinc. The effect of zinc on mitochondrial GSK-3β phosphorylation was not altered by the mitochondrial ATP-sensitive K+ channel blocker 5-hydroxydecanoic acid. Zinc applied at reperfusion reduced cell death in cells subjected to simulated ischemia/reperfusion, indicating that zinc can prevent reperfusion injury. However, zinc was not able to exert protection in cells transfected with the constitutively active GSK-3β (GSK-3β-S9A-HA) mutant, suggesting that zinc prevents reperfusion injury by inactivating GSK-3β. Cells transfected with the catalytically inactive GSK-3β (GSK-3β-KM-HA) also revealed a significant decrease in cell death, strongly supporting the essential role of GSK-3β inactivation in cardioprotection. Moreover, zinc prevented oxidant-induced mPTP opening through the inhibition of GSK-3β. Taken together, these data suggest that zinc prevents reperfusion injury by modulating the mPTP opening through the inactivation of GSK-3β. The PI3K/Akt signaling

  12. Increased Glycogen Synthase Kinase-3β and Hexose-6-Phosphate Dehydrogenase Expression in Adipose Tissue May Contribute to Glucocorticoid-Induced Mouse Visceral Adiposity

    PubMed Central

    Yan, Chaoying; Yang, Huabing; Wang, Ying; Dong, Yunzhou; Yu, Fei; Wu, Yong; Wang, Wei; Ume, Adaku; Lutfy, Kabirullah; Friedman, Theodore C.; Tian, Shiliu; Liu, Yanjun

    2016-01-01

    BACKGROUND Increased adiposity in visceral depots is a crucial feature associated with glucocorticoid (GC) excess. The action of GCs in target tissue is regulated by GC receptor (GR) and 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) coupled with hexose-6-phosphate dehydrogenase (H6pdh). Glycogen synthase kinase-3β (GSK3β) is known to be a crucial mediator of ligand-dependent gene transcription. We hypothesized that the major effects of corticosteroids on adipose fat accumulation are in part medicated by changes in GSK3β and H6pdh. METHODS We characterized the alterations of GSK3β and GC metabolic enzymes, and determined the impact of GR antagonist mifepristone on obesity-related genes and the expression of H6pdh and 11ß-HSD1 in adipose tissue of mice exposed to excess GC as well as in in vitro studies using 3T3-L1 adipocytes treated with GCs. RESULTS Corticosterone (CORT) exposure increased abdominal fat mass and induced expression of lipid synthase ACC and ACL with activation of GSK3β phosphorylation in abdominal adipose tissue of C57BL/6J mice. Increased pSer9 GSK3β was correlated with induction of H6pdh and 11ß-HSD1. Additionally, mifepristone treatment reversed the production of H6pdh and attenuated CORT-mediated production of 11ß-HSD1 and lipogenic gene expression with reduction of pSer9 GSK3β, thereby leading to improvement of phenotype of adiposity within adipose tissue in mice treated with excess GCs. Suppression of pSer9 GSK3β by mifepristone was accompanied by activation of pThr308 Akt and blockade of CORT-induced adipogenic transcriptor C/EBPα and PPARγ. In addition, mifepristone also attenuated CORT-mediated activation of IRE1α/XBP1. Additionally, reduction of H6pdh by shRNA showed comparable effects to mifepristone on attenuating CORT-induced expression of GC metabolic enzymes and improved lipid accumulation in vitro in 3T3-L1 adipocytes. CONCLUSION These findings suggest that elevated adipose GSK3β and H6pdh expression contribute

  13. 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

  14. 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)

  15. 5-imino-1,2,4-thiadiazoles: first small molecules as substrate competitive inhibitors of glycogen synthase kinase 3.

    PubMed

    Palomo, Valle; Perez, Daniel I; Perez, Concepcion; Morales-Garcia, Jose A; Soteras, Ignacio; Alonso-Gil, Sandra; Encinas, Arantxa; Castro, Ana; Campillo, Nuria E; Perez-Castillo, Ana; Gil, Carmen; Martinez, Ana

    2012-02-23

    Cumulative evidence strongly supports that glycogen synthase kinase-3 (GSK-3) is a pathogenic molecule when it is up-dysregulated, emerging as an important therapeutic target in severe unmet human diseases. GSK-3 specific inhibitors might be promising effective drugs for the treatment of devastating pathologies such as neurodegenerative diseases, stroke, and mood disorders. As GSK-3 has the ability to phosphorylate primed substrates, small molecules able to bind to this site should be perfect drug candidates, able to partially block the activity of the enzyme over some specific substrates. Here, we report substituted 5-imino-1,2,4-thiadiazoles as the first small molecules able to inhibit GSK-3 in a substrate competitive manner. These compounds are cell permeable, able to decrease inflammatory activation and to selectively differentiate neural stem cells. Overall, 5-imino-1,2,4-thiadiazoles are presented here as new molecules able to decrease neuronal cell death and to increase endogenous neurogenesis blocking the GSK-3 substrate site.

  16. 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 +/.

  17. Glycogen synthase kinase 3β in the nucleus accumbens core is critical for methamphetamine-induced behavioral sensitization.

    PubMed

    Xu, Chun-Mei; Wang, Jun; Wu, Ping; Xue, Yan-Xue; Zhu, Wei-Li; Li, Qian-Qian; Zhai, Hai-Feng; Shi, Jie; Lu, Lin

    2011-07-01

    As a ubiquitous serine/threonine protein kinase, glycogen synthase kinase 3β (GSK-3β) has been considered to be important in the synaptic plasticity that underlies dopamine-related behaviors and diseases. We recently found that GSK-3β activity in the nucleus accumbens (NAc) core is critically involved in cocaine-induced behavioral sensitization. The present study further explored the association between the changes in GSK-3β activity in the NAc and the chronic administration of methamphetamine. We also examined whether blocking GSK-3β activity in the NAc could alter the initiation and expression of methamphetamine (1 mg/kg, i.p.)-induced locomotor sensitization in rats using systemic administration of lithium chloride (LiCl, 100 mg/kg, i.p) and brain region-specific administration of the GSK-3β inhibitor SB216763 (1 ng/side). We found that GSK-3β activity increased in the NAc core, but not NAc shell, after chronic methamphetamine administration. The initiation and expression of methamphetamine-induced locomotor sensitization was attenuated by systemic administration of LiCl and direct infusion of SB216763 into the NAc core, but not NAc shell. These results indicate that GSK-3β activity in the NAc core mediates the initiation and expression of methamphetamine-induced locomotor sensitization, suggesting that GSK-3β may be a potential target for the treatment of psychostimulant addiction.

  18. p190A RhoGAP is a glycogen synthase kinase-3-beta substrate required for polarized cell migration.

    PubMed

    Jiang, Wei; Betson, Martha; Mulloy, Roseann; Foster, Rosemary; Lévay, Magdolna; Ligeti, Erzsébet; Settleman, Jeffrey

    2008-07-25

    The Rho GTPases are critical regulators of the actin cytoskeleton and are required for cell adhesion, migration, and polarity. Among the key Rho regulatory proteins in the context of cell migration are the p190 RhoGAPs (p190A and p190B), which function to modulate Rho signaling in response to integrin engagement. The p190 RhoGAPs undergo complex regulation, including phosphorylation by several identified kinases, interactions with phospholipids, and association with a variety of cellular proteins. Here, we have identified an additional regulatory mechanism unique to p190A RhoGAP that involves priming-dependent phosphorylation by glycogen synthase-3-beta (GSK-3beta), a kinase previously implicated in establishing cell polarity. We found that p190A-deficient fibroblasts exhibit a defect in directional cell migration reflecting a requirement for GSK-3beta-mediated phosphorylation of amino acids in the C-terminal "tail" of p190A. This phosphorylation leads to inhibition of p190A RhoGAP activity in vitro and in vivo. These studies identify p190A as a novel GSK-3beta substrate and reveal a mechanism by which GSK-3beta contributes to cellular polarization in directionally migrating cells via effects on Rho GTPase activity.

  19. 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

  20. Glycogen synthase kinase 3beta phosphorylates p21WAF1/CIP1 for proteasomal degradation after UV irradiation.

    PubMed

    Lee, Ji Young; Yu, Su Jin; Park, Yun Gyu; Kim, Joon; Sohn, Jeongwon

    2007-04-01

    UV irradiation has been reported to induce p21(WAF1/CIP1) protein degradation through a ubiquitin-proteasome pathway, but the underlying biochemical mechanism remains to be elucidated. Here, we show that ser-114 phosphorylation of p21 protein by glycogen synthase kinase 3beta (GSK-3beta) is required for its degradation in response to UV irradiation and that GSK-3beta activation is a downstream event in the ATR signaling pathway triggered by UV. UV transiently increased GSK-3beta activity, and this increase could be blocked by caffeine or by ATR small interfering RNA, indicating ATR-dependent activation of GSK-3beta. ser-114, located within the putative GSK-3beta target sequence, was phosphorylated by GSK-3beta upon UV exposure. The nonphosphorylatable S114A mutant of p21 was protected from UV-induced destabilization. Degradation of p21 protein by UV irradiation was independent of p53 status and prevented by proteasome inhibitors. In contrast to the previous report, the proteasomal degradation of p21 appeared to be ubiquitination independent. These data show that GSK-3beta is activated by UV irradiation through the ATR signaling pathway and phosphorylates p21 at ser-114 for its degradation by the proteasome. To our knowledge, this is the first demonstration of GSK-3beta as the missing link between UV-induced ATR activation and p21 degradation.

  1. Progesterone receptor A stability is mediated by glycogen synthase kinase-3β in the Brca1-deficient mammary gland.

    PubMed

    Wang, Shaohui; Li, Ying; Hsu, Pang-Hung; Lee, Sou-Ying; Kim, Yoon; Lee, Eva Y-H P

    2013-09-01

    Germ line mutations of the BRCA1 gene increase the risk of breast and ovarian cancer, but the basis of this tissue-specific tumor predisposition is not fully understood. Previously, we reported that the progesterone receptors are stabilized in Brca1-deficient mammary epithelial cells, and treating with anti-progesterone delays mammary tumorigenesis in Brca1/p53 conditional knock-out mice, suggesting that the progesterone has a critical role in breast carcinogenesis. To further explore how the stability of progesterone receptor is modulated, here, we have found that glycogen synthase kinase (GSK)-3β phosphorylation of progesterone receptor-A (PR-A) facilitates its ubiquitination. GSK-3β-mediated phosphorylation of serine 390 in PR-A regulates its subsequent ubiquitination and protein stability. Expression of PR-A(S390A) mutant in the human breast epithelial cells, MCF-10A, results in enhanced proliferation and formation of aberrant acini structure in the three-dimensional culture. Consistently, reduction of phosphorylation of serine 390 of PR-A and GSK-3β activity is observed in the Brca1-deficient mammary gland. Taken together, these results provide important aspects of tissue specificity of BRCA1-mediated suppression of breast carcinogenesis.

  2. Glycogen synthase kinase-3--a promising therapeutic target: Dr Hagit Eldar-Finkelman interviewed by Emma Quigley.

    PubMed

    Eldar-Finkelman, Hagit

    2006-04-01

    Dr Hagit Eldar-Finkelman (Sackler School of Medicine, Israel) was interviewed by Emma Quigley (Commissioning Editor, Expert Opinion on Therapeutic Targets) on 16th February 2006. Born in Jerusalem, Dr Eldar-Finkelman received her BSc in Chemistry in 1984 and both her MSc in Physical Chemistry (1986) and PhD in Life Science (1993) from the Weizmann Institute of Science. She was a recipient of the British Council Award, which allowed her to conduct research in biological nuclear magnetic resonance at the University of Oxford in the laboratory of Professor George K Radda. Following postdoctoral work at the School of Medicine of the University of Washington with Nobel Laureate Professor Edwin G Krebs, she became an Assistant Professor in the Department of Medicine at Harvard Medical School. Dr Eldar-Finkelman joined the Sackler School of Medicine at Tel Aviv University in 1999. Dr Eldar-Finkelman's research focuses on the molecular mechanisms regulating the protein kinase glycogen synthase kinase-3 (GSK-3), and their implications in negative regulation of signalling pathways. In particular, her work aims to develop specific inhibitors for GSK-3 and to test their functions in vitro and in vivo, considering the concept that such inhibitors may be useful in insulin resistance and Type 2 diabetes. These studies provide a conceptual basis for development of GSK-3 inhibitors and may lead to design of small molecules for treatment of diabetes and or neurodegenerative disorders.

  3. Glycogen Synthase Kinase 3β Is Positively Regulated by Protein Kinase Cζ-Mediated Phosphorylation Induced by Wnt Agonists

    PubMed Central

    Tejeda-Muñoz, Nydia; González-Aguilar, Héctor; Santoyo-Ramos, Paula; Castañeda-Patlán, M. Cristina

    2015-01-01

    The molecular events that drive Wnt-induced regulation of glycogen synthase kinase 3β (GSK-3β) activity are poorly defined. In this study, we found that protein kinase Cζ (PKCζ) and GSK-3β interact mainly in colon cancer cells. Wnt stimulation induced a rapid GSK-3β redistribution from the cytoplasm to the nuclei in malignant cells and a transient PKC-mediated phosphorylation of GSK-3β at a different site from serine 9. In addition, while Wnt treatment induced a decrease in PKC-mediated phosphorylation of GSK-3β in nonmalignant cells, in malignant cells, this phosphorylation was increased. Pharmacological inhibition and small interfering RNA (siRNA)-mediated silencing of PKCζ abolished all of these effects, but unexpectedly, it also abolished the constitutive basal activity of GSK-3β. In vitro activity assays demonstrated that GSK-3β phosphorylation mediated by PKCζ enhanced GSK-3β activity. We mapped Ser147 of GSK-3β as the site phosphorylated by PKCζ, i.e., its mutation into alanine abolished GSK-3β activity, resulting in β-catenin stabilization and increased transcriptional activity, whereas phosphomimetic replacement of Ser147 by glutamic acid maintained GSK-3β basal activity. Thus, we found that PKCζ phosphorylates GSK-3β at Ser147 to maintain its constitutive activity in resting cells and that Wnt stimulation modifies the phosphorylation of Ser147 to regulate GSK-3β activity in opposite manners in normal and malignant colon cells. PMID:26711256

  4. Glycogen synthase kinase 3 regulates PAX3-FKHR-mediated cell proliferation in human alveolar rhabdomyosarcoma cells

    SciTech Connect

    Zeng, Fu-Yue; Dong, Hanqing; Cui, Jimmy; Liu, Lingling; Chen, Taosheng

    2010-01-01

    Patients with alveolar rhabdomyosarcoma (ARMS) have poorer response to conventional chemotherapy and lower survival rates than those with embryonal RMS (ERMS). To identify compounds that preferentially block the growth of ARMS, we conducted a small-scale screen of 160 kinase inhibitors against the ARMS cell line Rh30 and ERMS cell line RD and identified inhibitors of glycogen synthase kinase 3 (GSK3), including TWS119 as ARMS-selective inhibitors. GSK3 inhibitors inhibited cell proliferation and induced apoptosis more effectively in Rh30 than RD cells. Ectopic expression of fusion protein PAX3-FKHR in RD cells significantly increased their sensitivity to TWS119. Down-regulation of GSK3 by GSK3 inhibitors or siRNA significantly reduced the transcriptional activity of PAX3-FKHR. These results suggest that GSK3 is directly involved in regulating the transcriptional activity of PAX3-FKHR. Also, GSK3 phosphorylated PAX3-FKHR in vitro, suggesting that GSK3 might regulate PAX3-FKHR activity via phosphorylation. These findings support a novel mechanism of PAX3-FKHR regulation by GSK3 and provide a novel strategy to develop GSK inhibitors as anti-ARMS therapies.

  5. Presenilin influences glycogen synthase kinase-3 β (GSK-3β) for kinesin-1 and dynein function during axonal transport.

    PubMed

    Dolma, Kunsang; Iacobucci, Gary J; Hong Zheng, Kan; Shandilya, Jayasha; Toska, Eneda; White, Joseph A; Spina, Elizabeth; Gunawardena, Shermali

    2014-03-01

    Within axons, molecular motors transport essential components required for neuronal growth and viability. Although many levels of control and regulation must exist for proper anterograde and retrograde transport of vital proteins, little is known about these mechanisms. We previously showed that presenilin (PS), a gene involved in Alzheimer's disease (AD), influences kinesin-1 and dynein function in vivo. Here, we show that these PS-mediated effects on motor protein function are via a pathway that involves glycogen synthase kinase-3β (GSK-3β). PS genetically interacts with GSK-3β in an activity-dependent manner. Excess of active GSK-3β perturbed axonal transport by causing axonal blockages, which were enhanced by reduction of kinesin-1 or dynein. These GSK-3β-mediated axonal defects do not appear to be caused by disruptions or alterations in microtubules (MTs). Excess of non-functional GSK-3β did not affect axonal transport. Strikingly, GSK-3β-activity-dependent axonal transport defects were enhanced by reduction of PS. Collectively, our findings suggest that PS and GSK-3β are required for normal motor protein function. Our observations propose a model, in which PS likely plays a role in regulating GSK-3β activity during transport. These findings have important implications for our understanding of the complex regulatory machinery that must exist in vivo and how this system is coordinated during the motility of vesicles within axons.

  6. TLR signalling affects sperm mitochondrial function and motility via phosphatidylinositol 3-kinase and glycogen synthase kinase-3α.

    PubMed

    Zhu, Xingxing; Shi, Dongyan; Li, Xiaoqian; Gong, Weijuan; Wu, Fengjiao; Guo, Xuejiang; Xiao, Hui; Liu, Lixin; Zhou, Hong

    2016-03-01

    Infection in male and female genital tracts can lead to infertility. The underlying mechanisms of this process remain unclear. Toll-like receptors (TLRs) recognize conserved structures and respond to pathogens by initiating signals that activate inflammatory gene transcription. Here, we demonstrate that TLR activation in sperm reduces sperm motility via signalling through myeloid differentiation factor 88 (MyD88), phosphatidylinositol 3-kinase (PI3K), and glycogen synthase kinase (GSK)-3α. Upon TLR activation, phosphorylated forms of PI3K and GSK3α were detected in the mitochondria, and the mitochondrial membrane potential was impaired in sperm. In addition, mitochondrial ATP levels were decreased after TLR agonist stimulation. Furthermore, blocking PI3K or GSK3α activation abrogated these effects and reversed the TLR-induced reduction in sperm motility. These results identify a previously unrecognized TLR signalling pathway that leads to dysfunctional sperm mitochondria, which reduce sperm motility. Our study reveals a novel mechanism by which pathogenic infection affects sperm motility and possibly leads to infertility.

  7. Glycogen synthase kinase-3 inhibition by lithium and beryllium suggests the presence of two magnesium binding sites.

    PubMed

    Ryves, W Jonathan; Dajani, Rana; Pearl, Laurence; Harwood, Adrian J

    2002-01-25

    Lithium inhibits (Li(+)) glycogen synthase kinase-3 (GSK-3) by competition for magnesium (Mg(2+)), but not ATP or substrate. Here, we show that the group II metal ion beryllium (Be(2+)) is a potent inhibitor of GSK-3 and competes for both Mg(2+) and ATP. Be(2+) also inhibits the related protein kinase cdc2 at similar potency, but not MAP kinase 2. To compare the actions of Li(+) and Be(2+) on GSK-3, we have devised a novel dual inhibition analysis. When Be(2+) and ADP are present together each interferes with the action of the other, indicating that both agents inhibit GSK-3 at the ATP binding site. In contrast, Li(+) exerts no interference with ADP inhibition or vice versa. We find, however, that Li(+) and Be(2+) do interfere with each other. These results suggest that Be(2+) competes for two distinct Mg(2+) binding sites: one is Li(+)-sensitive and the other, which is Li(+)-insensitive, binds the Mg:ATP complex.

  8. Inhibition of glycogen synthase kinase-3β enhances cognitive recovery after stroke: the role of TAK1

    PubMed Central

    Venna, Venugopal Reddy; Benashski, Sharon E.; Chauhan, Anjali

    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-3β (GSK-3β) is involved in several essential intracellular signaling pathways. Unlike many other kinases, GSK-3β is active only when dephosphorylated and activation promotes inflammation and apoptosis. In contrast, increased phosphorylation leads to reduced GSK-3β (pGSK-3β) activity. GSK-3β inhibition has beneficial effects on memory in other disease models. GSK-3β regulates both the 5′AMP-activated kinase (AMPK) and transforming growth factor-β-activated kinase (TAK1) pathways. In this work, we examined the effect of GSK-3β inhibition, both independently, in conjunction with a TAK inhibitor, and in AMPK-α2 deficient mice, after stroke to investigate mechanistic interactions between these pathways. GSK-3β inhibition was neuroprotective and ameliorated stroke-induced cognitive impairments. This was independent of AMPK signaling as the protective effects of GSK-3β inhibition were seen in AMPK deficient mice. However, GSK-3β inhibition provided no additive protection in mice treated with a TAK inhibitor suggesting that TAK1 is an upstream regulator of GSK-3β. Targeting GSK-3β could be a novel therapeutic strategy for post-stroke cognitive deficits. PMID:26077686

  9. Diabetes in Pregnancy Adversely Affects the Expression of Glycogen Synthase Kinase-3β in the Hippocampus of Rat Neonates.

    PubMed

    Hami, Javad; Karimi, Razieh; Haghir, Hossein; Gholamin, Mehran; Sadr-Nabavi, Ariane

    2015-10-01

    Diabetes during pregnancy causes a wide range of neurodevelopmental and neurocognitive abnormalities in offspring. Glycogen synthase kinase-3 (GSK-3) is widely expressed during brain development and regulates multiple cellular processes, and its dysregulation is implicated in the pathogenesis of diverse neurodegenerative and psychological diseases. This study was designed to examine the effects of maternal diabetes on GSK-3β messenger RNA (mRNA) expression and phosphorylation in the developing rat hippocampus. Female rats were maintained diabetic from a week before pregnancy through parturition, and male offspring was killed immediately after birth. We found a significant bilateral upregulation of GSK-3β mRNA expression in the hippocampus of pups born to diabetic mothers at P0, compared to controls. Moreover, at the same time point, there was a marked bilateral increase in the phosphorylation level of GSK-3β in the diabetic group. Unlike phosphorylation levels, there was a significant upregulation in hippocampal GSK-3β mRNA expression in the insulin-treated group, when compared to controls. The present study revealed that diabetes during pregnancy strongly influences the regulation of GSK-3β in the right/left developing hippocampi. These dysregulations may be part of the cascade of events through which diabetes during pregnancy affects the newborn's hippocampal structure and function.

  10. Inhibition of glycogen synthase kinase-3β enhances cognitive recovery after stroke: the role of TAK1.

    PubMed

    Venna, Venugopal Reddy; Benashski, Sharon E; Chauhan, Anjali; McCullough, Louise D

    2015-07-01

    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-3β (GSK-3β) is involved in several essential intracellular signaling pathways. Unlike many other kinases, GSK-3β is active only when dephosphorylated and activation promotes inflammation and apoptosis. In contrast, increased phosphorylation leads to reduced GSK-3β (pGSK-3β) activity. GSK-3β inhibition has beneficial effects on memory in other disease models. GSK-3β regulates both the 5'AMP-activated kinase (AMPK) and transforming growth factor-β-activated kinase (TAK1) pathways. In this work, we examined the effect of GSK-3β inhibition, both independently, in conjunction with a TAK inhibitor, and in AMPK-α2 deficient mice, after stroke to investigate mechanistic interactions between these pathways. GSK-3β inhibition was neuroprotective and ameliorated stroke-induced cognitive impairments. This was independent of AMPK signaling as the protective effects of GSK-3β inhibition were seen in AMPK deficient mice. However, GSK-3β inhibition provided no additive protection in mice treated with a TAK inhibitor suggesting that TAK1 is an upstream regulator of GSK-3β. Targeting GSK-3β could be a novel therapeutic strategy for post-stroke cognitive deficits.

  11. Glycogen Synthase Kinase 3 (GSK-3) influences epithelial barrier function by regulating Occludin, Claudin-1 and E-cadherin expression

    SciTech Connect

    Severson, Eric A.; Kwon, Mike; Hilgarth, Roland S.; Parkos, Charles A.; Nusrat, Asma

    2010-07-02

    The Apical Junctional Complex (AJC) encompassing the tight junction (TJ) and adherens junction (AJ) plays a pivotal role in regulating epithelial barrier function and epithelial cell proliferative processes through signaling events that remain poorly characterized. A potential regulator of AJC protein expression is Glycogen Synthase Kinase-3 (GSK-3). GSK-3 is a constitutively active kinase that is repressed during epithelial-mesenchymal transition (EMT). In the present study, we report that GSK-3 activity regulates the structure and function of the AJC in polarized model intestinal (SK-CO15) and kidney (Madin-Darby Canine Kidney (MDCK)) epithelial cells. Reduction of GSK-3 activity, either by small molecule inhibitors or siRNA targeting GSK-3 alpha and beta mRNA, resulted in increased permeability to both ions and bulk solutes. Immunofluorescence labeling and immunoblot analyses revealed that the barrier defects correlated with decreased protein expression of AJC transmembrane proteins Occludin, Claudin-1 and E-cadherin without influencing other TJ proteins, Zonula Occludens-1 (ZO-1) and Junctional Adhesion Molecule A (JAM-A). The decrease in Occludin and E-cadherin protein expression correlated with downregulation of the corresponding mRNA levels for these respective proteins following GSK-3 inhibition. These observations implicate an important role of GSK-3 in the regulation of the structure and function of the AJC that is mediated by differential modulation of mRNA transcription of key AJC proteins, Occludin, Claudin-1 and E-cadherin.

  12. TNF-α expression in neutrophils and its regulation by glycogen synthase kinase-3: a potentiating role for lithium.

    PubMed

    Giambelluca, Miriam S; Bertheau-Mailhot, Geneviève; Laflamme, Cynthia; Rollet-Labelle, Emmanuelle; Servant, Marc J; Pouliot, Marc

    2014-08-01

    Glycogen synthase kinase 3 (GSK-3) is associated with several cellular systems, including immune response. Lithium, a widely used pharmacological treatment for bipolar disorder, is a GSK-3 inhibitor. GSK-3α is the predominant isoform in human neutrophils. In this study, we examined the effect of GSK-3 inhibition on the production of TNF-α by neutrophils. In the murine air pouch model of inflammation, lithium chloride (LiCl) amplified TNF-α release. In lipopolysaccharide-stimulated human neutrophils, GSK-3 inhibitors mimicked the effect of LiCl, each potentiating TNF-α release after 4 h, in a concentration-dependent fashion, by up to a 3-fold increase (ED50 of 1 mM for lithium). LiCl had no significant effect on cell viability. A positive association was revealed between GSK-3 inhibition and prolonged activation of the p38/MNK1/eIF4E pathway of mRNA translation. Using lysine and arginine labeled with stable heavy isotopes followed by quantitative mass spectrometry, we determined that GSK-3 inhibition markedly increases (by more than 3-fold) de novo TNF-α protein synthesis. Our findings shed light on a novel mechanism of control of TNF-α expression in neutrophils with GSK-3 regulating mRNA translation and raise the possibility that lithium could be having a hitherto unforeseen effect on inflammatory diseases.

  13. Inhibition of glycogen synthase kinase-3 enhances the differentiation and reduces the proliferation of adult human olfactory epithelium neural precursors

    SciTech Connect

    Manceur, Aziza P.; Tseng, Michael; Holowacz, Tamara; Witterick, Ian; Weksberg, Rosanna; McCurdy, Richard D.; Warsh, Jerry J.; Audet, Julie

    2011-09-10

    The olfactory epithelium (OE) contains neural precursor cells which can be easily harvested from a minimally invasive nasal biopsy, making them a valuable cell source to study human neural cell lineages in health and disease. Glycogen synthase kinase-3 (GSK-3) has been implicated in the etiology and treatment of neuropsychiatric disorders and also in the regulation of murine neural precursor cell fate in vitro and in vivo. In this study, we examined the impact of decreased GSK-3 activity on the fate of adult human OE neural precursors in vitro. GSK-3 inhibition was achieved using ATP-competitive (6-bromoindirubin-3'-oxime and CHIR99021) or substrate-competitive (TAT-eIF2B) inhibitors to eliminate potential confounding effects on cell fate due to off-target kinase inhibition. GSK-3 inhibitors decreased the number of neural precursor cells in OE cell cultures through a reduction in proliferation. Decreased proliferation was not associated with a reduction in cell survival but was accompanied by a reduction in nestin expression and a substantial increase in the expression of the neuronal differentiation markers MAP1B and neurofilament (NF-M) after 10 days in culture. Taken together, these results suggest that GSK-3 inhibition promotes the early stages of neuronal differentiation in cultures of adult human neural precursors and provide insights into the mechanisms by which alterations in GSK-3 signaling affect adult human neurogenesis, a cellular process strongly suspected to play a role in the etiology of neuropsychiatric disorders.

  14. Structural and Functional Characterization of Nrf2 Degradation by the Glycogen Synthase Kinase 3/β-TrCP Axis

    PubMed Central

    Rada, Patricia; Rojo, Ana I.; Evrard-Todeschi, Nathalie; Innamorato, Nadia G.; Cotte, Axelle; Jaworski, Tomasz; Tobón-Velasco, Julio C.; Devijver, Herman; García-Mayoral, María Flor; Van Leuven, Fred; Hayes, John D.

    2012-01-01

    The transcription factor NF-E2-related factor 2 (Nrf2) is a master regulator of a genetic program, termed the phase 2 response, that controls redox homeostasis and participates in multiple aspects of physiology and pathology. Nrf2 protein stability is regulated by two E3 ubiquitin ligase adaptors, Keap1 and β-TrCP, the latter of which was only recently reported. Here, two-dimensional (2D) gel electrophoresis and site-directed mutagenesis allowed us to identify two serines of Nrf2 that are phosphorylated by glycogen synthase kinase 3β (GSK-3β) in the sequence DSGISL. Nuclear magnetic resonance studies defined key residues of this phosphosequence involved in docking to the WD40 propeller of β-TrCP, through electrostatic and hydrophobic interactions. We also identified three arginine residues of β-TrCP that participate in Nrf2 docking. Intraperitoneal injection of the GSK-3 inhibitor SB216763 led to increased Nrf2 and heme oxygenase-1 levels in liver and hippocampus. Moreover, mice with hippocampal absence of GSK-3β exhibited increased levels of Nrf2 and phase 2 gene products, reduced glutathione, and decreased levels of carbonylated proteins and malondialdehyde. This study establishes the structural parameters of the interaction of Nrf2 with the GSK-3/β-TrCP axis and its functional relevance in the regulation of Nrf2 by the signaling pathways that impinge on GSK-3. PMID:22751928

  15. 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.

  16. 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

  17. Targeted disruption of glycogen synthase kinase 3A (GSK3A) in mice affects sperm motility resulting in male infertility.

    PubMed

    Bhattacharjee, Rahul; Goswami, Suranjana; Dudiki, Tejasvi; Popkie, Anthony P; Phiel, Christopher J; Kline, Douglas; Vijayaraghavan, Srinivasan

    2015-03-01

    The signaling enzyme glycogen synthase kinase 3 (GSK3) exists as two isoforms-GSK3A and GSK3B. Protein phosphorylation by GSK3 has important signaling roles in several cells. In our past work, we found that both isoforms of GSK3 are present in mouse sperm and that catalytic GSK3 activity correlates with motility of sperm from several species. Here, we examined the role of Gsk3a in male fertility using a targeted gene knockout (KO) approach. The mutant mice are viable, but have a male infertility phenotype, while female fertility is unaffected. Testis weights of Gsk3a(-/-) mice are normal and sperm are produced in normal numbers. Although spermatogenesis is apparently unimpaired, sperm motility parameters in vitro are impaired. In addition, the flagellar waveform appears abnormal, characterized by low amplitude of flagellar beat. Sperm ATP levels were lower in Gsk3a(-/-) mice compared to wild-type animals. Protein phosphatase PP1 gamma2 protein levels were unaltered, but its catalytic activity was elevated in KO sperm. Remarkably, tyrosine phosphorylation of hexokinase and capacitation-associated changes in tyrosine phosphorylation of proteins are absent or significantly lower in Gsk3a(-/-) sperm. The GSK3B isoform was present and unaltered in testis and sperm of Gsk3a(-/-) mice, showing the inability of GSK3B to substitute for GSK3A in this context. Our studies show that sperm GSK3A is essential for male fertility. In addition, the GSK3A isoform, with its highly conserved glycine-rich N terminus in mammals, may have an isoform-specific role in its requirement for normal sperm motility and fertility.

  18. Decreased glycogen synthase kinase 3-beta levels and related physiological changes in Bacillus anthracis lethal toxin-treated macrophages.

    PubMed

    Tucker, Amy E; Salles, Isabelle I; Voth, Daniel E; Ortiz-Leduc, William; Wang, Han; Dozmorov, Igor; Centola, Michael; Ballard, Jimmy D

    2003-08-01

    The lethal factor (LF) component of Bacillus anthracis lethal toxin (LeTx) cleaves mitogen activated protein kinase kinases (MAPKKs) in a variety of different cell types, yet only macrophages are rapidly killed by this toxin. The reason for this selective killing is unclear, but suggests other factors may also be involved in LeTx intoxication. In the current study, DNA membrane arrays were used to identify broad changes in macrophage physiology after treatment with LeTx. Expression of genes regulated by MAPKK activity did not change significantly, yet a series of genes under glycogen synthase kinase-3-beta (GSK-3beta) regulation changed expression following LeTx treatment. Correlating with these transcriptional changes GSK-3beta was found to be below detectable levels in toxin-treated cells and an inhibitor of GSK-3beta, LiCl, sensitized resistant IC-21 macrophages to LeTx. In addition, zebrafish embryos treated with LeTx showed signs of delayed pigmentation and cardiac hypertrophy; both processes are subject to regulation by GSK-3beta. A putative compensatory response to loss of GSK-3beta was indicated by differential expression of three motor proteins following toxin treatment and Kif1C, a motor protein involved in sensitivity to LeTx, increased expression in toxin-sensitive cells yet decreased in resistant cells following toxin treatment. Differential expression of microtubule-associating proteins and a decrease in the level of cellular tubulin were detected in LeTx-treated cells, both of which can result from loss of GSK-3beta activity. These data provide new information on LeTx's overall influence on macrophage physiology and suggest loss of GSK-3beta contributes to cytotoxicity. PMID:12864812

  19. Beyond the brain: disrupted in schizophrenia 1 regulates pancreatic β-cell function via glycogen synthase kinase-3β.

    PubMed

    Jurczyk, Agata; Nowosielska, Anetta; Przewozniak, Natalia; Aryee, Ken-Edwin; DiIorio, Philip; Blodgett, David; Yang, Chaoxing; Campbell-Thompson, Martha; Atkinson, Mark; Shultz, Leonard; Rittenhouse, Ann; Harlan, David; Greiner, Dale; Bortell, Rita

    2016-02-01

    Individuals with schizophrenia and their first-degree relatives have higher rates of type 2 diabetes (T2D) than the general population (18-30 vs. 1.2-6.3%), independent of body mass index and antipsychotic medication, suggesting shared genetic components may contribute to both diseases. The cause of this association remains unknown. Mutations in disrupted in schizophrenia 1 (DISC1) increase the risk of developing psychiatric disorders [logarithm (base 10) of odds = 7.1]. Here, we identified DISC1 as a major player controlling pancreatic β-cell proliferation and insulin secretion via regulation of glycogen synthase kinase-3β (GSK3β). DISC1 expression was enriched in developing mouse and human pancreas and adult β- and ductal cells. Loss of DISC1 function, through siRNA-mediated depletion or expression of a dominant-negative truncation that models the chromosomal translocation of human DISC1 in schizophrenia, resulted in decreased β-cell proliferation (3 vs. 1%; P < 0.01), increased apoptosis (0.1 vs. 0.6%; P < 0.01), and glucose intolerance in transgenic mice. Insulin secretion was reduced (0.5 vs. 0.1 ng/ml; P < 0.05), and critical β-cell transcription factors Pdx1 and Nkx6.1 were significantly decreased. Impaired DISC1 allowed inappropriate activation of GSK3β in β cells, and antagonizing GSK3β (SB216763; IC50 = 34.3 nM) rescued the β-cell defects. These results uncover an unexpected role for DISC1 in normal β-cell physiology and suggest that DISC1 dysregulation contributes to T2D independently of its importance for cognition. PMID:26546129

  20. RNA Interference Silencing of Glycogen Synthase Kinase 3β Inhibites Tau Phosphorylation in Mice with Alzheimer Disease.

    PubMed

    Bian, Hong; Bian, Wei; Lin, Xiaoying; Ma, Zhaoyin; Chen, Wen; Pu, Ying

    2016-09-01

    To explore the effect of glycogen synthase kinase 3β (GSK-3β) silencing on Tau-5 phosphorylation in mice suffering Alzheimer disease (AD). GSK-3β was firstly silenced in human neuroblastoma SH-SY5Y cells using special lentivirus (LV) and the content of Tau (A-12), p-Tau (Ser396) and p-Tau (PHF-6) proteins. GSK-3β was also silenced in APP/PS1 mouse model of AD mice, which were divided into three groups (n = 10): AD, vehicle, and LV group. Ten C57 mice were used as control. The memory ability of mice was tested by square water maze, and the morphological changes of hippocampus and neuron death were analyzed by haematoxylin-eosin staining. Moreover, the levels of Tau and phosphorylated Tau (p-Tau) were detected by western blotting and immunohistochemistry, respectively. The lentivirus-mediated GSK-3β silencing system was successfully developed and silencing GSK-3β at the cellular level reduced Tau phosphorylation obviously. Moreover, GSK-3β silence significantly improved the memory ability of AD mice in LV group compared with AD group (P < 0.05) according to the latency periods and error numbers. As for the hippocampus morphology and neuron death, no significant change was observed between LV group and normal control. Immunohistochemical detection and western blotting revealed that the levels of Tau and p-Tau were significantly down-regulated after GSK-3β silence. Silencing GSK-3β may have a positive effect on inhibiting the pathologic progression of AD through down-regulating the level of p-Tau.

  1. Pulmonary artery smooth muscle hypertrophy: roles of glycogen synthase kinase-3β and p70 ribosomal S6 kinase

    PubMed Central

    Deng, Huan; Hershenson, Marc B.; Lei, Jing; Anyanwu, Anuli C.; Pinsky, David J.

    2010-01-01

    Increased medial arterial thickness is a structural change in pulmonary arterial hypertension (PAH). The role of smooth muscle hypertrophy in this process has not been well studied. Bone morphogenetic proteins (BMPs), transforming growth factor (TGF)-β1, serotonin (or 5-hydroxytryptamine; 5-HT), and endothelin (ET)-1 have been implicated in PAH pathogenesis. We examined the effect of these mediators on human pulmonary artery smooth muscle cell size, contractile protein expression, and contractile function, as well on the roles of glycogen synthase kinase (GSK)-3β and p70 ribosomal S6 kinase (p70S6K), two proteins involved in translational control, in this process. Unlike epidermal growth factor, BMP-4, TGF-β1, 5-HT, and ET-1 each increased smooth muscle cell size, contractile protein expression, fractional cell shortening, and GSK-3β phosphorylation. GSK-3β inhibition by lithium or SB-216763 increased cell size, protein synthesis, and contractile protein expression. Expression of a non-phosphorylatable GSK-3β mutant blocked BMP-4-, TGF-β1-, 5-HT-, and ET-1-induced cell size enlargement, suggesting that GSK-3β phosphorylation is required and sufficient for cellular hypertrophy. However, BMP-4, TGF-β1, 5-HT, and ET-1 stimulation was accompanied by an increase in serum response factor transcriptional activation but not eIF2 phosphorylation, suggesting that GSK-3β-mediated hypertrophy occurs via transcriptional, not translational, control. Finally, BMP-4, TGF-β1, 5-HT, and ET-1 treatment induced phosphorylation of p70S6K and ribosomal protein S6, and siRNAs against p70S6K and S6 blocked the hypertrophic response. We conclude that mediators implicated in the pathogenesis of PAH induce pulmonary arterial smooth muscle hypertrophy. Identification of the signaling pathways regulating vascular smooth muscle hypertrophy may define new therapeutic targets for PAH. PMID:20190034

  2. 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

  3. Selective enhancement of the uptake and bioactivity of a TAT-conjugated peptide inhibitor of glycogen synthase kinase-3.

    PubMed

    Manceur, Aziza P; Driscoll, Brandon D; Sun, Wei; Audet, Julie

    2009-03-01

    The use of cell-penetrating peptides as transduction vectors is a promising approach to deliver peptides and proteins into cells. However, the uptake and bioavailability of trans-activating transcriptor (TAT)-conjugated molecules vary depending on the cell type and the cargo. This study aimed to determine whether a low-voltage electrical pulse can enhance the TAT-mediated delivery of peptide cargoes in different cell types. In TF-1 and mouse embryonic stem cells, the uptake of a novel detachable TAT-conjugated glycogen synthase kinase-3 (GSK-3) peptide inhibitor was enhanced by an order of magnitude without affecting the cell viability. A similar increase in uptake was achieved in primary mouse bone marrow cells while maintaining >80% of their viability. Interestingly, under these low-voltage conditions, the uptake of a control peptide not conjugated to TAT was not significantly increased. A T-cell factor/lymphoid enhancer factor (TCF/LEF) luciferase reporter assay was also used to assess the bioactivity of the TAT construct. The results indicated that cells loaded with a low-voltage electrical pulse had a twofold increase in TCF/LEF activity, which was equivalent to a level of GSK-3 inhibition similar to that of cells treated with 20 mmol/l lithium or 500 nmol/l (2'Z,3'E)-6-bromoindirubin-3'-oxime. These results demonstrate the usefulness of low-voltage electrical pulses to enhance the uptake and bioactivity of TAT-conjugated molecules in different cell types. PMID:19107119

  4. Hydrosulfide attenuates acute myocardial ischemic injury through the glycogen synthase kinase-3β/β-catenin signaling pathway.

    PubMed

    Ge, Ning; Liu, Chao; Li, Guofeng; Xie, Lijun; Zhang, Qinzeng; Li, Liping; Hao, Na; Zhang, Jianxin

    2016-05-01

    The endogenous signaling gasotransmitter, hydrosulfide (H2S), has been shown to exert cardioprotective effects against acute myocardial infarction (AMI) due to ischemic injury. However, the mechanisms responsible for these effects are not yet fully understood. In this study, we investigated whether sodium hydrogen sulfide (NaHS), an H2S donor, attenuates acute myocardial ischemic injury through glycogen synthase kinase-3β (GSK-3β)/β-catenin signaling. For this purpose, we utilized an in vivo rat model of AMI by occluding the left anterior descending coronary artery. NaHS (0.39, 0.78 or 1.56 mg/kg, intraperitoneally), the GSK-3β inhibitor, SB216763 (0.6 mg/kg, intravenously), or 1% dimethylsulfoxide (2 ml/kg, intravenously) were administered to the rats. The results demonstrated that the administration of medium- and high-dose NaHS and SB216763 significantly improved rat cardiac function, as evidenced by an increase in the mean arterial pressure, left ventricular developed pressure, contraction and relaxation rates, as well as a decrease in left ventricular end-diastolic pressure. In addition, the administration of NaHS and SB216763 attenuated myocardial injury as reflected by a decrease in apoptotic cell death and in the serum lactate dehydrogenase concentrations, and prevented myocardial structural changes. The administration of NaHS and SB216763 increased the concentrations of phosphorylated (p-)GSK-3β, the p-GSK-3β/t-GSK-3β ratio and downstream protein β-catenin. Moreover, western blot and immunohistochemical analyses of apoptotic signaling pathway proteins further established the cardioprotective potential of NaHS, as reflected by the upregulation of Bcl-2 expression, the downregulation of Bax expression, and a decrease in the number of TUNEL-positive stained cells. These findings suggest that hydrosulfide exerts cardioprotective effects against AMI-induced apoptosis through the GSK-3β/β-catenin signaling pathway.

  5. Hydrosulfide attenuates acute myocardial ischemic injury through the glycogen synthase kinase-3β/β-catenin signaling pathway

    PubMed Central

    GE, NING; LIU, CHAO; LI, GUOFENG; XIE, LIJUN; ZHANG, QINZENG; LI, LIPING; HAO, NA; ZHANG, JIANXIN

    2016-01-01

    The endogenous signaling gasotransmitter, hydrosulfide (H2S), has been shown to exert cardioprotective effects against acute myocardial infarction (AMI) due to ischemic injury. However, the mechanisms responsible for these effects are not yet fully understood. In this study, we investigated whether sodium hydrogen sulfide (NaHS), an H2S donor, attenuates acute myocardial ischemic injury through glycogen synthase kinase-3β (GSK-3β)/β-catenin signaling. For this purpose, we utilized an in vivo rat model of AMI by occluding the left anterior descending coronary artery. NaHS (0.39, 0.78 or 1.56 mg/kg, intraperitoneally), the GSK-3β inhibitor, SB216763 (0.6 mg/kg, intravenously), or 1% dimethylsulfoxide (2 ml/kg, intravenously) were administered to the rats. The results demonstrated that the administration of medium- and high-dose NaHS and SB216763 significantly improved rat cardiac function, as evidenced by an increase in the mean arterial pressure, left ventricular developed pressure, contraction and relaxation rates, as well as a decrease in left ventricular end-diastolic pressure. In addition, the administration of NaHS and SB216763 attenuated myocardial injury as reflected by a decrease in apoptotic cell death and in the serum lactate dehydrogenase concentrations, and prevented myocardial structural changes. The administration of NaHS and SB216763 increased the concentrations of phosphorylated (p-)GSK-3β, the p-GSK-3β/t-GSK-3β ratio and downstream protein β-catenin. Moreover, western blot and immunohistochemical analyses of apoptotic signaling pathway proteins further established the cardioprotective potential of NaHS, as reflected by the upregulation of Bcl-2 expression, the downregulation of Bax expression, and a decrease in the number of TUNEL-positive stained cells. These findings suggest that hydrosulfide exerts cardioprotective effects against AMI-induced apoptosis through the GSK-3β/β-catenin signaling pathway. PMID:27035393

  6. 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

  7. 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.

  8. Beyond the brain: disrupted in schizophrenia 1 regulates pancreatic β-cell function via glycogen synthase kinase-3β.

    PubMed

    Jurczyk, Agata; Nowosielska, Anetta; Przewozniak, Natalia; Aryee, Ken-Edwin; DiIorio, Philip; Blodgett, David; Yang, Chaoxing; Campbell-Thompson, Martha; Atkinson, Mark; Shultz, Leonard; Rittenhouse, Ann; Harlan, David; Greiner, Dale; Bortell, Rita

    2016-02-01

    Individuals with schizophrenia and their first-degree relatives have higher rates of type 2 diabetes (T2D) than the general population (18-30 vs. 1.2-6.3%), independent of body mass index and antipsychotic medication, suggesting shared genetic components may contribute to both diseases. The cause of this association remains unknown. Mutations in disrupted in schizophrenia 1 (DISC1) increase the risk of developing psychiatric disorders [logarithm (base 10) of odds = 7.1]. Here, we identified DISC1 as a major player controlling pancreatic β-cell proliferation and insulin secretion via regulation of glycogen synthase kinase-3β (GSK3β). DISC1 expression was enriched in developing mouse and human pancreas and adult β- and ductal cells. Loss of DISC1 function, through siRNA-mediated depletion or expression of a dominant-negative truncation that models the chromosomal translocation of human DISC1 in schizophrenia, resulted in decreased β-cell proliferation (3 vs. 1%; P < 0.01), increased apoptosis (0.1 vs. 0.6%; P < 0.01), and glucose intolerance in transgenic mice. Insulin secretion was reduced (0.5 vs. 0.1 ng/ml; P < 0.05), and critical β-cell transcription factors Pdx1 and Nkx6.1 were significantly decreased. Impaired DISC1 allowed inappropriate activation of GSK3β in β cells, and antagonizing GSK3β (SB216763; IC50 = 34.3 nM) rescued the β-cell defects. These results uncover an unexpected role for DISC1 in normal β-cell physiology and suggest that DISC1 dysregulation contributes to T2D independently of its importance for cognition.

  9. Inhibition of glycogen synthase kinase (GSK)-3-β improves liver microcirculation and hepatocellular function after hemorrhagic shock.

    PubMed

    Jellestad, Lena; Fink, Tobias; Pradarutti, Sascha; Kubulus, Darius; Wolf, Beate; Bauer, Inge; Thiemermann, Chris; Rensing, Hauke

    2014-02-01

    Ischemia and reperfusion may cause liver injury and are characterized by hepatic microperfusion failure and a decreased hepatocellular function. Inhibition of glycogen synthase kinase (GSK)-3β, a serine-threonine kinase that has recently emerged as a key regulator in the modulation of the inflammatory response after stress events, may be protective in conditions like sepsis, inflammation and shock. Therefore, aim of the study was to assess the role of GSK-3β in liver microcirculation and hepatocellular function after hemorrhagic shock and resuscitation (H/R). Anesthetized male Sprague-Dawley rats underwent pretreatment with Ringer´s solution, vehicle (DMSO) or TDZD-8 (1 mg/kg), a selective GSK-3β inhibitor, 30 min before induction of hemorrhagic shock (mean arterial pressure 35±5 mmHg for 90 min) and were resuscitated with shed blood and Ringer´s solution (2h). 5h after resuscitation hepatic microcirculation was assessed by intravital microscopy. Propidium iodide (PI) positive cells, liver enzymes and alpha-GST were measured as indicators of hepatic injury. Liver function was estimated by assessment of indocyanine green plasma disappearance rate. H/R led to a significant decrease in sinusoidal diameters and impairment of liver function compared to sham operation. Furthermore, the number of PI positive cells in the liver as well as serum activities of liver enzymes and alpha-GST increased significantly after H/R. Pretreatment with TDZD-8 prevented the changes in liver microcirculation, hepatocellular injury and liver function after H/R. A significant rise in the plasma level of IL-10 was observed. Thus, inhibition of GSK-3β before hemorrhagic shock modulates the inflammatory response and improves hepatic microcirculation and hepatocellular function.

  10. Glycogen Synthase Kinase 3 Regulates Cell Death and Survival Signaling in Tumor Cells under Redox Stress1

    PubMed Central

    Venè, Roberta; Cardinali, Barbara; Arena, Giuseppe; Ferrari, Nicoletta; Benelli, Roberto; Minghelli, Simona; Poggi, Alessandro; Noonan, Douglas M.; Albini, Adriana; Tosetti, Francesca

    2014-01-01

    Targeting tumor-specific metabolic adaptations is a promising anticancer strategy when tumor defense mechanisms are restrained. Here, we show that redox-modulating drugs including the retinoid N-(4-hydroxyphenyl)retinamide (4HPR), the synthetic triterpenoid bardoxolone (2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid methyl ester), arsenic trioxide (As2O3), and phenylethyl isothiocyanate (PEITC), while affecting tumor cell viability, induce sustained Ser9 phosphorylation of the multifunctional kinase glycogen synthase kinase 3β (GSK3β). The antioxidant N-acetylcysteine decreased GSK3β phosphorylation and poly(ADP-ribose) polymerase cleavage induced by 4HPR, As2O3, and PEITC, implicating oxidative stress in these effects. GSK3β phosphorylation was associated with up-regulation of antioxidant enzymes, in particular heme oxygenase-1 (HO-1), and transient elevation of intracellular glutathione (GSH) in cells surviving acute stress, before occurrence of irreversible damage and death. Genetic inactivation of GSK3β or transfection with the non-phosphorylatable GSK3β-S9A mutant inhibited HO-1 induction under redox stress, while tumor cells resistant to 4HPR exhibited increased GSK3β phosphorylation, HO-1 expression, and GSH levels. The above-listed findings are consistent with a role for sustained GSK3β phosphorylation in a signaling network activating antioxidant effector mechanisms during oxidoreductive stress. These data underlie the importance of combination regimens of antitumor redox drugs with inhibitors of survival signaling to improve control of tumor development and progression and overcome chemoresistance. PMID:25246272

  11. Identification of novel scaffold of benzothiazepinones as non-ATP competitive glycogen synthase kinase-3β inhibitors through virtual screening.

    PubMed

    Zhang, Peng; Hu, Hai-Rong; Huang, Zhao-Hui; Lei, Jia-Yi; Chu, Yong; Ye, De-Yong

    2012-12-01

    Glycogen synthase kinase-3β (GSK-3β) is an important serine/threonine kinase that has been proved as a key target for neurodegenerative diseases and diabetes. Up to date, most of known inhibitors are bound to the ATP-binding pocket of GSK-3β, which might lead widespread effects due to the high homology between kinases. Recently, some of its non-ATP competitive inhibitors had been confirmed having therapeutical effects owing to their high selectivity. This finding opens a new pathway to study hopeful drugs for treatment of these diseases. However, it is still a challenge nowadays on how to efficiently find non-ATP competitors. Here, we successfully discovered a novel scaffold of benzothiazepinones (BTZs) as selective non-ATP competitive GSK-3β inhibitors through virtual screening approach. A 3D receptor model of substrate binding site of GSK-3β was constructed and applied to screen against drug-like Maybridge database through Autodock program. BTZ compounds were top ranked as efficient hits and were then synthesized for further screening. Among them, the representative compound 4j showed activity to GSK-3β (IC(50): 25 μM) in non-ATP competitive mechanism, and nearly no inhibitory effect on other 10 related protein kinases. Overall, the results point out that BTZ compounds might be useful in treatment of Alzheimer's disease and diabetes mellitus as novel GSK-3β inhibitors. It also suggests, on the other hand, that virtual screening would provide a valuable tool in combination with in vitro assays for the identification of novel selective and potent inhibitors.

  12. Glycogen synthase kinase 3 beta positively regulates Notch signaling in vascular smooth muscle cells: role in cell proliferation and survival.

    PubMed

    Guha, Shaunta; Cullen, John P; Morrow, David; Colombo, Alberto; Lally, Caitríona; Walls, Dermot; Redmond, Eileen M; Cahill, Paul A

    2011-09-01

    The role of glycogen synthase kinase 3 beta (GSK-3β) in modulating Notch control of vascular smooth muscle cell (vSMC) growth (proliferation and apoptosis) was examined in vitro under varying conditions of cyclic strain and validated in vivo following changes in medial tension and stress. Modulation of GSK-3β in vSMC following ectopic expression of constitutively active GSK-3β, siRNA knockdown and pharmacological inhibition with SB-216763 demonstrated that GSK-3β positively regulates Notch intracellular domain expression, CBF-1/RBP-Jκ transactivation and downstream target gene mRNA levels, while concomitantly promoting vSMC proliferation and inhibiting apoptosis. In contrast, inhibition of GSK-3β attenuated Notch signaling and decreased vSMC proliferation and survival. Exposure of vSMC to cyclic strain environments in vitro using both a Flexercell™ Tension system and a novel Sylgard™ phantom vessel following bare metal stent implantation revealed that cyclic strain inhibits GSK-3β activity independent of p42/p44 MAPK and p38 activation concomitant with reduced Notch signaling and decreased vSMC proliferation and survival. Exposure of vSMC to changes in medial strain microenvironments in vivo following carotid artery ligation revealed that enhanced GSK-3β activity was predominantly localized to medial and neointimal vSMC concomitant with increased Notch signaling, proliferating nuclear antigen and decreased Bax expression, respectively, as vascular remodeling progressed. GSK-3β is an important modulator of Notch signaling leading to altered vSMC cell growth where low strain/tension microenvironments prevail.

  13. Nitric Oxide Synthase and Breast Cancer: Role of TIMP-1 in NO-mediated Akt Activation

    PubMed Central

    Ridnour, Lisa A.; Barasch, Kimberly M.; Windhausen, Alisha N.; Dorsey, Tiffany H.; Lizardo, Michael M.; Yfantis, Harris G.; Lee, Dong H.; Switzer, Christopher H.; Cheng, Robert Y. S.; Heinecke, Julie L.; Brueggemann, Ernst; Hines, Harry B.; Khanna, Chand; Glynn, Sharon A.; Ambs, Stefan; Wink, David A.

    2012-01-01

    Prediction of therapeutic response and cancer patient survival can be improved by the identification of molecular markers including tumor Akt status. A direct correlation between NOS2 expression and elevated Akt phosphorylation status has been observed in breast tumors. Tissue inhibitor matrix metalloproteinase-1 (TIMP-1) has been proposed to exert oncogenic properties through CD63 cell surface receptor pathway initiation of pro-survival PI3k/Akt signaling. We employed immunohistochemistry to examine the influence of TIMP-1 on the functional relationship between NOS2 and phosphorylated Akt in breast tumors and found that NOS2-associated Akt phosphorylation was significantly increased in tumors expressing high TIMP-1, indicating that TIMP-1 may further enhance NO-induced Akt pathway activation. Moreover, TIMP-1 silencing by antisense technology blocked NO-induced PI3k/Akt/BAD phosphorylation in cultured MDA-MB-231 human breast cancer cells. TIMP-1 protein nitration and TIMP-1/CD63 co-immunoprecipitation was observed at NO concentrations that induced PI3k/Akt/BAD pro-survival signaling. In the survival analysis, elevated tumor TIMP-1 predicted poor patient survival. This association appears to be mainly restricted to tumors with high NOS2 protein. In contrast, TIMP-1 did not predict poor survival in patient tumors with low NOS2 expression. In summary, our findings suggest that tumors with high TIMP-1 and NOS2 behave more aggressively by mechanisms that favor Akt pathway activation. PMID:22957045

  14. Inhibition of invasion by glycogen synthase kinase-3 beta inhibitors through dysregulation of actin re-organisation via down-regulation of WAVE2.

    PubMed

    Yoshino, Yuki; Suzuki, Manami; Takahashi, Hidekazu; Ishioka, Chikashi

    2015-08-14

    Cancer cell invasion is a critical phenomenon in cancer pathogenesis. Glycogen synthase kinase-3β (GSK-3β) has been reported to regulate cancer cell invasion both negatively and positively. Thus, the net effect of GSK-3β on invasion is unclear. In this report, we showed that GSK-3β inhibitors induced dysregulation of the actin cytoskeleton and functional insufficiency of focal adhesion, which resulted in suppressed invasion. In addition, WAVE2, an essential molecule for actin fibre branching, was down-regulated after GSK-3β inhibition. Collectively, we propose that the WAVE2-actin cytoskeleton axis is an important target of GSK-3β inhibitors in cancer cell invasion.

  15. The Design and Synthesis of Potent and Selective Inhibitors of Trypanosoma brucei Glycogen Synthase Kinase 3 for the Treatment of Human African Trypanosomiasis

    PubMed Central

    2014-01-01

    Glycogen synthase kinase 3 (GSK3) is a genetically validated drug target for human African trypanosomiasis (HAT), also called African sleeping sickness. We report the synthesis and biological evaluation of aminopyrazole derivatives as Trypanosoma brucei GSK3 short inhibitors. Low nanomolar inhibitors, which had high selectivity over the off-target human CDK2 and good selectivity over human GSK3β enzyme, have been prepared. These potent kinase inhibitors demonstrated low micromolar levels of inhibition of the Trypanosoma brucei brucei parasite grown in culture. PMID:25198388

  16. 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

  17. 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

  18. Neuroprotective effect of hemeoxygenase-1/glycogen synthase kinase-3β modulators in 3-nitropropionic acid-induced neurotoxicity in rats.

    PubMed

    Khan, A; Jamwal, S; Bijjem, K R V; Prakash, A; Kumar, P

    2015-02-26

    The present study has been designed to explore the possible interaction between hemeoxygenase-1 (HO-1) and glycogen synthase kinase-3β (GSK-3β) pathway in 3-nitropropionic acid (3-NP)-induced neurotoxicity in rats. 3-NP produces neurotoxicity by inhibition of the mitochondrial complex II (enzyme succinate dehydrogenase) and by sensitizing the N-methyl-D-aspartate receptor. Recent studies have reported the therapeutic potential of HO-1/GSK-3β modulators in different neurodegenerative disorders. However, their exact role is yet to be explored. The present study is an attempt to investigate the effect of pharmacological modulation of HO-1/GSK-3β pathway against 3-NP-induced behavioral, biochemical and molecular alterations in rat. Behavioral observation, oxidative stress, pro-inflammatory [tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β)], HO-1 and GSK-3β activity were evaluated post 3-NP treatment. Findings of the present study demonstrate a significant alteration in the locomotor activity, motor coordination, oxidative burden (increased lipid peroxidation, nitrite concentration and decreased endogenous antioxidants), pro-inflammatory mediators [TNF-α, IL-1β], HO-1 and GSK-3β activity in 3-NP-treated animals. Further, administration of hemin (10- and 30-mg/kg; i.p.) and lithium chloride (LiCl) (25- and 50-mg/kg; i.p.) prevented the alteration in body weight, motor impairments, oxidative stress and cellular markers. In addition, combined administration of hemin (10-mg/kg) and LiCl (25-mg/kg) showed synergistic effect on 3-NP-treated rats. Pretreatment with Tin (IV) protoporphyrin (40 μM/kg), HO-1 inhibitor reversed the beneficial effect of LiCl and hemin. Outcomes of the present study suggest that HO-1 and GSK-3β enzymes are involved in the pathophysiology of HD. The modulators of both the pathways might be used as adjuvants or prophylactic therapy for the treatment of HD-like symptoms.

  19. 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

  20. Genistein activates endothelial nitric oxide synthase in broiler pulmonary arterial endothelial cells by an Akt-dependent mechanism

    PubMed Central

    Yang, Ying; Nie, Wei; Yuan, Jianmin; Zhang, Bingkun; Wang, Zhong

    2010-01-01

    Deregulation of endothelial nitric oxide synthase (eNOS) plays an important role in the development of multiple cardiovascular diseases. Our recent study demonstrated that genistein supplementation attenuates pulmonary arterial hypertension in broilers by restoration of endothelial function. In this study, we investigated the molecular mechanism by using broiler pulmonary arterial endothelial cells (PAECs). Our results showed that genistein stimulated a rapid phosphorylation of eNOS at Ser1179 which was associated with activation of eNOS/NO axis. Further study indicated that the activation of eNOS was not mediated through estrogen receptors or tyrosine kinase inhibition, but via a phosphatidylinositol 3-kinase (PI3K)/Akt-dependent signaling pathway, as the eNOS activity and related NO release were largely abolished by pharmacological inhibitors of PI3K or Akt. Thus, our findings revealed a critical function of Akt in mediating genistein-stimulated eNOS activity in PAECs, partially accounting for the beneficial effects of genistein on the development of cardiovascular diseases observed in animal models. PMID:20926919

  1. 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.

  2. Shear stress stimulates phosphorylation of endothelial nitric-oxide synthase at Ser1179 by Akt-independent mechanisms: role of protein kinase A

    NASA Technical Reports Server (NTRS)

    Boo, Yong Chool; Sorescu, George; Boyd, Nolan; Shiojima, Ichiro; Walsh, Kenneth; Du, Jie; Jo, Hanjoong

    2002-01-01

    Recently, we have shown that shear stress stimulates NO(*) production by the protein kinase B/Akt (Akt)-dependent mechanisms in bovine aortic endothelial cells (BAEC) (Go, Y. M., Boo, Y. C., Park, H., Maland, M. C., Patel, R., Pritchard, K. A., Jr., Fujio, Y., Walsh, K., Darley-Usmar, V., and Jo, H. (2001) J. Appl. Physiol. 91, 1574-1581). Akt has been believed to regulate shear-dependent production of NO(*) by directly phosphorylating endothelial nitric-oxide synthase (eNOS) at the Ser(1179) residue (eNOS-S(1179)), but a critical evaluation using specific inhibitors or dominant negative mutants (Akt(AA) or Akt(AAA)) has not been reported. In addition, other kinases, including protein kinase A (PKA) and AMP kinase have also shown to phosphorylate eNOS-S(1179). Here, we show that shear-dependent phosphorylation of eNOS-S(1179) is mediated by an Akt-independent, but a PKA-dependent, mechanism. Expression of Akt(AA) or Akt(AAA) in BAEC by using recombinant adenoviral constructs inhibited phosphorylation of eNOS-S(1179) if cells were stimulated by vascular endothelial growth factor (VEGF), but not by shear stress. As shown before, expression of Akt(AA) inhibited shear-dependent NO(*) production, suggesting that Akt is still an important regulator in NO production. Further studies showed that a selective inhibitor of PKA, H89, inhibited shear-dependent phosphorylation of eNOS-S(1179) and NO(*) production. In contrast, H89 did not inhibit phosphorylation of eNOS-S(1179) induced by expressing a constitutively active Akt mutant (Akt(Myr)) in BAEC, showing that the inhibitor did not affect the Akt pathway. 8-Bromo-cAMP alone phosphorylated eNOS-S(1179) within 5 min without activating Akt, in an H89-sensitive manner. Collectively, these results demonstrate that shear stimulates phosphorylation of eNOS-S(1179) in a PKA-dependent, but Aktindependent manner, whereas the NO(*) production is regulated by the mechanisms dependent on both PKA and Akt. A coordinated interaction

  3. Design, synthesis and biological evaluation of benzothiazepinones (BTZs) as novel non-ATP competitive inhibitors of glycogen synthase kinase-3β (GSK-3β).

    PubMed

    Zhang, Peng; Hu, Hai-Rong; Bian, Shi-Hui; Huang, Zhao-Hui; Chu, Yong; Ye, De-Yong

    2013-03-01

    Glycogen synthase kinase-3β (GSK-3β) plays a key role in type II diabetes and Alzheimer's diseases, to which non-ATP competitive inhibitors represent an effectively therapeutical approach due to their good specificity. Herein, a series of small molecules benzothiazepinones (BTZs) as novel non-ATP competitive inhibitors of GSK-3β have been designed and synthesized. The in vitro evaluation performed by luminescent assay showed most BTZ derivatives have inhibitory effects in micromolar scale. Among them compounds 6l, 6t and 6v have the IC50 values of 25.0 μM, 27.8 μM and 23.0 μM, respectively. Moreover 6v is devoid of any inhibitory activity in the assays to other thirteen protein kinases. Besides, SAR is analyzed and a hypothetical enzymatic binding mode is proposed by molecular docking study, which would be useful for new candidates design.

  4. The redox sensitive glycogen synthase kinase 3β suppresses the self-protective antioxidant response in podocytes upon oxidative glomerular injury.

    PubMed

    Li, Changbin; Ge, Yan; Peng, Ai; Gong, Rujun

    2015-11-24

    The redox sensitive glycogen synthase kinase (GSK) 3 has been recently implicated in the pathogenesis of proteinuric glomerulopathy. However, prior studies are less conclusive because they relied solely on chemical inhibitors of GSK3, which provide poor discrimination between the isoforms of GSK3 apart from potential off target activities. In murine kidneys, the β rather than the α isoform of GSK3 was predominantly expressed in glomeruli and distributed intensely in podocytes. By employing the doxycycline-activated Cre-loxP site specific gene targeting system, GSK3β was successfully knocked out (KO) selectively in podocytes in adult mice, resulting in a phenotype no different from control littermates. Electron microscopy of glomeruli in KO mice demonstrated more glycogen accumulation in podocytes but otherwise normal ultrastructures. Upon oxidative glomerular injury induced by protein overload, KO mice excreted significantly less albuminuria and had much attenuated podocytopathy and glomerular damage. The anti-proteinuric and glomerular protective effect was concomitant with diminished accumulation of reactive oxygen species in glomeruli in KO mice, which was likely secondary to a reinforced Nrf2 antioxidant response in podocytes. Collectively, our data suggest that GSK3β is dispensable for glomerular function and histology under normal circumstances but may serve as a therapeutic target for protecting from oxidative glomerular injuries.

  5. The redox sensitive glycogen synthase kinase 3β suppresses the self-protective antioxidant response in podocytes upon oxidative glomerular injury

    PubMed Central

    Li, Changbin; Ge, Yan; Peng, Ai; Gong, Rujun

    2015-01-01

    The redox sensitive glycogen synthase kinase (GSK) 3 has been recently implicated in the pathogenesis of proteinuric glomerulopathy. However, prior studies are less conclusive because they relied solely on chemical inhibitors of GSK3, which provide poor discrimination between the isoforms of GSK3 apart from potential off target activities. In murine kidneys, the β rather than the α isoform of GSK3 was predominantly expressed in glomeruli and distributed intensely in podocytes. By employing the doxycycline-activated Cre-loxP site specific gene targeting system, GSK3β was successfully knocked out (KO) selectively in podocytes in adult mice, resulting in a phenotype no different from control littermates. Electron microscopy of glomeruli in KO mice demonstrated more glycogen accumulation in podocytes but otherwise normal ultrastructures. Upon oxidative glomerular injury induced by protein overload, KO mice excreted significantly less albuminuria and had much attenuated podocytopathy and glomerular damage. The anti-proteinuric and glomerular protective effect was concomitant with diminished accumulation of reactive oxygen species in glomeruli in KO mice, which was likely secondary to a reinforced Nrf2 antioxidant response in podocytes. Collectively, our data suggest that GSK3β is dispensable for glomerular function and histology under normal circumstances but may serve as a therapeutic target for protecting from oxidative glomerular injuries. PMID:26567873

  6. 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

  7. Inhibition of glycogen synthase kinase-3β attenuates acute kidney injury in sodium taurocholate‑induced severe acute pancreatitis in rats.

    PubMed

    Zhao, Kailiang; Chen, Chen; Shi, Qiao; Deng, Wenhong; Zuo, Teng; He, Xiaobo; Liu, Tianyi; Zhao, Liang; Wang, Weixing

    2014-12-01

    The aim of the present study was to investigate the efficacy of 4‑benzyl‑2‑methyl‑1,2,4‑thiadiazolidine‑3,5‑dione (TDZD‑8), the selective inhibitor of glycogen synthase kinase‑3β (GSK‑3β), on the development of acute kidney injury in an experimental model of sodium taurocholate‑induced severe acute pancreatitis (SAP) in rats. The serum amylase, lipase, interleukin‑1β and interleukin‑6 levels, and the pancreatic pathological score were examined to determine the magnitude of pancreatitis injury. The serum creatinine and blood urea nitrogen levels, myeloperoxidase (MPO) activity and renal histological grading were measured to assess the magnitude of SAP‑induced acute kidney injury. The activation of nuclear factor‑κB (NF‑κB) was examined using an immunohistochemistry assay. The expression of GSK‑3β, phospho‑GSK‑3β (Ser9), tumour necrosis factor‑α (TNF‑α), intercellular adhesion molecule‑1 (ICAM‑1) and inducible nitric oxide synthase (iNOS) protein in the kidney was characterised using western blot analysis. TDZD‑8 attenuated (i) serum amylase, lipase and renal dysfunction; (ii) the serum concentrations of proinflammatory cytokines; (iii) pancreatic and renal pathological injury; (iv) renal MPO activity and (v) NF‑κB activation and TNF‑α, ICAM‑1 and iNOS protein expression in the kidney. The results obtained in the present study suggest that the inhibition of GSK‑3β attenuates renal disorders associated with SAP through the inhibition of NF‑κB activation and the downregulation of the expression of proinflammatory cytokines, TNF‑α, ICAM‑1 and iNOS in rats. Blocking GSK‑3β protein kinase activity may be a novel approach to the treatment of this inflammatory condition. PMID:25323773

  8. Inhibition of glycogen synthase kinase-3β by lithium chloride suppresses 6-hydroxydopamine-induced inflammatory response in primary cultured astrocytes.

    PubMed

    Wang, Hong-Mei; Zhang, Ting; Li, Qiang; Huang, Jian-Kang; Chen, Rong-Fu; Sun, Xiao-Jiang

    2013-11-01

    An increasing amount of evidence has emerged to suggest that neuroinflammatory process is involved in the pathogenesis of Parkinson's disease (PD). Activated microglia and astrocytes are found in the substantia nigra (SN) of Parkinson's disease brains as well as in animal models of Parkinson's disease. Although reactive astrocytes are involved in the progression of PD, the role of reactive astrocytes in neuroinflammation of PD has received limited attention to date. Recently, Glycogen synthase kinase-3β (GSK-3β) was identified as a crucial regulator of the inflammatory response. The purpose of this study was to explore the mechanism by which 6-hydroxydopamine (6-OHDA) induces inflammatory response in astrocytes and observe the anti-inflammatory effect of lithium chloride (LiCl) on 6-OHDA-treated astrocytes. In the present study, we found that glial fibrillary acidic protein (GFAP) was markedly upregulated in the presence of 6-OHDA. Moreover, our results revealed that proinflammatory molecules including inducible nitric oxide synthase (iNOS), nitric oxide (NO), cyclooxygenase-2(COX-2), prostaglandins E2 (PGE2), and tumor necrosis factor-α (TNF-α) were obviously increased in astrocytes exposed to 6-OHDA. Western blot analysis revealed that 6-OHDA significantly increased dephosphorylation/activation of GSK-3β as well as the nuclear translocation of nuclear factor-κB (NF-κB) p65. Besides, GSK-3β inhibitor LiCl and SB415286 inhibited the GSK-3β/NF-κB signaling pathway, leading to the reduction of proinflammatory molecules in 6-OHDA-activated astrocytes. These results confirmed that GSK-3β inhibitor LiCl and SB415286 provide protection against neuroinflammation in 6-OHDA-treated astrocytes. Therefore, GSK-3β may be a potential therapeutic target for the treatment of PD.

  9. Serine-threonine kinase 38 is regulated by glycogen synthase kinase-3 and modulates oxidative stress-induced cell death.

    PubMed

    Enomoto, Atsushi; Kido, Naoki; Ito, Michihiko; Takamatsu, Nobuhiko; Miyagawa, Kiyoshi

    2012-01-15

    Serine-threonine kinase 38 (STK38) is a member of the protein kinase A (PKA)/PKG/PKC-like family. In the present study, we investigated the regulatory mechanism of STK38 and assessed its role in the cellular stress response. Among various environmental stresses, STK38 was specifically activated by H(2)O(2), and the phosphatidylinositol 3-kinase inhibitor wortmannin or AKT inhibitor IV suppressed this activation. STK38 was also activated by a constitutively active AKT1 or by GSK-3β inhibitor VII. The phosphorylation level of GSK-3β was correlated with the STK38 activity, in response to various stimuli and in different cell lines. Co-immunoprecipitation analysis revealed that GSK-3β physically interacted with STK38 in cells. GSK-3β overexpression inhibited the H(2)O(2)-stimulated STK38 activity. GSK-3β phosphorylated STK38 on residues S6 and T7 in vitro, depending largely on a PKA-mediated priming phosphorylation of STK38 on residues S10 and S11, respectively. STK38's H(2)O(2)-stimulated activity was enhanced by alanine substitution at its priming sites and/or at S6 and T7, and it was partially reduced by a phosphomimetic mutation at S6 or T7. STK38 knockdown enhanced the H(2)O(2)-induced JNK phosphorylation and cell death. Our results indicate that that GSK-3β inhibits STK38's full activation, and suggest that STK38 activation is required to prevent cell death in response to oxidative stress.

  10. 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.

  11. Screening of inhibitors of glycogen synthase kinase-3β from traditional Chinese medicines using enzyme-immobilized magnetic beads combined with high-performance liquid chromatography.

    PubMed

    Li, Yunfang; Xu, Jia; Chen, Yu; Mei, Zhinan; Xiao, Yuxiu

    2015-12-18

    Glycogen synthase kinase-3β (GSK-3β) was immobilized on magnetic beads (MBs) by affinity method for the first time. The enzyme-immobilized MBs were coupled with high-performance liquid chromatography-ultraviolet (HPLC-UV) technique to establish a cost-effective and reliable method for screening of inhibitors of GSK-3β. A peptide substrate of GSK-3β containing a tyrosine residue was employed since it can be sensitively detected by UV detector at 214nm. The substrate and its phosphorylated product were separated by baseline within 10min. The enzyme activity was determined by the quantification of peak area of the product. Parameters including enzyme immobilization, enzyme reaction and the performance of immobilized-enzyme were investigated. The immobilized enzyme can be reused for 10 times and remain stable for 4 days at 4°C. The inhibitory activities of extracts of 15 traditional Chinese medicines (TCMs) were screened. As a result, three of them including Euonymus fortunei, Amygdalus communis and Garcinia xanthochymus were found possessing high inhibitory activities (inhibition rate >90%). From G. xanthochymus, a new inhibitor of GSK-3β, fukugetin, was discovered with an IC50 value of 3.18±0.07μM. Enzyme kinetics and molecular docking experiments further revealed the inhibitory mechanism, indicating fukugetin was a non-ATP competitive inhibitor interacting with the phosphate recognizing substrate binding site of GSK-3β.

  12. Effect of Imipramine, Paroxetine, and Lithium Carbonate on Neurobehavioral Changes of Streptozotocin in Rats: Impact on Glycogen Synthase Kinase-3 and Blood Glucose Level.

    PubMed

    Nadeem, Rania I; Ahmed, Hebatalla I; El-Denshary, Ezz-El-Din S

    2015-09-01

    Recent studies have demonstrated a scrutinized association of diabetes mellitus with depressive symptoms and major depression. Glycogen synthase kinase-3 (GSK-3) is a protein kinase enzyme constitutively active in non-stimulated cells and in multiple signalings. Independent lines of research provide a converging evidence for an involvement of GSK-3 in the regulation of behavior and hyperglycemia. The present study revealed that streptozotocin (STZ)-induced diabetic rats were found to show lengthened duration of immobility in the forced-swimming test (FST) and reduced locomotor and exploratory activities in the open-field test (OFT). Imipramine (15 mg/kg), Paroxetine (10 mg/kg) and lithium carbonate (36.94 mg/kg) for 14 days reduced immobility behavior in FST. Paroxetine and lithium carbonate increased the locomotor and exploratory activities, while imipramine decreased the locomotor activity in the OFT. Imipramine and lithium carbonate reduced the blood glucose level while paroxetine didn't alter it. STZ-induced diabetes increased GSK-3 gene expression which was determined using the reverse transcription-quantitative polymerase chain reaction test, while the three drugs decreased its expression. It can be concluded that lithium carbonate and imipramine can control both hyperglycemia and the associated symptoms of depression at the same time by inhibiting GSK-3 activity. On the other hand, paroxetine may only manage the depressive-like symptoms associated with diabetes through modulating the enzyme GSK-3, without changing blood glucose levels. PMID:26216050

  13. Glycogen synthase kinase 3 regulates expression of nuclear factor-erythroid-2 related transcription factor-1 (Nrf1) and inhibits pro-survival function of Nrf1

    SciTech Connect

    Biswas, Madhurima; Kwong, Erick K.; Park, Eujean; Nagra, Parminder; Chan, Jefferson Y.

    2013-08-01

    Nuclear factor E2-related factor-1 (Nrf1) is a basic leucine zipper transcription factor that is known to regulate antioxidant and cytoprotective gene expression. It was recently shown that Nrf1 is regulated by SCF–Fbw7 ubiquitin ligase. However our knowledge of upstream signals that targets Nrf1 for degradation by the UPS is not known. We report here that Nrf1 expression is negatively regulated by glycogen synthase kinase 3 (GSK3) in Fbw7-dependent manner. We show that GSK3 interacts with Nrf1 and phosphorylates the Cdc4 phosphodegron domain (CPD) in Nrf1. Mutation of serine residue in the CPD of Nrf1 to alanine (S350A), blocks Nrf1 from phosphorylation by GSK3, and stabilizes Nrf1. Knockdown of Nrf1 and expression of a constitutively active form of GSK3 results in increased apoptosis in neuronal cells in response to ER stress, while expression of the GSK3 phosphorylation resistant S350A–Nrf1 attenuates apoptotic cell death. Together these data suggest that GSK3 regulates Nrf1 expression and cell survival function in response to stress activation. Highlights: • The effect of GSK3 on Nrf1 expression was examined. • GSK3 destabilizes Nrf1 protein via Fbw7 ubiquitin ligase. • GSK3 binds and phosphorylates Nrf1. • Protection from stress-induced apoptosis by Nrf1 is inhibited by GSK3.

  14. Glycogen synthase kinase-3 (Gsk-3) plays a fundamental role in maintaining DNA methylation at imprinted loci in mouse embryonic stem cells.

    PubMed

    Meredith, Gavin D; D'Ippolito, Anthony; Dudas, Miroslav; Zeidner, Leigh C; Hostetter, Logan; Faulds, Kelsie; Arnold, Thomas H; Popkie, Anthony P; Doble, Bradley W; Marnellos, George; Adams, Christopher; Wang, Yulei; Phiel, Christopher J

    2015-06-01

    Glycogen synthase kinase-3 (Gsk-3) is a key regulator of multiple signal transduction pathways. Recently we described a novel role for Gsk-3 in the regulation of DNA methylation at imprinted loci in mouse embryonic stem cells (ESCs), suggesting that epigenetic changes regulated by Gsk-3 are likely an unrecognized facet of Gsk-3 signaling. Here we extend our initial observation to the entire mouse genome by enriching for methylated DNA with the MethylMiner kit and performing next-generation sequencing (MBD-Seq) in wild-type and Gsk-3α(-/-);Gsk-3β(-/-) ESCs. Consistent with our previous data, we found that 77% of known imprinted loci have reduced DNA methylation in Gsk-3-deficient ESCs. More specifically, we unambiguously identified changes in DNA methylation within regions that have been confirmed to function as imprinting control regions. In many cases, the reduced DNA methylation at imprinted loci in Gsk-3α(-/-);Gsk-3β(-/-) ESCs was accompanied by changes in gene expression as well. Furthermore, many of the Gsk-3-dependent, differentially methylated regions (DMRs) are identical to the DMRs recently identified in uniparental ESCs. Our data demonstrate the importance of Gsk-3 activity in the maintenance of DNA methylation at a majority of the imprinted loci in ESCs and emphasize the importance of Gsk-3-mediated signal transduction in the epigenome.

  15. Hyperinsulinemia enhances interleukin-17-induced inflammation to promote prostate cancer development in obese mice through inhibiting glycogen synthase kinase 3-mediated phosphorylation and degradation of interleukin-17 receptor.

    PubMed

    Liu, Sen; Zhang, Qiuyang; Chen, Chong; Ge, Dongxia; Qu, Yine; Chen, Rongyi; Fan, Yi-Ming; Li, Nan; Tang, Wendell W; Zhang, Wensheng; Zhang, Kun; Wang, Alun R; Rowan, Brian G; Hill, Steven M; Sartor, Oliver; Abdel-Mageed, Asim B; Myers, Leann; Lin, Qishan; You, Zongbing

    2016-03-22

    Interleukin-17 (IL-17) plays important roles in inflammation, autoimmune diseases, and some cancers. Obese people are in a chronic inflammatory state with increased serum levels of IL-17, insulin, and insulin-like growth factor 1 (IGF1). How these factors contribute to the chronic inflammatory status that promotes development of aggressive prostate cancer in obese men is largely unknown. We found that, in obese mice, hyperinsulinemia enhanced IL-17-induced expression of downstream proinflammatory genes with increased levels of IL-17 receptor A (IL-17RA), resulting in development of more invasive prostate cancer. Glycogen synthase kinase 3 (GSK3) constitutively bound to and phosphorylated IL-17RA at T780, leading to ubiquitination and proteasome-mediated degradation of IL-17RA, thus inhibiting IL-17-mediated inflammation. IL-17RA phosphorylation was reduced, while the IL-17RA levels were increased in the proliferative human prostate cancer cells compared to the normal cells. Insulin and IGF1 enhanced IL-17-induced inflammatory responses through suppressing GSK3, which was shown in the cultured cell lines in vitro and obese mouse models of prostate cancer in vivo. These findings reveal a mechanism underlying the intensified inflammation in obesity and obesity-associated development of aggressive prostate cancer, suggesting that targeting GSK3 may be a potential therapeutic approach to suppress IL-17-mediated inflammation in the prevention and treatment of prostate cancer, particularly in obese men.

  16. 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

  17. Protein Kinase A Opposes the Phosphorylation-dependent Recruitment of Glycogen Synthase Kinase 3β to A-kinase Anchoring Protein 220.

    PubMed

    Whiting, Jennifer L; Nygren, Patrick J; Tunquist, Brian J; Langeberg, Lorene K; Seternes, Ole-Morten; Scott, John D

    2015-08-01

    The proximity of an enzyme to its substrate can influence rate and magnitude of catalysis. A-kinase anchoring protein 220 (AKAP220) is a multivalent anchoring protein that can sequester a variety of signal transduction enzymes. These include protein kinase A (PKA) and glycogen synthase kinase 3β (GSK3β). Using a combination of molecular and cellular approaches we show that GSK3β phosphorylation of Thr-1132 on AKAP220 initiates recruitment of this kinase into the enzyme scaffold. We also find that AKAP220 anchors GSK3β and its substrate β-catenin in membrane ruffles. Interestingly, GSK3β can be released from the multienzyme complex in response to PKA phosphorylation on serine 9, which suppresses GSK3β activity. The signaling scaffold may enhance this regulatory mechanism, as AKAP220 has the capacity to anchor two PKA holoenzymes. Site 1 on AKAP220 (residues 610-623) preferentially interacts with RII, whereas site 2 (residues 1633-1646) exhibits a dual specificity for RI and RII. In vitro affinity measurements revealed that site 2 on AKAP220 binds RII with ∼10-fold higher affinity than site 1. Occupancy of both R subunit binding sites on AKAP220 could provide a mechanism to amplify local cAMP responses and enable cross-talk between PKA and GSK3β.

  18. 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.

  19. Glycogen synthase kinase 3 regulates expression of nuclear factor-erythroid-2 related transcription factor-1 (Nrf1) and inhibits pro-survival function of Nrf1.

    PubMed

    Biswas, Madhurima; Kwong, Erick K; Park, Eujean; Nagra, Parminder; Chan, Jefferson Y

    2013-08-01

    Nuclear factor E2-related factor-1 (Nrf1) is a basic leucine zipper transcription factor that is known to regulate antioxidant and cytoprotective gene expression. It was recently shown that Nrf1 is regulated by SCF-Fbw7 ubiquitin ligase. However our knowledge of upstream signals that targets Nrf1 for degradation by the UPS is not known. We report here that Nrf1 expression is negatively regulated by glycogen synthase kinase 3 (GSK3) in Fbw7-dependent manner. We show that GSK3 interacts with Nrf1 and phosphorylates the Cdc4 phosphodegron domain (CPD) in Nrf1. Mutation of serine residue in the CPD of Nrf1 to alanine (S350A), blocks Nrf1 from phosphorylation by GSK3, and stabilizes Nrf1. Knockdown of Nrf1 and expression of a constitutively active form of GSK3 results in increased apoptosis in neuronal cells in response to ER stress, while expression of the GSK3 phosphorylation resistant S350A-Nrf1 attenuates apoptotic cell death. Together these data suggest that GSK3 regulates Nrf1 expression and cell survival function in response to stress activation.

  20. Glycogen Synthase Kinase-3β (GSK3β) Negatively Regulates PTTG1/Human Securin Protein Stability, and GSK3β Inactivation Correlates with Securin Accumulation in Breast Tumors*

    PubMed Central

    Mora-Santos, Mar; Limón-Mortés, M. Cristina; Giráldez, Servando; Herrero-Ruiz, Joaquín; Sáez, Carmen; Japón, Miguel Á.; Tortolero, Maria; Romero, Francisco

    2011-01-01

    PTTG1, also known as securin, is an inactivating partner of separase, the major effector for chromosome segregation during mitosis. At the metaphase-to-anaphase transition, securin is targeted for proteasomal destruction by the anaphase-promoting complex or cyclosome, allowing activation of separase. In addition, securin is overexpressed in metastatic or genomically instable tumors, suggesting a relevant role for securin in tumor progression. Stability of securin is regulated by phosphorylation; some phosphorylated forms are degraded out of mitosis, by the action of the SKP1-CUL1-F-box protein (SCF) complex. The kinases targeting securin for proteolysis have not been identified, and mechanistic insight into the cause of securin accumulation in human cancers is lacking. Here, we demonstrate that glycogen synthase kinase-3β (GSK3β) phosphorylates securin to promote its proteolysis via SCFβTrCP E3 ubiquitin ligase. Importantly, a strong correlation between securin accumulation and GSK3β inactivation was observed in breast cancer tissues, indicating that GSK3β inactivation may account for securin accumulation in breast cancers. PMID:21757741

  1. Glycogen synthase kinase 3β inhibitors protect hippocampal neurons from radiation-induced apoptosis by regulating MDM2-p53 pathway

    PubMed Central

    Thotala, D K; Hallahan, D E; Yazlovitskaya, E M

    2012-01-01

    Exposure of the brain to ionizing radiation can cause neurocognitive deficiencies. The pathophysiology of these neurological changes is complex and includes radiation-induced apoptosis in the subgranular zone of the hippocampus. We have recently found that inhibition of glycogen synthase kinase 3β (GSK-3β) resulted in significant protection from radiation-induced apoptosis in hippocampal neurons. The molecular mechanisms of this cytoprotection include abrogation of radiation-induced accumulation of p53. Here we show that pretreatment of irradiated HT-22 hippocampal-derived neurons with small molecule inhibitors of GSK-3β SB216763 or SB415286, or with GSK-3β-specific shRNA resulted in accumulation of the p53-specific E3 ubiquitin ligase MDM2. Knockdown of MDM2 using specific shRNA or chemical inhibition of MDM2-p53 interaction prevented the protective changes triggered by GSK-3β inhibition in irradiated HT-22 neurons and restored radiation cytotoxicity. We found that this could be due to regulation of apoptosis by subcellular localization and interaction of GSK-3β, p53 and MDM2. These data suggest that the mechanisms of radioprotection by GSK-3β inhibitors in hippocampal neurons involve regulation of MDM2-dependent p53 accumulation and interactions between GSK-3β, MDM2 and p53. PMID:21738215

  2. Glycogen Synthase Kinase 3 Inactivation Drives T-bet-Mediated Downregulation of Co-receptor PD-1 to Enhance CD8+ Cytolytic T Cell Responses

    PubMed Central

    Taylor, Alison; Harker, James A.; Chanthong, Kittiphat; Stevenson, Philip G.; Zuniga, Elina I.; Rudd, Christopher E.

    2016-01-01

    Summary Despite the importance of the co-receptor PD-1 in T cell immunity, the upstream signaling pathway that regulates PD-1 expression has not been defined. Glycogen synthase kinase 3 (GSK-3, isoforms α and β) is a serine-threonine kinase implicated in cellular processes. Here, we identified GSK-3 as a key upstream kinase that regulated PD-1 expression in CD8+ T cells. GSK-3 siRNA downregulation, or inhibition by small molecules, blocked PD-1 expression, resulting in increased CD8+ cytotoxic T lymphocyte (CTL) function. Mechanistically, GSK-3 inactivation increased Tbx21 transcription, promoting enhanced T-bet expression and subsequent suppression of Pdcd1 (encodes PD-1) transcription in CD8+ CTLs. Injection of GSK-3 inhibitors in mice increased in vivo CD8+ OT-I CTL function and the clearance of murine gamma-herpesvirus 68 and lymphocytic choriomeningitis clone 13 and reversed T cell exhaustion. Our findings identify GSK-3 as a regulator of PD-1 expression and demonstrate the applicability of GSK-3 inhibitors in the modulation of PD-1 in immunotherapy. PMID:26885856

  3. A glycogen synthase kinase 3-beta promoter gene single nucleotide polymorphism is associated with age at onset and response to total sleep deprivation in bipolar depression.

    PubMed

    Benedetti, Francesco; Serretti, Alessandro; Colombo, Cristina; Lorenzi, Cristina; Tubazio, Viviana; Smeraldi, Enrico

    2004-09-23

    The molecular mechanisms driving the biological clock in the suprachiasmatic nucleus of the hypothalamus may play a role in mood disorders. A single nucleotide polymorphism (SNP) (-50T/C) falling into the effective promoter region (nt -171 to +29) of the gene coding for glycogen synthase kinase 3-beta (GSK3-beta) has been linked with different age at onset of bipolar illness. GSK3-beta codes for an enzyme which is a target for the action of lithium and valproic acid, and the inhibition of which causes antidepressant-like behaviors in a preclinical model. We studied the effect of this polymorphism on the acute response to total sleep deprivation of 60 depressed bipolar type I inpatients. Homozygotes for the mutant allele of GSK3-beta promoter (-50T/C) SNP showed a later onset of bipolar illness, and better acute effects of TSD treatment on perceived mood (as rated on VAS). Overall, these observations suggest a protective role for this genotype in respect to bipolar illness. Results warrant interest for the variants of genes pertaining to the molecular clock as possible endophenotypes of bipolar disorder, and for GSK3-beta as a target of a new class of antidepressant drugs, but caution ought to be taken in interpreting these preliminary results and future replication studies must be awaited because of the low frequency of the GSK3-beta*C/C genotype in the studied populations.

  4. A single nucleotide polymorphism in glycogen synthase kinase 3-beta promoter gene influences onset of illness in patients affected by bipolar disorder.

    PubMed

    Benedetti, Francesco; Bernasconi, Alessandro; Lorenzi, Cristina; Pontiggia, Adriana; Serretti, Alessandro; Colombo, Cristina; Smeraldi, Enrico

    2004-01-23

    Genetic studies in medicine exploited age of onset as a criterion to delineate subgroups of illness. Bipolar patients stratified with this criterion were shown to share clinical characteristics and patterns of inheritance of illness. The molecular mechanisms driving the biological clock in the suprachiasmatic nucleus of the hypothalamus may play a role in mood disorders. A single nucleotide polymorphism (SNP) (-50 T/C) falling into the effective promoter region (nt -171 to +29) of the gene coding for glycogen synthase kinase 3-beta (GSK3-beta) has been identified. GSK3-beta codes for an enzyme which is a target for the action of lithium and which is also known to regulate circadian rhythms in Drosophila. We studied the effect of this polymorphism on the age at onset of bipolar disorder type I. A homogeneous sample of 185 Italian patients affected by bipolar disorder was genotyped. Age at onset was retrospectively ascertained with best estimation procedures. No association was detected between GSK3-beta -50 T/C SNP and the presence of bipolar illness. Homozygotes for the wild variant (T/T) showed an earlier age at onset than carriers of the mutant allele (F=5.53, d.f.=2,182, P=0.0047). Results warrant interest for the variants of genes pertaining to the molecular clock as possible endophenotypes of bipolar disorder, but caution ought to be taken in interpreting these preliminary results and future replication studies must be awaited.

  5. Long-term response to lithium salts in bipolar illness is influenced by the glycogen synthase kinase 3-beta -50 T/C SNP.

    PubMed

    Benedetti, Francesco; Serretti, Alessandro; Pontiggia, Adriana; Bernasconi, Alessandro; Lorenzi, Cristina; Colombo, Cristina; Smeraldi, Enrico

    2005-03-01

    The molecular mechanisms driving the biological clock in the suprachiasmatic nucleus of the hypothalamus may play a role in mood disorders. A single nucleotide polymorphism (SNP) (-50 T/C) falling into the effective promoter region (nt -171 to +29) of the gene coding for glycogen synthase kinase 3-beta (GSK3-beta) has been linked with different age at onset of bipolar illness and with different antidepressant effects of total sleep deprivation. GSK3-beta codes for an enzyme which is a target for the action of lithium and possibly of valproic acid. We studied the effect of this polymorphism on the therapeutic response to lithium salts of 88 bipolar type I patients. Data about recurrence rate of mood episodes were collected for at least 2 years before lithium and 2 years on lithium. Results showed that homozygotes for the wild variant did not change their recurrence index while carriers of the mutant allele improved, thus supporting the hypothesis that GSK is a target for the therapeutic action of lithium. Results warrant interest for the variants of genes pertaining to the molecular clock as possible endophenotypes of bipolar disorder, but caution ought to be taken in interpreting these preliminary results and future replication studies must be awaited because of the low frequency of the GSK3-beta*C/C genotype in the studied populations.

  6. Hyperinsulinemia enhances interleukin-17-induced inflammation to promote prostate cancer development in obese mice through inhibiting glycogen synthase kinase 3-mediated phosphorylation and degradation of interleukin-17 receptor

    PubMed Central

    Chen, Chong; Ge, Dongxia; Qu, Yine; Chen, Rongyi; Fan, Yi-Ming; Li, Nan; Tang, Wendell W.; Zhang, Wensheng; Zhang, Kun; Wang, Alun R.; Rowan, Brian G.; Hill, Steven M.; Sartor, Oliver; Abdel, Asim B.; Myers, Leann; Lin, Qishan; You, Zongbing

    2016-01-01

    Interleukin-17 (IL-17) plays important roles in inflammation, autoimmune diseases, and some cancers. Obese people are in a chronic inflammatory state with increased serum levels of IL-17, insulin, and insulin-like growth factor 1 (IGF1). How these factors contribute to the chronic inflammatory status that promotes development of aggressive prostate cancer in obese men is largely unknown. We found that, in obese mice, hyperinsulinemia enhanced IL-17-induced expression of downstream proinflammatory genes with increased levels of IL-17 receptor A (IL-17RA), resulting in development of more invasive prostate cancer. Glycogen synthase kinase 3 (GSK3) constitutively bound to and phosphorylated IL-17RA at T780, leading to ubiquitination and proteasome-mediated degradation of IL-17RA, thus inhibiting IL-17-mediated inflammation. IL-17RA phosphorylation was reduced, while the IL-17RA levels were increased in the proliferative human prostate cancer cells compared to the normal cells. Insulin and IGF1 enhanced IL-17-induced inflammatory responses through suppressing GSK3, which was shown in the cultured cell lines in vitro and obese mouse models of prostate cancer in vivo. These findings reveal a mechanism underlying the intensified inflammation in obesity and obesity-associated development of aggressive prostate cancer, suggesting that targeting GSK3 may be a potential therapeutic approach to suppress IL-17-mediated inflammation in the prevention and treatment of prostate cancer, particularly in obese men. PMID:26871944

  7. Glycogen synthase kinase-3β facilitates cell apoptosis induced by high fluence low-power laser irradiation through acceleration of Bax translocation

    NASA Astrophysics Data System (ADS)

    Huang, Lei; Wu, Shengnan; Xing, Da

    2011-03-01

    Glycogen synthase kinase-3β (GSK-3β) is a critical activator of cell apoptosis induced by a diverse array of insults. However, the effects of GSK-3β on the human lung adenocarcinoma cell (ASTC-a-1) apoptosis induced by high fluence low-power laser irradiation (HF-LPLI) are not clear. Here, we showed that GSK-3β was constantly translocated from cytoplasm to nucleus and activated during HF-LPLI-induced cell apoptosis. In addition, we found that co-overexpression of YFP-GSK-3β and CFP-Bax in ASTC-a-1 cells accelerated both Bax translocations to mitochondria and cell apoptosis, compared to the cells expressed CFP-Bax only under HF-LPLI treatment, indicating that GSK-3β facilitated ASTC-a-1 cells apoptosis through acceleration mitochondrial translocation of Bax. Our results demonstrate that GSK-3β exerts some of its pro-apoptotic effects in ASTC-a-1 cells by regulating the mitochondrial localization of Bax, a key component of the intrinsic apoptotic cascade.

  8. Fine-tuning of NFκB by Glycogen Synthase Kinase 3β directs the fate of glomerular podocytes upon injury

    PubMed Central

    Bao, Hui; Ge, Yan; Peng, Ai; Gong, Rujun

    2014-01-01

    NFκB is regulated by a myriad of signaling cascades including glycogen synthase kinase (GSK) 3β and plays a Janus role in podocyte injury. In vitro, lipopolysaccharide or adriamycin elicited podocyte injury and cytoskeletal disruption, associated with NFκB activation and induced expression of NFκB target molecules, including pro-survival Bcl-xL and podocytopathic mediators like MCP-1, cathepsin L and B7-1. Broad range inhibition of NFκB diminished the expression of all NFκB target genes, restored cytoskeleton integrity, but potentiated apoptosis. In contrast, blockade of GSK3β by lithium or TDZD-8, mitigated the expression of podocytopathic mediators, ameliorated podocyte injury, but barely affected Bcl-xL expression or sensitized apoptosis. Mechanistically, GSK3β was sufficient and essential for RelA/p65 phosphorylation specifically at serine 467, which specifies the expression of selective NFκB target molecules, including podocytopathic mediators, but not Bcl-xL. In vivo, lithium or TDZD-8 therapy improved podocyte injury and proteinuria in mice treated with lipopolysaccharide or adriamycin, concomitant with suppression of podocytopathic mediators but retained Bcl-xL in glomerulus. Broad range inhibition of NFκB conferred similar but much weakened antiproteinuric and podoprotective effects accompanied with a blunted glomerular expression of Bcl-xL and marked podocyte apoptosis. Thus, the GSK3β dictated fine-tuning of NFκB may serve as a novel therapeutic target for podocytopathy. PMID:25629551

  9. 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

  10. Selective deletion of forebrain glycogen synthase kinase 3β reveals a central role in serotonin-sensitive anxiety and social behaviour

    PubMed Central

    Latapy, Camille; Rioux, Véronique; Guitton, Matthieu J.; Beaulieu, Jean-Martin

    2012-01-01

    Serotonin (5-HT) neurotransmission is thought to underlie mental illnesses, such as bipolar disorder, depression, autism and schizophrenia. Independent studies have indicated that 5-HT or drugs acting on 5-HT neurotransmission regulate the serine/threonine kinase glycogen synthase kinase 3β (GSK3β). Furthermore, GSK3β inhibition rescues behavioural abnormalities in 5-HT-deficient mice with a loss-of-function mutation equivalent to the human variant (R441H) of tryptophan hydroxylase 2. In an effort to define neuroanatomical correlates of GSK3β activity in the regulation of behaviour, we generated CamKIIcre-floxGSK3β mice in which the gsk3b gene is postnatally inactivated in forebrain pyramidal neurons. Behavioural characterization showed that suppression of GSK3β in these brain areas has anxiolytic and pro-social effects. However, while a global reduction of GSK2β expression reduced responsiveness to amphetamine and increased resilience to social defeat, these behavioural effects were not found in CamKIIcre-floxGSK3β mice. These findings demonstrate a dissociation of behavioural effects related to GSK3 inhibition, with forebrain GSK3β being involved in the regulation of anxiety and sociability while social preference, resilience and responsiveness to psychostimulants would involve a function of this kinase in subcortical areas such as the hippocampus and striatum. PMID:22826345

  11. Serine 1179 phosphorylation of endothelial nitric oxide synthase caused by 2,4,6-trinitrotoluene through PI3K/Akt signaling in endothelial cells

    SciTech Connect

    Sun Yang; Sumi, Daigo; Kumagai, Yoshito . E-mail: yk-em-tu@md.tsukuba.ac.jp

    2006-07-01

    Although 2,4,6-trinitrotoluene (TNT) has been found to uncouple nitric oxide synthase (NOS), thereby leading to reactive oxygen species (ROS), cellular response against TNT still remains unclear. Exposure of bovine aortic endothelial cells (BAECs) to TNT (100 {mu}M) resulted in serine 1179 phosphorylation of endothelial NOS (eNOS). With specific inhibitors (wortmannin and LY294002), we found that PI3K/Akt signaling participated in the eNOS phosphorylation caused by TNT, whereas the ERK pathway did not. ROS were generated following exposure of BAECs to TNT. However, TNT-mediated phosphorylation of either eNOS or Akt was drastically blocked by NAC and PEG-CAT. Interestingly, pretreatment with apocynin, a specific inhibitor for NADPH oxidase, diminished the phosphorylation of eNOS and Akt. These results suggest that TNT affects NADPH oxidase, thereby generating hydrogen peroxide, which is capable of activating PI3K/Akt signaling associated with eNOS Ser 1179 phosphorylation.

  12. 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.

  13. Identification and in vitro evaluation of new leads as selective and competitive glycogen synthase kinase-3β inhibitors through ligand and structure based drug design.

    PubMed

    Darshit, B S; Balaji, B; Rani, P; Ramanathan, M

    2014-09-01

    Glycogen synthase kinase-3β elicits multi-functional effects on intracellular signaling pathways, thereby making the kinase a therapeutic target in multiple pathologies. Hence, it is important to selectively inhibit GSK-3β over structurally and biologically similar targets, such as CDK5. The current study was designed to identify and evaluate novel ATP-competitive GSK-3β inhibitors. The study was designed to identify new leads by ligand based drug design, structure based drug design and in vitro evaluation. The best validated pharmacophore model (AADRRR) identified using LBDD was derived from a dataset of 135 molecules. There were 357 primary hits within the SPECS database using this pharmacophore model. A SBDD approach to the GSK-3β and CDK5 proteins was applied to all primary hits, and 5 selective inhibitors were identified for GSK-3β. GSK-3β and CDK5 in vitro kinase inhibition assays were performed with these molecules to confirm their selectivity for GSK-3β. The molecules showed IC50 values ranging from 0.825μM to 1.116μM and were 23- to 57-fold selective for GSK-3β. Of all the molecules, molecule 3 had the lowest IC50 value of 0.825μM. Our research identified molecules possessing benzothiophene, isoquinoline, thiazolidinedione imidazo-isoquinoline and quinazolinone scaffolds. Potency of these molecules may be due to H-bond interaction with backbone residues of Val135, Asp133 and side chain interaction with Tyr134. Selectivity over CDK5 may be due to side chain interactions with Asp200, backbone of Val61, ionic interaction with Lys60 and π-cationic interaction with Arg141. These selective molecules were also exhibited small atom hydrophobicity and H-bond interaction with water molecule.

  14. Cytoskeleton involvement in lithium-induced SH-SY5Y neuritogenesis and the role of glycogen synthase kinase 3β.

    PubMed

    Nciri, Riadh; Boujbiha, Mohamed Ali; Jbahi, Samira; Allagui, Mohamed Salah; Elfeki, Abdelfattah; Vincent, Christian; Croute, Françoise

    2015-06-01

    Lithium modulates signals impacting on the cytoskeleton, a dynamic system contributing to neural plasticity at multiple levels. In this study, SH-SY5Y human neuronal cells were cultured in the absence (C) or in presence (Li) of a 0.5 mM Li2CO3 (i.e. 1 mM lithium ion) for 25-50 weeks. We investigated the effect of this treatment on (1) morphological changes of cells observed using Hemalun eosin staining assay, (2) cytoskeletal changes by indirect immunofluorescence (IIF) staining of microtubules (α-tubulin) and heavy neurofilaments subunits (NF-H) and by measuring the expression rate changes of genes coding for receptor for activated C kinase (RACK1), casein kinase2 (CK2) and thymosine beta-10 using cDNA arrays technology, (3) cell adhesion properties by IIF staining of β-catenin protein. Besides, we have tried to understand the molecular mechanism of lithium action that triggers changes in cytoskeleton and neurites outgrowth. Thus, we examined the effect of this treatment on glycogen synthase kinase 3 (GSK3) expression and activity using western blotting of GSK3 and phosphorylated β-catenin, a downstream GSK3 target protein. Our results showed that lithium treatment reduces axon length, increases axonal spreading, enhances neurites growth and neurites branching with an increase of growth cone size. Moreover, genes coding for CK2 and thymosine beta-10 were significantly up-regulated, however, that coding for RACK1 was down-regulated. The most interesting result in this work is that mechanism underlying lithium action was not related to the inhibition of GSK3 activity. In fact, neither expression rate nor activity of this protein was changed.

  15. Synergistic neuroprotective effects of lithium and valproic acid or other histone deacetylase inhibitors in neurons: roles of glycogen synthase kinase-3 inhibition.

    PubMed

    Leng, Yan; Liang, Min-Huei; Ren, Ming; Marinova, Zoya; Leeds, Peter; Chuang, De-Maw

    2008-03-01

    Lithium and valproic acid (VPA) are two primary drugs used to treat bipolar mood disorder and have frequently been used in combination to treat bipolar patients resistant to monotherapy with either drug. Lithium, a glycogen synthase kinase-3 (GSK-3) inhibitor, and VPA, a histone deacetylase (HDAC) inhibitor, have neuroprotective effects. The present study was undertaken to demonstrate synergistic neuroprotective effects when both drugs were coadministered. Pretreatment of aging cerebellar granule cells with lithium or VPA alone provided little or no neuroprotection against glutamate-induced cell death. However, copresence of both drugs resulted in complete blockade of glutamate excitotoxicity. Combined treatment with lithium and VPA potentiated serine phosphorylation of GSK-3 alpha and beta isoforms and inhibition of GSK-3 enzyme activity. Transfection with GSK-3alpha small interfering RNA (siRNA) and/or GSK-3beta siRNA mimicked the ability of lithium to induce synergistic protection with VPA. HDAC1 siRNA or other HDAC inhibitors (phenylbutyrate, sodium butyrate or trichostatin A) also caused synergistic neuroprotection together with lithium. Moreover, combination of lithium and HDAC inhibitors potentiated beta-catenin-dependent, Lef/Tcf-mediated transcriptional activity. An additive increase in GSK-3 serine phosphorylation was also observed in mice chronically treated with lithium and VPA. Together, for the first time, our results demonstrate synergistic neuroprotective effects of lithium and HDAC inhibitors and suggest that GSK-3 inhibition is a likely molecular target for the synergistic neuroprotection. Our results may have implications for the combined use of lithium and VPA in treating bipolar disorder. Additionally, combined use of both drugs may be warranted for clinical trials to treat glutamate-related neurodegenerative diseases.

  16. A screen for transcription factor targets of glycogen synthase kinase-3 highlights an inverse correlation of NFκB and androgen receptor signaling in prostate cancer.

    PubMed

    Campa, Victor M; Baltziskueta, Eder; Bengoa-Vergniory, Nora; Gorroño-Etxebarria, Irantzu; Wesołowski, Radosław; Waxman, Jonathan; Kypta, Robert M

    2014-09-30

    Expression of Glycogen Synthase Kinase-3 (GSK-3) is elevated in prostate cancer and its inhibition reduces prostate cancer cell proliferation, in part by reducing androgen receptor (AR) signaling. However, GSK-3 inhibition can also activate signals that promote cell proliferation and survival, which may preclude the use of GSK-3 inhibitors in the clinic. To identify such signals in prostate cancer, we screened for changes in transcription factor target DNA binding activity in GSK-3-silenced cells. Among the alterations was a reduction in AR DNA target binding, as predicted from previous studies, and an increase in NFκB DNA target binding. Consistent with the latter, gene silencing of GSK-3 or inhibition using the GSK-3 inhibitor CHIR99021 increased basal NFκB transcriptional activity. Activation of NFκB was accompanied by an increase in the level of the NFκB family member RelB. Conversely, silencing RelB reduced activation of NFκB by CHIR99021. Furthermore, the reduction of prostate cancer cell proliferation by CHIR99021 was potentiated by inhibition of NFκB signaling using the IKK inhibitor PS1145. Finally, stratification of human prostate tumor gene expression data for GSK3 revealed an inverse correlation between NFκB-dependent and androgen-dependent gene expression, consistent with the results from the transcription factor target DNA binding screen. In addition, there was a correlation between expression of androgen-repressed NFκB target genes and reduced survival of patients with metastatic prostate cancer. These findings highlight an association between GSK-3/AR and NFκB signaling and its potential clinical importance in metastatic prostate cancer.

  17. Melatonin attenuated adipogenesis through reduction of the CCAAT/enhancer binding protein beta by regulating the glycogen synthase 3 beta in human mesenchymal stem cells.

    PubMed

    Rhee, Yun-Hee; Ahn, Jin-Chul

    2016-06-01

    Adipogenic differentiation is characterized by an increase in two major transcription factors: peroxisome proliferator-activated receptor gamma (PPARγ) and the CCAAT/enhancer binding protein alpha (C/EBPα). These two signals are influenced by C/EBPβ and C/EBPδ and cross-regulate each other's expression during the initial stages of adipogenesis. Melatonin has been known to act as not only a direct scavenger of free radicals but also an inhibitor of glycogen synthase kinase 3β (GSK-3β). Here, we report that melatonin inhibits the adipogenic differentiation of human mesenchymal stem cells (hMSCs) which is due to the regulations of C/EBPβ in the early stage of adipogenic differentiation. Melatonin reduced the lipid accumulation, adiponectin, and lipoprotein lipase (LPL) during the adipogenic differentiation of hMSCs. Since C/EBPβ has been associated with the activation of PPARγ and the consensus site of ERK/GSK-3β, PPARγ and β-catenin were detected by immunofluorescence staining after pretreatment of melatonin. Melatonin blocked the activation of PPARγ which induced the degradation of β-catenin. Melatonin also decreased the levels of cyclic adenosine-3,5-monophosphate (cAMP) and reactive oxygen species (ROS). The cAMP triggered the activity of C/EBPβ which is a critical inducer of PPARγ and C/EBPα activation in the early stage of adipogenic differentiation, and this is further affected by ROS production. The adipogenic marker proteins such as PPARγ, C/EBPα, C/EBPβ, and pERK were also decreased by melatonin. In summary, melatonin inhibited the cAMP synthesis through ROS reduction and the phosphorylation of the ERK/GSK-3β site which is known to be responsible for C/EBPβ activation for adipogenic differentiation in hMSCs. PMID:26797706

  18. A screen for transcription factor targets of Glycogen Synthase Kinase-3 highlights an inverse correlation of NFκB and Androgen Receptor Signaling in Prostate Cancer

    PubMed Central

    Campa, Victor M.; Baltziskueta, Eder; Bengoa-Vergniory, Nora; Gorroño-Etxebarria, Irantzu; Wesołowski, Radosław; Waxman, Jonathan; Kypta, Robert M.

    2014-01-01

    Expression of Glycogen Synthase Kinase-3 (GSK-3) is elevated in prostate cancer and its inhibition reduces prostate cancer cell proliferation, in part by reducing androgen receptor (AR) signaling. However, GSK-3 inhibition can also activate signals that promote cell proliferation and survival, which may preclude the use of GSK-3 inhibitors in the clinic. To identify such signals in prostate cancer, we screened for changes in transcription factor target DNA binding activity in GSK-3-silenced cells. Among the alterations was a reduction in AR DNA target binding, as predicted from previous studies, and an increase in NFκB DNA target binding. Consistent with the latter, gene silencing of GSK-3 or inhibition using the GSK-3 inhibitor CHIR99021 increased basal NFκB transcriptional activity. Activation of NFκB was accompanied by an increase in the level of the NFκB family member RelB. Conversely, silencing RelB reduced activation of NFκB by CHIR99021. Furthermore, the reduction of prostate cancer cell proliferation by CHIR99021 was potentiated by inhibition of NFκB signaling using the IKK inhibitor PS1145. Finally, stratification of human prostate tumor gene expression data for GSK3 revealed an inverse correlation between NFκB-dependent and androgen-dependent gene expression, consistent with the results from the transcription factor target DNA binding screen. In addition, there was a correlation between expression of androgen-repressed NFκB target genes and reduced survival of patients with metastatic prostate cancer. These findings highlight an association between GSK-3/AR and NFκB signaling and its potential clinical importance in metastatic prostate cancer. PMID:25327559

  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. 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)

  1. 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)

  2. Temozolomide downregulates P-glycoprotein expression in glioblastoma stem cells by interfering with the Wnt3a/glycogen synthase-3 kinase/β-catenin pathway

    PubMed Central

    Riganti, Chiara; Salaroglio, Iris Chiara; Caldera, Valentina; Campia, Ivana; Kopecka, Joanna; Mellai, Marta; Annovazzi, Laura; Bosia, Amalia; Ghigo, Dario; Schiffer, Davide

    2013-01-01

    Background Glioblastoma multiforme stem cells display a highly chemoresistant phenotype, whose molecular basis is poorly known. We aim to clarify this issue and to investigate the effects of temozolomide on chemoresistant stem cells. Methods A panel of human glioblastoma cultures, grown as stem cells (neurospheres) and adherent cells, was used. Results Neurospheres had a multidrug resistant phenotype compared with adherent cells. Such chemoresistance was overcome by apparently noncytotoxic doses of temozolomide, which chemosensitized glioblastoma cells to doxorubicin, vinblastine, and etoposide. This effect was selective for P-glycoprotein (Pgp) substrates and for stem cells, leading to an investigation of whether there was a correlation between the expression of Pgp and the activity of typical stemness pathways. We found that Wnt3a and ABCB1, which encodes for Pgp, were both highly expressed in glioblastoma stem cells and reduced by temozolomide. Temozolomide-treated cells had increased methylation of the cytosine–phosphate–guanine islands in the Wnt3a gene promoter, decreased expression of Wnt3a, disrupted glycogen synthase-3 kinase/β-catenin axis, reduced transcriptional activation of ABCB1, and a lower amount and activity of Pgp. Wnt3a overexpression was sufficient to transform adherent cells into neurospheres and to simultaneously increase proliferation and ABCB1 expression. On the contrary, glioblastoma stem cells silenced for Wnt3a lost the ability to form neurospheres and reduced at the same time the proliferation rate and ABCB1 levels. Conclusions Our work suggests that Wnt3a is an autocrine mediator of stemness, proliferation, and chemoresistance in human glioblastoma and that temozolomide may chemosensitize the stem cell population by downregulating Wnt3a signaling. PMID:23897632

  3. Effect of insulin and contraction on glycogen synthase phosphorylation and kinetic properties in epitrochlearis muscles from lean and obese Zucker rats.

    PubMed

    Lin, Fang Chin; Bolling, Astrid; Stuenæs, Jorid T; Cumming, Kristoffer T; Ingvaldsen, Ada; Lai, Yu-Chiang; Ivy, John L; Jensen, Jørgen

    2012-05-15

    In the present study, the effects of insulin and contraction on glycogen synthase (GS) kinetic properties and phosphorylation were investigated in epitrochlearis muscles from lean and obese Zucker rats. Total GS activity and protein expression were ~15% lower in epitrochlearis from obese rats compared with lean rats. Insulin-stimulated GS fractional activity and affinity for UDP-glucose were lower (higher K(m)) in muscles from obese rats. GS Ser(641) and Ser(645,649,653,657) phosphorylation was higher in insulin-stimulated muscles from obese rats, which agreed with lower GS activation. Contraction-mediated GS dephosphorylation of Ser(641), Ser(641+645), Ser(645,649,653,657), and Ser(7+10) was normal in muscles from obese Zucker rats, and GS fractional activity increased to similar levels in epitrochlearis muscles from lean and obese rats. GS affinity for UDP glucose was ~0.8, ~0.4, and ~0.1 mM with assay buffers containing 0, 0.17, and 12 mM glucose 6-phosphate, respectively. Contraction increased affinity for UDP-glucose (reduced K(m)) at a physiological concentration of glucose 6-phosphate (0.17 mM) to ~0.2 mM in muscles from both lean and obese rats. Interestingly, in the absence of glucose 6-phosphate in the assay buffer, contraction (and insulin) did not influence GS affinity for UDP-glucose, indicating that affinity is regulated by sensitivity for glucose 6-phosphate. In conclusion, contraction-mediated activation and dephosphorylation of GS were normal in muscles from obese Zucker rats, whereas insulin-mediated GS activation and dephosphorylation were impaired.

  4. 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

  5. Involvement of Glycogen Synthase Kinase-3β and Oxidation Status in the Loss of Cardioprotection by Postconditioning in Chronic Diabetic Male Rats

    PubMed Central

    Badalzadeh, Reza; Mohammadi, Mustafa; Yousefi, Bahman; Farajnia, Safar; Najafi, Moslem; Mohammadi, Shima

    2015-01-01

    Purpose: Diabetes mellitus as a main risk-factor of ischemic heart disease may interfere with postconditioning’scardioprotective effects. This study aimed to investigate the involvement of glycogen synthase kinase-3β (GSK-3β) and oxidation status in chronic diabetes-induced loss of cardioprotective effect of ischemic-postconditioning (IPostC) in Wistar rats. Methods: After 8 weeks of induction of diabetes by streptozotocin (50mg/kg), hearts of control and diabetic rats were isolated and mounted on a constant-pressure Langendorff system. All hearts were subjected to 30min regional ischemia followed by 60min reperfusion (by occluding and re-opening of left anterior descending coronary artery, respectively). IPostC was applied immediately at the onset of reperfusion. At the end of reperfusion, the infarct size of myocardium was measured via computerized planimetry. Myocardial contents of malondealdehyde and glutathione as indices of oxidative status were assayed spectrophotometrically and the total and phosphorylated forms of myocardial GSK-3β were quantified through western blotting. Results: IPostC reduced the infarct size of control hearts from 41±2.9% to 28±1.9% (P<0.05), whereas it could not induce significant changes in infarct size of diabetic animals (35±1.8% vs. 39±3.1%). IPostC-induced reduction in malondealdehyde and elevation in glutathione contents were significant only in control not in diabetic hearts. The total forms of GSK-3β were similar in all groups; however, the phosphorylation of GSK-3β (at Ser9) by IPostC was greater in control hearts than diabetics (P<0.01). Conclusion: The failure of cardioprotection by IPostC in diabetic hearts may be attributed to the loss of phosphorylation of GSK-3β and thereby increase in oxidative stress in diabetic states. PMID:26504753

  6. The role of glycogen synthase kinase-3β (GSK-3β) in endometrial carcinoma: A carcinogenesis, progression, prognosis, and target therapy marker

    PubMed Central

    Chen, Shuo; Sun, Kai-Xuan; Liu, Bo-Liang; Zong, Zhi-Hong; Zhao, Yang

    2016-01-01

    Purpose Glycogen synthase kinase-3β (GSK-3β) is a serine/threonine kinase involved in cancer development. Herein, we demonstrated the role of GSK-3β in endometrial cancer (EC) and identified new therapeutic targets. Results GSK-3β was overexpressed in EC tissues, and was positively correlated with International Federation of Gynecology and Obstetrics (FIGO) staging, dedifferentiation, and myometrial infiltration depth. Besides, GSK-3β overexpression predicted lower cumulative and relapse-free survival rate. si-GSK-3β transfection suppressed cell proliferation, migration, invasion, and promoted cell apoptosis through downregulating NF-kB, Cyclin D1 and MMP9 expression whereas upregulating P21 expression. Bioinformatic predictions and dual-luciferase reporter assays showed that GSK-3β was a possible target of miR-129. MiR-129 transfection reduced GSK-3β expression, and exhibited the same trend as si-GSK-3β transfection in cell function experiments. The nude mouse xenograft assay showed that miR-129 overexpression may suppress tumor growth through downregulating GSK-3β expression. Further studies showed that AZD1080, a GSK-3β inhibitor, could also inhibit EC cell proliferation, migration and invasion, while induced cell apoptosis through modulating relevant genes downstream of GSK-3β signaling. Experimental Design GSK-3β expression was determined in EC tissue and normal endometrial tissues by immunohistochemistry. After GSK-3β down-regulation by si-GSK-3β, microRNA-129 mimic transfection or GSK-3β inhibitor exposure, EC cell phenotypes and related molecules were examined. Conclusions Our results demonstrate for the first time that GSK-3β may be a novel and important therapeutic target for the treatment of endometrial carcinoma. GSK-3β inhibitor AZD1080 may be an effective drug for treating endometrial carcinoma. PMID:27050373

  7. 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.

  8. 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.

  9. Phosphorylation and inactivation of glycogen synthase kinase 3β (GSK3β) by dual-specificity tyrosine phosphorylation-regulated kinase 1A (Dyrk1A).

    PubMed

    Song, Woo-Joo; Song, Eun-Ah Christine; Jung, Min-Su; Choi, Sun-Hee; Baik, Hyung-Hwan; Jin, Byung Kwan; Kim, Jeong Hee; Chung, Sul-Hee

    2015-01-23

    Glycogen synthase kinase 3β (GSK3β) participates in many cellular processes, and its dysregulation has been implicated in a wide range of diseases such as obesity, type 2 diabetes, cancer, and Alzheimer disease. Inactivation of GSK3β by phosphorylation at specific residues is a primary mechanism by which this constitutively active kinase is controlled. However, the regulatory mechanism of GSK3β is not fully understood. Dual-specificity tyrosine phosphorylation-regulated kinase 1A (Dyrk1A) has multiple biological functions that occur as the result of phosphorylation of diverse proteins that are involved in metabolism, synaptic function, and neurodegeneration. Here we show that GSK3β directly interacts with and is phosphorylated by Dyrk1A. Dyrk1A-mediated phosphorylation at the Thr(356) residue inhibits GSK3β activity. Dyrk1A transgenic (TG) mice are lean and resistant to diet-induced obesity because of reduced fat mass, which shows an inverse correlation with the effect of GSK3β on obesity. This result suggests a potential in vivo association between GSK3β and Dyrk1A regarding the mechanism underlying obesity. The level of Thr(P)(356)-GSK3β was higher in the white adipose tissue of Dyrk1A TG mice compared with control mice. GSK3β activity was differentially regulated by phosphorylation at different sites in adipose tissue depending on the type of diet the mice were fed. Furthermore, overexpression of Dyrk1A suppressed the expression of adipogenic proteins, including peroxisome proliferator-activated receptor γ, in 3T3-L1 cells and in young Dyrk1A TG mice fed a chow diet. Taken together, these results reveal a novel regulatory mechanism for GSK3β activity and indicate that overexpression of Dyrk1A may contribute to the obesity-resistant phenotype through phosphorylation and inactivation of GSK3β. PMID:25477508

  10. Glycogen synthase kinase-3β inhibition depletes the population of prostate cancer stem/progenitor-like cells and attenuates metastatic growth

    PubMed Central

    Kroon, Jan; in 't Veld, Lars S.; Buijs, Jeroen T.; Cheung, Henry; van der Horst, Geertje; van der Pluijm, Gabri

    2014-01-01

    Cancer cells with stem or progenitor properties play a pivotal role in the initiation, recurrence and metastatic potential of solid tumors, including those of the human prostate. Cancer stem cells are generally more resistant to conventional therapies thus requiring the characterization of key pathways involved in the formation and/or maintenance of this malignant cellular subpopulation. To this end, we identified Glycogen Synthase Kinase-3β (GSK-3β) as a crucial kinase for the maintenance of prostate cancer stem/progenitor-like cells and pharmacologic inhibition of GSK-3β dramatically decreased the size of this cellular subpopulation. This was paralleled by impaired clonogenicity, decreased migratory potential and dramatic morphological changes. In line with our in vitro observations, treatment with a GSK-3β inhibitor leads to a complete loss of tumorigenicity and a decrease in metastatic potential in preclinical in vivo models. These observed anti-tumor effects appear to be largely Wnt-independent as simultaneous Wnt inhibition does not reverse the observed antitumor effects of GSK-3β blockage. We found that GSK-3β activity is linked to cytoskeletal protein F-actin and inhibition of GSK-3β leads to disturbance of F-actin polymerization. This may underlie the dramatic effects of GSK-3β inhibition on prostate cancer migration. Furthermore, GSK-3β inhibition led to strongly decreased expression of several integrin types including the cancer stem cell-associated α2β1 integrin. Taken together, our mechanistic observations highlight the importance of GSK-3β activity in prostate cancer stemness and may facilitate the development of novel therapy for advanced prostate cancer. PMID:25344861

  11. 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.

  12. Regulation of Ribosomal S6 Protein Kinase-p90rsk, Glycogen Synthase Kinase 3, and β-Catenin in Early Xenopus Development

    PubMed Central

    Torres, Monica A.; Eldar-Finkelman, Hagit; Krebs, Edwin G.; Moon, Randall T.

    1999-01-01

    β-Catenin is a multifunctional protein that binds cadherins at the plasma membrane, HMG box transcription factors in the nucleus, and several cytoplasmic proteins that are involved in regulating its stability. In developing embryos and in some human cancers, the accumulation of β-catenin in the cytoplasm and subsequently the nuclei of cells may be regulated by the Wnt-1 signaling cascade and by glycogen synthase kinase 3 (GSK-3). This has increased interest in regulators of both GSK-3 and β-catenin. Searching for kinase activities able to phosphorylate the conserved, inhibitory-regulatory GSK-3 residue serine 9, we found p90rsk to be a potential upstream regulator of GSK-3. Overexpression of p90rsk in Xenopus embryos leads to increased steady-state levels of total β-catenin but not of the free soluble protein. Instead, p90rsk overexpression increases the levels of β-catenin in a cell fraction containing membrane-associated cadherins. Consistent with the lack of elevation of free β-catenin levels, ectopic p90rsk was unable to rescue dorsal cell fate in embryos ventralized by UV irradiation. We show that p90rsk is a downstream target of fibroblast growth factor (FGF) signaling during early Xenopus development, since ectopic FGF signaling activates both endogenous and overexpressed p90rsk. Moreover, overexpression of a dominant negative FGF receptor, which blocks endogenous FGF signaling, leads to decreased p90rsk kinase activity. Finally, we report that FGF inhibits endogenous GSK-3 activity in Xenopus embryos. We hypothesize that FGF and p90rsk play heretofore unsuspected roles in modulating GSK-3 and β-catenin. PMID:9891076

  13. Role of PECAM-1 in the shear-stress-induced activation of Akt and the endothelial nitric oxide synthase (eNOS) in endothelial cells.

    PubMed

    Fleming, Ingrid; Fisslthaler, Beate; Dixit, Madhulika; Busse, Rudi

    2005-09-15

    The application of fluid shear stress to endothelial cells elicits the formation of nitric oxide (NO) and phosphorylation of the endothelial NO synthase (eNOS). Shear stress also elicits the enhanced tyrosine phosphorylation of endothelial proteins, especially of those situated in the vicinity of cell-cell contacts. Since a major constituent of these endothelial cell-cell contacts is the platelet endothelial cell adhesion molecule-1 (PECAM-1) we assessed the role of PECAM-1 in the activation of eNOS. In human endothelial cells, shear stress induced the tyrosine phosphorylation of PECAM-1 and enhanced the association of PECAM-1 with eNOS. Endothelial cell stimulation with shear stress elicited the phosphorylation of Akt and eNOS as well as of the AMP-activated protein kinase (AMPK). While the shear-stress-induced tyrosine phosphorylation of PECAM-1 as well as the serine phosphorylation of Akt and eNOS were abolished by the pre-treatment of cells with the tyrosine kinase inhibitor PP1 the phosphorylation of AMPK was unaffected. Down-regulation of PECAM-1 using a siRNA approach attenuated the shear-stress-induced phosphorylation of Akt and eNOS, as well as the shear-stress-induced accumulation of cyclic GMP levels while the shear-stress-induced phosphorylation of AMPK remained intact. A comparable attenuation of Akt and eNOS (but not AMPK) phosphorylation and NO production was also observed in endothelial cells generated from PECAM-1-deficient mice. These data indicate that the shear-stress-induced activation of Akt and eNOS in endothelial cells is modulated by the tyrosine phosphorylation of PECAM-1 whereas the shear-stress-induced phosphorylation of AMPK is controlled by an alternative signaling pathway. PMID:16118242

  14. 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

  15. Involvement of the PI3K/Akt/GSK3β pathway in photodynamic injury of neurons and glial cells

    NASA Astrophysics Data System (ADS)

    Komandirov, M. A.; Knyazeva, E. A.; Fedorenko, Y. P.; Rudkovskii, M. V.; Stetsurin, D. A.; Uzdensky, A. B.

    2011-03-01

    Photodynamic treatment causes intense oxidative stress and kills cells. It is currently used in neurooncology. However, along with tumor it damages surrounding healthy neuronal and glial cells. In order to study the possible role of the phosphatidylinositol 3-kinase/protein kinase Akt/glycogen synthase kinase-3β signaling pathway in photodynamic damage to normal neurons and glia, we used isolated crayfish stretch receptor that consists only of a single neuron surrounded by glial cells. It was photosensitized with alumophthalocyanine Photosens (100 nM). The laser diode (670nm, 0.4W/cm2) was used as a light source. Application of specific inhibitors of the enzymes involved in this pathway showed that phosphatidylinositol 3-kinase did not participate in photoinduced death of neurons and glia. Protein kinase Akt was involved in photoinduced necrosis but not in apoptosis of neurons and glia. Glycogen synthase kinase-3β participated in photoinduced apoptosis of glial cells and in necrosis of neurons. Therefore, the phosphatidylinositol 3-kinase/protein kinase Akt/glycogen synthase kinase-3β pathway was not involved as a whole in photodynamic injury of crayfish neurons and glial cells but its components, protein kinase Akt and glycogen synthase kinase-3β, independently and cell-specifically regulated photoinduced death of neurons and glial cells. These data showed that in this system necrosis was not non-regulated and catastrophic mode of cell death. It was controlled by some signaling proteins. The obtained results may be used for search of pharmacological agents that selectively modulate injury of normal neurons and glial cells during photodynamic therapy of brain tumors.

  16. Involvement of the PI3K/Akt/GSK3β pathway in photodynamic injury of neurons and glial cells

    NASA Astrophysics Data System (ADS)

    Komandirov, M. A.; Knyazeva, E. A.; Fedorenko, Y. P.; Rudkovskii, M. V.; Stetsurin, D. A.; Uzdensky, A. B.

    2010-10-01

    Photodynamic treatment causes intense oxidative stress and kills cells. It is currently used in neurooncology. However, along with tumor it damages surrounding healthy neuronal and glial cells. In order to study the possible role of the phosphatidylinositol 3-kinase/protein kinase Akt/glycogen synthase kinase-3β signaling pathway in photodynamic damage to normal neurons and glia, we used isolated crayfish stretch receptor that consists only of a single neuron surrounded by glial cells. It was photosensitized with alumophthalocyanine Photosens (100 nM). The laser diode (670nm, 0.4W/cm2) was used as a light source. Application of specific inhibitors of the enzymes involved in this pathway showed that phosphatidylinositol 3-kinase did not participate in photoinduced death of neurons and glia. Protein kinase Akt was involved in photoinduced necrosis but not in apoptosis of neurons and glia. Glycogen synthase kinase-3β participated in photoinduced apoptosis of glial cells and in necrosis of neurons. Therefore, the phosphatidylinositol 3-kinase/protein kinase Akt/glycogen synthase kinase-3β pathway was not involved as a whole in photodynamic injury of crayfish neurons and glial cells but its components, protein kinase Akt and glycogen synthase kinase-3β, independently and cell-specifically regulated photoinduced death of neurons and glial cells. These data showed that in this system necrosis was not non-regulated and catastrophic mode of cell death. It was controlled by some signaling proteins. The obtained results may be used for search of pharmacological agents that selectively modulate injury of normal neurons and glial cells during photodynamic therapy of brain tumors.

  17. CCN1 acutely increases nitric oxide production via integrin αvβ3-Akt-S6K-phosphorylation of endothelial nitric oxide synthase at the serine 1177 signaling axis.

    PubMed

    Hwang, Soojin; Lee, Hyeon-Ju; Kim, Gyungah; Won, Kyung-Jong; Park, Yoon Shin; Jo, Inho

    2015-12-01

    Although CCN1 (also known as cysteine-rich, angiogenic inducer 61, CYR61) has been reported to promote angiogenesis and neovascularization in endothelial cells (ECs), its effects on endothelial nitric oxide (NO) production have never been studied. Using human umbilical vein ECs, we investigated whether and how CCN1 regulates NO production. CCN1 acutely increased NO production in a time- and dose-dependent manner, which was accompanied by increased phosphorylation of endothelial NO synthase (eNOS) at serine 1177 (eNOS-Ser(1177)), but not that of eNOS-Thr(495) or eNOS-Ser(114). The level of total eNOS expression was unaltered. Treatment with either LY294002, a selective inhibitor of phosphoinositide 3-kinase known as an upstream kinase of Akt, or H-89, an inhibitor of protein kinase A, mitogen- and stress-activated protein kinase 1, Rho-associated protein kinase 2, and ribosomal protein S6 kinase (S6K), inhibited CCN1-stimulated eNOS-Ser(1177) phosphorylation and subsequent NO production. Ectopic expression of small interfering RNA against Akt and S6K significantly inhibited the effects of CCN1. Consistently, CCN1 increased the phosphorylation of Akt-Ser(473) and S6K-Thr(389). However, CCN1 did not alter the expression or secretion of VEGF, a known downstream factor of CCN1 and a potential upstream factor of Akt-mediated eNOS-Ser(1177) phosphorylation. Furthermore, neutralization of integrin αvβ3 with corresponding antibody completely reversed all of the observed effects of CCN1. Moreover, CCN1 increased acetylcholine-induced relaxation in the rat aortas. Finally, we also found that CCN1-stimulated eNOS-Ser(1177) phosphorylation and NO production are true for other types of EC tested. In conclusion, CCN1 acutely increases NO production via activation of a signaling axis in integrin αvβ3-Akt-S6K-eNOS-Ser(1177) phosphorylation, suggesting an important role for CCN1 in vasodilation.

  18. Neuroprotective effect of allicin against traumatic brain injury via Akt/endothelial nitric oxide synthase pathway-mediated anti-inflammatory and anti-oxidative activities.

    PubMed

    Chen, Wei; Qi, Jun; Feng, Feng; Wang, Mao-de; Bao, Gang; Wang, Tuo; Xiang, Mu; Xie, Wan-Fu

    2014-03-01

    Allicin, one of the main biologically active compounds derived from garlic, has been shown to exert various anti-oxidative and anti-inflammatory activities in in vitro and in vivo studies. Here, we sought to investigate the potential neuroprotective effects of allicin against traumatic brain injury (TBI) in rats. We found that allicin treatment (10 and 50mg/kg, not 1mg/kg) significantly reduced brain edema and motor functional deficits, as well as apoptotic neuronal cell death in injured cortex. These protective effects could be observed even if the administration was delayed to 4h after injury. Moreover, allicin treatment decreased the expression levels of MDA and protein carbonyl, preserved the endogenous antioxidant enzyme activities, and suppressed the expression of inflammatory cytokines. The results of Western blot analysis showed that allicin increased the phosphorylation of Akt and endothelial nitric oxide synthase (eNOS). Blocking Akt/eNOS pathway activation by specific inhibitor LY294002 (10μL, 10mmol/L) or L-NIO (0.5mg/kg) partly reversed the protective effects of allicin and its anti-inflammatory activities. The allicin induced anti-oxidative activity was partly prevented by LY294002, but not L-NIO. In summary, our data strongly suggested that allicin treatment at an appropriate dose can exert protective effect against TBI through Akt/eNOS pathway-mediated anti-inflammatory and anti-oxidative activities.

  19. Human stem cell osteoblastogenesis mediated by novel glycogen synthase kinase 3 inhibitors induces bone formation and a unique bone turnover biomarker profile in rats

    SciTech Connect

    Gilmour, Peter S.; O'Shea, Patrick J.; Fagura, Malbinder; Pilling, James E.; Sanganee, Hitesh; Wada, Hiroki; Courtney, Paul F.; Kavanagh, Stefan; Hall, Peter A.; Escott, K. Jane

    2013-10-15

    Wnt activation by inhibiting glycogen synthase kinase 3 (GSK-3) causes bone anabolism in rodents making GSK-3 a potential therapeutic target for osteoporotic and osteolytic metastatic bone disease. To understand the wnt pathway related to human disease translation, the ability of 3 potent inhibitors of GSK-3 (AZD2858, AR79, AZ13282107) to 1) drive osteoblast differentiation and mineralisation using human adipose-derived stem cells (hADSC) in vitro; and 2) stimulate rat bone formation in vivo was investigated. Bone anabolism/resorption was determined using clinically relevant serum biomarkers as indicators of bone turnover and bone formation assessed in femurs by histopathology and pQCT/μCT imaging. GSK-3 inhibitors caused β-catenin stabilisation in human and rat mesenchymal stem cells, stimulated hADSC commitment towards osteoblasts and osteogenic mineralisation in vitro. AZD2858 produced time-dependent changes in serum bone turnover biomarkers and increased bone mass over 28 days exposure in rats. After 7 days, AZD2858, AR79 or AZ13282107 exposure increased the bone formation biomarker P1NP, and reduced the resorption biomarker TRAcP-5b, indicating increased bone anabolism and reduced resorption in rats. This biomarker profile was differentiated from anabolic agent PTH{sub 1–34} or the anti-resorptive Alendronate-induced changes. Increased bone formation in cortical and cancellous bone as assessed by femur histopathology supported biomarker changes. 14 day AR79 treatment increased bone mineral density and trabecular thickness, and decreased trabecular number and connectivity assessed by pQCT/μCT. GSK-3 inhibition caused hADSC osteoblastogenesis and mineralisation in vitro. Increased femur bone mass associated with changes in bone turnover biomarkers confirmed in vivo bone formation and indicated uncoupling of bone formation and resorption. - Highlights: • Wnt modulation with 3 novel GSK-3 inhibitors alters bone growth. • Human stem cell osteoblastogenesis

  20. 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

  1. 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

  2. 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.

  3. Activated AKT pathway promotes establishment of endometriosis.

    PubMed

    Kim, Tae Hoon; Yu, Yanni; Luo, Lily; Lydon, John P; Jeong, Jae-Wook; Kim, J Julie

    2014-05-01

    The pathogenesis of endometriosis remains unclear, and relatively little is known about the mechanisms that promote establishment and survival of the disease. Previously, we demonstrated that v-akt murine thymoma viral oncogene homolog (AKT) activity was increased in endometriosis tissues and cells from ovarian endometriomas and that this increase promoted cell survival as well as decreased levels of progesterone receptor. The objective of this study was to demonstrate a role for AKT in the establishment of ectopic lesions. First, a dose-dependent inhibition of AKT in stromal cells from human ovarian endometriomas (OSIS) as well as endometrial stromal cells from disease-free patients (ESC) with the allosteric AKT inhibitor MK-2206 was demonstrated by decreased levels of phosphorylated (p)(Ser473)-AKT. Levels of the AKT target protein, p(Ser256)-forkhead box O1 were increased in OSIS cells, which decreased with MK-2206 treatment, whereas levels of p(Ser9)-glycogen synthase kinase 3β did not change in response to MK-2206. Although MK-2206 decreased viability of both OSIS and ESC in a dose-dependent manner, proliferation of OSIS cells was differentially decreased significantly compared with ESC. Next, the role of hyperactive AKT in the establishment of ectopic lesions was studied using the bigenic, PR(cre/+)Pten(f/+) heterozygous mouse. Autologous implantation of uterine tissues was performed in these mice. After 4 weeks, an average of 4 ± 0.33 lesions per Pten(f/+) mouse and 7.5 ± 0.43 lesions in the PR(cre/+)Pten(f/+) mouse were found. Histological examination of the lesions showed endometrial tissue-like morphology, which was similar in both the Pten(f/+) and PR(cre/+)Pten(f/+) mice. Treatment of mice with MK-2206 resulted in a significantly decreased number of lesions established. Immunohistochemical staining of ectopic lesions revealed decreased p(Ser473)-AKT and the proliferation marker Ki67 from MK-2206-treated mice compared with vehicle-treated mice

  4. Proteasome inhibition-induced p38 MAPK/ERK signaling regulates autophagy and apoptosis through the dual phosphorylation of glycogen synthase kinase 3{beta}

    SciTech Connect

    Choi, Cheol-Hee; Lee, Byung-Hoon; Ahn, Sang-Gun; Oh, Seon-Hee

    2012-02-24

    Highlights: Black-Right-Pointing-Pointer MG132 induces the phosphorylation of GSK3{beta}{sup Ser9} and, to a lesser extent, of GSK3{beta}{sup Thr390}. Black-Right-Pointing-Pointer MG132 induces dephosphorylation of p70S6K{sup Thr389} and phosphorylation of p70S6K{sup Thr421/Ser424}. Black-Right-Pointing-Pointer Inactivation of p38 dephosphorylates GSK3{beta}{sup Ser9} and phosphorylates GSK3{beta}{sup Thr390}. Black-Right-Pointing-Pointer Inactivation of p38 phosphorylates p70S6K{sup Thr389} and increases the phosphorylation of p70S6K{sup Thr421/Ser424}. Black-Right-Pointing-Pointer Inactivation of p38 decreases autophagy and increases apoptosis induced by MG132. -- Abstract: Proteasome inhibition is a promising approach for cancer treatment; however, the underlying mechanisms involved have not been fully elucidated. Here, we show that proteasome inhibition-induced p38 mitogen-activated protein kinase regulates autophagy and apoptosis by modulating the phosphorylation status of glycogen synthase kinase 3{beta} (GSK3{beta}) and 70 kDa ribosomal S6 kinase (p70S6K). The treatment of MDA-MB-231 cells with MG132 induced endoplasmic reticulum stress through the induction of ATF6a, PERK phosphorylation, and CHOP, and apoptosis through the cleavage of Bax and procaspase-3. MG132 caused the phosphorylation of GSK3{beta} at Ser{sup 9} and, to a lesser extent, Thr{sup 390}, the dephosphorylation of p70S6K at Thr{sup 389}, and the phosphorylation of p70S6K at Thr{sup 421} and Ser{sup 424}. The specific p38 inhibitor SB203080 reduced the p-GSK3{beta}{sup Ser9} and autophagy through the phosphorylation of p70S6K{sup Thr389}; however, it augmented the levels of p-ERK, p-GSK3{beta}{sup Thr390}, and p-70S6K{sup Thr421/Ser424} induced by MG132, and increased apoptotic cell death. The GSK inhibitor SB216763, but not lithium, inhibited the MG132-induced phosphorylation of p38, and the downstream signaling pathway was consistent with that in SB203580-treated cells. Taken together, our

  5. Arctigenin, a Potent Ingredient of Arctium lappa L., Induces Endothelial Nitric Oxide Synthase and Attenuates Subarachnoid Hemorrhage-Induced Vasospasm through PI3K/Akt Pathway in a Rat Model.

    PubMed

    Chang, Chih-Zen; Wu, Shu-Chuan; Chang, Chia-Mao; Lin, Chih-Lung; Kwan, Aij-Lie

    2015-01-01

    Upregulation of protein kinase B (PKB, also known as Akt) is observed within the cerebral arteries of subarachnoid hemorrhage (SAH) animals. This study is of interest to examine Arctigenin, a potent antioxidant, on endothelial nitric oxide synthase (eNOS) and Akt pathways in a SAH in vitro study. Basilar arteries (BAs) were obtained to examine phosphatidylinositol-3-kinase (PI3K), phospho-PI3K, Akt, phospho-Akt (Western blot) and morphological examination. Endothelins (ETs) and eNOS evaluation (Western blot and immunostaining) were also determined. Arctigenin treatment significantly alleviates disrupted endothelial cells and tortured internal elastic layer observed in the SAH groups (p < 0.01). The reduced eNOS protein and phospho-Akt expression in the SAH groups were relieved by the treatment of Arctigenin (p < 0.01). This result confirmed that Arctigenin might exert dural effects in preventing SAH-induced vasospasm through upregulating eNOS expression via the PI3K/Akt signaling pathway and attenuate endothelins after SAH. Arctigenin shows therapeutic promise in the treatment of cerebral vasospasm following SAH.

  6. Arctigenin, a Potent Ingredient of Arctium lappa L., Induces Endothelial Nitric Oxide Synthase and Attenuates Subarachnoid Hemorrhage-Induced Vasospasm through PI3K/Akt Pathway in a Rat Model

    PubMed Central

    Chang, Chih-Zen; Wu, Shu-Chuan; Chang, Chia-Mao; Lin, Chih-Lung; Kwan, Aij-Lie

    2015-01-01

    Upregulation of protein kinase B (PKB, also known as Akt) is observed within the cerebral arteries of subarachnoid hemorrhage (SAH) animals. This study is of interest to examine Arctigenin, a potent antioxidant, on endothelial nitric oxide synthase (eNOS) and Akt pathways in a SAH in vitro study. Basilar arteries (BAs) were obtained to examine phosphatidylinositol-3-kinase (PI3K), phospho-PI3K, Akt, phospho-Akt (Western blot) and morphological examination. Endothelins (ETs) and eNOS evaluation (Western blot and immunostaining) were also determined. Arctigenin treatment significantly alleviates disrupted endothelial cells and tortured internal elastic layer observed in the SAH groups (p < 0.01). The reduced eNOS protein and phospho-Akt expression in the SAH groups were relieved by the treatment of Arctigenin (p < 0.01). This result confirmed that Arctigenin might exert dural effects in preventing SAH-induced vasospasm through upregulating eNOS expression via the PI3K/Akt signaling pathway and attenuate endothelins after SAH. Arctigenin shows therapeutic promise in the treatment of cerebral vasospasm following SAH. PMID:26539501

  7. Arctigenin, a Potent Ingredient of Arctium lappa L., Induces Endothelial Nitric Oxide Synthase and Attenuates Subarachnoid Hemorrhage-Induced Vasospasm through PI3K/Akt Pathway in a Rat Model.

    PubMed

    Chang, Chih-Zen; Wu, Shu-Chuan; Chang, Chia-Mao; Lin, Chih-Lung; Kwan, Aij-Lie

    2015-01-01

    Upregulation of protein kinase B (PKB, also known as Akt) is observed within the cerebral arteries of subarachnoid hemorrhage (SAH) animals. This study is of interest to examine Arctigenin, a potent antioxidant, on endothelial nitric oxide synthase (eNOS) and Akt pathways in a SAH in vitro study. Basilar arteries (BAs) were obtained to examine phosphatidylinositol-3-kinase (PI3K), phospho-PI3K, Akt, phospho-Akt (Western blot) and morphological examination. Endothelins (ETs) and eNOS evaluation (Western blot and immunostaining) were also determined. Arctigenin treatment significantly alleviates disrupted endothelial cells and tortured internal elastic layer observed in the SAH groups (p < 0.01). The reduced eNOS protein and phospho-Akt expression in the SAH groups were relieved by the treatment of Arctigenin (p < 0.01). This result confirmed that Arctigenin might exert dural effects in preventing SAH-induced vasospasm through upregulating eNOS expression via the PI3K/Akt signaling pathway and attenuate endothelins after SAH. Arctigenin shows therapeutic promise in the treatment of cerebral vasospasm following SAH. PMID:26539501

  8. 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.

  9. A Switch in Akt Isoforms Is Required for Notch-Induced Snail1 Expression and Protection from Cell Death

    PubMed Central

    Frías, Alex; Lambies, Guillem; Viñas-Castells, Rosa; Martínez-Guillamon, Catalina; Dave, Natàlia

    2015-01-01

    Notch activation in aortic endothelial cells (ECs) takes place at embryonic stages during cardiac valve formation and induces endothelial-to-mesenchymal transition (EndMT). Using aortic ECs, we show here that active Notch expression promotes EndMT, resulting in downregulation of vascular endothelial cadherin (VE-cadherin) and upregulation of mesenchymal genes such as those for fibronectin and Snail1/2. In these cells, transforming growth factor β1 exacerbates Notch effects by increasing Snail1 and fibronectin activation. When Notch-downstream pathways were analyzed, we detected an increase in glycogen synthase kinase 3β (GSK-3β) phosphorylation and inactivation that facilitates Snail1 nuclear retention and protein stabilization. However, the total activity of Akt was downregulated. The discrepancy between Akt activity and GSK-3β phosphorylation is explained by a Notch-induced switch in the Akt isoforms, whereby Akt1, the predominant isoform expressed in ECs, is decreased and Akt2 transcription is upregulated. Mechanistically, Akt2 induction requires the stimulation of the β-catenin/TCF4 transcriptional complex, which activates the Akt2 promoter. Active, phosphorylated Akt2 translocates to the nucleus in Notch-expressing cells, resulting in GSK-3β inactivation in this compartment. Akt2, but not Akt1, colocalizes in the nucleus with lamin B in the nuclear envelope. In addition to promoting GSK-3β inactivation, Notch downregulates Forkhead box O1 (FoxO1), another Akt2 nuclear substrate. Moreover, Notch protects ECs from oxidative stress-induced apoptosis through an Akt2- and Snail1-dependent mechanism. PMID:26711268

  10. 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

  11. PI3K-C2γ is a Rab5 effector selectively controlling endosomal Akt2 activation downstream of insulin signalling

    PubMed Central

    Braccini, Laura; Ciraolo, Elisa; Campa, Carlo C.; Perino, Alessia; Longo, Dario L.; Tibolla, Gianpaolo; Pregnolato, Marco; Cao, Yanyan; Tassone, Beatrice; Damilano, Federico; Laffargue, Muriel; Calautti, Enzo; Falasca, Marco; Norata, Giuseppe D.; Backer, Jonathan M.; Hirsch, Emilio

    2015-01-01

    In the liver, insulin-mediated activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway is at the core of metabolic control. Multiple PI3K and Akt isoenzymes are found in hepatocytes and whether isoform-selective interplays exist is currently unclear. Here we report that insulin signalling triggers the association of the liver-specific class II PI3K isoform γ (PI3K-C2γ) with Rab5-GTP, and its recruitment to Rab5-positive early endosomes. In these vesicles, PI3K-C2γ produces a phosphatidylinositol-3,4-bisphosphate pool specifically required for delayed and sustained endosomal Akt2 stimulation. Accordingly, loss of PI3K-C2γ does not affect insulin-dependent Akt1 activation as well as S6K and FoxO1-3 phosphorylation, but selectively reduces Akt2 activation, which specifically inhibits glycogen synthase activity. As a consequence, PI3K-C2γ-deficient mice display severely reduced liver accumulation of glycogen and develop hyperlipidemia, adiposity as well as insulin resistance with age or after consumption of a high-fat diet. Our data indicate PI3K-C2γ supports an isoenzyme-specific forking of insulin-mediated signal transduction to an endosomal pool of Akt2, required for glucose homeostasis. PMID:26100075

  12. 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

  13. 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

  14. Hypoxia activates Akt and induces phosphorylation of GSK-3 in PC12 cells.

    PubMed

    Beitner-Johnson, D; Rust, R T; Hsieh, T C; Millhorn, D E

    2001-01-01

    Akt is a serine/threonine kinase that has been shown to play a central role in promoting cell survival and opposing apoptosis. We evaluated the effect of hypoxia on Akt in rat pheochromocytoma (PC12) cells. PC12 cells were exposed to varying levels of hypoxia, including 21%, 15%, 10%, 5%, and 1% O(2). Hypoxia dramatically increased phosphorylation of Akt (Ser(473)). This effect peaked after 6 h exposure to hypoxia, but persisted strongly for up to 24 h. Phosphorylation of Akt was paralleled with a progressive increase in phosphorylation of glycogen synthase kinase-3 (GSK-3), one of its downstream substrates. The effect of hypoxia on phosphorylation of Akt was completely blocked by pretreatment of the cells with wortmannin (100 nM), indicating that this effect is mediated by phosphatidylinositol 3-kinase (P13K). In contrast, whereas hypoxia also strongly induced phosphorylation of the transcription factors CREB and EPAS1, these effects persisted in the presence of wortmannin. Thus, hypoxia regulates both P13K-dependent and P13K-independent signaling pathways. Furthermore, activation of the P13K and Akt signaling pathways may be one mechanism by which cells adapt and survive under conditions of hypoxia. PMID:11257444

  15. Human recombinant H2 relaxin induces AKT and GSK3β phosphorylation and HTR-8/SVneo cell proliferation.

    PubMed

    Astuti, Yoni; Nakabayashi, Koji; Deguchi, Masashi; Ebina, Yasuhiko; Yamada, Hideto

    2015-01-01

    Relaxin is essential for trophoblast development during pregnancy. Evidence shows that relaxin increases trophoblast cell migration capacity. Here, we show the effect of relaxin on protein kinase B (AKT) activation and glycogen synthase kinase 3-beta (GSK3β) inactivation as well as on the proliferation of HTR-8/SVneo cells, a model of human extravillous trophoblast (EVT). HTR-8/SVneo cells were treated with different doses of human recombinant (rH2) relaxin in serum-deprived conditions and treated for increasing time with 1 ng/mL of rH2 relaxin. Western blot analysis was performed to detect pAKT, AKT, pGSK3β, GSK3β, and actin expression. Proliferation of HTR-8/SVneo cells was analyzed by MTS assay. rH2 relaxin treatment increased the ratio of pAKT/AKT, pGSK3β/GSK3β, and proliferation in HTR-8/SVneo cells. Furthermore, AKT and GSK3β activation by rH2 relaxin was inhibited by a phosphoinositide 3-kinase (PI3K) inhibitor. This study suggests that rH2 relaxin induces AKT and GSK3β phosphorylation as well as proliferation in HTR-8/SVneo cells. PMID:25868609

  16. The A-Kinase Anchoring Protein (AKAP) Glycogen Synthase Kinase 3β Interaction Protein (GSKIP) Regulates β-Catenin through Its Interactions with Both Protein Kinase A (PKA) and GSK3β.

    PubMed

    Dema, Alessandro; Schröter, Micha Friedemann; Perets, Ekaterina; Skroblin, Philipp; Moutty, Marie Christine; Deàk, Veronika Anita; Birchmeier, Walter; Klussmann, Enno

    2016-09-01

    The A-kinase anchoring protein (AKAP) GSK3β interaction protein (GSKIP) is a cytosolic scaffolding protein binding protein kinase A (PKA) and glycogen synthase kinase 3β (GSK3β). Here we show that both the AKAP function of GSKIP, i.e. its direct interaction with PKA, and its direct interaction with GSK3β are required for the regulation of β-catenin and thus Wnt signaling. A cytoplasmic destruction complex targets β-catenin for degradation and thus prevents Wnt signaling. Wnt signals cause β-catenin accumulation and translocation into the nucleus, where it induces Wnt target gene expression. GSKIP facilitates control of the β-catenin stabilizing phosphorylation at Ser-675 by PKA. Its interaction with GSK3β facilitates control of the destabilizing phosphorylation of β-catenin at Ser-33/Ser-37/Thr-41. The influence of GSKIP on β-catenin is explained by its scavenger function; it recruits the kinases away from the destruction complex without forming a complex with β-catenin. The regulation of β-catenin by GSKIP is specific for this AKAP as AKAP220, which also binds PKA and GSK3β, did not affect Wnt signaling. We find that the binding domain of AKAP220 for GSK3β is a conserved GSK3β interaction domain (GID), which is also present in GSKIP. Our findings highlight an essential compartmentalization of both PKA and GSK3β by GSKIP, and ascribe a function to a cytosolic AKAP-PKA interaction as a regulatory factor in the control of canonical Wnt signaling. Wnt signaling controls different biological processes, including embryonic development, cell cycle progression, glycogen metabolism, and immune regulation; deregulation is associated with diseases such as cancer, type 2 diabetes, inflammatory, and Alzheimer's and Parkinson's diseases.

  17. A novel PKB/Akt inhibitor, MK-2206, effectively inhibits insulin-stimulated glucose metabolism and protein synthesis in isolated rat skeletal muscle.

    PubMed

    Lai, Yu-Chiang; Liu, Yang; Jacobs, Roxane; Rider, Mark H

    2012-10-01

    PKB (protein kinase B), also known as Akt, is a key component of insulin signalling. Defects in PKB activation lead to insulin resistance and metabolic disorders, whereas PKB overactivation has been linked to tumour growth. Small-molecule PKB inhibitors have thus been developed for cancer treatment, but also represent useful tools to probe the roles of PKB in insulin action. In the present study, we examined the acute effects of two allosteric PKB inhibitors, MK-2206 and Akti 1/2 (Akti) on PKB signalling in incubated rat soleus muscles. We also assessed the effects of the compounds on insulin-stimulated glucose uptake, glycogen and protein synthesis. MK-2206 dose-dependently inhibited insulin-stimulated PKB phosphorylation, PKBβ activity and phosphorylation of PKB downstream targets (including glycogen synthase kinase-3α/β, proline-rich Akt substrate of 40 kDa and Akt substrate of 160 kDa). Insulin-stimulated glucose uptake, glycogen synthesis and glycogen synthase activity were also decreased by MK-2206 in a dose-dependent manner. Incubation with high doses of MK-2206 (10 μM) inhibited insulin-induced p70 ribosomal protein S6 kinase and 4E-BP1 (eukaryotic initiation factor 4E-binding protein-1) phosphorylation associated with increased eEF2 (eukaryotic elongation factor 2) phosphorylation. In contrast, Akti only modestly inhibited insulin-induced PKB and mTOR (mammalian target of rapamycin) signalling, with little or no effect on glucose uptake and protein synthesis. MK-2206, rather than Akti, would thus be the tool of choice for studying the role of PKB in insulin action in skeletal muscle. The results point to a key role for PKB in mediating insulin-stimulated glucose uptake, glycogen synthesis and protein synthesis in skeletal muscle.

  18. Hyperphosphorylation of glucosyl C6 carbons and altered structure of glycogen in the neurodegenerative epilepsy Lafora disease.

    PubMed

    Nitschke, Felix; Wang, Peixiang; Schmieder, Peter; Girard, Jean-Marie; Awrey, Donald E; Wang, Tony; Israelian, Johan; Zhao, XiaoChu; Turnbull, Julie; Heydenreich, Matthias; Kleinpeter, Erich; Steup, Martin; Minassian, Berge A

    2013-05-01

    Laforin or malin deficiency causes Lafora disease, characterized by altered glycogen metabolism and teenage-onset neurodegeneration with intractable and invariably fatal epilepsy. Plant starches possess small amounts of metabolically essential monophosphate esters. Glycogen contains similar phosphate amounts, which are thought to originate from a glycogen synthase error side reaction and therefore lack any specific function. Glycogen is also believed to lack monophosphates at glucosyl carbon C6, an essential phosphorylation site in plant starch metabolism. We now show that glycogen phosphorylation is not due to a glycogen synthase side reaction, that C6 is a major glycogen phosphorylation site, and that C6 monophosphates predominate near centers of glycogen molecules and positively correlate with glycogen chain lengths. Laforin or malin deficiency causes C6 hyperphosphorylation, which results in malformed long-chained glycogen that accumulates in many tissues, causing neurodegeneration in brain. Our work advances the understanding of Lafora disease pathogenesis and suggests that glycogen phosphorylation has important metabolic function.

  19. Phosphorylations of Serines 21/9 in Glycogen Synthase Kinase 3α/β Are Not Required for Cell Lineage Commitment or WNT Signaling in the Normal Mouse Intestine

    PubMed Central

    Hey, Fiona; Giblett, Susan; Forrest, Stephanie; Herbert, Chelsea; Pritchard, Catrin

    2016-01-01

    The WNT signalling pathway controls many developmental processes and plays a key role in maintenance of intestine renewal and homeostasis. Glycogen Synthase Kinase 3 (GSK3) is an important component of the WNT pathway and is involved in regulating β-catenin stability and expression of WNT target genes. The mechanisms underpinning GSK3 regulation in this context are not completely understood, with some evidence suggesting this occurs through inhibitory N-terminal serine phosphorylation in a similar way to GSK3 inactivation in insulin signaling. To investigate this in a physiologically relevant context, we have analysed the intestinal phenotype of GSK3 knockin mice in which N-terminal serines 21/9 of GSK3α/β have been mutated to non-phosphorylatable alanine residues. We show that these knockin mutations have very little effect on overall intestinal integrity, cell lineage commitment, β-catenin localization or WNT target gene expression although a small increase in apoptosis at villi tips is observed. Our results provide in vivo evidence that GSK3 is regulated through mechanisms independent of N-terminal serine phosphorylation in order for β-catenin to be stabilised. PMID:27284979

  20. 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.

  1. Mutational Analysis of Glycogen Synthase Kinase 3β Protein Kinase Together with Kinome-Wide Binding and Stability Studies Suggests Context-Dependent Recognition of Kinases by the Chaperone Heat Shock Protein 90

    PubMed Central

    Pasculescu, Adrian; Dai, Anna Yue; Williton, Kelly; Taylor, Lorne; Savitski, Mikhail M.; Bantscheff, Marcus; Woodgett, James R.; Pawson, Tony; Colwill, Karen

    2016-01-01

    The heat shock protein 90 (HSP90) and cell division cycle 37 (CDC37) chaperones are key regulators of protein kinase folding and maturation. Recent evidence suggests that thermodynamic properties of kinases, rather than primary sequences, are recognized by the chaperones. In concordance, we observed a striking difference in HSP90 binding between wild-type (WT) and kinase-dead (KD) glycogen synthase kinase 3β (GSK3β) forms. Using model cell lines stably expressing these two GSK3β forms, we observed no interaction between WT GSK3β and HSP90, in stark contrast to KD GSK3β forming a stable complex with HSP90 at a 1:1 ratio. In a survey of 91 ectopically expressed kinases in DLD-1 cells, we compared two parameters to measure HSP90 dependency: static binding and kinase stability following HSP90 inhibition. We observed no correlation between HSP90 binding and reduced stability of a kinase after pharmacological inhibition of HSP90. We expanded our stability study to >50 endogenous kinases across four cell lines and demonstrated that HSP90 dependency is context dependent. These observations suggest that HSP90 binds to its kinase client in a particular conformation that we hypothesize to be associated with the nucleotide-processing cycle. Lastly, we performed proteomics profiling of kinases and phosphopeptides in DLD-1 cells to globally define the impact of HSP90 inhibition on the kinome. PMID:26755559

  2. 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.

  3. 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.

  4. 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

  5. A novel adamantyl benzylbenzamide derivative, AP736, inhibits melanogenesis in B16F10 mouse melanoma cells via glycogen synthase kinase 3β phosphorylation.

    PubMed

    Shin, Hong-Ju; Oh, Chang Taek; Kwon, Tae-Rin; Beak, Heung Soo; Joo, Yung Hyup; Kim, Jeong-Hwan; Lee, Chang Seok; Lee, John Hwan; Kim, Beom Joon; Shin, Song Seok; Park, Eun-Seok

    2015-11-01

    Recently, much effort has been made to develop effective dermatological depigmenting compounds. In this study, we investigated the novel candidate compound, AP736 (an adamantyl benzylbenzamide derivative), and its effects on melanogenesis in B16F10 melanoma cells, as well as the mechanisms involved. AP736 has been reported to exert anti-melanogenic effects in melanocytes in vitro and in artificial skin equivalents through the inhibition of key melanogenic enzymes and the suppression of the cAMP-protein kinase A (PKA)-cAMP response element‑binding protein (CREB) signaling pathway. Thus, we examined another pathway of melanogenesis involving the effects of AP736 on the glycogen synthesis kinase 3β (GSK3β) pathway. Melanin content and tyrosinase activity were measured using a spectrophotometer after the cells were treated with AP736. The AP736-induced activation of signaling pathways was examined by western blot analysis. We confirmed that AP736 decreased melanin production in a dose-dependent manner; however, it did not directly inhibit tyrosinase, the rate-limiting melanogenic enzyme. The expression of microphthalmia-associated transcription factor, tyrosinase, and related signal transduction pathways was also investigated. The Wnt signaling pathway is deeply involved in melanogenesis; therefore, phosphorylation by GSK3β was assessed following treatment with AP736. AP736 induced GSK3β phosphorylation (inactivation), but it did not alter the level of β-catenin. Furthermore, the expression of α-melanocyte-stimulating hormone-induced tyrosinase was downregulated by AP736. Our data suggest that AP736 exerts hypopigmentary effects through the downregulation of tyrosinase via GSK3β phosphorylation. PMID:26398893

  6. Candidate tumor suppressor B-cell translocation gene 3 impedes neoplastic progression by suppression of AKT

    PubMed Central

    Cheng, Y-C; Chen, P-H; Chiang, H-Y; Suen, C-S; Hwang, M-J; Lin, T-Y; Yang, H-C; Lin, W-C; Lai, P-L; Shieh, S-Y

    2015-01-01

    BTG3 (B-cell translocation gene 3) is a p53 target that also binds and inhibits E2F1. Although it connects two major growth-regulatory pathways functionally and is downregulated in human cancers, whether and how BTG3 acts as a tumor suppressor remain largely uncharacterized. Here we present evidence that BTG3 binds and suppresses AKT, a kinase frequently deregulated in cancers. BTG3 ablation results in increased AKT activity that phosphorylates and inhibits glycogen synthase kinase 3β. Consequently, we also observed elevated β-catenin/T-cell factor activity, upregulation of mesenchymal markers, and enhanced cell migration. Consistent with these findings, BTG3 overexpression suppressed tumor growth in mouse xenografts, and was associated with diminished AKT phosphorylation and reduced β-catenin in tissue specimens. Significantly, a short BTG3-derived peptide was identified, which recapitulates these effects in vitro and in cells. Thus, our study provides mechanistic insights into a previously unreported AKT inhibitory pathway downstream of p53. The identification of an AKT inhibitory peptide also unveils a new avenue for cancer therapeutics development. PMID:25569101

  7. Modulation of curcumin-induced Akt phosphorylation and apoptosis by PI3K inhibitor in MCF-7 cells

    SciTech Connect

    Kizhakkayil, Jaleel; Thayyullathil, Faisal; Chathoth, Shahanas; Hago, Abdulkader; Patel, Mahendra; Galadari, Sehamuddin

    2010-04-09

    Curcumin has been shown to induce apoptosis in various malignant cancer cell lines. One mechanism of curcumin-induced apoptosis is through the PI3K/Akt signaling pathway. Akt, also known as protein kinase B (PKB), is a member of the family of phosphatidylinositol 3-OH-kinase regulated Ser/Thr kinases. The active Akt regulates cell survival and proliferation; and inhibits apoptosis. In this study we found that curcumin induces apoptotic cell death in MCF-7 cells, as assessed by MTT assay, DNA ladder formation, PARP cleavage, p53 and Bax induction. At apoptotic inducing concentration, curcumin induces a dramatic Akt phosphorylation, accompanied by an increased phosphorylation of glycogen synthase kinase 3{beta} (GSK3{beta}), which has been considered to be a pro-growth signaling molecule. Combining curcumin with PI3K inhibitor, LY290042, synergizes the apoptotic effect of curcumin. The inhibitor LY290042 was capable of attenuating curcumin-induced Akt phosphorylation and activation of GSK3{beta}. All together, our data suggest that blocking the PI3K/Akt survival pathway sensitizes the curcumin-induced apoptosis in MCF-7 cells.

  8. Bufalin reverses intrinsic and acquired drug resistance to cisplatin through the AKT signaling pathway in gastric cancer cells.

    PubMed

    Zhao, Hongyan; Zhao, Dali; Jin, Huilin; Li, Hongwei; Yang, Xiaoying; Zhuang, Liwei; Liu, Tiefu

    2016-08-01

    Cisplatin is the most common chemotherapeutic agent for gastric cancer (GC), however it activates AKT, which contributes to intrinsic and acquired resistance. Bufalin, a traditional Chinese medicine, shows significant anticancer activity by inhibiting the AKT pathway. It was therefore hypothesized that bufalin could counteract cisplatin resistance in GC cells. SGC7901, MKN‑45 and BGC823 human GC cells were cultured under normoxic and hypoxic conditions. Effects of cisplatin and bufalin on GC cells were measured by a cell counting kit, apoptosis was analyzed by flow cytometry, and immunoblotting was used to detect proteins associated with the AKT signaling pathway. It was demonstrated that bufalin synergized with cisplatin to inhibit proliferation and promote apoptosis of GC cells by diminishing the activation of cisplatin-induced AKT under normoxic and hypoxic conditions. Bufalin also inhibits cisplatin-activated molecules downstream of AKT that affect proliferation and apoptosis, including glycogen synthase kinase, mammalian target of rapamycin, ribosomal protein S6 Kinase and eukaryotic translation initiation factor-4E-binding protein-1. To investigate acquired cisplatin resistance, a cisplatin‑resistant cell line SGC7901‑CR was used. It was demonstrated that bufalin reversed acquired cisplatin resistance and significantly induced apoptosis through the AKT pathway. These results imply that bufalin could extend the therapeutic effect of cisplatin on GC cells when administered in combination. PMID:27357249

  9. Lithium-induced activation of Akt and CaM kinase II contributes to its neuroprotective action in a rat microsphere embolism model.

    PubMed

    Sasaki, Takuya; Han, Feng; Shioda, Norifumi; Moriguchi, Shigeki; Kasahara, Jiro; Ishiguro, Koichi; Fukunaga, Kohji

    2006-09-01

    Lithium used in bipolar mood disorder therapy protects neurons from brain ischemic cell death. Here, we documented that lithium administration under microsphere-embolism (ME)-induced brain ischemia restored decreased protein kinase B (Akt) and Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) activities 24 h after ischemia in rat brain. Akt activation was associated with increased phosphorylation of its potential targets forkhead transcription factor (FKHR) and glycogen synthase kinase-3beta (GSK-3beta). In parallel with decreased CaMKII autophosphorylation, we also found marked dephosphorylation of tau proteins 24-72 h after ME. Increased protein phosphatase 2A (PP2A) activity was found 24 h after ME. Inhibition of increased PP2A activity by lithium treatment apparently mediated restored tau phosphorylation. Taken together, activation of Akt and CaMKII by lithium was associated with neuroprotective activity in ME-induced neuronal injury.

  10. 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.

  11. 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

  12. Fine-Tuning of the RIG-I-Like Receptor/Interferon Regulatory Factor 3-Dependent Antiviral Innate Immune Response by the Glycogen Synthase Kinase 3/β-Catenin Pathway

    PubMed Central

    Khan, Kashif Aziz; Dô, Florence; Marineau, Alexandre; Doyon, Priscilla; Clément, Jean-François; Woodgett, James R.; Doble, Bradley W.

    2015-01-01

    Induction of an antiviral innate immune response relies on pattern recognition receptors, including retinoic acid-inducible gene 1-like receptors (RLR), to detect invading pathogens, resulting in the activation of multiple latent transcription factors, including interferon regulatory factor 3 (IRF3). Upon sensing of viral RNA and DNA, IRF3 is phosphorylated and recruits coactivators to induce type I interferons (IFNs) and selected sets of IRF3-regulated IFN-stimulated genes (ISGs) such as those for ISG54 (Ifit2), ISG56 (Ifit1), and viperin (Rsad2). Here, we used wild-type, glycogen synthase kinase 3α knockout (GSK-3α−/−), GSK-3β−/−, and GSK-3α/β double-knockout (DKO) embryonic stem (ES) cells, as well as GSK-3β−/− mouse embryonic fibroblast cells in which GSK-3α was knocked down to demonstrate that both isoforms of GSK-3, GSK-3α and GSK-3β, are required for this antiviral immune response. Moreover, the use of two selective small-molecule GSK-3 inhibitors (CHIR99021 and BIO-acetoxime) or ES cells reconstituted with the catalytically inactive versions of GSK-3 isoforms showed that GSK-3 activity is required for optimal induction of antiviral innate immunity. Mechanistically, GSK-3 isoform activation following Sendai virus infection results in phosphorylation of β-catenin at S33/S37/T41, promoting IRF3 DNA binding and activation of IRF3-regulated ISGs. This study identifies the role of a GSK-3/β-catenin axis in antiviral innate immunity. PMID:26100021

  13. Inhibition of cyclin-dependent kinase 5 but not of glycogen synthase kinase 3-β prevents neurite retraction and tau hyperphosphorylation caused by secretable products of human T-cell leukemia virus type I-infected lymphocytes.

    PubMed

    Maldonado, Horacio; Ramírez, Eugenio; Utreras, Elias; Pando, María E; Kettlun, Ana M; Chiong, Mario; Kulkarni, Ashok B; Collados, Lucía; Puente, Javier; Cartier, Luis; Valenzuela, María A

    2011-09-01

    Human T-cell leukemia virus type I (HTLV-I)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a neurodegenerative disease characterized by selective loss of axons and myelin in the corticospinal tracts. This central axonopathy may originate from the impairment of anterograde axoplasmic transport. Previous work showed tau hyperphosphorylation at T(181) in cerebrospinal fluid of HAM/TSP patients. Similar hyperphosphorylation occurs in SH-SY5Y cells incubated with supernatant from MT-2 cells (HTLV-I-infected lymphocytes secreting viral proteins, including Tax) that produce neurite shortening. Tau phosphorylation at T(181) is attributable to glycogen synthase kinase 3-β (GSK3-β) and cyclin-dependent kinase 5 (CDK5) activation. Here we investigate whether neurite retraction in the SH-SY5Y model associates with concurrent changes in other tau hyperphosphorylable residues. Threonine 181 turned out to be the only tau hyperphosphorylated residue. We also evaluate the role of GSK3-β and CDK5 in this process by using specific kinase inhibitors (LiCl, TDZD-8, and roscovitine). Changes in both GSK3-β active and inactive forms were followed by measuring the regulatory phosphorylable sites (S(9) and Y(216) , inactivating and activating phosphorylation, respectively) together with changes in β-catenin protein levels. Our results showed that LiCl and TDZD-8 were unable to prevent MT-2 supernatant-mediated neurite retraction and also that neither Y(216) nor S(9) phosphorylations were changed in GSK3-β. Thus, GSK3-β seems not to play a role in T(181) hyperphosphorylation. On the other hand, the CDK5 involvement in tau phosphorylation was confirmed by both the increase in its enzymatic activity and the absence of MT-2 neurite retraction in the presence of roscovitine or CDK5 siRNA transfection. PMID:21671254

  14. Early glycogen synthase kinase-3β and protein phosphatase 2A independent tau dephosphorylation during global brain ischaemia and reperfusion following cardiac arrest and the role of the adenosine monophosphate kinase pathway.

    PubMed

    Majd, Shohreh; Power, John H T; Koblar, Simon A; Grantham, Hugh J M

    2016-08-01

    Abnormal tau phosphorylation (p-tau) has been shown after hypoxic damage to the brain associated with traumatic brain injury and stroke. As the level of p-tau is controlled by Glycogen Synthase Kinase (GSK)-3β, Protein Phosphatase 2A (PP2A) and Adenosine Monophosphate Kinase (AMPK), different activity levels of these enzymes could be involved in tau phosphorylation following ischaemia. This study assessed the effects of global brain ischaemia/reperfusion on the immediate status of p-tau in a rat model of cardiac arrest (CA) followed by cardiopulmonary resuscitation (CPR). We reported an early dephosphorylation of tau at its AMPK sensitive residues, Ser(396) and Ser(262) after 2 min of ischaemia, which did not recover during the first two hours of reperfusion, while the tau phosphorylation at GSK-3β sensitive but AMPK insensitive residues, Ser(202) /Thr(205) (AT8), as well as the total amount of tau remained unchanged. Our data showed no alteration in the activities of GSK-3β and PP2A during similar episodes of ischaemia of up to 8 min and reperfusion of up to 2 h, and 4 weeks recovery. Dephosphorylation of AMPK followed the same pattern as tau dephosphorylation during ischaemia/reperfusion. Catalase, another AMPK downstream substrate also showed a similar pattern of decline to p-AMPK, in ischaemic/reperfusion groups. This suggests the involvement of AMPK in changing the p-tau levels, indicating that tau dephosphorylation following ischaemia is not dependent on GSK-3β or PP2A activity, but is associated with AMPK dephosphorylation. We propose that a reduction in AMPK activity is a possible early mechanism responsible for tau dephosphorylation.

  15. 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.

  16. Dopamine D2 receptor-mediated Akt/PKB signalling: initiation by the D2S receptor and role in quinpirole-induced behavioural activation.

    PubMed

    Chen, Han-Ting; Ruan, Nan-Yu; Chen, Jin-Chung; Lin, Tzu-Yung

    2012-09-24

    The short and long isoforms of the dopamine D2 receptor (D2S and D2L respectively) are highly expressed in the striatum. Functional D2 receptors activate an intracellular signalling pathway that includes a cAMP-independent route involving Akt/GSK3 (glycogen synthase kinase 3). To investigate the Akt/GSK3 response to the seldom-studied D2S receptor, we established a rat D2S receptor-expressing cell line [HEK (human embryonic kidney)-293/rD2S]. We found that in HEK-293/rD2S cells, the D2/D3 agonists bromocriptine and quinpirole significantly induced Akt and GSK3 phosphorylation, as well as ERK1/2 (extracellular-signal-regulated kinase 1/2) activation. The D2S receptor-induced Akt signals were profoundly inhibited by the internalization blockers monodansyl cadaverine and concanavalin A. Activation of the D2S receptor in HEK-293/rD2S cells appeared to trigger Akt/phospho-Akt translocation to the cell membrane. In addition to our cell culture experiments, we studied D2 receptor-dependent Akt in vivo by systemic administration of the D2/D3 agonist quinpirole. The results show that quinpirole evoked Akt-Ser473 phosphorylation in the ventral striatum. Furthermore, intra-accumbens administration of wortmannin, a PI3K (phosphoinositide 3-kinase) inhibitor, significantly suppressed the quinpirole-evoked behavioural activation. Overall, we demonstrate that activation of the dopamine D2S receptor stimulates Akt/GSK3 signalling. In addition, in vivo Akt activity in the ventral striatum appears to play an important role in systemic D2/D3 agonist-induced behavioural activation.

  17. Dopamine D2 receptor-mediated Akt/PKB signalling: initiation by the D2S receptor and role in quinpirole-induced behavioural activation

    PubMed Central

    Chen, Han-Ting; Ruan, Nan-Yu; Chen, Jin-Chung; Lin, Tzu-Yung

    2012-01-01

    The short and long isoforms of the dopamine D2 receptor (D2S and D2L respectively) are highly expressed in the striatum. Functional D2 receptors activate an intracellular signalling pathway that includes a cAMP-independent route involving Akt/GSK3 (glycogen synthase kinase 3). To investigate the Akt/GSK3 response to the seldom-studied D2S receptor, we established a rat D2S receptor-expressing cell line [HEK (human embryonic kidney)-293/rD2S]. We found that in HEK-293/rD2S cells, the D2/D3 agonists bromocriptine and quinpirole significantly induced Akt and GSK3 phosphorylation, as well as ERK1/2 (extracellular-signal-regulated kinase 1/2) activation. The D2S receptor-induced Akt signals were profoundly inhibited by the internalization blockers monodansyl cadaverine and concanavalin A. Activation of the D2S receptor in HEK-293/rD2S cells appeared to trigger Akt/phospho-Akt translocation to the cell membrane. In addition to our cell culture experiments, we studied D2 receptor-dependent Akt in vivo by systemic administration of the D2/D3 agonist quinpirole. The results show that quinpirole evoked Akt-Ser473 phosphorylation in the ventral striatum. Furthermore, intra-accumbens administration of wortmannin, a PI3K (phosphoinositide 3-kinase) inhibitor, significantly suppressed the quinpirole-evoked behavioural activation. Overall, we demonstrate that activation of the dopamine D2S receptor stimulates Akt/GSK3 signalling. In addition, in vivo Akt activity in the ventral striatum appears to play an important role in systemic D2/D3 agonist-induced behavioural activation. PMID:22909302

  18. Decreased phospho-Akt signaling in a mouse model of total parenteral nutrition: a potential mechanism for the development of intestinal mucosal atrophy

    PubMed Central

    Feng, Yongjia; McDunn, Jonathan E.

    2010-01-01

    Total parenteral nutrition (TPN) leads to a decline in phosphatidylinositol 3-kinase (PI3K)/phospho-Akt (p-Akt) activity, affecting downstream signaling, reducing epithelial cell (EC) proliferation, and contributing to intestinal mucosal atrophy. We hypothesized that promoting Akt activity would prevent these changes. We used a novel Akt-activating peptide, TCL1 (a head-to-tail dimer of the Akt-binding domain of T-cell lymphoma-1), or an inactive mutant sequence TCL1G conjugated to a transactivator of transcription peptide sequence to promote intracellular uptake. Four groups of mice were studied, enteral nutrition group (control), control mice given a functioning TCL1 (control + TCL1), TPN mice given TCL1G (control peptide, TPN + TCL1G); and TPN mice given TCL1. TPN mice given TCL1G showed a significant decrease in jejunal EC p-Akt (Ser473 and Thr308) abundance, whereas TPN + TCL1 mice showed increased p-Akt (Ser473) abundance. Phosphorylation of β-catenin and glycogen synthase kinase-3β (downstream targets of Akt signaling) were also decreased in the TPN + TCL1G group and completely prevented in the TPN + TCL1 group. Use of TCL1 nearly completely prevented the decline in EC proliferation seen in the TPN + TCL1G group, as well as partly returned EC apoptosis levels close to controls. The mammalian target of rapamycin pathway demonstrated a similar reduction in activity in the TPN + TCL1G group that was significantly prevented in the TPN + TCL1 group. These results support a significant loss of PI3K/p-Akt signaling upon replacing enteral nutrition with TPN, and prevention of this loss demonstrates the key importance of PI3K/p-Akt signaling in maintaining gut integrity including EC proliferation and reduction in apoptosis. PMID:20299605

  19. 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

  20. 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.

  1. The Phosphoinositide-3-Kinase–Akt Signaling Pathway Is Important for Staphylococcus aureus Internalization by Endothelial Cells ▿

    PubMed Central

    Oviedo-Boyso, Javier; Cortés-Vieyra, Ricarda; Huante-Mendoza, Alejandro; Yu, Hong B.; Valdez-Alarcón, Juan J.; Bravo-Patiño, Alejandro; Cajero-Juárez, Marcos; Finlay, B. Brett; Baizabal-Aguirre, Víctor M.

    2011-01-01

    Internalization of Staphylococcus aureus in bovine endothelial cells (BEC) is increased by tumor necrosis factor alpha stimulation and NF-κB activation. Because the phosphoinositide-3-kinase (PI3K)–Akt signaling pathway also modulates NF-κB activity, we considered whether the internalization of S. aureus by BEC is associated with the activity of PI3K and Akt. We found a time- and multiplicity of infection-dependent phosphorylation of Akt on Ser473 in BEC infected with S. aureus. This phosphorylation was inhibited by LY294002 (LY), indicating the participation of PI3K. Inhibition of either PI3K with LY or wortmannin, or Akt with SH-5, strongly reduced the internalization of S. aureus. Transfection of BEC with a dominant-negative form of the Akt gene significantly decreased S. aureus internalization, whereas transfection with the constitutively active mutant increased the number of internalized bacterium. Inhibition of PDK1 activity with OSU-03012 did not affect the level of S. aureus internalization, demonstrating that phosphorylation of Akt on Thr308 is not important for this process. Compared to the untreated control, the adherence of S. aureus to the surface of BEC was unaltered when cells were transfected or incubated with the pharmacological inhibitors. Furthermore, Akt activation by internalized S. aureus triggered a time-dependent phosphorylation of glycogen synthase kinase-3α (GSK-3α) on Ser21 and GSK-3β on Ser9 that was partially inhibited with SH-5. Finally, treatment of BEC with LY prior to S. aureus infection inhibited the NF-κB p65 subunit phosphorylation on Ser536, indicating the involvement of PI3K. These results suggest that PI3K-Akt activity is important for the internalization of S. aureus and phosphorylation of GSK-3α, GSK-3β, and NF-κB. PMID:21844240

  2. Resistin disrupts glycogen synthesis under high insulin and high glucose levels by down-regulating the hepatic levels of GSK3β.

    PubMed

    Song, Rongjing; Wang, Xi; Mao, Yiqing; Li, Hui; Li, Zhixin; Xu, Wei; Wang, Rong; Guo, Tingting; Jin, Ling; Zhang, Xiaojing; Zhang, Yizhuang; Zhou, Na; Hu, Ruobi; Jia, Jianwei; Lei, Zhen; Irwin, David M; Niu, Gang; Tan, Huanran

    2013-10-15

    The effect of mouse resistin on hepatic insulin resistance in vivo and in vitro, and its possible molecular mechanism were examined. Focusing on liver glycogen metabolism and gluconeogenesis, which are important parts of glucose metabolism, in primary cultures of rat hepatocytes we found that glycogen content was significantly lower (P<0.05) after treatment with recombinant murine resistin only in the presence of insulin plus glucose stimulation. Protein levels of factors in the insulin signaling pathway involved in glycogen synthesis were examined by Western blot analysis, with the only significant change observed being the level of phosphorylated (at Ser 9) glycogen synthase kinase-3β (GSK-3β) (P<0.001). No differences in the protein levels for the insulin receptor β (IRβ), insulin receptor substrates (IRS1 and IRS2), phosphatidylinositol 3-kinase (PI3K), protein kinase B (Akt) or their phosphorylated forms were observed between control and resistin treated primary rat hepatocytes. In a mouse model with high liver-specific expression of resistin, fasting blood glucose levels and liver glycogen content changed. Fasting blood glucose levels were significantly higher (P<0.001) in the model mice, compared to the control mice, while the glycogen content of the liver tissue was about 60% of that of the control mice (P<0.05). The gluconeogenic response was not altered between the experimental and control mice. The level of phosphorylated GSK-3β in the liver tissue was also decreased (P<0.05) in the model mice, consistent with the results from the primary rat hepatocytes. Our results suggest that resistin reduces the levels of GSK-3β phosphorylated at Ser 9 leading to impaired hepatic insulin action in primary rat hepatocytes and in a mouse model with high liver-specific expression of resistin. PMID:23860320

  3. Copper ions strongly activate the phosphoinositide-3-kinase/Akt pathway independent of the generation of reactive oxygen species.

    PubMed

    Ostrakhovitch, Elena A; Lordnejad, Mohammad Reza; Schliess, Freimut; Sies, Helmut; Klotz, Lars-Oliver

    2002-01-15

    Copper is implicated in metabolic disorders, such as Wilson's disease or Alzheimer's disease. Analysis of signaling pathways regulating cellular survival and function in response to a copper stress is crucial for understanding the pathogenesis of such diseases. Exposure of human skin fibroblasts or HeLa cells to Cu(2+) resulted in a dose- and time-dependent activation of the antiapoptotic kinase Akt/protein kinase B, starting at concentrations as low as 3 microM. Only Cu(II), but not Cu(I), had this effect. Activation of Akt was accompanied by phosphorylation of a downstream target of Akt, glycogen synthase kinase-3. Inhibitors of phosphoinositide-3-kinase (PI3K) completely blocked activation of Akt by Cu(2+), indicating a requirement of PI3K for Cu(2+)-induced activation of Akt. Indeed, cellular PI3K activity was strongly enhanced after exposure to Cu(2+). Copper ions may lead to the formation of reactive oxygen species, such as hydrogen peroxide. Activation of Akt by hydrogen peroxide or growth factors is known to proceed via the activation growth factor receptors. In line with this, pretreatment with inhibitors of growth factor receptor tyrosine kinases blocked activation of Akt by hydrogen peroxide and growth factors, as did a src-family tyrosine kinase inhibitor or the broad-spectrum tyrosine kinase inhibitor genistein. Activation of Akt by Cu(2+), however, remained unimpaired, implying (i) that tyrosine kinase activation is not involved in Cu(2+) activation of Akt and (ii) that activation of the PI3K/Akt pathway by Cu(2+) is initiated independently of that induced by reactive oxygen species. Comparison of the time course of the oxidation of 2',7'-dichlorodihydrofluorescein in copper-treated cells with that of Akt activation led to the conclusion that production of hydroperoxides cannot be an upstream event in copper-induced Akt activation. Rather, both activation of Akt and generation of ROS are proposed to occur in parallel, regulating cell survival after a

  4. 6-Br-5methylindirubin-3'oxime (5-Me-6-BIO) targeting the leishmanial glycogen synthase kinase-3 (GSK-3) short form affects cell-cycle progression and induces apoptosis-like death: exploitation of GSK-3 for treating leishmaniasis.

    PubMed

    Xingi, Evangelia; Smirlis, Despina; Myrianthopoulos, Vassilios; Magiatis, Prokopios; Grant, Karen M; Meijer, Laurent; Mikros, Emmanuel; Skaltsounis, Alexios-Leandros; Soteriadou, Ketty

    2009-10-01

    Indirubins known to target mammalian cyclin-dependent kinases (CDKs) and glycogen synthase kinase (GSK-3) were tested for their antileishmanial activity. 6-Br-indirubin-3'-oxime (6-BIO), 6-Br-indirubin-3'acetoxime and 6-Br-5methylindirubin-3'oxime (5-Me-6-BIO) were the most potent inhibitors of Leishmania donovani promastigote and amastigote growth (half maximal inhibitory concentration (IC(50)) values < or =1.2 microM). Since the 6-Br substitution on the indirubin backbone greatly enhances the selectivity for mammalian GSK-3 over CDKs, we identified the leishmanial GSK-3 homologues, a short (LdGSK-3s) and a long one, focusing on LdGSK-3s which is closer to human GSK-3beta, for further studies. Kinase assays showed that 5-Me-6-BIO inhibited LdGSK-3s more potently than CRK3 (the CDK1 homologue in Leishmania), whilst 6-BIO was more selective for CRK3. Promastigotes treated with 5-Me-6-BIO accumulated in the S and G2/M cell-cycle phases and underwent apoptosis-like death. Interestingly, these phenotypes were completely reversed in parasites over-expressing LdGSK-3s. This finding strongly supports that LdGSK-3s is: (i) the intracellular target of 5-Me-6-BIO, and (ii) involved in cell-cycle control and in pathways leading to apoptosis-like death. 6-BIO treatment induced a G2/M arrest, consistent with inhibition of CRK3 and apoptosis-like death. These effects were partially reversed in parasites over-expressing LdGSK-3s suggesting that in vivo 6-BIO may also target LdGSK-3s. Molecular docking of 5-Me-6-BIO in CRK3 and 6-BIO in human GSK-3beta and LdGSK-3s active sites predict the existence of functional/structural differences that are sufficient to explain the observed difference in their affinity. In conclusion, LdGSK-3s is validated as a potential drug target in Leishmania and could be exploited for the development of selective indirubin-based leishmanicidals. PMID:19445946

  5. 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.

  6. 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

  7. Glycogen Synthase Kinase-3β Plays a Pro-Apoptotic Role in β-Adrenergic Receptor-Stimulated Apoptosis in Adult Rat Ventricular Myocytes: Role of β1 Integrins

    PubMed Central

    Menon, Bindu; Johnson, Jennifer N.; Ross, Robert S.; Singh, Mahipal; Singh, Krishna

    2007-01-01

    β-adrenergic receptor (β-AR) stimulation induces apoptosis in adult rat ventricular myocytes (ARVM). β1 integrin signaling plays a protective role in β-AR-stimulated apoptosis. Glycogen synthase kinase-3β (GSK-3β), a multifunctional serine/threonine kinase, negatively regulates cardiac hypertrophy. Here we show that β-AR stimulation (isoproterenol; 15 min) increases tyr216 phosphorylation and GSK-3β activity. Inclusion of LiCl, inhibitor of GSK-3β, in the reaction mix or expression of catalytically inactive GSK-3β (KM-GSK) inhibited β-AR-stimulated GSK-3β activity. Inhibition of tyrosine kinase using genistein or chelation of intracellular Ca2+ using BAPTA-AM inhibited β-AR-stimulated increases in tyr216 phosphorylation and GSK-3β activity. Inhibition of GSK-3β using pharmacological inhibitors or infection with KM-GSK decreased β-AR-stimulated cytosolic cytochrome C release and apoptosis. Expression of β1 integrins increased ser9 phosphorylation and inhibited β-AR-stimulated increase in GSK-3β activity. Wortmannin, inhibitor of PI3-kinase, reversed the effects of β1 integrins on GSK-3β activity and apoptosis. Purified active matrix metalloproteinase-2 (MMP-2), shown to interfere with β1 integrin signaling, increased GSK-3β activity, while inhibition of MMP-2 inhibited β-AR-stimulated increases in GSK-3β activity. β-AR stimulation induced nuclear accumulation of GSK-3β. β-AR stimulation (3 h) increased the expression of transcription factor Gadd153 (growth arrest- and DNA damage-inducible gene 153). These data suggest that β-AR stimulation increases GSK-3β activity. Activation of GSK-3β plays a pro-apoptotic role in β-AR stimulated apoptosis via the involvement of mitochondrial death pathway. β1 integrins inactivate GSK-3β and play an anti-apoptotic role via the involvement of PI3-kinase pathway. The apoptotic effects of GSK-3β may be mediated, at least in part, via its nuclear localization and induction of pro-apoptotic genes

  8. [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

  9. 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

  10. 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.

  11. Inhibitors of glycogen synthase 3 kinase

    DOEpatents

    Schultz, Peter; Ring, David B.; Harrison, Stephen D.; Bray, Andrew M.

    2006-05-30

    Compounds of formula 1: ##STR00001## wherein R.sub.1 is alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or heteroaralkyl, substituted with 0 3 substituents selected from lower alkyl, halo, hydroxy, lower alkoxy, amino, lower alkyl-amino, and nitro; R.sub.2 is hydroxy, amino, or lower alkoxy; R.sub.3 is H, lower alkyl, lower acyl, lower alkoxy-acyl, or amino-acyl; R.sub.4 is H or lower alkyl; and pharmaceutically acceptable salts and esters thereof; are effective inhibitors of GSK3.

  12. Inhibitors of glycogen synthase 3 kinase

    DOEpatents

    Schultz, Peter; Ring, David B.; Harrison, Stephen D.; Bray, Andrew M.

    2000-01-01

    Compounds of formula 1: ##STR1## wherein R.sub.1 is alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or heteroaralkyl, substituted with 0-3 substituents selected from lower alkyl, halo, hydroxy, lower alkoxy, amino, lower alkyl-amino, and nitro; R.sub.2 is hydroxy, amino, or lower alkoxy; R.sub.3 is H, lower alkyl, lower acyl, lower alkoxy-acyl, or amnino-acyl; R.sub.4 is H or lower alkyl; and pharmaceutically acceptable salts and esters thereof; are effective inhibitors of GSK3.

  13. Phosphatidylinositol 3-Kinase Couples Localised Calcium Influx to Activation of Akt in Central Nerve Terminals.

    PubMed

    Nicholson-Fish, Jessica C; Cousin, Michael A; Smillie, Karen J

    2016-03-01

    The efficient retrieval of synaptic vesicle membrane and cargo in central nerve terminals is dependent on the efficient recruitment of a series of endocytosis modes by different patterns of neuronal activity. During intense neuronal activity the dominant endocytosis mode is activity-dependent endocytosis (ADBE). Triggering of ADBE is linked to calcineurin-mediated dynamin I dephosphorylation since the same stimulation intensities trigger both. Dynamin I dephosphorylation is maximised by a simultaneous inhibition of its kinase glycogen synthase kinase 3 (GSK3) by the protein kinase Akt, however it is unknown how increased neuronal activity is transduced into Akt activation. To address this question we determined how the activity-dependent increases in intracellular free calcium ([Ca(2+)]i) control activation of Akt. This was achieved using either trains of high frequency action potentials to evoke localised [Ca(2+)]i increases at active zones, or a calcium ionophore to raise [Ca(2+)]i uniformly across the nerve terminal. Through the use of either non-specific calcium channel antagonists or intracellular calcium chelators we found that Akt phosphorylation (and subsequent GSK3 phosphorylation) was dependent on localised [Ca(2+)]i increases at the active zone. In an attempt to determine mechanism, we antagonised either phosphatidylinositol 3-kinase (PI3K) or calmodulin. Activity-dependent phosphorylation of both Akt and GSK3 was arrested on inhibition of PI3K, but not calmodulin. Thus localised calcium influx in central nerve terminals activates PI3K via an unknown calcium sensor to trigger the activity-dependent phosphorylation of Akt and GSK3.

  14. 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

  15. 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

  16. 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.

  17. 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.

  18. PTEN Contributes to Profound PI3K/Akt Signaling Pathway Deregulation in Dystrophin-Deficient Dog Muscle

    PubMed Central

    Feron, Marie; Guevel, Laetitia; Rouger, Karl; Dubreil, Laurence; Arnaud, Marie-Claire; Ledevin, Mireille; Megeney, Lynn A.; Cherel, Yan; Sakanyan, Vehary

    2009-01-01

    Duchenne muscular dystrophy is the most common and severe form of muscular dystrophy, and although the genetic basis of this disease is well defined, the overall mechanisms that define its pathogenesis remain obscure. Alterations in individual signaling pathways have been described, but little information is available regarding their putative implications in Duchenne muscular dystrophy pathogenesis. Here, we studied the status of various major signaling pathways in the Golden Retriever muscular dystrophy dog that specifically reproduces the full spectrum of human pathology. Using antibody arrays, we found that Akt1, glycogen synthase kinase-3β (GSK3β), 70-kDa ribosomal protein S6 kinase (p70S6K), extracellular signal-regulated kinases 1/2, and p38δ and p38γ kinases all exhibited decreased phosphorylation in muscle from a 4-month-old animal with Golden Retriever muscular dystrophy, revealing a deep alteration of the phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen-activated protein kinase pathways. Immunohistochemistry analysis revealed the presence of muscle fibers exhibiting a cytosolic accumulation of Akt1, GSK3β, and phosphatidylinositol-3,4,5-trisphosphate 3-phosphatase (PTEN), an enzyme counteracting PI3K-mediated Akt activation. Enzymatic assays established that these alterations in phosphorylation and expression levels were associated with decreased Akt and increased GSK3β and PTEN activities. PTEN/GSK3β-positive fibers were also observed in muscle sections from 3- and 36-month-old animals, indicating long-term PI3K/Akt pathway alteration. Collectively, our data suggest that increased PTEN expression and activity play a central role in PI3K/Akt/GSK3β and p70S6K pathway modulation, which could exacerbate the consequences of dystrophin deficiency. PMID:19264909

  19. Lycium barbarum Polysaccharides Protect against Trimethyltin Chloride-Induced Apoptosis via Sonic Hedgehog and PI3K/Akt Signaling Pathways in Mouse Neuro-2a Cells

    PubMed Central

    Zhao, Wanyun; Pan, Xiaoqi; Li, Tao; Zhang, Changchun; Shi, Nian

    2016-01-01

    Trimethyltin chloride (TMT) is a classic neurotoxicant that can cause severe neurodegenerative diseases. Some signaling pathways involving cell death play pivotal roles in the central nervous system. In this study, the role of Sonic Hedgehog (Shh) and PI3K/Akt pathways in TMT-induced apoptosis and protective effect of Lycium barbarum polysaccharides (LBP) on mouse neuro-2a (N2a) cells were investigated. Results showed that TMT treatment significantly enhanced apoptosis, upregulated proapoptotic Bax, downregulated antiapoptotic Bcl-2 expression, and increased caspase-3 activity in a dose-dependent manner in N2a cells. TMT induced oxidative stress in cells, performing reactive oxygen species (ROS) and malondialdehyde (MDA) excessive generation, and superoxide dismutase (SOD) activity reduction. TMT significantly decreased phosphorylated glycogen synthase kinase-3β (GSK-3β) and inhibited Shh and PI3K/Akt pathways. However, the addition of LBP upregulated GSK-3β phosphorylation, activated Shh and PI3K/Akt pathways, and eventually reduced apoptosis and oxidative stress caused by TMT. The interaction between Shh and PI3K/Akt pathways was clarified by specific PI3K inhibitor LY294002 or Shh inhibitor GDC-0449. Moreover, LY294002 and GDC-0449 pretreatment both induced phosphorylated GSK-3β downregulation and significantly promoted apoptosis induced by TMT. These results suggest that LBP could reduce TMT-induced N2a cells apoptosis by regulating GSK-3β phosphorylation, Shh, and PI3K/Akt signaling pathways. PMID:27143997

  20. Differential regulation of AKT, MAPK and GSK3β during C2-ceramide-induced neuronal death.

    PubMed

    Arboleda, Gonzalo; Cárdenas, Yolanda; Rodríguez, Yeldy; Morales, Luis Carlos; Matheus, Luisa; Arboleda, Humberto

    2010-12-01

    Evidence has implicated apoptosis as a mechanism underlying cell demise in diverse neurodegenerative diseases including Parkinson's disease (PD). Endogenous toxins and other stress signals activate the sphingomyelin pathway increasing the levels of ceramide, an important regulator of cell death. In the present paper we have analysed the contribution of PI3K/AKT-GSK3β and MAPK (ERK and JNK) pathways to cell death in a catecholaminergic cell line following exposure to C(2)-ceramide. We also explored the potential neuroprotective action of insulin-like growth factor-1 (IGF-1) and neurotrophin-3 (NT3). We demonstrated that C(2)-ceramide-induced cell death is associated to an early decrease in phosphorylation (inhibition) of PI3K/AKT and ERK, followed by phosphorylation (activation) of JNK and de-phosphorylation (activation) of glycogen synthase kinase-3 beta (GSK3β). NT3 and IGF-1 increased survival at early time points, but only IGF-1 is capable to attenuate C(2)-ceramide-mediated neuronal death, and this neuroprotection is associated to strong and permanent activation of AKT and inhibition of GSK3β. In conclusion, C(2)-ceramide initiates a series of events including an early inactivation of PI3K/AKT and ERK pathways followed by activation of JNK and activation of GSK3β and neuronal death, changes that are counteracted by IGF-1.

  1. Dichloroacetate Stimulates Glycogen Accumulation in Primary Hepatocytes through an Insulin-Independent Mechanism

    SciTech Connect

    Lingohr, Melissa K.; Bull, Richard J.; Kato-Weinstein, Junko; Thrall, Brian D. )

    2002-01-01

    Dichloroacetate (DCA), a by-product of water chlorination, causes liver cancer in B6C3F1 mice. A hallmark response observed in mice exposed to carcinogenic doses of DCA is an accumulation of hepatic glycogen content. To distinguish whether the in vivo glycogenic effect of DCA was dependent on insulin and insulin signaling proteins, experiments were conducted in isolated hepatocytes where insulin concentrations could be controlled. In hepatocytes isolated from male B6C3F1 mice, DCA increased glycogen levels in a dose-related manner, independently of insulin. The accumulation of hepatocellular glycogen induced by DCA was not the result of decreased glycogenolysis, since DCA had no effect on the rate of glucagon-stimulated glycogen breakdown. Glycogen accumulation caused by DCA treatment was not hindered by inhibitors of extracellular-regulated protein kinase kinase (Erk1/2 kinase or MEK) or p70 kDa S6 protein kinase (p70(S6K)), but was completely blocked by the phosphatidylinositol 3-kinase (PI3K) inhibitors, LY294002 and wortmannin. Similarly, insulin-stimulated glycogen deposition was not influenced by the Erk1/2 kinase inhibitor, PD098509, or the p70(S6K) inhibitor, rapamycin. Unlike DCA-stimulated glycogen deposition, PI3K-inhibition only partially blocked the glycogenic effect of insulin. DCA did not cause phosphorylation of the downstream PI3K target protein, protein kinase B (PKB/Akt). The phosphorylation of PKB/Akt did not correlate to insulin-stimulated glycogenesis either. Similar to insulin, DCA in the medium decreased IR expression in isolated hepatocytes. The results indicate DCA increases hepatocellular glycogen accumulation through a PI3K-dependent mechanism that does not involve PKB/Akt and is, at least in part, different from the classical insulin-stimulated glycogenesis pathway. Somewhat surprisingly, insulin-stimulated glycogenesis also appears not to involve PKB/Akt in isolated murine hepatocytes.

  2. 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.

  3. 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

  4. Adipose tissue glycogen accumulation is associated with obesity-linked inflammation in humans

    PubMed Central

    Ceperuelo-Mallafré, Victòria; Ejarque, Miriam; Serena, Carolina; Duran, Xavier; Montori-Grau, Marta; Rodríguez, Miguel Angel; Yanes, Oscar; Núñez-Roa, Catalina; Roche, Kelly; Puthanveetil, Prasanth; Garrido-Sánchez, Lourdes; Saez, Enrique; Tinahones, Francisco J.; Garcia-Roves, Pablo M.; Gómez-Foix, Anna Ma; Saltiel, Alan R.; Vendrell, Joan; Fernández-Veledo, Sonia

    2015-01-01

    Objective Glycogen metabolism has emerged as a mediator in the control of energy homeostasis and studies in murine models reveal that adipose tissue might contain glycogen stores. Here we investigated the physio(patho)logical role of glycogen in human adipose tissue in the context of obesity and insulin resistance. Methods We studied glucose metabolic flux of hypoxic human adipoctyes by nuclear magnetic resonance and mass spectrometry-based metabolic approaches. Glycogen synthesis and glycogen content in response to hypoxia was analyzed in human adipocytes and macrophages. To explore the metabolic effects of enforced glycogen deposition in adipocytes and macrophages, we overexpressed PTG, the only glycogen-associated regulatory subunit (PP1-GTS) reported in murine adipocytes. Adipose tissue gene expression analysis was performed on wild type and homozygous PTG KO male mice. Finally, glycogen metabolism gene expression and glycogen accumulation was analyzed in adipose tissue, mature adipocytes and resident macrophages from lean and obese subjects with different degrees of insulin resistance in 2 independent cohorts. Results We show that hypoxia modulates glucose metabolic flux in human adipocytes and macrophages and promotes glycogenesis. Enforced glycogen deposition by overexpression of PTG re-orients adipocyte secretion to a pro-inflammatory response linked to insulin resistance and monocyte/lymphocyte migration. Furthermore, glycogen accumulation is associated with inhibition of mTORC1 signaling and increased basal autophagy flux, correlating with greater leptin release in glycogen-loaded adipocytes. PTG-KO mice have reduced expression of key inflammatory genes in adipose tissue and PTG overexpression in M0 macrophages induces a pro-inflammatory and glycolytic M1 phenotype. Increased glycogen synthase expression correlates with glycogen deposition in subcutaneous adipose tissue of obese patients. Glycogen content in subcutaneous mature adipocytes is associated

  5. Variations in Glycogen Synthesis in Human Pluripotent Stem Cells with Altered Pluripotent States.

    PubMed

    Chen, Richard J; Zhang, Guofeng; Garfield, Susan H; Shi, Yi-Jun; Chen, Kevin G; Robey, Pamela G; Leapman, Richard D

    2015-01-01

    Human pluripotent stem cells (hPSCs) represent very promising resources for cell-based regenerative medicine. It is essential to determine the biological implications of some fundamental physiological processes (such as glycogen metabolism) in these stem cells. In this report, we employ electron, immunofluorescence microscopy, and biochemical methods to study glycogen synthesis in hPSCs. Our results indicate that there is a high level of glycogen synthesis (0.28 to 0.62 μg/μg proteins) in undifferentiated human embryonic stem cells (hESCs) compared with the glycogen levels (0 to 0.25 μg/μg proteins) reported in human cancer cell lines. Moreover, we found that glycogen synthesis was regulated by bone morphogenetic protein 4 (BMP-4) and the glycogen synthase kinase 3 (GSK-3) pathway. Our observation of glycogen bodies and sustained expression of the pluripotent factor Oct-4 mediated by the potent GSK-3 inhibitor CHIR-99021 reveals an altered pluripotent state in hPSC culture. We further confirmed glycogen variations under different naïve pluripotent cell growth conditions based on the addition of the GSK-3 inhibitor BIO. Our data suggest that primed hPSCs treated with naïve growth conditions acquire altered pluripotent states, similar to those naïve-like hPSCs, with increased glycogen synthesis. Furthermore, we found that suppression of phosphorylated glycogen synthase was an underlying mechanism responsible for altered glycogen synthesis. Thus, our novel findings regarding the dynamic changes in glycogen metabolism provide new markers to assess the energetic and various pluripotent states in hPSCs. The components of glycogen metabolic pathways offer new assays to delineate previously unrecognized properties of hPSCs under different growth conditions.

  6. 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.

  7. 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

  8. Ferulic acid regulates the AKT/GSK-3β/CRMP-2 signaling pathway in a middle cerebral artery occlusion animal model.

    PubMed

    Gim, Sang-A; Sung, Jin-Hee; Shah, Fawad-Ali; Kim, Myeong-Ok; Koh, Phil-Ok

    2013-06-01

    Ferulic acid, a component of the plants Angelica sinensis (Oliv.) Diels and Ligusticum chuanxiong Hort, exerts a neuroprotective effect by regulating various signaling pathways. This study showed that ferulic acid treatment prevents the injury-induced increase of collapsin response mediator protein 2 (CRMP-2) in focal cerebral ischemia. Glycogen synthase kinase-3β (GSK-3β) regulates CRMP-2 function through phosphorylation of CRMP-2. Moreover, the pro-apoptotic activity of GSK-3β is inactivated by phosphorylation by Akt. This study investigated whether ferulic acid modulates the expression of CRMP-2 and its upstream targets, Akt and GSK-3β, in focal cerebral ischemia. Male rats were treated immediately with ferulic acid (100 mg/kg, i.v.) or vehicle after middle cerebral artery occlusion (MCAO), and then cerebral cortices were collected 24 hr after MCAO. MCAO resulted in decreased levels of phospho-Akt and phospho-GSK-3β, while ferulic acid treatment prevented the decrease in the levels of these proteins. Moreover, phospho-CRMP-2 and CRMP-2 levels increased during MCAO, whereas ferulic acid attenuated these injury-induced increases. These results demonstrate that ferulic acid regulates the Akt/GSK-3β/CRMP-2 signaling pathway in focal cerebral ischemic injury, thereby protecting against brain injury. PMID:23825478

  9. Resveratrol induces cell cycle arrest in human gastric cancer MGC803 cells via the PTEN-regulated PI3K/Akt signaling pathway.

    PubMed

    Jing, Xiaoping; Cheng, Weiwei; Wang, Shiying; Li, Pin; He, Li

    2016-01-01

    Resveratrol is a polyphenolic compound that is extracted from Polygonum cuspidatum and is used in traditional Chinese medicine. Previous data have shown that resveratrol inhibits the growth of human gastric cancer. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] and trypan blue assays showed that resveratrol significantly decreased the survival rate of MGC803 cells in a concentration- and time-dependent manner. Our flow cytometric analysis showed that resveratrol treatment arrested the cells at the G0/G1 phase of the cell cycle. Furthermore, western blotting demonstrated that resveratrol decreased the protein expression of phospho-glycogen synthase kinase 3β (p-GSK3β), cyclin D1, phospho-phosphatase and tensin homologue (p-PTEN), phospho-phosphatidylinositol 3'-OH kinase (p-PI3K), and phospho-protein kinase B (p-PKB/Akt). We also found that resveratrol inhibited the progression of the cell cycle in MGC803 cells by repressing p-PI3K and p-Akt expression. Meanwhile, resveratrol did not decrease the phosphorylation level of Akt when the PTEN gene expression was knocked down by an siRNA in the MGC803 cells. Taken together, these results suggest that resveratrol induced cell cycle arrest in human gastric cancer MGC803 cells by regulating the PTEN/PI3K/Akt signaling pathway.

  10. Resveratrol induces cell cycle arrest in human gastric cancer MGC803 cells via the PTEN-regulated PI3K/Akt signaling pathway.

    PubMed

    Jing, Xiaoping; Cheng, Weiwei; Wang, Shiying; Li, Pin; He, Li

    2016-01-01

    Resveratrol is a polyphenolic compound that is extracted from Polygonum cuspidatum and is used in traditional Chinese medicine. Previous data have shown that resveratrol inhibits the growth of human gastric cancer. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] and trypan blue assays showed that resveratrol significantly decreased the survival rate of MGC803 cells in a concentration- and time-dependent manner. Our flow cytometric analysis showed that resveratrol treatment arrested the cells at the G0/G1 phase of the cell cycle. Furthermore, western blotting demonstrated that resveratrol decreased the protein expression of phospho-glycogen synthase kinase 3β (p-GSK3β), cyclin D1, phospho-phosphatase and tensin homologue (p-PTEN), phospho-phosphatidylinositol 3'-OH kinase (p-PI3K), and phospho-protein kinase B (p-PKB/Akt). We also found that resveratrol inhibited the progression of the cell cycle in MGC803 cells by repressing p-PI3K and p-Akt expression. Meanwhile, resveratrol did not decrease the phosphorylation level of Akt when the PTEN gene expression was knocked down by an siRNA in the MGC803 cells. Taken together, these results suggest that resveratrol induced cell cycle arrest in human gastric cancer MGC803 cells by regulating the PTEN/PI3K/Akt signaling pathway. PMID:26530632

  11. Matrine inhibits the growth and induces apoptosis of osteosarcoma cells in vitro by inactivating the Akt pathway.

    PubMed

    Xu, Gong-Ping; Zhao, Wei; Zhuang, Jin-Peng; Zu, Jia-Ning; Wang, Duan-Yang; Han, Fei; Zhang, Zhi-Peng; Yan, Jing-Long

    2015-03-01

    Matrine, a natural product, has been demonstrated to be a promising chemotherapeutic drug for some cancers. Using flow cytometric analysis of the cell cycle and apoptosis, we found that matrine inhibited the proliferation and induced apoptosis in the human osteosarcoma (OS) cell lines MG63, HOS, U2OS, and SAOS2 in vitro in a dose-dependent manner. We therefore assessed the role of the serine/threonine kinase Akt in the regulation of matrine-mediated cell growth inhibition and apoptosis induction in human OS cell lines. After treatment for 48 h, matrine induced G0/G1-stage cell cycle arrest in MG63, U2OS, and SAOS2 cells associated with an increase in the expression of p27(Kip1) and a decrease in the expression of Akt, glycogen synthase kinase 3 (GSK3)-β (Ser9), and cyclin D1. Furthermore, the pro-apoptotic factor Bax was upregulated. Overall, our findings suggest that matrine may be an effective anti-osteosarcoma drug due to its ability to inhibit proliferation and induce apoptosis in OS cells, possibly through the involvement of Akt signaling.

  12. β2-Glycoprotein I Inhibits Vascular Endothelial Growth Factor-Induced Angiogenesis by Suppressing the Phosphorylation of Extracellular Signal-Regulated Kinase 1/2, Akt, and Endothelial Nitric Oxide Synthase.

    PubMed

    Chiu, Wen-Chin; Chiou, Tzeon-Jye; Chung, Meng-Ju; Chiang, An-Na

    2016-01-01

    Angiogenesis is the process of new blood vessel formation, and it plays a key role in various physiological and pathological conditions. The β2-glycoprotein I (β2-GPI) is a plasma glycoprotein with multiple biological functions, some of which remain to be elucidated. This study aimed to identify the contribution of 2-GPI on the angiogenesis induced by vascular endothelial growth factor (VEGF), a pro-angiogenic factor that may regulate endothelial remodeling, and its underlying mechanism. Our results revealed that β2-GPI dose-dependently decreased the VEGF-induced increase in endothelial cell proliferation, using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and the bromodeoxyuridine (BrdU) incorporation assays. Furthermore, incubation with both β2-GPI and deglycosylated β2-GPI inhibited the VEGF-induced tube formation. Our results suggest that the carbohydrate residues of β2-GPI do not participate in the function of anti-angiogenesis. Using in vivo Matrigel plug and angioreactor assays, we show that β2-GPI remarkably inhibited the VEGF-induced angiogenesis at a physiological concentration. Moreover, β2-GPI inhibited the VEGF-induced phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), Akt, and endothelial nitric oxide synthase (eNOS). In summary, our in vitro and in vivo data reveal for the first time that β2-GPI inhibits the VEGF-induced angiogenesis and highlights the potential for β2-GPI in anti-angiogenic therapy. PMID:27579889

  13. β2-Glycoprotein I Inhibits Vascular Endothelial Growth Factor-Induced Angiogenesis by Suppressing the Phosphorylation of Extracellular Signal-Regulated Kinase 1/2, Akt, and Endothelial Nitric Oxide Synthase

    PubMed Central

    Chiu, Wen-Chin; Chiou, Tzeon-Jye; Chung, Meng-Ju; Chiang, An-Na

    2016-01-01

    Angiogenesis is the process of new blood vessel formation, and it plays a key role in various physiological and pathological conditions. The β2-glycoprotein I (β2-GPI) is a plasma glycoprotein with multiple biological functions, some of which remain to be elucidated. This study aimed to identify the contribution of 2-GPI on the angiogenesis induced by vascular endothelial growth factor (VEGF), a pro-angiogenic factor that may regulate endothelial remodeling, and its underlying mechanism. Our results revealed that β2-GPI dose-dependently decreased the VEGF-induced increase in endothelial cell proliferation, using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and the bromodeoxyuridine (BrdU) incorporation assays. Furthermore, incubation with both β2-GPI and deglycosylated β2-GPI inhibited the VEGF-induced tube formation. Our results suggest that the carbohydrate residues of β2-GPI do not participate in the function of anti-angiogenesis. Using in vivo Matrigel plug and angioreactor assays, we show that β2-GPI remarkably inhibited the VEGF-induced angiogenesis at a physiological concentration. Moreover, β2-GPI inhibited the VEGF-induced phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), Akt, and endothelial nitric oxide synthase (eNOS). In summary, our in vitro and in vivo data reveal for the first time that β2-GPI inhibits the VEGF-induced angiogenesis and highlights the potential for β2-GPI in anti-angiogenic therapy. PMID:27579889

  14. Krüppel-like factor 14 increases insulin sensitivity through activation of PI3K/Akt signal pathway.

    PubMed

    Yang, Min; Ren, Yan; Lin, Zhimin; Tang, Chenchen; Jia, Yanjun; Lai, Yerui; Zhou, Tingting; Wu, Shaobo; Liu, Hua; Yang, Gangyi; Li, Ling

    2015-11-01

    Genome-wide association studies (GWAS) have shown that Krüppel-like factor 14 (KLF14) is associated with type 2 diabetes mellitus (T2DM). However, no report has demonstrated a relationship between KLF14 and glucose metabolism. The aim of this study was to determine whether KLF14 is associated with glucose metabolism and insulin signaling in vitro. The mRNA and protein expressions of KLF14 were determined by Real-time PCR and Western blotting. Glucose uptake was assessed by 2-[(3)H]-deoxyglucose (2-DG) uptake. Western blotting was used to identify the activation of insulin signaling proteins. KLF14 mRNA and protein in fat and muscle were significantly decreased in HFD-fed mice, db/db mice and T2DM patients. Overexpression of KLF14 enhanced insulin-stimulated glucose uptake and the activation of Akt kinase in Hepa1-6 cells. The phosphorylation of insulin receptor (InsR), insulin receptor substrate-1(IRS-1), glycogen synthase kinase-3β (GSK-3β) and Akt also elevated significantly by up-regulation of KLF14. KLF14 overexpression in Hepa1-6 cells prevented the inhibition of glucose uptake and Akt phosphorylation induced by high glucose and/or high insulin, or T2DM serum. However, KLF14's ability to increase glucose uptake and Akt activation was significantly attenuated by LY294002, a PI3-kinase inhibitor. These data suggested that KLF14 could increase insulin sensitivity probably through the PI3K/Akt pathway. PMID:26226221

  15. 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.

  16. The AKT-mTOR signalling pathway in kidney cancer tissues

    NASA Astrophysics Data System (ADS)

    Spirina, L. V.; Usynin, Y. A.; Kondakova, I. V.; Yurmazov, Z. A.; Slonimskaya, E. M.; Kolegova, E. S.

    2015-11-01

    An increased expression of phospho-AKT, m-TOR, glycogen regulator GSK-3-beta and transcription inhibitor 4E-BP1 was observed in kidney cancer tissues. Tumor size growth was associated with a high level of c-Raf and low content of phospho-m-TOR. Cancer metastasis development led to a decreased PTEN and phospho-AKT expression.

  17. Diosgenin and 5-Methoxypsoralen Ameliorate Insulin Resistance through ER-α/PI3K/Akt-Signaling Pathways in HepG2 Cells.

    PubMed

    Fang, Ke; Dong, Hui; Jiang, Shujun; Li, Fen; Wang, Dingkun; Yang, Desen; Gong, Jing; Huang, Wenya; Lu, Fuer

    2016-01-01

    To determine the effects and the underlying mechanism of diosgenin (DSG) and 5-methoxypsoralen (5-MOP), two main active components in the classical Chinese prescription Hu-Lu-Ba-Wan (HLBW), on insulin resistance, HepG2 cells were incubated in medium containing insulin. Treatments with DSG, 5-MOP, and their combination were performed, respectively. The result showed that the incubation of HepG2 cells with high concentration insulin markedly decreased glucose consumption and glycogen synthesis. However, treatment with DSG, 5-MOP, or their combination significantly reversed the condition and increased the phosphorylated expression of estrogen receptor-α (ERα), sarcoma (Src), Akt/protein kinase B, glycogen synthase kinase-3β (GSK-3β), and the p85 regulatory subunit of phosphatidylinositol 3-kinase p85 (PI3Kp85). At the transcriptional level, expression of the genes mentioned above also increased except for the negative regulation of GSK-3β mRNA. The increased expression of glucose transport-4 (GLUT-4) was meanwhile observed through immunofluorescence. Nevertheless, the synergistic effect of DSG and 5-MOP on improving glycometabolism was not obvious in the present study. These results suggested that DSG and 5-MOP may improve insulin resistance through an ER-mediated PI3K/Akt activation pathway which may be a new strategy for type 2 diabetes mellitus, especially for women in an estrogen-deficient condition. PMID:27656241

  18. Diosgenin and 5-Methoxypsoralen Ameliorate Insulin Resistance through ER-α/PI3K/Akt-Signaling Pathways in HepG2 Cells

    PubMed Central

    Dong, Hui; Jiang, Shujun; Li, Fen; Wang, Dingkun; Yang, Desen; Gong, Jing; Huang, Wenya

    2016-01-01

    To determine the effects and the underlying mechanism of diosgenin (DSG) and 5-methoxypsoralen (5-MOP), two main active components in the classical Chinese prescription Hu-Lu-Ba-Wan (HLBW), on insulin resistance, HepG2 cells were incubated in medium containing insulin. Treatments with DSG, 5-MOP, and their combination were performed, respectively. The result showed that the incubation of HepG2 cells with high concentration insulin markedly decreased glucose consumption and glycogen synthesis. However, treatment with DSG, 5-MOP, or their combination significantly reversed the condition and increased the phosphorylated expression of estrogen receptor-α (ERα), sarcoma (Src), Akt/protein kinase B, glycogen synthase kinase-3β (GSK-3β), and the p85 regulatory subunit of phosphatidylinositol 3-kinase p85 (PI3Kp85). At the transcriptional level, expression of the genes mentioned above also increased except for the negative regulation of GSK-3β mRNA. The increased expression of glucose transport-4 (GLUT-4) was meanwhile observed through immunofluorescence. Nevertheless, the synergistic effect of DSG and 5-MOP on improving glycometabolism was not obvious in the present study. These results suggested that DSG and 5-MOP may improve insulin resistance through an ER-mediated PI3K/Akt activation pathway which may be a new strategy for type 2 diabetes mellitus, especially for women in an estrogen-deficient condition.

  19. Diosgenin and 5-Methoxypsoralen Ameliorate Insulin Resistance through ER-α/PI3K/Akt-Signaling Pathways in HepG2 Cells

    PubMed Central

    Dong, Hui; Jiang, Shujun; Li, Fen; Wang, Dingkun; Yang, Desen; Gong, Jing; Huang, Wenya

    2016-01-01

    To determine the effects and the underlying mechanism of diosgenin (DSG) and 5-methoxypsoralen (5-MOP), two main active components in the classical Chinese prescription Hu-Lu-Ba-Wan (HLBW), on insulin resistance, HepG2 cells were incubated in medium containing insulin. Treatments with DSG, 5-MOP, and their combination were performed, respectively. The result showed that the incubation of HepG2 cells with high concentration insulin markedly decreased glucose consumption and glycogen synthesis. However, treatment with DSG, 5-MOP, or their combination significantly reversed the condition and increased the phosphorylated expression of estrogen receptor-α (ERα), sarcoma (Src), Akt/protein kinase B, glycogen synthase kinase-3β (GSK-3β), and the p85 regulatory subunit of phosphatidylinositol 3-kinase p85 (PI3Kp85). At the transcriptional level, expression of the genes mentioned above also increased except for the negative regulation of GSK-3β mRNA. The increased expression of glucose transport-4 (GLUT-4) was meanwhile observed through immunofluorescence. Nevertheless, the synergistic effect of DSG and 5-MOP on improving glycometabolism was not obvious in the present study. These results suggested that DSG and 5-MOP may improve insulin resistance through an ER-mediated PI3K/Akt activation pathway which may be a new strategy for type 2 diabetes mellitus, especially for women in an estrogen-deficient condition. PMID:27656241

  20. Hypoglycemic effect of D-chiro-inositol in type 2 diabetes mellitus rats through the PI3K/Akt signaling pathway.

    PubMed

    Gao, Yun-Feng; Zhang, Meng-Na; Wang, Tian-Xin; Wu, Tian-Chen; Ai, Ru-Dan; Zhang, Ze-Sheng

    2016-09-15

    In this investigation, a model of type 2 diabetes mellitus (T2DM) was used on Sprague-Dawley (SD) rats to clarify more details of the mechanism in the therapy of T2DM. D-chiro-inositol (DCI) was administrated to the diabetic rats as two doses [30, 60 mg/(kg·body weight·day)]. The biochemical indices revealed that DCI had a positive effect on hypoglycemic activity and promoted the glycogen synthesis. The rats in DCI high-dosage group had a blood glucose reduction rate of 21.5% after 5 weeks of treatment, and had insulin content in serum about 15.3 ± 2.37 mIU/L which was significantly decreased than diabetes control group. Real-time polymerase chain reaction (RT-PCR) results revealed that DCI gave a positive regulation on glycogen synthase (GS) and protein glucose transporter-4 (Glut4). Western blotting suggested that DCI could up-regulated the expression of the phosphatidylinositol-3-kinase (PI3K) p85, PI3Kp110, GS as well as the phosphorylation of protein kinase B (Akt) both in the liver and the skeletal muscle. The results also revealed that DCI enhanced the Glut4 expression on skeletal muscle. Above all, DCI played a positive role in regulating insulin-mediated glucose uptake through the PI3K/Akt signaling pathway in T2DM rats.

  1. Pioglitazone Protected against Cardiac Hypertrophy via Inhibiting AKT/GSK3β and MAPK Signaling Pathways

    PubMed Central

    Wei, Wen-Ying; Ma, Zhen-Guo; Xu, Si-Chi; Zhang, Ning; Tang, Qi-Zhu

    2016-01-01

    Peroxisome proliferator activated receptor γ (PPARγ) has been closely involved in the process of cardiovascular diseases. This study was to investigate whether pioglitazone (PIO), a PPARγ agonist, could protect against pressure overload-induced cardiac hypertrophy. Mice were orally given PIO (2.5 mg/kg) from 1 week after aortic banding and continuing for 7 weeks. The morphological examination and biochemical analysis were used to evaluate the effects of PIO. Neonatal rat ventricular cardiomyocytes were also used to verify the protection of PIO against hypertrophy in vitro. The results in our study demonstrated that PIO remarkably inhibited hypertrophic response induced by aortic banding in vivo. Besides, PIO also suppressed cardiac fibrosis in vivo. PIO treatment also inhibited the activation of protein kinase B (AKT)/glycogen synthase kinase-3β (GSK3β) and mitogen-activated protein kinase (MAPK) in the heart. In addition, PIO alleviated angiotensin II-induced hypertrophic response in vitro. In conclusion, PIO could inhibit cardiac hypertrophy via attenuation of AKT/GSK3β and MAPK pathways. PMID:27110236

  2. 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.

  3. 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

  4. 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.

  5. 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

  6. 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

  7. 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.

  8. Transactivation of lifeguard (LFG) by Akt-/LEF-1 pathway in MCF-7 and MDA-MB 231 human breast cancer cells.

    PubMed

    Bucan, Vesna; Adili, Mehran Y; Choi, Claudia Y U; Eddy, Mau-Thek; Vogt, Peter M; Reimers, Kerstin

    2010-07-01

    Lifeguard (LFG) has been identified as a molecule that uniquely inhibits death mediated by Fas. The molecular function of human LFG and its regulation in carcinogenesis is uncertain. In our study, we investigated the potential regulation of LFG expression by Akt/LEF-1 pathway. The Glycogen synthase kinase-3 (GSK3) can be regulated by different signaling pathways including those mediated by protein kinase Akt. Inhibition of GSK3beta subunits activity results in the stabilisation of the beta-catenin protein and its accumulation in the nucleus, where it associates with members of the TCF/LEF-1 family of transcription factors to mediate gene transcription. In Western blots, RT-PCR and by small interfering RNA directed against LEF-1, we demonstrated that LFG expression correlates with GSK3beta and LEF-1 activation. Moreover, we showed that LFG mRNA was down-regulated after transfection with siRNA against LEF-1 in MDA-MB-231 cells. Our results therefore identify LFG as a target of the Akt/LEF-1 pathway in MDA-MB-231 breast tumour cells, a regulation which could play a key role in breast tumour progression.

  9. Age-related neuroinflammation and changes in AKT-GSK-3β and WNT/ β-CATENIN signaling in rat hippocampus

    PubMed Central

    Orellana, Ana Maria Marques; Vasconcelos, Andrea Rodrigues; Leite, Jacqueline Alves; de Sá Lima, Larissa; Andreotti, Diana Zukas; Munhoz, Carolina Demarchi; Kawamoto, Elisa Mitiko; Scavone, Cristoforo

    2015-01-01

    Aging is a multifactorial process associated with an increased susceptibility to neurodegenerative disorders which can be related to chronic inflammation. Chronic inflammation, however, can be characterized by the persistent elevated glucocorticoid (GCs) levels, activation of the proinflammatory transcription factor NF-кB, as well as an increase in cytokines. Interestingly, both NF-кB and cytokines can be even modulated by Glycogen Synthase Kinase 3 beta (GSK-3β) activity, which is a key protein that can intermediate inflammation and metabolism, once it has a critical role in AKT signaling pathway, and can also intermediate WNT/β-CATENIN signaling pathway. The aim of this study was to verify age-related changes in inflammatory status, as well as in the AKT and WNT signaling pathways. Results showed an age-related increase in neuroinflammation as indicated by NF-кB activation, TNF-α and GCs increased levels, a decrease in AKT activation and an increase in GSK-3β activity in both 12- and 24- month old animals. Aging also seems to induce a progressive decrease in canonical WNT/β-CATENIN signaling pathway once there is a decrease in DVL-2 levels and in the transcription of Axin2 gene. Little is known about the DVL-2 regulation as well as its roles in WNT signaling pathway, but for the first time it was suggested that DVL-2 expression can be changed along aging. PMID:26647069

  10. Age-related neuroinflammation and changes in AKT-GSK-3β and WNT/ β-CATENIN signaling in rat hippocampus.

    PubMed

    Orellana, Ana Maria Marques; Vasconcelos, Andrea Rodrigues; Leite, Jacqueline Alves; de Sá Lima, Larissa; Andreotti, Diana Zukas; Munhoz, Carolina Demarchi; Kawamoto, Elisa Mitiko; Scavone, Cristoforo

    2015-12-01

    Aging is a multifactorial process associated with an increased susceptibility to neurodegenerative disorders which can be related to chronic inflammation. Chronic inflammation, however, can be characterized by the persistent elevated glucocorticoid (GCs) levels, activation of the proinflammatory transcription factor NF-кB, as well as an increase in cytokines. Interestingly, both NF-кB and cytokines can be even modulated by Glycogen Synthase Kinase 3 beta (GSK-3β) activity, which is a key protein that can intermediate inflammation and metabolism, once it has a critical role in AKT signaling pathway, and can also intermediate WNT/β-CATENIN signaling pathway. The aim of this study was to verify age-related changes in inflammatory status, as well as in the AKT and WNT signaling pathways. Results showed an age-related increase in neuroinflammation as indicated by NF-кB activation, TNF-α and GCs increased levels, a decrease in AKT activation and an increase in GSK-3β activity in both 12- and 24- month old animals. Aging also seems to induce a progressive decrease in canonical WNT/β-CATENIN signaling pathway once there is a decrease in DVL-2 levels and in the transcription of Axin2 gene. Little is known about the DVL-2 regulation as well as its roles in WNT signaling pathway, but for the first time it was suggested that DVL-2 expression can be changed along aging.

  11. Mouse hippocampal phosphorylation footprint induced by generalized seizures: Focus on ERK, mTORC1 and Akt/GSK-3 pathways.

    PubMed

    Gangarossa, Giuseppe; Sakkaki, Sophie; Lory, Philippe; Valjent, Emmanuel

    2015-12-17

    Exacerbated hippocampal activity has been associated to critical modifications of the intracellular signaling pathways. We have investigated rapid hippocampal adaptive responses induced by maximal electroshock seizure (MES). Here, we demonstrate that abnormal and exacerbated hippocampal activity induced by MES triggers specific and temporally distinct patterns of phosphorylation of extracellular signal-related kinase (ERK), mammalian target of rapamycin complex (mTORC) and Akt/glycogen synthase kinase-3 (Akt/GSK-3) pathways in the mouse hippocampus. While the ERK pathway is transiently activated, the mTORC1 cascade follows a rapid inhibition followed by a transient activation. This rebound of mTORC1 activity leads to the selective phosphorylation of p70S6K, which is accompanied by an enhanced phosphorylation of the ribosomal subunit S6. In contrast, the Akt/GSK-3 pathway is weakly altered. Finally, MES triggers a rapid upregulation of several plasticity-associated genes as a consequence exacerbated hippocampal activity. The results reported in the present study are reminiscent of the one observed in other models of generalized seizures, thus defining a common molecular footprint induced by intense and aberrant hippocampal activities.

  12. 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

  13. GLP1 protects cardiomyocytes from palmitate-induced apoptosis via Akt/GSK3b/b-catenin pathway.

    PubMed

    Ying, Ying; Zhu, Huazhang; Liang, Zhen; Ma, Xiaosong; Li, Shiwei

    2015-12-01

    Activation of apoptosis in cardiomyocytes by saturated palmitic acids contributes to cardiac dysfunction in diabetic cardiomyopathy. Beta-catenin (b-catenin) is a transcriptional regulator of several genes involved in survival/anti-apoptosis. However, its role in palmitate-induced cardiomyocyte apoptosis remains unclear. Glucagon-like peptide 1 (GLP1) has been shown to exhibit potential cardioprotective properties. This study was designed to evaluate the role of b-catenin signalling in palmitate-induced cardiomyocyte apoptosis and the molecular mechanism underlying the protective effects of GLP1 on palmitate-stressed cardiomyocytes. Exposure of neonatal rat cardiomyocytes to palmitate increased the fatty acid transporter CD36-mediated intracellular lipid accumulation and cardiomyocyte apoptosis, decreased accumulation and nuclear translocation of active b-catenin, and reduced expression of b-catenin target protein survivin and BCL2. These detrimental effects of palmitate were significantly attenuated by GLP1 co-treatment. However, the anti-apoptotic effects of GLP1 were markedly abolished when b-catenin was silenced with a specific short hairpin RNA. Furthermore, analysis of the upstream molecules and mechanisms responsible for GLP1-associated cardiac protection revealed that GLP1 restored the decreased phosphorylation of protein kinase B (Akt) and glycogen synthase kinase-3b (GSK3b) in palmitate-stimulated cardiomyocytes. In contrast, inhibition of Akt with an Akt-specific inhibitor MK2206 or blockade of GLP1 receptor (GLP1R) with a competitive antagonist exendin-(9-39) significantly abrogated the GLP1-mediated activation of GSK3b/b-catenin signalling, leading to increased apoptosis in palmitate-stressed cardiomyocytes. Collectively, our results demonstrated for the first time that the attenuated b-catenin signalling may contribute to palmitate-induced cardiomyocyte apoptosis, while GLP1 can protect cardiomyocytes from palmitate-induced apoptosis through

  14. GLP1 protects cardiomyocytes from palmitate-induced apoptosis via Akt/GSK3b/b-catenin pathway

    PubMed Central

    Ying, Ying; Zhu, Huazhang; Liang, Zhen; Ma, Xiaosong; Li, Shiwei

    2015-01-01

    Activation of apoptosis in cardiomyocytes by saturated palmitic acids contributes to cardiac dysfunction in diabetic cardiomyopathy. Beta-catenin (b-catenin) is a transcriptional regulator of several genes involved in survival/anti-apoptosis. However, its role in palmitate-induced cardiomyocyte apoptosis remains unclear. Glucagon-like peptide 1 (GLP1) has been shown to exhibit potential cardioprotective properties. This study was designed to evaluate the role of b-catenin signalling in palmitate-induced cardiomyocyte apoptosis and the molecular mechanism underlying the protective effects of GLP1 on palmitate-stressed cardiomyocytes. Exposure of neonatal rat cardiomyocytes to palmitate increased the fatty acid transporter CD36-mediated intracellular lipid accumulation and cardiomyocyte apoptosis, decreased accumulation and nuclear translocation of active b-catenin, and reduced expression of b-catenin target protein survivin and BCL2. These detrimental effects of palmitate were significantly attenuated by GLP1 co-treatment. However, the anti-apoptotic effects of GLP1 were markedly abolished when b-catenin was silenced with a specific short hairpin RNA. Furthermore, analysis of the upstream molecules and mechanisms responsible for GLP1-associated cardiac protection revealed that GLP1 restored the decreased phosphorylation of protein kinase B (Akt) and glycogen synthase kinase-3b (GSK3b) in palmitate-stimulated cardiomyocytes. In contrast, inhibition of Akt with an Akt-specific inhibitor MK2206 or blockade of GLP1 receptor (GLP1R) with a competitive antagonist exendin-(9–39) significantly abrogated the GLP1-mediated activation of GSK3b/b-catenin signalling, leading to increased apoptosis in palmitate-stressed cardiomyocytes. Collectively, our results demonstrated for the first time that the attenuated b-catenin signalling may contribute to palmitate-induced cardiomyocyte apoptosis, while GLP1 can protect cardiomyocytes from palmitate-induced apoptosis through

  15. Blood glucose fluctuation accelerates renal injury involved to inhibit the AKT signaling pathway in diabetic rats.

    PubMed

    Ying, Changjiang; Zhou, Xiaoyan; Chang, Zhenzhen; Ling, Hongwei; Cheng, Xingbo; Li, Wei

    2016-07-01

    Blood glucose fluctuation is associated with diabetic nephropathy. However, the mechanism by which blood glucose fluctuation accelerates renal injury is not fully understood. The aim of the present study was to assess the effects of blood glucose fluctuation on diabetic nephropathy in rats and investigate its underlying mechanism. Diabetes in the rats was induced by a high sugar, high-fat diet, and a single dose of STZ (35 mg/kg)-injected intraperitoneally. Unstable blood sugar models were induced by subcutaneous insulin injection and intravenous glucose injection alternately. Body weight, glycosylated hemoglobin A1c (HbAlc), blood urea nitrogen (BUN), serum creatinine (Scr), and Creatinine clearance (Ccr) were assessed. T-SOD activity and MDA level were measured by assay kit. Change in renal tissue ultrastructure was observed by light microscopy and electron microscopy. Phosphorylated ser/thr protein kinase (p-AKT) (phosphor-Ser473), phosphorylated glycogen synthase kinase-3 beta (p-GSK-3β) (phosphor-Ser9), Bcl-2-associated X protein (BAX), B cell lymphoma/leukemia 2 (BCL-2), and cleaved-cysteinyl aspartate-specific proteinase-3 (caspase-3) levels were detected by immunohistochemistry and Western blot. We observed that BUN and Scr were increased in diabetic rats, and Ccr was decreased. Furthermore, blood glucose fluctuations could exacerbate the Ccr changes. Renal tissue ultrastructure was also seriously injured by glucose variability in diabetic rats. In addition, glucose fluctuation increased the oxidative stress of renal tissue. Moreover, fluctuating blood glucose decreased p-AKT level and BCL-2, and increased p-GSK-3β, BAX, cleaved-caspase-3 levels, and ratio of BAX/BCL-2 in the kidneys of diabetic rats. In conclusion, these results suggest that blood glucose fluctuation accelerated renal injury is due, at least in part to its oxidative stress promoting and inhibiting the AKT signaling pathway in diabetic rats. PMID:26860515

  16. Neuroprotective effect of astaxanthin against glutamate-induced cytotoxicity in HT22 cells: Involvement of the Akt/GSK-3β pathway.

    PubMed

    Wen, X; Huang, A; Hu, J; Zhong, Z; Liu, Y; Li, Z; Pan, X; Liu, Z

    2015-09-10

    Oxidative stress (OS) mediated the pathogenesis of Alzheimer's disease (AD). Astaxanthin (ATX) has been reported to exert antioxidant activities as well as neuroprotective effects in vivo and in vitro. But it is still unknown whether the Akt/glycogen synthase kinase-3β (GSK-3β) signaling mediated the neuroprotective effect of ATX in HT22 cells. Flow cytometric analysis was used to evaluate reactive oxygen species (ROS) generation. Caspase and PARP activity was measured. The expressions of heme oxygenase-1 (HO-1), nuclear factor-E2-related factor 2 (Nrf2), Bcl-2, Bax, apoptosis-inducing factor (AIF), cytochrome-c (Cyto-c), p-Akt and p-GSK-3β were evaluated to elucidate the underlying mechanism. Our results showed that ATX significantly attenuated glutamate-induced cell viability loss and lactate dehydrogenase (LDH) release, decreased the expression of caspase-3/8/9 activity and cleaved PARP, and suppressed the intracellular accumulation of ROS in HT22 cells after exposure to glutamate. ATX also increased the mitochondrial expression of AIF, Cyto-c as well as Bax while decreased Bcl-2. Moreover, ATX also induced the HO-1 expression in a dose and time-dependent manner, increased the antioxidant-responsive element (ARE) activity and nuclear Nrf2 expression. Furthermore, treatment with ATX restored the p-Akt and p-GSK-3β (Ser9) as well as HO-1 expression reduced by glutamate. This protective effect was partially blocked by the inhibitors lithium chloride treatment in HT22, indicating the involvement of Akt/GSK-3β inactivation during the neuroprotective effect of ATX. Our results provide the first evidence that ATX can protect glutamate-induced cytotoxicity in HT22 via attenuating caspase activation and mitochondrial dysfunction and modulating the Akt/GSK-3β signaling, indicating ATX may be useful for the treatment of neurodegenerative disorders such as AD. PMID:26197224

  17. Substance P promotes the recovery of oxidative stress-damaged retinal pigmented epithelial cells by modulating Akt/GSK-3β signaling

    PubMed Central

    Baek, Sang-Min; Yu, Seung-Young; Son, Youngsook

    2016-01-01

    Purpose Senescence of the retina causes an accumulation of reactive oxygen species (ROS). Oxidative stress associated with ROS can damage RPE cells, leading to neovascularization and severe ocular disorders, including age-related macular degeneration (AMD). Thus, the early treatment of the damage caused by oxidative stress is critical for preventing the development of ocular diseases such as AMD. In this study, we examined the role of substance P (SP) in the recovery of RPE cells damaged by oxidative stress. Methods To induce oxidative stress, RPE cells were treated with H2O2 at various doses. Recovery from oxidative stress was studied following treatment with SP by analyzing cell viability, cell proliferation, cell apoptosis, and Akt/glycogen synthase kinase (GSK)-3β activation in RPE cells in vitro. Results H2O2 treatment reduced cellular viability in a dose-dependent manner. SP inhibited the reduction of cell viability due to H2O2 and caused increased cell proliferation and decreased cell apoptosis. Cell survival under oxidative stress requires the activation of Akt signaling that enables cells to resist oxidative stress-induced damage. SP treatment activated Akt/GSK-3β signaling in RPE cells, which were damaged due to oxidative stress, and the inhibition of Akt signaling in SP-treated RPE cells prevented SP-induced recovery. Pretreatment with the neurokinin 1 receptor (NK1R) antagonist reduced the recovery effect of SP on damaged RPE cells. Conclusions SP can protect RPE cells from oxidant-induced cell death by activating Akt/GSK-3β signaling via NK1R. This study suggests the possibility of SP as a treatment for oxidative stress-related diseases. PMID:27582624

  18. α-Lipoic acid interaction with dopamine D2 receptor-dependent activation of the Akt/GSK-3β signaling pathway induced by antipsychotics: potential relevance for the treatment of schizophrenia.

    PubMed

    Deslauriers, Jessica; Desmarais, Christian; Sarret, Philippe; Grignon, Sylvain

    2013-05-01

    Chronic administration of antipsychotics has been associated with dopamine D2 receptor (D2R) upregulation and tardive dyskinesia. We have previously shown that haloperidol, a first-generation antipsychotic (FGA), exerted an increase in D2R expression and oxidative stress and that (±)-α-lipoic acid reversed its effect. Previous studies have implicated the Akt/glycogen synthase kinase-3β (GSK-3β) signaling pathway in antipsychotic action. These findings led us to examine whether the Akt/GSK-3β pathway was involved in D2R upregulation and oxidative stress elicited by antipsychotics and, in (±)-α-lipoic acid-induced reversal of these phenomena, in SH-SY5Y cells. Antipsychotics increased phosphorylation of Akt and GSK-3β, and additive effects were observed with (±)-α-lipoic acid. GSK-3β inhibitors reversed haloperidol-induced overexpression of D2R mRNA levels but did not affect haloperidol-induced oxidative stress. Sustained antipsychotic treatment increased β-arrestin-2 and D2R receptor interaction. Regarding Akt/GSK-3β downstream targets, antipsychotics increased β-catenin levels, whereas (±)-α-lipoic acid induced an elevation of mTOR activation. These results suggest (1) that the effect of antipsychotics on the Akt/GSK-3β pathway in SH-SY5Y cells is reminiscent of their in vivo action, (2) that (±)-α-lipoic acid partially synergizes with antipsychotic drugs (APDs) on the same pathway, and (3) that the Akt/GSK-3β signaling cascade is not involved in the preventive effect of (±)-α-lipoic acid on antipsychotics-induced D2R upregulation.

  19. Lithium protection of phencyclidine-induced neurotoxicity in developing brain: the role of phosphatidylinositol-3 kinase/Akt and mitogen-activated protein kinase kinase/extracellular signal-regulated kinase signaling pathways.

    PubMed

    Xia, Yan; Wang, Cheng Z; Liu, Jie; Anastasio, Noelle C; Johnson, Kenneth M

    2008-09-01

    Phencyclidine (PCP) and other N-methyl-D-aspartate (NMDA) receptor antagonists have been shown to be neurotoxic to developing brains and to result in schizophrenia-like behaviors later in development. Prevention of both effects by antischizophrenic drugs suggests the validity of PCP neurodevelopmental toxicity as a heuristic model of schizophrenia. Lithium is used for the treatment of bipolar and schizoaffective disorders and has recently been shown to have neuroprotective properties. The present study used organotypic corticostriatal slices taken from postnatal day 2 rat pups to investigate the protective effect of lithium and the role of the phosphatidylinositol-3 kinase (PI-3K)/Akt and mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (MEK/ERK) pathways in PCP-induced cell death. Lithium pretreatment dose-dependently reduced PCP-induced caspase-3 activation and DNA fragmentation in layers II to IV of the cortex. PCP elicited time-dependent inhibition of the MEK/ERK and PI-3K/Akt pathways, as indicated by dephosphorylation of ERK1/2 and Akt. The proapoptotic factor glycogen synthase kinase (GSK)-3beta was also dephosphorylated at serine 9 and thus activated. Lithium prevented PCP-induced inhibition of the two pathways and activation of GSK-3beta. Furthermore, blocking either PI-3K/Akt or MEK/ERK pathway abolished the protective effect of lithium, whereas inhibiting GSK-3beta activity mimicked the protective effect of lithium. However, no cross-talk between the two pathways was found. Finally, specific GSK-3beta inhibition did not prevent PCP-induced dephosphorylation of Akt and ERK. These data strongly suggest that the protective effect of lithium against PCP-induced neuroapoptosis is mediated through independent stimulation of the PI-3K/Akt and ERK pathways and suppression of GSK-3beta activity.

  20. Human cardiac stem cells exhibit mesenchymal features and are maintained through Akt/GSK-3{beta} signaling

    SciTech Connect

    Tateishi, Kento |; Ashihara, Eishi; Honsho, Shoken |; Takehara, Naofumi; Nomura, Tetsuyaital |; Takahashi, Tomosaburo; Ueyama, Tomomi; Yamagishi, Masaaki; Yaku, Hitoshi; Matsubara, Hiroaki |. E-mail: matsubah@koto.kpu-m.ac.jp; Oh, Hidemasa . E-mail: hidemasa@kuhp.kyoto-u.ac.jp

    2007-01-19

    Recent evidence suggested that human cardiac stem cells (hCSCs) may have the clinical application for cardiac repair; however, their characteristics and the regulatory mechanisms of their growth have not been fully investigated. Here, we show the novel property of hCSCs with respect to their origin and tissue distribution in human heart, and demonstrate the signaling pathway that regulates their growth and survival. Telomerase-active hCSCs were predominantly present in the right atrium and outflow tract of the heart (infant > adult) and had a mesenchymal cell-like phenotype. These hCSCs expressed the embryonic stem cell markers and differentiated into cardiomyocytes to support cardiac function when transplanted them into ischemic myocardium. Inhibition of Akt pathway impaired the hCSC proliferation and induced apoptosis, whereas inhibition of glycogen synthase kinase-3 (GSK-3) enhanced their growth and survival. We conclude that hCSCs exhibit mesenchymal features and that Akt/GSK-3{beta} may be crucial modulators for hCSC maintenance in human heart.

  1. 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.

  2. 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

  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. Role of Sulindac and Celecoxib in the regulation of angiogenesis during the early neoplasm of colon: exploring PI3-K/PTEN/Akt pathway to the canonical Wnt/β-catenin signaling.

    PubMed

    Vaish, Vivek; Sanyal, Sankar Nath

    2012-07-01

    Angiogenesis refers to the generation of new blood vasculature from the nearby pre-existing one and is regulated by a balance between the pro- and anti-angiogenic factors. During carcinogenesis, pro-angiogenic factors dominate and initialize the growth of new blood capillaries to provide nutrition, growth factors and overcome hypoxia inside the tumor microenvironment. In the present study, we have observed the role of Phosphatidylinositol-3-kinase (PI3-K)/Phophatase and tensin homolog deleted on chromosome ten (PTEN)/Akt (Protein kinase B) pathway and canonical Wnt/β-catenin downstream signaling in the regulation of various pro-angiogenic molecules including the vascular endocrine growth factor-A (VEGF-A), matix metalloproteinases (MMPs), inducible nitric oxide synthase (iNOS) and chemokines for the progression of experimental colorectal cancer with 1,2-dimethylhydrazine dihydrochloride (DMH) and anti-angiogenic effects of two non-steroidal anti-inflammatory drugs (NSAIDs) viz. Sulindac and Celecoxib. Morphological and histopathological studies were performed to analyze the tumorigenic modifications while flow cytometry for the relative quantification of apoptotic events. Transcriptional and translational modifications of biomolecules were analyzed via Reverse Transcriptase-and quantitative Real Time PCR, Western immoblotting and immunoflurescence, respectively. In vitro phosphorylation, gelatin zymography and nitric oxide (NO) assay were performed to observe the activation states of Akt, MMPs and iNOS enzyme, respectively. Dysregultion in Akt activation, and thereby, aberrant signaling of β-catenin along with the production of NO could positively regulate tumor angiogenesis. NSAIDs can overcome these carcinogenic effects by controlling various key check points including higher PTEN and glycogen synthase kinase-3β (GSK-3β) expression and repressing Akt, MMPs and iNOS activation while inducing apoptosis among the cancer cells.

  5. 4-Hydroxyisoleucine improves hepatic insulin resistance by restoring glycogen synthesis in vitro

    PubMed Central

    Lu, Furong; Cai, Qin; Zafar, Mohammad Ishraq; Cai, Lun; Du, Wen; Jian, Liumeng; Li, Liping; Gao, Feng

    2015-01-01

    Introduction/Aims: Hydroxyisoleucine (4-HIL), derived from fenugreek seeds, improves insulin resistance in 3T3-L1 adipocytes and L6 myotubes. However, the effects of 4-HIL on liver glycogen synthesis and hepatic insulin resistance have not been described. The aim of this study was to investigate the effects of 4-HIL on glycogen synthesis in a tumor necrosis factor-α (TNF-α)-induced insulin resistance model using HepG2 cells. Materials and methods: HepG2 cells were divided into eight groups: control; TNF-α; and 5, 10, or 20 μM 4-HIL without or with TNF-α. Glycogen and protein expression were evaluated using a glycogen assay kit and western blotting, respectively. Results: Glycogen levels did not differ between the 4-HIL groups and control (P>0.05), but were decreased significantly in the TNF-α group (P<0.05), indicating the establishment of insulin-resistant HepG2 cells. Adding 20 μM 4-HIL to TNF-α-treated cells increased glycogen levels (P<0.05). Relative to the control group, the P-IRS-1/IRS-1 and P-JNK/JNK ratios were increased (P<0.001) in the TNF-α group, whereas the P-AKT/AKT and P-GSK/GSK ratios were decreased (P<0.001). When 20 μM 4-HIL was added to TNF-α-treated cells, the P-IRS-1/IRS-1 and P-JNK/JNK ratios decreased (P<0.001 and P<0.05, respectively), whereas the P-AKT/AKT and P-GSK/GSK ratios increased (P<0.05 and P<0.001). Conclusions: 4-HIL directly or indirectly reversed TNF-α reduced glycogen levels by inhibiting JNK and IRS-1 (Ser307) phosphorylation and increasing AKT (Ser473) and GSK-3 phosphorylation. These findings demonstrate that 4-HIL modulates hepatic insulin resistance at the molecular level, and suggest that it is a novel potential therapeutic agent for the treatment of insulin resistance in patients with diabetes. PMID:26309514

  6. 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.

  7. 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.

  8. 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.

  9. 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

  10. Inhibition of fatty acid synthase suppresses U-2 OS cell invasion and migration via downregulating the activity of HER2/PI3K/AKT signaling pathway in vitro

    SciTech Connect

    Wang, Tao Fang; Wang, Heng; Peng, Ai Fen; Luo, Qing Feng; Liu, Zhi Li; Zhou, Rong Ping; Gao, Song; Zhou, Yang; Chen, Wen Zhao

    2013-10-18

    Highlights: •We investigate the relationship between FASN and HER2 or p-HER2 by IHC in OS tissues. •We construct FASN-specific RNAi plasmid. •Inhibiting FASN down-regulates HER2/PI3K/AKT cell signaling in U-2 OS. •Inhibiting FASN blocks U-2 OS cell invasion and migration. -- Abstract: FASN plays an important role in the malignant phenotype of various tumors. Our previous studies show that inhibition FASN could induce apoptosis and inhibit proliferation in human osteosarcoma (OS) cell in vivo and vitro. The aim in this study was to investigate the effect of inhibition FASN on the activity of HER2/PI3K/AKT axis and invasion and migration of OS cell. The expression of FASN, HER2 and p-HER2(Y1248) proteins was detected by immunohistochemistry in OS tissues from 24 patients with pulmonary metastatic disease, and the relationship between FASN and p-HER2 as well as HER2 was investigated. The results showed that there was a positive correlation between FASN and HER2 as well as p-HER2 protein expression. The U-2 OS cells were transfected with either the FASN specific RNAi plasmid or the negative control RNAi plasmid. FASN mRNA was measured by RT-PCR. Western blot assays was performed to examine the protein expression of FASN, HER2, p-HER2(Y1248), PI3K, Akt and p-Akt (Ser473). Migration and invasion of cells were investigated by wound healing and transwell invasion assays. The results showed that the activity of HER2/PI3K/AKT signaling pathway was suppressed by inhibiting FASN. Meanwhile, the U-2OS cells migration and invasion were also impaired by inhibiting the activity of FASN/HER2/PI3K/AKT. Our results indicated that inhibition of FASN suppresses OS cell invasion and migration via down-regulation of the “HER2/PI3K/AKT” axis in vitro. FASN blocker may be a new therapeutic strategy in OS management.

  11. CD147 modulates androgen receptor activity through the Akt/Gsk-3β/β-catenin/AR pathway in prostate cancer cells

    PubMed Central

    Fang, Fang; Qin, Yingxin; Hao, Feng; Li, Qiang; Zhang, Wei; Zhao, Chen; Chen, Shuang; Zhao, Liangzhong; Wang, Liguo; Cai, Jianhui

    2016-01-01

    The androgen signaling pathway serves an important role in the development of prostate cancer. β-Catenin is an androgen receptor (AR) cofactor and augments AR signaling. Glycogen synthase kinase-3β (GSK-3β), a target of phosphorylated serine/threonine protein kinase B (p-Akt), regulates β-catenin stability. In addition, β-catenin, a coregulator of AR, physically interacts with AR and enhances AR-mediated target gene transcription. The multifunctional glycoprotein cluster of differentiation (CD) 147 is highly expressed on the cell surface of the majority of cancer cells, and it promotes tumor invasion, metastasis and growth. In the present study, the molecular effects of CD147 on the Akt/GSK-3β/β-catenin/AR signaling network were investigated in LNCaP cells. Using short hairpin-mediated RNA knockdown of CD147 in LNCaP cells, it was demonstrated that downregulation of CD147 resulted in inhibitory phosphorylation of GSK-3β, and then promoted degeneration of β-catenin and reduced nuclear accumulation of β-catenin. In addition, immunoprecipitation studies demonstrated that CD147 downregulation decreased the formation of a complex between β-catenin and AR. It was shown that CD147 knockdown suppressed the expression of the AR target gene prostate-specific antigen and the growth of AR-positive LNCaP cells. Furthermore, inhibition of PI3K/Akt with LY294002 augmented CD147-mediated function. The present study indicates that the PI3K/Akt pathway may facilitate CD147-mediated activation of the AR pathway. PMID:27446405

  12. Helicobacter pylori VacA-induced Inhibition of GSK3 through the PI3K/Akt Signaling Pathway*S⃞

    PubMed Central

    Nakayama, Masaaki; Hisatsune, Junzo; Yamasaki, Eiki; Isomoto, Hajime; Kurazono, Hisao; Hatakeyama, Masanori; Azuma, Takeshi; Yamaoka, Yoshio; Yahiro, Kinnosuke; Moss, Joel; Hirayama, Toshiya

    2009-01-01

    Helicobacter pylori VacA toxin contributes to the pathogenesis and severity of gastric injury. We found that incubation of AZ-521 cells with VacA resulted in phosphorylation of protein kinase B (Akt) and glycogen synthase kinase-3β (GSK3β) through a PI3K-dependent pathway. Following phosphorylation and inhibition of GSK3β,β-catenin was released from a GSK3β/β-catenin complex, with subsequent nuclear translocation. Methyl-β-cyclodextrin (MCD) and phosphatidylinositol-specific phospholipase C (PI-PLC), but not 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) and bafilomycin A1, inhibited VacA-induced phosphorylation of Akt, indicating that it does not require VacA internalization and is independent of vacuolation. VacA treatment of AZ-521 cells transfected with TOPtkLuciferase reporter plasmid or control FOPtkLucifease reporter plasmid resulted in activation of TOPtkLuciferase, but not FOPtkLucifease. In addition, VacA transactivated the β-catenin-dependent cyclin D1 promoter in a luciferase reporter assay. Infection of AZ-521 cells by a vacA mutant strain of H. pylori failed to induce phosphorylation of Akt and GSK3β, or release of β-catenin from a GSK3β/β-catenin complex. Taken together, these results support the conclusion that VacA activates the PI3K/Akt signaling pathway, resulting in phosphorylation and inhibition of GSK3β, and subsequent translocation ofβ-catenin to the nucleus, consistent with effects of VacA on β-catenin-regulated transcriptional activity. These data introduce the possibility that Wnt-dependent signaling might play a role in the pathogenesis of H. pylori infection, including the development of gastric cancer. PMID:18996844

  13. Altered regulation of Akt signaling with murine cerebral malaria, effects on long-term neuro-cognitive function, restoration with lithium treatment.

    PubMed

    Dai, Minxian; Freeman, Brandi; Shikani, Henry J; Bruno, Fernando Pereira; Collado, J Elias; Macias, Rolando; Reznik, Sandra E; Davies, Peter; Spray, David Conover; Tanowitz, Herbert Bernard; Weiss, Louis Martin; Desruisseaux, Mahalia Sabrina

    2012-01-01

    Neurological and cognitive impairment persist in more than 20% of cerebral malaria (CM) patients long after successful anti-parasitic treatment. We recently reported that long term memory and motor coordination deficits are also present in our experimental cerebral malaria model (ECM). We also documented, in a murine model, a lack of obvious pathology or inflammation after parasite elimination, suggesting that the long-term negative neurological outcomes result from potentially reversible biochemical and physiological changes in brains of ECM mice, subsequent to acute ischemic and inflammatory processes. Here, we demonstrate for the first time that acute ECM results in significantly reduced activation of protein kinase B (PKB or Akt) leading to decreased Akt phosphorylation and inhibition of the glycogen kinase synthase (GSK3β) in the brains of mice infected with Plasmodium berghei ANKA (PbA) compared to uninfected controls and to mice infected with the non-neurotrophic P. berghei NK65 (PbN). Though Akt activation improved to control levels after chloroquine treatment in PbA-infected mice, the addition of lithium chloride, a compound which inhibits GSK3β activity and stimulates Akt activation, induced a modest, but significant activation of Akt in the brains of infected mice when compared to uninfected controls treated with chloroquine with and without lithium. In addition, lithium significantly reversed the long-term spatial and visual memory impairment as well as the motor coordination deficits which persisted after successful anti-parasitic treatment. GSK3β inhibition was significantly increased after chloroquine treatment, both in lithium and non-lithium treated PbA-infected mice. These data indicate that acute ECM is associated with abnormalities in cell survival pathways that result in neuronal damage. Regulation of Akt/GSK3β with lithium reduces neuronal degeneration and may have neuroprotective effects in ECM. Aberrant regulation of Akt/GSK3β signaling

  14. Altered Regulation of Akt Signaling with Murine Cerebral Malaria, Effects on Long-Term Neuro-Cognitive Function, Restoration with Lithium Treatment

    PubMed Central

    Bruno, Fernando Pereira; Collado, J. Elias; Macias, Rolando; Reznik, Sandra E.; Davies, Peter; Spray, David Conover; Tanowitz, Herbert Bernard; Weiss, Louis Martin; Desruisseaux, Mahalia Sabrina

    2012-01-01

    Neurological and cognitive impairment persist in more than 20% of cerebral malaria (CM) patients long after successful anti-parasitic treatment. We recently reported that long term memory and motor coordination deficits are also present in our experimental cerebral malaria model (ECM). We also documented, in a murine model, a lack of obvious pathology or inflammation after parasite elimination, suggesting that the long-term negative neurological outcomes result from potentially reversible biochemical and physiological changes in brains of ECM mice, subsequent to acute ischemic and inflammatory processes. Here, we demonstrate for the first time that acute ECM results in significantly reduced activation of protein kinase B (PKB or Akt) leading to decreased Akt phosphorylation and inhibition of the glycogen kinase synthase (GSK3β) in the brains of mice infected with Plasmodium berghei ANKA (PbA) compared to uninfected controls and to mice infected with the non-neurotrophic P. berghei NK65 (PbN). Though Akt activation improved to control levels after chloroquine treatment in PbA-infected mice, the addition of lithium chloride, a compound which inhibits GSK3β activity and stimulates Akt activation, induced a modest, but significant activation of Akt in the brains of infected mice when compared to uninfected controls treated with chloroquine with and without lithium. In addition, lithium significantly reversed the long-term spatial and visual memory impairment as well as the motor coordination deficits which persisted after successful anti-parasitic treatment. GSK3β inhibition was significantly increased after chloroquine treatment, both in lithium and non-lithium treated PbA-infected mice. These data indicate that acute ECM is associated with abnormalities in cell survival pathways that result in neuronal damage. Regulation of Akt/GSK3β with lithium reduces neuronal degeneration and may have neuroprotective effects in ECM. Aberrant regulation of Akt/GSK3β signaling

  15. Cannabinoid receptor agonist WIN55,212-2 and fatty acid amide hydrolase inhibitor URB597 may protect against cognitive impairment in rats of chronic cerebral hypoperfusion via PI3K/AKT signaling.

    PubMed

    Su, Shao-Hua; Wang, Yue-Qing; Wu, Yi-Fang; Wang, Da-Peng; Lin, Qi; Hai, Jian

    2016-10-15

    The present study further investigated the protective effects of cannabinoid receptor agonist WIN55,212-2 (WIN) and fatty acid amide hydrolase (FAAH) inhibitor URB597 (URB) on chronic cerebral hypoperfusion (CCH)-induced cognitive impairment in rats. Spatial learning and memory were assessed with the Morris water maze and by measuring Long-term potentiation. The expression of microtubule-associated protein-2 (MAP)-2, growth-associated protein-43 (GAP)-43, synaptophysin, cannabinoid receptor 1 (CB1), brain-derived neurotrophic factor (BDNF), FAAH, N-acylphosphatidylethanolamine phospholipase D(NAPE-PLD) and monoacyl glycerol lipase (MGL) as well as phosphoinositide 3-kinase (PI3K)/AKT signaling pathway molecules and downstream targets including AKT, phosphorylated (p-)AKT, cyclic AMP response element- binding protein (CREB), p-CREB, Bcl-2-associated death protein (BAD), p-BAD, glycogen synthase kinase (GSK)-3β, p-GSK-3β, forkhead box protein (FOXO) 3A and p-FOXO3A was determined by western blotting. WIN and URB treatment improved learning and memory performance, effects that were abolished by co-administration of the PI3K/AKT inhibitor LY294002. Moreover, WIN and URB reversed the decreases in MAP-2 and synaptophysin expression resulting from CCH, and stimulated BDNF and CB1 expression as well as CREB, FOXO3A, GSK-3β, and BAD phosphorylation, confirming that WIN and URB mediate neuroprotection by preventing neuronal apoptosis and improving cognition via PI3K/AKT signaling. These findings suggest that WIN and URB are promising agents for therapeutic management of CCH. PMID:27424778

  16. Oxidized LDL at low concentration promotes in-vitro angiogenesis and activates nitric oxide synthase through PI3K/Akt/eNOS pathway in human coronary artery endothelial cells

    SciTech Connect

    Yu, Shan; Wong, Siu Ling; Lau, Chi Wai; Huang, Yu; Yu, Cheuk-Man

    2011-04-01

    Research highlights: {yields} Low-concentration oxidized LDL enhances angiogenesis through nitric oxide (NO). {yields} Oxidized LDL increases intracellular NO levels via eNOS phosphorylation. {yields} Akt/PI3K signaling mediates oxidized LDL-induced eNOS phosphorylation. -- Abstract: It has long been considered that oxidized low-density lipoprotein (oxLDL) causes endothelial dysfunction and is remarkably related to the development of atherosclerosis. However, the effect of oxLDL at very low concentration (<10 {mu}g/ml) on the endothelial cells remains speculative. Nitric oxide (NO) has a crucial role in the endothelial cell function. In this study, we investigated the effect of oxLDL at low concentration on NO production and proliferation, migration, tube formation of the human coronary artery endothelial cells (HCAEC). Results showed that oxLDL at 5 {mu}g/ml enhanced HCAEC proliferation, migration and tube formation. These phenomena were accompanied by an increased intracellular NO production. L-NAME (a NOS inhibitor), LY294002 and wortmannin (PI3K inhibitors) could abolish oxLDL-induced angiogenic effects and prevent NO production in the HCAEC. The phosphorylation of Akt, PI3K and eNOS were up-regulated by oxLDL, which was attenuated by LY294002. Our results suggested that oxLDL at low concentration could promote in-vitro angiogenesis and activate nitric oxide synthesis through PI3K/Akt/eNOS pathway in HCAEC.

  17. Urocortin 3 activates AMPK and AKT pathways and enhances glucose disposal in rat skeletal muscle

    PubMed Central

    Roustit, Manon M; Vaughan, Joan M; Jamieson, Pauline M; Cleasby, Mark E

    2014-01-01

    Insulin resistance (IR) in skeletal muscle is an important component of both type 2 diabetes and the syndrome of sarcopaenic obesity, for which there are no effective therapies. Urocortins (UCNs) are not only well established as neuropeptides but also have their roles in metabolism in peripheral tissues. We have shown recently that global overexpression of UCN3 resulted in muscular hypertrophy and resistance to the adverse metabolic effects of a high-fat diet. Herein, we aimed to establish whether short-term local UCN3 expression could enhance glucose disposal and insulin signalling in skeletal muscle. UCN3 was found to be expressed in right tibialis cranialis and extensor digitorum longus muscles of rats by in vivo electrotransfer and the effects studied vs the contralateral muscles after 1 week. No increase in muscle mass was detected, but test muscles showed 19% larger muscle fibre diameter (P=0.030), associated with increased IGF1 and IGF1 receptor mRNA and increased SER256 phosphorylation of forkhead transcription factor. Glucose clearance into the test muscles after an intraperitoneal glucose load was increased by 23% (P=0.018) per unit mass, associated with increased GLUT1 (34% increase; P=0.026) and GLUT4 (48% increase; P=0.0009) proteins, and significantly increased phosphorylation of insulin receptor substrate-1, AKT, AKT substrate of 160 kDa, glycogen synthase kinase-3β, AMP-activated protein kinase and its substrate acetyl coA carboxylase. Thus, UCN3 expression enhances glucose disposal and signalling in muscle by an autocrine/paracrine mechanism that is separate from its pro-hypertrophic effects, implying that such a manipulation may have promised for the treatment of IR syndromes including sarcopaenic obesity. PMID:25122003

  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

    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. 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

  20. Quercetin Protects against Okadaic Acid-Induced Injury via MAPK and PI3K/Akt/GSK3β Signaling Pathways in HT22 Hippocampal Neurons.

    PubMed

    Jiang, Wei; Luo, Tao; Li, Sheng; Zhou, Yue; Shen, Xiu-Yin; He, Feng; Xu, Jie; Wang, Hua-Qiao

    2016-01-01

    Increasing evidence shows that oxidative stress and the hyperphosphorylation of tau protein play essential roles in the progression of Alzheimer's disease (AD). Quercetin is a major flavonoid that has anti-oxidant, anti-cancer and anti-inflammatory properties. We investigated the neuroprotective effects of quercetin to HT22 cells (a cell line from mouse hippocampal neurons). We found that Okadaic acid (OA) induced the hyperphosphorylation of tau protein at Ser199, Ser396, Thr205, and Thr231 and produced oxidative stress to the HT22 cells. The oxidative stress suppressed the cell viability and decreased the levels of lactate dehydrogenase (LDH), superoxide dismutase (SOD), mitochondria membrane potential (MMP) and Glutathione peroxidase (GSH-Px). It up-regulated malondialdehyde (MDA) production and intracellular reactive oxygen species (ROS). In addition, phosphoinositide 3 kinase/protein kinase B/Glycogen synthase kinase3β (PI3K/Akt/GSK3β) and mitogen activated protein kinase (MAPK) were also involved in this process. We found that pre-treatment with quercetin can inhibited OA-induced the hyperphosphorylation of tau protein and oxidative stress. Moreover, pre-treatment with quercetin not only inhibited OA-induced apoptosis via the reduction of Bax, and up-regulation of cleaved caspase 3, but also via the inhibition of PI3K/Akt/GSK3β, MAPKs and activation of NF-κB p65. Our findings suggest the therapeutic potential of quercetin to treat AD. PMID:27050422

  1. Caffeine inhibits adipogenesis through modulation of mitotic clonal expansion and the AKT/GSK3 pathway in 3T3-L1 adipocytes.

    PubMed

    Kim, Ah-Reum; Yoon, Bo Kyung; Park, Hyounkyoung; Seok, Jo Woon; Choi, Hyeonjin; Yu, Jung Hwan; Choi, Yoonjeong; Song, Su Jin; Kim, Ara; Kim, Jae-Woo

    2016-02-01

    Caffeine has been proposed to have several beneficial effects on obesity and its related metabolic diseases; however, how caffeine affects adipocyte differentiation has not been elucidated. In this study, we demonstrated that caffeine suppressed 3T3-L1 adipocyte differentiation and inhibited the expression of CCAAT/enhancer binding protein (C/EBP)α and peroxisome proliferator-activated receptor (PPAR)γ, two main adipogenic transcription factors. Anti-adipogenic markers, such as preadipocyte secreted factor (Pref)-1 and Krüppel-like factor 2, remained to be expressed in the presence of caffeine. Furthermore, 3T3-L1 cells failed to undergo typical mitotic clonal expansion in the presence of caffeine. Investigation of hormonal signaling revealed that caffeine inhibited the activation of AKT and glycogen synthase kinase (GSK) 3 in a dose-dependent manner, but not extracellular signal-regulated kinase (ERK). Our data show that caffeine is an anti-adipogenic bioactive compound involved in the modulation of mitotic clonal expansion during adipocyte differentiation through the AKT/GSK3 pathway. [BMB Reports 2016; 49(2): 111-115].

  2. Quercetin Protects against Okadaic Acid-Induced Injury via MAPK and PI3K/Akt/GSK3β Signaling Pathways in HT22 Hippocampal Neurons

    PubMed Central

    Li, Sheng; Zhou, Yue; Shen, Xiu-Yin; He, Feng; Xu, Jie; Wang, Hua-Qiao

    2016-01-01

    Increasing evidence shows that oxidative stress and the hyperphosphorylation of tau protein play essential roles in the progression of Alzheimer’s disease (AD). Quercetin is a major flavonoid that has anti-oxidant, anti-cancer and anti-inflammatory properties. We investigated the neuroprotective effects of quercetin to HT22 cells (a cell line from mouse hippocampal neurons). We found that Okadaic acid (OA) induced the hyperphosphorylation of tau protein at Ser199, Ser396, Thr205, and Thr231 and produced oxidative stress to the HT22 cells. The oxidative stress suppressed the cell viability and decreased the levels of lactate dehydrogenase (LDH), superoxide dismutase (SOD), mitochondria membrane potential (MMP) and Glutathione peroxidase (GSH-Px). It up-regulated malondialdehyde (MDA) production and intracellular reactive oxygen species (ROS). In addition, phosphoinositide 3 kinase/protein kinase B/Glycogen synthase kinase3β (PI3K/Akt/GSK3β) and mitogen activated protein kinase (MAPK) were also involved in this process. We found that pre-treatment with quercetin can inhibited OA-induced the hyperphosphorylation of tau protein and oxidative stress. Moreover, pre-treatment with quercetin not only inhibited OA-induced apoptosis via the reduction of Bax, and up-regulation of cleaved caspase 3, but also via the inhibition of PI3K/Akt/GSK3β, MAPKs and activation of NF-κB p65. Our findings suggest the therapeutic potential of quercetin to treat AD. PMID:27050422

  3. 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. ...

  4. Role of brain glycogen in the response to hypoxia and in susceptibility to epilepsy

    PubMed Central

    López-Ramos, Juan C.; Duran, Jordi; Gruart, Agnès; Guinovart, Joan J.; Delgado-García, José M.

    2015-01-01

    Although glycogen is the only carbohydrate reserve of the brain, its overall contribution to brain functions remains unclear. It has been proposed that glycogen participates in the preservation of such functions during hypoxia. Several reports also describe a relationship between brain glycogen and susceptibility to epilepsy. To address these issues, we used our brain-specific Glycogen Synthase knockout (GYS1Nestin-KO) mouse to study the functional consequences of glycogen depletion in the brain under hypoxic conditions and susceptibility to epilepsy. GYS1Nestin-KO mice presented significantly different power spectra of hippocampal local field potentials (LFPs) than controls under hypoxic conditions. In addition, they showed greater excitability than controls for paired-pulse facilitation evoked at the hippocampal CA3–CA1 synapse during experimentally induced hypoxia, thereby suggesting a compensatory switch to presynaptic mechanisms. Furthermore, GYS1Nestin-KO mice showed greater susceptibility to hippocampal seizures and myoclonus following the administration of kainate and/or a brief train stimulation of Schaffer collaterals. We conclude that brain glycogen could play a protective role both in hypoxic situations and in the prevention of brain seizures. PMID:26578889

  5. Molecular mechanism underlying Akt activation in zinc-induced cardioprotection

    PubMed Central

    Lee, SungRyul; Chanoit, Guillaume; McIntosh, Rachel; Zvara, David A.; Xu, Zhelong

    2009-01-01

    Our previous study demonstrated that zinc prevents cardiac reperfusion injury by targeting the mitochondrial permeability transition pore (mPTP) via Akt and glycogen synthetase kinase 3β (GSK-3β). We aimed to address the mechanism by which zinc activates Akt. Treatment of H9c2 cells with ZnCl2 (10 μM) in the presence of the zinc ionophore pyrithione (4 μM) for 20 min enhanced Akt phosphorylation (Ser473), indicating that zinc can rapidly activate Akt. Zinc did not alter either phosphatase and tensin homolog deleted on chromosome 10 (PTEN) phosphorylation and total PTEN protein levels or PTEN oxidation, implying that PTEN may not play a role in the action of zinc. However, zinc-induced Akt phosphorylation was blocked by both the nonselective receptor tyrosine kinase (RTK) inhibitor genistein and the selective insulin-like growth factor-1 RTK (IGF-1RTK) inhibitor AG1024, indicating that zinc activates Akt via IGF-1RTK. Zinc-induced phosphorylation of protein tyrosine and Ser/Thr was also abolished by AG1024. In addition, zinc markedly enhanced phosphorylation of IGF-1 receptor (IGF-1R), which was again reversed by genistein and AG1024. A confocal imaging study revealed that AG1024 abolished the preventive effect of zinc on oxidant-induced mPTP opening, confirming that IGF-1RTK plays a role in zinc-induced cardioprotection. Furthermore, zinc decreased the activity of protein phosphatase 2A (PP2A), a major protein Ser/Thr phosphatase, implying that protein Ser/Thr phosphatases may also play a role in the action of zinc on Akt activity. Taken together, these findings demonstrate that exogenous zinc activates Akt via IGF-1RTK and prevents the mPTP opening in cardiac cells. Inactivation of Ser/Thr protein phosphatases may also contribute to zinc-induced Akt activation. PMID:19525380

  6. 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

  7. Neuroprotective effect of ginsenoside Rg1 prevents cognitive impairment induced by isoflurane anesthesia in aged rats via antioxidant, anti-inflammatory and anti-apoptotic effects mediated by the PI3K/AKT/GSK-3β pathway.

    PubMed

    Zhang, Yini; Zhang, Zhao; Wang, Haitang; Cai, Nan; Zhou, Shuang; Zhao, Yaoping; Chen, Xue; Zheng, Shaoqiang; Si, Qi; Zhang, Wei

    2016-09-01

    Ginsenoside Rg1 is the primary active substance in ginseng, and it has multiple pharmacological actions. Investigations on the pharmacologic action of ginsenoside Rg1 have developed, with a particular focus on the regulation of metabolism. The present study hypothesized that the neuroprotective effects of ginsenoside Rg1 prevent cognitive impairment induced by isoflurane anesthesia via antioxidant, anti‑inflammatory and anti‑apoptotic effects, mediated by the phosphoinositide 3‑kinase (PI3K)/AKT/glycogen synthase kinase‑3β (GSK‑3β) pathway in aged rats. Sprague‑Dawley rats were divided into isoflurane and ginsenoside Rg1 groups and were treated with 20 mg/kg ginsenoside Rg1 for 7 days. Morris water maze was performed to analyze the cognitive function of the rats. Enzyme‑linked immunosorbent assays were used to analyze the levels of malondialdehyde, glutathione, interleukin (IL)‑1β, IL‑6 and caspase 3. The protein expression levels of AKT, GSK 3β, p21WAF1/CIP1 and p53 were measured using western blot analysis. Ginsenoside Rg1 significantly improved cognitive function, and exhibited antioxidant and anti‑inflammatory effects, demonstrating the neuroprotective effects of ginsenoside Rg1 against the effect of isoflurane anesthesia in the rats. In addition, ginsenoside Rg1 significantly reduced caspase‑3 activity, upregulated the expression of PI3K/AKT/GSK‑3β and downregulated the mRNA expression levels of p21WAF1/CIP1 and p53 in the aged rats exposed to isoflurane anesthesia. The data obtained in the present study provided evidence that the neuroprotective effects of ginsenoside Rg1 prevented the cognitive impairment induced by isoflurane anesthesia via antioxidant, anti‑inflammatory and anti‑apoptotic effects, mediated by the PI3K/AKT/GSK‑3β pathway.

  8. PTEN Overexpression Cooperates With Lithium to Reduce the Malignancy and to Increase Cell Death by Apoptosis via PI3K/Akt Suppression in Colorectal Cancer Cells.

    PubMed

    de Araujo, Wallace Martins; Robbs, Bruno Kaufmann; Bastos, Lilian G; de Souza, Waldemir F; Vidal, Flávia C B; Viola, João P B; Morgado-Diaz, Jose A

    2016-02-01

    Lithium is a well-established non-competitive inhibitor of glycogen synthase kinase-3β (GSK-3β), a kinase that is involved in several cellular processes related to cancer progression. GSK-3β is regulated upstream by PI3K/Akt, which is negatively modulated by PTEN. The role that lithium plays in cancer is controversial because lithium can activate or inhibit survival signaling pathways depending on the cell type. In this study, we analyzed the mechanisms by which lithium can modulate events related to colorectal cancer (CRC) progression and evaluated the role that survival signaling pathways such as PI3K/Akt and PTEN play in this context. We show that the administration of lithium decreased the proliferative potential of CRC cells in a GSK-3β-independent manner but induced the accumulation of cells in G2/M phase. Furthermore, high doses of lithium increased apoptosis, which was accompanied by decreased proteins levels of Akt and PTEN. Then, cells that were induced to overexpress PTEN were treated with lithium; we observed that low doses of lithium strongly increased apoptosis. Additionally, PTEN overexpression reduced proliferation, but this effect was minor compared with that in cells treated with lithium alone. Furthermore, we demonstrated that PTEN overexpression and lithium treatment separately reduced cell migration, colony formation, and invasion, and these effects were enhanced when lithium treatment and PTEN overexpression were combined. In conclusion, our findings indicate that PTEN overexpression and lithium treatment cooperate to reduce the malignancy of CRC cells and highlight lithium and PTEN as potential candidates for studies to identify new therapeutic approaches for CRC treatment.

  9. PTEN permits acute increases in D3-phosphoinositide levels following TCR stimulation but inhibits distal signaling events by reducing the basal activity of Akt.

    PubMed

    Seminario, Maria-Cristina; Precht, Patricia; Bunnell, Stephen C; Warren, Sarah E; Morris, Christa M; Taub, Dennis; Wange, Ronald L

    2004-11-01

    Phosphoinositide 3-kinase (PI3K) is important in TCR signaling. PI3K generates phosphatidylinositol 3, 4, 5-trisphosphate (PI-3,4,5-P3), which regulates membrane localization and/or activity of multiple signaling proteins. PTEN (phosphatase and tensin homologue deleted on chromosome 10) opposes PI3K, reversing this reaction. Maintaining the balance between these two enzymes is important for normal T cell function. Here we use the PTEN-null Jurkat T cell line to address the role of PTEN in modulating proximal and distal TCR-signaling events. PTEN expression at levels that restored low basal Akt phosphorylation (an indicator of PI-3,4,5-P3 levels), but which were not themselves cytotoxic, had minimal effect on TCR-stimulated activation of phospholipase Cgamma1 and Ca2+ flux, but reduced the duration of extracellular signal-regulated kinase (Erk) activation. Distal signaling events, including nuclear factor of activated T cells (NFAT) activation, CD69 expression and IL-2 production, were all inhibited by PTEN expression. Notably, PTEN did not block TCR-stimulated PI-3,4,5-P3 accumulation. The effect of PTEN on distal TCR signaling events was strongly correlated with the loss of the constitutive Akt activation and glycogen synthase kinase-3 (GSK3) inhibition that is typical of Jurkat cells, and could be reversed by expression of activated Akt or pharmacologic inhibition of GSK3. These results suggest that PTEN acts in T cells primarily to control basal PI-3,4,5-P3 levels, rather than opposing PI3K acutely during TCR stimulation.

  10. Hydrogen Sulfide Prevents Synaptic Plasticity from VD-Induced Damage via Akt/GSK-3β Pathway and Notch Signaling Pathway in Rats.

    PubMed

    Liu, Chunhua; Xu, Xiaxia; Gao, Jing; Zhang, Tao; Yang, Zhuo

    2016-08-01

    Our previous study has demonstrated that hydrogen sulfide (H2S) attenuates neuronal injury induced by vascular dementia (VD) in rats, but the mechanism is still poorly understood. In this study, we aimed to investigate whether the neuroprotection of H2S was associated with synaptic plasticity and try to interpret the potential underlying mechanisms. Adult male Wistar rats were suffered the ligation of bilateral common carotid arteries. At 24 h after surgery, rats were administered intraperitoneally with sodium hydrosulfide (NaHS, 5.6 mg·kg(-1)·day(-1)), a H2S donor, for 3 weeks in the VD+NaHS group and treated intraperitoneally with saline in the VD group respectively. Our results demonstrated that NaHS significantly decreased the level of glutamate. It obviously ameliorated cognitive flexibility as well as the spatial learning and memory abilities by Morris water maze. Moreover, NaHS significantly improved the long-term depression (LTD), and was able to elevate the expression of N-methyl-D-aspartate receptor subunit 2A, which plays a pivotal role in synaptic plasticity. Interestingly, NaHS decreased the phosphorylation of Akt, and it could maintain the activity of glycogen synthase kinase-3β (GSK-3β). Surprisingly, NaHS triggered the canonical Notch pathway by increasing expressions of Jagged-1 and Hes-1. These findings suggest that NaHS prevents synaptic plasticity from VD-induced damage partly via Akt/GSK-3β pathway and Notch signaling pathway.Hydrogen sulfide modulated the ratio of NMDAR 2A/2B and improved the synaptic plasticity via Akt/GSK-3β pathway and Notch signaling pathway in VD rats.

  11. Akt/GSK3β signaling is involved in fipronil-induced apoptotic cell death of human neuroblastoma SH-SY5Y cells.

    PubMed

    Lee, Jeong Eun; Kang, Jin Sun; Ki, Yeo-Woon; Lee, Sang-Hun; Lee, Soo-Jin; Lee, Kyung Suk; Koh, Hyun Chul

    2011-04-25

    Fipronil (FPN) is a phenylpyrazole insecticide acted on insect gamma-aminobutyric acid (GABA) receptors. Although action of FPN is restricted on insect neuronal or muscular transmitter system, a few studies have assessed the effects of this neurotoxicant on neuronal cell death. To determine the mechanisms underlying FPN-induced neuronal cell death, we investigated whether reactive oxygen species (ROS) plays a role in FPN-induced apoptosis, using an in vitro model of human dopaminergic SH-SY5Y cells. FPN was cytotoxic to these cells and its cytotoxicity showed a concentration-dependent manner. Additionally, FPN treatment significantly decreased the tyrosine hydroxylase (TH) expression without change of glutamic acid decarboxylase 65 (GAD65) expression. FPN-induced dopaminergic cell death involved in increase of ROS generation since pretreatment with N-acetyl cysteine (NAC), an anti-oxidant, reduced cell death. After FPN treatment, dopamine (DA) levels decreased significantly in both cell and culture media, and oxidative effects of DA were blocked by NAC pretreatment. We showed that cell death in response to FPN was due to apoptosis since FPN increased cytochrome c release into the cytosol and activated caspase-3. It also led to nuclear accumulation of p53 and reduced the level of Bcl-2 protein in a concentration-dependent manner. Additionally, FPN altered the level of Akt/glycogen synthase kinase-3 (GSK3β) phosphorylation. FPN reduced the Akt phosphorylation on Ser473, and in parallel with the inactivation of Akt, phosphorylation of GSK3β on Ser9 which inactivates GSK3β, decreased after treatment with FPN. Furthermore, inhibition of the GSK3β signal protected the cell against FPN-induced cell death. These results suggest that regulation of GSK3β activity may control the apoptosis induced by FPN-induced oxidative stress associated with neuronal cell death.

  12. AKT/SGK-sensitive phosphorylation of GSK3 in the regulation of L-selectin and perforin expression as well as activation induced cell death of T-lymphocytes

    SciTech Connect

    Bhavsar, Shefalee K.; Merches, Katja; Bobbala, Diwakar; Lang, Florian

    2012-08-17

    Highlights: Black-Right-Pointing-Pointer Akt/SGK dependent phosphorylation of GSK3{alpha},{beta} regulates T lymphocytes. Black-Right-Pointing-Pointer T cells from mice expressing Akt/SGK insensitive GSK3{alpha},{beta} (gsk3{sup KI}) release less IL-2. Black-Right-Pointing-Pointer CD4{sup +} cells from gsk3{sup KI} mice express less CD62L. Black-Right-Pointing-Pointer CD8{sup +} cells from gsk3{sup KI} mice are relatively resistant to activation induced cell death. Black-Right-Pointing-Pointer Perforin expression is enhanced in gsk3{sup KI} T cells. -- Abstract: Survival and function of T-lymphocytes critically depends on phosphoinositide (PI) 3 kinase. PI3 kinase signaling includes the PKB/Akt and SGK dependent phosphorylation and thus inhibition of glycogen synthase kinase GSK3{alpha},{beta}. Lithium, a known unspecific GSK3 inhibitor protects against experimental autoimmune encephalomyelitis. The present study explored, whether Akt/SGK-dependent regulation of GSK3 activity is a determinant of T cell survival and function. Experiments were performed in mutant mice in which Akt/SGK-dependent GSK3{alpha},{beta} inhibition was disrupted by replacement of the serine residue in the respective SGK/Akt-phosphorylation consensus sequence by alanine (gsk3{sup KI}). T cells from gsk3{sup KI} mice were compared to T cells from corresponding wild type mice (gsk3{sup WT}). As a result, in gsk3{sup KI} CD4{sup +} cells surface CD62L (L-selectin) was significantly less abundant than in gsk3{sup WT} CD4{sup +} cells. Upon activation in vitro T cells from gsk3{sup KI} mice reacted with enhanced perforin production and reduced activation induced cell death. Cytokine production was rather reduced in gsk3{sup KI} T cells, suggesting that GSK3 induces effector function in CD8{sup +} T cells. In conclusion, PKB/Akt and SGK sensitive phosphorylation of GSK3{alpha},{beta} is a potent regulator of perforin expression and activation induced cell death in T lymphocytes.

  13. 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.

  14. 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

  15. 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.

  16. 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.

  17. PTH-related protein upregulates integrin {alpha}6{beta}4 expression and activates Akt in breast cancer cells

    SciTech Connect

    Shen Xiaoli; Falzon, Miriam . E-mail: mfalzon@utmb.edu

    2006-11-15

    Breast cancer is the most common carcinoma that metastasizes to bone. Tumor-produced parathyroid hormone-related protein (PTHrP), a known stimulator of osteoclastic bone resorption, is a major mediator of the osteolytic process in breast cancer. We have previously shown that PTHrP increases breast cancer cell proliferation, survival, migration, and pro-invasive integrin {alpha}6{beta}4 expression. To determine the role of integrin {alpha}6{beta}4 in these PTHrP-mediated effects, we utilized two strategies to modulate expression of the {alpha}6 and {beta}4 subunits in parental and PTHrP-overexpressing MDA-MB-231 and MCF-7 cells: overexpression of {alpha}6{beta}4 by transfection with constructs encoding the {alpha}6 and {beta}4 subunits, and suppression of endogenous {alpha}6{beta}4 expression by transfection with siRNAs targeting these subunits. We now show that the effects of PTHrP are mediated via upregulation of integrin {alpha}6{beta}4 expression. We also show that integrin {alpha}6{beta}4 expression is modulated at the mRNA level, indicating a transcriptional and/or post-transcriptional mechanism of action for PTHrP. PTHrP expression also increased the levels of phosphorylated Akt, with a consequent increase in the levels of phosphorylated (inactive) glycogen synthase kinase-3 (GSK-3). The role of PTHrP in breast cancer growth and metastasis may thus be mediated via upregulation of integrin {alpha}6{beta}4 expression and Akt activation, with consequent inactivation of GSK-3.

  18. 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

  19. Effects of dexmedetomidine postconditioning on myocardial ischemia and the role of the PI3K/Akt-dependent signaling pathway in reperfusion injury

    PubMed Central

    CHENG, XIANG YANG; GU, XIAO YU; GAO, QIN; ZONG, QIAO FENG; LI, XIAO HONG; ZHANG, YE

    2016-01-01

    The present study aimed to determine whether post-ischemic treatment with dexmedetomidine (DEX) protected the heart against acute myocardial ischemia/reperfusion (I/R)-induced injury in rats. The phosphatidylinositol-3 kinase/protein kinase B(PI3K/Akt)-dependent signaling pathway was also investigated. Male Sprague Dawley rats (n=64) were subjected to ligation of the left anterior descending artery (LAD), which produced ischemia for 25 min, followed by reperfusion. Following LAD ligation, rats were treated with DEX (5, 10 and 20 µg/kg) or underwent post-ischemic conditioning, which included three cycles of ischemic insult. In order to determine the role of the PI3K/Akt signaling pathway, wortmannin (Wort), a PI3K inhibitor, was used to treat a group of rats that had also been treated with DEX (20 µg/kg). Post-reperfusion, lactate dehydrogenase (LDH), cardiac troponin I (cTnI), creatine kinase isoenzymes (CK-MB), superoxide dismutase (SOD) and malondialdehyde (MDA) serum levels were measured using an ultraviolet spectrophotometer. The protein expression levels of phosphorylated (p)-Akt, Ser9-p-glycogen synthase kinase-3β (p-GSK-3β) and cleaved caspase-3 were detected in heart tissue by western blotting. The mRNA expression levels of B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X protein (Bax) were detected using reverse transcription-polymerase chain reaction. At the end of the experiment, the hearts were removed and perfused in an isolated perfusion heart apparatus with Evans blue (1%) in order to determine the non-ischemic areas. The risk and infarct areas of the heart were not dyed. As expected, I/R induced myocardial infarction, as determined by the increased serum levels of cTnI, CK-MB and MDA, and the decreased levels of SOD. Post-ischemic treatment with DEX increased the expression levels of p-Akt and p-GSK-3β, whereas caspase-3 expression was reduced following DEX treatment compared with in the I/R group. Compared with the I/R group, the ratio of Bcl

  20. Biocatalytic role of potato starch synthase III for α-glucan biosynthesis in Synechocystis sp. PCC6803 mutants.

    PubMed

    Yoo, Sang-Ho; Lee, Byung-Hoo; Li, Li; Perris, Shayani D N; Spalding, Martin H; Han, Sang Yun; Jane, Jay-lin

    2015-11-01

    A potato starch synthase III (PSSIII) was expressed in the Synechocystis mutants deficient in either glycogen synthase I (M1) or II (M2) to replenish α-(1,4) linkage synthesizing activity, resulting in new mutants, PM1 and PM2, respectively. These mutants were applied to study the role of exogenous plant starch synthase for starch/glycogen biosynthesis mechanism established in the cyanobacteria. The remaining glycogen synthase genes in PM1 and PM2 were further disrupted to make the mutants PM12 and PM21 which contained PSSIII as the sole glycogen/starch synthase. Among wild type and mutants, there were no significant differences in the amount of α-glucan produced. All the mutants harboring active PSSIII produced α-glucans with relatively much shorter and less longer α-1,4 chains than wild-type glycogen, which was exactly in accordance with the increase in glycogen branching enzyme activity. In fact, α-glucan structure of PM1 was very similar to those of PM12 and PM21, and PM2 had more intermediate chains than M2. This result suggests PSSIII may have distributive elongation property during α-glucan synthesis. In conclusion, the Synechocystis as an expression model system of plant enzymes can be applied to determine the role of starch synthesizing enzymes and their association during α-glucan synthesis. PMID:26358554

  1. Akt isoforms in vascular disease.

    PubMed

    Yu, Haixiang; Littlewood, Trevor; Bennett, Martin

    2015-08-01

    The mammalian serine/threonine Akt kinases comprise three closely related isoforms: Akt1, Akt2 and Akt3. Akt activation has been implicated in both normal and disease processes, including in development and metabolism, as well as cancer and cardiovascular disease. Although Akt signalling has been identified as a promising therapeutic target in cancer, its role in cardiovascular disease is less clear. Importantly, accumulating evidence suggests that the three Akt isoforms exhibit distinct tissue expression profiles, localise to different subcellular compartments, and have unique modes of activation. Consistent with in vitro findings, genetic studies in mice show distinct effects of individual Akt isoforms on the pathophysiology of cardiovascular disease. This review summarises recent studies of individual Akt isoforms in atherosclerosis, vascular remodelling and aneurysm formation, to provide a comprehensive overview of Akt function in vascular disease.

  2. Akt isoforms in vascular disease

    PubMed Central

    Yu, Haixiang; Littlewood, Trevor; Bennett, Martin

    2015-01-01

    The mammalian serine/threonine Akt kinases comprise three closely related isoforms: Akt1, Akt2 and Akt3. Akt activation has been implicated in both normal and disease processes, including in development and metabolism, as well as cancer and cardiovascular disease. Although Akt signalling has been identified as a promising therapeutic target in cancer, its role in cardiovascular disease is less clear. Importantly, accumulating evidence suggests that the three Akt isoforms exhibit distinct tissue expression profiles, localise to different subcellular compartments, and have unique modes of activation. Consistent with in vitro findings, genetic studies in mice show distinct effects of individual Akt isoforms on the pathophysiology of cardiovascular disease. This review summarises recent studies of individual Akt isoforms in atherosclerosis, vascular remodelling and aneurysm formation, to provide a comprehensive overview of Akt function in vascular disease. PMID:25929188

  3. Pseudouridine synthases.

    PubMed

    Hamma, Tomoko; Ferré-D'Amaré, Adrian R

    2006-11-01

    Pseudouridine synthases are the enzymes responsible for the most abundant posttranscriptional modification of cellular RNAs. These enzymes catalyze the site-specific isomerization of uridine residues that are already part of an RNA chain, and appear to employ both sequence and structural information to achieve site specificity. Crystallographic analyses have demonstrated that all pseudouridine synthases share a common core fold and active site structure and that this core is modified by peripheral domains, accessory proteins, and guide RNAs to give rise to remarkable substrate versatility.

  4. 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 ...

  5. Selective inhibition of PTEN preserves ischaemic post-conditioning cardioprotection in STZ-induced Type 1 diabetic rats: role of the PI3K/Akt and JAK2/STAT3 pathways.

    PubMed

    Xue, Rui; Lei, Shaoqing; Xia, Zhong-yuan; Wu, Yang; Meng, Qingtao; Zhan, Liying; Su, Wating; Liu, Huimin; Xu, Jinjin; Liu, Zhenzhen; Zhou, Bin; Xia, Zhengyuan

    2016-03-01

    Patients with diabetes are vulnerable to MI/R (myocardial ischaemia/reperfusion) injury, but are not responsive to IPostC (ischaemic post-conditioning) which activates PI3K (phosphoinositide 3-kinase)/Akt (also known as PKB or protein kinase B) and JAK2 (Janus kinase 2)/STAT3 (signal transducer and activator of transcription 3) pathways to confer cardioprotection. We hypothesized that increased cardiac PTEN (phosphatase and tensin homologue deleted on chromosome 10), a major negative regulator of PI3K/Akt, is responsible for the loss of diabetic heart sensitivity to IPostC cardioprotecton. In STZ (streptozotocin)-induced Type 1 diabetic rats subjected to MI/R (30 min coronary occlusion and 120 min reperfusion), the post-ischaemic myocardial infarct size, CK-MB (creatine kinase-MB) and 15-F2t-isoprostane release, as well as cardiac PTEN expression were significantly higher than those in non-diabetic controls, concomitant with more severe cardiac dysfunction and lower cardiac Akt, STAT3 and GSK-3β (glycogen synthase kinase 3β) phosphorylation. IPostC significantly attenuated post-ischaemic infarct size, decreased PTEN expression and further increased Akt, STAT3 and GSK-3β phosphorylation in non-diabetic, but not in diabetic rats. Application of the PTEN inhibitor BpV (bisperoxovanadium) (1.0 mg/kg) restored IPostC cardioprotection in diabetic rats. HPostC (hypoxic post-conditioning) in combination with PTEN gene knockdown, but not HPostC alone, significantly reduced H/R (hypoxia/reoxygenation) injury in cardiac H9c2 cells exposed to high glucose as was evident from reduced apoptotic cell death and JC-1 monomer in cells, accompanied by increased phosphorylation of Akt, STAT3 and GSK-3β. PTEN inhibition/gene knockdown mediated restoration of IPostC/HPostC cardioprotection was completely reversed by the PI3K inhibitor wortmannin, and partially reversed by the JAK2 inhibitor AG490. Increased cardiac PTEN, by impairing PI3K/Akt and JAK2/STAT3 pathways, is a major

  6. 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

  7. 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.

  8. 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.

  9. 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.

  10. A Genome-wide Screen for Neurospora crassa Transcription Factors Regulating Glycogen Metabolism*

    PubMed Central

    Gonçalves, Rodrigo Duarte; Cupertino, Fernanda Barbosa; Freitas, Fernanda Zanolli; Luchessi, Augusto Ducati; Bertolini, Maria Célia

    2011-01-01

    Transcription factors play a key role in transcription regulation as they recognize and directly bind to defined sites in promoter regions of target genes, and thus modulate differential expression. The overall process is extremely dynamic, as they have to move through the nucleus and transiently bind to chromatin in order to regulate gene transcription. To identify transcription factors that affect glycogen accumulation in Neurospora crassa, we performed a systematic screen of a deletion strains set generated by the Neurospora Knockout Project and available at the Fungal Genetics Stock Center. In a wild-type strain of N. crassa, glycogen content reaches a maximal level at the end of the exponential growth phase, but upon heat stress the glycogen content rapidly drops. The gene encoding glycogen synthase (gsn) is transcriptionally down-regulated when the mycelium is exposed to the same stress condition. We identified 17 deleted strains having glycogen accumulation profiles different from that of the wild-type strain under both normal growth and heat stress conditions. Most of the transcription factors identified were annotated as hypothetical protein, however some of them, such as the PacC, XlnR, and NIT2 proteins, were biochemically well-characterized either in N. crassa or in other fungi. The identification of some of the transcription factors was coincident with the presence of DNA-binding motifs specific for the transcription factors in the gsn 5′-flanking region, and some of these DNA-binding motifs were demonstrated to be functional by Electrophoretic Mobility Shift Assay (EMSA) experiments. Strains knocked-out in these transcription factors presented impairment in the regulation of gsn expression, suggesting that the transcription factors regulate glycogen accumulation by directly regulating gsn gene expression. Five selected mutant strains showed defects in cell cycle progression, and two transcription factors were light-regulated. The results indicate

  11. 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

  12. 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).

  13. 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

  14. 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

  15. 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.

  16. Proteomic Identification of 14-3-3ζ as an Adapter for IGF-1 and Akt/GSK-3β Signaling and Survival of Renal Mesangial Cells

    PubMed Central

    Singh, Lalit P.; Jiang, Yan; Cheng, Davis W.

    2007-01-01

    Recently we demonstrated that IGF-1 expression is increased in the diabetic kidney and that it may involve in renal hypertrophy and extracellular matrix protein (ECM) accumulation in mesangial cells as seen in diabetic glomerulopathy. The present study investigates the molecular mechanism(s) of IGF-1 and Akt/glycogen synthase kinase-3beta (GSK-3β) signaling pathway in the regulation of fibronectin and cyclin D1 expression and survival of renal mesangial cells. A proteomic approach is also employed to identify protein targets of IGF-1 signaling via GSK-3β inhibition in mesangial cells. We show that IGF-1 (100 ng/ml) significantly increases the protein kinase Akt/PKB activity (1.5-2-fold, p<0.05) within 1-5 minutes, which is completely blocked by the presence of 100 nM Wortmannin (phosphatidyl-inositol 3-kinase inhibitor). Akt activation is coupled with Ser9 phosphorylation and inactivation of its down-stream target GSK-3β. IGF-1 increases the cyclic AMP-responsive element (CRE) binding transcription factor CREB phosphorylation at Ser 133 and CRE-binding activity in mesangial cells, which parallels cyclin D1 and fibronectin expressions. Both proteins are known to have CRE-sequences in their promoter regions upstream of the transcription start site. Suppression of GSK-3β by SB216763 (100 nM) increases CREB phosphorylation, cyclin D1 and fibronectin levels. Two dimensional gel electrophoresis followed by MALDI-TOF mass spectrometric analysis of mesangial proteins reveals that IGF-1 treatment or an inhibition of GSK-3β increases the expression of the phosphorylated Ser/Thr binding signal adapter protein 14-3-3ζ. Immuno-precipitation of 14-3-3ζ followed by Western blotting validates the association of phosphorylated GSK-3β with 14-3-3ζ in renal mesangial cells. Stable expression of a constitutively active GSK-3β(Ser9Ala) induces cell death while overexpression of HA-tagged 14-3-3ζ increases cell viability as measured by MTT assays. These results indicate that

  17. 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.

  18. 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.

  19. Hydrogen sulfide preconditioning protects rat liver against ischemia/reperfusion injury by activating Akt-GSK-3β signaling and inhibiting mitochondrial permeability transition.

    PubMed

    Zhang, Qingqing; Fu, Hailong; Zhang, Hao; Xu, Fengying; Zou, Zui; Liu, Meng; Wang, Quanxing; Miao, Mingyong; Shi, Xueyin

    2013-01-01

    Hydrogen sulfide (H2S) is the third most common endogenously produced gaseous signaling molecule, but its impact on hepatic ischemia/reperfusion (I/R) injury, especially on mitochondrial function, remains unclear. In this study, rats were randomized into Sham, I/R, ischemia preconditioning (IPC) or sodium hydrosulfide (NaHS, an H2S donor) preconditioning groups. To establish a model of segmental (70%) warm hepatic ischemia, the hepatic artery, left portal vein and median liver lobes were occluded for 60 min and then unclamped to allow reperfusion. Preconditioning with 12.5, 25 or 50 μmol/kg NaHS prior to the I/R insult significantly increased serum H2S levels, and, similar to IPC, NaHS preconditioning decreased alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in the plasma and prevented hepatocytes from undergoing I/R-induced necrosis. Moreover, a sub-toxic dose of NaHS (25 μmol/kg) did not disrupt the systemic hemodynamics but dramatically inhibited mitochondrial permeability transition pore (MPTP) opening and thus prevented mitochondrial-related cell death and apoptosis. Mechanistic studies revealed that NaHS preconditioning markedly increased the expression of phosphorylated protein kinase B (p-Akt), phosphorylated glycogen synthase kinase-3 beta (p-GSK-3β) and B-cell lymphoma-2 (Bcl-2) and decreased the release of mitochondrial cytochrome c and cleaved caspase-3/9 levels. Therefore, NaHS administration prior to hepatic I/R ameliorates mitochondrial and hepatocellular damage through the inhibition of MPTP opening and the activation of Akt-GSK-3β signaling. Furthermore, this study provides experimental evidence for the clinical use of H2S to reduce liver damage after perioperative I/R injury. PMID:24058562

  20. Hydrogen Sulfide Preconditioning Protects Rat Liver against Ischemia/Reperfusion Injury by Activating Akt-GSK-3β Signaling and Inhibiting Mitochondrial Permeability Transition

    PubMed Central

    Zhang, Hao; Xu, Fengying; Zou, Zui; Liu, Meng; Wang, Quanxing; Miao, Mingyong; Shi, Xueyin

    2013-01-01

    Hydrogen sulfide (H2S) is the third most common endogenously produced gaseous signaling molecule, but its impact on hepatic ischemia/reperfusion (I/R) injury, especially on mitochondrial function, remains unclear. In this study, rats were randomized into Sham, I/R, ischemia preconditioning (IPC) or sodium hydrosulfide (NaHS, an H2S donor) preconditioning groups. To establish a model of segmental (70%) warm hepatic ischemia, the hepatic artery, left portal vein and median liver lobes were occluded for 60 min and then unclamped to allow reperfusion. Preconditioning with 12.5, 25 or 50 μmol/kg NaHS prior to the I/R insult significantly increased serum H2S levels, and, similar to IPC, NaHS preconditioning decreased alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in the plasma and prevented hepatocytes from undergoing I/R-induced necrosis. Moreover, a sub-toxic dose of NaHS (25 μmol/kg) did not disrupt the systemic hemodynamics but dramatically inhibited mitochondrial permeability transition pore (MPTP) opening and thus prevented mitochondrial-related cell death and apoptosis. Mechanistic studies revealed that NaHS preconditioning markedly increased the expression of phosphorylated protein kinase B (p-Akt), phosphorylated glycogen synthase kinase-3 beta (p-GSK-3β) and B-cell lymphoma-2 (Bcl-2) and decreased the release of mitochondrial cytochrome c and cleaved caspase-3/9 levels. Therefore, NaHS administration prior to hepatic I/R ameliorates mitochondrial and hepatocellular damage through the inhibition of MPTP opening and the activation of Akt-GSK-3β signaling. Furthermore, this study provides experimental evidence for the clinical use of H2S to reduce liver damage after perioperative I/R injury. PMID:24058562

  1. HDAC inhibition prevents white matter injury by modulating microglia/macrophage polarization through the GSK3β/PTEN/Akt axis.

    PubMed

    Wang, Guohua; Shi, Yejie; Jiang, Xiaoyan; Leak, Rehana K; Hu, Xiaoming; Wu, Yun; Pu, Hongjian; Li, Wei-Wei; Tang, Bo; Wang, Yun; Gao, Yanqin; Zheng, Ping; Bennett, Michael V L; Chen, Jun

    2015-03-01

    Severe traumatic brain injury (TBI) elicits destruction of both gray and white matter, which is exacerbated by secondary proinflammatory responses. Although white matter injury (WMI) is strongly correlated with poor neurological status, the maintenance of white matter integrity is poorly understood, and no current therapies protect both gray and white matter. One candidate approach that may fulfill this role is inhibition of class I/II histone deacetylases (HDACs). Here we demonstrate that the HDAC inhibitor Scriptaid protects white matter up to 35 d after TBI, as shown by reductions in abnormally dephosphorylated neurofilament protein, increases in myelin basic protein, anatomic preservation of myelinated axons, and improved nerve conduction. Furthermore, Scriptaid shifted microglia/macrophage polarization toward the protective M2 phenotype and mitigated inflammation. In primary cocultures of microglia and oligodendrocytes, Scriptaid increased expression of microglial glycogen synthase kinase 3 beta (GSK3β), which phosphorylated and inactivated phosphatase and tensin homologue (PTEN), thereby enhancing phosphatidylinositide 3-kinases (PI3K)/Akt signaling and polarizing microglia toward M2. The increase in GSK3β in microglia and their phenotypic switch to M2 was associated with increased preservation of neighboring oligodendrocytes. These findings are consistent with recent findings that microglial phenotypic switching modulates white matter repair and axonal remyelination and highlight a previously unexplored role for HDAC activity in this process. Furthermore, the functions of GSK3β may be more subtle than previously thought, in that GSK3β can modulate microglial functions via the PTEN/PI3K/Akt signaling pathway and preserve white matter homeostasis. Thus, inhibition of HDACs in microglia is a potential future therapy in TBI and other neurological conditions with white matter destruction. PMID:25691750

  2. Cross-talk between the two divergent insulin signaling pathways is revealed by the protein kinase B (Akt)-mediated phosphorylation of adapter protein APS on serine 588.

    PubMed

    Katsanakis, Kostas D; Pillay, Tahir S

    2005-11-11

    The APS adapter protein is recruited to the autophosphorylated kinase domain of the insulin receptor and initiates the phosphatidylinositol 3-kinase (PI3K)-independent pathway of insulin-stimulated glucose transport by recruiting CAP and c-Cbl. In this study, we have identified APS as a novel substrate for protein kinase B/Akt using an antibody that exhibits insulin-dependent immunoreactivity with a phosphospecific antibody raised against the protein kinase B substrate consensus sequence RXRXX(pS/pT) and a phosphospecific antibody that recognizes serine 21/9 of glycogen synthase kinase-3alpha/beta. This phosphorylation of APS is observed in both 3T3-L1 adipocytes and transfected cells. The insulin-stimulated serine phosphorylation of APS was inhibited by a PI3-kinase inhibitor, LY290004, a specific protein kinase B (PKB) inhibitor, deguelin, and knockdown of Akt. Serine 588 of APS is contained in a protein kinase B consensus sequence for phosphorylation conserved in APS across multiple species but not found in other members of this family, including SH2-B and Lnk. Mutation of serine 588 to alanine abolished the insulin-stimulated serine phosphorylation of APS and prevented the localization of APS to membrane ruffles. A glutathione S-transferase fusion protein containing amino acids 534-621 of APS was phosphorylated by purified PKB in vitro, and mutation of serine 588 abolished the PKB-mediated phosphorylation of APS in vitro. Taken together, this study identifies APS as a novel physiological substrate for PKB and the first serine phosphorylation site on APS. These data therefore reveal the molecular cross-talk between the insulin-activated PI3-kinase-dependent and -independent pathways previously thought to be distinct and divergent.

  3. 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

  4. 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

  5. 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)

  6. 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

  7. 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.

  8. 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.

  9. 5-AIQ inhibits H{sub 2}O{sub 2}-induced apoptosis through reactive oxygen species scavenging and Akt/GSK-3β signaling pathway in H9c2 cardiomyocytes

    SciTech Connect

    Park, Eun-Seok; Kang, Jun Chul; Kang, Do-Hyun; Jang, Yong Chang; Yi, Kyu Yang; Chung, Hun-Jong; Park, Jong Seok; Kim, Bokyung; Feng, Zhong-Ping; Shin, Hwa-Sup

    2013-04-01

    Poly(adenosine 5′-diphosphate ribose) polymerase (PARP) is a nuclear enzyme activated by DNA strand breaks and plays an important role in the tissue injury associated with ischemia and reperfusion. The aim of the present study was to investigate the protective effect of 5-aminoisoquinolinone (5-AIQ), a PARP inhibitor, against oxidative stress-induced apoptosis in H9c2 cardiomyocytes. 5-AIQ pretreatment significantly protected against H{sub 2}O{sub 2}-induced cell death, as determined by the XTT assay, cell counting, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay, and Western blot analysis of apoptosis-related proteins such as caspase-3, Bax, and Bcl-2. Upregulation of antioxidant enzymes such as manganese superoxide dismutase and catalase accompanied the protective effect of 5-AIQ on H{sub 2}O{sub 2}-induced cell death. Our data also showed that 5-AIQ pretreatment protected H9c2 cells from H{sub 2}O{sub 2}-induced apoptosis by triggering activation of Akt and glycogen synthase kinase-3β (GSK-3β), and that the protective effect of 5-AIQ was diminished by the PI3K inhibitor LY294002 at a concentration that effectively abolished 5-AIQ-induced Akt and GSK-3β activation. In addition, inhibiting the Akt/GSK-3β pathway by LY294002 significantly attenuated the 5-AIQ-mediated decrease in cleaved caspase-3 and Bax activation and H9c2 cell apoptosis induction. Taken together, these results demonstrate that 5-AIQ prevents H{sub 2}O{sub 2}-induced apoptosis in H9c2 cells by reducing intracellular reactive oxygen species production, regulating apoptosis-related proteins, and activating the Akt/GSK-3β pathway. - Highlights: ► 5-AIQ, a PARP inhibitor, decreased H{sub 2}O{sub 2}-induced H9c2 cell death and apoptosis. ► 5-AIQ upregulated antioxidant Mn-SOD and catalase, while decreasing ROS production. ► 5-AIQ decreased H{sub 2}O{sub 2}-induced increase in cleaved caspase-3 and Bax and decrease in Bcl2. ► 5-AIQ activated Akt and GSK-3

  10. 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

  11. 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

  12. 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

  13. 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

  14. 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.

  15. 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.

  16. 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.

  17. 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

  18. 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.

  19. 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.

  20. Molecular mechanisms of environmental enrichment: impairments in Akt/GSK3β, neurotrophin-3 and CREB signaling.

    PubMed

    Hu, Yuan-Shih; Long, Nancy; Pigino, Gustavo; Brady, Scott T; Lazarov, Orly

    2013-01-01

    Experience of mice in a complex environment enhances neurogenesis and synaptic plasticity in the hippocampus of wild type and transgenic mice harboring familial Alzheimer's disease (FAD)-linked APPswe/PS1ΔE9. In FAD mice, this experience also reduces levels of tau hyperphosphorylation and oligomeric β-amyloid. Although environmental enrichment has significant effects on brain plasticity and neuropathology, the molecular mechanisms underlying these effects are unknown. Here we show that environmental enrichment upregulates the Akt pathway, leading to the downregulation of glycogen synthase kinase 3β (GSK3β), in wild type but not FAD mice. Several neurotrophic signaling pathways are activated in the hippocampus of both wild type and FAD mice, including brain derived neurotrophic factor (BDNF) and nerve growth factor (NGF), and this increase is accompanied by the upregulation of the BDNF receptor, tyrosine kinase B (TrkB). Interestingly, neurotrophin-3 (NT-3) is upregulated in the brains of wild type mice but not FAD mice, while insulin growth factor-1 (IGF-1) is upregulated exclusively in the brains of FAD mice. Upregulation of neurotrophins is accompanied by the increase of N-Methyl-D-aspartic acid (NMDA) receptors in the hippocampus following environmental enrichment. Most importantly, we observed a significant increase in levels of cAMP response element- binding (CREB) transcripts in the hippocampus of wild type and FAD mice following environmental enrichment. However, CREB phosphorylation, a critical step for the initiation of learning and memory-required gene transcription, takes place in the hippocampus of wild type but not of FAD mice. These results suggest that experience of wild type mice in a complex environmental upregulates critical signaling that play a major role in learning and memory in the hippocampus. However, in FAD mice, some of these pathways are impaired and cannot be rescued by environmental enrichment.

  1. Molecular Mechanisms of Environmental Enrichment: Impairments in Akt/GSK3β, Neurotrophin-3 and CREB Signaling

    PubMed Central

    Hu, Yuan-Shih; Long, Nancy; Pigino, Gustavo; Brady, Scott T.; Lazarov, Orly

    2013-01-01

    Experience of mice in a complex environment enhances neurogenesis and synaptic plasticity in the hippocampus of wild type and transgenic mice harboring familial Alzheimer's disease (FAD)-linked APPswe/PS1ΔE9. In FAD mice, this experience also reduces levels of tau hyperphosphorylation and oligomeric β-amyloid. Although environmental enrichment has significant effects on brain plasticity and neuropathology, the molecular mechanisms underlying these effects are unknown. Here we show that environmental enrichment upregulates the Akt pathway, leading to the downregulation of glycogen synthase kinase 3β (GSK3β), in wild type but not FAD mice. Several neurotrophic signaling pathways are activated in the hippocampus of both wild type and FAD mice, including brain derived neurotrophic factor (BDNF) and nerve growth factor (NGF), and this increase is accompanied by the upregulation of the BDNF receptor, tyrosine kinase B (TrkB). Interestingly, neurotrophin-3 (NT-3) is upregulated in the brains of wild type mice but not FAD mice, while insulin growth factor-1 (IGF-1) is upregulated exclusively in the brains of FAD mice. Upregulation of neurotrophins is accompanied by the increase of N-Methyl-D-aspartic acid (NMDA) receptors in the hippocampus following environmental enrichment. Most importantly, we observed a significant increase in levels of cAMP response element- binding (CREB) transcripts in the hippocampus of wild type and FAD mice following environmental enrichment. However, CREB phosphorylation, a critical step for the initiation of learning and memory-required gene transcription, takes place in the hippocampus of wild type but not of FAD mice. These results suggest that experience of wild type mice in a complex environmental upregulates critical signaling that play a major role in learning and memory in the hippocampus. However, in FAD mice, some of these pathways are impaired and cannot be rescued by environmental enrichment. PMID:23700479

  2. 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.

  3. Apelin ameliorates TNF-α-induced reduction of glycogen synthesis in the hepatocytes through G protein-coupled receptor APJ.

    PubMed

    Chu, Jiaojiao; Zhang, Hangxiang; Huang, Xiuqing; Lin, Yajun; Shen, Tao; Chen, Beidong; Man, Yong; Wang, Shu; Li, Jian

    2013-01-01

    Apelin, a novel adipokine, is the specific endogenous ligand of G protein-coupled receptor APJ. Consistent with its putative role as an adipokine, apelin has been linked to states of insulin resistance. However, the function of apelin in hepatic insulin resistance, a vital part of insulin resistance, and its underlying mechanisms still remains unclear. Here we define the impacts of apelin on TNF-α-induced reduction of glycogen synthesis in the hepatocytes. Our studies indicate that apelin reversed TNF-α-induced reduction of glycogen synthesis in HepG2 cells, mouse primary hepatocytes and liver tissues of C57BL/6J mice by improving JNK-IRS1-AKT-GSK pathway. Moreover, Western blot revealed that APJ, but not apelin, expressed in the hepatocytes and liver tissues of mice. We found that F13A, a competitive antagonist for G protein-coupled receptor APJ, suppressed the effects of apelin on TNF-α-induced reduction of glycogen synthesis in the hepatocytes, suggesting APJ is involved in the function of apelin. In conclusion, we show novel evidence suggesting that apelin ameliorates TNF-α-induced reduction of glycogen synthesis in the hepatocytes through G protein-coupled receptor APJ. Apelin appears as a beneficial adipokine with anti-insulin resistance properties, and thus as a promising therapeutic target in metabolic disorders.

  4. 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.

  5. 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 ...

  6. 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 ...

  7. 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 ...

  8. 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

  9. 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

  10. Activation of the PI3K-Akt pathway by human T cell leukemia virus type 1 (HTLV-1) oncoprotein Tax increases Bcl3 expression, which is associated with enhanced growth of HTLV-1-infected T cells

    SciTech Connect

    Saito, Kousuke; Saito, Mineki; Taniura, Naoko; Okuwa, Takako; Ohara, Yoshiro

    2010-08-01

    Bcl3 is a member of the I{kappa}B family that regulates genes involved in cell proliferation and apoptosis. Recent reports indicated that Bcl3 is overexpressed in HTLV-1-infected T cells via Tax-mediated transactivation, and acts as a negative regulator of viral transcription. However, the role of Bcl3 in cellular signal transduction and the growth of HTLV-1-infected T cells have not been reported. In this study, we showed that the knockdown of Bcl3 by short hairpin RNA inhibited the growth of HTLV-1-infected T cells. Although phosphatidylinositol-3 kinase (PI3K) inhibitor reduced Bcl3 expression, inactivation of glycogen synthase kinase 3 (GSK3), an effector kinase of the PI3K/Akt signaling pathway, restored Bcl3 expression in Tax-negative but not in Tax-positive T cells. Our results indicate that the overexpression of Bcl3 in HTLV-1-infected T cells is regulated not only by transcriptional but also by post-transcriptional mechanisms, and is involved in overgrowth of HTLV-1-infected T cells.

  11. The Akt1-eNOS axis illustrates the specificity of kinase-substrate relationships in vivo.

    PubMed

    Schleicher, Michael; Yu, Jun; Murata, Takahisa; Derakhshan, Berhad; Atochin, Dimitriy; Qian, Li; Kashiwagi, Satoshi; Di Lorenzo, Annarita; Harrison, Kenneth D; Huang, Paul L; Sessa, William C

    2009-01-01

    Akt1 is critical for many in vivo functions; however, the cell-specific substrates responsible remain to be defined. Here, we examine the importance of endothelial nitric oxide synthase (eNOS) as an Akt1 substrate by generating Akt1-deficient mice (Akt1(-/-) mice) carrying knock-in mutations (serine to aspartate or serine to alanine substitutions) of the critical Akt1 phosphorylation site on eNOS (serine 1176) that render the enzyme "constitutively active" or "less active." The eNOS mutations did not influence several phenotypes in Akt1(-/-) mice; however, the defective postnatal angiogenesis characteristic of Akt1(-/-) mice was rescued by crossing the Akt1(-/-) mice with mice carrying the constitutively active form of eNOS, but not by crossing with mice carrying the less active eNOS mutant. This genetic rescue resulted in the stabilization of hypoxia-inducible factor 1alpha (HIF-1alpha) and increased production of HIF-1alpha-responsive genes in vivo and in vitro. Thus, Akt1 regulates angiogenesis largely through phosphorylation of eNOS and NO-dependent signaling. PMID:19654415

  12. The Akt1-eNOS axis illustrates the specificity of kinase-substrate relationships in vivo.

    PubMed

    Schleicher, Michael; Yu, Jun; Murata, Takahisa; Derakhshan, Berhad; Atochin, Dimitriy; Qian, Li; Kashiwagi, Satoshi; Di Lorenzo, Annarita; Harrison, Kenneth D; Huang, Paul L; Sessa, William C

    2009-08-04

    Akt1 is critical for many in vivo functions; however, the cell-specific substrates responsible remain to be defined. Here, we examine the importance of endothelial nitric oxide synthase (eNOS) as an Akt1 substrate by generating Akt1-deficient mice (Akt1(-/-) mice) carrying knock-in mutations (serine to aspartate or serine to alanine substitutions) of the critical Akt1 phosphorylation site on eNOS (serine 1176) that render the enzyme "constitutively active" or "less active." The eNOS mutations did not influence several phenotypes in Akt1(-/-) mice; however, the defective postnatal angiogenesis characteristic of Akt1(-/-) mice was rescued by crossing the Akt1(-/-) mice with mice carrying the constitutively active form of eNOS, but not by crossing with mice carrying the less active eNOS mutant. This genetic rescue resulted in the stabilization of hypoxia-inducible factor 1alpha (HIF-1alpha) and increased production of HIF-1alpha-responsive genes in vivo and in vitro. Thus, Akt1 regulates angiogenesis largely through phosphorylation of eNOS and NO-dependent signaling.

  13. 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

  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. Effect of carbohydrate-protein supplementation postexercise on rat muscle glycogen synthesis and phosphorylation of proteins controlling glucose storage.

    PubMed

    Hara, Daisuke; Morrison, Paul J; Ding, Zhenping; Ivy, John L

    2011-10-01

    To examine whether addition of protein to a carbohydrate supplement enhances muscle glycogen synthesis, we compared the muscle glycogen concentrations of rats that had been depleted of their muscle glycogen stores with a 3-hour swim and immediately supplemented with a placebo (Con), carbohydrate (CHO), or carbohydrate plus protein supplement (C+P). Rats were given either 0.9 g carbohydrate per kilogram body mass for the CHO group or 0.9 g carbohydrate + 0.3 g protein per kilogram body mass for the C+P groups. Muscle samples of the red and white quadriceps were excised immediately, 30 minutes, or 90 minutes postexercise. Glycogen concentration of the C+P group was greater than that of the CHO group at 90 minutes postexercise in both red (C+P, 28.3 ± 2.6 µmol/g vs CHO, 22.4 ± 2.0 µmol/g; P < .05) and white (C+P, 24.9 ± 2.4 µmol/g vs CHO, 17.64 ± 1.5 µmol/g; P < .01) quadriceps. Protein kinase B phosphorylation was greater in the C+P-30 group (the number following treatment group abbreviation refers to time [in minutes] of euthanasia following exercise) than the sedentary control and exercised control groups in red quadriceps at 30 minutes and in white quadriceps at 90 minutes postexercise. This difference was not observed in the CHO group. Phosphorylation of glycogen synthase was significantly reduced 30 minutes postexercise and returned to baseline levels by 90 minutes postexercise in both CHO- and C+P-supplemented groups, with no difference between supplements. These results demonstrated that the addition of protein to a carbohydrate supplement will enhance the rate of muscle glycogen restoration postexercise and may involve facilitation of the glucose transport process.

  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. Co-activation of AKT and c-Met triggers rapid hepatocellular carcinoma development via the mTORC1/FASN pathway in mice

    PubMed Central

    Hu, Junjie; Che, Li; Li, Lei; Pilo, Maria G.; Cigliano, Antonio; Ribback, Silvia; Li, Xiaolei; Latte, Gavinella; Mela, Marta; Evert, Matthias; Dombrowski, Frank; Zheng, Guohua; Chen, Xin; Calvisi, Diego F.

    2016-01-01

    Activation of the AKT/mTOR cascade and overexpression of c-Met have been implicated in the development of human hepatocellular carcinoma (HCC). To elucidate the functional crosstalk between the two pathways, we generated a model characterized by the combined expression of activated AKT and c-Met in the mouse liver. Co-expression of AKT and c-Met triggered rapid liver tumor development and mice required to be euthanized within 8 weeks after hydrodynamic injection. At the molecular level, liver tumors induced by AKT/c-Met display activation of AKT/mTOR and Ras/MAPK cascades as well as increased lipogenesis and glycolysis. Since a remarkable lipogenic phenotype characterizes liver lesions from AKT/c-Met mice, we determined the requirement of lipogenesis in AKT/c-Met driven hepatocarcinogenesis using conditional Fatty Acid Synthase (FASN) knockout mice. Of note, hepatocarcinogenesis induced by AKT/c-Met was fully inhibited by FASN ablation. In human HCC samples, coordinated expression of FASN, activated AKT, and c-Met proteins was detected in a subgroup of biologically aggressive tumors. Altogether, our study demonstrates that co-activation of AKT and c-Met induces HCC development that depends on the mTORC1/FASN pathway. Suppression of mTORC1 and/or FASN might be highly detrimental for the growth of human HCC subsets characterized by concomitant induction of the AKT and c-Met cascades. PMID:26857837

  18. 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.

  19. 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.

  20. 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.

  1. MYOCARDIAL AKT: THE OMNIPRESENT NEXUS

    PubMed Central

    Sussman, Mark A.; Völkers, Mirko; Fischer, Kimberlee; Bailey, Brandi; Cottage, Christopher T.; Din, Shabana; Gude, Natalie; Avitabile, Daniele; Alvarez, Roberto; Sundararaman, Balaji; Quijada, Pearl; Mason, Matt; Konstandin, Mathias H.; Malhowski, Amy; Cheng, Zhaokang; Khan, Mohsin; McGregor, Michael

    2013-01-01

    One of the greatest examples of integrated signal transduction is revealed by examination of effects mediated by AKT kinase in myocardial biology. Positioned at the intersection of multiple afferent and efferent signals, AKT exemplifies a molecular sensing node that coordinates dynamic responses of the cell in literally every aspect of biological responses. The balanced and nuanced nature of homeostatic signaling is particularly essential within the myocardial context, where regulation of survival, energy production, contractility, and response to pathological stress all flow through the nexus of AKT activation or repression. Equally important, the loss of regulated AKT activity is primarily the cause or consequence of pathological conditions leading to remodeling of the heart and eventual decompensation. This review presents an overview compendium of the complex world of myocardial AKT biology gleaned from more than a decade of research. Summarization of the widespread influence that AKT exerts upon myocardial responses leaves no doubt that the participation of AKT in molecular signaling will need to be reckoned with as a seemingly omnipresent regulator of myocardial molecular biological responses. PMID:21742795

  2. Loss of the starvation-induced gene Rack1 leads to glycogen deficiency and impaired autophagic responses in Drosophila

    PubMed Central

    Érdi, Balázs; Nagy, Péter; Zvara, Ágnes; Varga, Ágnes; Pircs, Karolina; Ménesi, Dalma; Puskás, László G.; Juhász, Gábor

    2012-01-01

    Autophagy delivers cytoplasmic material for lysosomal degradation in eukaryotic cells. Starvation induces high levels of autophagy to promote survival in the lack of nutrients. We compared genome-wide transcriptional profiles of fed and starved control, autophagy-deficient Atg7 and Atg1 null mutant Drosophila larvae to search for novel regulators of autophagy. Genes involved in catabolic processes including autophagy were transcriptionally upregulated in all cases. We also detected repression of genes involved in DNA replication in autophagy mutants compared with control animals. The expression of Rack1 (receptor of activated protein kinase C 1) increased 4.1- to 5.5-fold during nutrient deprivation in all three genotypes. The scaffold protein Rack1 plays a role in a wide range of processes including translation, cell adhesion and migration, cell survival and cancer. Loss of Rack1 led to attenuated autophagic response to starvation, and glycogen stores were decreased 11.8-fold in Rack1 mutant cells. Endogenous Rack1 partially colocalized with GFP-Atg8a and early autophagic structures on the ultrastructural level, suggesting its involvement in autophagosome formation. Endogenous Rack1 also showed a high degree of colocalization with glycogen particles in the larval fat body, and with Shaggy, the Drosophila homolog of glycogen synthase kinase 3B (GSK-3B). Our results, for the first time, demonstrated the fundamental role of Rack1 in autophagy and glycogen synthesis. PMID:22562043

  3. 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

  4. 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

  5. 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.

  6. 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

  7. 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.

  8. Stilbene Synthase and Chalcone Synthase 1

    PubMed Central

    Rolfs, Claus-Henning; Kindl, Helmut

    1984-01-01

    Cultured cells of Picea excelsa capable of forming stilbenes and flavanoids have been established. Unlike needles of intact plants containing piceatannol (3,3′,4′,5-tetrahydroxystilbene) and stilbene glycosides the cultured cells converted phenylalanine and p-coumaric acid primarily into resveratrol monomethyl ether (3,4′-dihydroxy-5-methoxystilbene) and naringenin. Partially purified enzyme preparations were assayed for chalcone synthase as well as for stilbene synthase activity converting malonyl-CoA plus p-coumaroyl-CoA into 3,4′,5-trihydroxystilbene (resveratrol). Although stilbene synthase and chalcone synthase use the same substrates and exhibit similar molecular properties, i.e. molecular weight and subunit molecular weight, they are two different proteins. This difference was demonstrated by gel electrophoresis and by means of monospecific antibodies. PMID:16663649

  9. 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.

  10. Protein kinase B/Akt activates c-Jun NH(2)-terminal kinase by increasing NO production in response to shear stress

    NASA Technical Reports Server (NTRS)

    Go, Y. M.; Boo, Y. C.; Park, H.; Maland, M. C.; Patel, R.; Pritchard, K. A. Jr; Fujio, Y.; Walsh, K.; Darley-Usmar, V.; Jo, H.

    2001-01-01

    Laminar shear stress activates c-Jun NH(2)-terminal kinase (JNK) by the mechanisms involving both nitric oxide (NO) and phosphatidylinositide 3-kinase (PI3K). Because protein kinase B (Akt), a downstream effector of PI3K, has been shown to phosphorylate and activate endothelial NO synthase, we hypothesized that Akt regulates shear-dependent activation of JNK by stimulating NO production. Here, we examined the role of Akt in shear-dependent NO production and JNK activation by expressing a dominant negative Akt mutant (Akt(AA)) and a constitutively active mutant (Akt(Myr)) in bovine aortic endothelial cells (BAEC). As expected, pretreatment of BAEC with the PI3K inhibitor (wortmannin) prevented shear-dependent stimulation of Akt and NO production. Transient expression of Akt(AA) in BAEC by using a recombinant adenoviral construct inhibited the shear-dependent stimulation of NO production and JNK activation. However, transient expression of Akt(Myr) by using a recombinant adenoviral construct did not induce JNK activation. This is consistent with our previous finding that NO is required, but not sufficient on its own, to activate JNK in response to shear stress. These results and our previous findings strongly suggest that shear stress triggers activation of PI3K, Akt, and endothelial NO synthase, leading to production of NO, which (along with O(2-), which is also produced by shear) activates Ras-JNK pathway. The regulation of Akt, NO, and JNK by shear stress is likely to play a critical role in its antiatherogenic effects.

  11. 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.

  12. Ganoderma atrum polysaccharide improves aortic relaxation in diabetic rats via PI3K/Akt pathway.

    PubMed

    Zhu, Ke-Xue; Nie, Shao-Ping; Li, Chuan; Gong, Deming; Xie, Ming-Yong

    2014-03-15

    A newly identified polysaccharide (PSG-1) has been purified from Ganoderma atrum. The study was to investigate the protective effect of PSG-1 on diabetes-induced endothelial dysfunction in rat aorta. Rats were fed a high fat diet for 8 weeks and then injected with a low dose of streptozotocin to induce type 2 diabetes. The diabetic rats were orally treated with PSG-1 for 4 weeks. It was found that administration of PSG-1 significantly reduced levels of fasting blood glucose, improved endothelium-dependent aortic relaxation, increased levels of phosphoinositide 3-kinase (PI3K), phospho-Akt (p-Akt), endothelial nitric oxide synthase (eNOS) and nitric oxide in the aorta from diabetic rats, compared to un-treated diabetics. These results suggested that the protective effects of PSG-1 against endothelial dysfunction may be related to activation of the PI3K/Akt/eNOS pathway.

  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. 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

  15. 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 du