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Sample records for impaired glycogen synthase

  1. 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

  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. Hypoxia Promotes Glycogen Accumulation through Hypoxia Inducible Factor (HIF)-Mediated Induction of Glycogen Synthase 1

    PubMed Central

    Pescador, Nuria; Garcia-Rocha, Mar; Ortiz-Barahona, Amaya; Vazquez, Silvia; Ordoñez, Angel; Cuevas, Yolanda; Saez-Morales, David; Garcia-Bermejo, Maria Laura; Landazuri, Manuel O.; Guinovart, Joan; del Peso, Luis

    2010-01-01

    When oxygen becomes limiting, cells reduce mitochondrial respiration and increase ATP production through anaerobic fermentation of glucose. The Hypoxia Inducible Factors (HIFs) play a key role in this metabolic shift by regulating the transcription of key enzymes of glucose metabolism. Here we show that oxygen regulates the expression of the muscle glycogen synthase (GYS1). Hypoxic GYS1 induction requires HIF activity and a Hypoxia Response Element within its promoter. GYS1 gene induction correlated with a significant increase in glycogen synthase activity and glycogen accumulation in cells exposed to hypoxia. Significantly, knockdown of either HIF1α or GYS1 attenuated hypoxia-induced glycogen accumulation, while GYS1 overexpression was sufficient to mimic this effect. Altogether, these results indicate that GYS1 regulation by HIF plays a central role in the hypoxic accumulation of glycogen. Importantly, we found that hypoxia also upregulates the expression of UTP:glucose-1-phosphate urydylyltransferase (UGP2) and 1,4-α glucan branching enzyme (GBE1), two enzymes involved in the biosynthesis of glycogen. Therefore, hypoxia regulates almost all the enzymes involved in glycogen metabolism in a coordinated fashion, leading to its accumulation. Finally, we demonstrated that abrogation of glycogen synthesis, by knock-down of GYS1 expression, impairs hypoxic preconditioning, suggesting a physiological role for the glycogen accumulated during chronic hypoxia. In summary, our results uncover a novel effect of hypoxia on glucose metabolism, further supporting the central importance of metabolic reprogramming in the cellular adaptation to hypoxia. PMID:20300197

  4. 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.

  5. 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.

  6. Altered Wnt Signaling Pathway in Cognitive Impairment Caused by Chronic Intermittent Hypoxia: Focus on Glycogen Synthase Kinase-3β and β-catenin

    PubMed Central

    Pan, Yue-Ying; Deng, Yan; Xie, Sheng; Wang, Zhi-Hua; Wang, Yu; Ren, Jie; Liu, Hui-Guo

    2016-01-01

    Background: Cognitive impairment is a severe complication caused by obstructive sleep apnea (OSA). The mechanisms of causation are still unclear. The Wnt/β-catenin signaling pathway is involved in cognition, and abnormalities in it are implicated in neurological disorders. Here, we explored the Wnt/β-catenin signaling pathway abnormalities caused by chronic intermittent hypoxia (CIH), the most characteristic pathophysiological component of OSA. Methods: We divided 32 4-week-old male C57/BL mice into four groups of eight each: a CIH + normal saline (NS) group, CIH + LiCl group, sham CIH + NS group, and a sham CIH + LiCl group. The spatial learning performance of each group was assessed by using the Morris water maze (MWM). Protein expressions of glycogen synthase kinase-3β (GSK-3β) and β-catenin in the hippocampus were examined using the Western blotting test. EdU labeling and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling staining methods were used, respectively, to determine the proliferation and apoptosis of neurons in the hippocampal dentate gyrus region. Results: Mice exposed to CIH showed impaired spatial learning performance in the MWM, including increased mean escape latencies to reach the target platform, decreased mean times passing through the target platform and mean duration in the target quadrant. The GSK-3β activity increased, and expression of β-catenin decreased significantly in the hippocampus of the CIH-exposed mice. Besides, CIH significantly increased hippocampal neuronal apoptosis, with an elevated apoptosis index. Meanwhile, LiCl decreased the activity of GSK-3β and increased the expression of β-catenin and partially reversed the spatial memory deficits in MWM and the apoptosis caused by CIH. Conclusions: Wnt/β-catenin signaling pathway abnormalities possibly play an important role in the development of cognitive deficits among mice exposed to CIH and that LiCl might attenuate CIH-induced cognitive

  7. 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.

  8. Hexokinase 2, Glycogen Synthase and Phosphorylase Play a Key Role in Muscle Glycogen Supercompensation

    PubMed Central

    Irimia, José M.; Rovira, Jordi; Nielsen, Jakob N.; Guerrero, Mario; Wojtaszewski, Jørgen F. P.; Cussó, Roser

    2012-01-01

    Background Glycogen-depleting exercise can lead to supercompensation of muscle glycogen stores, but the biochemical mechanisms of this phenomenon are still not completely understood. Methods Using chronic low-frequency stimulation (CLFS) as an exercise model, the tibialis anterior muscle of rabbits was stimulated for either 1 or 24 hours, inducing a reduction in glycogen of 90% and 50% respectively. Glycogen recovery was subsequently monitored during 24 hours of rest. Results In muscles stimulated for 1 hour, glycogen recovered basal levels during the rest period. However, in those stimulated for 24 hours, glycogen was supercompensated and its levels remained 50% higher than basal levels after 6 hours of rest, although the newly synthesized glycogen had fewer branches. This increase in glycogen correlated with an increase in hexokinase-2 expression and activity, a reduction in the glycogen phosphorylase activity ratio and an increase in the glycogen synthase activity ratio, due to dephosphorylation of site 3a, even in the presence of elevated glycogen stores. During supercompensation there was also an increase in 5′-AMP-activated protein kinase phosphorylation, correlating with a stable reduction in ATP and total purine nucleotide levels. Conclusions Glycogen supercompensation requires a coordinated chain of events at two levels in the context of decreased cell energy balance: First, an increase in the glucose phosphorylation capacity of the muscle and secondly, control of the enzymes directly involved in the synthesis and degradation of the glycogen molecule. However, supercompensated glycogen has fewer branches. PMID:22860128

  9. Molecular Basis of Impaired Glycogen Metabolism during Ischemic Stroke and Hypoxia

    PubMed Central

    Hossain, Mohammed Iqbal; Roulston, Carli Lorraine; Stapleton, David Ian

    2014-01-01

    Background Ischemic stroke is the combinatorial effect of many pathological processes including the loss of energy supplies, excessive intracellular calcium accumulation, oxidative stress, and inflammatory responses. The brain's ability to maintain energy demand through this process involves metabolism of glycogen, which is critical for release of stored glucose. However, regulation of glycogen metabolism in ischemic stroke remains unknown. In the present study, we investigate the role and regulation of glycogen metabolizing enzymes and their effects on the fate of glycogen during ischemic stroke. Results Ischemic stroke was induced in rats by peri-vascular application of the vasoconstrictor endothelin-1 and forebrains were collected at 1, 3, 6 and 24 hours post-stroke. Glycogen levels and the expression and activity of enzymes involved in glycogen metabolism were analyzed. We found elevated glycogen levels in the ipsilateral hemispheres compared with contralateral hemispheres at 6 and 24 hours (25% and 39% increase respectively; P<0.05). Glycogen synthase activity and glycogen branching enzyme expression were found to be similar between the ipsilateral, contralateral, and sham control hemispheres. In contrast, the rate-limiting enzyme for glycogen breakdown, glycogen phosphorylase, had 58% lower activity (P<0.01) in the ipsilateral hemisphere (24 hours post-stroke), which corresponded with a 48% reduction in cAMP-dependent protein kinase A (PKA) activity (P<0.01). In addition, glycogen debranching enzyme expression 24 hours post-stroke was 77% (P<0.01) and 72% lower (P<0.01) at the protein and mRNA level, respectively. In cultured rat primary cerebellar astrocytes, hypoxia and inhibition of PKA activity significantly reduced glycogen phosphorylase activity and increased glycogen accumulation but did not alter glycogen synthase activity. Furthermore, elevated glycogen levels provided metabolic support to astrocytes during hypoxia. Conclusion Our study has

  10. Immunohistochemical localization of glycogen synthase and GSK3β: control of glycogen content in retina.

    PubMed

    Pérezleón, Jorge Alberto; Osorio-Paz, Ixchel; Francois, Liliana; Salceda, Rocío

    2013-05-01

    Glycogen has an important role in energy handling in several brain regions. In the brain, glycogen is localized in astrocytes and its role in several normal and pathological processes has been described, whereas in the retina, glycogen metabolism has been scarcely investigated. The enzyme glycogen phosphorylase has been located in retinal Müller cells; however the cellular location of glycogen synthase (GS) and its regulatory partner, glycogen synthase kinase 3β (GSK3β), has not been investigated. Our aim was to localize these enzymes in the rat retina by immunofluorescence techniques. We found both GS and GSK3β in Müller cells in the synaptic layers, and within the inner segments of photoreceptor cells. The presence of these enzymes in Müller cells suggests that glycogen could be regulated within the retina as in other tissues. Indeed, we showed that glycogen content in the whole retina in vitro was increased by high glucose concentrations, glutamate, and insulin. In contrast, retina glycogen levels were not modified by norepinephrine nor by depolarization with high KCl concentrations. Insulin also induced an increase in glycogen content in cultured Müller cells. The effect of insulin in both, whole retina and cultured Müller cells was blocked by inhibitors of phosphatidyl-inositol 3-kinase, strongly suggesting that glycogen content in retina is modulated by the insulin signaling pathway. The expression of GS and GSK3β in the synaptic layers and photoreceptor cells suggests an important role of GSK3β regulating glycogen synthase in neurons, which opens multiple feasible roles of insulin within the retina. PMID:23512644

  11. Structural basis for the recruitment of glycogen synthase by glycogenin

    PubMed Central

    Zeqiraj, Elton; Tang, Xiaojing; Hunter, Roger W.; García-Rocha, Mar; Judd, Andrew; Deak, Maria; von Wilamowitz-Moellendorff, Alexander; Kurinov, Igor; Guinovart, Joan J.; Tyers, Mike; Sakamoto, Kei; Sicheri, Frank

    2014-01-01

    Glycogen is a primary form of energy storage in eukaryotes that is essential for glucose homeostasis. The glycogen polymer is synthesized from glucose through the cooperative action of glycogen synthase (GS), glycogenin (GN), and glycogen branching enzyme and forms particles that range in size from 10 to 290 nm. GS is regulated by allosteric activation upon glucose-6-phosphate binding and inactivation by phosphorylation on its N- and C-terminal regulatory tails. GS alone is incapable of starting synthesis of a glycogen particle de novo, but instead it extends preexisting chains initiated by glycogenin. The molecular determinants by which GS recognizes self-glucosylated GN, the first step in glycogenesis, are unknown. We describe the crystal structure of Caenorhabditis elegans GS in complex with a minimal GS targeting sequence in GN and show that a 34-residue region of GN binds to a conserved surface on GS that is distinct from previously characterized allosteric and binding surfaces on the enzyme. The interaction identified in the GS-GN costructure is required for GS–GN interaction and for glycogen synthesis in a cell-free system and in intact cells. The interaction of full-length GS-GN proteins is enhanced by an avidity effect imparted by a dimeric state of GN and a tetrameric state of GS. Finally, the structure of the N- and C-terminal regulatory tails of GS provide a basis for understanding phosphoregulation of glycogen synthesis. These results uncover a central molecular mechanism that governs glycogen metabolism. PMID:24982189

  12. Subcutaneous administration of liraglutide ameliorates learning and memory impairment by modulating tau hyperphosphorylation via the glycogen synthase kinase-3β pathway in an amyloid β protein induced alzheimer disease mouse model.

    PubMed

    Qi, Liqin; Ke, Linfang; Liu, Xiaohong; Liao, Lianming; Ke, Sujie; Liu, Xiaoying; Wang, Yanping; Lin, Xiaowei; Zhou, Yu; Wu, Lijuan; Chen, Zhou; Liu, Libin

    2016-07-15

    Type 2 diabetes mellitus is a risk factor for Alzheimer's disease (AD). The glucagon-like peptide-1 analog liraglutide, a novel long-lasting incretin hormone, has been used to treat type 2 diabetes mellitus. In addition, liraglutide has been shown to be neurotrophic and neuroprotective. Here, we investigated the effects of liraglutide on amyloid β protein (Aβ)-induced AD in mice and explored its mechanism of action. The results showed that subcutaneous administration of liraglutide (25nmol/day), once daily for 8 weeks, prevented memory impairments in the Y Maze and Morris Water Maze following Aβ1-42 intracerebroventricular injection, and alleviated the ultra-structural changes of pyramidal neurons and chemical synapses in the hippocampal CA1 region. Furthermore, liraglutide reduced Aβ1-42-induced tau phosphorylation via the protein kinase B and glycogen synthase kinase-3β pathways. Thus liraglutide may alleviate cognitive impairment in AD by at least decreasing the phosphorylation of tau. PMID:27131827

  13. Enhanced Symbiotic Performance by Rhizobium tropici Glycogen Synthase Mutants

    PubMed Central

    Marroquí, Silvia; Zorreguieta, Angeles; Santamaría, Carmen; Temprano, Francisco; Soberón, Mario; Megías, Manuel; Downie, J. Allan

    2001-01-01

    We isolated a Tn5-induced Rhizobium tropici mutant that has enhanced capacity to oxidize N,N-dimethyl-p-phenylendiamine (DMPD) and therefore has enhanced respiration via cytochrome oxidase. The mutant had increased levels of the cytochromes c1 and CycM and a small increase in the amount of cytochrome aa3. In plant tests, the mutant increased the dry weight of Phaseolus vulgaris plants by 20 to 38% compared with the control strain, thus showing significantly enhanced symbiotic performance. The predicted product of the mutated gene is homologous to glycogen synthases from several bacteria, and the mutant lacked glycogen. The DNA sequence of the adjacent gene region revealed six genes predicted to encode products homologous to the following gene products from Escherichia coli: glycogen phosphorylase (glgP), glycogen branching enzyme (glgB), ADP glucose pyrophosphorylase (glgC), glycogen synthase (glgA), phosphoglucomutase (pgm), and glycogen debranching enzyme (glgX). All six genes are transcribed in the same direction, and analysis with lacZ gene fusions suggests that the first five genes are organized in one operon, although pgm appears to have an additional promoter; glgX is transcribed independently. Surprisingly, the glgA mutant had decreased levels of high-molecular-weight exopolysaccharide after growth on glucose, but levels were normal after growth on galactose. A deletion mutant was constructed in order to generate a nonpolar mutation in glgA. This mutant had a phenotype similar to that of the Tn5 mutant, indicating that the enhanced respiration and symbiotic nitrogen fixation and decreased exopolysaccharide were due to mutation of glgA and not to a polar effect on a downstream gene. PMID:11208782

  14. Dietary whey protein hydrolysates increase skeletal muscle glycogen levels via activation of glycogen synthase in mice.

    PubMed

    Kanda, Atsushi; Morifuji, Masashi; Fukasawa, Tomoyuki; Koga, Jinichiro; Kanegae, Minoru; Kawanaka, Kentaro; Higuchi, Mitsuru

    2012-11-14

    Previously, we have shown that consuming carbohydrate plus whey protein hydrolysates (WPHs) replenished muscle glycogen after exercise more effectively than consuming intact whey protein or branched-chain amino acids (BCAAs). The mechanism leading to superior glycogen replenishment after consuming WPH is unclear. In this 5 week intervention, ddY mice were fed experimental diets containing WPH, a mixture of whey amino acids (WAAs), or casein (control). After the intervention, gastrocnemius muscle glycogen levels were significantly higher in the WPH group (4.35 mg/g) than in the WAA (3.15 mg/g) or control (2.51 mg/g) groups. In addition, total glycogen synthase (GS) protein levels were significantly higher in the WPH group (153%) than in the WAA (89.2%) or control groups, and phosphorylated GS levels were significantly decreased in the WPH group (51.4%). These results indicate that dietary WPH may increase the muscle glycogen content through increased GS activity. PMID:23113736

  15. 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…

  16. 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.

  17. Inhibition of Glycogen Synthase Kinase-3β Improves Tolerance to Ischemia in Hypertrophied Hearts

    PubMed Central

    Barillas, Rodrigo; Friehs, Ingeborg; Cao-Danh, Hung; Martinez, Joseph F.; del Nido, Pedro J.

    2012-01-01

    Background Hypertrophied myocardium is more susceptible to ischemia/reperfusion injury, in part owing to impaired insulin-mediated glucose uptake. Glycogen synthase kinase-3β (GSK-3β) is a key regulatory enzyme in glucose metabolism that, when activated, phosphorylates/inactivates target enzymes of the insulin signaling pathway. Glycogen synthase kinase-3β is regulated upstream by Akt-1. We sought to determine whether GSK-3β is activated in ischemic hypertrophied myocardium owing to impaired Akt-1 function, and whether inhibition with lithium (Li) or indirubin-3′-monoxime,5-iodo- (IMI), a specific inhibitor, improves post-ischemic myocardial recovery by improving glucose metabolism. Methods Pressure-overload hypertrophy was achieved by aortic banding in neonatal rabbits. At 6 weeks, isolated hypertrophied hearts underwent 30 minutes of normothermic ischemia and reperfusion with or without GSK-3β inhibitor (0.1 mM Li; 1 µM IMI) as cardioplegic additives. Cardiac function was measured before and after ischemia. Expression, activity of Akt-1 and GSK-3β, and lactate were determined at end-ischemia. Results Contractile function after ischemia was better preserved in hypertrophied hearts treated with GSK-3β inhibitors. Activity of Akt-1 was significantly impaired in hypertrophied myocardium at end-ischemia. Glycogen synthase kinase-3β enzymatic activity at end-ischemia was increased in hypertrophied hearts and was blocked by Li or IMI concomitant with significantly increased lactate production, indicating increased glycolysis. Conclusions Regulatory inhibition of GSK-3β by Akt-1 in hypertrophied hearts is impaired, leading to activation during ischemia. Inhibition of GSK-3β by Li or IMI improves tolerance to ischemia/reperfusion injury in hypertrophied myocardium. The likely protective mechanism is an increase in insulin-mediated glucose uptake, resulting in greater substrate availability for glycolysis during ischemia and early reperfusion. PMID:17588398

  18. Glycogen synthase activation in human skeletal muscle: effects of diet and exercise.

    PubMed

    Kochan, R G; Lamb, D R; Lutz, S A; Perrill, C V; Reimann, E M; Schlender, K K

    1979-06-01

    We investigated the role of glycogen synthase in supranormal resynthesis (supercompensation) of skeletal muscle glycogen after exhaustive exercise. Six healthy men exercised 60 min by cycling with one leg at 75% VO2max, recovered 3 days on a low-carbohydrate diet, exercised again, and recovered 4 days on high-carbohydrate diet. Glycogen and glycogen synthase activities at several glucose-6-phosphate (G6P) concentrations were measured in biopsy samples of m. vastus lateralis. Dietary alterations alone did not affect glycogen, whereas exercise depleted glycogen stores. After the second exercise bout, glycogen returned to normal within 24 h and reached supercompensated levels by 48 h of recovery. Glycogen synthase activation state strikingly increased after exercise in exercised muscle and remained somewhat elevated for the first 48 h of recovery in both muscles. We suggest that 1) forms of glycogen synthase intermediate to I (G6P-independent) and D (G6P-dependent) forms are present in vivo, and 2) glycogen supercompensation can in part be explained by the formation of intermediate forms of glycogen synthase that exhibit relatively low activity ratios, but an increased sensitivity to activation by G6P. PMID:109015

  19. 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

  20. Transient down-regulation and restoration of glycogen synthase levels in axotomized rat facial motoneurons.

    PubMed

    Takezawa, Yosuke; Kohsaka, Shinichi; Nakajima, Kazuyuki

    2014-10-24

    In adult rats, transection of the facial nerve causes a functional down-regulation of motoneurons and glial activation/proliferation. It has not been clear how energy-supplying systems are regulated in an axotomized facial nucleus. Here we investigated the regulation of molecules involved in glycogen degradation/synthesis in axotomized facial nuclei in rats. Immunoblotting revealed that the amounts of glycogen phosphorylase in the contralateral and ipsilateral nuclei were unchanged for the first 14 days, whereas the amount of glycogen synthase in the axotomized facial nuclei was significantly decreased from days 7-14 post-insult. A quantitative analysis estimated that the glycogen synthase levels in the transected nucleus were reduced to approx. 50% at 14 days post-injury. An immunohistochemical study showed that the injured motoneurons had decreased expressions of glycogen synthase proteins. The glycogen synthase levels in the axotomized facial nucleus had returned to control levels by 5 weeks post-insult, as had the cholinergic markers. The immunohistochemical study also revealed the recovery of glycogen synthase levels at the later stage. The glycogen phosphorylase levels in the injured nucleus were not significantly changed during weeks 3-5 post-insult. Taken together, these results demonstrated that the injured facial motoneurons transiently reduced glycogen synthase levels at around 1-2 weeks post-insult, but restored the levels at 4-5 weeks post-insult. PMID:25152465

  1. 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

  2. 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

  3. Glycogen synthase kinase 3 in Wnt signaling pathway and cancer.

    PubMed

    Tejeda-Muñoz, Nydia; Robles-Flores, Martha

    2015-12-01

    Glycogen synthase kinase 3 (GSK-3) was first discovered in 1980 as one of the key enzymes of glycogen metabolism. Since then, GSK-3 has been revealed as one of the master regulators of a diverse range of signaling pathways, including those activated by Wnts, participating in the regulation of numerous cellular functions, suggesting that its activity is tightly regulated. Numerous studies have pointed to an association of GSK-3 dysregulation with the onset and progression of human diseases, including diabetes mellitus, obesity, inflammation, neurological illnesses, and cancer. Therefore, GSK-3 is recognized as an attractive therapeutic target in multiple disorders. However, the great number of substrates that are phosphorylated by GSK-3 has raised the question of whether this limits its feasibility as a therapeutic target because of the potential disruption of many cellular processes and also by the fear that inhibition of GSK-3 may stimulate or aid in malignant transformation, as GSK-3 can phosphorylate pro-oncogenic factors. This mini review focuses on the role played by GSK-3 in Wnt signaling pathway and cancer using as model colon cancer. PMID:26600003

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

    PubMed

    François, J; Villanueva, M E; Hers, H G

    1988-06-15

    The addition of glucose to a suspension of yeast initiated glycogen synthesis and ethanol formation. Other effects of the glucose addition were a transient rise in the concentration of cyclic AMP and a more prolonged increase in the concentration of hexose 6-monophosphate and of fructose 2,6-bisphosphate. The activity of glycogen synthase increased about 4-fold and that of glycogen phosphorylase decreased 3-5-fold. These changes could be reversed by the removal of glucose from the medium and induced again by a new addition of the sugar. These effects of glucose were also obtained with glucose derivatives known to form the corresponding 6-phosphoester. Similar changes in glycogen synthase and glycogen phosphorylase activity were induced by glucose in a thermosensitive mutant deficient in adenylate cyclase (cdc35) when incubated at the permissive temperature of 26 degrees C, but were much more pronounced at the nonpermissive temperature of 35 degrees C. Under the latter condition, glycogen synthase was nearly fully activated and glycogen phosphorylase fully inactivated. Such large effects of glucose were, however, not seen in another adenylate-cyclase-deficient mutant (cyr1), able to incorporate exogenous cyclic AMP. When a nitrogen source or uncouplers were added to the incubation medium after glucose, they had effects on glycogen metabolism and on the activity of glycogen synthase and glycogen phosphorylase which were directly opposite to those of glucose. By contrast, like glucose, these agents also caused, under most experimental conditions, a detectable rise in cyclic AMP concentration and a series of cyclic-AMP-dependent effects such as an activation of phosphofructokinase 2 and of trehalase and an increase in the concentration of fructose 2,6-bisphosphate and in the rate of glycolysis. Under all experimental conditions, the rate of glycolysis was proportional to the concentration of fructose 2,6-bisphosphate. Uncouplers, but not a nitrogen source, also induced

  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. PMID:17062013

  6. 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

  7. 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

  8. Platelet-derived growth factor (PDGF) stimulates glycogen synthase activity in 3T3 cells

    SciTech Connect

    Chan, C.P.; Bowen-Pope, D.F.; Ross, R.; Krebs, E.G.

    1986-05-01

    Hormonal regulation of glycogen synthase, an enzyme that can be phosphorylated on multiple sites, is often associated with changes in its phosphorylation state. Enzyme activation is conventionally monitored by determining the synthase activity ratio ((activity in the absence of glucose 6-P)/(activity in the presence of glucose 6-P)). Insulin causes an activation of glycogen synthase with a concomitant decrease in its phosphate content. In a previous report, the authors showed that epidermal growth factor (EGF) increases the glycogen synthase activity ratio in Swiss 3T3 cells. The time and dose-dependency of this response was similar to that of insulin. Their recent results indicate that PDGF also stimulates glycogen synthase activity. Enzyme activation was maximal after 30 min. of incubation with PDGF; the time course observed was very similar to that with insulin and EGF. At 1 ng/ml (0.03nM), PDGF caused a maximal stimulation of 4-fold in synthase activity ratio. Half-maximal stimulation was observed at 0.2 ng/ml (6 pM). The time course of changes in enzyme activity ratio closely followed that of /sup 125/I-PDGF binding. The authors data suggest that PDGF, as well as EFG and insulin, may be important in regulating glycogen synthesis through phosphorylation/dephosphorylation mechanisms.

  9. Pivotal role of glycogen synthase kinase-3: A therapeutic target for Alzheimer's disease.

    PubMed

    Maqbool, Mudasir; Mobashir, Mohammad; Hoda, Nasimul

    2016-01-01

    Neurodegenerative diseases are among the most challenging diseases with poorly known mechanism of cause and paucity of complete cure. Out of all the neurodegenerative diseases, Alzheimer's disease is the most devastating and loosening of thinking and judging ability disease that occurs in the old age people. Many hypotheses came forth in order to explain its causes. In this review, we have enlightened Glycogen Synthase Kinase-3 which has been considered as a concrete cause for Alzheimer's disease. Plaques and Tangles (abnormal structures) are the basic suspects in damaging and killing of nerve cells wherein Glycogen Synthase Kinase-3 has a key role in the formation of these fatal accumulations. Various Glycogen Synthase Kinase-3 inhibitors have been reported to reduce the amount of amyloid-beta as well as the tau hyperphosphorylation in both neuronal and nonneuronal cells. Additionally, Glycogen Synthase Kinase-3 inhibitors have been reported to enhance the adult hippocampal neurogenesis in vivo as well as in vitro. Keeping the chemotype of the reported Glycogen Synthase Kinase-3 inhibitors in consideration, they may be grouped into natural inhibitors, inorganic metal ions, organo-synthetic, and peptide like inhibitors. On the basis of their mode of binding to the constituent enzyme, they may also be grouped as ATP, nonATP, and allosteric binding sites competitive inhibitors. ATP competitive inhibitors were known earlier inhibitors but they lack efficient selectivity. This led to find the new ways for the enzyme inhibition. PMID:26562543

  10. Identification of glycogen synthase as a new substrate for stress-activated protein kinase 2b/p38beta.

    PubMed

    Kuma, Yvonne; Campbell, David G; Cuenda, Ana

    2004-04-01

    The endogenous glycogen synthase in extracts from mouse skeletal muscle, liver and brain bound specifically to SAPK2b (stress-activated protein kinase 2b)/p38b, but not to other members of the group of SAPK/p38 kinases. Glycogen synthase was phosphorylated in vitro more efficiently by SAPK2b/p38b than by SAPK2a/p38a, SAPK3/p38g or SAPK4/p38d. SAPK2b/p38b phosphorylated glycogen synthase in vitro at residues Ser644, Ser652, Thr718 and Ser724, two of which (Ser644 and Ser652) are also phosphorylated by glycogen synthase kinase 3. Thr718 and Ser724 are novel sites not known to be phosphorylated by other protein kinases. Glycogen synthase becomes phosphorylated at Ser644 in response to osmotic shock; this phosphorylation is prevented by pretreatment of the cells with SB 203580, which inhibits SAPK2a/p38a and SAPK2b/p38b activity. In vitro, phosphorylation of glycogen synthase by SAPK2b/p38b alone had no significant effect on its activity, indicating that phosphorylation at residue Ser644 itself is insufficient to decrease glycogen synthase activity. However, after phosphorylation by SAPK2b/p38b, subsequent phosphorylation at Ser640 by glycogen synthase kinase 3 decreased the activity of glycogen synthase. This decrease was not observed when SAPK2b/p38b activity was blocked with SB 203580. These results suggest that SAPK2b/p38b may be a priming kinase that allows glycogen synthase kinase 3 to phosphorylate Ser640 and thereby inhibit glycogen synthase activity. PMID:14680475

  11. [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

  12. Glycogen synthase (GYS1) mutation causes a novel skeletal muscle glycogenosis

    PubMed Central

    McCue, Molly E; Valberg, Stephanie J; Miller, Michael B; Wade, Claire; DiMauro, Salvatore; Akman, Hasan O; Mickelson, James R

    2008-01-01

    Summary We describe a gain of function mutation in the skeletal muscle glycogen synthase gene that is responsible for a novel myopathy, and is highly prevalent in multiple breeds of horses because it arose before the founding of many modern breeds. Polysaccharide Storage Myopathy (PSSM) is a novel glycogenosis in horses characterized by abnormal glycogen accumulation in skeletal muscle and muscle damage with exertion. It is unlike glycogen storage diseases resulting from known defects in glycogenolysis, glycolysis and glycogen synthesis that have been described in humans and domestic animals. A genome wide association identified GYS1, encoding skeletal muscle glycogen synthase (GS), as a candidate gene for PSSM. DNA sequence analysis revealed a mutation resulting in an arginine to histidine substitution in a highly conserved region of GS. Functional analysis demonstrated an elevated GS activity in PSSM horses and haplotype analysis and allele age estimation demonstrated that this mutation is identical by descent among horse breeds. This is the first report of a gain of function mutation in GYS1 resulting in a glycogenosis. PMID:18358695

  13. Hepatic Glycogen Supercompensation Activates AMP-Activated Protein Kinase, Impairs Insulin Signaling, and Reduces Glycogen Deposition in the Liver

    PubMed Central

    Winnick, Jason J.; An, Zhibo; Ramnanan, Christopher J.; Smith, Marta; Irimia, Jose M.; Neal, Doss W.; Moore, Mary Courtney; Roach, Peter J.; Cherrington, Alan D.

    2011-01-01

    OBJECTIVE The objective of this study was to determine how increasing the hepatic glycogen content would affect the liver’s ability to take up and metabolize glucose. RESEARCH DESIGN AND METHODS During the first 4 h of the study, liver glycogen deposition was stimulated by intraportal fructose infusion in the presence of hyperglycemic-normoinsulinemia. This was followed by a 2-h hyperglycemic-normoinsulinemic control period, during which the fructose infusion was stopped, and a 2-h experimental period in which net hepatic glucose uptake (NHGU) and disposition (glycogen, lactate, and CO2) were measured in the absence of fructose but in the presence of a hyperglycemic-hyperinsulinemic challenge including portal vein glucose infusion. RESULTS Fructose infusion increased net hepatic glycogen synthesis (0.7 ± 0.5 vs. 6.4 ± 0.4 mg/kg/min; P < 0.001), causing a large difference in hepatic glycogen content (62 ± 9 vs. 100 ± 3 mg/g; P < 0.001). Hepatic glycogen supercompensation (fructose infusion group) did not alter NHGU, but it reduced the percent of NHGU directed to glycogen (79 ± 4 vs. 55 ± 6; P < 0.01) and increased the percent directed to lactate (12 ± 3 vs. 29 ± 5; P = 0.01) and oxidation (9 ± 3 vs. 16 ± 3; P = NS). This change was associated with increased AMP-activated protein kinase phosphorylation, diminished insulin signaling, and a shift in glycogenic enzyme activity toward a state discouraging glycogen accumulation. CONCLUSIONS These data indicate that increases in hepatic glycogen can generate a state of hepatic insulin resistance, which is characterized by impaired glycogen synthesis despite preserved NHGU. PMID:21270252

  14. A protein kinase screen of Neurospora crassa mutant strains reveals that the SNF1 protein kinase promotes glycogen synthase phosphorylation.

    PubMed

    Candido, Thiago De Souza; Gonçalves, Rodrigo Duarte; Felício, Ana Paula; Freitas, Fernanda Zanolli; Cupertino, Fernanda Barbosa; De Carvalho, Ana Carolina Gomes Vieira; Bertolini, Maria Célia

    2014-12-15

    Glycogen functions as a carbohydrate reserve in a variety of organisms and its metabolism is highly regulated. The activities of glycogen synthase and glycogen phosphorylase, the rate-limiting enzymes of the synthesis and degradation processes, respectively, are regulated by allosteric modulation and reversible phosphorylation. To identify the protein kinases affecting glycogen metabolism in Neurospora crassa, we performed a screen of 84 serine/threonine kinase knockout strains. We identified multiple kinases that have already been described as controlling glycogen metabolism in different organisms, such as NcSNF1, NcPHO85, NcGSK3, NcPKA, PSK2 homologue and NcATG1. In addition, many hypothetical kinases have been implicated in the control of glycogen metabolism. Two kinases, NcIME-2 and NcNIMA, already functionally characterized but with no functions related to glycogen metabolism regulation, were also identified. Among the kinases identified, it is important to mention the role of NcSNF1. We showed in the present study that this kinase was implicated in glycogen synthase phosphorylation, as demonstrated by the higher levels of glycogen accumulated during growth, along with a higher glycogen synthase (GSN) ±glucose 6-phosphate activity ratio and a lesser set of phosphorylated GSN isoforms in strain Ncsnf1KO, when compared with the wild-type strain. The results led us to conclude that, in N. crassa, this kinase promotes phosphorylation of glycogen synthase either directly or indirectly, which is the opposite of what is described for Saccharomyces cerevisiae. The kinases also play a role in gene expression regulation, in that gdn, the gene encoding the debranching enzyme, was down-regulated by the proteins identified in the screen. Some kinases affected growth and development, suggesting a connection linking glycogen metabolism with cell growth and development. PMID:25253091

  15. 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

  16. 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

  17. 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

  18. Fgk3 glycogen synthase kinase is important for development, pathogenesis, and stress responses in Fusarium graminearum.

    PubMed

    Qin, Jun; Wang, Guanghui; Jiang, Cong; Xu, Jin-Rong; Wang, Chenfang

    2015-01-01

    Wheat scab caused by Fusarium graminearum is an important disease. In a previous study, the FGK3 glycogen synthase kinase gene orthologous to mammalian GSK3 was identified as an important virulence factor. Although GSK3 orthologs are well-conserved, none of them have been functionally characterized in fungal pathogens. In this study, we further characterized the roles of FGK3 gene. The Δfgk3 mutant had pleiotropic defects in growth rate, conidium morphology, germination, and perithecium formation. It was non-pathogenic in infection assays and blocked in DON production. Glycogen accumulation was increased in the Δfgk3 mutant, confirming the inhibitory role of Fgk3 on glycogen synthase. In FGK3-GFP transformants, GFP signals mainly localized to the cytoplasm in conidia but to the cytoplasm and nucleus in hyphae. Moreover, the expression level of FGK3 increased in response to cold, H2O2, and SDS stresses. In the Δfgk3 mutant, cold, heat, and salt stresses failed to induce the expression of the stress response-related genes FgGRE2, FgGPD1, FgCTT1, and FgMSN2. In the presence of 80 mM LiCl, a GSK3 kinase inhibitor, the wild type displayed similar defects to the Δfgk3 mutant. Overall, our results indicate that FGK3 is important for growth, conidiogenesis, DON production, pathogenicity, and stress responses in F. graminearum. PMID:25703795

  19. Glycogen Synthase Kinase 3β Interaction Protein Functions as an A-kinase Anchoring Protein*

    PubMed Central

    Hundsrucker, Christian; Skroblin, Philipp; Christian, Frank; Zenn, Hans-Michael; Popara, Viola; Joshi, Mangesh; Eichhorst, Jenny; Wiesner, Burkhard; Herberg, Friedrich W.; Reif, Bernd; Rosenthal, Walter; Klussmann, Enno

    2010-01-01

    A-kinase anchoring proteins (AKAPs) include a family of scaffolding proteins that target protein kinase A (PKA) and other signaling proteins to cellular compartments and thereby confine the activities of the associated proteins to distinct regions within cells. AKAPs bind PKA directly. The interaction is mediated by the dimerization and docking domain of regulatory subunits of PKA and the PKA-binding domain of AKAPs. Analysis of the interactions between the dimerization and docking domain and various PKA-binding domains yielded a generalized motif allowing the identification of AKAPs. Our bioinformatics and peptide array screening approaches based on this signature motif identified GSKIP (glycogen synthase kinase 3β interaction protein) as an AKAP. GSKIP directly interacts with PKA and GSK3β (glycogen synthase kinase 3β). It is widely expressed and facilitates phosphorylation and thus inactivation of GSK3β by PKA. GSKIP contains the evolutionarily conserved domain of unknown function 727. We show here that this domain of GSKIP and its vertebrate orthologues binds both PKA and GSK3β and thereby provides a mechanism for the integration of PKA and GSK3β signaling pathways. PMID:20007971

  20. 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

  1. 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 ...

  2. 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

  3. Role of glycogen synthase kinase-3 beta in the inflammatory response caused by bacterial pathogens

    PubMed Central

    2012-01-01

    Glycogen synthase kinase 3β (GSK3β) plays a fundamental role during the inflammatory response induced by bacteria. Depending on the pathogen and its virulence factors, the type of cell and probably the context in which the interaction between host cells and bacteria takes place, GSK3β may promote or inhibit inflammation. The goal of this review is to discuss recent findings on the role of the inhibition or activation of GSK3β and its modulation of the inflammatory signaling in monocytes/macrophages and epithelial cells at the transcriptional level, mainly through the regulation of nuclear factor-kappaB (NF-κB) activity. Also included is a brief overview on the importance of GSK3 in non-inflammatory processes during bacterial infection. PMID:22691598

  4. Glycogen synthase kinase-3 is involved in regulation of ribosome biogenesis in yeast.

    PubMed

    Yabuki, Yukari; Kodama, Yushi; Katayama, Masako; Sakamoto, Akiko; Kanemaru, Hirofumi; Wan, Kun; Mizuta, Keiko

    2014-01-01

    Secretory defects cause transcriptional repression of both ribosomal proteins and ribosomal RNA genes in Saccharomyces cerevisiae. Rrs1, a trans-acting factor that participates in ribosome biogenesis, is involved in the signaling pathway induced by secretory defects. Here, we found that Rrs1 interacts with two homologs of the glycogen synthase kinase-3 (GSK-3), Rim11, and Mrk1. Rrs1 possesses a repetitive consensus amino acid sequence for phosphorylation by GSK-3, and mutation of this sequence abolished the interaction of Rrs1 with Rim11 and Mrk1. Although this mutation did not affect vegetative cell growth or secretory response, disruption of all four genes encoding GSK-3 homologs, especially Mck1, diminished the transcriptional repression of ribosomal protein genes in response to secretory defects. Among the four GSK-3 kinases, Mck1 appears to be the primary mediator of this response, while the other GSK-3 kinases contribute redundantly. PMID:25035982

  5. 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.

  6. Seasonal, tissue-specific regulation of Akt/protein kinase B and glycogen synthase in hibernators.

    PubMed

    Hoehn, Kyle L; Hudachek, Susan F; Summers, Scott A; Florant, Gregory L

    2004-03-01

    Yellow-bellied marmots (Marmota flaviventris) exhibit a circannual cycle of hyperphagia and nutrient storage in the summer followed by hibernation in the winter. This annual cycle of body mass gain and loss is primarily due to large-scale accumulation of lipid in the summer, which is then mobilized and oxidized for energy during winter. The rapid and predictable change in body mass makes these animals ideal for studies investigating the molecular basis for body weight regulation. In the study described herein, we monitored seasonal changes in the protein levels and activity of a central regulator of anabolic metabolism, the serine-threonine kinase Akt-protein kinase B (Akt/PKB), during the months accompanying maximal weight gain and entry into hibernation (June-November). Interestingly, under fasting conditions, Akt/PKB demonstrated a tissue-specific seasonal activation. Specifically, although Akt/PKB levels did not change, the activity of Akt/PKB (isoforms 1/alpha and 2/beta) in white adipose tissue (WAT) increased significantly in July. Moreover, glycogen synthase, which lies downstream of Akt/PKB on a linear pathway linking the enzyme to the stimulation of glycogen synthesis, demonstrated a similar pattern of seasonal activation. By contrast, Akt/PKB activity in skeletal muscle peaked much later (i.e., September). These data suggest the existence of a novel, tissue-specific mechanism regulating Akt/PKB activation during periods of marked anabolism. PMID:14656767

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

    PubMed Central

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

    2015-01-01

    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. PMID:25527037

  8. Phosphorylation of sites 3 and 2 in rabbit skeletal muscle glycogen synthase by a multifunctional protein kinase (ATP-citrate lyase kinase)

    SciTech Connect

    Sheorain, V.S.; Ramakrishna, S.; Benjamin, W.B.; Soderling, T.R.

    1985-10-05

    A multifunctional protein kinase, purified from rat liver as ATP-citrate lyase kinase, has been identified as a glycogen synthase kinase. This kinase catalyzed incorporation of up to 1.5 mol of and)2numberSPO4/mol of synthase subunit associated with a decrease in the glycogen synthase activity ratio from 0.85 to a value of 0.15. Approximately 65-70% of the TUPO4 was incorporated into site 3 and 30-35% into site 2 as determined by reverse phase high performance liquid chromatography. This multifunctional kinase was distinguished from glycogen synthase kinase-3 on the basis of nucleotide and protein substrate specificities. Since the phosphate contents in glycogen synthase of sites 3 and 2 are altered in diabetes and by insulin administration, the possible involvement of the multifunctional kinase was explored. Glycogen synthase purified from diabetic rabbits was phosphorylated in vitro by this multifunctional kinase at only 10% of the rate compared to synthase purified from control rabbits. Treatment of the diabetics with insulin restored the synthase to a form that was readily phosphorylated in vitro.

  9. Insulin induces an increase in cytosolic glucose levels in 3T3-L1 cells with inhibited glycogen synthase activation.

    PubMed

    Chowdhury, Helena H; Kreft, Marko; Jensen, Jørgen; Zorec, Robert

    2014-01-01

    Glucose is an important source of energy for mammalian cells and enters the cytosol via glucose transporters. It has been thought for a long time that glucose entering the cytosol is swiftly phosphorylated in most cell types; hence the levels of free glucose are very low, beyond the detection level. However, the introduction of new fluorescence resonance energy transfer-based glucose nanosensors has made it possible to measure intracellular glucose more accurately. Here, we used the fluorescent indicator protein (FLIPglu-600µ) to monitor cytosolic glucose dynamics in mouse 3T3-L1 cells in which glucose utilization for glycogen synthesis was inhibited. The results show that cells exhibit a low resting cytosolic glucose concentration. However, in cells with inhibited glycogen synthase activation, insulin induced a robust increase in cytosolic free glucose. The insulin-induced increase in cytosolic glucose in these cells is due to an imbalance between the glucose transported into the cytosol and the use of glucose in the cytosol. In untreated cells with sensitive glycogen synthase activation, insulin stimulation did not result in a change in the cytosolic glucose level. This is the first report of dynamic measurements of cytosolic glucose levels in cells devoid of the glycogen synthesis pathway. PMID:25279585

  10. 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.

  11. Lithium inhibits invasion of glioma cells; possible involvement of glycogen synthase kinase-3

    PubMed Central

    Nowicki, Michal O.; Dmitrieva, Nina; Stein, Andrew M.; Cutter, Jennifer L.; Godlewski, Jakub; Saeki, Yoshinaga; Nita, Masayuki; Berens, Michael E.; Sander, Leonard M.; Newton, Herbert B.; Chiocca, E. Antonio; Lawler, Sean

    2008-01-01

    Therapies targeting glioma cells that diffusely infiltrate normal brain are highly sought after. Our aim was to identify novel approaches to this problem using glioma spheroid migration assays. Lithium, a currently approved drug for the treatment of bipolar illnesses, has not been previously examined in the context of glioma migration. We found that lithium treatment potently blocked glioma cell migration in spheroid, wound-healing, and brain slice assays. The effects observed were dose dependent and reversible, and worked using every glioma cell line tested. In addition, there was little effect on cell viability at lithium concentrations that inhibit migration, showing that this is a specific effect. Lithium treatment was associated with a marked change in cell morphology, with cells retracting the long extensions at their leading edge. Examination of known targets of lithium showed that inositol monophosphatase inhibition had no effect on glioma migration, whereas inhibition of glycogen synthase kinase-3 (GSK-3) did. This suggested that the effects of lithium on glioma cell migration could possibly be mediated through GSK-3. Specific pharmacologic GSK-3 inhibitors and siRNA knockdown of GSK-3α or GSK-3β isoforms both reduced cell motility. These data outline previously unidentified pathways and inhibitors that may be useful for the development of novel anti-invasive therapeutics for the treatment of brain tumors. PMID:18715951

  12. Inhibitors of Glycogen Synthase Kinase 3 with Exquisite Kinome-Wide Selectivity and Their Functional Effects.

    PubMed

    Wagner, Florence F; Bishop, Joshua A; Gale, Jennifer P; Shi, Xi; Walk, Michelle; Ketterman, Joshua; Patnaik, Debasis; Barker, Doug; Walpita, Deepika; Campbell, Arthur J; Nguyen, Shannon; Lewis, Michael; Ross, Linda; Weïwer, Michel; An, W Frank; Germain, Andrew R; Nag, Partha P; Metkar, Shailesh; Kaya, Taner; Dandapani, Sivaraman; Olson, David E; Barbe, Anne-Laure; Lazzaro, Fanny; Sacher, Joshua R; Cheah, Jaime H; Fei, David; Perez, Jose; Munoz, Benito; Palmer, Michelle; Stegmaier, Kimberly; Schreiber, Stuart L; Scolnick, Edward; Zhang, Yan-Ling; Haggarty, Stephen J; Holson, Edward B; Pan, Jen Q

    2016-07-15

    The mood stabilizer lithium, the first-line treatment for bipolar disorder, is hypothesized to exert its effects through direct inhibition of glycogen synthase kinase 3 (GSK3) and indirectly by increasing GSK3's inhibitory serine phosphorylation. GSK3 comprises two highly similar paralogs, GSK3α and GSK3β, which are key regulatory kinases in the canonical Wnt pathway. GSK3 stands as a nodal target within this pathway and is an attractive therapeutic target for multiple indications. Despite being an active field of research for the past 20 years, many GSK3 inhibitors demonstrate either poor to moderate selectivity versus the broader human kinome or physicochemical properties unsuitable for use in in vitro systems or in vivo models. A nonconventional analysis of data from a GSK3β inhibitor high-throughput screening campaign, which excluded known GSK3 inhibitor chemotypes, led to the discovery of a novel pyrazolo-tetrahydroquinolinone scaffold with unparalleled kinome-wide selectivity for the GSK3 kinases. Taking advantage of an uncommon tridentate interaction with the hinge region of GSK3, we developed highly selective and potent GSK3 inhibitors, BRD1652 and BRD0209, which demonstrated in vivo efficacy in a dopaminergic signaling paradigm modeling mood-related disorders. These new chemical probes open the way for exclusive analyses of the function of GSK3 kinases in multiple signaling pathways involved in many prevalent disorders. PMID:27128528

  13. Rapid Detection of Glycogen Synthase Kinase-3 Activity in Mouse Sperm Using Fluorescent Gel Shift Electrophoresis.

    PubMed

    Choi, Hoseok; Choi, Bomi; Seo, Ju Tae; Lee, Kyung Jin; Gye, Myung Chan; Kim, Young-Pil

    2016-01-01

    Assaying the glycogen synthase kinase-3 (GSK3) activity in sperm is of great importance because it is closely implicated in sperm motility and male infertility. While a number of studies on GSK3 activity have relied on labor-intensive immunoblotting to identify phosphorylated GSK3, here we report the simple and rapid detection of GSK3 activity in mouse sperm using conventional agarose gel electrophoresis and a fluorescent peptide substrate. When a dye-tethered and prephosphorylated (primed) peptide substrate for GSK3 was employed, a distinct mobility shift in the fluorescent bands on the agarose was observed by GSK3-induced phosphorylation of the primed peptides. The GSK3 activity in mouse testes and sperm were quantifiable by gel shift assay with low sample consumption and were significantly correlated with the expression levels of GSK3 and p-GSK3. We suggest that our assay can be used for reliable and rapid detection of GSK3 activity in cells and tissue extracts. PMID:27092510

  14. 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

  15. Glycogen synthase kinase 3 phosphorylates RBL2/p130 during quiescence.

    PubMed

    Litovchick, Larisa; Chestukhin, Anton; DeCaprio, James A

    2004-10-01

    Phosphorylation of the retinoblastoma-related or pocket proteins RB1/pRb, RBL1/p107, and RBL2/p130 regulates cell cycle progression and exit. While all pocket proteins are phosphorylated by cyclin-dependent kinases (CDKs) during the G1/S-phase transition, p130 is also specifically phosphorylated in G0-arrested cells. We have previously identified several phosphorylated residues that match the consensus site for glycogen synthase kinase 3 (GSK3) in the G0 form of p130. Using small-molecule inhibitors of GSK3, site-specific mutants of p130, and phospho-specific antibodies, we demonstrate here that GSK3 phosphorylates p130 during G0. Phosphorylation of p130 by GSK3 contributes to the stability of p130 but does not affect its ability to interact with E2F4 or cyclins. Regulation of p130 by GSK3 provides a novel link between growth factor signaling and regulation of the cell cycle progression and exit. PMID:15456871

  16. Rapid Detection of Glycogen Synthase Kinase-3 Activity in Mouse Sperm Using Fluorescent Gel Shift Electrophoresis

    PubMed Central

    Choi, Hoseok; Choi, Bomi; Seo, Ju Tae; Lee, Kyung Jin; Gye, Myung Chan; Kim, Young-Pil

    2016-01-01

    Assaying the glycogen synthase kinase-3 (GSK3) activity in sperm is of great importance because it is closely implicated in sperm motility and male infertility. While a number of studies on GSK3 activity have relied on labor-intensive immunoblotting to identify phosphorylated GSK3, here we report the simple and rapid detection of GSK3 activity in mouse sperm using conventional agarose gel electrophoresis and a fluorescent peptide substrate. When a dye-tethered and prephosphorylated (primed) peptide substrate for GSK3 was employed, a distinct mobility shift in the fluorescent bands on the agarose was observed by GSK3-induced phosphorylation of the primed peptides. The GSK3 activity in mouse testes and sperm were quantifiable by gel shift assay with low sample consumption and were significantly correlated with the expression levels of GSK3 and p-GSK3. We suggest that our assay can be used for reliable and rapid detection of GSK3 activity in cells and tissue extracts. PMID:27092510

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

    PubMed Central

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

    2016-01-01

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

  18. Aberrant glycogen synthase kinase 3β in the development of pancreatic cancer

    PubMed Central

    Shimasaki, Takeo; Kitano, Ayako; Motoo, Yoshiharu; Minamoto, Toshinari

    2012-01-01

    Development and progression of pancreatic cancer involves general metabolic disorder, local chronic inflammation, and multistep activation of distinct oncogenic molecular pathways. These pathologic processes result in a highly invasive and metastatic tumor phenotype that is a major obstacle to curative surgical intervention, infusional gemcitabine-based chemotherapy, and radiation therapy. Many clinical trials with chemical compounds and therapeutic antibodies targeting growth factors, angiogenic factors, and matrix metalloproteinases have failed to demonstrate definitive therapeutic benefits to refractory pancreatic cancer patients. Glycogen synthase kinase 3β (GSK3β), a serine/threonine protein kinase, has emerged as a therapeutic target in common chronic and progressive diseases, including cancer. Here we review accumulating evidence for a pathologic role of GSK3β in promoting tumor cell survival, proliferation, invasion, and resistance to chemotherapy and radiation in pancreatic cancer. We also discuss the putative involvement of GSK3β in mediating metabolic disorder, local inflammation, and molecular alteration leading to pancreatic cancer development. Taken together, we highlight potential therapeutic as well as preventive effects of GSK3β inhibition in pancreatic cancer. PMID:23230392

  19. Role of glycogen synthase kinase-3β inhibitor AZD1080 in ovarian cancer

    PubMed Central

    Chen, Shuo; Sun, Kai-Xuan; Feng, Miao-Xiao; Sang, Xiu-Bo; Liu, Bo-Liang; Zhao, Yang

    2016-01-01

    Background Glycogen synthase kinase-3β (GSK-3β) is a multifunctional serine/threonine kinase that plays an important role in cancer tumorigenesis and progression. We investigated the role of the GSK-3β inhibitor AZD1080 in ovarian cancer cell lines. Methods A2780 and OVCAR3 ovarian cancer cell lines were exposed to AZD1080, after which cell proliferation, cell cycle, invasion, and migration assays were performed. Phalloidin staining was used to observe lamellipodia formation. Reverse transcription polymerase chain reaction and Western blot were used to assess the respective mRNA and protein expression levels of GSK-3β, CDK2, CDK1, cyclin D1, matrix metalloproteinase-9 (MMP9), and Bcl-xL. Results AZD1080 exposure suppressed ovarian cancer cell proliferation, invasion, migration, and lamellipodia formation, and induced G1 arrest, which was concentration dependent. AZD1080 also significantly downregulated GSK-3β, CDK2, CDK1, cyclin D1, MMP9, and Bcl-xL expression at both mRNA and protein levels. Conclusion Taken together, our results demonstrate that the GSK-3β inhibitor AZD1080 suppresses ovarian cancer development and therefore may indicate a new direction for ovarian cancer treatment. PMID:27051274

  20. Glycogen Synthase Kinase-3 promotes cell survival, growth and PAX3 levels in human melanoma cells

    PubMed Central

    Kubic, Jennifer D.; Mascarenhas, Joseph B.; Iizuka, Takumi; Wolfgeher, Don; Lang, Deborah

    2012-01-01

    Glycogen Synthase Kinase-3 (GSK-3) is a serine/threonine kinase involved in a diverse range of cellular processes. GSK-3 exists in two isoforms, GSK-3α and GSK-3β, which possess some functional redundancy but also play distinct roles depending on developmental and cellular context. In this report we found that GSK-3 actively promoted cell growth and survival in melanoma cells, and blocking this activity with small molecule inhibitor SB216763 or gene-specific siRNA decreased proliferation, increased apoptosis and altered cellular morphology. These alterations coincided with loss of PAX3, a transcription factor implicated in proliferation, survival and migration of developing melanoblasts. We further found that PAX3 directly interacted with and was phosphorylated in vitro on a number of residues by GSK-3β. In melanoma cells, direct inhibition of PAX3 lead to cellular changes that paralleled the response to GSK-3 inhibition. Maintenance of PAX3 expression protected melanoma cells from the anti-tumor effects of SB216763. These data support a model wherein GSK-3 regulates proliferation and morphology of melanoma through phosphorylation and increased levels of PAX3. PMID:22679108

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

    PubMed

    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

  2. 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. PMID:25001420

  3. 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

  4. Improving the glycosyltransferase activity of Agrobacterium tumefaciens glycogen synthase by fusion of N-terminal starch binding domains (SBDs).

    PubMed

    Martín, Mariana; Wayllace, Nahuel Z; Valdez, Hugo A; Gomez-Casati, Diego F; Busi, María V

    2013-10-01

    Glycogen and starch, the major storage carbohydrate in most living organisms, result mainly from the action of starch or glycogen synthases (SS or GS, respectively, EC 2.4.1.21). SSIII from Arabidopsis thaliana is an SS isoform with a particular modular organization: the C-terminal highly conserved glycosyltransferase domain is preceded by a unique specific region (SSIII-SD) which contains three in tandem starch binding domains (SBDs, named D1, D2 and D3) characteristic of polysaccharide degrading enzymes. N-terminal SBDs have a probed regulatory role in SSIII activity, showing starch binding ability and modulating the catalytic properties of the enzyme. On the other hand, GS from Agrobacterium tumefaciens has a simple primary structure organization, characterized only by the highly conserved glycosyltransferase domain and lacking SBDs. To further investigate the functional role of A. thaliana SSIII-SD, three chimeric proteins were constructed combining the SBDs from A. thaliana with the GS from A. tumefaciens. Recombinant proteins were expressed in and purified to homogeneity from Escherichia coli cells in order to be kinetically characterized. Furthermore, we tested the ability to restore in vivo glycogen biosynthesis in transformed E. coli glgA(-) cells, deficient in GS. Results show that the D3-GS chimeric enzyme showed increased capacity of glycogen synthesis in vivo with minor changes in its kinetics parameters compared to GS. PMID:23796574

  5. Apolipoprotein E and beta-amyloid (1-42) regulation of glycogen synthase kinase-3beta.

    PubMed

    Cedazo-Mínguez, A; Popescu, B O; Blanco-Millán, J M; Akterin, S; Pei, J-J; Winblad, B; Cowburn, R F

    2003-12-01

    Glycogen synthase kinase-3beta (GSK-3beta) is implicated in regulating apoptosis and tau protein hyperphosphorylation in Alzheimer's disease (AD). We investigated the effects of two key AD molecules, namely apoE (E3 and E4 isoforms) and beta-amyloid (Abeta) 1-42 on GSK-3beta and its major upstream regulators, intracellular calcium and protein kinases C and B (PKC and PKB) in human SH-SY5Y neuroblastoma cells. ApoE3 induced a mild, transient, Ca2+-independent and early activation of GSK-3beta. ApoE4 effects were biphasic, with an early strong GSK-3beta activation that was partially dependent on extracellular Ca2+, followed by a GSK-3beta inactivation. ApoE4 also activated PKC-alpha and PKB possibly giving the subsequent GSK-3beta inhibition. Abeta(1-42) effects were also biphasic with a strong activation dependent partially on extracellular Ca2+ followed by an inactivation. Abeta(1-42) induced an early and potent activation of PKC-alpha and a late decrease of PKB activity. ApoE4 and Abeta(1-42) were more toxic than apoE3 as shown by MTT reduction assays and generation of activated caspase-3. ApoE4 and Abeta(1-42)-induced early activation of GSK-3beta could lead to apoptosis and tau hyperphosphorylation. A late inhibition of GSK-3beta through activation of upstream kinases likely compensates the effects of apoE4 and Abeta(1-42) on GSK-3beta, the unbalanced regulation of which may contribute to AD pathology. PMID:14622095

  6. Identification of a Glycogen Synthase Kinase-3β Inhibitor that Attenuates Hyperactivity in CLOCK Mutant Mice

    PubMed Central

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

    2012-01-01

    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-3β (GSK-3β) plays a central role in regulating circadian rhythms, and lithium is known to be a direct inhibitor of GSK-3β. We designed a series of second generation benzofuran-3-yl-(indol-3-yl)maleimides containing a piperidine ring that possess IC50 values in the range of 4 to 680 nm against human GSK-3β. 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 mgkg−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 mgkg−1) and the antipsychotic haloperidol (1 mgkg−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-3β in the treatment of manic episodes of bipolar/mood disorders, thus further validating GSK-3β as a relevant therapeutic target in the identification of new therapies for bipolar patients. PMID:21732538

  7. 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

  8. Identification and regulation of glycogen synthase kinase-3 during bovine embryo development.

    PubMed

    Aparicio, I M; Garcia-Herreros, M; Fair, T; Lonergan, P

    2010-07-01

    The aim of this study was to examine the presence and regulation of glycogen synthase kinase-3alpha (GSK3A) and GSK-3beta (GSK3B) in bovine embryos and their possible roles in embryo development. Our results show that GSK3A and GSK3B are present in bovine embryos at the two-cell stage to the hatched blastocyst stage. Bovine embryo development was associated with an increase in the phosphorylation of both isoforms, being statistically significant at blastocyst and hatched blastocyst stages, compared with earlier stages. Inhibition of GSK3 with CT99021 (3 microM) resulted in a significant increase in the percentage and quality of blastocysts, while inhibition of GSK3 with lithium chloride (LiCl; 20 mM) significantly reduced at the proportion of eight-cell embryos on day 3 and inhibited blastocyst formation. The use of LY294002 (10 microM), a specific inhibitor of phosphatidylinositol-3 kinase, also produced a significant decrease in embryo development. In addition, treatment with LiCl and LY294002 produced a significant decrease in the serine phosphorylation of both isoforms of GSK3. Finally, CT99021 and LiCl reduced the phosphorylation of beta-catenin on Ser45 in two-cell embryos, while LY294002 increased it. Despite the fact that LiCl inhibited GSK3 activity, as demonstrated by beta-catenin phosphorylation, its effects on the bovine embryo could be mediated through other signaling pathways leading finally to a decrease in the phosphorylation of GSK3 and a reduction in embryo development. Therefore, in conclusion, GSK3A/B serine phosphorylation was positively correlated with embryo development, indicating the importance of an accurate regulation of GSK3 activity during developmental stages to achieve normal bovine embryo development. PMID:20427566

  9. 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.

  10. 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

  11. 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

  12. Decreased glycogen synthase kinase-3 levels and activity contribute to Huntington's disease.

    PubMed

    Fernández-Nogales, Marta; Hernández, Félix; Miguez, Andrés; Alberch, Jordi; Ginés, Silvia; Pérez-Navarro, Esther; Lucas, José J

    2015-09-01

    Huntington's disease (HD) is a hereditary neurodegenerative disorder characterized by brain atrophy particularly in striatum leading to personality changes, chorea and dementia. Glycogen synthase kinase-3 (GSK-3) is a serine/threonine kinase in the crossroad of many signaling pathways that is highly pleiotropic as it phosphorylates more than hundred substrates including structural, metabolic, and signaling proteins. Increased GSK-3 activity is believed to contribute to the pathogenesis of neurodegenerative diseases like Alzheimer's disease and GSK-3 inhibitors have been postulated as therapeutic agents for neurodegeneration. Regarding HD, GSK-3 inhibitors have shown beneficial effects in cell and invertebrate animal models but no evident efficacy in mouse models. Intriguingly, those studies were performed without interrogating GSK-3 level and activity in HD brain. Here we aim to explore the level and also the enzymatic activity of GSK-3 in the striatum and other less affected brain regions of HD patients and of the R6/1 mouse model to then elucidate the possible contribution of its alteration to HD pathogenesis by genetic manipulation in mice. We report a dramatic decrease in GSK-3 levels and activity in striatum and cortex of HD patients with similar results in the mouse model. Correction of the GSK-3 deficit in HD mice, by combining with transgenic mice with conditional GSK-3 expression, resulted in amelioration of their brain atrophy and behavioral motor and learning deficits. Thus, our results demonstrate that decreased brain GSK-3 contributes to HD neurological phenotype and open new therapeutic opportunities based on increasing GSK-3 activity or attenuating the harmful consequences of its decrease. PMID:26082469

  13. Mechanisms underlying impaired GLUT-4 translocation in glycogen-supercompensated muscles of exercised rats.

    PubMed

    Kawanaka, K; Nolte, L A; Han, D H; Hansen, P A; Holloszy, J O

    2000-12-01

    Exercise training induces an increase in GLUT-4 in muscle. We previously found that feeding rats a high-carbohydrate diet after exercise, with muscle glycogen supercompensation, results in a decrease in insulin responsiveness so severe that it masks the effect of a training-induced twofold increase in GLUT-4 on insulin-stimulated muscle glucose transport. One purpose of this study was to determine whether insulin signaling is impaired. Maximally insulin-stimulated phosphatidylinositol (PI) 3-kinase activity was not significantly reduced, whereas protein kinase B (PKB) phosphorylation was approximately 50% lower (P < 0.01) in muscles of chow-fed, than in those of fasted, exercise-trained rats. Our second purpose was to determine whether contraction-stimulated glucose transport is also impaired. The stimulation of glucose transport and the increase in cell surface GLUT-4 induced by contractions were both decreased by approximately 65% in glycogen-supercompensated muscles of trained rats. The contraction-stimulated increase in AMP kinase activity, which has been implicated in the activation of glucose transport by contractions, was approximately 80% lower in the muscles of the fed compared with the fasted rats 18 h after exercise. These results show that both the insulin- and contraction-stimulated pathways for muscle glucose transport activation are impaired in glycogen-supercompensated muscles and provide insight regarding possible mechanisms. PMID:11093919

  14. Glycogen synthase kinase 3beta (GSK3beta) mediates 6-hydroxydopamine-induced neuronal death.

    PubMed

    Chen, Gang; Bower, Kimberly A; Ma, Cuiling; Fang, Shengyun; Thiele, Carol J; Luo, Jia

    2004-07-01

    The causes of sporadic Parkinson's disease (PD) are poorly understood. 6-Hydroxydopamine (6-OHDA), a PD mimetic, is widely used to model this neurodegenerative disorder in vitro and in vivo; however, the underlying mechanisms remain incompletely elucidated. We demonstrate here that 6-OHDA evoked endoplasmic reticulum (ER) stress, which was characterized by an up-regulation in the expression of GRP78 and GADD153 (Chop), cleavage of procaspase-12, and phosphorylation of eukaryotic initiation factor-2 alpha in a human dopaminergic neuronal cell line (SH-SY5Y) and cultured rat cerebellar granule neurons (CGNs). Glycogen synthase kinase-3 beta (GSK3beta) responds to ER stress, and its activity is regulated by phosphorylation. 6-OHDA significantly inhibited phosphorylation of GSK3beta at Ser9, whereas it induced hyperphosphorylation of Tyr216 with little effect on GSK3beta expression in SH-SY5Y cells and PC12 cells (a rat dopamine cell line), as well as CGNs. Furthermore, 6-OHDA decreased the expression of cyclin D1, a substrate of GSK3beta, and dephosphorylated Akt, the upstream signaling component of GSK3beta. Protein phosphatase 2A (PP2A), an ER stress-responsive phosphatase, was involved in 6-OHDA-induced GSK3beta dephosphorylation (Ser9). Blocking GSK3beta activity by selective inhibitors (lithium, TDZD-8, and L803-mts) prevented 6-OHDA-induced cleavage of caspase-3 and poly(ADP-ribose) polymerase (PARP), DNA fragmentations and cell death. With a tetracycline (Tet)-controlled TrkB inducible system, we demonstrated that activation of TrkB in SH-SY5Y cells alleviated 6-OHDA-induced GSK3beta dephosphorylation (Ser9) and ameliorated 6-OHDA neurotoxicity. TrkB activation also protected CGNs against 6-OHDA-induced damage. Although antioxidants also offered neuroprotection, they had little effect on 6-OHDA-induced GSK3beta activation. These results suggest that GSK3beta is a critical intermediate in pro-apoptotic signaling cascades that are associated with

  15. 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

  16. Aldose reductase modulates cardiac glycogen synthase kinase-3β phosphorylation during ischemia-reperfusion

    PubMed Central

    Abdillahi, Mariane; Ananthakrishnan, Radha; Vedantham, Srinivasan; Shang, Linshan; Zhu, Zhengbin; Rosario, Rosa; Zirpoli, Hylde; Bohren, Kurt M.; Gabbay, Kenneth H.

    2012-01-01

    Earlier studies have demonstrated that aldose reductase (AR) plays a key role in mediating ischemia-reperfusion (I/R) injury. Our objective was to investigate if AR mediates I/R injury by influencing phosphorylation of glycogen synthase kinase-3β (p-GSK3β). To investigate this issue, we used three separate models to study the effects of stress injury on the heart. Hearts isolated from wild-type (WT), human expressing AR transgenic (ARTg), and AR knockout (ARKO) mice were perfused with/without GSK3β inhibitors (SB-216763 and LiCl) and subjected to I/R. Ad-human AR (Ad-hAR)-expressing HL-1 cardiac cells were exposed to hypoxia (0.5% O2) and reoxygenation (20.9% O2) conditions. I/R in a murine model of transient occlusion and reperfusion of the left anterior descending coronary artery (LAD) was used to study if p-GSK3β was affected through increased AR flux. Lactate dehydrogenase (LDH) release and left ventricular developed pressure (LVDP) were measured. LVDP was decreased in hearts from ARTg mice compared with WT and ARKO after I/R, whereas LDH release and apoptotic markers were increased (P < 0.05). p-GSK3β was decreased in ARTg hearts compared with WT and ARKO (P < 0.05). In ARKO, p-GSK3β and apoptotic markers were decreased compared with WT (P < 0.05). WT and ARTg hearts perfused with GSK3β inhibitors improved p-GSK3β expression and LVDP and exhibited decreased LDH release, apoptosis, and mitochondrial pore opening (P < 0.05). Ad-hAR-expressing HL-1 cardiac cells, exposed to hypoxia (0.5% O2) and reoxygenation (20.9% O2), had greater LDH release compared with control HL-1 cells (P < 0.05). p-GSK3β was decreased and correlated with increased apoptotic markers in Ad-hAR HL-1 cells (P < 0.05). Treatment with phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) inhibitor increased injury demonstrated by increased LDH release in ARTg, WT, and ARKO hearts and in Ad-hAR-expressing HL-1 cells. Cells treated with protein kinase C (PKC) α/β inhibitor

  17. Aldose reductase modulates cardiac glycogen synthase kinase-3β phosphorylation during ischemia-reperfusion.

    PubMed

    Abdillahi, Mariane; Ananthakrishnan, Radha; Vedantham, Srinivasan; Shang, Linshan; Zhu, Zhengbin; Rosario, Rosa; Zirpoli, Hylde; Bohren, Kurt M; Gabbay, Kenneth H; Ramasamy, Ravichandran

    2012-08-01

    Earlier studies have demonstrated that aldose reductase (AR) plays a key role in mediating ischemia-reperfusion (I/R) injury. Our objective was to investigate if AR mediates I/R injury by influencing phosphorylation of glycogen synthase kinase-3β (p-GSK3β). To investigate this issue, we used three separate models to study the effects of stress injury on the heart. Hearts isolated from wild-type (WT), human expressing AR transgenic (ARTg), and AR knockout (ARKO) mice were perfused with/without GSK3β inhibitors (SB-216763 and LiCl) and subjected to I/R. Ad-human AR (Ad-hAR)-expressing HL-1 cardiac cells were exposed to hypoxia (0.5% O(2)) and reoxygenation (20.9% O(2)) conditions. I/R in a murine model of transient occlusion and reperfusion of the left anterior descending coronary artery (LAD) was used to study if p-GSK3β was affected through increased AR flux. Lactate dehydrogenase (LDH) release and left ventricular developed pressure (LVDP) were measured. LVDP was decreased in hearts from ARTg mice compared with WT and ARKO after I/R, whereas LDH release and apoptotic markers were increased (P < 0.05). p-GSK3β was decreased in ARTg hearts compared with WT and ARKO (P < 0.05). In ARKO, p-GSK3β and apoptotic markers were decreased compared with WT (P < 0.05). WT and ARTg hearts perfused with GSK3β inhibitors improved p-GSK3β expression and LVDP and exhibited decreased LDH release, apoptosis, and mitochondrial pore opening (P < 0.05). Ad-hAR-expressing HL-1 cardiac cells, exposed to hypoxia (0.5% O(2)) and reoxygenation (20.9% O(2)), had greater LDH release compared with control HL-1 cells (P < 0.05). p-GSK3β was decreased and correlated with increased apoptotic markers in Ad-hAR HL-1 cells (P < 0.05). Treatment with phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) inhibitor increased injury demonstrated by increased LDH release in ARTg, WT, and ARKO hearts and in Ad-hAR-expressing HL-1 cells. Cells treated with protein kinase C (PKC)

  18. Adenomatous polyposis coli (APC) regulates multiple signaling pathways by enhancing glycogen synthase kinase-3 (GSK-3) activity.

    PubMed

    Valvezan, Alexander J; Zhang, Fang; Diehl, J Alan; Klein, Peter S

    2012-02-01

    Glycogen synthase kinase-3 (GSK-3) is essential for many signaling pathways and cellular processes. As Adenomatous Polyposis Coli (APC) functions in many of the same processes, we investigated a role for APC in the regulation of GSK-3-dependent signaling. We find that APC directly enhances GSK-3 activity. Furthermore, knockdown of APC mimics inhibition of GSK-3 by reducing phosphorylation of glycogen synthase and by activating mTOR, revealing novel roles for APC in the regulation of these enzymes. Wnt signaling inhibits GSK-3 through an unknown mechanism, and this results in both stabilization of β-catenin and activation of mTOR. We therefore hypothesized that Wnts may regulate GSK-3 by disrupting the interaction between APC and the Axin-GSK-3 complex. We find that Wnts rapidly induce APC dissociation from Axin, correlating with β-catenin stabilization. Furthermore, Axin interaction with the Wnt co-receptor LRP6 causes APC dissociation from Axin. We propose that APC regulates multiple signaling pathways by enhancing GSK-3 activity, and that Wnts induce APC dissociation from Axin to reduce GSK-3 activity and activate downstream signaling. APC regulation of GSK-3 also provides a novel mechanism for Wnt regulation of multiple downstream effectors, including β-catenin and mTOR. PMID:22184111

  19. ADIPOCYTES FROM WOMEN WITH POLYCYSTIC OVARY SYNDROME DEMONSTRATE ALTERED PHOSPHORYLATION AND ACTIVITY OF GLYCOGEN SYNTHASE KINASE 3

    PubMed Central

    Chang, Wendy; Goodarzi, Mark O.; Williams, Heith; Magoffin, Denis A.; Pall, Marita; Azziz, Ricardo

    2009-01-01

    Objective To test the hypothesis that an abnormality in glycogen synthase kinase-3 (GSK3) is a pathogenic factor in PCOS. Design Prospective experimental study (adipocytes). Setting Tertiary care academic medical center and teaching hospital Patients Patients with PCOS and healthy controls. Interventions Blood sampling, physical exam, biopsy of subcutaneous lower abdominal fat. Main Outcome Measure(s) Glucose transport and protein levels and phosphorylation state of GSK3α and GSK3β in adipocytes, assessment of GSK3β activity. Results Basal protein levels of glycogen synthase kinase (GSK3α and GSK3β) did not differ between controls and women with PCOS, nor did basal or insulin-stimulated levels of serine phosphorylated GSK3α. However, in adipocytes of PCOS women insulin stimulation was not associated with increased serine phosphorylation of GSK3β, in contrast to controls. Tyrosine phosphorylation of GSK3β was also higher in PCOS compared to controls. Consistent with the phosphorylation data, GSK3β activity was elevated in PCOS adipocytes. Conclusions These data suggest GSK3β is hyperactivated and resistant to downregulation by insulin in PCOS. Using physiologic approaches, we demonstrated that abnormal GSK3β regulation is a potential mechanism for the insulin resistance seen in some women with PCOS, which may contribute to their development of the syndrome. PMID:18178198

  20. 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.

  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. Antisense Oligonucleotide-mediated Suppression of Muscle Glycogen Synthase 1 Synthesis as an Approach for Substrate Reduction Therapy of Pompe Disease.

    PubMed

    Clayton, Nicholas P; Nelson, Carol A; Weeden, Timothy; Taylor, Kristin M; Moreland, Rodney J; Scheule, Ronald K; Phillips, Lucy; Leger, Andrew J; Cheng, Seng H; Wentworth, Bruce M

    2014-01-01

    Pompe disease is an autosomal recessive disorder caused by a deficiency of acid α-glucosidase (GAA; EC 3.2.1.20) and the resultant progressive lysosomal accumulation of glycogen in skeletal and cardiac muscles. Enzyme replacement therapy using recombinant human GAA (rhGAA) has proven beneficial in addressing several aspects of the disease such as cardiomyopathy and aberrant motor function. However, residual muscle weakness, hearing loss, and the risks of arrhythmias and osteopenia persist despite enzyme therapy. Here, we evaluated the relative merits of substrate reduction therapy (by inhibiting glycogen synthesis) as a potential adjuvant strategy. A phosphorodiamidate morpholino oligonucleotide (PMO) designed to invoke exon skipping and premature stop codon usage in the transcript for muscle specific glycogen synthase (Gys1) was identified and conjugated to a cell penetrating peptide (GS-PPMO) to facilitate PMO delivery to muscle. GS-PPMO systemic administration to Pompe mice led to a dose-dependent decrease in glycogen synthase transcripts in the quadriceps, and the diaphragm but not the liver. An mRNA response in the heart was seen only at the higher dose tested. Associated with these decreases in transcript levels were correspondingly lower tissue levels of muscle specific glycogen synthase and activity. Importantly, these reductions resulted in significant decreases in the aberrant accumulation of lysosomal glycogen in the quadriceps, diaphragm, and heart of Pompe mice. Treatment was without any overt toxicity, supporting the notion that substrate reduction by GS-PPMO-mediated inhibition of muscle specific glycogen synthase represents a viable therapeutic strategy for Pompe disease after further development. PMID:25350581

  3. 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

  4. The glycogen synthase 2 gene (Gys2) displays parallel evolution between Old World and New World fruit bats.

    PubMed

    Qian, Yamin; Fang, Tao; Shen, Bin; Zhang, Shuyi

    2014-01-01

    Frugivorous and nectarivorous bats rely largely on hepatic glycogenesis and glycogenolysis for postprandial blood glucose disposal and maintenance of glucose homeostasis during short time starvation, respectively. The glycogen synthase 2 encoded by the Gys2 gene plays a critical role in liver glycogen synthesis. To test whether the Gys2 gene has undergone adaptive evolution in bats with carbohydrate-rich diets in relation to their insect-eating sister taxa, we sequenced the coding region of the Gys2 gene in a number of bat species, including three Old World fruit bats (OWFBs) (Pteropodidae) and two New World fruit bats (NWFBs) (Phyllostomidae). Our results showed that the Gys2 coding sequences are highly conserved across all bat species we examined, and no evidence of positive selection was detected in the ancestral branches leading to OWFBs and NWFBs. Our explicit convergence test showed that posterior probabilities of convergence between several branches of OWFBs, and the NWFBs were markedly higher than that of divergence. Three parallel amino acid substitutions (Q72H, K371Q, and E666D) were detected among branches of OWFBs and NWFBs. Tests for parallel evolution showed that two parallel substitutions (Q72H and E666D) were driven by natural selection, while the K371Q was more likely to be fixed randomly. Thus, our results suggested that the Gys2 gene has undergone parallel evolution on amino acid level between OWFBs and NWFBs in relation to their carbohydrate metabolism. PMID:24258790

  5. Insulin alters cAMP-activated lipolysis but not cAMP-inhibited glycogen synthase in permeabilized adipocytes

    SciTech Connect

    Mooney, R.A.; Wisniewski, J.L.

    1986-05-01

    Lipolysis and, to a lesser extent, glycogen synthase activity are regulated in adipocytes by cellular cAMP and counter-regulated by insulin. These activities were measured in situ in digitonin (20 ..mu..g/ml) permeabilized rat adipocytes. Incorporation of /sup 3/H UDP-glucose into endogenous glycogen in the presence of KF, EDTA and 10mM glucose-6-phosphate was the basis of the G.S. assay. Cellular GS activity determined by this technique was 1.4 +/- 0.2 fold greater than that of matched homogenates. Insulin treatment of intact cells prior to permeabilization increased GS activity ratio (-/+ G-6-P) 2.5 fold when subsequently measured by the in situ assay. Following digitonin permeabilization, addition of cAMP to the suspension medium increased lipolysis 7 fold and decreased GS activity ratio to 0.38 +/- 0.01 from a basal value of 0.44 +/- 0.06. ATP had a negligible effect on lipolysis but decreased GS to 0.16 +/- 0.04. ATP plus cAMP was only slightly more effective on GS than ATP alone. Insulin at 10/sup -9/M inhibited cAMP-dependent lipolysis by 27% but had no effect on the cAMP- or ATP-dependent decrease in GS. These results suggest that insulin's counter-regulatory mechanisms on these two cAMP-dependent processes may be different.

  6. Focal adhesion kinase-mediated activation of glycogen synthase kinase 3β regulates IL-33 receptor internalization and IL-33 signaling

    PubMed Central

    Zhao, Jing; Wei, Jianxin; Bowser, Rachel K; Traister, Russell S; Fan, Ming-Hui; Zhao, Yutong

    2014-01-01

    IL-33, a relatively new member of the IL-1 cytokine family, plays a crucial role in allergic inflammation and acute lung injury. ST2L, the receptor for IL-33, is expressed on immune effector cells and lung epithelia, and plays a critical role in triggering inflammation. We have previously shown that ST2L stability is regulated by the ubiquitin-proteasome system, however its upstream internalization has not been studied. Here, we demonstrate that glycogen synthase kinase 3β (GSK3β) regulates ST2L internalization and IL-33 signaling. IL-33 treatment induced ST2L internalization, an effect was attenuated by inhibition or downregulation of GSK3β. GSK3β was found to interact with ST2L on serine residue 446 in response to IL-33 treatment. GSK3β binding site mutant (ST2LS446A) and phosphorylation site mutant (ST2LS442A) are resistant to IL-33-induced ST2L internalization. We also found that IL-33 activated focal adhesion kinase (FAK). Inhibition of FAK impaired IL-33-induced GSK3β activation and ST2L internalization. Further, inhibition of ST2L internalization enhanced IL-33-induced cytokine release in lung epithelial cells. These results suggest that modulation of the ST2L internalization by FAK/GSK3β might serve as a unique strategy to lessen pulmonary inflammation. PMID:25472995

  7. Glycogen synthase kinase-3β regulates tumor necrosis factor-related apoptosis inducing ligand (TRAIL)-induced apoptosis via the NF-κB pathway in hepatocellular carcinoma

    PubMed Central

    FU, KAI; PAN, HUAZHENG; LIU, SHIHAI; LV, JING; WAN, ZHAOJUN; LI, JIAO; SUN, QING; LIANG, JUN

    2015-01-01

    Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is known for its ability to selectively induce apoptosis in malignant cells. However, human hepatocellular carcinoma (HCC) cells display resistance to TRAIL-induced cell death. The present study investigated whether TRAIL-induced apoptosis in HCC cells was enhanced by the administration of an inhibitor of glycogen synthase kinase-3β (GSK-3β) or by short hairpin RNA-mediated inhibition of GSK-3β. The results of the current study demonstrated that inhibition of GSK-3β significantly impairs the expression of the nuclear factor-κB (NF-κB) target genes Bcl-xL and clAP2 in HCC cells (P<0.05). This indicates that GSK-3β may regulate NF-κB target genes involved in cell survival. Furthermore, knockdown of Bcl-xL significantly enhanced the sensitizing effect of GSK-3β inhibitor on TRAIL-induced apoptosis (P<0.05). Overall, the present study provides a rationale for further exploration of GSK-3β inhibition combined with TRAIL as a novel treatment for HCC. PMID:26788169

  8. Potential Role of Glycogen Synthase Kinase-3β in Regulation of Myocardin Activity in Human Vascular Smooth Muscle Cells.

    PubMed

    Zhou, Yi-Xia; Shi, Zhan; Singh, Pavneet; Yin, Hao; Yu, Yan-Ni; Li, Long; Walsh, Michael P; Gui, Yu; Zheng, Xi-Long

    2016-02-01

    Glycogen synthase kinase (GSK)-3β, a serine/threonine kinase with an inhibitory role in glycogen synthesis in hepatocytes and skeletal muscle, is also expressed in cardiac and smooth muscles. Inhibition of GSK-3β results in cardiac hypertrophy through reducing phosphorylation and increasing transcriptional activity of myocardin, a transcriptional co-activator for serum response factor. Myocardin plays critical roles in differentiation of smooth muscle cells (SMCs). This study, therefore, aimed to examine whether and how inhibition of GSK-3β regulates myocardin activity in human vascular SMCs. Treatment of SMCs with the GSK-3β inhibitors AR-A014418 and TWS 119 significantly reduced endogenous myocardin activity, as indicated by lower expression of myocardin target genes (and gene products), CNN1 (calponin), TAGLN1 (SM22), and ACTA2 (SM α-actin). In human SMCs overexpressing myocardin through the T-REx system, treatment with either GSK-3β inhibitor also inhibited the expression of CNN1, TAGLN1, and ACTA2. These effects of GSK-3β inhibitors were mimicked by transfection with GSK-3β siRNA. Notably, both AR-A014418 and TWS 119 decreased the serine/threonine phosphorylation of myocardin. The chromatin immunoprecipitation assay showed that AR-A014418 treatment reduced myocardin occupancy of the promoter of the myocardin target gene ACTA2. Overexpression of a dominant-negative GSK-3β mutant in myocardin-overexpressing SMCs reduced the expression of calponin, SM22, and SM α-actin. As expected, overexpression of constitutively active or wild-type GSK-3β in SMCs without myocardin overexpression increased expression of these proteins. In summary, our results indicate that inhibition of GSK-3β reduces myocardin transcriptional activity, suggesting a role for GSK-3β in myocardin transcriptional activity and smooth muscle differentiation. PMID:26129946

  9. 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

  10. Glycogen synthase kinase 3 is part of the molecular machinery regulating the adaptive response to LPS stimulation in microglial cells.

    PubMed

    Ajmone-Cat, Maria Antonietta; D'Urso, Maria Cristina; di Blasio, Giorgia; Brignone, Maria Stefania; De Simone, Roberta; Minghetti, Luisa

    2016-07-01

    Repeated stimulation of TLR4 signaling by lipopolysaccharide (LPS) in microglia induces a state of tolerance/sensitization consisting in the reprogramming of the expression of pro-inflammatory genes in favor of anti-inflammatory ones. The molecular mechanisms underlying this adaptive response are far to be elucidated. Glycogen synthase kinase 3 (GSK3) has emerged as crucial regulator of TLR signaling, mediating the balance between pro- and anti-inflammatory functions in both periphery and central nervous system. The present study extends this notion identifying GSK3 as part of the molecular machinery regulating the LPS-adaptive response in microglial cells, by using primary microglial cultures and organotypic hippocampal slices (OHSCs). We found that lithium chloride (LiCl), a widely used GSK3 inhibitor and the mainstay treatment for bipolar disorder, reinforced the LPS adaptive response by enhancing both downregulation of pro-inflammatory genes (inducible nitric oxide synthase, interleukin 1β, interleukin 6, tumor necrosis factor α), and upregulation of genes typically associated to anti-inflammatory functions (interleukin 10 and MRC1). The effects of GSK3 inhibition were mimicked by Wnt3a, added exogenously, and reversed by Inhibitor of Wnt-Response-1-endo, a pharmacological disruptor of the canonical Wnt/β-catenin pathway, and GW9662, a selective peroxisome proliferator activated receptor γ antagonist, suggesting that these two pathways are involved in the regulation of LPS-tolerance/sensitization by GSK. Finally, LiCl treatment of OHSCs enhanced the protective functional consequences of the microglial adaptive response to LPS on oligodendrocyte maturation, as indicated by MBP mRNA upregulation. These results further indicate GSK3 as key component in the orchestration of neuroinflammation and target for neuroprotective strategies. PMID:26593276

  11. 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

  12. 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 +/.

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

    PubMed Central

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

    2015-01-01

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

  14. 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.

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

    PubMed

    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. PMID:19995556

  16. 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.

  17. Lithium Regulates Keratinocyte Proliferation Via Glycogen Synthase Kinase 3 and NFAT2 (Nuclear Factor of Activated T Cells 2)

    PubMed Central

    Hampton, Philip J; Jans, Ralph; Flockhart, Ross J; Parker, Graeme; Reynolds, Nick J

    2012-01-01

    Certain environmental factors including drugs exacerbate or precipitate psoriasis. Lithium is the commonest cause of drug-induced psoriasis but underlying mechanisms are currently unknown. Lithium inhibits glycogen synthase kinase 3 (GSK-3). As lithium does not exacerbate other T-cell-mediated chronic inflammatory diseases, we investigated whether lithium may be acting directly on epidermal keratinocytes by inhibiting GSK-3. We report that lithium-induced keratinocyte proliferation at therapeutically relevant doses (1–2 mM) and increased the proportion of cells in S phase of the cell cycle. Inhibition of GSK-3 in keratinocytes by retroviral transduction of GSK-binding protein (an endogenous inhibitory protein) or through a highly selective pharmacological inhibitor also resulted in increased keratinocyte proliferation. Nuclear factor of activated T cells (NFAT) is an important substrate for GSK-3 and for cyclosporin, an effective treatment for psoriasis that inhibits NFAT activation in keratinocytes as well as in lymphocytes. Both lithium and genetic/pharmacological inhibition of GSK-3 resulted in increased nuclear localization of NFAT2 (NFATc1) and increased NFAT transcriptional activation. Finally, retroviral transduction of NFAT2 increased keratinocyte proliferation whereas siRNA-mediated knockdown of NFAT2 reduced keratinocyte proliferation and decreased epidermal thickness in an organotypic skin equivalent model. Taken together, these data identify GSK-3 and NFAT2 as key regulators of keratinocyte proliferation and as potential molecular targets relevant to lithium-provoked psoriasis. J. Cell. Physiol. 227: 1529–1537, 2012. © 2011 Wiley Periodicals, Inc. PMID:21678407

  18. 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. PMID:24803542

  19. Interaction of yeast repressor-activator protein Ume6p with glycogen synthase kinase 3 homolog Rim11p.

    PubMed Central

    Malathi, K; Xiao, Y; Mitchell, A P

    1997-01-01

    Meiosis and expression of early meiotic genes in the budding yeast Saccharomyces cerevisiae depend upon Rim11p, Ume6p, and Ime1p. Rim11p (also called Mds1p and ScGSK3) is a protein kinase related to glycogen synthase kinase 3 (GSK3); Ume6p is an architectural transcription factor; and Imelp is a Ume6p-binding protein that provides a transcriptional activation domain. Rim11p is required for Ime1p-Ume6p interaction, and prior studies have shown that Rim11p binds to and phosphorylates Ime1p. We show here that Rim11p binds to and phosphorylates Ume6p, as well. Amino acid substitutions in Ume6p that alter a consensus GSK3 site reduce or abolish Rim11p-Ume6p interaction and Rim11p-dependent phosphorylation, and they cause defects in interaction between Ume6p and Ime1p and in meiotic gene expression. Therefore, interaction between Rim11p and Ume6p, resulting in phosphorylation of Ume6p, is required for Ime1p-Ume6p complex formation. Rim11p, like metazoan GSK3beta, phosphorylates both interacting subunits of a target protein complex. PMID:9372955

  20. The Canonical Wnt Signal Restricts the Glycogen Synthase Kinase 3/Fbw7-Dependent Ubiquitination and Degradation of Eya1 Phosphatase

    PubMed Central

    Sun, Ye

    2014-01-01

    Haploinsufficiency of Eya1 causes the branchio-oto-renal (BOR) syndrome, and abnormally high levels of Eya1 are linked to breast cancer progression and poor prognosis. Therefore, regulation of Eya1 activity is key to its tissue-specific functions and oncogenic activities. Here, we show that Eya1 is posttranslationally modified by ubiquitin and that its ubiquitination level is self-limited to prevent premature degradation. Eya1 has an evolutionarily conserved CDC4 phosphodegron (CPD) signal, a target site of glycogen synthase kinase 3 (GSK3) kinase and Fbw7 ubiquitin ligase, which is required for Eya1 ubiquitination. Genetic deletion of Fbw7 and pharmacological inhibition of GSK3 significantly decrease Eya1 ubiquitination. Conversely, activation of the phosphatidylinositol 3-kinase (PI3K)/Akt and the canonical Wnt signal suppresses Eya1 ubiquitination. Compound Eya1+/−; Wnt9b+/− mutants exhibit an increased penetrance of renal defect, indicating that they function in the same genetic pathway in vivo. Together, these findings reveal that the canonical Wnt and PI3K/Akt signal pathways restrain the GSK3/Fbw7-dependent Eya1 ubiquitination, and they further suggest that dysregulation of this novel axis contributes to tumorigenesis. PMID:24752894

  1. 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. PMID:22257026

  2. Cocaine regulates protein kinase B and glycogen synthase kinase-3 activity in selective regions of rat brain

    PubMed Central

    SA, Perrine; JS, Miller; EM, Unterwald

    2008-01-01

    Protein kinase B (Akt) signaling regulates dopamine-mediated locomotor behaviors. Here the ability of cocaine to regulate Akt and glycogen synthase kinase-3 (GSK3) was studied. Rats were injected with cocaine or saline in a binge-pattern, which consisted of 3 daily injections of 15 mg/kg cocaine or 1 ml/kg saline spaced one hour apart for 1, 3 or 14 days. Amygdala, nucleus accumbens, caudate putamen and hippocampus tissues were dissected 30 minutes following the last injection and analyzed for phosphorylated and total Akt and GSK3(α & β) protein levels using Western blot analysis. Phosphorylation of Akt on the threonine-308 residue was significantly reduced in the nucleus accumbens and increased in the amygdala after 1 day of cocaine treatment; however, these effects were not accompanied by a significant decrease in GSK3 phosphorylation. Phosphorylation of Akt and GSK3 were significantly reduced after 14 days of cocaine administration, an effect that was only observed in the amygdala. Cocaine did not alter Akt or GSK3 phosphorylation in the caudate putamen or hippocampus. The findings in nucleus accumbens may reflect dopaminergic motor-stimulant activity caused by acute cocaine, whereas the effects in amygdala may be associated with changes in emotional state that occur after acute and chronic cocaine exposure. PMID:18717814

  3. Endoplasmic Reticulum Stress Accelerates p53 Degradation by the Cooperative Actions of Hdm2 and Glycogen Synthase Kinase 3β

    PubMed Central

    Pluquet, Olivier; Qu, Li-Ke; Baltzis, Dionissios; Koromilas, Antonis E.

    2005-01-01

    Inactivation of the tumor suppressor p53 by degradation is a mechanism utilized by cells to adapt to endoplasmic reticulum (ER) stress. However, the mechanisms of p53 destabilization by ER stress are not known. We demonstrate here that the E3 ubiquitin-ligase Hdm2 is essential for the nucleocytoplasmic transport and proteasome-dependent degradation of p53 in ER-stressed cells. We also demonstrate that p53 phosphorylation at S315 and S376 is required for its nuclear export and degradation by Hdm2 without interfering with the ubiquitylation process. Furthermore, we show that p53 destabilization in unstressed cells utilizes the cooperative action of Hdm2 and glycogen synthase kinase 3β, a process that is enhanced in cells exposed to ER stress. In contrast to other stress pathways that stabilize p53, our findings further substantiate a negative role of ER stress in p53 activation with important implications for the function of the tumor suppressor in cells with a dysfunctional ER. PMID:16227590

  4. 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. PMID:16548769

  5. 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

  6. 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.

  7. Progesterone Receptor A Stability Is Mediated by Glycogen Synthase Kinase-3β in the Brca1-deficient Mammary Gland*

    PubMed Central

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

    2013-01-01

    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-AS390A 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. PMID:23880761

  8. 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

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

    PubMed

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

    2016-03-01

    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

  10. Inhibition of glycogen synthase kinase-3 beta induces apoptosis and mitotic catastrophe by disrupting centrosome regulation in cancer cells

    PubMed Central

    Yoshino, Yuki; Ishioka, Chikashi

    2015-01-01

    Glycogen synthase kinase-3 beta (GSK-3β) has been investigated as a therapeutic target for numerous human diseases including cancer because of their diverse cellular functions. Although GSK-3β inhibitors have been investigated as anticancer reagents, precise biological mechanisms remain to be determined. In this study, we investigated the anticancer effects of GSK-3β inhibitors on cancer cell lines and observed centrosome dysregulation which resulted in abnormal mitosis. Mitotic checkpoints sensed the mitotic abnormalities and induced apoptosis. For cells that were inherently resistant to apoptosis, cell death distinct from apoptosis was induced. After GSK-3β inhibitor treatment, these cells exhibited characteristic features of mitotic catastrophe, including distended and multivesiculated nuclei and inappropriate reductions in cyclin B1 expression. This suggested that mitotic catastrophe was an alternative mechanism in cells resistant to apoptosis. Although the role of GSK-3β in centrosomes has not yet been clarified, phosphorylated GSK-3β was localised in centrosomes. From these data, GSK-3β seems to regulate centrosome function. Thus, we propose that centrosome dysregulation is an important mechanism for the anticancer effects of GSK-3β inhibitors and that mitotic catastrophe serves as a safe-guard system to remove cells with any mitotic abnormalities induced by GSK-3β inhibition. PMID:26292722

  11. New applications for known drugs: Human glycogen synthase kinase 3 inhibitors as modulators of Aspergillus fumigatus growth.

    PubMed

    Sebastián, Víctor; Manoli, Maria-Tsampika; Pérez, Daniel I; Gil, Carmen; Mellado, Emilia; Martínez, Ana; Espeso, Eduardo A; Campillo, Nuria E

    2016-06-30

    Invasive aspergillosis (IA) is one of the most severe forms of fungi infection. IA disease is mainly due to Aspergillus fumigatus, an air-borne opportunistic pathogen. Mortality rate caused by IA is still very high (50-95%), because of difficulty in early diagnostics and reduced antifungal treatment options, thus new and efficient drugs are necessary. The aim of this work is, using Aspergillus nidulans as non-pathogen model, to develop efficient drugs to treat IA. The recent discovered role of glycogen synthase kinase-3 homologue, GskA, in A. fumigatus human infection and our previous experience on human GSK-3 inhibitors focus our attention on this kinase as a target for the development of antifungal drugs. With the aim to identify effective inhibitors of colonial growth of A. fumigatus we use A. nidulans as an accurate model for in vivo and in silico studies. Several well-known human GSK-3β inhibitors were tested for inhibition of A. nidulans colony growth. Computational tools as docking studies and binding site prediction was used to explain the different biological profile of the tested inhibitors. Three of the five tested hGSK3β inhibitors are able to reduce completely the colonial growth by covalent bind to the enzyme. Therefore these compounds may be useful in different applications to eradicate IA. PMID:27131621

  12. 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

  13. Deficiency of sphingomyelin synthase-1 but not sphingomyelin synthase-2 causes hearing impairments in mice.

    PubMed

    Lu, Mei-Hong; Takemoto, Makoto; Watanabe, Ken; Luo, Huan; Nishimura, Masataka; Yano, Masato; Tomimoto, Hidekazu; Okazaki, Toshiro; Oike, Yuichi; Song, Wen-Jie

    2012-08-15

    Sphingomyelin (SM) is a sphingolipid reported to function as a structural component of plasma membranes and to participate in signal transduction. The role of SM metabolism in the process of hearing remains controversial. Here, we examined the role of SM synthase (SMS), which is subcategorized into the family members SMS1 and SMS2, in auditory function. Measurements of auditory brainstem response (ABR) revealed hearing impairment in SMS1−/− mice in a low frequency range (4–16 kHz). As a possible mechanism of this impairment, we found that the stria vascularis (SV) in these mice exhibited atrophy and disorganized marginal cells. Consequently, SMS1−/− mice exhibited significantly smaller endocochlear potentials (EPs). As a possible mechanism for EP reduction, we found altered expression patterns and a reduced level of KCNQ1 channel protein in the SV of SMS1−/− mice. These mice also exhibited reduced levels of distortion product otoacoustic emissions. Quantitative comparison of the SV atrophy, KCNQ1 expression, and outer hair cell density at the cochlear apical and basal turns revealed no location dependence, but more macrophage invasion into the SV was observed in the apical region than the basal region, suggesting a role of cochlear location-dependent oxidative stress in producing the frequency dependence of hearing loss in SMS1−/− mice. Elevated ABR thresholds, decreased EPs, and abnormal KCNQ1 expression patterns in SMS1−/− mice were all found to be progressive with age. Mice lacking SMS2, however, exhibited neither detectable hearing loss nor changes in their EPs. Taken together, our results suggest that hearing impairments occur in SMS1−/− but not SMS2−/− mice. Defects in the SV with subsequent reductions in EPs together with hair cell dysfunction may account, at least partially, for hearing impairments in SMS1−/− mice. PMID:22641779

  14. Impairment of calcium mobilization in phagocytic cells in glycogen storage disease type 1b.

    PubMed

    Korchak, H M; Garty, B Z; Stanley, C A; Baker, L; Douglas, S D; Kilpatrick, L

    1993-01-01

    Patients with glycogen storage disease (GSD) type 1b, in contrast to patients with GSD 1a, are susceptible to recurrent bacterial infections suggesting defective phagocytic function. We have demonstrated a selective defect in respiratory burst activity but not in degranulation by phagocytic cells in GSD 1b but not in GSD 1a. The respiratory burst abnormality in phagocytic cells from GSD 1b patients was associated with impaired calcium mobilization, whereas these processes were normal in GSD 1a patients. Therefore, the alteration in calcium mobilization was an indication of a signalling defect in phagocytic cells from GSD 1b. However, calcium mobilization was normal in lymphocytes, indicating that defective calcium mobilization was not a global finding in circulating leukocytes, but was specific to phagocytic cells. Calcium mobilization in response to ionomycin was reduced suggesting decreased calcium stores in GSD 1b neutrophils. Therefore, altered phagocytic cell function in GSD 1b patients appears to be associated with diminished calcium mobilization and defective calcium stores. This defective calcium signalling was associated with a selective defect in respiratory burst activity but not degranulation. PMID:8319725

  15. Impaired clearance of accumulated lysosomal glycogen in advanced Pompe disease despite high-level vector-mediated transgene expression

    PubMed Central

    Sun, Baodong; Zhang, Haoyue; Bird, Andrew; Li, Songtao; Young, Sarah P.; Koeberl, Dwight D.

    2013-01-01

    Background Infantile-onset glycogen storage disease type II (GSD-II; Pompe disease; MIM 232300) causes death early in childhood from cardiorespiratory failure in absence of effective treatment, whereas late-onset Pompe disease causes a progressive skeletal myopathy. The limitations of enzyme replacement therapy could potentially be addressed with adeno-associated virus (AAV) vector-mediated gene therapy. Methods AAV vectors containing tissue-specific regulatory cassettes, either liver-specific or muscle-specific, were administered to 12 and 17 month old Pompe disease mice to evaluate the efficacy of gene therapy in advanced Pompe disease. Biochemical correction was evaluated through GAA activity and glycogen content analyses of the heart and skeletal muscle. Western blotting, urinary biomarker, and Rotarod performance were evaluated following vector administration. Results The AAV vector containing the liver-specific regulatory cassette secreted high-level hGAA into the blood and corrected glycogen storage in the heart and diaphragm. The biochemical correction of the heart and diaphragm was associated with efficacy, as reflected by increased Rotarod performance; however, the clearance of glycogen from skeletal muscles was relatively impaired, in comparison with younger Pompe disease mice. An alternative vector containing a muscle-specific regulatory cassette transduced skeletal muscle with high efficiency, but also failed to achieve complete clearance of accumulated glycogen. Decreased transduction of the heart and liver in older mice, especially in females, was implicated as a cause for reduced efficacy in advanced Pompe disease. Conclusion The impaired efficacy of AAV vector-mediated gene therapy in old Pompe disease mice emphasized the need for early treatment to achieve full efficacy. PMID:19621331

  16. Efficacy of small-molecule glycogen synthase kinase-3 inhibitors in the postnatal rat model of tau hyperphosphorylation

    PubMed Central

    Selenica, M-L; Jensen, H S; Larsen, A K; Pedersen, M L; Helboe, L; Leist, M; Lotharius, J

    2007-01-01

    Background and purpose: Glycogen synthase kinase-3 (GSK-3) affects neuropathological events associated with Alzheimeŕs disease (AD) such as hyperphosphorylation of the protein, tau. GSK-3β expression, enzyme activity and tau phosphorylated at AD-relevant epitopes are elevated in juvenile rodent brains. Here, we assess five GSK-3β inhibitors and lithium in lowering phosphorylated tau (p-tau) and GSK-3β enzyme activity levels in 12-day old postnatal rats. Experimental approach: Brain levels of inhibitors following treatment in vivo were optimized based on pharmacokinetic data. At optimal doses, p-tau (Ser396) levels in brain tissue was measured by immunoblotting and correlated with GSK-3β enzyme activities in the same tissues. Effects of GSK inhibitors on p-tau, GSK-3β activities and cell death were measured in a human neuronal cell line (LUHMES). Key results: Lithium and CHIR98014 reduced tau phosphorylation (Ser396) in the cortex and hippocampus of postnatal rats, while Alsterpaullone and SB216763 were effective only in hippocampus. AR-A014418 and Indirubin-3′-monoxime were ineffective in either brain region. Inhibition of p-tau in brain required several-fold higher levels of GSK inhibitors than the IC50 values obtained in recombinant or cell-based GSK-3β enzyme activity assays. The inhibitory effect on GSK-3β activity ex vivo correlated with protection against cell death and decrease of p-tau- in LUHMES cells, using low μM inhibitor concentrations. Conclusions and Implications: Selective small-molecule inhibitors of GSK-3 reduce tau phosphorylation in vivo. These findings corroborate earlier suggestions that GSK-3β may be an attractive target for disease-modification in AD and related conditions where tau phosphorylation is believed to contribute to disease pathogenesis. PMID:17906685

  17. 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

  18. 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

  19. Phosphorylation of tau by glycogen synthase kinase 3beta affects the ability of tau to promote microtubule self-assembly.

    PubMed Central

    Utton, M A; Vandecandelaere, A; Wagner, U; Reynolds, C H; Gibb, G M; Miller, C C; Bayley, P M; Anderton, B H

    1997-01-01

    To study the effects of phosphorylation by glycogen synthase kinase-3beta (GSK-3beta) on the ability of the microtubule-associated protein tau to promote microtubule self-assembly, tau isoform 1 (foetal tau) and three mutant forms of this tau isoform were investigated. The three mutant forms of tau had the following serine residues, known to be phosphorylated by GSK-3, replaced with alanine residues so as to preclude their phosphorylation: (1) Ser-199 and Ser-202 (Ser-199/202-->Ala), (2) Ser-235 (Ser-235-->Ala) and (3) Ser-396 and Ser-404 (Ser-396/404-->Ala). Wild-type tau and the mutant forms of tau were phosphorylated with GSK-3beta, and their ability to promote microtubule self-assembly was compared with the corresponding non-phosphorylated tau species. In the non-phosphorylated form, wild-type tau and all of the mutants affected the mean microtubule length and number concentrations of assembled microtubules in a manner consistant with enhanced microtubule nucleation. Phosphorylation of these tau species with GSK-3beta consistently reduced the ability of a given tau species to promote microtubule self-assembly, although the affinity of the tau for the microtubules was not greatly affected by phosphorylation since the tau species remained largely associated with the microtubules. This suggests that the regulation of microtubule assembly can be controlled by phosphorylation of tau at sites accessible to GSK-3beta by a mechanism that does not necessarily involve the dissociation of tau from the microtubules. PMID:9169608

  20. 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

  1. 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

  2. Genetic and Pharmacologic Targeting of Glycogen Synthase Kinase 3β Reinforces the Nrf2 Antioxidant Defense against Podocytopathy.

    PubMed

    Zhou, Sijie; Wang, Pei; Qiao, Yingjin; Ge, Yan; Wang, Yingzi; Quan, Songxia; Yao, Ricky; Zhuang, Shougang; Wang, Li Juan; Du, Yong; Liu, Zhangsuo; Gong, Rujun

    2016-08-01

    Evidence suggests that the glycogen synthase kinase 3 (GSK3)-dictated nuclear exclusion and degradation of Nrf2 is pivotal in switching off the self-protective antioxidant stress response after injury. Here, we examined the mechanisms underlying this regulation in glomerular disease. In primary podocytes, doxorubicin elicited cell death and actin cytoskeleton disorganization, concomitant with overactivation of GSK3β (the predominant GSK3 isoform expressed in glomerular podocytes) and minimal Nrf2 activation. SB216763, a highly selective small molecule inhibitor of GSK3, exerted a protective effect that depended on the potentiated Nrf2 antioxidant response, marked by increased Nrf2 expression and nuclear accumulation and augmented production of the Nrf2 target heme oxygenase-1. Ectopic expression of the kinase-dead mutant of GSK3β in cultured podocytes reinforced the doxorubicin-induced Nrf2 activation and prevented podocyte injury. Conversely, a constitutively active GSK3β mutant blunted the doxorubicin-induced Nrf2 response and exacerbated podocyte injury, which could be abolished by treatment with SB216763. In murine models of doxorubicin nephropathy or nephrotoxic serum nephritis, genetic targeting of GSK3β by doxycycline-inducible podocyte-specific knockout or pharmacologic targeting by SB216763 significantly attenuated albuminuria and ameliorated histologic signs of podocyte injury, including podocytopenia, loss of podocyte markers, podocyte de novo expression of desmin, and ultrastructural lesions of podocytopathy (such as foot process effacement). This beneficial outcome was likely attributable to an enhanced Nrf2 antioxidant response in glomerular podocytes because the selective Nrf2 antagonist trigonelline abolished the proteinuria-reducing and podocyte-protective effect. Collectively, our results suggest the GSK3β-regulated Nrf2 antioxidant response as a novel therapeutic target for protecting podocytes and treating proteinuric glomerulopathies. PMID

  3. OP33GLYCOGEN SYNTHASE KINASE INHIBITORS REDUCE 3D MIGRATION OF PATIENT DERIVED GLIOBLASTOMA MULTIFORME STEM CELLS

    PubMed Central

    Tams, Daniel M.; Murray, Clare; Barry, Simon T.; Lawler, Sean; Bruning-Richardson, Anke; Short, Susan

    2014-01-01

    INTRODUCTION: Glioblastoma multiforme (GBM) is a fast growing, highly invasive malignant brain tumour. Inhibition of tumour cell migration into normal brain tissue represents a major target for treatment. Glycogen synthase kinase (GSK-3) inhibition has been associated with reduced GBM invasion in in vitro and in vivo models. Targeting this pathway with established and/or novel drugs may elucidate more effective treatment combinations. METHOD: The effect of GSK-3 inhibitors BIO, AZD2858, AZ1293 and AZ1080 on GBM migration was assessed in patient derived GBM stem cells (GBM-1) and two established cell lines (U251 and U87) using a 3D collagen based assay. Multiple drug concentrations were investigated with up to 72 hours exposure. A migration index was determined using aggregate core size and cell migration area. Immunohistochemistry and immunocytochemistry were used to assess cell morphology and cytoskeletal changes. RESULTS: All compounds inhibit migration in this model. AZD2858 was the most potent, causing significant effects at 1 micro molar. All compounds were cytotoxic at between 10 and 20 micro molar. Cytoskeletal and nuclear abnormalities were noted following drug exposure in all cell lines. These data suggest that possible mechanisms for the anti-migratory effect of these compounds include effects on F-actin localization and microtubule polarity. Inhibition of migration and cell architecture changes occurred at non-toxic doses. CONCLUSION: Inhibition of GSK3 significantly reduced migration of this highly invasive tumour. It is evident from these data that inhibiting the complex biological mechanisms driven by GSK3 may aid treatment of GBM through a number of different mechanisms.

  4. Lovastatin modulates glycogen synthase kinase-3β pathway and inhibits mossy fiber sprouting after pilocarpine-induced status epilepticus.

    PubMed

    Lee, Chun-Yao; Jaw, Thomas; Tseng, Huan-Chin; Chen, I-Chun; Liou, Horng-Huei

    2012-01-01

    This study was undertaken to assay the effect of lovastatin on the glycogen synthase kinase-3 beta (GSK-3β) and collapsin responsive mediator protein-2 (CRMP-2) signaling pathway and mossy fiber sprouting (MFS) in epileptic rats. MFS in the dentate gyrus (DG) is an important feature of temporal lobe epilepsy (TLE) and is highly related to the severity and the frequency of spontaneous recurrent seizures. However, the molecular mechanism of MFS is mostly unknown. GSK-3β and CRMP-2 are the genes responsible for axonal growth and neuronal polarity in the hippocampus, therefore this pathway is a potential target to investigate MFS. Pilocarpine-induced status epilepticus animal model was taken as our researching material. Western blot, histological and electrophysiological techniques were used as the studying tools. The results showed that the expression level of GSK-3β and CRMP-2 were elevated after seizure induction, and the administration of lovastatin reversed this effect and significantly reduced the extent of MFS in both DG and CA3 region in the hippocampus. The alteration of expression level of GSK-3β and CRMP-2 after seizure induction proposes that GSK-3β and CRMP-2 are crucial for MFS and epiletogenesis. The fact that lovastatin reversed the expression level of GSK-3β and CRMP-2 indicated that GSK-3β and CRMP-2 are possible to be a novel mechanism of lovatstain to suppress MFS and revealed a new therapeutic target and researching direction for studying the mechanism of MFS and epileptogenesis. PMID:22761705

  5. 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. PMID:27255602

  6. Glycogen synthase kinase-3 inhibition attenuates fibroblast activation and development of fibrosis following renal ischemia-reperfusion in mice

    PubMed Central

    Singh, Shailendra P.; Tao, Shixin; Fields, Timothy A.; Webb, Sydney; Harris, Raymond C.; Rao, Reena

    2015-01-01

    ABSTRACT Glycogen synthase kinase-3β (GSK3β) is a serine/threonine protein kinase that plays an important role in renal tubular injury and regeneration in acute kidney injury. However, its role in the development of renal fibrosis, often a long-term consequence of acute kidney injury, is unknown. Using a mouse model of renal fibrosis induced by ischemia-reperfusion injury, we demonstrate increased GSK3β expression and activity in fibrotic kidneys, and its presence in myofibroblasts in addition to tubular epithelial cells. Pharmacological inhibition of GSK3 using TDZD-8 starting before or after ischemia-reperfusion significantly suppressed renal fibrosis by reducing the myofibroblast population, collagen-1 and fibronectin deposition, inflammatory cytokines, and macrophage infiltration. GSK3 inhibition in vivo reduced TGF-β1, SMAD3 activation and plasminogen activator inhibitor-1 levels. Consistently in vitro, TGF-β1 treatment increased GSK3β expression and GSK3 inhibition abolished TGF-β1-induced SMAD3 activation and α-smooth muscle actin (α-SMA) expression in cultured renal fibroblasts. Importantly, overexpression of constitutively active GSK3β stimulated α-SMA expression even in the absence of TGF-β1 treatment. These results suggest that TGF-β regulates GSK3β, which in turn is important for TGF-β–SMAD3 signaling and fibroblast-to-myofibroblast differentiation. Overall, these studies demonstrate that GSK3 could promote renal fibrosis by activation of TGF-β signaling and the use of GSK3 inhibitors might represent a novel therapeutic approach for progressive renal fibrosis that develops as a consequence of acute kidney injury. PMID:26092126

  7. Valproate Inhibits Methamphetamine Induced Hyperactivity via Glycogen Synthase Kinase 3β Signaling in the Nucleus Accumbens Core

    PubMed Central

    Xing, Bo; Liang, Xiao-ping; Liu, Peng; Zhao, Yan; Chu, Zheng; Dang, Yong-hui

    2015-01-01

    Valproate (VPA) has recently been shown to influence the behavioral effects of psycho-stimulants. Although glycogen synthase kinase 3β (GSK3β) signaling in the nucleus accumbens (NAc) plays a key role in mediating dopamine (DA)-dependent behaviors, there is less direct evidence that how VPA acts on the GSK3β signaling in the functionally distinct sub-regions of the NAc, the NAc core (NAcC) and the NAc shell (NAcSh), during psycho-stimulant-induced hyperactivity. In the present study, we applied locomotion test after acute methamphetamine (MA) (2 mg/kg) injection to identify the locomotor activity of rats received repeated VPA (300 mg/kg) pretreatment. We next measured phosphor-GSK3β at serine 9 and total GSK3β levels in NAcC and NAcSh respectively to determine the relationship between the effect of VPA on MA-induced hyperlocomotor and changes in GSK3β activity. We further investigated whether microinjection of VPA (300 μg/0.5 μl/side, once daily for 7 consecutive days) into NAcC or NAcSh could affect hyperactivity induced by MA. Our data indicated that repeated VPA treatment attenuated MA-induced hyperlocomotor, and the effect was associated with decreased levels of phosphorylated GSK3β at Ser 9 in the NAcC. Moreover, repeated bilateral intra-NAcC, but not intra-NAcSh VPA treatment, significantly attenuated MA-induced hyperactivity. Our results suggested that GSK3β activity in NAcC contributes to the inhibitory effects of VPA on MA-induced hyperactivity. PMID:26030405

  8. Glycogen Synthase Kinase-3β (GSK3β) Binds to and Promotes the Actions of p53*

    PubMed Central

    Watcharasit, Piyajit; Bijur, Gautam N.; Song, Ling; Zhu, Jianhui; Chen, Xinbin; Jope, Richard S.

    2006-01-01

    The recent discovery of direct interactions between two important regulators of cell fate, the tumor suppressor p53 and glycogen synthase kinase-3β (GSK3β), led us to examine the mechanism and outcomes of this interaction. Two regions of p53 were identified that regulate its binding to GSK3β. Deletion of the p53 activation domain-1 (AD1), but not mutations that prevent MDM2 binding through the AD1 domain, enhanced GSK3β binding to p53, indicating that the AD1 domain interferes with p53 binding to GSK3β. Deletion of the p53 basic domain (BD) abrogated GSK3β binding, and a ten amino acid region within the C-terminal BD domain was identified as necessary for binding to GSK3β. GSK3β activity was not required for p53 binding, but inhibition of GSK3β stabilized the association, suggesting a transient interaction during which active GSK3β promotes actions of p53. This regulatory role of GSK3β was demonstrated by large reductions of p53-induced increases in the levels of MDM2, p21, and Bax when GSK3β was inhibited. Besides promoting p53-mediated transcription, GSK3β also contributed to mitochondrial p53 apoptotic signaling. After DNA damage, mitochondrial GSK3β co-immunoprecipitated with p53 and was activated, and inhibition of GSK3β blocked cytochrome c release and caspase-3 activation. Thus, GSK3β interacts with p53 in both the nucleus and mitochondria and promotes its actions at both sites. PMID:14523002

  9. Glycogen metabolism in humans.

    PubMed

    Adeva-Andany, María M; González-Lucán, Manuel; Donapetry-García, Cristóbal; Fernández-Fernández, Carlos; Ameneiros-Rodríguez, Eva

    2016-06-01

    In the human body, glycogen is a branched polymer of glucose stored mainly in the liver and the skeletal muscle that supplies glucose to the blood stream during fasting periods and to the muscle cells during muscle contraction. Glycogen has been identified in other tissues such as brain, heart, kidney, adipose tissue, and erythrocytes, but glycogen function in these tissues is mostly unknown. Glycogen synthesis requires a series of reactions that include glucose entrance into the cell through transporters, phosphorylation of glucose to glucose 6-phosphate, isomerization to glucose 1-phosphate, and formation of uridine 5'-diphosphate-glucose, which is the direct glucose donor for glycogen synthesis. Glycogenin catalyzes the formation of a short glucose polymer that is extended by the action of glycogen synthase. Glycogen branching enzyme introduces branch points in the glycogen particle at even intervals. Laforin and malin are proteins involved in glycogen assembly but their specific function remains elusive in humans. Glycogen is accumulated in the liver primarily during the postprandial period and in the skeletal muscle predominantly after exercise. In the cytosol, glycogen breakdown or glycogenolysis is carried out by two enzymes, glycogen phosphorylase which releases glucose 1-phosphate from the linear chains of glycogen, and glycogen debranching enzyme which untangles the branch points. In the lysosomes, glycogen degradation is catalyzed by α-glucosidase. The glucose 6-phosphatase system catalyzes the dephosphorylation of glucose 6-phosphate to glucose, a necessary step for free glucose to leave the cell. Mutations in the genes encoding the enzymes involved in glycogen metabolism cause glycogen storage diseases. PMID:27051594

  10. 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

  11. 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

  12. 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

  13. A negative feedback control of transforming growth factor-beta signaling by glycogen synthase kinase 3-mediated Smad3 linker phosphorylation at Ser-204.

    PubMed

    Millet, Caroline; Yamashita, Motozo; Heller, Mary; Yu, Li-Rong; Veenstra, Timothy D; Zhang, Ying E

    2009-07-24

    Through the action of its membrane-bound type I receptor, transforming growth factor-beta (TGF-beta) elicits a wide range of cellular responses that regulate cell proliferation, differentiation, and apo ptosis. Many of these signaling responses are mediated by Smad proteins. As such, controlling Smad activity is crucial for proper signaling by TGF-beta and its related factors. Here, we show that TGF-beta induces phosphorylation at three sites in the Smad3 linker region in addition to the two C-terminal residues, and glycogen synthase kinase 3 is responsible for phosphorylation at one of these sites, namely Ser-204. Alanine substitution at Ser-204 and/or the neighboring Ser-208, the priming site for glycogen synthase kinase 3 in vivo activity, strengthened the affinity of Smad3 to CREB-binding protein, suggesting that linker phosphorylation may be part of a negative feedback loop that modulates Smad3 transcriptional activity. Thus, our findings reveal a novel aspect of the Smad3 signaling mechanism that controls the final amplitude of cellular responses to TGF-beta. PMID:19458083

  14. A Negative Feedback Control of Transforming Growth Factor-β Signaling by Glycogen Synthase Kinase 3-mediated Smad3 Linker Phosphorylation at Ser-204*

    PubMed Central

    Millet, Caroline; Yamashita, Motozo; Heller, Mary; Yu, Li-Rong; Veenstra, Timothy D.; Zhang, Ying E.

    2009-01-01

    Through the action of its membrane-bound type I receptor, transforming growth factor-β (TGF-β) elicits a wide range of cellular responses that regulate cell proliferation, differentiation, and apo pto sis. Many of these signaling responses are mediated by Smad proteins. As such, controlling Smad activity is crucial for proper signaling by TGF-β and its related factors. Here, we show that TGF-β induces phos pho ryl a tion at three sites in the Smad3 linker region in addition to the two C-terminal residues, and glycogen synthase kinase 3 is responsible for phos pho ryl a tion at one of these sites, namely Ser-204. Alanine substitution at Ser-204 and/or the neighboring Ser-208, the priming site for glycogen synthase kinase 3 in vivo activity, strengthened the affinity of Smad3 to CREB-binding protein, suggesting that linker phos pho ryl a tion may be part of a negative feedback loop that modulates Smad3 transcriptional activity. Thus, our findings reveal a novel aspect of the Smad3 signaling mechanism that controls the final amplitude of cellular responses to TGF-β. PMID:19458083

  15. Testicular Metabolic Reprogramming in Neonatal Streptozotocin-Induced Type 2 Diabetic Rats Impairs Glycolytic Flux and Promotes Glycogen Synthesis

    PubMed Central

    Rato, L.; Alves, M. G.; Dias, T. R.; Cavaco, J. E.; Oliveira, Pedro F.

    2015-01-01

    Defects in testicular metabolism are directly implicated with male infertility, but most of the mechanisms associated with type 2 diabetes- (T2DM) induced male infertility remain unknown. We aimed to evaluate the effects of T2DM on testicular glucose metabolism by using a neonatal-streptozotocin- (n-STZ) T2DM animal model. Plasma and testicular hormonal levels were evaluated using specific kits. mRNA and protein expression levels were assessed by real-time PCR and Western Blot, respectively. Testicular metabolic profile was assessed by 1H-NMR spectroscopy. T2DM rats showed increased glycemic levels, impaired glucose tolerance and hyperinsulinemia. Both testicular and serum testosterone levels were decreased, whereas those of 17β-estradiol were not altered. Testicular glycolytic flux was not favored in testicles of T2DM rats, since, despite the increased expression of both glucose transporters 1 and 3 and the enzyme phosphofructokinase 1, lactate dehydrogenase activity was severely decreased contributing to lower testicular lactate content. However, T2DM enhanced testicular glycogen accumulation, by modulating the availability of the precursors for its synthesis. T2DM also affected the reproductive sperm parameters. Taken together these results indicate that T2DM is able to reprogram testicular metabolism by enhancing alternative metabolic pathways, particularly glycogen synthesis, and such alterations are associated with impaired sperm parameters. PMID:26064993

  16. Testicular Metabolic Reprogramming in Neonatal Streptozotocin-Induced Type 2 Diabetic Rats Impairs Glycolytic Flux and Promotes Glycogen Synthesis.

    PubMed

    Rato, L; Alves, M G; Dias, T R; Cavaco, J E; Oliveira, Pedro F

    2015-01-01

    Defects in testicular metabolism are directly implicated with male infertility, but most of the mechanisms associated with type 2 diabetes- (T2DM) induced male infertility remain unknown. We aimed to evaluate the effects of T2DM on testicular glucose metabolism by using a neonatal-streptozotocin- (n-STZ) T2DM animal model. Plasma and testicular hormonal levels were evaluated using specific kits. mRNA and protein expression levels were assessed by real-time PCR and Western Blot, respectively. Testicular metabolic profile was assessed by (1)H-NMR spectroscopy. T2DM rats showed increased glycemic levels, impaired glucose tolerance and hyperinsulinemia. Both testicular and serum testosterone levels were decreased, whereas those of 17β-estradiol were not altered. Testicular glycolytic flux was not favored in testicles of T2DM rats, since, despite the increased expression of both glucose transporters 1 and 3 and the enzyme phosphofructokinase 1, lactate dehydrogenase activity was severely decreased contributing to lower testicular lactate content. However, T2DM enhanced testicular glycogen accumulation, by modulating the availability of the precursors for its synthesis. T2DM also affected the reproductive sperm parameters. Taken together these results indicate that T2DM is able to reprogram testicular metabolism by enhancing alternative metabolic pathways, particularly glycogen synthesis, and such alterations are associated with impaired sperm parameters. PMID:26064993

  17. 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. PMID:26116771

  18. Discovery of a Highly Selective Glycogen Synthase Kinase-3 Inhibitor (PF-04802367) That Modulates Tau Phosphorylation in the Brain: Translation for PET Neuroimaging.

    PubMed

    Liang, Steven H; Chen, Jinshan Michael; Normandin, Marc D; Chang, Jeanne S; Chang, George C; Taylor, Christine K; Trapa, Patrick; Plummer, Mark S; Para, Kimberly S; Conn, Edward L; Lopresti-Morrow, Lori; Lanyon, Lorraine F; Cook, James M; Richter, Karl E G; Nolan, Charlie E; Schachter, Joel B; Janat, Fouad; Che, Ye; Shanmugasundaram, Veerabahu; Lefker, Bruce A; Enerson, Bradley E; Livni, Elijahu; Wang, Lu; Guehl, Nicolas J; Patnaik, Debasis; Wagner, Florence F; Perlis, Roy; Holson, Edward B; Haggarty, Stephen J; El Fakhri, Georges; Kurumbail, Ravi G; Vasdev, Neil

    2016-08-01

    Glycogen synthase kinase-3 (GSK-3) regulates multiple cellular processes in diabetes, oncology, and neurology. N-(3-(1H-1,2,4-triazol-1-yl)propyl)-5-(3-chloro-4-methoxyphenyl)oxazole-4-carboxamide (PF-04802367 or PF-367) has been identified as a highly potent inhibitor, which is among the most selective antagonists of GSK-3 to date. Its efficacy was demonstrated in modulation of tau phosphorylation in vitro and in vivo. Whereas the kinetics of PF-367 binding in brain tissues are too fast for an effective therapeutic agent, the pharmacokinetic profile of PF-367 is ideal for discovery of radiopharmaceuticals for GSK-3 in the central nervous system. A (11) C-isotopologue of PF-367 was synthesized and preliminary PET imaging studies in non-human primates confirmed that we have overcome the two major obstacles for imaging GSK-3, namely, reasonable brain permeability and displaceable binding. PMID:27355874

  19. Silencing Glycogen Synthase Kinase-3β Inhibits Acetaminophen Hepatotoxicity and Attenuates JNK Activation and Loss of Glutamate Cysteine Ligase and Myeloid Cell Leukemia Sequence 1*

    PubMed Central

    Shinohara, Mie; Ybanez, Maria D.; Win, Sanda; Than, Tin Aung; Jain, Shilpa; Gaarde, William A.; Han, Derick; Kaplowitz, Neil

    2010-01-01

    Previously we demonstrated that c-Jun N-terminal kinase (JNK) plays a central role in acetaminophen (APAP)-induced liver injury. In the current work, we examined other possible signaling pathways that may also contribute to APAP hepatotoxicity. APAP treatment to mice caused glycogen synthase kinase-3β (GSK-3β) activation and translocation to mitochondria during the initial phase of APAP-induced liver injury (∼1 h). The silencing of GSK-3β, but not Akt-2 (protein kinase B) or glycogen synthase kinase-3α (GSK-3α), using antisense significantly protected mice from APAP-induced liver injury. The silencing of GSK-3β affected several key pathways important in conferring protection against APAP-induced liver injury. APAP treatment was observed to promote the loss of glutamate cysteine ligase (GCL, rate-limiting enzyme in GSH synthesis) in liver. The silencing of GSK-3β decreased the loss of hepatic GCL, and promoted greater GSH recovery in liver following APAP treatment. Silencing JNK1 and -2 also prevented the loss of GCL. APAP treatment also resulted in GSK-3β translocation to mitochondria and the degradation of myeloid cell leukemia sequence 1 (Mcl-1) in mitochondrial membranes in liver. The silencing of GSK-3β reduced Mcl-1 degradation caused by APAP treatment. The silencing of GSK-3β also resulted in an inhibition of the early phase (0–2 h), and blunted the late phase (after 4 h) of JNK activation and translocation to mitochondria in liver following APAP treatment. Taken together our results suggest that activation of GSK-3β is a key mediator of the initial phase of APAP-induced liver injury through modulating GCL and Mcl-1 degradation, as well as JNK activation in liver. PMID:20061376

  20. Glycogen synthase and phosphofructokinase protein and mRNA levels in skeletal muscle from insulin-resistant patients with non-insulin-dependent diabetes mellitus.

    PubMed Central

    Vestergaard, H; Lund, S; Larsen, F S; Bjerrum, O J; Pedersen, O

    1993-01-01

    In patients with non-insulin-dependent diabetes mellitus (NIDDM) and matched control subjects we examined the interrelationships between in vivo nonoxidative glucose metabolism and glucose oxidation and the muscle activities, as well as the immunoreactive protein and mRNA levels of the rate-limiting enzymes in glycogen synthesis and glycolysis, glycogen synthase (GS) and phosphofructokinase (PFK), respectively. Analysis of biopsies of quadriceps muscle from 19 NIDDM patients and 19 control subjects showed in the basal state a 30% decrease (P < 0.005) in total GS activity and a 38% decrease (P < 0.001) in GS mRNA/microgram DNA in NIDDM patients, whereas the GS protein level was normal. The enzymatic activity and protein and mRNA levels of PFK were all normal in diabetic patients. In subgroups of NIDDM patients and control subjects an insulin-glucose clamp in combination with indirect calorimetry was performed. The rate of insulin-stimulated nonoxidative glucose metabolism was decreased by 47% (P < 0.005) in NIDDM patients, whereas the glucose oxidation rate was normal. The PFK activity, protein level, and mRNA/microgram DNA remained unchanged. The relative activation of GS by glucose-6-phosphate was 33% lower (P < 0.02), whereas GS mRNA/micrograms DNA was 37% lower (P < 0.05) in the diabetic patients after 4 h of hyperinsulinemia. Total GS immunoreactive mass remained normal. In conclusion, qualitative but not quantitative posttranslational abnormalities of the GS protein in muscle determine the reduced insulin-stimulated nonoxidative glucose metabolism in NIDDM. Images PMID:8514849

  1. 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. PMID:25394244

  2. Akt2 influences glycogen synthase activity in human skeletal muscle through regulation of NH2-terminal (sites 2 + 2a) phosphorylation

    PubMed Central

    Birk, Jesper B.; Richter, Erik A.; Ribel-Madsen, Rasmus; Pehmøller, Christian; Hansen, Bo Falck; Beck-Nielsen, Henning; Hirshman, Michael F.; Goodyear, Laurie J.; Vaag, Allan; Poulsen, Pernille; Wojtaszewski, Jørgen F. P.

    2013-01-01

    Type 2 diabetes is characterized by reduced muscle glycogen synthesis. The key enzyme in this process, glycogen synthase (GS), is activated via proximal insulin signaling, but the exact molecular events remain unknown. Previously, we demonstrated that phosphorylation of Thr308 on Akt (p-Akt-Thr308), Akt2 activity, and GS activity in muscle were positively associated with insulin sensitivity. Here, in the same study population, we determined the influence of several upstream elements in the canonical PI3K signaling on muscle GS activation. One-hundred eighty-one nondiabetic twins were examined with the euglycemic hyperinsulinemic clamp combined with excision of muscle biopsies. Insulin signaling was evaluated at the levels of the insulin receptor, IRS-1-associated PI3K (IRS-1-PI3K), Akt, and GS employing activity assays and phosphospecific Western blotting. The insulin-stimulated GS activity was positively associated with p-Akt-Thr308 (P = 0.01) and Akt2 activity (P = 0.04) but not p-Akt-Ser473 or IRS-1-PI3K activity. Furthermore, p-Akt-Thr308 and Akt2 activity were negatively associated with NH2-terminal GS phosphorylation (P = 0.001 for both), which in turn was negatively associated with insulin-stimulated GS activity (P < 0.001). We found no association between COOH-terminal GS phosphorylation and Akt or GS activity. Employing whole body Akt2-knockout mice, we validated the necessity for Akt2 in insulin-mediated GS activation. However, since insulin did not affect NH2-terminal phosphorylation in mice, we could not use this model to validate the observed association between GS NH2-terminal phosphorylation and Akt activity in humans. In conclusion, our study suggests that although COOH-terminal dephosphorylation is likely necessary for GS activation, Akt2-dependent NH2-terminal dephosphorylation may be the site for “fine-tuning” insulin-mediated GS activation in humans. PMID:23321478

  3. Mitochondrial ATP synthase activity is impaired by suppressed O-GlcNAcylation in Alzheimer's disease.

    PubMed

    Cha, Moon-Yong; Cho, Hyun Jin; Kim, Chaeyoung; Jung, Yang Ouk; Kang, Min Jueng; Murray, Melissa E; Hong, Hyun Seok; Choi, Young-Joo; Choi, Heesun; Kim, Dong Kyu; Choi, Hyunjung; Kim, Jisoo; Dickson, Dennis W; Song, Hyun Kyu; Cho, Jin Won; Yi, Eugene C; Kim, Jungsu; Jin, Seok Min; Mook-Jung, Inhee

    2015-11-15

    Glycosylation with O-linked β-N-acetylglucosamine (O-GlcNAc) is one of the protein glycosylations affecting various intracellular events. However, the role of O-GlcNAcylation in neurodegenerative diseases such as Alzheimer's disease (AD) is poorly understood. Mitochondrial adenosine 5'-triphosphate (ATP) synthase is a multiprotein complex that synthesizes ATP from ADP and Pi. Here, we found that ATP synthase subunit α (ATP5A) was O-GlcNAcylated at Thr432 and ATP5A O-GlcNAcylation was decreased in the brains of AD patients and transgenic mouse model, as well as Aβ-treated cells. Indeed, Aβ bound to ATP synthase directly and reduced the O-GlcNAcylation of ATP5A by inhibition of direct interaction between ATP5A and mitochondrial O-GlcNAc transferase, resulting in decreased ATP production and ATPase activity. Furthermore, treatment of O-GlcNAcase inhibitor rescued the Aβ-induced impairment in ATP production and ATPase activity. These results indicate that Aβ-mediated reduction of ATP synthase activity in AD pathology results from direct binding between Aβ and ATP synthase and inhibition of O-GlcNAcylation of Thr432 residue on ATP5A. PMID:26358770

  4. 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

  5. Glycogen synthase kinase-3-mediated phosphorylation of serine 73 targets sterol response element binding protein-1c (SREBP-1c) for proteasomal degradation

    PubMed Central

    Dong, Qingming; Giorgianni, Francesco; Beranova-Giorgianni, Sarka; Deng, Xiong; O'Meally, Robert N.; Bridges, Dave; Park, Edwards A.; Cole, Robert N.; Elam, Marshall B.; Raghow, Rajendra

    2015-01-01

    Sterol regulatory element binding protein-1c (SREBP-1c) is a key transcription factor that regulates genes involved in the de novo lipid synthesis and glycolysis pathways. The structure, turnover and transactivation potential of SREBP-1c are regulated by macronutrients and hormones via a cascade of signalling kinases. Using MS, we have identified serine 73 as a novel glycogen synthase kinase-3 (GSK-3) phosphorylation site in the rat SREBP-1c purified from McA-RH7777 hepatoma cells. Our site-specific mutagenesis strategy revealed that the turnover of SREBP-1c, containing wild type, phospho-null (serine to alanine) or phospho-mimetic (serine to aspartic acid) substitutions, was differentially regulated. We show that the S73D mutant of pSREBP-1c, that mimicked a state of constitutive phosphorylation, dissociated from the SREBP-1c–SCAP complex more readily and underwent GSK-3-dependent proteasomal degradation via SCFFbw7 ubiquitin ligase pathway. Pharmacologic inhibition of GSK-3 or knockdown of GSK-3 by siRNA prevented accelerated degradation of SREBP-1c. As demonstrated by MS, SREBP-1c was phosphorylated in vitro by GSK-3β at serine 73. Phosphorylation of serine 73 also occurs in the intact liver. We propose that GSK-3-mediated phosphorylation of serine 73 in the rat SREBP-1c and its concomitant destabilization represents a novel mechanism involved in the inhibition of de novo lipid synthesis in the liver. PMID:26589965

  6. Overexpression of interleukin-18 protein reduces viability and induces apoptosis of tongue squamous cell carcinoma cells by activation of glycogen synthase kinase-3β signaling

    PubMed Central

    LIU, WEIWEI; HU, MIN; WANG, YUMEI; SUN, BAOZHEN; GUO, YU; XU, ZHIMIN; LI, JIA; HAN, BING

    2015-01-01

    The aim of this study was to investigate the effects of interleukin-18 (IL-18) expression on regulating the viability and apoptosis of tongue squamous cell carcinoma (TSCC) cells in vitro and examine the underlying molecular events. Human IL-18 cDNA was cloned into the vector pcDNA3.1 (+) and transfected into CRL-1623™ cells. Quantitative reverse transcription-PCR (RT-qPCR), western blot analysis, immunofluorescence, cell viability MTT assay, flow cytometric Annexin V/propidium iodide (PI), Giemsa staining, and caspase-3 activity assay were performed. The data showed that overexpression of IL-18 protein reduced TSCC cell viability by inducing apoptosis. Compared with cells transfected with the control vector, IL-18 expression activated caspase-3, -7, and -9 by inducing their cleavage and increased the expression of interferon (IFN)-γ and cytochrome c mRNA, but reduced cyclin D1 and A1 expression in TSCC cells. IL-18 expression upregulated the expression and phosphorylation of glycogen synthase kinase (GSK)-3β protein in CRL1623 cells, whereas the selective GSK-3β inhibitor kenpaullone antagonized the effects of IL-18 protein on TSCC cells in vitro. The results indicated that IL-18 played an important role in the inhibition of TSCC cell growth and may be further investigated as a novel therapeutic target against TSCC. PMID:25591548

  7. 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. PMID:26610618

  8. Glycogen Synthase Kinase-3β and Caspase-2 Mediate Ceramide- and Etoposide-Induced Apoptosis by Regulating the Lysosomal-Mitochondrial Axis

    PubMed Central

    Lin, Chiou-Feng; Tsai, Cheng-Chieh; Huang, Wei-Ching; Wang, Yu-Chih; Tseng, Po-Chun; Tsai, Tsung-Ting; Chen, Chia-Ling

    2016-01-01

    Glycogen synthase kinase-3β (GSK-3β) regulates the sequential activation of caspase-2 and caspase-8 before mitochondrial apoptosis. Here, we report the regulation of Mcl-1 destabilization and cathepsin D-regulated caspase-8 activation by GSK-3β and caspase-2. Treatment with either the ceramide analogue C2-ceramide or the topoisomerase II inhibitor etoposide sequentially induced lysosomal membrane permeabilization (LMP), the reduction of mitochondrial transmembrane potential, and apoptosis. Following LMP, cathepsin D translocated from lysosomes to the cytoplasm, whereas inhibiting cathepsin D blocked mitochondrial apoptosis. Furthermore, cathepsin D caused the activation of caspase-8 but not caspase-2. Inhibiting GSK-3β and caspase-2 blocked Mcl-1 destabilization, LMP, cathepsin D re-localization, caspase-8 activation, and mitochondrial apoptosis. Expression of Mcl-1 was localized to the lysosomes, and forced expression of Mcl-1 prevented apoptotic signaling via the lysosomal-mitochondrial pathway. These results demonstrate the importance of GSK-3β and caspase-2 in ceramide- and etoposide-induced apoptosis through mechanisms involving Mcl-1 destabilization and the lysosomal-mitochondrial axis. PMID:26727221

  9. 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.

  10. Protective Effects of Kaempferol against Myocardial Ischemia/Reperfusion Injury in Isolated Rat Heart via Antioxidant Activity and Inhibition of Glycogen Synthase Kinase-3β

    PubMed Central

    Zhou, Mingjie; Ren, Huanhuan; Han, Jichun; Wang, Wenjuan; Zheng, Qiusheng; Wang, Dong

    2015-01-01

    Objective. This study aimed to evaluate the protective effect of kaempferol against myocardial ischemia/reperfusion (I/R) injury in rats. Method. Left ventricular developed pressure (LVDP) and its maximum up/down rate (±dp/dtmax) were recorded as myocardial function. Infarct size was detected with 2,3,5-triphenyltetrazolium chloride staining. Cardiomyocyte apoptosis was determined using terminal deoxynucleotidyl nick-end labeling (TUNEL). The levels of creatine kinase (CK), lactate dehydrogenase (LDH), malondialdehyde (MDA), superoxide dismutase (SOD), glutathione/glutathione disulfide (GSH/GSSG) ratio, and tumor necrosis factor-alpha (TNF-α) were determined using enzyme linked immunosorbent assay (ELISA). Moreover, total glycogen synthase kinase-3β (GSK-3β), phospho-GSK-3β (P-GSK-3β), precaspase-3, cleaved caspase-3, and cytoplasm cytochrome C were assayed using Western blot analysis. Results. Pretreatment with kaempferol significantly improved the recovery of LVDP and ±dp/dtmax, as well as increased the levels of SOD and P-GSK-3β and GSH/GSSG ratio. However, the pretreatment reduced myocardial infarct size and TUNEL-positive cell rate, as well as decreased the levels of cleaved caspase-3, cytoplasm cytochrome C, CK, LDH, MDA, and TNF-α. Conclusion. These results suggested that kaempferol provides cardioprotection via antioxidant activity and inhibition of GSK-3β activity in rats with I/R. PMID:26265983

  11. 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. PMID:26088133

  12. Glycogen synthase kinase 3 inhibitors induce the canonical WNT/β-catenin pathway to suppress growth and self-renewal in embryonal rhabdomyosarcoma

    PubMed Central

    Chen, Eleanor Y.; DeRan, Michael T.; Ignatius, Myron S.; Grandinetti, Kathryn Brooke; Clagg, Ryan; McCarthy, Karin M.; Lobbardi, Riadh M.; Brockmann, Jillian; Keller, Charles; Wu, Xu; Langenau, David M.

    2014-01-01

    Embryonal rhabdomyosarcoma (ERMS) is a common pediatric malignancy of muscle, with relapse being the major clinical challenge. Self-renewing tumor-propagating cells (TPCs) drive cancer relapse and are confined to a molecularly definable subset of ERMS cells. To identify drugs that suppress ERMS self-renewal and induce differentiation of TPCs, a large-scale chemical screen was completed. Glycogen synthase kinase 3 (GSK3) inhibitors were identified as potent suppressors of ERMS growth through inhibiting proliferation and inducing terminal differentiation of TPCs into myosin-expressing cells. In support of GSK3 inhibitors functioning through activation of the canonical WNT/β-catenin pathway, recombinant WNT3A and stabilized β-catenin also enhanced terminal differentiation of human ERMS cells. Treatment of ERMS-bearing zebrafish with GSK3 inhibitors activated the WNT/β-catenin pathway, resulting in suppressed ERMS growth, depleted TPCs, and diminished self-renewal capacity in vivo. Activation of the canonical WNT/β-catenin pathway also significantly reduced self-renewal of human ERMS, indicating a conserved function for this pathway in modulating ERMS self-renewal. In total, we have identified an unconventional tumor suppressive role for the canonical WNT/β-catenin pathway in regulating self-renewal of ERMS and revealed therapeutic strategies to target differentiation of TPCs in ERMS. PMID:24706870

  13. Hypoxic inactivation of glycogen synthase kinase-3β promotes gastric tumor growth and angiogenesis by facilitating hypoxia-inducible factor-1 signaling.

    PubMed

    Ko, Young San; Cho, Sung Jin; Park, Jinju; Choi, Yiseul; Lee, Jae-Seon; Youn, Hong-Duk; Kim, Woo Ho; Kim, Min A; Park, Jong-Wan; Lee, Byung Lan

    2016-09-01

    Since the molecular mechanism of hypoxic adaptation in cancer cells is cell-type specific, we investigated whether glycogen synthase kinase-3β (GSK-3β) activation is involved in hypoxia-induced gastric tumor promotion. Stable gastric cancer cell lines (SNU-638, SNU-484, MKN1, and MKN45) were cultured under hypoxic conditions. Cells overexpressing wild-type GSK-3β (WT-GSK-3β) or kinase-dead mutant of GSK-3β (KD-GSK-3β) were generated and used for cell culture and animal studies. In cell culture experiments, hypoxia decreased GSK-3β activation in gastric cancer cells. Cell viability and the expressions of HIF-1α protein and VEGF mRNA in gastric cancer cells were higher in KD-GSK-3β transfectants than in WT-GSK-3β transfectants under hypoxic conditions, but not under normoxic conditions. Gastric cancer xenografts showed that tumor growth, microvessel area, HIF-1α activation, and VEGF expression were higher in KD-GSK-3β tumors than in WT-GSK-3β tumors in vivo. In addition, the expression of hypoxia-induced HIF-1α protein was regulated by GSK-3β at the translational level. Our data suggest that GSK-3β is involved in hypoxic adaptation of gastric cancer cells as an inhibitory upstream regulator of the HIF-1α/VEGF signaling pathway. PMID:27365055

  14. Quantitative Phosphoproteomic Study Reveals that Protein Kinase A Regulates Neural Stem Cell Differentiation Through Phosphorylation of Catenin Beta-1 and Glycogen Synthase Kinase 3β.

    PubMed

    Wang, Shuxin; Li, Zheyi; Shen, Hongyan; Zhang, Zhong; Yin, Yuxin; Wang, Qingsong; Zhao, Xuyang; Ji, Jianguo

    2016-08-01

    Protein phosphorylation is central to the understanding of multiple cellular signaling pathways responsible for regulating the self-renewal and differentiation of neural stem cells (NSCs). Here we performed a large-scale phosphoproteomic analysis of rat fetal NSCs using strong cation exchange chromatography prefractionation and citric acid-assisted two-step enrichment with TiO2 strategy followed by nanoLC-MS/MS analysis. Totally we identified 32,546 phosphosites on 5,091 phosphoproteins, among which 23,945 were class I phosphosites, and quantified 16,000 sites during NSC differentiation. More than 65% of class I phosphosites were novel when compared with PhosphoSitePlus database. Quantification results showed that the early and late stage of NSC differentiation differ greatly. We mapped 69 changed phosphosites on 20 proteins involved in Wnt signaling pathway, including S552 on catenin beta-1 (Ctnnb1) and S9 on glycogen synthase kinase 3β (Gsk3β). Western blotting and real-time PCR results proved that Wnt signaling pathway plays critical roles in NSC fate determination. Furthermore, inhibition and activation of PKA dramatically affected the phosphorylation state of Ctnnb1 and Gsk3β, which regulates the differentiation of NSCs. Our data provides a valuable resource for studying the self-renewal and differentiation of NSCs. Stem Cells 2016;34:2090-2101. PMID:27097102

  15. 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

  16. 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.

  17. Enhancement of paclitaxel-induced breast cancer cell death via the glycogen synthase kinase-3β-mediated B-cell lymphoma 2 regulation

    PubMed Central

    Noh, Kyung Tae; Cha, Gil Sun; Kang, Tae Heung; Cho, Joon; Jung, In Duk; Kim, Kwang-Youn; Ahn, Soon-Cheol; You, Ji Chang; Park, Yeong-Min

    2016-01-01

    Glycogen synthase kinase-3β (GSK-3β) is a serine/threonine protein kinase that is known to mediate cancer cell death. Here, we show that B-cell lymphoma 2 (Bcl-2), an anti-apoptotic protein, is regulated by GSK-3β and that GSK-3β-mediated regulation of Bcl-2 is crucial for mitochondrial-dependent cell death in paclitaxel-stimulated cells. We demonstrate that MCF7 GSK-3β siRNA cells are more sensitive to cell death than MCF7 GFP control cells and that in the absence of GSK-3β, Bcl-2 levels are reduced, a result enhanced by paclitaxel. Paclitaxel-induced JNK (c-Jun N-terminal kinase) activation is critical for Bcl-2 modulation. In the absence of GSK-3β, Bcl-2 was unstable in an ubiquitination-dependent manner in both basal- and paclitaxel-treated cells. Furthermore, we demonstrate that GSK-3β-mediated regulation of Bcl-2 influences cytochrome C release and mitochondrial membrane potential. Taken together, our data suggest that GSK-3β-dependent regulation of Bcl-2 is crucial for mitochondria-dependent cell death in paclitaxel-mediated breast cancer therapy. [BMB Reports 2016; 49(1): 51-56] PMID:26246283

  18. Enhancement of paclitaxel-induced breast cancer cell death via the glycogen synthase kinase-3β-mediated B-cell lymphoma 2 regulation.

    PubMed

    Noh, Kyung Tae; Cha, Gil Sun; Kang, Tae Heung; Cho, Joon; Jung, In Duk; Kim, Kwang-Youn; Ahn, Soon-Cheol; You, Ji Chang; Park, Yeong-Min

    2016-01-01

    Glycogen synthase kinase-3β (GSK-3β) is a serine/threonine protein kinase that is known to mediate cancer cell death. Here, we show that B-cell lymphoma 2 (Bcl-2), an anti-apoptotic protein, is regulated by GSK-3β and that GSK-3β-mediated regulation of Bcl-2 is crucial for mitochondrial-dependent cell death in paclitaxel-stimulated cells. We demonstrate that MCF7 GSK-3β siRNA cells are more sensitive to cell death than MCF7 GFP control cells and that in the absence of GSK-3β, Bcl-2 levels are reduced, a result enhanced by paclitaxel. Paclitaxel-induced JNK (c-Jun N-terminal kinase) activation is critical for Bcl-2 modulation. In the absence of GSK-3β, Bcl-2 was unstable in an ubiquitination-dependent manner in both basal- and paclitaxeltreated cells. Furthermore, we demonstrate that GSK-3β-mediated regulation of Bcl-2 influences cytochrome C release and mitochondrial membrane potential. Taken together, our data suggest that GSK-3β-dependent regulation of Bcl-2 is crucial for mitochondria-dependent cell death in paclitaxel-mediated breast cancer therapy. PMID:26246283

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

    PubMed

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

    2016-02-16

    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

  20. 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

  1. Glycogen synthase kinase-3β antagonizes ROS-induced hepatocellular carcinoma cell death through suppression of the apoptosis signal-regulating kinase 1.

    PubMed

    Zhang, Na; Liu, Lu; Dou, Yueying; Song, Danqing; Deng, Hongbin

    2016-07-01

    Glycogen synthase kinase-3β (GSK-3β), a multifunctional kinase, is an important regulator of cancer cell survival. Apoptosis signal-regulating kinase 1 (ASK1) is also a key factor for controlling several cellular events including the cell cycle, senescence, and apoptosis, in response to reactive oxygen species (ROS). The role of GSK-3β regulating the activity and protein level of ASK1 in the cancer cells remains largely unexplored. In this study, we showed that GSK-3β inhibits ROS-induced hepatocellular carcinoma cell death by suppressing ASK1. We first found that ectopic expression of GSK-3β suppressed hydrogen peroxide (H2O2)-induced cell death in HepG2 cells and knockdown of endogenous GSK-3β expression exhibited opposite effects. Moreover, GSK-3β expression clearly inhibited H2O2-induced phosphorylation of ASK1 in HepG2 cells, in association with a decrease in ASK1 protein level. Further exploration revealed that GSK-3β induced ubiquitination and proteasome-dependent degradation of ASK1 via inhibition of ubiquitin-specific protease USP9X. Our results thus suggest that GSK-3β is a key factor involved in ASK1 activation and ROS-induced cell death. PMID:27221474

  2. Protein kinase R-like endoplasmic reticulum kinase and glycogen synthase kinase-3α/β regulate foam cell formation[S

    PubMed Central

    McAlpine, Cameron S.; Werstuck, Geoff H.

    2014-01-01

    Evidence suggests a causative role for endoplasmic reticulum (ER) stress in the development of atherosclerosis. This study investigated the potential role of glycogen synthase kinase (GSK)-3α/β in proatherogenic ER stress signaling. Thp1-derived macrophages were treated with the ER stress-inducing agents, glucosamine, thapsigargin, or palmitate. Using small-molecule inhibitors of specific unfolded protein response (UPR) signaling pathways, we found that protein kinase R-like ER kinase (PERK), but not inositol requiring enzyme 1 or activating transcription factor 6, is required for the activation of GSK3α/β by ER stress. GSK3α/β inhibition or siRNA-directed knockdown attenuated ER stress-induced expression of distal components of the PERK pathway. Macrophage foam cells within atherosclerotic plaques and isolated macrophages from ApoE−/− mice fed a diet supplemented with the GSK3α/β inhibitor valproate had reduced levels of C/EBP homologous protein (CHOP). GSK3α/β inhibition blocked ER stress-induced lipid accumulation and the upregulation of genes associated with lipid metabolism. In primary mouse macrophages, PERK inhibition blocked ER stress-induced lipid accumulation, whereas constitutively active S9A-GSK3β promoted foam cell formation and CHOP expression, even in cells treated with a PERK inhibitor. These findings suggest that ER stress-PERK-GSK3α/β signaling promotes proatherogenic macrophage lipid accumulation. PMID:25183803

  3. 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

  4. Inhibition of Glycogen Synthase Kinase-3β (GSK-3β) as potent therapeutic strategy to ameliorates L-dopa-induced dyskinesia in 6-OHDA parkinsonian rats

    PubMed Central

    Xie, Cheng-long; Lin, Jing-Ya; Wang, Mei-Hua; Zhang, Yu; Zhang, Su-fang; Wang, Xi-Jin; Liu, Zhen-Guo

    2016-01-01

    Levodopa (L-dopa) is the dominating therapy drug for exogenous dopaminergic substitution and can alleviate most of the manifestations of Parkinson’s disease (PD), but long-term therapy is associated with the emergence of L-dopa-induced dyskinesia (LID). Evidence points towards an involvement of Glycogen Synthase Kinase-3β (GSK-3β) in development of LID. In the present study, we found that animals rendered dyskinetic by L-dopa treatment, administration of TDZD8 (2mg/kg) obviously prevented the severity of AIM score, as well as improvement in motor function (P < 0.05). Moreover, the TDZD8-induced reduction in dyskinetic behavior correlated with a reduction in molecular correlates of LID. TDZD8 reduced the phosphorylation levels of tau, DARPP32, ERK and PKA protein, which represent molecular markers of LID, as well as reduced L-dopa-induced FosB mRNA and PPEB mRNA levels in the lesioned striatum. In addition, we found that TDZD8 antidyskinetic properties were overcome by D1 receptor, as pretreatment with SKF38393 (5 mg/kg, 10 mg/kg, reapectively), a D1 receptor agonist, blocked TDZD8 antidyskinetic actions. This study supported the hypothesis that GSK-3β played an important role in the development and expression of LID. Inhibition of GSK-3β with TDZD8 reduced the development of ALO AIM score and associated molecular changes in 6-OHDA-lesioned rats. PMID:26997328

  5. 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

  6. Glycogen synthase kinase-3 controls IL-10 expression in CD4+ effector T-cell subsets through epigenetic modification of the IL-10 promoter

    PubMed Central

    Hill, Elaine V; Ng, T H Sky; Burton, Bronwen R; Oakley, Charly M; Malik, Karim; Wraith, David C

    2015-01-01

    The serine/threonine kinase glycogen synthase kinase-3 (GSK3) plays an important role in balancing pro- and anti-inflammatory cytokines. We have examined the role of GSK3 in production of IL-10 by subsets of CD4+ T helper cells. Treatment of naive murine CD4+ T cells with GSK3 inhibitors did not affect their production of IL-10. However, treatment of Th1 and Th2 cells with GSK3 inhibitors dramatically increased production of IL-10. GSK3 inhibition also led to upregulation of IL-10 among Th1, Th2, and Th17 subsets isolated from human blood. The encephalitogenic potential of GSK3 inhibitor treated murine Th1 cells was significantly reduced in adoptive transfer experiments by an IL-10-dependent mechanism. Analysis of the murine IL-10 promoter in response to inhibition of GSK3 in Th1 cells showed modification to a transcriptionally active state indicated by changes in histone H3 acetylation and methylation. Additionally, GSK3 inhibition increased expression of the transcription factors c-Maf, Nfil3, and GATA3, correlating with the increase in IL-10. These findings are important in the context of autoimmune disease since they show that it is possible to reprogram disease-causing cells through GSK3 inhibition. PMID:25627813

  7. Glycogen synthase kinase-3-mediated phosphorylation of serine 73 targets sterol response element binding protein-1c (SREBP-1c) for proteasomal degradation.

    PubMed

    Dong, Qingming; Giorgianni, Francesco; Beranova-Giorgianni, Sarka; Deng, Xiong; O'Meally, Robert N; Bridges, Dave; Park, Edwards A; Cole, Robert N; Elam, Marshall B; Raghow, Rajendra

    2016-01-01

    Sterol regulatory element binding protein-1c (SREBP-1c) is a key transcription factor that regulates genes involved in the de novo lipid synthesis and glycolysis pathways. The structure, turnover and transactivation potential of SREBP-1c are regulated by macronutrients and hormones via a cascade of signalling kinases. Using MS, we have identified serine 73 as a novel glycogen synthase kinase-3 (GSK-3) phosphorylation site in the rat SREBP-1c purified from McA-RH7777 hepatoma cells. Our site-specific mutagenesis strategy revealed that the turnover of SREBP-1c, containing wild type, phospho-null (serine to alanine) or phospho-mimetic (serine to aspartic acid) substitutions, was differentially regulated. We show that the S73D mutant of pSREBP-1c, that mimicked a state of constitutive phosphorylation, dissociated from the SREBP-1c-SCAP complex more readily and underwent GSK-3-dependent proteasomal degradation via SCF(Fbw7) ubiquitin ligase pathway. Pharmacologic inhibition of GSK-3 or knockdown of GSK-3 by siRNA prevented accelerated degradation of SREBP-1c. As demonstrated by MS, SREBP-1c was phosphorylated in vitro by GSK-3β at serine 73. Phosphorylation of serine 73 also occurs in the intact liver. We propose that GSK-3-mediated phosphorylation of serine 73 in the rat SREBP-1c and its concomitant destabilization represents a novel mechanism involved in the inhibition of de novo lipid synthesis in the liver. PMID:26589965

  8. Shaggy/glycogen synthase kinase 3β and phosphorylation of Sarah/regulator of calcineurin are essential for completion of Drosophila female meiosis

    PubMed Central

    Takeo, Satomi; Swanson, Selene K.; Nandanan, Kavyasree; Nakai, Yasuhiro; Aigaki, Toshiro; Washburn, Michael P.; Florens, Laurence; Hawley, R. Scott

    2012-01-01

    The Ca2+/Calmodulin-dependent phosphatase calcineurin is essential for exit from meiotic arrest at metaphases I and II in Drosophila and Xenopus oocytes. We previously found that Sarah, the Drosophila homolog of regulator of calcineurin, acts as a positive regulator of calcineurin and is required to complete anaphase I of female meiosis. Here, we undertook biochemical approaches, including MS and posttranslational modification analyses, to better understand the mechanism by which Sarah regulates calcineurin. A search for phosphorylated residues revealed that Sarah is highly phosphorylated at Ser100, Thr102, and Ser219 in both ovaries and activated eggs and that Ser215 is phosphorylated only in activated eggs. Functional analyses using mutant forms of Sarah showed that phosphorylation at Ser215, a consensus phosphorylation site for glycogen synthase kinase 3β (GSK-3β) and its priming kinase site Ser219, are essential for Sarah function. Furthermore, germ-line clones homozygous for a null allele of shaggy (Drosophila GSK-3β) both fail to complete meiosis and lack phosphorylation of Sarah at Ser215, suggesting that the phosphorylation of Sarah by Shaggy/GSK-3β is required to complete meiosis. Our findings suggest a mechanism in which Shaggy/GSK-3β activates calcineurin through Sarah phosphorylation on egg activation in Drosophila. PMID:22421435

  9. 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

  10. Hyperhomocysteinaemia in rats is associated with erectile dysfunction by impairing endothelial nitric oxide synthase activity

    PubMed Central

    Jiang, Weijun; Xiong, Lei; Bin Yang; Li, Weiwei; Zhang, Jing; Zhou, Qing; Wu, Qiuyue; Li, Tianfu; Zhang, Cui; Zhang, Mingchao; Xia, Xinyi

    2016-01-01

    To investigate the effect of hyperhomocysteinaemia (HHCy) on penile erectile function in a rat model, a methionine-rich diet was used in which erectile function, the reproductive system, and nitric oxide synthase were characterized. The intracavernous pressure, apomorphine experiments, measurement of oxidative stress, hematoxylin and eosin staining, immunohistochemistry analysis, reverse transcription-polymerase chain reactions and measurement of endothelial nitric oxide synthase activity were utilized. Our results showed that erections in the middle-dose, high-dose, and interference (INF) groups were significantly lower than the control (P < 0.05). INF group, being fed with vitamins B and folic acid, demonstrated markedly improved penile erections compared with the middle-dose group (P < 0.05). HHCy-induced eNOS and phospho-eNOS protein expression was reduced and the antioxidant effect was markedly impaired. The data of the present data provide evidence that HHCy is a vascular risk factor for erectile dysfunction by impairing cavernosa endothelial nitric oxide synthase activity. Intake of vitamins B can alleviate this abnormality. PMID:27221552

  11. Hyperhomocysteinaemia in rats is associated with erectile dysfunction by impairing endothelial nitric oxide synthase activity.

    PubMed

    Jiang, Weijun; Xiong, Lei; Bin Yang; Li, Weiwei; Zhang, Jing; Zhou, Qing; Wu, Qiuyue; Li, Tianfu; Zhang, Cui; Zhang, Mingchao; Xia, Xinyi

    2016-01-01

    To investigate the effect of hyperhomocysteinaemia (HHCy) on penile erectile function in a rat model, a methionine-rich diet was used in which erectile function, the reproductive system, and nitric oxide synthase were characterized. The intracavernous pressure, apomorphine experiments, measurement of oxidative stress, hematoxylin and eosin staining, immunohistochemistry analysis, reverse transcription-polymerase chain reactions and measurement of endothelial nitric oxide synthase activity were utilized. Our results showed that erections in the middle-dose, high-dose, and interference (INF) groups were significantly lower than the control (P < 0.05). INF group, being fed with vitamins B and folic acid, demonstrated markedly improved penile erections compared with the middle-dose group (P < 0.05). HHCy-induced eNOS and phospho-eNOS protein expression was reduced and the antioxidant effect was markedly impaired. The data of the present data provide evidence that HHCy is a vascular risk factor for erectile dysfunction by impairing cavernosa endothelial nitric oxide synthase activity. Intake of vitamins B can alleviate this abnormality. PMID:27221552

  12. AMP-Activated Protein Kinase and Glycogen Synthase Kinase 3β Modulate the Severity of Sepsis-Induced Lung Injury

    PubMed Central

    Liu, Zhongyu; Bone, Nathaniel; Jiang, Shaoning; Park, Dae Won; Tadie, Jean-Marc; Deshane, Jessy; Rodriguez, Cilina Ann; Pittet, Jean-Francois; Abraham, Edward; Zmijewski, Jaroslaw W

    2015-01-01

    Alterations in metabolic and bioenergetic homeostasis contribute to sepsis-mediated organ injury. However, how AMP-activated protein kinase (AMPK), a major sensor and regulator of energy expenditure and production, affects development of organ injury and loss of innate capacity during polymicrobial sepsis remains unclear. In the present experiments, we found that cross-talk between the AMPK and GSK3β signaling pathways controls chemotaxis and the ability of neutrophils and macrophages to kill bacteria ex vivo. In mice with polymicrobial abdominal sepsis or more severe sepsis induced by the combination of hemorrhage and intraabdominal infection, administration of the AMPK activator metformin or the GSK3β inhibitor SB216763 reduced the severity of acute lung injury (ALI). Improved survival in metformin-treated septic mice was correlated with preservation of mitochondrial complex V (ATP synthase) function and increased amounts of ETC complex III and IV. Although immunosuppression is a consequence of sepsis, metformin effectively increased innate immune capacity to eradicate P. aeruginosa in the lungs of septic mice. We also found that AMPK activation diminished accumulation of the immunosuppressive transcriptional factor HIF-1α as well as the development of endotoxin tolerance in LPS-treated macrophages. Furthermore, AMPK-dependent preservation of mitochondrial membrane potential also prevented LPS-mediated dysfunction of neutrophil chemotaxis. These results indicate that AMPK activation reduces the severity of polymicrobial sepsis-induced lung injury and prevents the development of sepsis-associated immunosuppression. PMID:26650187

  13. Glycogen synthase kinase 3β sustains invasion of glioblastoma via the focal adhesion kinase, Rac1, and c-Jun N-terminal kinase-mediated pathway.

    PubMed

    Chikano, Yuri; Domoto, Takahiro; Furuta, Takuya; Sabit, Hemragul; Kitano-Tamura, Ayako; Pyko, Ilya V; Takino, Takahisa; Sai, Yoshimichi; Hayashi, Yutaka; Sato, Hiroshi; Miyamoto, Ken-ichi; Nakada, Mitsutoshi; Minamoto, Toshinari

    2015-02-01

    The failure of current treatment options for glioblastoma stems from their inability to control tumor cell proliferation and invasion. Biologically targeted therapies offer great hope and one promising target is glycogen synthase kinase-3β (GSK3β), implicated in various diseases, including cancer. We previously reported that inhibition of GSK3β compromises the survival and proliferation of glioblastoma cells, induces their apoptosis, and sensitizes them to temozolomide and radiation. Here, we explore whether GSK3β also contributes to the highly invasive nature of glioblastoma. The effects of GSK3β inhibition on migration and invasion of glioblastoma cells were examined by wound-healing and Transwell assays, as well as in a mouse model of glioblastoma. We also investigated changes in cellular microarchitectures, cytoskeletal components, and proteins responsible for cell motility and invasion. Inhibition of GSK3β attenuated the migration and invasion of glioblastoma cells in vitro and that of tumor cells in a mouse model of glioblastoma. These effects were associated with suppression of the molecular axis involving focal adhesion kinase, guanine nucleotide exchange factors/Rac1 and c-Jun N-terminal kinase. Changes in cellular phenotypes responsible for cell motility and invasion were also observed, including decreased formation of lamellipodia and invadopodium-like microstructures and alterations in the subcellular localization, and activity of Rac1 and F-actin. These changes coincided with decreased expression of matrix metalloproteinases. Our results confirm the potential of GSK3β as an attractive therapeutic target against glioblastoma invasion, thus highlighting a second role in this tumor type in addition to its involvement in chemo- and radioresistance. PMID:25504636

  14. STRE- and cAMP-independent transcriptional induction of Saccharomyces cerevisiae GSY2 encoding glycogen synthase during diauxic growth on glucose.

    PubMed

    Parrou, J L; Enjalbert, B; François, J

    1999-10-01

    It has been shown that the so-called stationary phase GSY2 gene encoding glycogen synthase was induced as the cells left the exponential phase of growth, while glucose and all other nutrients were still plentiful in the medium (Parrou et al., 1999). Since this effect was essentially controlled at the transcriptional level, we looked for the cis- and trans-acting elements required for this specific growth-related genetic event. We demonstrated that mutations of the HAP2/3/4 binding site and of the two STress-Responsive cis-Elements (STRE) did not abolish the early induction of GSY2, although the latter mutation led to a 20-fold drop in the transcriptional activity of the promoter, as determined from lacZ gene fusions. Insertion of a DNA fragment (from -390 to -167 bp, relative to the ATG) of the promoter lacking the two STREs, upstream to the TATA box of a CYC1-lacZ fusion gene, allowed this reporter gene to be induced with a kinetic similar to that of GSY2-lacZ. Mutations in BCY1, which results in a hyperactive protein kinase A, did not alleviate the early induction, while causing a five- to 10-fold reduction in the transcriptional activity of GSY2. In addition, the repressive effect of protein kinase A was quantitatively conserved when both STREs were mutated in GSY2 promoter, indicating that the negative control of gene expression by the RAS-cAMP signalling pathway does not act solely through STREs. Taken together, these results are indicative of an active process that couples growth control to dynamic glucose consumption. PMID:10514565

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

    SciTech Connect

    Chow Winsion; Hou Guangpei; Bendeck, Michelle P.

    2008-10-01

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

  16. 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

  17. Tanshinone I Enhances Neurogenesis in the Mouse Hippocampal Dentate Gyrus via Increasing Wnt-3, Phosphorylated Glycogen Synthase Kinase-3β and β-Catenin Immunoreactivities.

    PubMed

    Chen, Bai Hui; Park, Joon Ha; Cho, Jeong Hwi; Kim, In Hye; Lee, Jae Chul; Lee, Tae-Kyeong; Ahn, Ji Hyeon; Tae, Hyun Jin; Shin, Bich Na; Kim, Jong-Dai; Kang, Il Jun; Won, Moo-Ho; Lee, Yun Lyul

    2016-08-01

    Tanshinone I (TsI), a lipophilic diterpene extracted from Danshan (Radix Salvia miltiorrhizae), exerts neuroprotection in cerebrovascular diseases including transient ischemic attack. In this study, we examined effects of TsI on cell proliferation and neuronal differentiation in the subgranular zone (SGZ) of the mouse dentate gyrus (DG) using Ki-67, BrdU and doublecortin (DCX) immunohistochemistry. Mice were treated with 1 and 2 mg/kg TsI for 28 days. In the 1 mg/kg TsI-treated-group, distribution patterns of BrdU, Ki-67 and DCX positive ((+)) cells in the SGZ were similar to those in the vehicle-treated-group. However, in the 2 mg/kg TsI-treated-group, double labeled BrdU(+)/NeuN(+) cells, which are mature neurons, as well as Ki-67(+), DCX(+) and BrdU(+) cells were significantly increased compared with those in the vehicle-treated-group. On the other hand, immunoreactivities and protein levels of Wnt-3, β-catenin and serine-9-glycogen synthase kinase-3β (p-GSK-3β), which are related with morphogenesis, were significantly increased in the granule cell layer of the DG only in the 2 mg/kg TsI-treated-group. Therefore, these findings indicate that TsI can promote neurogenesis in the mouse DG and that the neurogenesis is related with increases of Wnt-3, p-GSK-3β and β-catenin immunoreactivities. PMID:27053301

  18. Glycogen synthase kinase-3β (GSK3β) inhibition suppresses the inflammatory response to Francisella infection and protects against tularemia in mice

    PubMed Central

    Zhang, Ping; Katz, Jenny; Michalek, Suzanne M.

    2011-01-01

    Francisella tularensis, the causative agent of tularemia, is currently considered a category A bioterrorism agent due to its high virulence. Infection with F. tularensis results in an inflammatory response that plays an important role in the pathogenesis of the disease; however, the cellular mechanisms regulating this response are poorly understood. Glycogen synthase kinase-3β (GSK3β) is a serine/threonine protein kinase that has recently emerged as a key regulatory switch in the modulation of the inflammatory response. In this study, we investigated the effect of GSK3β inhibition in regulating F. tularensis LVS-induced inflammatory responses. F. tularensis LVS infection of murine peritoneal macrophages induced a TLR2 dependent phosphorylation of GSK3β. Inhibition of GSK3β resulted in a significant decrease in the production of pro-inflammatory cytokine IL-6, IL-12p40 and TNF-α, as well as a significant increase in the production of the anti-inflammatory cytokine IL-10. GSK3β regulated the F. tularensis LVS-induced cytokine response by differentially affecting the activation of transcription factors NF-κB and CREB. Inhibition of GSK3β by lithium in vivo suppressed the inflammatory response in mice infected with F. tularensis LVS and conferred a survival advantage. In addition, we show that the production of IFN-γ contributed to the development of tularemia and to the fatal outcome of the infected animals, depending on the timing and the relative level of the IFN-γ produced. IFN-γ potentiated F. tularensis LVS-induced cytokine production by increasing GSK3β activity and the nuclear translocation of NF-κB. Taken together, these results demonstrate a regulatory function of GSK3β in modulating inflammatory responses that can be detrimental to the host during an F. tularensis LVS infection, and suggest that inhibition of GSK3β may represent a novel therapeutic approach in the treatment of tularemia. PMID:18929413

  19. Stimulation of EphB2 attenuates tau phosphorylation through PI3K/Akt-mediated inactivation of glycogen synthase kinase-3β

    PubMed Central

    Jiang, Jun; Wang, Zhi-Hao; Qu, Min; Gao, Di; Liu, Xiu-Ping; Zhu, Ling-Qiang; Wang, Jian-Zhi

    2015-01-01

    Abnormal tau hyperphosphorylation is an early pathological marker of Alzheimer’s disease (AD), however, the upstream factors that regulate tau phosphorylation are not illustrated and there is no efficient strategy to arrest tau hyperphosphorylation. Here, we find that activation of endogenous EphB2 receptor by ligand stimulation (ephrinB1/Fc) or by ectopic expression of EphB2 plus the ligand stimulation induces a remarkable tau dephosphorylation at multiple AD-associated sites in SK-N-SH cells and human embryonic kidney cells that stably express human tau (HEK293-tau). In cultured hippocampal neurons and the hippocampus of human tau transgenic mice, dephosphorylation of tau proteins was also detected by stimulation of EphB2 receptor. EphB2 activation inhibits glycogen synthase kinase-3β (GSK-3β), a crucial tau kinase, and activates phosphatidylinositol-3-kinase (PI3K)/Akt both in vitro and in vivo, whereas simultaneous inhibition of PI3K or upregulation of GSK-3β abolishes the EphB2 stimulation-induced tau dephosphorylation. Finally, we confirm that ephrinB1/Fc treatment induces tyrosine phosphorylation (activation) of EphB2, while deletion of the tyrosine kinase domain (VM) of EphB2 eliminates the receptor stimulation-induced GSK-3β inhibition and tau dephosphorylation. We conclude that activation of EphB2 receptor kinase arrests tau hyperphosphorylation through PI3K-/Akt-mediated GSK-3β inhibition. Our data provide a novel membranous target to antagonize AD-like tau pathology. PMID:26119563

  20. 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.

  1. Glycogen synthase kinase-3β inhibition in the medial prefrontal cortex mediates paradoxical amphetamine action in a mouse model of ADHD

    PubMed Central

    Yen, Yi-Chun; Gassen, Nils C.; Zellner, Andreas; Rein, Theo; Landgraf, Rainer; Wotjak, Carsten T.; Anderzhanova, Elmira

    2015-01-01

    Psychostimulants show therapeutic efficacy in the treatment of attention-deficit hyperactivity disorder (ADHD). It is generally assumed that they ameliorate ADHD symptoms via interfering with monoaminergic signaling. We combined behavioral pharmacology, neurochemistry and molecular analyses to identify mechanisms underlying the paradoxical calming effect of amphetamine in low trait anxiety behavior (LAB) mice, a novel multigenetic animal model of ADHD. Amphetamine (1 mg/kg) and methylphenidate (10 mg/kg) elicited similar dopamine and norepinephrine release in the medial prefrontal cortex (mPFC) and in the striatum of LAB mice. In contrast, amphetamine decreased, while methylphenidate increased locomotor activity. This argues against changes in dopamine and/or norepinephrine release as mediators of amphetamine paradoxical effects. Instead, the calming activity of amphetamine corresponded to the inhibition of glycogen synthase kinase 3β (GSK3β) activity, specifically in the mPFC. Accordingly, not only systemic administration of the GSK3β inhibitor TDZD-8 (20 mg/kg), but also local microinjections of TDZD-8 and amphetamine into the mPFC, but not into the striatum, decreased locomotor activity in LAB mice. Amphetamine effects seem to depend on NMDA receptor signaling, since pre- or co-treatment with MK-801 (0.3 mg/kg) abolished the effects of amphetamine (1 mg/kg) on the locomotion and on the phosphorylation of GSK3β at the level of the mPFC. Taken together, the paradoxical calming effect of amphetamine in hyperactive LAB mice concurs with a decreased GSK3β activity in the mPFC. This effect appears to be independent of dopamine or norepinephrine release, but contingent on NMDA receptor signaling. PMID:25852508

  2. Inhibition of glycogen synthase kinase 3β promotes autophagy to protect mice from acute liver failure mediated by peroxisome proliferator-activated receptor α

    PubMed Central

    Ren, F; Zhang, L; Zhang, X; Shi, H; Wen, T; Bai, L; Zheng, S; Chen, Y; Chen, D; Li, L; Duan, Z

    2016-01-01

    Our previous studies have demonstrated that inhibition of glycogen synthase kinase 3β (GSK3β) activity protects mice from acute liver failure (ALF), whereas its protective and regulatory mechanism remains elusive. Autophagy is a recently recognized rudimentary cellular response to inflammation and injury. The aim of the present study was to test the hypothesis that inhibition of GSK3β mediates autophagy to inhibit liver inflammation and protect against ALF. In ALF mice model induced by d-galactosamine (d-GalN) and lipopolysaccharide (LPS), autophagy was repressed compared with normal control, and d-GalN/LPS can directly induce autophagic flux in the progression of ALF mice. Autophagy activation by rapamycin protected against liver injury and its inhibition by 3-methyladenine (3-MA) or autophagy gene 7 (Atg7) small interfering RNA (siRNA) exacerbated liver injury. The protective effect of GSK3β inhibition on ALF mice model depending on the induction of autophagy, because that inhibition of GSK3β promoted autophagy in vitro and in vivo, and inhibition of autophagy reversed liver protection and inflammation of GSK3β inhibition. Furthermore, inhibition of GSK3β increased the expression of peroxisome proliferator-activated receptor α (PPARα), and the downregulated PPARα by siRNA decreased autophagy induced by GSK3β inhibition. More importantly, the expressions of autophagy-related gene and PPARα are significantly downregulated and the activity of GSK3β is significantly upregulated in liver of ALF patients with hepatitis B virus. Thus, we have demonstrated the new pathological mechanism of ALF that the increased GSK3β activity suppresses autophagy to promote the occurrence and development of ALF by inhibiting PPARα pathway. PMID:27010852

  3. 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

  4. Foscarnet, an inhibitor of the sodium-phosphate cotransporter NaPi-IIa, inhibits phosphorylation of glycogen synthase kinase-3β by lithium in the rat kidney cortex.

    PubMed

    Uwai, Yuichi; Kawasaki, Tatsuya; Nabekura, Tomohiro

    2016-06-01

    Lithium, which is used in the treatment of and prophylaxis for bipolar disease, inhibits glycogen synthase kinase-3β (GSK3β) by producing its phosphorylated form (p-GSK3β). GSK3β plays a role in apoptosis and some kinds of acute kidney injuries, and the formation of p-GSK3β is considered to contribute to protection against acute kidney injury. We previously reported that the sodium-phosphate cotransporter NaPi-IIa (SLC34A1) mediated the reabsorption of lithium in the rat kidney. In the present study, the phosphorylation status of GSK3β in the kidney cortex of rats administered lithium chloride and foscarnet, a typical inhibitor of NaPi-IIa, was examined using Western blotting. Under a 2-h infusion of lithium chloride, the plasma concentration of lithium was 1.06 mEq/l, and its renal clearance was calculated as 1.18 ml/min/kg, which was 29.6% of creatinine clearance. The abundance of p-GSK3β in the kidney cortex was augmented by the administration of lithium. The simultaneous infusion of foscarnet increased the renal clearance of lithium and its ratio to creatinine clearance as well as the urinary excretion of phosphate. Foscarnet also inhibited the lithium-induced phosphorylation of GSK3β. These results suggest that the reabsorption of lithium by NaPi-IIa triggers the phosphorylation of GSK3β in the rat kidney cortex. PMID:27238574

  5. 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

  6. 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

  7. A Screen for Extracellular Signal-Regulated Kinase-Primed Glycogen Synthase Kinase 3 Substrates Identifies the p53 Inhibitor iASPP

    PubMed Central

    Woodard, Crystal; Liao, Gangling; Goodwin, C. Rory; Hu, Jianfei; Xie, Zhi; dos Reis, Thaila F.; Newman, Rob; Rho, Heesool; Qian, Jiang

    2015-01-01

    ABSTRACT The Kaposi's sarcoma-associated herpesvirus (KSHV) LANA protein is essential for the replication and maintenance of virus genomes in latently KSHV-infected cells. LANA also drives dysregulated cell growth through a multiplicity of mechanisms that include altering the activity of the cellular kinases extracellular signal-regulated kinase (ERK) and glycogen synthase kinase 3 (GSK-3). To investigate the potential impact of these changes in enzyme activity, we used protein microarrays to identify cell proteins that were phosphorylated by the combination of ERK and GSK-3. The assays identified 58 potential ERK-primed GSK-3 substrates, of which 23 had evidence for in vivo phosphorylation in mass spectrometry databases. Two of these, SMAD4 and iASPP, were selected for further analysis and were confirmed as ERK-primed GSK-3 substrates. Cotransfection experiments revealed that iASPP, but not SMAD4, was targeted for degradation in the presence of GSK-3. iASPP interferes with apoptosis induced by p53 family members. To determine the importance of iASPP to KSHV-infected-cell growth, primary effusion lymphoma (PEL) cells were treated with an iASPP inhibitor in the presence or absence of the MDM2 inhibitor Nutlin-3. Drug inhibition of iASPP activity induced apoptosis in BC3 and BCBL1 PEL cells but did not induce poly(ADP-ribose) polymerase (PARP) cleavage in virus-negative BJAB cells. The effect of iASPP inhibition was additive with that of Nutlin-3. Interfering with iASPP function is therefore another mechanism that can sensitize KSHV-positive PEL cells to cell death. IMPORTANCE KSHV is associated with several malignancies, including primary effusion lymphoma (PEL). The KSHV-encoded LANA protein is multifunctional and promotes both cell growth and resistance to cell death. LANA is known to activate ERK and limit the activity of another kinase, GSK-3. To discover ways in which LANA manipulation of these two kinases might impact PEL cell survival, we screened a human

  8. Lupus Nephritis: Glycogen Synthase Kinase 3β Promotion of Renal Damage Through Activation of the NLRP3 Inflammasome in Lupus-Prone Mice

    PubMed Central

    Zhao, Jijun; Wang, Hongyue; Huang, Yuefang; Zhang, Hui; Wang, Shuang; Gaskin, Felicia; Yang, Niansheng; Fu, Shu Man

    2015-01-01

    Objective Glycogen synthase kinase 3β (GSK-3β) has been demonstrated to be involved in immune and inflammatory responses via multiple signaling pathways, leading to the production of proinflammatory cytokines. The purpose of this study was to investigate the role of GSK-3β in the pathogenesis of lupus nephritis in 2 mouse models. Methods Thiadiazolidinone 8 (TDZD-8), a selective inhibitor of GSK-3β, was administered intraperitoneally to 12-week-old MRL/lpr mice for 8 weeks or to 22-week-old (NZB × NZW)F1 mice for 12 weeks. The expression of GSK-3β and NLRP3 inflammasome components was analyzed. Proteinuria, biochemical parameters, proinflammatory cytokines, anti–double-stranded DNA (anti-dsDNA) antibody levels, and renal pathology were examined. In vitro, the effect of GSK-3β–directed small interfering RNA (siRNA) on NLRP3 inflammasome activation was evaluated in bone marrow–derived macrophages (BMMs) from the mice and in the J774A.1 macrophage cell line. Results The incidence of severe proteinuria and renal inflammation was significantly attenuated in both models, with a significant reduction in anti-dsDNA antibody production, immune complex deposition in the kidney, and circulating proinflammatory cytokine levels. TDZD-8 inhibited the activation of GSK-3β and caspase 1, with a concomitant decrease in interleukin-1β (IL-1β) synthesis. In vitro, GSK-3β siRNA transfection of mouse BMMs and the J774A.1 cell line with GSK-3β siRNA inhibited the expression of GSK-3β, the activation of caspase 1, and the production of IL-1β. Conclusion These results show that GSK-3β promotes lupus nephritis at least partly by activating the NLRP3/IL-1β pathway. The linking of GSK-3β to the NLRP3/IL-1β pathway is a novel observation in our study. Our results suggest that the GSK-3β/NLRP3/IL-1β pathway may be a potential therapeutic target for lupus in humans. PMID:25512114

  9. ER stress-mediated apoptosis induced by celastrol in cancer cells and important role of glycogen synthase kinase-3β in the signal network.

    PubMed

    Feng, L; Zhang, D; Fan, C; Ma, C; Yang, W; Meng, Y; Wu, W; Guan, S; Jiang, B; Yang, M; Liu, X; Guo, D

    2013-01-01

    HeLa cells treated with celastrol, a natural compound with inhibitive effect on proteasome, exhibited increase in apoptotic rate and characteristics of apoptosis. To clarify the signal network activated by celastrol to induce apoptosis, both the direct target proteins and undirect target proteins of celastrol were searched in the present study. Proteasome catalytic subunit β1 was predicted by computational analysis to be a possible direct target of celastrol and confirmed by checking direct effect of celastrol on the activity of recombinant human proteasome subunit β1 in vitro. Undirect target-related proteins of celastrol were searched using proteomic studies including two-dimensional electrophoresis (2-DE) analysis and iTRAQ-based LC-MS analysis. Possible target-related proteins of celastrol such as endoplasmic reticulum protein 29 (ERP29) and mitochondrial import receptor Tom22 (TOM22) were found by 2-DE analysis of total cellular protein expression profiles. Further study showed that celastrol induced ER stress and ER stress inhibitor could ameliorate cell death induced by celastrol. Celastrol induced translocation of Bax into the mitochondria, which might be related to the upregulation of BH-3-only proteins such as BIM and the increase in the expression level of TOM22. To further search possible target-related proteins of celastrol in ER and ER-related fractions, iTRAQ-based LC-MS method was use to analyze protein expression profiles of ER/microsomal vesicles-riched fraction of cells with or without celastrol treatment. Based on possible target-related proteins found in both 2-DE analysis and iTRAQ-based LC-MS analysis, protein-protein interaction (PPI) network was established using bioinformatic analysis. The important role of glycogen synthase kinase-3β (GSK3β) in the signal cascades of celastrol was suggested. Pretreatment of LiCL, an inhibitor of GSK3β, could significantly ameliorate apoptosis induced by celastrol. On the basis of the results of the

  10. 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

  11. Insulin resistance after a 72-h fast is associated with impaired AS160 phosphorylation and accumulation of lipid and glycogen in human skeletal muscle

    PubMed Central

    Vendelbo, M. H.; Clasen, B. F. F.; Treebak, J. T.; Møller, L.; Krusenstjerna-Hafstrøm, T.; Madsen, M.; Nielsen, T. S.; Stødkilde-Jørgensen, H.; Pedersen, S. B.; Jørgensen, J. O. L.; Goodyear, L. J.; Wojtaszewski, J. F. P.; Møller, N.

    2012-01-01

    During fasting, human skeletal muscle depends on lipid oxidation for its energy substrate metabolism. This is associated with the development of insulin resistance and a subsequent reduction of insulin-stimulated glucose uptake. The underlying mechanisms controlling insulin action on skeletal muscle under these conditions are unresolved. In a randomized design, we investigated eight healthy subjects after a 72-h fast compared with a 10-h overnight fast. Insulin action on skeletal muscle was assessed by a hyperinsulinemic euglycemic clamp and by determining insulin signaling to glucose transport. In addition, substrate oxidation, skeletal muscle lipid content, regulation of glycogen synthesis, and AMPK signaling were assessed. Skeletal muscle insulin sensitivity was reduced profoundly in response to a 72-h fast and substrate oxidation shifted to predominantly lipid oxidation. This was associated with accumulation of both lipid and glycogen in skeletal muscle. Intracellular insulin signaling to glucose transport was impaired by regulation of phosphorylation at specific sites on AS160 but not TBC1D1, both key regulators of glucose uptake. In contrast, fasting did not impact phosphorylation of AMPK or insulin regulation of Akt, both of which are established upstream kinases of AS160. These findings show that insulin resistance in muscles from healthy individuals is associated with suppression of site-specific phosphorylation of AS160, without Akt or AMPK being affected. This impairment of AS160 phosphorylation, in combination with glycogen accumulation and increased intramuscular lipid content, may provide the underlying mechanisms for resistance to insulin in skeletal muscle after a prolonged fast. PMID:22028408

  12. Glycogen metabolism in humans☆☆☆

    PubMed Central

    Adeva-Andany, María M.; González-Lucán, Manuel; Donapetry-García, Cristóbal; Fernández-Fernández, Carlos; Ameneiros-Rodríguez, Eva

    2016-01-01

    In the human body, glycogen is a branched polymer of glucose stored mainly in the liver and the skeletal muscle that supplies glucose to the blood stream during fasting periods and to the muscle cells during muscle contraction. Glycogen has been identified in other tissues such as brain, heart, kidney, adipose tissue, and erythrocytes, but glycogen function in these tissues is mostly unknown. Glycogen synthesis requires a series of reactions that include glucose entrance into the cell through transporters, phosphorylation of glucose to glucose 6-phosphate, isomerization to glucose 1-phosphate, and formation of uridine 5ʹ-diphosphate-glucose, which is the direct glucose donor for glycogen synthesis. Glycogenin catalyzes the formation of a short glucose polymer that is extended by the action of glycogen synthase. Glycogen branching enzyme introduces branch points in the glycogen particle at even intervals. Laforin and malin are proteins involved in glycogen assembly but their specific function remains elusive in humans. Glycogen is accumulated in the liver primarily during the postprandial period and in the skeletal muscle predominantly after exercise. In the cytosol, glycogen breakdown or glycogenolysis is carried out by two enzymes, glycogen phosphorylase which releases glucose 1-phosphate from the linear chains of glycogen, and glycogen debranching enzyme which untangles the branch points. In the lysosomes, glycogen degradation is catalyzed by α-glucosidase. The glucose 6-phosphatase system catalyzes the dephosphorylation of glucose 6-phosphate to glucose, a necessary step for free glucose to leave the cell. Mutations in the genes encoding the enzymes involved in glycogen metabolism cause glycogen storage diseases. PMID:27051594

  13. 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

  14. Regulation of glucose and glycogen metabolism during and after exercise.

    PubMed

    Jensen, Thomas E; Richter, Erik A

    2012-03-01

    Utilization of carbohydrate in the form of intramuscular glycogen stores and glucose delivered from plasma becomes an increasingly important energy substrate to the working muscle with increasing exercise intensity. This review gives an update on the molecular signals by which glucose transport is increased in the contracting muscle followed by a discussion of glycogen mobilization and synthesis by the action of glycogen phosphorylase and glycogen synthase, respectively. Finally, this review deals with the signalling relaying the well-described increased sensitivity of glucose transport to insulin in the post-exercise period which can result in an overshoot of intramuscular glycogen resynthesis post exercise (glycogen supercompensation). PMID:22199166

  15. Regulation of glucose and glycogen metabolism during and after exercise

    PubMed Central

    Jensen, Thomas E; Richter, Erik A

    2012-01-01

    Utilization of carbohydrate in the form of intramuscular glycogen stores and glucose delivered from plasma becomes an increasingly important energy substrate to the working muscle with increasing exercise intensity. This review gives an update on the molecular signals by which glucose transport is increased in the contracting muscle followed by a discussion of glycogen mobilization and synthesis by the action of glycogen phosphorylase and glycogen synthase, respectively. Finally, this review deals with the signalling relaying the well-described increased sensitivity of glucose transport to insulin in the post-exercise period which can result in an overshoot of intramuscular glycogen resynthesis post exercise (glycogen supercompensation). PMID:22199166

  16. Radiosynthesis and preliminary PET evaluation of glycogen synthase kinase 3β (GSK-3β) inhibitors containing [(11)C]methylsulfanyl, [(11)C]methylsulfinyl or [(11)C]methylsulfonyl groups.

    PubMed

    Kumata, Katsushi; Yui, Joji; Xie, Lin; Zhang, Yiding; Nengaki, Nobuki; Fujinaga, Masayuki; Yamasaki, Tomoteru; Shimoda, Yoko; Zhang, Ming-Rong

    2015-08-15

    Three compounds 1-3 containing methyl-sufanyl, sufinyl, or sulfonyl groups are strong inhibitors of glycogen synthase kinase 3β (GSK-3β), an enzyme associated with Alzheimer's disease. We labeled 1-3 with (11)C for a positron emission tomography (PET) brain imaging study. A novel thiophenol precursor 4 for radiosynthesis was prepared by reacting sulfoxide 2 with trifluoroacetic anhydride. [(11)C]1 was synthesized by reacting 4 with [(11)C]methyl iodide in 52 ± 5% radiochemical yield (n = 5, based on [(11)C]CO2, corrected for decay). Oxidation of [(11)C]1 with Oxone® produced [(11)C]2 and [(11)C]3, respectively. PET with [(11)C]1 and [(11)C]3 showed 2 fold higher brain uptake of radioactivity in a mouse model of cold water stress in which GSK-3β expression was increased, than in the controls. PMID:26067173

  17. Cross-talk between glycogen synthase kinase 3β (GSK3β) and p38MAPK regulates myocyte enhancer factor 2 (MEF2) activity in skeletal and cardiac muscle.

    PubMed

    Dionyssiou, M G; Nowacki, N B; Hashemi, S; Zhao, J; Kerr, A; Tsushima, R G; McDermott, J C

    2013-01-01

    Characterizing the signaling network that controls MEF2 transcription factors is crucial for understanding skeletal and cardiac muscle gene expression. Glycogen synthase kinase 3β (GSK3β) regulates MEF2 activity indirectly through reciprocal regulation of p38MAPK. Cross-talk between GSK3β and p38MAPK regulates MEF2 activity in skeletal and cardiac muscle. Understanding cross-talk in the signaling network converging at MEF2 control has therapeutic implications in cardiac and skeletal muscle pathology. Glycogen synthase kinase 3β (GSK3β) is a known regulator of striated muscle gene expression suppressing both myogenesis and cardiomyocyte hypertrophy. Since myocyte enhancer factor 2 (MEF2) proteins are key transcriptional regulators in both systems, we assessed whether MEF2 is a target for GSK3β. Pharmacological inhibition of GSK3β resulted in enhanced MEF2A/D expression and transcriptional activity in skeletal myoblasts and cardiac myocytes. Even though in silico analysis revealed GSK3β consensus (S/T)XXX(S/T) sites on MEF2A, a subsequent in vitro kinase assay revealed that MEF2A is only a weak substrate. However, we did observe a posttranslational modification in MEF2A in skeletal myoblasts treated with a GSK3β inhibitor which coincided with increased p38MAPK phosphorylation, a potent MEF2A activator, indicating that GSK3β inhibition may de-repress p38MAPK. Heart specific excision of GSK3β in mice also resulted in up-regulation of p38MAPK activity. Interestingly, upon pharmacological p38MAPK inhibition (SB203580), GSK3β inhibition loses its effect on MEF2 transcriptional activity suggesting potent cross-talk between the two pathways. Thus we have documented that cross-talk between p38MAPK and GSK3β signaling converges on MEF2 activity having potential consequences for therapeutic modulation of cardiac and skeletal muscle gene expression. PMID:23137781

  18. Impaired cellulose synthase guidance leads to stem torsion and twists phyllotactic patterns in Arabidopsis.

    PubMed

    Landrein, Benoît; Lathe, Rahul; Bringmann, Martin; Vouillot, Cyril; Ivakov, Alexander; Boudaoud, Arezki; Persson, Staffan; Hamant, Olivier

    2013-05-20

    The parallel alignment of stiff cellulose microfibrils in plant-cell walls mediates anisotropic growth. This is largely controlled by cortical microtubules, which drive the insertion and trajectory of the cellulose synthase (CESA) complex at the plasma membrane. The CESA interactive protein 1 (CSI1) acts as a physical linker between CESA and cortical microtubules. Here we show that the inflorescence stems of csi1 mutants exhibit subtle right-handed torsion. Because cellulose deposition is largely uncoupled from cortical microtubules in csi1, we hypothesize that strictly transverse deposition of microfibrils in the wild-type is replaced by a helical orientation of uniform handedness in the mutant and that the helical microfibril alignment generates torsion. Interestingly, both elastic and viscous models for an expanding cell predict that a net helical orientation of microfibrils gives rise to a torque. We indeed observed tilted microfibrils in csi1 cells, and the torsion was almost absent in a csi1 prc1 background with impaired cellulose synthesis. In addition, the stem torsion led to a novel bimodal and robust phyllotactic pattern in the csi1 mutant, illustrating how growth perturbations can replace one robust mathematical pattern with a different, equally robust pattern. PMID:23623553

  19. 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

  20. 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

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

    PubMed

    Henriksen, E J; Kirby, C R; Tischler, M E

    1989-06-01

    The time course of glycogen changes in soleus muscle recovering from 3 days of nonweight bearing by hindlimb suspension was investigated. Within 15 min and up to 2 h, muscle glycogen decreased. Coincidentally, muscle glucose 6-phosphate and the fractional activity of glycogen phosphorylase, measured at the fresh muscle concentrations of AMP, increased. Increased fractional activity of glycogen synthase during this time was likely the result of greater glucose 6-phosphate and decreased glycogen. From 2 to 4 h, when the synthase activity remained elevated and the phosphorylase activity declined, glycogen levels increased (glycogen supercompensation). A further increase of glycogen up to 24 h did not correlate with the enzyme activities. Between 24 and 72 h, glycogen decreased to control values, possibly initiated by high phosphorylase activity at 24 h. At 12 and 24 h, the inverse relationship between glycogen concentration and the synthase activity ratio was lost, indicating that reloading transiently uncoupled glycogen control of this enzyme. These data suggest that the activities of glycogen synthase and phosphorylase, when measured at physiological effector levels, likely provide the closest approximation to the actual enzyme activities in vivo. Measurements made in this way effectively explained the majority of the changes in the soleus glycogen content during recovery from nonweight bearing. PMID:2501291

  2. Anti-dengue virus nonstructural protein 1 antibodies cause NO-mediated endothelial cell apoptosis via ceramide-regulated glycogen synthase kinase-3β and NF-κB activation.

    PubMed

    Chen, Chia-Ling; Lin, Chiou-Feng; Wan, Shu-Wen; Wei, Li-Shiung; Chen, Mei-Chun; Yeh, Trai-Ming; Liu, Hsiao-Sheng; Anderson, Robert; Lin, Yee-Shin

    2013-08-15

    Immunopathogenetic mechanisms of dengue virus (DENV) infection are involved in hemorrhagic syndrome resulting from thrombocytopenia, coagulopathy, and vasculopathy. We have proposed a mechanism of molecular mimicry in which Abs against DENV nonstructural protein 1 (NS1) cross-react with human endothelial cells and cause NF-κB-regulated immune activation and NO-mediated apoptosis. However, the signaling pathway leading to NF-κB activation after the binding of anti-DENV NS1 Abs to endothelial cells is unresolved. In this study, we found that anti-DENV NS1 Abs caused the formation of lipid raftlike structures, and that disrupting lipid raft formation by methyl-β-cyclodextrin decreased NO production and apoptosis. Treatment with anti-DENV NS1 Abs elevated ceramide generation in lipid rafts. Pharmacological inhibition of acid sphingomyelinase (aSMase) decreased anti-DENV NS1 Ab-mediated ceramide and NO production, as well as apoptosis. Exogenous ceramide treatment induced biogenesis of inducible NO synthase (iNOS)/NO and apoptosis through an NF-κB-regulated manner. Furthermore, activation of glycogen synthase kinase-3β (GSK-3β) was required for ceramide-induced NF-κB activation and iNOS expression. Notably, anti-DENV NS1 Abs caused GSK-3β-mediated NF-κB activation and iNOS expression, which were regulated by aSMase. Moreover, pharmacological inhibition of GSK-3β reduced hepatic endothelial cell apoptosis in mice passively administered anti-DENV NS1 Abs. These results suggest that anti-DENV NS1 Abs bind to the endothelial cell membrane and cause NO production and apoptosis via a mechanism involving the aSMase/ceramide/GSK-3β/NF-κB/iNOS/NO signaling pathway. PMID:23851680

  3. 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. PMID:26486317

  4. Glycogen synthase kinase (GSK) 3β phosphorylates and protects nuclear myosin 1c from proteasome-mediated degradation to activate rDNA transcription in early G1 cells.

    PubMed

    Sarshad, Aishe A; Corcoran, Martin; Al-Muzzaini, Bader; Borgonovo-Brandter, Laura; Von Euler, Anne; Lamont, Douglas; Visa, Neus; Percipalle, Piergiorgio

    2014-06-01

    Nuclear myosin 1c (NM1) mediates RNA polymerase I (pol I) transcription activation and cell cycle progression by facilitating PCAF-mediated H3K9 acetylation, but the molecular mechanism by which NM1 is regulated remains unclear. Here, we report that at early G1 the glycogen synthase kinase (GSK) 3β phosphorylates and stabilizes NM1, allowing for NM1 association with the chromatin. Genomic analysis by ChIP-Seq showed that this mechanism occurs on the rDNA as active GSK3β selectively occupies the gene. ChIP assays and transmission electron microscopy in GSK3β-/- mouse embryonic fibroblasts indicated that at G1 rRNA synthesis is suppressed due to decreased H3K9 acetylation leading to a chromatin state incompatible with transcription. We found that GSK3β directly phosphorylates the endogenous NM1 on a single serine residue (Ser-1020) located within the NM1 C-terminus. In G1 this phosphorylation event stabilizes NM1 and prevents NM1 polyubiquitination by the E3 ligase UBR5 and proteasome-mediated degradation. We conclude that GSK3β-mediated phosphorylation of NM1 is required for pol I transcription activation. PMID:24901984

  5. Galangin suppresses the proliferation of β-catenin response transcription-positive cancer cells by promoting adenomatous polyposis coli/Axin/glycogen synthase kinase-3β-independent β-catenin degradation.

    PubMed

    Gwak, Jungsug; Oh, Jingyo; Cho, Munju; Bae, Soo Kyung; Song, Im-Sook; Liu, Kwang-Hyeon; Jeong, Yongsu; Kim, Dong-Eun; Chung, Young-Hwa; Oh, Sangtaek

    2011-06-01

    Galangin is a naturally occurring bioflavonoid with anticancer activity against certain human cancers, yet little is known about its mechanism of action. Here, we used a chemical biology approach to reveal that galangin suppresses β-catenin response transcription (CRT), which is aberrantly up-regulated in colorectal and liver cancers, by promoting the degradation of intracellular β-catenin. Inhibition of glycogen synthase kinase-3β (GSK-3β) activity or mutation of the GSK-3β-targeted sequence from β-catenin was unable to abrogate the galangin-mediated degradation of β-catenin. In addition, galangin down-regulated the intracellular β-catenin levels in cancer cells with inactivating mutations of adenomatous polyposis coli (APC) or Axin, which are components of the β-catenin destruction complex. Galangin repressed the expression of β-catenin/T-cell factor-dependent genes, such as cyclin D1 and c-myc, and thus inhibited the proliferation of CRT-positive cancer cells. Structure-activity data indicated that the major structural requirements for galangin-mediated β-catenin degradation are hydroxyl groups at positions 3, 5, and 7. Our findings suggest that galangin exerts its anticancer activity by promoting APC/Axin/GSK-3β-independent proteasomal degradation of β-catenin. PMID:21406604

  6. 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

  7. Identification of a Maleimide-Based Glycogen Synthase Kinase-3 (GSK-3) Inhibitor, BIP-135, That Prolongs the Median Survival Time of Δ7 SMA KO Mouse Model of Spinal Muscular Atrophy

    PubMed Central

    2011-01-01

    The discovery of upregulated glycogen synthase kinase-3 (GSK-3) in various pathological conditions has led to the development of a host of chemically diverse small molecule GSK-3 inhibitors, such as BIP-135. GSK-3 inhibition emerged as an alternative therapeutic target for treating spinal muscular atrophy (SMA) when a number of GSK-3 inhibitors were shown to elevate survival motor neuron (SMN) levels in vitro and to rescue motor neurons when their intrinsic SMN level was diminished by SMN-specific short hairpin RNA (shRNA). Despite their cellular potency, the in vivo efficacy of GSK-3 inhibitors has yet to be evaluated in an animal model of SMA. Herein, we disclose that a potent and reasonably selective GSK-3 inhibitor, namely BIP-135, was tested in a transgenic Δ7 SMA KO mouse model of SMA and found to prolong the median survival of these animals. In addition, this compound was shown to elevate the SMN protein level in SMA patient-derived fibroblast cells as determined by Western blot, and was neuroprotective in a cell-based, SMA-related model of oxidative stress-induced neurodegeneration. PMID:22348181

  8. From a natural product lead to the identification of potent and selective benzofuran-3-yl-(indol-3-yl)maleimides as glycogen synthase kinase 3beta inhibitors that suppress proliferation and survival of pancreatic cancer cells.

    PubMed

    Gaisina, Irina N; Gallier, Franck; Ougolkov, Andrei V; Kim, Ki H; Kurome, Toru; Guo, Songpo; Holzle, Denise; Luchini, Doris N; Blond, Sylvie Y; Billadeau, Daniel D; Kozikowski, Alan P

    2009-04-01

    Recent studies have demonstrated that glycogen synthase kinase 3beta (GSK-3beta) is overexpressed in human colon and pancreatic carcinomas, contributing to cancer cell proliferation and survival. Here, we report the design, synthesis, and biological evaluation of benzofuran-3-yl-(indol-3-yl)maleimides, potent GSK-3beta inhibitors. Some of these compounds show picomolar inhibitory activity toward GSK-3beta and an enhanced selectivity against cyclin-dependent kinase 2 (CDK-2). Selected GSK-3beta inhibitors were tested in the pancreatic cancer cell lines MiaPaCa-2, BXPC-3, and HupT3. We determined that some of these compounds, namely compounds 5, 6, 11, 20, and 26, demonstrate antiproliferative activity against some or all of the pancreatic cancer cells at low micromolar to nanomolar concentrations. We found that the treatment of pancreatic cancer cells with GSK-3beta inhibitors 5 and 26 resulted in suppression of GSK-3beta activity and a distinct decrease of the X-linked inhibitor of apoptosis (XIAP) expression, leading to significant apoptosis. The present data suggest a possible role for GSK-3beta inhibitors in cancer therapy, in addition to their more prominent applications in CNS disorders. PMID:19338355

  9. Synthesis and preliminary characterization of radioiodinated benzofuran-3-yl-(indol-3-yl)maleimide derivatives as potential SPECT imaging probes for the detection of glycogen synthase kinase-3β (GSK-3β) in the brain.

    PubMed

    Ono, Masahiro; Kitada, Ayane; Watanabe, Hiroyuki; Miyazaki, Anna; Kimura, Hiroyuki; Saji, Hideo

    2016-06-30

    We report on the synthesis and preliminary characterization of two radioiodinated benzofuran-3-yl-(indol-3-yl)maleimides, 3-(benzofuran-3-yl)-4-(5-[(125) I]iodo-1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione ([(125) I]5), and 3-(5-[(125) I]iodo-1-methyl-1H-indol-3-yl)-4-(6-methoxybenzofuran-3-yl)-1H-pyrrole-2,5-dione ([(125) I]6), as the first potential SPECT imaging probes targeting glycogen synthase kinase-3β (GSK-3β). In this study, we used (125) I as a surrogate of (123) I because of its ease of use. The radioiodinated ligands were prepared from the corresponding tributyltin precursors through an iododestannylation reaction using hydrogen peroxide as an oxidant with a radiochemical yield of 10-30%. In vitro binding experiments suggested that both compounds show high affinity for GSK-3β at a level similar to a known GSK-3β inhibitor. Biodistribution studies with normal mice revealed that the radioiodinated compounds display sufficient uptake into (1.8%ID/g at 10 min postinjection) and clearance from the brain (1.0%ID/g at 60 min postinjection). These preliminary results suggest that the further optimization of radioiodinated benzofuran-3-yl-(indol-3-yl)maleimide derivatives may facilitate the development of clinically useful SPECT imaging probes for the in vivo detection of GSK-3β. PMID:27126914

  10. 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

  11. Exercise Training-Induced Adaptations Associated with Increases in Skeletal Muscle Glycogen Content

    PubMed Central

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

    2012-01-01

    Chronic exercise training results in numerous skeletal muscle adaptations, including increases in insulin sensitivity and glycogen content. To understand the mechanism for 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. Compared to 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 3 (GSK3). 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 RGL(GM), another regulatory PP1 subunit, significantly decreased after 4 and 7 weeks of training. Unlike PP1, GSK3 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 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 PP1activity. PMID:23206309

  12. Glycogen metabolism in rat heart muscle cultures after hypoxia.

    PubMed

    Vigoda, Ayelet; Mamedova, Liaman K; Shneyvays, Vladimir; Katz, Abram; Shainberg, Asher

    2003-12-01

    Elevated glycogen levels in heart have been shown to have cardioprotective effects against ischemic injury. We have therefore established a model for elevating glycogen content in primary rat cardiac cells grown in culture and examined potential mechanisms for the elevation (glycogen supercompensation). Glycogen was depleted by exposing the cells to hypoxia for 2 h in the absence of glucose in the medium. This was followed by incubating the cells with 28 mM glucose in normoxia for up to 120 h. Hypoxia decreased glycogen content to about 15% of control, oxygenated cells. This was followed by a continuous increase in glycogen in the hypoxia treated cells during the 120 h recovery period in normoxia. By 48 h after termination of hypoxia, the glycogen content had returned to baseline levels and by 120 h glycogen was about 150% of control. The increase in glycogen at 120 h was associated with comparable relative increases in glucose uptake (approximately 180% of control) and the protein level of the glut-1 transporter (approximately 170% of control), whereas the protein level of the glut-4 transporter was decreased to < 10% of control. By 120 h, the hypoxia-treated cells also exhibited marked increases in the total (approximately 170% of control) and fractional activity of glycogen synthase (control, approximately 15%; hypoxia-treated, approximately 30%). Concomitantly, the hypoxia-treated cells also exhibited marked decreases in the total (approximately 50% of control) and fractional activity of glycogen phosphorylase (control, approximately 50%; hypoxia-treated, approximately 25%). Thus, we have established a model of glycogen supercompensation in cultures of cardiac cells that is explained by concerted increases in glucose uptake and glycogen synthase activity and decreases in phosphorylase activity. This model should prove useful in studying the cardioprotective effects of glycogen. PMID:14674711

  13. Glycogen: the forgotten cerebral energy store.

    PubMed

    Gruetter, Rolf

    2003-10-15

    The brain contains a significant amount of glycogen that is an order of magnitude smaller than that in muscle, but several-fold higher than the cerebral glucose content. Although the precise role of brain glycogen to date is unknown, it seems affected by focal activation, neurotransmitters, and overall electrical activity and hormones. Based on its relatively low concentration, the role of brain glycogen as a significant energy store has been discounted. This work reviews recent experimental evidence that brain glycogen is an important reserve of glucose equivalents: (1) glial glycogen can provide the majority of the glucose supply deficit during hypoglycemia for more than 100 min, consistent with the proposal that glial lactate is a fuel for neurons; (2) glycogen concentrations may be as high as 10 micromol/g, substantially higher than was thought previously; (3) glucose cycling in and out of glycogen amounts to approximately 1% of the cerebral metabolic rate of glucose (CMRglc) in human and rat brain, amounting to an effective stability of glycogen in the resting awake brain during euglycemia and hyperglycemia, (4) brain glycogen metabolism/concentrations are insulin/glucose sensitive; and (5) after a single episode of hypoglycemia, brain glycogen levels rebound to levels that exceed the pre-hypoglycemic concentrations (supercompensation). This experimental evidence supports the proposal that brain glycogen may be involved in the development of diabetes complications, specifically impaired glucose sensing (hypoglycemia unawareness) observed clinically in some diabetes patients under insulin treatment. It is proposed further that brain glycogen becomes important in any metabolic state where supply transiently cannot meet demand, such conditions that could occur during prolonged focal activation, sleep deprivation, seizures, and mild hypoxia. PMID:14515346

  14. Impaired learning in rats in a 14-unit T-maze by 7-nitroindazole, a neuronal nitric oxide synthase inhibitor, is attenuated by the nitric oxide donor, molsidomine.

    PubMed

    Meyer, R C; Spangler, E L; Patel, N; London, E D; Ingram, D K

    1998-01-01

    In previous experiments, it was demonstrated that systemic or central administration of the nitric oxide synthase (NO synthase) inhibitor, NG-nitro-L-arginine (N-Arg), produced dose-dependent learning impairments in rats in a 14-unit T-maze; and that sodium nitroprusside, a NO donor, could attenuate the impairment. Since N-Arg is not specific for neuronal NO synthase and produces hypertension, it is possible that effects on the cardiovasculature may have contributed to the impaired maze performance. In the present experiment, we have investigated the maze performance of 3-4 months old male Fischer-344 rats following treatment with 7-nitroindazole, a NO synthase inhibitor that is selective for neuronal NO synthase and does not produce hypertension. In addition, we examined the effects of the NO donor, molsidomine, which is much longer acting than sodium nitroprusside. Rats were pretrained to avoid footshock in a straight runway and received training in a 14-unit T-maze 24 h later. In an initial dose-response study, rats received intraperitoneal (i.p.) injections of either 7-nitroindazole (25, 50, or 65 mg/kg) or peanut oil 30 min prior to maze training. 7-nitroindazole produced significant, dose-dependent maze acquisition deficits, with 65 mg/kg producing the greatest learning impairment. This dose of 7-nitroindazole had no significant effect on systolic blood pressure. Following the dose-response study, rats were given i.p. injections of either 7-nitroindazole (70 mg/kg) plus saline, 7-nitroindazole (70 mg/kg) plus the NO donor, molsidomine (2 or 4 mg/kg), or peanut oil plus saline as controls. Both doses of molsidomine significantly attenuated the learning deficit induced by 7-nitroindazole relative to controls. These findings represent the first evidence that impaired learning produced by inhibition of neuronal NO synthase can be overcome by systemic administration of a NO donor. PMID:9489851

  15. Behavioral impairments and changes of nitric oxide and inducible nitric oxide synthase in the brains of molarless KM mice.

    PubMed

    Pang, Qian; Hu, Xingxue; Li, Xinya; Zhang, Jianjun; Jiang, Qingsong

    2015-02-01

    More studies showed that as a common disorder in senior population, loss of teeth could adversely affect human cognitive function, and nitric oxide (NO) might play an important role in the cognitive function. However, the underlying mechanism has not yet been well-established. The objectives of this study are to evaluate behavior changes of KM mice after loss of molars, and levels of NO and inducible nitric oxide synthase (iNOS) in the brain in molarless condition. It is hypothesized that loss of molars of the mice tested results in the cognitive impairments and that the process is mediated by NO in the brain through the signaling pathways. Morris water maze is used to test the behavioral changes after 8 weeks of the surgery. The changes of NO and iNOS are evaluated by using Griess assay, western blot, and immunohistochemistry method. The results show that 8 weeks after loss of molars, the spatial learning and memory of KM mice impair and the levels of NO and iNOS in mice hippocampus increase. These findings suggest that molar extraction is associated with the behavioral impairment, and that the changes of NO and iNOS in the hippocampus may be involved in the behavioral changes in the molarless condition. PMID:25447296

  16. Protective effects of Phyllanthus emblica against myocardial ischemia-reperfusion injury: the role of PI3-kinase/glycogen synthase kinase 3β/β-catenin pathway.

    PubMed

    Thirunavukkarasu, Mahesh; Selvaraju, Vaithinathan; Tapias, Leonidas; Sanchez, Juan A; Palesty, J Alexander; Maulik, Nilanjana

    2015-12-01

    Clinical studies of Phyllanthus emblica (P. emblica) have shown that it increases production of nitric oxide, glutathione, and high-density lipoprotein (HDL); decreases low-density lipoprotein (LDL), total cholesterol, triglycerides, and high-sensitivity C-reactive protein (hsCRP); and significantly inhibits platelet aggregation. The following study was designed to examine the effect of P. emblica treatment on myocardial ischemia-reperfusion (I/R) injury and identify the molecular targets and its underlying mechanism(s). Experimental animals were divided into four groups: control sham (CS), P. emblica sham (PS), control I/R (CIR), and P. emblica I/R (PIR). Rats in the P. emblica groups were gavaged with aqueous P. emblica solution (100 mg/kg body weight) for 30 days. After 30 days of gavaging, the I/R group underwent I/R surgery (45-min ischemia) followed by 4 or 30 days of reperfusion. Rats in the sham group underwent surgery without ligation. Left ventricular tissue samples, 4 and 30 days after I/R, were used for Western blot analysis and immunohistochemistry, respectively. Western blot analysis showed upregulation of phosphorylated Akt and GSK3-β and increased nuclear translocation of β-catenin in the PIR group versus CIR. PIR rats also indicated reduced 3-nitrotyrosine and Caspase-3 expression. Increased phosphorylation of endothelial nitric oxide synthase (p-eNOS) and upregulation of anti-apoptotic protein Bcl-2 were found in the PIR group. Echocardiography showed increased ejection fraction and fractional shortening and decreased left ventricular internal diameter in experimental subjects compared to controls. There was decreased fibrosis in P. emblica-treated rats compared to controls. The results of this study indicate that P. emblica is capable of upregulating the PI3K/Akt/GSK3β/β-catenin cardioprotective pathway, thereby preserving cardiac tissue during ischemia-reperfusion injury. PMID:26342597

  17. Convergence of 3′,5′-Cyclic Adenosine 5′-Monophosphate/Protein Kinase A and Glycogen Synthase Kinase-3β/β-Catenin Signaling in Corpus Luteum Progesterone Synthesis

    PubMed Central

    Roy, Lynn; McDonald, Claudia A.; Jiang, Chao; Maroni, Dulce; Zeleznik, Anthony J.; Wyatt, Todd A.; Hou, Xiaoying; Davis, John S.

    2009-01-01

    Progesterone secretion by the steroidogenic cells of the corpus luteum (CL) is essential for reproduction. Progesterone synthesis is under the control of LH, but the exact mechanism of this regulation is unknown. It is established that LH stimulates the LH receptor/choriogonadotropin receptor, a G-protein coupled receptor, to increase cAMP and activate cAMP-dependent protein kinase A (PKA). In the present study, we tested the hypothesis that cAMP/PKA-dependent regulation of the Wnt pathway components glycogen synthase kinase (GSK)-3β and β-catenin contributes to LH-dependent steroidogenesis in luteal cells. We observed that LH via a cAMP/PKA-dependent mechanism stimulated the phosphorylation of GSK3β at N-terminal Ser9 causing its inactivation and resulted in the accumulation of β-catenin. Overexpression of N-terminal truncated β-catenin (Δ90 β-catenin), which lacks the phosphorylation sites responsible for its destruction, significantly augmented LH-stimulated progesterone secretion. In contrast, overexpression of a constitutively active mutant of GSK3β (GSK-S9A) reduced β-catenin levels and inhibited LH-stimulated steroidogenesis. Chromatin immunoprecipitation assays demonstrated the association of β-catenin with the proximal promoter of the StAR gene, a gene that expresses the steroidogenic acute regulatory protein, which is a cholesterol transport protein that controls a rate-limiting step in steroidogenesis. Collectively these data suggest that cAMP/PKA regulation of GSK3β/β-catenin signaling may contribute to the acute increase in progesterone production in response to LH. PMID:19819952

  18. 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. PMID:27177932

  19. 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

  20. Mechanism of sphingosine-1-phosphate induced cardioprotection against I/R injury in diabetic rat heart: Possible involvement of glycogen synthase kinase 3β and mitochondrial permeability transition pore.

    PubMed

    Rana, Ajay; Sharma, Saurabh

    2016-02-01

    There is growing evidence that diabetes mellitus causes attenuation of the bioactive metabolite of membrane sphingolipids, sphingosine-1-phosphate, and this may be a key mechanism in the decreased cardioprotective effect of ischaemic preconditioning (IPC) in the diabetic heart. Thus, this study has been designed to investigate the role and pharmacological potential of sphingosine-1-phosphate in diabetic rat heart. Diabetes was produced in Wistar rats by administration of a low dose of streptozotocin (STZ) (35 mg/kg, i.p., once) and feeding a high fat diet (HFD) for 6 weeks. Isolated rat heart was subjected to 30 min ischaemia followed by 120 min of reperfusion (I/R). The heart was subjected to pre-ischaemic treatment (before ischaemia for 20 min) and pharmacological preconditioning with the S1P agonist FTY720 (0.6 μmol/L) with and without atractyloside (an mPTP opener; in the last episode of reperfusion before I/R). Myocardial infarction was assessed in terms of increase in lactate dehydrogenase (LDH), creatinine kinase-MB (CK-MB), myeloperoxidase (MPO) level and infarct size (triphenyltetrazolium chloride staining). Immunohistochemistry analysis was done for assessment of tumour necrosis factor (TNF)-α and glycogen synthase kinase (GSK)-3β level in cardiac tissue. Pre-ischaemic treatment and pharmacological preconditioning with FTY720 significantly decreased I/R-induced myocardial infarction, TNF-alpha, GSK-3β level and release of LDH and CK-MB as compared to control group. The cardioprotective effect of S1P agonist was significantly attenuated by atractyloside. It may be concluded that S1P agonist FTY720 prevents the diabetic heart from ischaemic reperfusion injury, possibly through inhibition of GSK-3β and regulation of opening of mitochondrial permeability transition pore. PMID:26582369

  1. Lithium Chloride Dependent Glycogen Synthase Kinase 3 Inactivation Links Oxidative DNA Damage, Hypertrophy and Senescence in Human Articular Chondrocytes and Reproduces Chondrocyte Phenotype of Obese Osteoarthritis Patients

    PubMed Central

    Platano, Daniela; Cattini, Luca; Trisolino, Giovanni; Mariani, Erminia; Borzì, Rosa Maria

    2015-01-01

    functional impairment via induction of hypertrophy and senescence. Indeed, GSK3β inactivation is responsible for ROS production, triggering oxidative stress and DNA damage response. PMID:26618897

  2. Mechanism of glycogen supercompensation in rat skeletal muscle cultures.

    PubMed

    Mamedova, Liaman K; Shneyvays, Vladimir; Katz, Abram; Shainberg, Asher

    2003-08-01

    A model to study glycogen supercompensation (the significant increase in glycogen content above basal level) in primary rat skeletal muscle culture was established. Glycogen was completely depleted in differentiated myotubes by 2 h of electrical stimulation or exposure to hypoxia during incubation in medium devoid of glucose. Thereafter, cells were incubated in medium containing glucose, and glycogen supercompensation was clearly observed in treated myotubes after 72 h. Peak glycogen levels were obtained after 120 h, averaging 2.5 and 4 fold above control values in the stimulated- and hypoxia-treated cells, respectively. Glycogen synthase activity increased and phosphorylase activity decreased continuously during 120 h of recovery in the treated cells. Rates of 2-deoxyglucose uptake were significantly elevated in the treated cells at 96 and 120 h, averaging 1.4-2 fold above control values. Glycogenin content increased slightly in the treated cells after 48 h (1.2 fold vs. control) and then increased considerably, achieving peak values after 120 h (2 fold vs. control). The results demonstrate two phases of glycogen supercompensation: the first phase depends primarily on activation of glycogen synthase and inactivation of phosphorylase; the second phase includes increases in glucose uptake and glycogenin level. PMID:12962138

  3. Polyglucosan Molecules Induce Mitochondrial Impairment and Apoptosis in Germ Cells Without Affecting the Integrity and Functionality of Sertoli Cells.

    PubMed

    Villarroel-Espíndola, Franz; Tapia, Cynthia; González-Stegmaier, Roxana; Concha, Ilona I; Slebe, Juan Carlos

    2016-10-01

    Glycogen is the main storage form of glucose; however, the accumulation of glycogen-like glucose polymers can lead to degeneration and cellular death. Previously, we reported that the accumulation of glycogen in testis of transgenic animals overexpressing a constitutively active form of glycogen synthase enhances the apoptosis of pre-meiotic male germ cells and a complete disorganization of the seminiferous tubules. Here we sought to further identify the effects of glycogen storage in cells from the seminiferous tubules and the mechanism behind the pro-apoptotic activity induced by its accumulation. Using an in vitro culture of Sertoli cells (line 42GPA9) and spermatocyte-like cells (line GC-1) expressing a superactive form of glycogen synthase or the Protein Targeting to Glycogen (PTG), we found that glycogen synthesized in both cell lines is poorly branched. In addition, the immunodetection of key molecules of apoptotic events suggests that cellular death induced by polyglucosan molecules affects GC-1 cells, but not 42GPA9 cells by mitochondrial impairment and activation of an intrinsic apoptotic pathway. Furthermore, we analyzed the effects of glycogen deposition during the establishment of an in vitro blood-testis barrier. The results using a non-permeable fluorescent molecule showed that, in conditions of over-synthesis of glycogen, 42GPA9 cells do not lose their capacity to generate an impermeable barrier and the levels of connexin43, occludin, and ZO1 proteins were not affected. These results suggest that the accumulation of polyglucosan molecules has a selective effect-triggered by the intrinsic activation of the apoptotic pathway-in germ cells without directly affecting Sertoli cells. J. Cell. Physiol. 231: 2142-2152, 2016. © 2016 Wiley Periodicals, Inc. PMID:26790645

  4. Increased Oxidative Stress Impairs Adipose Tissue Function in Sphingomyelin Synthase 1 Null Mice

    PubMed Central

    Nishimura, Naotaka; Gotoh, Tomomi; Watanabe, Ken; Ikeda, Kazutaka; Garan, Yohei; Taguchi, Ryo; Node, Koichi; Okazaki, Toshiro; Oike, Yuichi

    2013-01-01

    Sphingomyelin synthase 1 (SMS1) catalyzes the conversion of ceramide to sphingomyelin. Here, we found that SMS1 null mice showed lipodystrophic phenotype. Mutant mice showed up-regulation of plasma triglyceride concentrations accompanied by reduction of white adipose tissue (WAT) as they aged. Lipoprotein lipase (LPL) activity was severely reduced in mutant mice. In vivo analysis indicated that fatty acid uptake in WAT but not in liver decreased in SMS1 null compared to wild-type mice. In vitro analysis using cultured cell revealed that SMS1 depletion reduced fatty acid uptake. Proteins extracted from WAT of mutant mice were severely modified by oxidative stress, and up-regulation of mRNAs related to apoptosis, redox adjustment, mitochondrial stress response and mitochondrial biogenesis was observed. ATP content of WAT was reduced in SMS1 null mice. Blue native gel analysis indicated that accumulation of mitochondrial respiratory chain complexes was reduced. These results suggest that WAT of SMS1 null mice is severely damaged by oxidative stress and barely functional. Indeed, mutant mice treated with the anti-oxidant N-acetyl cysteine (NAC) showed partial recovery of lipodystrophic phenotypes together with normalized plasma triglyceride concentrations. Altogether, our data suggest that SMS1 is crucial to control oxidative stress in order to maintain WAT function. PMID:23593476

  5. Increased oxidative stress impairs adipose tissue function in sphingomyelin synthase 1 null mice.

    PubMed

    Yano, Masato; Yamamoto, Tadashi; Nishimura, Naotaka; Gotoh, Tomomi; Watanabe, Ken; Ikeda, Kazutaka; Garan, Yohei; Taguchi, Ryo; Node, Koichi; Okazaki, Toshiro; Oike, Yuichi

    2013-01-01

    Sphingomyelin synthase 1 (SMS1) catalyzes the conversion of ceramide to sphingomyelin. Here, we found that SMS1 null mice showed lipodystrophic phenotype. Mutant mice showed up-regulation of plasma triglyceride concentrations accompanied by reduction of white adipose tissue (WAT) as they aged. Lipoprotein lipase (LPL) activity was severely reduced in mutant mice. In vivo analysis indicated that fatty acid uptake in WAT but not in liver decreased in SMS1 null compared to wild-type mice. In vitro analysis using cultured cell revealed that SMS1 depletion reduced fatty acid uptake. Proteins extracted from WAT of mutant mice were severely modified by oxidative stress, and up-regulation of mRNAs related to apoptosis, redox adjustment, mitochondrial stress response and mitochondrial biogenesis was observed. ATP content of WAT was reduced in SMS1 null mice. Blue native gel analysis indicated that accumulation of mitochondrial respiratory chain complexes was reduced. These results suggest that WAT of SMS1 null mice is severely damaged by oxidative stress and barely functional. Indeed, mutant mice treated with the anti-oxidant N-acetyl cysteine (NAC) showed partial recovery of lipodystrophic phenotypes together with normalized plasma triglyceride concentrations. Altogether, our data suggest that SMS1 is crucial to control oxidative stress in order to maintain WAT function. PMID:23593476

  6. 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

  7. Protein ingestion does not impair exercise-induced AMPK signalling when in a glycogen-depleted state: implications for train-low compete-high.

    PubMed

    Taylor, Conor; Bartlett, Jonathan D; van de Graaf, Christian Soler; Louhelainen, Jari; Coyne, Vicki; Iqbal, Zafar; Maclaren, Don P M; Gregson, Warren; Close, Graeme L; Morton, James P

    2013-06-01

    The aim of the present study was to test the hypothesis that consuming protein does not attenuate AMPK signalling when exercise is commenced in a glycogen-depleted state. After performing a glycogen-depleting protocol the evening before, the subsequent morning ten active men performed 45 min steady-state cycling at 50 % of peak power output (PPO) followed by an exercise capacity test (1-min intervals at 80 % PPO interspersed with 1-min periods at 40 % PPO). In a repeated measures design, subjects consumed 20 g of a casein hydrolysate solution (PRO) 45 min before exercise, 10 g during and a further 20 g immediately post-exercise, or an equivalent volume of a non-calorie taste matched placebo (PLA). Resting (PRO = 134 ± 29; PLA = 136 ± 28 mmol kg(-1)) and post-exercise muscle glycogen (PRO = 43 ± 16; PLA = 47 ± 18 mmol kg(-1)) was not different (P > 0.05) between trials nor was exercise capacity (PRO = 26 ± 9; PLA = 25 ± 10 min, P > 0.05). Phosphorylation of AMPK(Thr172) increased threefold immediately post-exercise (P < 0.05) and PGC1-mRNA increased sixfold at 3 h post-exercise (P < 0.05), though there were no differences between conditions (P > 0.05). In contrast, there was a trend (P = 0.08) for a divergent response in eEF2(Thr56) phosphorylation such that 1.5 fold increases post- and 3 h post-exercise in PLA were blunted with PRO, thus indicative of greater eEF2 activation. We conclude that athletes who deliberately incorporate training phases with reduced muscle glycogen into their training programmes may consume protein before, during and after exercise without negating signalling through the AMPK cascade. PMID:23263742

  8. 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

  9. Mice Lacking Inducible Nitric Oxide Synthase Demonstrate Impaired Killing of Porphyromonas gingivalis

    PubMed Central

    Gyurko, Robert; Boustany, Gabriel; Huang, Paul L.; Kantarci, Alpdogan; Van Dyke, Thomas E.; Genco, Caroline A.; Gibson III, Frank C.

    2003-01-01

    Porphyromonas gingivalis is a primary etiological agent of generalized severe periodontitis, and emerging data suggest the importance of reactive oxygen and nitrogen species in periodontal tissue damage, as well as in microbial killing. Since nitric oxide (NO) released from inducible NO synthase (iNOS) has been shown to possess immunomodulatory, cytotoxic, and antibacterial effects in experimental models, we challenged iNOS-deficient (iNOS−/−) mice with P. gingivalis by using a subcutaneous chamber model to study the specific contribution of NO to host defense during P. gingivalis infection. iNOS−/− mice inoculated with P. gingivalis developed skin lesions and chamber rejection with higher frequency and to a greater degree than similarly challenged C57BL/6 wild-type (WT) mice. Chamber fluid from iNOS−/− mice possessed significantly more P. gingivalis than that of WT mice. The immunoglobulin G responses to P. gingivalis in serum was similar in WT and iNOS−/− mice, and the inductions of tumor necrosis factor alpha, interleukin-1β and interleukin-6, and prostaglandin E2 were comparable between the two mouse strains. Although no differences in total leukocyte counts in chamber fluids were observed between iNOS−/− and WT mice, the percentage of dead polymorphonuclear leukocytes (PMNs) was significantly greater in iNOS−/− mouse chamber fluids than that of WT samples. Interestingly, casein-elicited PMNs from iNOS−/− mice released more superoxide than did WT PMNs when stimulated with P. gingivalis. These results indicate that modulation of superoxide levels is a mechanism by which NO influences PMN function and that NO is an important element of the host defense against P. gingivalis. PMID:12933833

  10. Phosphorylation of Leukotriene C4 Synthase at Serine 36 Impairs Catalytic Activity.

    PubMed

    Ahmad, Shabbir; Ytterberg, A Jimmy; Thulasingam, Madhuranayaki; Tholander, Fredrik; Bergman, Tomas; Zubarev, Roman; Wetterholm, Anders; Rinaldo-Matthis, Agnes; Haeggström, Jesper Z

    2016-08-26

    Leukotriene C4 synthase (LTC4S) catalyzes the formation of the proinflammatory lipid mediator leukotriene C4 (LTC4). LTC4 is the parent molecule of the cysteinyl leukotrienes, which are recognized for their pathogenic role in asthma and allergic diseases. Cellular LTC4S activity is suppressed by PKC-mediated phosphorylation, and recently a downstream p70S6k was shown to play an important role in this process. Here, we identified Ser(36) as the major p70S6k phosphorylation site, along with a low frequency site at Thr(40), using an in vitro phosphorylation assay combined with mass spectrometry. The functional consequences of p70S6k phosphorylation were tested with the phosphomimetic mutant S36E, which displayed only about 20% (20 μmol/min/mg) of the activity of WT enzyme (95 μmol/min/mg), whereas the enzyme activity of T40E was not significantly affected. The enzyme activity of S36E increased linearly with increasing LTA4 concentrations during the steady-state kinetics analysis, indicating poor lipid substrate binding. The Ser(36) is located in a loop region close to the entrance of the proposed substrate binding pocket. Comparative molecular dynamics indicated that Ser(36) upon phosphorylation will pull the first luminal loop of LTC4S toward the neighboring subunit of the functional homotrimer, thereby forming hydrogen bonds with Arg(104) in the adjacent subunit. Because Arg(104) is a key catalytic residue responsible for stabilization of the glutathione thiolate anion, this phosphorylation-induced interaction leads to a reduction of the catalytic activity. In addition, the positional shift of the loop and its interaction with the neighboring subunit affect active site access. Thus, our mutational and kinetic data, together with molecular simulations, suggest that phosphorylation of Ser(36) inhibits the catalytic function of LTC4S by interference with the catalytic machinery. PMID:27365393

  11. 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.

  12. Liver Glycogen Loading Dampens Glycogen Synthesis Seen in Response to Either Hyperinsulinemia or Intraportal Glucose Infusion

    PubMed Central

    Winnick, Jason J.; An, Zhibo; Kraft, Guillaume; Ramnanan, Christopher J.; Irimia, Jose M.; Smith, Marta; Lautz, Margaret; Roach, Peter J.; Cherrington, Alan D.

    2013-01-01

    The purpose of this study was to determine the effect of liver glycogen loading on net hepatic glycogen synthesis during hyperinsulinemia or hepatic portal vein glucose infusion in vivo. Liver glycogen levels were supercompensated (SCGly) in two groups (using intraportal fructose infusion) but not in two others (Gly) during hyperglycemic-normoinsulinemia. Following a 2-h control period during which fructose infusion was stopped, there was a 2-h experimental period in which the response to hyperglycemia plus either 4× basal insulin (INS) or portal vein glucose infusion (PoG) was measured. Increased hepatic glycogen reduced the percent of glucose taken up by the liver that was deposited in glycogen (74 ± 3 vs. 53 ± 5% in Gly+INS and SCGly+INS, respectively, and 72 ± 3 vs. 50 ± 6% in Gly+PoG and SCGly+PoG, respectively). The reduction in liver glycogen synthesis in SCGly+INS was accompanied by a decrease in both insulin signaling and an increase in AMPK activation, whereas only the latter was observed in SCGly+PoG. These data indicate that liver glycogen loading impairs glycogen synthesis regardless of the signal used to stimulate it. PMID:22923473

  13. 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. PMID:12383944

  14. 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.

  15. 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.

  16. A functional glycogen biosynthesis pathway in Lactobacillus acidophilus: expression and analysis of the glg operon

    PubMed Central

    Goh, Yong Jun; Klaenhammer, Todd R

    2013-01-01

    Glycogen metabolism contributes to energy storage and various physiological functions in some prokaryotes, including colonization persistence. A role for glycogen metabolism is proposed on the survival and fitness of Lactobacillus acidophilus, a probiotic microbe, in the human gastrointestinal environment. L. acidophilus NCFM possesses a glycogen metabolism (glg) operon consisting of glgBCDAP-amy-pgm genes. Expression of the glg operon and glycogen accumulation were carbon source- and growth phase-dependent, and were repressed by glucose. The highest intracellular glycogen content was observed in early log-phase cells grown on trehalose, which was followed by a drastic decrease of glycogen content prior to entering stationary phase. In raffinose-grown cells, however, glycogen accumulation gradually declined following early log phase and was maintained at stable levels throughout stationary phase. Raffinose also induced an overall higher temporal glg expression throughout growth compared with trehalose. Isogenic ΔglgA (glycogen synthase) and ΔglgB (glycogen-branching enzyme) mutants are glycogen-deficient and exhibited growth defects on raffinose. The latter observation suggests a reciprocal relationship between glycogen synthesis and raffinose metabolism. Deletion of glgB or glgP (glycogen phosphorylase) resulted in defective growth and increased bile sensitivity. The data indicate that glycogen metabolism is involved in growth maintenance, bile tolerance and complex carbohydrate utilization in L. acidophilus. PMID:23879596

  17. A functional glycogen biosynthesis pathway in Lactobacillus acidophilus: expression and analysis of the glg operon.

    PubMed

    Goh, Yong Jun; Klaenhammer, Todd R

    2013-09-01

    Glycogen metabolism contributes to energy storage and various physiological functions in some prokaryotes, including colonization persistence. A role for glycogen metabolism is proposed on the survival and fitness of Lactobacillus acidophilus, a probiotic microbe, in the human gastrointestinal environment. L. acidophilus NCFM possesses a glycogen metabolism (glg) operon consisting of glgBCDAP-amy-pgm genes. Expression of the glg operon and glycogen accumulation were carbon source- and growth phase-dependent, and were repressed by glucose. The highest intracellular glycogen content was observed in early log-phase cells grown on trehalose, which was followed by a drastic decrease of glycogen content prior to entering stationary phase. In raffinose-grown cells, however, glycogen accumulation gradually declined following early log phase and was maintained at stable levels throughout stationary phase. Raffinose also induced an overall higher temporal glg expression throughout growth compared with trehalose. Isogenic ΔglgA (glycogen synthase) and ΔglgB (glycogen-branching enzyme) mutants are glycogen-deficient and exhibited growth defects on raffinose. The latter observation suggests a reciprocal relationship between glycogen synthesis and raffinose metabolism. Deletion of glgB or glgP (glycogen phosphorylase) resulted in defective growth and increased bile sensitivity. The data indicate that glycogen metabolism is involved in growth maintenance, bile tolerance and complex carbohydrate utilization in L. acidophilus. PMID:23879596

  18. 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.

  19. Glutamate-induced activation of nitric oxide synthase is impaired in cerebral cortex in vivo in rats with chronic liver failure.

    PubMed

    Rodrigo, Regina; Erceg, Slaven; Rodriguez-Diaz, Jesus; Saez-Valero, Javier; Piedrafita, Blanca; Suarez, Isabel; Felipo, Vicente

    2007-07-01

    It has been proposed that impairment of the glutamate-nitric oxide-cyclic guanosine monophosphate (cGMP) pathway in brain contributes to cognitive impairment in hepatic encephalopathy. The aims of this work were to assess whether the function of this pathway and of nitric oxide synthase (NOS) are altered in cerebral cortex in vivo in rats with chronic liver failure due to portacaval shunt (PCS) and whether these alterations are due to hyperammonemia. The glutamate-nitric oxide-cGMP pathway function and NOS activation by NMDA was analysed by in vivo microdialysis in cerebral cortex of PCS and control rats and in rats with hyperammonemia without liver failure. Similar studies were done in cortical slices from these rats and in cultured cortical neurons exposed to ammonia. Basal NOS activity, nitrites and cGMP are increased in cortex of rats with hyperammonemia or liver failure. These increases seem due to increased inducible nitric oxide synthase expression. NOS activation by NMDA is impaired in cerebral cortex in both animal models and in neurons exposed to ammonia. Chronic liver failure increases basal NOS activity, nitric oxide and cGMP but reduces activation of NOS induced by NMDA receptors activation. Hyperammonemia is responsible for both effects which will lead, independently, to alterations contributing to neurological alterations in hepatic encephalopathy. PMID:17286583

  20. 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

  1. Are there errors in glycogen biosynthesis and is laforin a repair enzyme?

    PubMed Central

    Roach, Peter J.

    2016-01-01

    Glycogen, a branched polymer of glucose, is well known as a cellular reserve of metabolic energy and/or biosynthetic precursors. Besides glucose, however, glycogen contains small amounts of covalent phosphate, present as C2 and C3 phosphomonoesters. Current evidence suggests that the phosphate is introduced by the biosynthetic enzyme glycogen synthase as a rare alternative to its normal catalytic addition of glucose units. The phosphate can be removed by the laforin phosphatase, whose mutation causes a fatal myoclonus epilepsy called Lafora disease. The hypothesis is that glycogen phosphorylation can be considered a catalytic error and laforin a repair enzyme. PMID:21930129

  2. 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. PMID:25865793

  3. 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

  4. Neuronal Nitric-Oxide Synthase Deficiency Impairs the Long-Term Memory of Olfactory Fear Learning and Increases Odor Generalization

    ERIC Educational Resources Information Center

    Pavesi, Eloisa; Heldt, Scott A.; Fletcher, Max L.

    2013-01-01

    Experience-induced changes associated with odor learning are mediated by a number of signaling molecules, including nitric oxide (NO), which is predominantly synthesized by neuronal nitric oxide synthase (nNOS) in the brain. In the current study, we investigated the role of nNOS in the acquisition and retention of conditioned olfactory fear. Mice…

  5. ANTISENSE INHIBITION OF NADH-GLUTAMATE SYNTHASE IMPAIRS CARBON/NITROGEN ASSIMILATION IN NODULES OF ALFALFA (MEDICAGO SATIVA)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Legumes acquire significant amounts of nitrogen for growth from symbiotic nitrogen fixation in root nodules. The glutamine synthetase (GS)/NADH-dependent glutamate synthase (NADH-GOGAT) cycle catalyzes initial nitrogen assimilation. This report describes the impacts of specific reduction on nodule N...

  6. RNA interference-based gene silencing of phytoene synthase impairs growth, carotenoids, and plastid phenotype in Oncidium hybrid orchid.

    PubMed

    Liu, Jian-Xin; Chiou, Chung-Yi; Shen, Chin-Hui; Chen, Peng-Jen; Liu, Yao-Chung; Jian, Chin-Der; Shen, Xiao-Lan; Shen, Fu-Quan; Yeh, Kai-Wun

    2014-01-01

    Phytoene synthase (PSY) is the first rate-limiting regulatory enzyme in the carotenoid biosynthesis pathway. In order to modify the floral color pattern by reducing carotenoid contents, a phytoene synthase-RNAi construct was delivered into protocorm-like body (PLB) of Oncidium hybrid orchid. The transgenic orchids show down-regulated level of PSY and geranyl synthase gene. They displayed semi-dwarf phenotype and brilliant green leaves. The microscopic anatomy revealed development-arrested plastids with rare grana. The total carotenoid content was decreased and the efficiency of the photosynthetic electron transport was declined. The chlorophyll level and the expression of chlorophyll biosynthetic genes, such as OgGLUTR and OgCS were dramatically reduced. HPLC analysis showed that the endogenous level of gibberellic acid and abscisic acid in the dwarf transformants are 4-fold lower than in wild type plants. In addition, chilling tolerance of the transgenic Oncidium plants was reduced. The data showed that down-regulation of PSY resulted in alterations of gene expression in enzymes involved in many metabolic pathways, such as carotenoid, gibberellic acid, abscisic acid and chlorophyll biosynthetic pathway as well as causes predominant defects in plant growth and development. PMID:25221736

  7. Diverse effects of two allosteric inhibitors on the phosphorylation state of glycogen phosphorylase in hepatocytes.

    PubMed Central

    Latsis, Theodore; Andersen, Birgitte; Agius, Loranne

    2002-01-01

    Two distinct allosteric inhibitors of glycogen phosphorylase, 1,4-dideoxy-1,4-imino-D-arabinitol (DAB) and CP-91149 (an indole-2-carboxamide), were investigated for their effects on the phosphorylation state of the enzyme in hepatocytes in vitro. CP-91149 induced inactivation (dephosphorylation) of phosphorylase in the absence of hormones and partially counteracted the phosphorylation caused by glucagon. Inhibition of glycogenolysis by CP-91149 can be explained by dephosphorylation of phosphorylase a. This was associated with activation of glycogen synthase and stimulation of glycogen synthesis. DAB, in contrast, induced a small degree of phosphorylation of phosphorylase. This was associated with inactivation of glycogen synthase and inhibition of glycogen synthesis. Despite causing phosphorylation (activation) of phosphorylase, DAB is a very potent inhibitor of glycogenolysis in both the absence and presence of glucagon. This is explained by allosteric inhibition of phosphorylase a, which overrides the increase in activation state. In conclusion, two potent phosphorylase inhibitors exert different effects on glycogen metabolism in intact hepatocytes as a result of opposite effects on the phosphorylation state of both phosphorylase and glycogen synthase. PMID:12186629

  8. 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

  9. Stimulation of glycogen synthesis by heat shock in L6 skeletal-muscle cells: regulatory role of site-specific phosphorylation of glycogen-associated protein phosphatase 1.

    PubMed Central

    Moon, Byoung; Duddy, Noreen; Ragolia, Louis; Begum, Najma

    2003-01-01

    Recent evidence suggests that glycogen-associated protein phosphatase 1 (PP-1(G)) is essential for basal and exercise-induced glycogen synthesis, which is mediated in part by dephosphorylation and activation of glycogen synthase (GS). In the present study, we examined the potential role of site-specific phosphorylation of PP-1(G) in heat-shock-induced glycogen synthesis. L6 rat skeletal-muscle cells were stably transfected with wild-type PP-1(G) or with PP-1(G) mutants in which site-1 (S1) Ser(48) and site-2 (S2) Ser(67) residues were substituted with Ala. Cells expressing wild-type and PP-1(G) mutants, S1, S2 and S1/S2, were examined for potential alterations in glycogen synthesis after a 60 min heat shock at 45 degrees C, followed by analysis of [(14)C]glucose incorporation into glycogen at 37 degrees C. PP-1(G) S1 mutation caused a 90% increase in glycogen synthesis on heat-shock treatment, whereas the PP-1(G) S2 mutant was not sensitive to heat stress. The S1/S2 double mutant was comparable with wild-type, which showed a 30% increase over basal. Heat-shock-induced glycogen synthesis was accompanied by increased PP-1 and GS activities. The highest activation was observed in S1 mutant. Heat shock also resulted in a rapid and sustained Akt/ glycogen synthase kinase 3 beta (GSK-3 beta) phosphorylation. Wortmannin blocked heat-shock-induced Akt/GSK-3 beta phosphorylation, prevented 2-deoxyglucose uptake and abolished the heat-shock-induced glycogen synthesis. Muscle glycogen levels regulate GS activity and glycogen synthesis and were found to be markedly depleted in S1 mutant on heat-shock treatment, suggesting that PP-1(G) S1 Ser phosphorylation may inhibit glycogen degradation during thermal stimulation, as S1 mutation resulted in excessive glycogen synthesis on heat-shock treatment. In contrast, PP-1(G) S2 Ser phosphorylation may promote glycogen breakdown under stressful conditions. Heat-shock-induced glycogenesis appears to be mediated via phosphoinositide 3

  10. 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