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

    PubMed Central

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

    2011-01-01

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

  3. Impaired glucose tolerance and predisposition to the fasted state in liver glycogen synthase knock-out mice.

    PubMed

    Irimia, Jose M; Meyer, Catalina M; Peper, Caron L; Zhai, Lanmin; Bock, Cheryl B; Previs, Stephen F; McGuinness, Owen P; DePaoli-Roach, Anna; Roach, Peter J

    2010-04-23

    Conversion to glycogen is a major fate of ingested glucose in the body. A rate-limiting enzyme in the synthesis of glycogen is glycogen synthase encoded by two genes, GYS1, expressed in muscle and other tissues, and GYS2, primarily expressed in liver (liver glycogen synthase). Defects in GYS2 cause the inherited monogenic disease glycogen storage disease 0. We have generated mice with a liver-specific disruption of the Gys2 gene (liver glycogen synthase knock-out (LGSKO) mice), using Lox-P/Cre technology. Conditional mice carrying floxed Gys2 were crossed with mice expressing Cre recombinase under the albumin promoter. The resulting LGSKO mice are viable, develop liver glycogen synthase deficiency, and have a 95% reduction in fed liver glycogen content. They have mild hypoglycemia but dispose glucose less well in a glucose tolerance test. Fed, LGSKO mice also have a reduced capacity for exhaustive exercise compared with mice carrying floxed alleles, but the difference disappears after an overnight fast. Upon fasting, LGSKO mice reach within 4 h decreased blood glucose levels attained by control floxed mice only after 24 h of food deprivation. The LGSKO mice maintain this low blood glucose for at least 24 h. Basal gluconeogenesis is increased in LGSKO mice, and insulin suppression of endogenous glucose production is impaired as assessed by euglycemic-hyperinsulinemic clamp. This observation correlates with an increase in the liver gluconeogenic enzyme phosphoenolpyruvate carboxykinase expression and activity. This mouse model mimics the pathophysiology of glycogen storage disease 0 patients and highlights the importance of liver glycogen stores in whole body glucose homeostasis.

  4. Nuclear glycogen and glycogen synthase kinase 3.

    PubMed

    Ragano-Caracciolo, M; Berlin, W K; Miller, M W; Hanover, J A

    1998-08-19

    Glycogen is the principal storage form of glucose in animal cells. It accumulates in electron-dense cytoplasmic granules and is synthesized by glycogen synthase (GS), the rate-limiting enzyme of glycogen deposition. Glycogen synthase kinase-3 (GSK-3) is a protein kinase that phosphorylates GS. Two nearly identical forms of GSK-3 exist: GSK-3 alpha and GSK-3 beta. Both are constitutively active in resting cells and their activity can be modulated by hormones and growth factors. GSK-3 is implicated in the regulation of many physiological responses in mammalian cells by phosphorylating substrates including neuronal cell adhesion molecule, neurofilaments, synapsin I, and tau. Recent observations point to functions for glycogen and glycogen metabolism in the nucleus. GSK-3 phosphorylates several transcription factors, and we have recently shown that it modifies the major nuclear pore protein p62. It also regulates PK1, a protein kinase required for maintaining the interphase state and for DNA replication in cycling Xenopus egg extracts. Recently, glycogen was shown to be required for nuclear reformation in vitro using ovulated Xenopus laevis egg lysates. Because neither glycogen nor GSK-3 has been localized to the nuclear envelope or intranuclear sites, glycogen and GSK-3 activites were measured in rat liver nuclei and nuclear reformation extracts. Significant quantities of glycogen-like material co-purified with the rat-liver nuclear envelope. GSK-3 is also highly enriched in the glycogen pellet of egg extracts of Xenopus that is required for nuclear assembly in vitro. Based on the finding that enzymes of glycogen metabolism copurify with glycogen, we propose that glycogen may serve a structural role as a scaffold for nuclear assembly and sequestration of critical kinases and phosphatases in the nucleus. Copyright 1998 Academic Press.

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

    PubMed Central

    Eldar-Finkelman, Hagit; Krebs, Edwin G.

    1997-01-01

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

  6. Multiple defects in muscle glycogen synthase activity contribute to reduced glycogen synthesis in non-insulin dependent diabetes mellitus.

    PubMed Central

    Thorburn, A W; Gumbiner, B; Bulacan, F; Brechtel, G; Henry, R R

    1991-01-01

    To define the mechanisms of impaired muscle glycogen synthase and reduced glycogen formation in non-insulin dependent diabetes mellitus (NIDDM), glycogen synthase activity was kinetically analyzed during the basal state and three glucose clamp studies (insulin approximately equal to 300, 700, and 33,400 pmol/liter) in eight matched nonobese NIDDM and eight control subjects. Muscle glycogen content was measured in the basal state and following clamps at insulin levels of 33,400 pmol/liter. NIDDM subjects had glucose uptake matched to controls in each clamp by raising serum glucose to 15-20 mmol/liter. The insulin concentration required to half-maximally activate glycogen synthase (ED50) was approximately fourfold greater for NIDDM than control subjects (1,004 +/- 264 vs. 257 +/- 110 pmol/liter, P less than 0.02) but the maximal insulin effect was similar. Total glycogen synthase activity was reduced approximately 38% and glycogen content was approximately 30% lower in NIDDM. A positive correlation was present between glycogen content and glycogen synthase activity (r = 0.51, P less than 0.01). In summary, defects in muscle glycogen synthase activity and reduced glycogen content are present in NIDDM. NIDDM subjects also have less total glycogen synthase activity consistent with reduced functional mass of the enzyme. These findings and the correlation between glycogen synthase activity and glycogen content support the theory that multiple defects in glycogen synthase activity combine to cause reduced glycogen formation in NIDDM. PMID:1899428

  7. Exercise with calorie restriction improves insulin sensitivity and glycogen synthase activity in obese postmenopausal women with impaired glucose tolerance

    PubMed Central

    Ortmeyer, Heidi K.; Sorkin, John D.

    2012-01-01

    Our objective was to compare the effects of in vivo insulin on skeletal muscle glycogen synthase (GS) activity in normal (NGT) vs. impaired glucose-tolerant (IGT) obese postmenopausal women and to determine whether an increase in insulin activation of GS is associated with an improvement in insulin sensitivity (M) following calorie restriction (CR) and/or aerobic exercise plus calorie restriction (AEX + CR) in women with NGT and IGT. We did a longitudinal, clinical intervention study of CR compared with AEX + CR. Overweight and obese women, 49–76 yr old, completed 6 mo of CR (n = 46) or AEX + CR (n = 50) with V̇o2 max, body composition, and glucose tolerance testing. Hyperinsulinemic euglycemic (80 mU·m−2·min−1) clamps (n = 73) and skeletal muscle biopsies (before and during clamp) (n = 58) were performed before and after the interventions (n = 50). After 120 min of hyperinsulinemia during the clamp, GS fractional activity and insulin's effect to increase GS fractional activity (insulin − basal) were significantly lower in IGT vs. NGT (P < 0.01) at baseline. GS total activity increased during the clamp in NGT (P < 0.05), but not IGT, at baseline. CR and AEX + CR resulted in a significant 8% weight loss with reductions in total fat mass, visceral fat, subcutaneous fat, and intramuscular fat. Overall, M increased (P < 0.01), and the change in M (postintervention − preintervention) was associated with the change in insulin-stimulated GS fractional activity (partial r = 0.44, P < 0.005). In IGT, the change (postintervention − preintervention) in insulin-stimulated GS total activity was greater following AEX + CR than CR alone (P < 0.05). In IGT, insulin-stimulated GS-independent (P < 0.005) and fractional activity (P = 0.06) increased following AEX + CR. We conclude that the greatest benefits at the whole body and cellular level (insulin activation of GS) in older women at highest risk for diabetes are derived from a lifestyle intervention that

  8. 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

  9. Subcutaneous liraglutide ameliorates methylglyoxal-induced Alzheimer-like tau pathology and cognitive impairment by modulating tau hyperphosphorylation and glycogen synthase kinase-3β

    PubMed Central

    Qi, Liqin; Chen, Zhou; Wang, Yanping; Liu, Xiaoying; Liu, Xiaohong; Ke, Linfang; Zheng, Zhongjie; Lin, Xiaowei; Zhou, Yu; Wu, Lijuan; Liu, Libin

    2017-01-01

    Memory deterioration and synapse damage with accumulation of β-amyloid and hyperphosphorylated tau are hallmark lesions of Alzheimer’s disease (AD). Methylglyoxal (MG), a key intermediate of glucose metabolism, is elevated in AD brains and modifies Aβ42, increasing misfolding and leading to the accumulation of senile plaques. Liraglutide, an analog of glucagon-like peptide-1 (GLP-1), is neurotrophic and neuroprotective. However, whether liraglutide can protect against AD-like memory-related deficits and tau hyperphosphorylation caused by MG in vivo is not known. Here, we report that MG induces tau hyperphosphorylation and causes ultrastructural hippocampal damage and cognitive impairment in C57BL/6J mice. Liraglutide reduced these effects via activation of the protein kinase B and glycogen synthase kinase-3β pathways. Our data reveal that liraglutide may alleviate AD-like cognitive impairment by decreasing the phosphorylation of tau. PMID:28337257

  10. Kinetic studies on muscle glycogen synthase.

    PubMed

    Salsas, E; Larner, J

    1975-05-10

    Using the I form of rabbit muscle glycogen synthase essentially free of glycogen, the kinetics and mechanism of action was investigated. No evidence for an exchange between [14C]UDP and UDP-glucose was found. The bisubstrate kinetics of the enzyme for UDP-glucose and glycogen, as well as for UDP-glucose and maltose, was determined. An intersecting pattern in the double reciprocal plot (velocity versus substrate concentration) suggestive of a sequential mechanism (ordered or random) was found in all cases. The K-m for UDP-glucose (45 to 48 mM) was the same with either maltose or glycogen as acceptor. The K-m for maltose (230 mM) and for glycogen (1.5 mug/ml) differed.

  11. 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.

  12. 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.

  13. 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

  14. 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.

  15. 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

  16. Inhibition of glycogen synthesis by increased lipid availability is associated with subcellular redistribution of glycogen synthase.

    PubMed

    Taylor, A J; Ye, J-M; Schmitz-Peiffer, C

    2006-01-01

    Increased lipid availability is associated with diminished insulin-stimulated glucose uptake and glycogen synthesis in muscle, but it is not clear whether alterations in glycogen synthase activity itself play a direct role. Because intracellular localization of this enzyme is involved in its regulation, we investigated whether fat oversupply causes an inhibitory redistribution. We examined the recovery of glycogen synthase in subcellular fractions from muscle of insulin-resistant, fat-fed rats and chow-fed controls, either maintained in the basal state or after a euglycaemic-hyperinsulinaemic clamp. Although glycogen synthase protein and activity were mostly recovered in an insoluble fraction, insulin caused translocation of activity from the smaller soluble pool to the insoluble fraction. Fat-feeding, which led to a reduction in glycogen synthesis during the clamp, was associated with a depletion in the soluble pool, consistent with an important role for this component. A similar depletion was also observed in cytosolic fractions of muscles from obese db/db mice, another model of lipid-induced insulin resistance. To investigate this in more detail, we employed lipid-pretreated L6 myotubes, which exhibited a reduction in insulin-stimulated glycogen synthesis independently of alterations in glucose flux or insulin signalling through protein kinase B. In control cells, insulin caused redistribution of a minor cytosolic pool of glycogen synthase to an insoluble fraction, which was again forestalled by lipid pretreatment. Glycogen synthase recovered in the insoluble fraction from pre-treated cells exhibited a low fractional velocity that was not increased in response to insulin. Our results suggest that the initial localization of glycogen synthase in a soluble pool plays an important role in glycogen synthesis, and that its sequestration in an insulin-resistant insoluble pool may explain in part the reduced glycogen synthesis caused by lipid oversupply.

  17. Kinetic mechanism of rabbit muscle glycogen synthase I.

    PubMed

    Gold, A M

    1980-08-05

    The kinetic mechanism of rabbit muscle glycogen synthase I was investigated by determining isotope-exchange rates at chemical equilibrium between uridine diphosphoglucose (UDPG) and glycogen and between UDPG and uridine 5'-diphosphate (UDP). The rates were followed simultaneously by use of UDPG labeled with 14C in the glucose moiety and with 3H in the uracil group. They were found to be independent of the concentrations of glycogen and the UDPG-UDP pair, averaging 6 X 10(-9) mol min-1 mg-1, with a ratio of UDPG-glycogen exchange to UDPG-UDP exchange of 0.85-0.95. The conclusion is that glycogen synthase has a rapid equilibrium random bi bi mechanism. The previously reported slow activation of glycogen-free synthase in the presence of glycogen was examined kinetically. The activation rate appears to be independent of glycogen concentration over a wide range, while the maximum activation is related to the third or fourth root of the glycogen concentration. This suggest that the slow bimolecular reaction mechanism proposed for human polymorphonuclear leucocyte glycogen synthase I [Sølling, H., & Esmann, V. (1977) Eur. J. Biochem. 81, 129] does not apply to rabbit muscle synthase I. The rate of exchange of glycogen molecules in the complex between glycogen and rabbit muscle synthase I under conditions where the enzyme is catalytically active was estimated by a novel method. The enzyme-glycogen complex was treated with [glucose-14C]UDPG and glycogen of different molecular weight. The distribution of isotope between the two forms of glycogen was determined after their separation by agarose gel chromatography. A rate constant of 0.3 min-1 was estimated for the exchange. It can be calculated, on the basis of the specific activity of the enzyme (20 mumol min-1 mg-1) and its action pattern, that hundreds of individual chains in the glycogen molecule must be available to the enzyme during the average lifetime of the complex. A mechanism is proposed for this process.

  18. Allosteric regulation of glycogen synthase in liver. A physiological dilemma.

    PubMed

    Nuttall, F Q; Gannon, M C

    1993-06-25

    Glycogen synthase catalyzes the transfer of the glucosyl moiety from UDP-glucose to the terminal branch of the glycogen molecule and is considered to be the rate-limiting enzyme for glycogen synthesis. However, under ideal assay conditions, i.e. 37 degrees C with saturating concentrations of UDP-glucose and the activator, glucose-6-P, the maximal catalytic activity of glycogen synthase was only 78% of the in vivo glycogen synthetic rate. Using concentrations of UDP-glucose and glucose-6-P likely to be present in vivo, the rate was only approximately 30%. This prompted us to reassess a possible role of allosteric effectors on synthase activity. Glycogen synthase was assayed at 37 degrees C using dilute, pH 7.0, buffered extracts, initial rate conditions, and UDP-glucose and glucose-6-P concentrations, which approximate those calculated to be present in total liver cell water. Several allosteric effectors were tested. Magnesium and AMP had little effect on activity. Pi, ADP, ATP, and UTP inhibited activity. When a combination of effectors were added at concentrations approximating those present in cell water, synthase activity could account for only 2% of the glycogen synthetic rate. Thus, although allosteric effectors are likely to be playing a major role in regulating synthase enzymic activity in liver cells, to date, a metabolite that can stimulate activity and/or overcome nucleotide inhibition has yet to be identified. If such a metabolite cannot be identified, an additional or alternative pathway for glycogen synthesis must be considered.

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

    PubMed Central

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

    1996-01-01

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

  20. 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.

  1. Insulin stimulation of glycogen synthase in cultured human diploid fibroblasts.

    PubMed

    Hidaka, H; Howard, B V; Kosmakos, F C; Fields, R M; Craig, J W; Bennett, P H; Larner, J

    1980-10-01

    The effect of insulin on glycogen synthase activity in human diploid fibroblasts has been studied. As little as 2 X 10(-10) M insulin increased the glycogen synthase / activity without changing the total activity. Stimulation occurred within 5 min and became maximal in 30 min. A half-maximal increase of / activity was achieved at 3 X 10(-9) M insulin. Glucose starvation increased the magnitude of response of glycogen synthase to insulin but did not change the insulin concentration necessary to give a half-maximal stimulation. Glucose increased the basal level of / activity in human diploid fibroblasts; the effect of insulin was additive. During in vitro senescence the total glycogen synthase activity declined, but the concentration of insulin that produced a half-maximal stimulation remained unchanged. These data indicate that regulation of glycogen synthase activity in human diploid fibroblasts is responsive to physiologic insulin levels and that the system provides a useful model for the in vitro study of insulin sensitivity.

  2. Glycogen synthase from the parabasalian parasite Trichomonas vaginalis: An unusual member of the starch/glycogen synthase family.

    PubMed

    Wilson, Wayne A; Pradhan, Prajakta; Madhan, Nayasha; Gist, Galen C; Brittingham, Andrew

    2017-07-01

    Trichomonas vaginalis, a parasitic protist, is the causative agent of the common sexually-transmitted infection trichomoniasis. The organism has long been known to synthesize substantial glycogen as a storage polysaccharide, presumably mobilizing this compound during periods of carbohydrate limitation, such as might be encountered during transmission between hosts. However, little is known regarding the enzymes of glycogen metabolism in T. vaginalis. We had previously described the identification and characterization of two forms of glycogen phosphorylase in the organism. Here, we measure UDP-glucose-dependent glycogen synthase activity in cell-free extracts of T. vaginalis. We then demonstrate that the TVAG_258220 open reading frame encodes a glycosyltransferase that is presumably responsible for this synthetic activity. We show that expression of TVAG_258220 in a yeast strain lacking endogenous glycogen synthase activity is sufficient to restore glycogen accumulation. Furthermore, when TVAG_258220 is expressed in bacteria, the resulting recombinant protein has glycogen synthase activity in vitro, transferring glucose from either UDP-glucose or ADP-glucose to glycogen and using both substrates with similar affinity. This protein is also able to transfer glucose from UDP-glucose or ADP-glucose to maltose and longer oligomers of glucose but not to glucose itself. However, with these substrates, there is no evidence of processivity and sugar transfer is limited to between one and three glucose residues. Taken together with our earlier work on glycogen phosphorylase, we are now well positioned to define both how T. vaginalis synthesizes and utilizes glycogen, and how these processes are regulated. Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  3. Shared control of hepatic glycogen synthesis by glycogen synthase and glucokinase.

    PubMed Central

    Gomis, R R; Ferrer, J C; Guinovart, J J

    2000-01-01

    We have used recombinant adenoviruses (AdCMV-RLGS and AdCMV-GK) to overexpress the liver isoforms of glycogen synthase (GS) and glucokinase (GK) in primary cultured rat hepatocytes. Glucose activated overexpressed GS in a dose-dependent manner and caused the accumulation of larger amounts of glycogen in the AdCMV-RLGS-treated hepatocytes. The concentration of intermediate metabolites of the glycogenic pathway, such as glucose 6-phosphate (Glc-6-P) and UDP-glucose, were not significantly altered. GK overexpression also conferred on the hepatocyte an enhanced capacity to synthesize glycogen in response to glucose, as described previously [Seoane, Gómez-Foix, O'Doherty, Gómez-Ara, Newgard and Guinovart (1996) J. Biol. Chem. 271, 23756-23760], although, in this case, they accumulated Glc-6-P. When GS and GK were simultaneously overexpressed, the accumulation of glycogen was enhanced in comparison with cells overexpressing either GS or GK. Our results are consistent with the hypothesis that liver GS catalyses the rate-limiting step of hepatic glycogen synthesis. However, hepatic glycogen deposition from glucose is submitted to a system of shared control in which the 'controller', GS, is, in turn, controlled by GK. This control is indirectly exerted through Glc-6-P, which 'switches on' GS dephosphorylation and activation. PMID:11042138

  4. In vivo regulation of muscle glycogen synthase and the control of glycogen synthesis.

    PubMed Central

    Shulman, R G; Bloch, G; Rothman, D L

    1995-01-01

    The activity of glycogen synthase (GSase; EC 2.4.1.11) is regulated by covalent phosphorylation. Because of this regulation, GSase has generally been considered to control the rate of glycogen synthesis. This hypothesis is examined in light of recent in vivo NMR experiments on rat and human muscle and is found to be quantitatively inconsistent with the data under conditions of glycogen synthesis. Our first experiments showed that muscle glycogen synthesis was slower in non-insulin-dependent diabetics compared to normals and that their defect was in the glucose transporter/hexokinase (GT/HK) part of the pathway. From these and other in vivo NMR results a quantitative model is proposed in which the GT/HK steps control the rate of glycogen synthesis in normal humans and rat muscle. The flux through GSase is regulated to match the proximal steps by "feed forward" to glucose 6-phosphate, which is a positive allosteric effector of all forms of GSase. Recent in vivo NMR experiments specifically designed to test the model are analyzed by metabolic control theory and it is shown quantitatively that the GT/HK step controls the rate of glycogen synthesis. Preliminary evidence favors the transporter step. Several conclusions are significant: (i) glucose transport/hexokinase controls the glycogen synthesis flux; (ii) the role of covalent phosphorylation of GSase is to adapt the activity of the enzyme to the flux and to control the metabolite levels not the flux; (iii) the quantitative data needed for inferring and testing the present model of flux control depended upon advances of in vivo NMR methods that accurately measured the concentration of glucose 6-phosphate and the rate of glycogen synthesis. Images Fig. 1 PMID:7567971

  5. Mutations in the liver glycogen synthase gene in children with hypoglycemia due to glycogen storage disease type 0.

    PubMed Central

    Orho, M; Bosshard, N U; Buist, N R; Gitzelmann, R; Aynsley-Green, A; Blümel, P; Gannon, M C; Nuttall, F Q; Groop, L C

    1998-01-01

    Glycogen storage disease type 0 (GSD-0) is a rare form of fasting hypoglycemia presenting in infancy or early childhood and accompanied by high blood ketones and low alanine and lactate concentrations. Although feeding relieves symptoms, it often results in postprandial hyperglycemia and hyperlactatemia. The glycogen synthase (GS) activity has been low or immeasurable in liver biopsies, whereas the liver glycogen content has been only moderately decreased. To investigate whether mutations in the liver GS gene (GYS2) on chromosome 12p12.2 were involved in GSD-0, we determined the exon-intron structure of the GYS2 gene and examined nine affected children from five families for linkage of GSD-0 to the GYS2 gene. Mutation screening of the 16 GYS2 exons was done by single-strand conformational polymorphism (SSCP) and direct sequencing. Liver GS deficiency was diagnosed from liver biopsies (GS activity and glycogen content). GS activity in the liver of the affected children was extremely low or nil, resulting in subnormal glycogen content. After suggestive linkage to the GYS2 gene had been established (LOD score = 2.9; P < 0.01), mutation screening revealed several different mutations in these families, including a premature stop codon in exon 5 (Arg246X), a 5'-donor splice site mutation in intron 6 (G+1T--> CT), and missense mutations Asn39Ser, Ala339Pro, His446Asp, Pro479Gln, Ser483Pro, and Met491Arg. Seven of the affected children carried mutations on both alleles. The mutations could not be found in 200 healthy persons. Expression of the mutated enzymes in COS7 cells indicated severely impaired GS activity. In conclusion, the results demonstrate that GSD-0 is caused by different mutations in the GYS2 gene. PMID:9691087

  6. 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.

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

    PubMed

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

    2016-10-01

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

  8. 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…

  9. 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…

  10. Glycogen synthase kinase 3 in the world of cell migration.

    PubMed

    Sun, Tong; Rodriguez, Marbelys; Kim, Leung

    2009-12-01

    Glycogen synthase kinase 3 (GSK3) is one of the few master switch kinases that regulate many aspects of cell functions. Recent studies on cell polarization and migration have shown that GSK3 is also essential for proper regulation of these processes. GSK3 influences cell migration as one of the regulators of the spatiotemporally controlled dynamics of the actin cytoskeleton, microtubules, and cell-to-matrix adhesions. In this mini-review, the effects of GSK3 on these three aspects of cell migration will be discussed.

  11. Relationship between single nucleotide polymorphism of glycogen synthase gene of Pacific oyster Crassostrea gigas and its glycogen content

    NASA Astrophysics Data System (ADS)

    Liu, Siwei; Li, Qi; Yu, Hong; Kong, Lingfeng

    2017-02-01

    Glycogen is important not only for the energy supplementary of oysters, but also for human consumption. High glycogen content can improve the stress survival of oyster. A key enzyme in glycogenesis is glycogen synthase that is encoded by glycogen synthase gene GYS. In this study, the relationship between single nucleotide polymorphisms (SNPs) in coding regions of Crassostrea gigas GYS (Cg-GYS) and individual glycogen content was investigated with 321 individuals from five full-sib families. Single-strand conformation polymorphism (SSCP) procedure was combined with sequencing to confirm individual SNP genotypes of Cg-GYS. Least-square analysis of variance was performed to assess the relationship of variation in glycogen content of C. gigas with single SNP genotype and SNP haplotype. As a consequence, six SNPs were found in coding regions to be significantly associated with glycogen content ( P < 0.01), from which we constructed four main haplotypes due to linkage disequilibrium. Furthermore, the most effective haplotype H2 (GAGGAT) had extremely significant relationship with high glycogen content ( P < 0.0001). These findings revealed the potential influence of Cg-GYS polymorphism on the glycogen content and provided molecular biological information for the selective breeding of good quality traits of C. gigas.

  12. 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

  13. Glycogen synthase can use glucose as an acceptor.

    PubMed

    Salsas, E; Larner, J

    1975-03-10

    Glycogen synthase purified to homogeneity from rabbit skeletal muscle is essentially free of carbohydrate and shows no activity in the absence of added acceptor. It can use glucose as a substrate converting it to a glucose disaccharide in the presence of UDP-glucose as cosubstrate. The reaction is dependent on time, and on UDP-glucose, glucose, and enzyme concentrations. The product of the single step reaction co-chromatographs in two solvent systems with maltose. The glucose disaccharide produced in the reaction with UDP-[14-C]glucose and nonradioactive glucose as well as with nonradioactive UDP-glucoes and [14-C]glucose is labeled asymmetrically. The linkage is characterized as alpha-1,4 and therefore the disaccharide is identified as maltose.

  14. Hyperactivity: glycogen synthase kinase-3 as a therapeutic target.

    PubMed

    Mines, Marjelo A

    2013-05-15

    The diagnosis of hyperactivity-associated disorders has increased within the past few years. The prevalence of hyperactivity-associated disorders is indicative of the need to more fully understand the underlying causes and to develop improved therapeutic interventions. There is increasing evidence that glycogen synthase kinase-3 (GSK3) mediates locomotor hyperactivity in a number of animal models, and therefore may be a potential target for therapeutic intervention in hyperactivity-associated behaviors. In this review, we discuss 1) the effect of manipulations of GSK3 in the absence of drugs and disorders on locomotor activity, 2) the role of GSK3 in drug-induced hyperactivity in rodents, and 3) regulation of locomotor activity by GSK3 in transgenic mouse models related to specific disorders. These studies link GSK3 regulation and activity to hyperactivity-associated behaviors and disease pathologies.

  15. Immunochemical Study of the Effect of F2Glc on Glycogen Synthase Translocation and Glycogen Synthesis in Isolated Rat Hepatocytes.

    PubMed

    Fernández-Novell, J M; Díaz-Lobo, M

    2017-09-16

    The compound 2-deoxy-2-fluoro-α-D-glucopyranosyl fluoride (F2Glc), which is a nonmetabolized superior glucose analogue, is a potent inhibitor of glycogen phosphorylase and pharmacological properties are reported. Glycogen phosphorylase (GP) and glycogen synthase (GS) are responsible of the degradation and synthesis, respectively, of glycogen which is a polymer of glucose units that provides a readily available source of energy in mammals. GP and GS are two key enzymes that modulate cellular glucose and glycogen levels; therefore, these proteins are suggested as potential targets for the treatment of diseases related to glycogen metabolism disorders. We studied by Western Blot technique that F2Glc decreased GP activity, and we also showed that F2Glc did not affect GS activity and its translocation from a uniform cytosolic distribution to the hepatocyte periphery, which is crucial for glycogen synthesis, using immunoblotting and immunofluorescence labeling techniques. F2Glc specifically inhibits glycogenolysis pathway and permits a greater deposition of glycogen. These observations open up the possibility of further develop drugs that act specifically on GP. The ability to selectively inhibit GP, which is a key enzyme for the release of glucose from the hepatic glycogen reserve, may represent a new approach for the treatment of hyperglycemia in type 2 diabetes.

  16. Hepatic overexpression of a constitutively active form of liver glycogen synthase improves glucose homeostasis.

    PubMed

    Ros, Susana; Zafra, Delia; Valles-Ortega, Jordi; García-Rocha, Mar; Forrow, Stephen; Domínguez, Jorge; Calbó, Joaquim; Guinovart, Joan J

    2010-11-26

    In this study, we tested the efficacy of increasing liver glycogen synthase to improve blood glucose homeostasis. The overexpression of wild-type liver glycogen synthase in rats had no effect on blood glucose homeostasis in either the fed or the fasted state. In contrast, the expression of a constitutively active mutant form of the enzyme caused a significant lowering of blood glucose in the former but not the latter state. Moreover, it markedly enhanced the clearance of blood glucose when fasted rats were challenged with a glucose load. Hepatic glycogen stores in rats overexpressing the activated mutant form of liver glycogen synthase were enhanced in the fed state and in response to an oral glucose load but showed a net decline during fasting. In order to test whether these effects were maintained during long term activation of liver glycogen synthase, we generated liver-specific transgenic mice expressing the constitutively active LGS form. These mice also showed an enhanced capacity to store glycogen in the fed state and an improved glucose tolerance when challenged with a glucose load. Thus, we conclude that the activation of liver glycogen synthase improves glucose tolerance in the fed state without compromising glycogenolysis in the postabsorptive state. On the basis of these findings, we propose that the activation of liver glycogen synthase may provide a potential strategy for improvement of glucose tolerance in the postprandial state.

  17. Glycogen synthase kinase-3: cryoprotection and glycogen metabolism in the freeze-tolerant wood frog.

    PubMed

    Dieni, Christopher A; Bouffard, Melanie C; Storey, Kenneth B

    2012-02-01

    The terrestrial anuran Rana sylvatica tolerates extended periods of whole-body freezing during the winter. Freezing survival is facilitated by extensive glycogen hydrolysis and distribution of high concentrations of the cryoprotectant glucose into blood and all tissues. As glycogenesis is both an energy-expensive process and counter-productive to maintaining sustained high cryoprotectant levels, we proposed that glycogen synthase kinase-3 (GSK-3) would be activated when wood frogs froze and would phosphorylate its downstream substrates to inactivate glycogen synthesis. Western blot analysis determined that the amount of phosphorylated (inactive) GSK-3 decreased in all five tissues tested in 24 h frozen frogs compared with unfrozen controls. Total GSK-3 protein levels did not change, with the exception of heart GSK-3, indicating that post-translational modification was the primary regulatory mechanism for this kinase. Kinetic properties of skeletal muscle GSK-3 from control and frozen frogs displayed differential responses to a temperature change (22 versus 4°C) and high glucose. For example, when assayed at 4°C, the K(m) for the GSK-3 substrate peptide was ∼44% lower for frozen frogs than the corresponding value in control frogs, indicating greater GSK-3 affinity for its substrates in the frozen state. This indicates that at temperatures similar to the environment encountered by frogs, GSK-3 in frozen frogs will phosphorylate its downstream targets more readily than in unfrozen controls. GSK-3 from skeletal muscle of control frogs was also allosterically regulated. AMP and phosphoenolpyruvate activated GSK-3 whereas inhibitors included glucose, glucose 6-phosphate, pyruvate, ATP, glutamate, glutamine, glycerol, NH(4)Cl, NaCl and KCl. The combination of phosphorylation and allosteric control argues for a regulatory role of GSK-3 in inactivating glycogenesis to preserve high glucose cryoprotectant levels throughout each freezing bout.

  18. 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.

  19. Pivotal roles of glycogen synthase-3 in hepatocellular carcinoma.

    PubMed

    Cervello, Melchiorre; Augello, Giuseppa; Cusimano, Antonella; Emma, Maria Rita; Balasus, Daniele; Azzolina, Antonina; McCubrey, James A; Montalto, Giuseppe

    2017-08-01

    Hepatocellular carcinoma (HCC) is one of the most common cancers in the world, and represents the second most frequently cancer and third most common cause of death from cancer worldwide. At advanced stage, HCC is a highly aggressive tumor with a poor prognosis and with very limited response to common therapies. Therefore, there is still the need for new effective and well-tolerated therapeutic strategies. Molecular-targeted therapies hold promise for HCC treatment. One promising molecular target is the multifunctional serine/threonine kinase glycogen synthase kinase 3 (GSK-3). The roles of GSK-3β in HCC remain controversial, several studies suggested a possible role of GSK-3β as a tumor suppressor gene in HCC, whereas, other studies indicate that GSK-3β is a potential therapeutic target for this neoplasia. In this review, we will focus on the different roles that GSK-3 plays in HCC and its interaction with signaling pathways implicated in the pathogenesis of HCC, such as Insulin-like Growth Factor (IGF), Notch, Wnt/β-catenin, Hedgehog (HH), and TGF-β pathways. In addition, the pivotal roles of GSK3 in epithelial-mesenchymal transition (EMT), invasion and metastasis will be also discussed. Copyright © 2017 Elsevier Ltd. All rights reserved.

  20. The role of glycogen synthase kinase-3beta in schizophrenia.

    PubMed

    Koros, Eliza; Dorner-Ciossek, Cornelia

    2007-09-01

    Glycogen synthase kinase (GSK)-3beta is recognized as a ubiquitous multifunctional enzyme involved in the modulation of many aspects of neuronal function. Inhibitory control of GSK-3beta has been identified to be crucial for the phosphoinositide 3'-kinase (PI3K)-protein kinase B (Akt)-mediated cell survival. Several lines of evidence converge in implicating abnormal GSK-3beta activity in the pathogenesis of schizophrenia. Preclinical evidence showing that both typical and atypical antipsychotics can indirectly inhibit the activity of GSK-3beta, has pointed to GSK-3beta as a possible therapeutic target for schizophrenia. It is well known that GSK-3beta can be indirectly inhibited via regulation of several intracellular signaling cascades, including the canonical Wnt, Reelin and tyrosine kinase receptor (Trk)-PI3K-Akt. Recently, direct inhibition of GSK-3beta has emerged as a possible option in the pharmacotherapy of several neuropsychiatric disorders. There is, however, a number of issues that need to be considered regarding therapeutic utility of GSK-3beta inhibitors. This article reviews the evidence supporting the possible role of aberrant GSK-3beta in the pathogenesis of schizophrenia and thus suggesting GSK-3beta to be a potential therapeutic target for this disorder.

  1. 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.

  2. 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

  3. Additive effect of contraction and insulin on glucose uptake and glycogen synthase in muscle with different glycogen contents.

    PubMed

    Lai, Yu-Chiang; Zarrinpashneh, Elham; Jensen, Jørgen

    2010-05-01

    Insulin and contraction regulate glucose uptake and glycogen synthase (GS) via distinct mechanisms in skeletal muscles, and an additive effect has been reported. Glycogen content is known to influence both contraction- and insulin-stimulated glucose uptake and GS activity. Our study reports that contraction and insulin additively stimulate glucose uptake in rat epitrochlearis muscles with normal (NG) and high (HG) glycogen contents, but the additive effect was only partial. In muscles with low glycogen (LG) content no additive effect was seen, but glucose uptake was higher in LG than in NG and HG during contraction, insulin stimulation, and when the two stimuli were combined. In LG, contraction-stimulated AMP-activated protein kinase (AMPK) activity and insulin-stimulated PKB phosphorylation were higher than in NG and HG, but phosphorylation of Akt substrate of 160 kDa was not elevated correspondingly. GLUT4 content was 50% increased in LG (rats fasted 24 h), which may explain the increased glucose uptake. Contraction and insulin also additively increased GS fractional activity in NG and HG but not in LG. GS fractional activity correlated most strongly with GS Ser641 phosphorylation (R -0.94, P<0.001). GS fractional activity also correlated with GS Ser7,10 phosphorylation, but insulin did not reduce GS Ser7,10 phosphorylation. In conclusion, an additive effect of contraction and insulin on glucose uptake and GS activity occurs in muscles with normal and high glycogen content but not in muscles with low glycogen content. Furthermore, contraction, insulin, and glycogen content all regulate GS Ser641 phosphorylation and GS fractional activity in concert.

  4. Purification, characterization and partial amino acid sequence of glycogen synthase from Saccharomyces cerevisiae.

    PubMed Central

    Carabaza, A; Arino, J; Fox, J W; Villar-Palasi, C; Guinovart, J J

    1990-01-01

    Glycogen synthase from Saccharomyces cerevisiae was purified to homogeneity. The enzyme showed a subunit molecular mass of 80 kDa. The holoenzyme appears to be a tetramer. Antibodies developed against purified yeast glycogen synthase inactivated the enzyme in yeast extracts and allowed the detection of the protein in Western blots. Amino acid analysis showed that the enzyme is very rich in glutamate and/or glutamine residues. The N-terminal sequence (11 amino acid residues) was determined. In addition, selected tryptic-digest peptides were purified by reverse-phase h.p.l.c. and submitted to gas-phase sequencing. Up to eight sequences (79 amino acid residues) could be aligned with the human muscle enzyme sequence. Levels of identity range between 37 and 100%, indicating that, although human and yeast glycogen synthases probably share some conserved regions, significant differences in their primary structure should be expected. Images Fig. 1. Fig. 2. Fig. 3. PMID:2114092

  5. Hypohydration Does Not Impair Skeletal Muscle Glycogen Resynthesis After Exercise

    DTIC Science & Technology

    1991-01-01

    AD-A262 085 9 3 -0 6 4 1 1 Hypohydration does not impair skeletal muscle glycogen resynthesis after exercise P. DARRELL NEUFER , MICHAEL N. SAWKA...5007 NEUFER , P. DARRELL, MICHAEL N. SAWKA, ANDREW J. for the restoration of preexercise glycogen levels, More- YOUNG, MARK D. QUIGLEY, WILLIAM A...Scand. S. Clin. Lab. Invest. 38:5.57 -560. 1978. tory Integratiue (’omp. Physiol. 14l: R25--R31, 198.3.19. NEUFER . P. D., A. J. YOUNG, AND M. N. SAWKA

  6. GLYCOGEN SYNTHASE KINASE-3 REGULATES MICROGLIAL MIGRATION, INFLAMMATION, AND INFLAMMATION-INDUCED NEUROTOXICITY

    PubMed Central

    Yuskaitis, Christopher J.; Jope, Richard S.

    2009-01-01

    Microglia play a prominent role in the brain’s inflammatory response to injury or infection by migrating to affected locations, secreting inflammatory molecules, and phagocytosing damaged tissue. However, because severe or chronic neuroinflammation exacerbates many neurological conditions, controlling microglia actions may provide therapeutic benefits in a diverse array of diseases. Since glycogen synthase kinase-3 (GSK3) promotes inflammatory responses in peripheral immune cells, we investigated if inhibitors of GSK3 attenuated microglia responses to inflammatory stimuli. Treatment of BV-2 microglia with GSK3 inhibitors greatly reduced the migration of microglia in both a scratch assay and in a transwell migration assay. Treatment of BV-2 microglia with lipopolysaccharide (LPS) stimulated the production of interleukin-6 and increased the expression of inducible nitric oxide synthase (iNOS) and NO production. Each of these microglia responses to inflammatory stimulation were greatly attenuated by GSK3 inhibitors. However, GSK3 inhibitors did not cause a general impairment of microglia functions, as the LPS-induced stimulated expression of cylcooxygenase-2 was unaltered. Regulation of microglia functions were also evident in cultured mouse hippocampal slices where GSK3 inhibitors reduced cytokine production and microglial migration, and provided protection from inflammation-induced neuronal toxicity. These findings demonstrate that GSK3 promotes microglial responses to inflammation and that the utilization of GSK3 inhibitors provides a means to limit the inflammatory actions of microglia. PMID:19007880

  7. Isolation of a glycogen synthase I kinase that is independent of adenosine 3':5'-monophosphate.

    PubMed Central

    Schlender, K K; Reimann, E M

    1975-01-01

    Three protein kinases (ATP:protein phosphotransferase, EC 2.7.1.37) were detected when the soluble fraction of rabbit kidney medulla was chromatographed on DEAE-cellulose with a linear NaC1 gradient. The first two kinases eluted (Peak 1 and Peak II) were cyclic-AMP-dependent, wheras Peak III was cyclic-AMP-independent. A procedure was developed to separate the catalytic subunit of Peak II cyclic-AMP-dependent protein kinase (representing the bulk of the histone kinase activity) from Peak III protein kinase. In contrast to the catalytic subunit, Peak III protein kinase phosphorylated casein more rapidly than histone. Peak III was insensitive to the heat-stable protein inhibitor of cyclic-AMP-dependent protein kinases and appeared to have a higher requirement for ATP than did the catalytic subunit. Peak III catalyzed the conversion of glycogen synthase (UDPglucose:glycogen alpha-4-glucosyltransferase, EC 2.4.1.11) from the I (glucose-6-phosphate-independent) to the D (glucose-6-phosphate-dependent) form. This conversion was dependent on Mg-2+ and ATP and was unaffected by cyclic AMP, cyclic GMP, or the protein inhibitor. Glycogen synthase I in the soluble fraction of kidney medulla could be converted to the D form by endogenous glycogen synthase I kinase if Mg-2+ and ATP were added. Most of this glycogen synthase I kinase activity was unaffected by cyclic AMP or by the protein inhibitor, suggesting that Peak III may be of major importance in the regulation of glycogen synthase in vivo. PMID:166380

  8. A highly prevalent equine glycogen storage disease is explained by constitutive activation of a mutant glycogen synthase.

    PubMed

    Maile, C A; Hingst, J R; Mahalingan, K K; O'Reilly, A O; Cleasby, M E; Mickelson, J R; McCue, M E; Anderson, S M; Hurley, T D; Wojtaszewski, J F P; Piercy, R J

    2017-01-01

    Equine type 1 polysaccharide storage myopathy (PSSM1) is associated with a missense mutation (R309H) in the glycogen synthase (GYS1) gene, enhanced glycogen synthase (GS) activity and excessive glycogen and amylopectate inclusions in muscle. Equine muscle biochemical and recombinant enzyme kinetic assays in vitro and homology modelling in silico, were used to investigate the hypothesis that higher GS activity in affected horse muscle is caused by higher GS expression, dysregulation, or constitutive activation via a conformational change. PSSM1-affected horse muscle had significantly higher glycogen content than control horse muscle despite no difference in GS expression. GS activity was significantly higher in muscle from homozygous mutants than from heterozygote and control horses, in the absence and presence of the allosteric regulator, glucose 6 phosphate (G6P). Muscle from homozygous mutant horses also had significantly increased GS phosphorylation at sites 2+2a and significantly higher AMPKα1 (an upstream kinase) expression than controls, likely reflecting a physiological attempt to reduce GS enzyme activity. Recombinant mutant GS was highly active with a considerably lower Km for UDP-glucose, in the presence and absence of G6P, when compared to wild type GS, and despite its phosphorylation. Elevated activity of the mutant enzyme is associated with ineffective regulation via phosphorylation rendering it constitutively active. Modelling suggested that the mutation disrupts a salt bridge that normally stabilises the basal state, shifting the equilibrium to the enzyme's active state. This study explains the gain of function pathogenesis in this highly prevalent polyglucosan myopathy. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. Piperidine and pyrrolidine analogs as glycogen synthase activators; a patent evaluation (WO2011058122).

    PubMed

    Uto, Yoshikazu

    2012-01-01

    A small series of piperidine analogs that effectively activate glycogen synthase (GS) was prepared in WO2011058122. The treatment or prophylaxis of metabolic disease and disorders by these novel GS activators is claimed. These compounds represent further variations around a structural motif explored in the prior patent publications by Roche.

  10. Chloroquine stimulates glucose uptake and glycogen synthase in muscle cells through activation of Akt.

    PubMed

    Halaby, Marie-Jo; Kastein, Brandon K; Yang, Da-Qing

    2013-06-14

    Chloroquine is a pharmaceutical agent that has been widely used to treat patients with malaria. Chloroquine has also been reported to have hypoglycemic effects on humans and animal models of diabetes. Despite many previous studies, the mechanism responsible for its hypoglycemic effect is still unclear. Chloroquine was recently reported to be an activator of ATM, the protein deficient in the Ataxia-telagiectasia (A-T) disease. Since ATM is also known as an insulin responsive protein that mediates Akt activation, we tested the effect of chloroquine on the activity of Akt and its downstream targets. In L6 muscle cells treated with insulin and chloroquine, the phosphorylation of Akt and glucose uptake were dramatically increased compared to cells treated with insulin alone, suggesting that chloroquine is a potent activator of Akt and glucose uptake in these cells. We also found that the reduction of insulin-mediated Akt activity in muscle tissues of insulin resistant rats was partially reversed by chloroquine treatment. Moreover, insulin-mediated phosphorylation of glycogen synthase kinase-3β in L6 cells was greatly enhanced by chloroquine. A substantial decrease in phosphorylation of glycogen synthase was also observed in chloroquine-treated L6 cells, indicating enhanced activity of glycogen synthase. Taken together, our results not only show that chloroquine is a novel activator of Akt that stimulates glucose uptake and glycogen synthase, but also validate chloroquine as a potential therapeutic agent for patients with type 2 diabetes mellitus. Copyright © 2013 Elsevier Inc. All rights reserved.

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

    PubMed

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

    2008-05-01

    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.

  12. [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

  13. Examination of methylphenidate-mediated behavior regulation by glycogen synthase kinase-3 in mice

    PubMed Central

    Mines, Marjelo A.; Beurel, Eleonore; Jope, Richard S.

    2012-01-01

    Abnormalities in dopaminergic activity have been implicated in psychiatric diseases, such as attention deficit hyperactivity disorder (ADHD), and are treated with therapeutic stimulants, commonly methylphenidate or amphetamine. Amphetamine administration increases glycogen synthase kinase-3 (GSK3) activation, which is necessary for certain acute behavioral responses to amphetamine, including increased locomotor activity and impaired sensorimotor gating. Here, we tested if modulating GSK3 by administration of the GSK3 inhibitor lithium or expression of constitutively active GSK3 altered behavioral responses to methylphenidate administered to mice acutely or daily for 8 days. Methylphenidate or amphetamine was administered to mice intraperitoneally for 1 or 8 days. Open-field activity and pre-pulse inhibition (PPI) were measured. In contrast to lithium’s blockade of acute amphetamine-induced locomotor hyperactivity, lithium treatment did not significantly reduce methylphenidate-induced locomotor hyperactivity in wild-type mice after acute or 8 days of repeated methylphenidate administration. Lithium treatment significantly increased the impairment in PPI caused by methylphenidate, but significantly reduced the amphetamine-induced PPI deficit. In GSK3 knockin mice, expression of constitutively active GSK3β, but not GSK3α, significantly increased locomotor hyperactivity after acute methylphenidate treatment, and significantly impaired PPI, preventing further methylphenidate-induced impairment of PPI that was evident in wild-type mice and GSK3α knockin mice. Lithium does not counteract locomotor activity and PPI responses to methylphenidate as it does these responses to amphetamine, indicating that different mechanisms mediate these behavioral responses to methylphenidate and amphetamine. Only active GSK3β, not GSK3α, modulates behavioral responses to MPH, indicating selectivity in the actions of GSK3 isoforms. PMID:23099259

  14. Glucose-induced glycogenesis in the liver involves the glucose-6-phosphate-dependent dephosphorylation of glycogen synthase.

    PubMed Central

    Cadefau, J; Bollen, M; Stalmans, W

    1997-01-01

    Non-metabolized glucose derivatives may cause inactivation of phosphorylase but, unlike glucose, they are unable to elicit activation of glycogen synthase in isolated hepatocytes. We report here that, after the previous inactivation of phosphorylase by one of these glucose derivatives (2-deoxy-2-fluoro-alpha-glucosyl fluoride), glycogen synthase was progressively activated by addition of increasing concentrations of glucose. Under these conditions, the degree of activation of glycogen synthase was linearly correlated with the intracellular glucose-6-phosphate (Glc-6-P) concentration. Addition of glucosamine, an inhibitor of glucokinase, decreased both parameters in parallel. Further experiments using an inhibitor of either protein kinases (5-iodotubercidin) or protein phosphatases (microcystin) in isolated hepatocytes indicated that Glc-6-P does not affect glycogen-synthase kinase activity but enhances the glycogen-synthase phosphatase reaction. Experiments in vitro showed that the synthase phosphatase activity of glycogen-bound type-1 protein phosphatase was increased by physiological concentrations of Glc-6-P (0.1-0.5 mM), but not by 2.5 mM fructose-6-P, fructose-1-P or glucose-1-P. At physiological ionic strength, the glycogen-associated synthase phosphatase activity was nearly entirely Glc-6-P-dependent, but Glc-6-P did not relieve the strong inhibitory effect of phosphorylase a. The large stimulatory effects of 2.5 mM Glc-6-P, with glycogen synthase b and phosphorylase a as substrates, appeared to be mostly substrate-directed, while the modest effects observed with casein and histone IIA pointed to an additional stimulation of glycogen-bound protein phosphatase-1 by Glc-6-P. We conclude that glucose elicits hepatic synthase phosphatase activity both by removal of the inhibitor, phosphorylase a, and by generation of the stimulator, Glc-6-P. PMID:9148744

  15. Glucose-induced glycogenesis in the liver involves the glucose-6-phosphate-dependent dephosphorylation of glycogen synthase.

    PubMed

    Cadefau, J; Bollen, M; Stalmans, W

    1997-03-15

    Non-metabolized glucose derivatives may cause inactivation of phosphorylase but, unlike glucose, they are unable to elicit activation of glycogen synthase in isolated hepatocytes. We report here that, after the previous inactivation of phosphorylase by one of these glucose derivatives (2-deoxy-2-fluoro-alpha-glucosyl fluoride), glycogen synthase was progressively activated by addition of increasing concentrations of glucose. Under these conditions, the degree of activation of glycogen synthase was linearly correlated with the intracellular glucose-6-phosphate (Glc-6-P) concentration. Addition of glucosamine, an inhibitor of glucokinase, decreased both parameters in parallel. Further experiments using an inhibitor of either protein kinases (5-iodotubercidin) or protein phosphatases (microcystin) in isolated hepatocytes indicated that Glc-6-P does not affect glycogen-synthase kinase activity but enhances the glycogen-synthase phosphatase reaction. Experiments in vitro showed that the synthase phosphatase activity of glycogen-bound type-1 protein phosphatase was increased by physiological concentrations of Glc-6-P (0.1-0.5 mM), but not by 2.5 mM fructose-6-P, fructose-1-P or glucose-1-P. At physiological ionic strength, the glycogen-associated synthase phosphatase activity was nearly entirely Glc-6-P-dependent, but Glc-6-P did not relieve the strong inhibitory effect of phosphorylase a. The large stimulatory effects of 2.5 mM Glc-6-P, with glycogen synthase b and phosphorylase a as substrates, appeared to be mostly substrate-directed, while the modest effects observed with casein and histone IIA pointed to an additional stimulation of glycogen-bound protein phosphatase-1 by Glc-6-P. We conclude that glucose elicits hepatic synthase phosphatase activity both by removal of the inhibitor, phosphorylase a, and by generation of the stimulator, Glc-6-P.

  16. 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

  17. Pre-eclampsia is associated with an increase in trophoblast glycogen content and glycogen synthase activity, similar to that found in hydatidiform moles.

    PubMed Central

    Arkwright, P D; Rademacher, T W; Dwek, R A; Redman, C W

    1993-01-01

    Pre-eclampsia is a placental disorder, but until now, biochemical details of dysfunction have been lacking. During an analysis of the oligosaccharide content of syncytiotrophoblast microvesicles purified from the placental chorionic villi of 10 primigravid women with proteinuric pre-eclampsia, we found an excess of glycogen breakdown products. Further investigation revealed a 10-fold increase in glycogen content (223 +/- 117 micrograms glycogen/mg protein), when compared with controls matched for gestational age at delivery (23 +/- 18 micrograms glycogen/mg protein) (P < 0.01). This was confirmed by examination of electron micrographs of chorionic villous tissue stained for glycogen. The increase in glycogen content was associated with 16 times more glycogen synthase (1,323 +/- 1,013 relative to 83 +/- 96 pmol glucose/mg protein per min) (P < 0.001), and a threefold increase in glycogen phosphorylase activity (2,280 +/- 1,360 relative to 700 +/- 540 pmol glucose/mg protein per min; P < 0.05). Similar changes in glycogen metabolism were found in trophoblast microvesicles derived from hydatidiform moles. Glycogen accumulation in villous syncytiotrophoblast may be a metabolic marker of immaturity of this cell which is unable to divide. The implications of these findings with regard to the pathogenesis of pre-eclampsia are discussed. Images PMID:8514882

  18. Amylin impairment of insulin effects on glycogen synthesis and phosphoenolpyruvate carboxykinase gene expression in rat primary cultured hepatocytes.

    PubMed Central

    Baqué, S; Guinovart, J J; Gómez-Foix, A M

    1994-01-01

    The ability of amylin to impair hepatic insulin action is controversial. We have found that the effect of amylin in primary cultured hepatocytes is strongly dependent on the culture conditions. Only in hepatocytes preincubated in the presence of fetal serum did amylin, at concentrations ranging from 1 to 100 nM, reduce insulin-stimulated glycogen synthesis rate and glycogen accumulation without showing direct effects. Neither basal glycogen synthase nor glycogen phosphorylase activity was modified by amylin treatment. Nevertheless, amylin (100 nM) blocked the activation of glycogen synthase by insulin. Amylin also proved capable of opposing the reduction in the expression of the phosphoenolpyruvate carboxykinase (PEPCK) gene induced by insulin, whereas the basal mRNA level of PEPCK was unaffected by amylin treatment. Thus, these results show that, in cultured rat hepatocytes, amylin is indeed able to interfere with insulin regulation of glycogenesis and PEPCK gene expression, favouring the hypothesis that amylin may modulate liver sensitivity to insulin. Images Figure 3 PMID:7998979

  19. Hepatic glycogen supercompensation activates AMP-activated protein kinase, impairs insulin signaling, and reduces glycogen deposition in the liver.

    PubMed

    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

    2011-02-01

    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. 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 CO(2)) were measured in the absence of fructose but in the presence of a hyperglycemic-hyperinsulinemic challenge including portal vein glucose infusion. 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. 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.

  20. Hypohydration does not impair skeletal muscle glycogen resynthesis after exercise.

    PubMed

    Neufer, P D; Sawka, M N; Young, A J; Quigley, M D; Latzka, W A; Levine, L

    1991-04-01

    The purpose of this investigation was to examine the effects of moderate hypohydration (HY) on skeletal muscle glycogen resynthesis after exhaustive exercise. On two occasions, eight males completed 2 h of intermittent cycle ergometer exercise (4 bouts of 17 min at 60% and 3 min at 80% of maximal O2 consumption/10 min rest) to reduce muscle glycogen concentrations (control values 711 +/- 41 mumol/g dry wt). During one trial, cycle exercise was followed by several hours of light upper body exercise in the heat without fluid replacement to induce HY (-5% body wt); in the second trial, sufficient water was ingested during the upper body exercise and heat exposure to maintain euhydration (EU). In both trials, 400 g of carbohydrate were ingested at the completion of exercise and followed by 15 h of rest while the desired hydration level was maintained. Muscle biopsy samples were obtained from the vastus lateralis immediately after intermittent cycle exercise (T1) and after 15 h of rest (T2). During the HY trial, the muscle water content was lower (P less than 0.05) at T1 and T2 (288 +/- 9 and 265 +/- 5 ml/100 g dry wt, respectively; NS) than during EU (313 +/- 8 and 301 +/- 4 ml/100 g dry wt, respectively; NS). Muscle glycogen concentration was not significantly different during EU and HY at T1 (200 +/- 35 vs. 251 +/- 50 mumol/g dry wt) or T2 (452 +/- 34 vs. 491 +/- 35 mumol/g dry wt). These data indicate that, despite reduced water content during the first 15 h after heavy exercise, skeletal muscle glycogen resynthesis is not impaired.

  1. 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.

  2. Benzisoxazole analogs as glycogen synthase activators, a patent evaluation (WO2011057956).

    PubMed

    Uto, Yoshikazu

    2012-06-01

    A small series of benzisoxazole analogs that effectively activate glycogen synthase (GS) was prepared in WO2011057956. These novel GS activators are claimed to be beneficial for the treatment or prophylaxis of metabolic disease and disorders. The 1,2-benzisoxazole-3-ol moiety is utilized in the present patent as a bioisoster of benzoic acid, which has often been employed in prior examples of the GS activators.

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

    PubMed

    Bhanot, Haymanti; Reddy, Mamatha M; Nonami, Atsushi; Weisberg, Ellen L; Bonal, Dennis; Kirschmeier, Paul T; Salgia, Sabrina; Podar, Klaus; Galinsky, Ilene; Chowdary, Tirumala K; Neuberg, Donna; Tonon, Giovanni; Stone, Richard M; Asara, John; Griffin, James D; Sattler, Martin

    2015-07-01

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

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

    PubMed Central

    Nonami, Atsushi; Weisberg, Ellen L.; Bonal, Dennis; Kirschmeier, Paul T.; Salgia, Sabrina; Podar, Klaus; Galinsky, Ilene; Chowdary, Tirumala K.; Neuberg, Donna; Tonon, Giovanni; Stone, Richard M.; Asara, John; Griffin, James D.; Sattler, Martin

    2015-01-01

    The rapid proliferation of myeloid leukemia cells is highly dependent on increased glucose metabolism. Through an unbiased metabolomics analysis of leukemia cells, we found that the glycogenic precursor UDP-D-glucose is pervasively upregulated, despite low glycogen levels. Targeting the rate-limiting glycogen synthase 1 (GYS1) not only decreased glycolytic flux but also increased activation of the glycogen-responsive AMPK (AMP kinase), leading to significant growth suppression. Further, genetic and pharmacological hyper-activation of AMPK was sufficient to induce the changes observed with GYS1 targeting. Cancer genomics data also indicate that elevated levels of the glycogenic enzymes GYS1/2 or GBE1 (glycogen branching enzyme 1) are associated with poor survival in AML. These results suggest a novel mechanism whereby leukemic cells sustain aberrant proliferation by suppressing excess AMPK activity through elevated glycogenic flux and provide a therapeutic entry point for targeting leukemia cell metabolism. PMID:25703587

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

    PubMed Central

    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

  6. Analysis of Mesorhizobium loti glycogen operon: effect of phosphoglucomutase (pgm) and glycogen synthase (g/gA) null mutants on nodulation of Lotus tenuis.

    PubMed

    Lepek, Viviana C; D'Antuono, Alejandra L; Tomatis, Pablo E; Ugalde, Juan E; Giambiagi, Susana; Ugalde, Rodolfo A

    2002-04-01

    The phosphoglucomutase (pgm) gene codes for a key enzyme required for the formation of UDP-glucose and ADP-glucose, the sugar donors for the biosynthesis of glucose containing polysaccharides. A Mesorhizobium loti pgm null mutant obtained in this study contains an altered form of lipopolysaccharide (LPS), lacks exopolysaccharide (EPS), beta cyclic glucan, and glycogen and is unable to nodulate Lotus tenuis. The nonnodulating phenotype of the pgm mutant was not due to the absence of glycogen, since a glycogen synthase (glgA) null mutant effectively nodulates this legume. In M. loti, pgm is part of the glycogen metabolism gene cluster formed by GlgP (glycogen phosphorylase), glgB (glycogen branching), glgC (ADP-glucose pyrophosphorylase), glgA, pgm, and glgX (glycogen debranching). The genes are transcribed as a single transcript from glgP to at least pgm under the control of a strong promoter (promoter I) upstream of glgP. An alternative promoter (promoter II), mapping in a 154-bp DNA fragment spanning 85 bp upstream of the glgA start codon and the first 69 bp of the glgA coding region, controls the expression of glgA and pgm, independently of the rest of the upstream genes. Primer extension experiments showed that transcription starts 19 bp upstream of the glgA start codon.

  7. Aerobic Exercise Plus Weight Loss Improves Insulin Sensitivity and Increases Skeletal Muscle Glycogen Synthase Activity in Older Men

    PubMed Central

    Katzel, Leslie I.; Prior, Steven J.; McLenithan, John C.; Goldberg, Andrew P.; Ortmeyer, Heidi K.

    2014-01-01

    The purpose of this study was to determine the effects of 6-month aerobic exercise training + weight loss (AEX + WL) on basal and insulin activation of glycogen synthase, basal citrate synthase activity, and Akt and AS160 phosphorylation in older, overweight/obese insulin-resistant men (n = 14; 63 ± 2 years; body mass index, 32 ± kg/m2). Muscle samples of the vastus lateralis were collected before and during a 3-hour 80 mU/m2/min hyperinsulinemic-euglycemic clamp. AEX + WL increased VO2max by 11% (p < .05) and decreased body weight (−9%, p < .001). AEX + WL increased basal citrate synthase activity by 46% (p < .01) and insulin activation of independent (2.9-fold) and fractional (2.3-fold) activities (both p < .001) of glycogen synthase. AEX + WL had no effect on phosphorylation of Akt or AS160. Glucose utilization (M) improved 25% (p < .01), and the change tended to be related to the increase in insulin activation of glycogen synthase fractional activity (r = .50, p = .08) following AEX + WL. In summary, AEX + WL has a robust effect on insulin activation of skeletal muscle glycogen synthase activity that likely contributes to improved glucose utilization in older insulin-resistant men. PMID:24357038

  8. Aerobic exercise plus weight loss improves insulin sensitivity and increases skeletal muscle glycogen synthase activity in older men.

    PubMed

    Ryan, Alice S; Katzel, Leslie I; Prior, Steven J; McLenithan, John C; Goldberg, Andrew P; Ortmeyer, Heidi K

    2014-07-01

    The purpose of this study was to determine the effects of 6-month aerobic exercise training + weight loss (AEX + WL) on basal and insulin activation of glycogen synthase, basal citrate synthase activity, and Akt and AS160 phosphorylation in older, overweight/obese insulin-resistant men (n = 14; 63 ± 2 years; body mass index, 32 ± kg/m(2)). Muscle samples of the vastus lateralis were collected before and during a 3-hour 80 mU/m(2)/min hyperinsulinemic-euglycemic clamp. AEX + WL increased VO2max by 11% (p < .05) and decreased body weight (-9%, p < .001). AEX + WL increased basal citrate synthase activity by 46% (p < .01) and insulin activation of independent (2.9-fold) and fractional (2.3-fold) activities (both p < .001) of glycogen synthase. AEX + WL had no effect on phosphorylation of Akt or AS160. Glucose utilization (M) improved 25% (p < .01), and the change tended to be related to the increase in insulin activation of glycogen synthase fractional activity (r = .50, p = .08) following AEX + WL. In summary, AEX + WL has a robust effect on insulin activation of skeletal muscle glycogen synthase activity that likely contributes to improved glucose utilization in older insulin-resistant men.

  9. Evidence of Positive Selection for a Glycogen Synthase (GYS1) Mutation in Domestic Horse Populations

    PubMed Central

    2014-01-01

    A dominantly inherited gain-of-function mutation in the glycogen synthase (GYS1) gene, resulting in excess skeletal muscle glycogen, has been identified in more than 30 horse breeds. This mutation is associated with the disease Equine Polysaccharide Storage Myopathy Type 1, yet persists at high frequency in some breeds. Under historical conditions of daily work and limited feed, excess muscle glycogen may have been advantageous, driving the increase in frequency of this allele. Fine-scale DNA sequencing in 80 horses and genotype assays in 279 horses revealed a paucity of haplotypes carrying the mutant allele when compared with the wild-type allele. Additionally, we found increased linkage disequilibrium, measured by relative extended haplotype homozygosity, in haplotypes carrying the mutation compared with haplotypes carrying the wild-type allele. Coalescent simulations of Belgian horse populations demonstrated that the high frequency and extended haplotype associated with the GYS1 mutation were unlikely to have arisen under neutrality or due to population demography. In contrast, in Quarter Horses, elevated relative extended haplotype homozygosity was associated with multiple haplotypes and may be the result of recent population expansion or a popular sire effect. These data suggest that the GYS1 mutation underwent historical selection in the Belgian, but not in the Quarter Horse. PMID:24215078

  10. 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

  11. Monte Carlo method based QSAR modeling of maleimide derivatives as glycogen synthase kinase-3β inhibitors.

    PubMed

    Živković, Jelena V; Trutić, Nataša V; Veselinović, Jovana B; Nikolić, Goran M; Veselinović, Aleksandar M

    2015-09-01

    The Monte Carlo method was used for QSAR modeling of maleimide derivatives as glycogen synthase kinase-3β inhibitors. The first QSAR model was developed for a series of 74 3-anilino-4-arylmaleimide derivatives. The second QSAR model was developed for a series of 177 maleimide derivatives. QSAR models were calculated with the representation of the molecular structure by the simplified molecular input-line entry system. Two splits have been examined: one split into the training and test set for the first QSAR model, and one split into the training, test and validation set for the second. The statistical quality of the developed model is very good. The calculated model for 3-anilino-4-arylmaleimide derivatives had following statistical parameters: r(2)=0.8617 for the training set; r(2)=0.8659, and r(m)(2)=0.7361 for the test set. The calculated model for maleimide derivatives had following statistical parameters: r(2)=0.9435, for the training, r(2)=0.9262 and r(m)(2)=0.8199 for the test and r(2)=0.8418, r(av)(m)(2)=0.7469 and ∆r(m)(2)=0.1476 for the validation set. Structural indicators considered as molecular fragments responsible for the increase and decrease in the inhibition activity have been defined. The computer-aided design of new potential glycogen synthase kinase-3β inhibitors has been presented by using defined structural alerts.

  12. 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

  13. Glycogen synthase kinase 3beta as a target for the therapy of shock and inflammation.

    PubMed

    Dugo, Laura; Collin, Marika; Thiemermann, Christoph

    2007-02-01

    After the discovery that glycogen synthase kinase (GSK) 3beta plays a fundamental role in the regulation of the activity of nuclear factor kappaB, a number of studies have investigated the effects of this protein kinase in the regulation of the inflammatory process. The GSK-3beta inhibition, using genetically modified cells and chemically different pharmacological inhibitors, affects the regulation of various inflammatory mediators in vitro and in vivo. Insulin, an endogenous inhibitor of GSK-3 in the pathway leading to the regulation of glycogen synthase activity, has recently been clinically used in the therapy for septic shock. The beneficial anti-inflammatory effects of insulin in preclinical and clinical studies could possibly be due, at least in part, to the inhibition of GSK-3 and not directly correlated to the regulation of blood glucose. We describe the latest studies describing the effects of GSK-3 inhibition as potential target of the therapy for diseases associated with inflammation, ischemia/reperfusion, and shock.

  14. Transcriptional regulation of pig GYS1 gene by glycogen synthase kinase 3β (GSK3β).

    PubMed

    Wang, Yilin; Wang, Yan; Zhong, Tao; Guo, Jiazhong; Li, Li; Zhang, Hongping; Wang, Linjie

    2017-01-01

    Glycogen synthase kinase 3β (GSK3β) is a ubiquitous serine/threonine kinase and has important roles in glycogen metabolism biosynthesis. Studies have revealed that GSK3β can directly regulate the glycogen synthase activity, yet little is known about the regulation of GSK3β on GYS1 gene transcription. Here, we show that overexpression of GSK3β decreased the mRNA expression level of GYS1. Then we cloned approximately 1.5 kb of pig GYS1 gene promoter region, generated sequential deletion constructs, and evaluated their activity. A gradual increase of the promoter activity was seen with increasing length of the promoter sequence, reaching its highest activity to the sequence corresponding to nt -350 to +224, and then decreased. However, the activities of constructed promoter fragments show different responses to GSK3β co-transfection. By analyzing a series of GYS1 promoter reporter constructs, we have defined two crucial regions (-1488 to -539, -350 to -147) that are responsible for GSK3β-induced transcriptional repression. Furthermore, the ChIP results revealed that only the first and second NF-κB sites of GYS1 promoter could bind to p65, and overexpression of GSK3β induced a significant decrease in p65 binding to the second NF-κB binding site, suggesting that GSK3β may regulate expression of GYS1 gene through binding to the second rather than the first NF-κB site. These data suggest that the NF-κB plays important roles in the transcriptional activity of pig GYS1 gene regulated by GSK3β.

  15. Ambient pH controls glycogen levels by regulating glycogen synthase gene expression in Neurospora crassa. New insights into the pH signaling pathway.

    PubMed

    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 pacC(KO) 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.

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

    PubMed Central

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

    2015-01-01

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

  17. Chelation of extracellular calcium-induced cell death was prevented by glycogen synthase kinase-3 inhibitors in PC12 cells.

    PubMed

    Takadera, Tsuneo; Ohtsuka, Megumi; Aoki, Haruka

    2010-03-01

    Calcium ion is a secondary messenger that mediates a variety of physiological responses of neurons, including cell survival responses. To determine the role of calcium in regulating neuronal survival and death, we examined whether chelation of extracellular calcium with EGTA induces caspase-dependent apoptotic cell death and whether glycogen synthase kinase-3 is involved in EGTA-induced cell death in PC12 cells. EGTA increased apoptotic cell death with morphological changes characterized by cell shrinkage and nuclear condensation and fragmentation accompanied by caspase activation. EGTA increased GRP78 protein expression, suggesting that EGTA induces ER stress. Glycogen synthase kinase-3 inhibitors prevented EGTA-induced apoptosis. In addition, nerve growth factor and insulin growth factor-I completely blocked EGTA-induced cell death. Moreover, caspase-3 activation was inhibited by glycogen synthase kinase-3 inhibitors. These results suggest that chelation of extracellular calcium with EGTA induces caspase-dependent apoptosis, and the activation of glycogen synthase kinase-3 is involved in the death of PC12 cells.

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

    USDA-ARS?s Scientific Manuscript database

    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 ...

  19. Skeletal muscle glycogen synthase subcellular localization: effects of insulin and PPAR-alpha agonist (K-111) administration in rhesus monkeys.

    PubMed

    Ortmeyer, Heidi K; Adall, Yohannes; Marciani, Karina R; Katsiaras, Andreas; Ryan, Alice S; Bodkin, Noni L; Hansen, Barbara C

    2005-06-01

    Insulin covalently and allosterically regulates glycogen synthase (GS) and may also cause the translocation of GS from glycogen-poor to glycogen-rich locations. We examined the possible role of subcellular localization of GS and glycogen in insulin activation of GS in skeletal muscle of six obese monkeys and determined whether 1) insulin stimulation during a hyperinsulinemic euglycemic clamp and/or peroxisome proliferator-activated receptor (PPAR)-alpha agonist treatment (K-111, 3 mg.kg(-1).day(-1); Kowa) induced translocation of GS and 2) translocation of GS was associated with insulin activation of GS. GS and glycogen were present in all fractions obtained by differential centrifugation, except for the cytosolic fraction, under both basal and insulin-stimulated conditions. We found no evidence for translocation of GS by insulin. GS total (GST) activity was strongly associated with glycogen content (r = 0.70, P < 0.001). Six weeks of treatment with K-111 increased GST activity in all fractions, except the cytosolic fraction, and mean GST activity, GS independent activity, and glycogen content were significantly higher in the insulin-stimulated samples compared with basal samples, effects not seen with vehicle. The increase in GST activity was strongly related to the increase in glycogen content during the hyperinsulinemic euglycemic clamp after K-111 administration (r = 0.74, P < 0.001). Neither GS protein expression nor GS gene expression was affected by insulin or by K-111 treatment. We conclude that 1) in vivo insulin does not cause translocation of GS from a glycogen-poor to a glycogen-rich location in primate skeletal muscle and 2) the mechanism of action of K-111 to improve insulin sensitivity includes an increase in GST activity without an increase in GS gene or protein expression.

  20. Regulation of persistent sodium currents by glycogen synthase kinase 3 encodes daily rhythms of neuronal excitability

    NASA Astrophysics Data System (ADS)

    Paul, Jodi R.; Dewoskin, Daniel; McMeekin, Laura J.; Cowell, Rita M.; Forger, Daniel B.; Gamble, Karen L.

    2016-11-01

    How neurons encode intracellular biochemical signalling cascades into electrical signals is not fully understood. Neurons in the central circadian clock in mammals provide a model system to investigate electrical encoding of biochemical timing signals. Here, using experimental and modelling approaches, we show how the activation of glycogen synthase kinase 3 (GSK3) contributes to neuronal excitability through regulation of the persistent sodium current (INaP). INaP exhibits a day/night difference in peak magnitude and is regulated by GSK3. Using mathematical modelling, we predict and confirm that GSK3 activation of INaP affects the action potential afterhyperpolarization, which increases the spontaneous firing rate without affecting the resting membrane potential. Together, these results demonstrate a crucial link between the molecular circadian clock and electrical activity, providing examples of kinase regulation of electrical activity and the propagation of intracellular signals in neuronal networks.

  1. 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.

  2. Glycogen synthase kinase 3 alpha phosphorylates and regulates the osteogenic activity of Osterix.

    PubMed

    Li, Hongyan; Jeong, Hyung Min; Choi, You Hee; Lee, Sung Ho; Jeong, Hye Gwang; Jeong, Tae Cheon; Lee, Kwang Youl

    2013-05-10

    Osteoblast-specific transcription factor Osterix is a zinc-finger transcription factor that required for osteoblast differentiation and new bone formation. The function of Osterix can be modulated by post-translational modification. Glycogen synthase kinase 3 alpha (GSK3α) is a multifunctional serine/threonine protein kinase that plays a role in the Wnt signaling pathways and is implicated in the control of several regulatory proteins and transcription factors. In the present study, we investigated how GSK3α regulates Osterix during osteoblast differentiation. Wide type GSK3α up-regulated the protein level, protein stability and transcriptional activity of Osterix. These results suggest that GSK3α regulates osteogenic activity of Osterix.

  3. Regulation of persistent sodium currents by glycogen synthase kinase 3 encodes daily rhythms of neuronal excitability

    PubMed Central

    Paul, Jodi R.; DeWoskin, Daniel; McMeekin, Laura J.; Cowell, Rita M.; Forger, Daniel B.; Gamble, Karen L.

    2016-01-01

    How neurons encode intracellular biochemical signalling cascades into electrical signals is not fully understood. Neurons in the central circadian clock in mammals provide a model system to investigate electrical encoding of biochemical timing signals. Here, using experimental and modelling approaches, we show how the activation of glycogen synthase kinase 3 (GSK3) contributes to neuronal excitability through regulation of the persistent sodium current (INaP). INaP exhibits a day/night difference in peak magnitude and is regulated by GSK3. Using mathematical modelling, we predict and confirm that GSK3 activation of INaP affects the action potential afterhyperpolarization, which increases the spontaneous firing rate without affecting the resting membrane potential. Together, these results demonstrate a crucial link between the molecular circadian clock and electrical activity, providing examples of kinase regulation of electrical activity and the propagation of intracellular signals in neuronal networks. PMID:27841351

  4. 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

  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. Proteomic Analysis of Ribosomes: Translational Control of mRNA populations by Glycogen Synthase GYS1

    PubMed Central

    Fuchs, Gabriele; Diges, Camille; Kohlstaedt, Lori A.; Wehner, Karen A.; Sarnow, Peter

    2011-01-01

    Ribosomes exist as a heterogenous pool of macromolecular complexes composed of ribosomal RNA molecules, ribosomal proteins and numerous associated, “non-ribosomal” proteins. To identify non-ribosomal proteins that may modulate ribosome activity, we examined the composition of translationally active and inactive ribosomes using a proteomic multidimensional protein identification technology. Notably, the phosphorylated isoform of glycogen synthase, GYS1, was preferentially associated with elongating ribosomes. Depletion of GYS1 affected the translation of a subset of cellular mRNAs, some of which encode proteins that modulate protein biosynthesis. These findings argue that GYS1 abundance, and by virtue of its ribosomal association, provide a feedback loop between the energy state of the cells and the translation machinery. PMID:21570405

  7. Expression and regulation of glycogen synthase kinase 3 in human neutrophils.

    PubMed

    Giambelluca, Miriam S; Cloutier, Nathalie; Rollet-Labelle, Emmanuelle; Boilard, Eric; Pouliot, Marc

    2013-11-01

    Glycogen synthase kinase 3 (GSK-3) is a serine/threonine kinase involved in the regulation of cellular processes ranging from glycogen metabolism to cell cycle regulation. Its two known isoforms, α and β, are differentially expressed in tissues throughout the body and exert distinct but often overlapping functions. GSK-3 is typically active in resting cells, inhibition by phosphorylation of Ser21 (GSK-3α) or Ser9 (GSK-3β) being the most common regulatory mechanism. GSK-3 activity has been linked recently with immune system function, yet little is known about the role of this enzyme in neutrophils, the most abundant leukocyte type. In the present study, we examined GSK-3 expression and regulation in human neutrophils. GSK-3α was found to be the predominant isoform, it was constitutively expressed and cell stimulation with different agonists did not alter its expression. Stimulation by fMLP, LPS, GM-CSF, Fcγ receptor engagement, or adenosine A2A receptor engagement all resulted in phosphorylation of Ser21. The use of metabolic inhibitors revealed that combinations of Src kinase, PKC, PI3K/AKT, ERK/RSK and PKA signaling pathways could mediate phosphorylation, depending on the agonist. Neither PLC nor p38 were involved. We conclude that GSK-3α is the main isoform expressed in neutrophils and that many different pathways can converge to inhibit GSK-3α activity via Ser21-phosphorylation. GSK-3α thus might be a hub of cellular regulation.

  8. Hypothalamic glycogen synthase kinase 3β has a central role in the regulation of food intake and glucose metabolism.

    PubMed

    Benzler, Jonas; Ganjam, Goutham K; Krüger, Manon; Pinkenburg, Olaf; Kutschke, Maria; Stöhr, Sigrid; Steger, Juliane; Koch, Christiane E; Ölkrug, Rebecca; Schwartz, Michael W; Shepherd, Peter R; Grattan, David R; Tups, Alexander

    2012-10-01

    GSK3β (glycogen synthase kinase 3β) is a ubiquitous kinase that plays a key role in multiple intracellular signalling pathways, and increased GSK3β activity is implicated in disorders ranging from cancer to Alzheimer's disease. In the present study, we provide the first evidence of increased hypothalamic signalling via GSK3β in leptin-deficient Lep(ob/ob) mice and show that intracerebroventricular injection of a GSK3β inhibitor acutely improves glucose tolerance in these mice. The beneficial effect of the GSK3β inhibitor was dependent on hypothalamic signalling via PI3K (phosphoinositide 3-kinase), a key intracellular mediator of both leptin and insulin action. Conversely, neuron-specific overexpression of GSK3β in the mediobasal hypothalamus exacerbated the hyperphagia, obesity and impairment of glucose tolerance induced by a high-fat diet, while having little effect in controls fed standard chow. These results demonstrate that increased hypothalamic GSK3β signalling contributes to deleterious effects of leptin deficiency and exacerbates high-fat diet-induced weight gain and glucose intolerance.

  9. 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.

  10. Analysis of the in vivo phosphorylation state of rabbit skeletal muscle glycogen synthase by fast-atom-bombardment mass spectrometry.

    PubMed

    Poulter, L; Ang, S G; Gibson, B W; Williams, D H; Holmes, C F; Caudwell, F B; Pitcher, J; Cohen, P

    1988-08-15

    The in vivo phosphorylation state of glycogen synthase was re-examined by fast-atom-bombardment mass spectrometry and a procedure in which phosphoserine residues are first converted to S-ethylcysteine. In animals injected with the beta-adrenergic antagonist propranolol, the phosphorylation sites in the N-terminal (N) and C-terminal (C) cyanogen bromide peptides were identified as the serine residues at N7, the region C28-C39, C42, C46 and C100. In animals injected with adrenalin, the phosphorylation of N7 increased from 0.6 to 0.8 mol/mol, the region C28-C39 from 0.7 to 1.2 mol/mol and C100 from 0.3 to 0.6 mol/mol. The phosphorylation states of C42 (0.7 mol/mol) and C46 (0.9 mol/mol) were unchanged. In addition, two further serine residues became phosphorylated at positions N10 (0.5 mol/mol) and C87 (0.5 mol/mol), which were not phosphorylated in the absence of adrenalin. Residues N10 and C42 have not been recognized as in vivo sites of phosphorylation previously. The results suggest that N10 is phosphorylated by a novel protein kinase which may be activated by cyclic-AMP-dependent protein kinase. The phosphorylation of C42 is likely to be catalysed by glycogen synthase kinase 3. The protein kinases responsible for phosphorylating N7, the region C28-C39, C46, C87 and C100 in vivo and the molecular mechanisms by which adrenalin inactivates glycogen synthase in vivo are discussed. Residue N3, a major site phosphorylated by casein kinase-I in vitro is not phosphorylated in vivo. This and other evidence indicates that casein kinase-I is not a glycogen synthase kinase in vivo.

  11. Unfolded protein response activates glycogen synthase kinase-3 via selective lysosomal degradation.

    PubMed

    Nijholt, Diana A T; Nölle, Anna; van Haastert, Elise S; Edelijn, Hessel; Toonen, Ruud F; Hoozemans, Jeroen J M; Scheper, Wiep

    2013-07-01

    The unfolded protein response (UPR) is a stress response that is activated upon disturbed homeostasis in the endoplasmic reticulum. In Alzheimer's disease, as well as in other tauopathies, the UPR is activated in neurons that contain early tau pathology. A recent genome-wide association study identified genetic variation in a UPR transducer as a risk factor for tauopathy, supporting a functional connection between UPR activation and tau pathology. Here we show that UPR activation increases the activity of the major tau kinase glycogen synthase kinase (GSK)-3 in vitro via a selective removal of inactive GSK-3 phosphorylated at Ser(21/9). We demonstrate that this is mediated by the autophagy/lysosomal pathway. In brain tissue from patients with different tauopathies, lysosomal accumulations of pSer(21/9) GSK-3 are found in neurons with markers for UPR activation. Our data indicate that UPR activation increases the activity of GSK-3 by a novel mechanism, the lysosomal degradation of the inactive pSer(21/9) GSK-3. This may provide a functional explanation for the close association between UPR activation and early tau pathology in neurodegenerative diseases.

  12. Role and regulation of Glycogen synthase kinase-3 beta in bovine spermatozoa.

    PubMed

    Belenky, Michael; Breitbart, Haim

    2017-01-01

    The serine/threonine kinase Glycogen synthase kinase 3 (GSK-3) is a master switch that regulates a multitude of cellular pathways, including the acrosome reaction in sperm. In epididymal sperm cells, for example, GSK-3 activity correlates with inhibition of motility-yet no direct pathways connecting GSK-3 activation with loss of motility have been described. Indeed, the details of how GSK-3 is regulated during sperm capacitation and the acrosome reaction remains obscure. To this end, we addressed the involvement of the GSK-3 beta isoform in several known pathways that contribute to motility and the acrosome reaction. We established that Protein kinase A (PKA) is the main regulator of GSK-3β in sperm, as pre-treatment of cells with a GSK-3 inhibitor prior to addition of H89, an inhibitor of PKA, attenuated the motility loss induced by blocking PKA activity. Both induced and spontaneous acrosome reactions also occurred less frequently in sperm treated with GSK-3 inhibitors. Finally, we observed a slow decline in phosphorylation of GSK-3β on Ser 9, which represents an inhibited state, during sperm capacitation; this phenotype is reversed during the induced acrosome reaction, in parallel to activation of Protein phosphatase 1. These results suggest that maintenance of sperm motility and acrosome reaction timing are mediated by PKA through the regulation of GSK-3 beta activity. Mol. Reprod. Dev. 84: 8-18, 2017. © 2016 Wiley Periodicals, Inc.

  13. Inhibition of Glycogen Synthase Kinase 3 Accelerated Liver Regeneration after Acetaminophen-Induced Hepatotoxicity in Mice.

    PubMed

    Bhushan, Bharat; Poudel, Samikshya; Manley, Michael W; Roy, Nairita; Apte, Udayan

    2017-03-01

    Overdose of acetaminophen (APAP) is the leading cause of acute liver failure (ALF) in the United States. Timely initiation of compensatory liver regeneration after APAP hepatotoxicity is critical for final recovery, but the mechanisms of liver regeneration after APAP-induced ALF have not been extensively explored yet. Previous studies from our laboratory have demonstrated that activation of β-catenin signaling after APAP overdose is associated with timely liver regeneration. Herein, we investigated the role of glycogen synthase kinase 3 (GSK3) in liver regeneration after APAP hepatotoxicity using a pharmacological inhibition strategy in mice. Treatment with specific GSK3 inhibitor (L803-mts), starting from 4 hours after 600 mg/kg dose of APAP, resulted in early initiation of liver regeneration in a dose-dependent manner, without modifying the peak regenerative response. Acceleration of liver regeneration was not secondary to alteration of APAP-induced hepatotoxicity, which remained unchanged after GSK3 inhibition. Early cell cycle initiation in hepatocytes after GSK3 inhibition was because of rapid induction of cyclin D1 and phosphorylation of retinoblastoma protein. This was associated with increased activation of β-catenin signaling after GSK3 inhibition. Taken together, our study has revealed a novel role of GSK3 in liver regeneration after APAP overdose and identified GSK3 as a potential therapeutic target to improve liver regeneration after APAP-induced ALF. Copyright © 2017 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  14. The PPLA motif of glycogen synthase kinase 3beta is required for interaction with Fe65.

    PubMed

    Lee, Eun Jeoung; Hyun, Sunghee; Chun, Jaesun; Shin, Sung Hwa; Lee, Kyung Eun; Yeon, Kwang Hum; Park, Tae Yoon; Kang, Sang Sun

    2008-07-31

    Glycogen synthase kinase 3beta (GSK 3 beta) is a serine/ threonine kinase that phosphorylates substrates such as beta-catenin and is involved in a variety of biological processes, including embryonic development, metabolism, tumorigenesis, and cell death. Here, we present evidence that human GSK 3beta is associated with Fe65, which has the characteristics of an adaptor protein, possessing a WW domain, and two phosphotyrosine interaction domains, PID1 and PID2. The GSK 3beta catalytic domain also contains a putative WW domain binding motif ((371)PPLA(374)), and we observed, using a pull down approach and co-immuno-precipitation, that it interacts physically with Fe65 via this motif. In addition, we detected co-localization of GSK 3beta and Fe65 by confocal microscopy, and this co-localization was disrupted by mutation of the putative WW domain binding motif of GSK 3beta.Finally, in transient transfection assays interaction of GSK 3 beta (wt) with Fe65 induced substantial cell apoptosis, whereas interaction with the GSK 3beta AALA mutant ((371)AALA(374)) did not, and we noted that phosphorylation of the Tyr 216 residue of the GSK 3beta AALA mutant was significantly reduced compared to that of GSK 3beta wild type. Thus, our observations indicate that GSK 3beta binds to Fe65 through its (371)PPLA(374) motif and that this interaction regulates apoptosis and phosphorylation of Tyr 216 of GSK 3beta.

  15. Glycogen synthase kinase-3/Shaggy mediates ethanol-induced excitotoxic cell death of Drosophila olfactory neurons

    PubMed Central

    French, Rachael L.; Heberlein, Ulrike

    2009-01-01

    It has long been known that heavy alcohol consumption leads to neuropathology and neuronal death. While the response of neurons to an ethanol insult is strongly influenced by genetic background, the underlying mechanisms are poorly understood. Here, we show that even a single intoxicating exposure to ethanol causes non-cell-autonomous apoptotic death specifically of Drosophila olfactory neurons, which is accompanied by a loss of a behavioral response to the smell of ethanol and a blackening of the third antennal segment. The Drosophila homolog of glycogen synthase kinase-3 (GSK-3)β, Shaggy, is required for ethanol-induced apoptosis. Consistent with this requirement, the GSK-3β inhibitor lithium protects against the neurotoxic effects of ethanol, indicating the possibility for pharmacological intervention in cases of alcohol-induced neurodegeneration. Ethanol-induced death of olfactory neurons requires both their neural activity and functional NMDA receptors. This system will allow the investigation of the genetic and molecular basis of ethanol-induced apoptosis in general and provide an understanding of the molecular role of GSK-3β in programmed cell death. PMID:19923438

  16. Molecular Pathways: Revisiting Glycogen Synthase Kinase-3β as a target for the treatment of cancer.

    PubMed

    Walz, Amy; Ugolkov, Andrey; Chandra, Sunandana; Kozikowski, Alan; Carneiro, Benedito A; O'Halloran, Thomas V; Giles, Francis J; Billadeau, Daniel D; Mazar, Andrew P

    2017-01-04

    Glycogen synthase kinase-3β (GSK-3β), a serine/threonine protein kinase, is a complex regulator of numerous cellular functions. GSK-3β is a unique kinase which is constitutively active in resting and non-stimulated cells. GSK-3β has been implicated in a wide range of diseases including neurodegeneration, inflammation and fibrosis, noninsulin-dependent diabetes mellitus, and cancer. It is a regulator of NF-κB-mediated survival of cancer cells, which provided a rationale for the development of GSK-3 inhibitors targeting malignant tumors. Recent studies, many of them reported over the past decade, have identified GSK-3β as a potential therapeutic target in at over 15 different types of cancer. Whereas only active GSK-3β is expressed in cancer cell nucleus, aberrant nuclear accumulation of GSK-3β has been identified as a hallmark of cancer cells in malignant tumors of different origin. This review focuses on the preclinical and clinical development of GSK-3 inhibitors, and the potential therapeutic impact of targeting GSK-3β in human cancer.

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

    PubMed Central

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

    2016-01-01

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

  18. Glycogen Synthase Kinase 3β Dictates Podocyte Motility and Focal Adhesion Turnover by Modulating Paxillin Activity

    PubMed Central

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

    2015-01-01

    Aberrant focal adhesion turnover is centrally involved in podocyte actin cytoskeleton disorganization and foot process effacement. The structural and dynamic integrity of focal adhesions is orchestrated by multiple cell signaling molecules, including glycogen synthase kinase 3β (GSK3β), a multitasking kinase lately identified as a mediator of kidney injury. However, the role of GSK3β in podocytopathy remains obscure. In doxorubicin (Adriamycin)-injured podocytes, lithium, a GSK3β inhibitor and neuroprotective mood stabilizer, obliterated the accelerated focal adhesion turnover, rectified podocyte hypermotility, and restored actin cytoskeleton integrity. Mechanistically, lithium counteracted the doxorubicin-elicited GSK3β overactivity and the hyperphosphorylation and overactivation of paxillin, a focal adhesion–associated adaptor protein. Moreover, forced expression of a dominant negative kinase dead mutant of GSK3β highly mimicked, whereas ectopic expression of a constitutively active GSK3β mutant abolished, the effect of lithium in doxorubicin-injured podocytes, suggesting that the effect of lithium is mediated, at least in part, through inhibition of GSK3β. Furthermore, paxillin interacted with GSK3β and served as its substrate. In mice with doxorubicin nephropathy, a single low dose of lithium ameliorated proteinuria and glomerulosclerosis. Consistently, lithium therapy abrogated GSK3β overactivity, blunted paxillin hyperphosphorylation, and reinstated actin cytoskeleton integrity in glomeruli associated with an early attenuation of podocyte foot process effacement. Thus, GSK3β-modulated focal adhesion dynamics might serve as a novel therapeutic target for podocytopathy. PMID:25239564

  19. GLUT4 and Glycogen Synthase Are Key Players in Bed Rest–Induced Insulin Resistance

    PubMed Central

    Biensø, Rasmus S.; Ringholm, Stine; Kiilerich, Kristian; Aachmann-Andersen, Niels-Jacob; Krogh-Madsen, Rikke; Guerra, Borja; Plomgaard, Peter; van Hall, Gerrit; Treebak, Jonas T.; Saltin, Bengt; Lundby, Carsten; Calbet, Jose A.L.; Pilegaard, Henriette; Wojtaszewski, Jørgen F.P.

    2012-01-01

    To elucidate the molecular mechanisms behind physical inactivity–induced insulin resistance in skeletal muscle, 12 young, healthy male subjects completed 7 days of bed rest with vastus lateralis muscle biopsies obtained before and after. In six of the subjects, muscle biopsies were taken from both legs before and after a 3-h hyperinsulinemic euglycemic clamp performed 3 h after a 45-min, one-legged exercise. Blood samples were obtained from one femoral artery and both femoral veins before and during the clamp. Glucose infusion rate and leg glucose extraction during the clamp were lower after than before bed rest. This bed rest–induced insulin resistance occurred together with reduced muscle GLUT4, hexokinase II, protein kinase B/Akt1, and Akt2 protein level, and a tendency for reduced 3-hydroxyacyl-CoA dehydrogenase activity. The ability of insulin to phosphorylate Akt and activate glycogen synthase (GS) was reduced with normal GS site 3 but abnormal GS site 2+2a phosphorylation after bed rest. Exercise enhanced insulin-stimulated leg glucose extraction both before and after bed rest, which was accompanied by higher GS activity in the prior-exercised leg than the rested leg. The present findings demonstrate that physical inactivity–induced insulin resistance in muscle is associated with lower content/activity of key proteins in glucose transport/phosphorylation and storage. PMID:22403297

  20. Aberrant spindle dynamics and cytokinesis in Dictyostelium discoideum cells that lack glycogen synthase kinase 3.

    PubMed

    Harwood, Adrian J; Forde-Thomas, Josephine E; Williams, Hazel; Samereier, Matthias; Müller-Taubenberger, Annette

    2013-01-01

    Eukaryotic cell division requires the co-ordinated assembly and disassembly of the mitotic spindle, accurate chromosome segregation and temporal control of cytokinesis to generate two daughter cells. While the absolute details of these processes differ between organisms, there are evolutionarily conserved core components common to all eukaryotic cells, whose identification will reveal the key processes that control cell division. Glycogen synthase kinase 3 (GSK-3) is a major protein kinase found throughout the eukaryotes and regulates many processes, including cell differentiation, growth, motility and apoptosis. In animals, GSK-3 associates with mitotic spindles and its inhibition causes mis-regulation of chromosome segregation. Two suppressor screens in yeast point to a more general effect of GSK-3 on cell division, however the direct role of GSK-3 in control of mitosis has not been explored outside the animal kingdom. Here we report that the Dictyostelium discoideum GSK-3 orthologue, GskA, associates with the mitotic spindle during cell division, as seen for its mammalian counterparts. Dictyostelium possesses only a single GSK-3 gene that can be deleted to eliminate all GSK-3 activity. We found that gskA-null mutants failed to elongate their mitotic spindle and were unable to divide in shaking culture, but have no chromosome segregation defect. These results suggest further conservation for the role of GSK-3 in the regulation of spindle dynamics during mitosis, but also reveal differences in the mechanisms ensuring accurate chromosome segregation.

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

    PubMed Central

    2012-01-01

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

  2. Inhibition of glycogen synthase kinase-3 increases the cytotoxicity of enzastaurin.

    PubMed

    Rovedo, Mark A; Krett, Nancy L; Rosen, Steven T

    2011-07-01

    Cutaneous T-cell lymphomas (CTCL) represent a spectrum of several distinct non-Hodgkin's lymphomas that are characterized by an invasion of the skin by malignant, clonal lymphocytes. Our laboratory has previously demonstrated that the protein kinase C (PKC) β inhibitor Enzastaurin increases apoptosis in malignant lymphocytes of CTCL. These results directly led to a clinical trial for Enzastaurin in CTCL in which it was well tolerated and showed modest activity. To ascertain a means of improving the efficacy of Enzastaurin, we investigated complementary signaling pathways and identified glycogen synthase kinase-3 (GSK3) as important in survival signaling in CTCL. Enzastaurin combined with GSK3 inhibitors demonstrated an enhancement of cytotoxicity. Treatment with a combination of Enzastaurin and the GSK3 inhibitor AR-A014418 resulted in upregulation of β-catenin total protein and β-catenin-mediated transcription. Inhibition of β-catenin-mediated transcription or small hairpin RNA (shRNA) knockdown of β-catenin decreased the cytotoxic effects of Enzastaurin plus AR-A014418. In addition, treatment with Enzastaurin and AR-A014418 decreased the mRNA levels and surface expression of CD44. shRNA knockdown of β-catenin also restored CD44 surface expression. Our observations provide a rationale for the combined targeting of PKC and GSK3 signaling pathways in CTCL to enhance the therapeutic outcome.

  3. Glycogen Synthase Kinase-3β Haploinsufficiency Mimics the Behavioral and Molecular Effects of Lithium

    PubMed Central

    O’Brien, W. Timothy; Harper, Amber DeAra; Jové, Fernando; Woodgett, James R.; Maretto, Silvia; Piccolo, Stefano; Klein, Peter S.

    2016-01-01

    Lithium is widely used to treat bipolar disorder, but its mechanism of action in this disorder is unknown. Several molecular targets of lithium have been identified, but these putative targets have not been shown to be responsible for the behavioral effects of lithium in vivo. A robust model for the effects of chronic lithium on behavior in mice would greatly facilitate the characterization of lithium action. We describe behaviors in mice that are robustly affected by chronic lithium. Remarkably, these lithium-sensitive behaviors are also observed in mice lacking one copy of the gene encoding glycogen synthase kinase-3β (Gsk-3β), a well established direct target of lithium. In addition, chronic lithium induces molecular changes consistent with inhibition of GSK-3 within regions of the brain that are paralleled in Gsk-3β+/− heterozygous mice. We also show that lithium therapy activates Wnt signaling in vivo, as measured by increased Wnt-dependent gene expression in the amygdala, hippocampus, and hypothalamus. These observations support a central role for GSK-3β in mediating behavioral responses to lithium. PMID:15282284

  4. Inhibition of glycogen synthase (casein) kinase-1 by different divalent metal ions

    SciTech Connect

    Singh, T.J.

    1987-05-01

    The specificity of glycogen synthase (casein) kinase-1 (CK-1) for different divalent metal ions was explored in this study. Of the eight metal ions (Mg, Mn, Zn, Cu, Ca, Ni, Co, Fe) tested only Mg/sup 2 +/ supported significant kinase activity. Several of the other metals, however, inhibited the Mg/sup 2 +/-stimulated kinase activity. Half-maximal inhibition by Zn, Mn, Co, Fe and Ni were observed at 50, 55, 110, 125 and 300 ..mu..M, respectively. Kinetic analyses using Zn/sup 2 +/ indicate that at a constant (ATP) Zn/sup 2 +/ increases the K/sub m/ for casein, the V/sub max/ being unaffected +/- Zn/sup 2 +/. At a constant (casein), Zn/sup 2 +/ decreased the V/sub max/ for ATP, the K/sub m/ remaining unchanged +/- Zn/sup 2 +/. The inhibition of CK-1 by the different metals is reversed by EGTA or EDTA. These results suggest that divalent metal ions such as Zn/sup 2 +/ and Mn/sup 2 +/ that do not support CK-1 activity may serve as inhibitors of the kinase in vivo.

  5. Inhibiting glycogen synthase kinase-3 mitigates the hematopoietic acute radiation syndrome in mice.

    PubMed

    Lee, Chang-Lung; Lento, William E; Castle, Katherine D; Chao, Nelson J; Kirsch, David G

    2014-05-01

    Exposure to a nuclear accident or radiological attack can cause death from acute radiation syndrome (ARS), which results from radiation injury to vital organs such as the hematopoietic system. However, the U.S. Food and Drug Administration (FDA) has not approved any medical countermeasures for this specific purpose. With growing concern over nuclear terrorism, there is an urgent need to develop small molecule deliverables that mitigate mortality from ARS. One emerging modulator of hematopoietic stem/progenitor cell (HSPC) activity is glycogen synthase kinase-3 (GSK-3). The inhibition of GSK-3 has been shown to augment hematopoietic repopulation in mouse models of bone marrow transplantation. In this study, we performed an in vitro screen using irradiated bone marrow mononuclear cells (BM-MNCs) to test the effects of four GSK-3 inhibitors: CHIR99021; 6-Bromoindirubin-3'-oxime (BIO); SB415286; and SB216763. This screen showed that SB216763 significantly increased the frequency of c-Kit(+) Lin(-) Sca1(+) (KLS) cells and hematopoietic colony-forming cells in irradiated BM-MNCs. Importantly, administration of a single dose of SB216763 to C57BL/6J mice by subcutaneous injection 24 h after total-body irradiation significantly improved hematopoietic recovery and mitigated hematopoietic ARS. Collectively, our results demonstrate that the GSK-3 inhibitor SB216763 is an effective medical countermeasure against acute radiation injury of the hematopoietic system.

  6. Inhibiting Glycogen Synthase Kinase-3 Mitigates the Hematopoietic Acute Radiation Syndrome in Mice

    PubMed Central

    Lee, Chang-Lung; Lento, William E.; Castle, Katherine D.; Chao, Nelson J.; Kirsch, David G.

    2014-01-01

    Exposure to a nuclear accident or radiological attack can cause death from acute radiation syndrome (ARS), which results from radiation injury to vital organs such as the hematopoietic system. However, the U.S. Food and Drug Administration (FDA) has not approved any medical countermeasures for this specific purpose. With growing concern over nuclear terrorism, there is an urgent need to develop small molecule deliverables that mitigate mortality from ARS. One emerging modulator of hematopoietic stem/progenitor cell (HSPC) activity is glycogen synthase kinase-3 (GSK-3). The inhibition of GSK-3 has been shown to augment hematopoietic repopulation in mouse models of bone marrow transplantation. In this study, we performed an in vitro screen using irradiated bone marrow mononuclear cells (BM-MNCs) to test the effects of four GSK-3 inhibitors: CHIR99021; 6-Bromoindirubin-3′-oxime (BIO); SB415286; and SB216763. This screen showed that SB216763 significantly increased the frequency of c-Kit+ Lin− Sca1+ (KLS) cells and hematopoietic colony-forming cells in irradiated BM-MNCs. Importantly, administration of a single dose of SB216763 to C57BL/6J mice by subcutaneous injection 24 h after total-body irradiation significantly improved hematopoietic recovery and mitigated hematopoietic ARS. Collectively, our results demonstrate that the GSK-3 inhibitor SB216763 is an effective medical countermeasure against acute radiation injury of the hematopoietic system. PMID:24720754

  7. Glycogen synthase kinase 3β and cyclin D1 expression in cervical carcinogenesis

    PubMed Central

    Park, Hyunsoo; Lee, Myunghwa; Kim, Dae Woon; Hong, Seo Yoo

    2016-01-01

    Objective Glycogen synthase kinase 3β (GSK3β) is a pluripotent protein kinase involved in the development of cancers through regulation of numerous oncogenic molecules. Cyclin D1, an important regulator of G1 to S phase transition in various cells, is one of target proteins that GSK3β regulate. Our objective was to assess the expression of GSK3β and cyclin D1 in cervical neoplasm of different histologic grades and to identify their correlation in cervical carcinogenesis. Methods Immunohistochemical analysis of GSK3β and cyclin D1 was performed in a total of 137 patients with 12 normal, 62 cervical intraepithelial neoplasia (CIN) (31 CIN1 and 31 CIN3) and 63 invasive cancers including 56 squamous cell carcinomas and 7 adenocarcinomas. Results The expression of GSK3β increased in parallel with the lesion grade, while that of cyclin D1 decreased with severity of the lesion (P<0.001). There was a significant inverse correlation between GSK3β and cyclin D1 expression in overall cervical neoplasia (Φ=-0.413, P<0.001). GSK3β expression was higher in squamous cell carcinoma than in adenocarcinoma (P=0.049). Conclusion These results suggest that the expressional increase in GSK3β plays a role in cervical carcinogenesis and has inverse correlation with cyclin D1 expression in this process. In addition, GSK3β expression appears to be associated with the histologic type of cervical cancer, especially squamous cell carcinoma. PMID:27896249

  8. 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

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

    PubMed Central

    Polter, Abigail M.; Li, Xiaohua

    2011-01-01

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

  10. Identification of glycogen synthase kinase 3α as a therapeutic target in melanoma

    PubMed Central

    Madhunapantula, SubbaRao V.; Sharma, Arati; Gowda, Raghavendra; Robertson, Gavin P.

    2014-01-01

    Summary Deregulated expression or activity of kinases can lead to melanomas, but often the particular kinase isoform causing the effect is not well established, making identification and validation of different isoforms regulating disease development especially important. To accomplish this objective, an siRNA screen was undertaken that which identified glycogen synthase kinase 3α (GSK3α) as an important melanoma growth regulator. Melanocytes and melanoma cell lines representing various stages of melanoma tumor progression expressed both GSK3α and GSK3β, but analysis of tumors in patients with melanoma showed elevated expression of GSK3α in 72% of samples, which was not observed for GSK3β. Furthermore, 80% of tumors in patients with melanoma expressed elevated levels of catalytically active phosphorylated GSK3α (pGSK3αY279), but not phosphorylated GSK3β (pGSK3βY216). siRNA-mediated reduction in GSK3α protein levels reduced melanoma cell survival and proliferation, sensitized cells to apoptosis-inducing agents and decreased xenografted tumor development by up to 56%. Mechanistically, inhibiting GSK3α expression using siRNA or the pharmacological agent AR-A014418 arrested melanoma cells in the G0/G1 phase of the cell cycle and induced apoptotic death to retard tumorigenesis. Therefore, GSK3α is a key therapeutic target in melanoma. PMID:24034838

  11. Glycogen synthase kinase-3β promotes cyst expansion in polycystic kidney disease

    PubMed Central

    Tao, Shixin; Kakade, Vijayakumar; Woodgett, James; Pandey, Pankaj; Suderman, Erin; Rajagopal, Madhumitha; Rao, Reena

    2014-01-01

    Polycystic kidney diseases (PKDs) are inherited disorders characterized by the formation of fluid filled renal cysts. Elevated cAMP levels in PKDs stimulate progressive cyst enlargement involving cell proliferation and transepithelial fluid secretion often leading to end stage renal disease. The glycogen synthase kinase-3 (GSK3) family of protein kinases consists of GSK3α and GSK3β isoforms and plays a crucial role in multiple cellular signaling pathways. We previously found that GSK3β, a regulator of cell proliferation, is also crucial for cAMP generation and vasopressin mediated urine concentration by the kidneys. However, the role of GSK3β in the pathogenesis of PKDs is not known. Here we found that GSK3β expression and activity were markedly up-regulated and associated with cyst-lining epithelia in the kidneys of mice and humans with PKD. Renal collecting duct specific gene knockout of GSK3β or pharmacological inhibition of GSK3 effectively slowed the progression of PKD in mouse models of autosomal recessive or autosomal dominant PKD. GSK3 inactivation inhibited cAMP generation and cell proliferation resulting in reduced cyst expansion, improved renal function and extended lifespan. GSK3β inhibition also reduced pERK, c-Myc and Cyclin-D1, known mitogens in proliferation of cystic epithelial cells. Thus, GSK3β plays a novel functional role in PKD pathophysiology and its inhibition may be therapeutically useful to slow cyst expansion and progression of PKD. PMID:25629553

  12. 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

    2016-04-16

    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.

  13. AJS1669, a novel small-molecule muscle glycogen synthase activator, improves glucose metabolism and reduces body fat mass in mice.

    PubMed

    Nakano, Kazuhiro; Takeshita, Sen; Kawasaki, Noriko; Miyanaga, Wataru; Okamatsu, Yoriko; Dohi, Mizuki; Nakagawa, Tadakiyo

    2017-04-01

    Impaired glycogen synthesis and turnover are common in insulin resistance and type 2 diabetes. As glycogen synthase (GS) is a key enzyme involved in the synthetic process, it presents a promising therapeutic target for the treatment of type 2 diabetes. In the present study, we identified a novel, potent and orally available GS activator AJS1669 {sodium 2-[[5-[[4-(4,5-difluoro-2-methylsulfanyl-phenyl)phenoxy] methyl]furan-2-carbonyl]-(2-furylmethyl)amino] acetate}. In vitro, we performed a glycogen synthase 1 (GYS1) activation assay for screening GS activators and identified that the activity of AJS1669 was further potentiated in the presence of glucose-6-phosphate (G6P). In vivo, we used ob/ob mice to evaluate the novel anti-diabetic effects of AJS1669 by measuring basal blood glucose levels, glucose tolerance and body fat mass index. Repeated administration of AJS1669 over 4 weeks reduced blood glucose and hemoglobin A1c (HbA1c) levels in ob/ob mice. AJS1669 also improved glucose tolerance in a dose-dependent manner, and decreased body fat mass. The mRNA levels of genes involved in mitochondrial fatty acid oxidation and mitochondrial biogenesis were elevated in skeletal muscle tissue following AJS1669 treatment. Hepatic tissue of treated mice also exhibited elevated expression of genes associated with fatty acid oxidation. In contrast to ob/ob mice, in C57Bl/6 mice AJS1669 administration did not alter body weight or reduce glucose levels. These results demonstrate that pharmacological agents that activate GYS1, the main GS subtype found in skeletal muscle, have potential for use as novel treatments for diabetes that improve glucose metabolism in skeletal muscle.

  14. AJS1669, a novel small-molecule muscle glycogen synthase activator, improves glucose metabolism and reduces body fat mass in mice

    PubMed Central

    Nakano, Kazuhiro; Takeshita, Sen; Kawasaki, Noriko; Miyanaga, Wataru; Okamatsu, Yoriko; Dohi, Mizuki; Nakagawa, Tadakiyo

    2017-01-01

    Impaired glycogen synthesis and turnover are common in insulin resistance and type 2 diabetes. As glycogen synthase (GS) is a key enzyme involved in the synthetic process, it presents a promising therapeutic target for the treatment of type 2 diabetes. In the present study, we identified a novel, potent and orally available GS activator AJS1669 {sodium 2-[[5-[[4-(4,5-difluoro-2-methylsulfanyl-phenyl) phenoxy] methyl]furan-2-carbonyl]-(2-furylmethyl)amino] acetate}. In vitro, we performed a glycogen synthase 1 (GYS1) activation assay for screening GS activators and identified that the activity of AJS1669 was further potentiated in the presence of glucose-6-phosphate (G6P). In vivo, we used ob/ob mice to evaluate the novel anti-diabetic effects of AJS1669 by measuring basal blood glucose levels, glucose tolerance and body fat mass index. Repeated administration of AJS1669 over 4 weeks reduced blood glucose and hemoglobin A1c (HbA1c) levels in ob/ob mice. AJS1669 also improved glucose tolerance in a dose-dependent manner, and decreased body fat mass. The mRNA levels of genes involved in mitochondrial fatty acid oxidation and mitochondrial biogenesis were elevated in skeletal muscle tissue following AJS1669 treatment. Hepatic tissue of treated mice also exhibited elevated expression of genes associated with fatty acid oxidation. In contrast to ob/ob mice, in C57Bl/6 mice AJS1669 administration did not alter body weight or reduce glucose levels. These results demonstrate that pharmacological agents that activate GYS1, the main GS subtype found in skeletal muscle, have potential for use as novel treatments for diabetes that improve glucose metabolism in skeletal muscle. PMID:28290602

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

    PubMed Central

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

    1996-01-01

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

  16. 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.

  17. Impaired glycogen breakdown and synthesis in phosphoglucomutase 1 deficiency.

    PubMed

    Preisler, Nicolai; Cohen, Jonathan; Vissing, Christoffer Rasmus; Madsen, Karen Lindhardt; Heinicke, Katja; Sharp, Lydia Jane; Phillips, Lauren; Romain, Nadine; Park, Sun Young; Newby, Marta; Wyrick, Phil; Mancias, Pedro; Galbo, Henrik; Vissing, John; Haller, Ronald Gerald

    2017-08-25

    We investigated metabolism and physiological responses to exercise in an 18-year-old woman with multiple congenital abnormalities and exertional muscle fatigue, tightness, and rhabdomyolysis. We studied biochemistry in muscle and fibroblasts, performed mutation analysis, assessed physiological responses to forearm and cycle-ergometer exercise combined with stable-isotope techniques and indirect calorimetry, and evaluated the effect of IV glucose infusion and oral sucrose ingestion on the exercise response. Phosphoglucomutase type 1 (PGM1) activity in muscle and fibroblasts was severely deficient and PGM1 in muscle was undetectable by Western blot. The patient was compound heterozygous for missense (R422W) and nonsense (Q530X) mutations in PGM1. Forearm exercise elicited no increase in lactate, but an exaggerated increase in ammonia, and provoked a forearm contracture. Comparable to patients with McArdle disease, the patient developed a 'second wind' with a spontaneous fall in exercise heart rate and perceived exertion. Like in McArdle disease, this was attributable to an increase in muscle oxidative capacity. Carbohydrate oxidation was blocked during exercise, and the patient had exaggerated oxidation of fat to fuel exercise. Exercise heart rate and perceived exertion were lower after IV glucose and oral sucrose. Muscle glycogen level was low normal. The second wind phenomenon has been considered to be pathognomonic for McArdle disease, but we demonstrate that it can also be present in PGM1 deficiency. We show that severe loss of PGM1 activity causes blocked muscle glycogenolysis that mimics McArdle disease, but may also limit glycogen synthesis, which broadens the phenotypic spectrum of this disorder. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. Lithium and neuropsychiatric therapeutics: neuroplasticity via glycogen synthase kinase-3beta, beta-catenin, and neurotrophin cascades.

    PubMed

    Wada, Akihiko

    2009-05-01

    Mood disorders are not merely attributed to the functional defect of neurotransmission, but also are due to the structural impairment of neuroplasticity. Chronic stress decreases neurotrophin levels, precipitating or exacerbating depression; conversely, antidepressants increase expression of various neurotrophins (e.g., brain-derived neurotrophic factor and vascular endothelial growth factor), thereby blocking or reversing structural and functional pathologies via promoting neurogenesis. Since the worldwide approval of lithium therapy in 1970, lithium has been used for its anti-manic, antidepressant, and anti-suicidal effects, yet the therapeutic mechanisms at the cellular level remain not-fully defined. During the last five years, multiple lines of evidence have shown that the mood stabilization and neurogenesis by lithium are due to the lithium-induced inhibition of glycogen synthase kinase-3beta (GSK-3beta), allowing accumulation of beta-catenin and beta-catenin-dependent gene transcriptional events. Altered levels of GSK-3beta and beta-catenin are associated with various neuropsychiatric and neurodegenerative diseases, while various classical neuropsychiatric drugs inhibit GSK-3beta and up-regulate beta-catenin expression. In addition, evidence has emerged that insulin-like growth factor-I enhances antidepression, anti-anxiety, memory, neurogenesis, and angiogenesis; antidepressants up-regulate expression of insulin-like growth factor-I, while insulin-like growth factor-I up-regulates brain-derived neurotrophic factor expression and its receptor TrkB level, as well as brain-derived neurotrophic factor-induced synaptic protein levels. More importantly, physical exercise and healthy diet raise transport of peripheral circulating insulin-like growth factor I into the brain, reinforcing the expression of neurotrophins (e.g., brain-derived neurotrophic factor) and the strength of cell survival signalings (e.g., phosphoinositide 3-kinase / Akt / GSK-3beta pathway

  19. Regulation of skeletal muscle plasticity by glycogen synthase kinase-3β: a potential target for the treatment of muscle wasting.

    PubMed

    Verhees, Koen J P; Pansters, Nicholas A M; Schols, Annemie M W J; Langen, Ramon C J

    2013-01-01

    Muscle wasting is a prevalent and disabling condition in chronic disease and cancer and has been associated with increased mortality and impaired efficacy of surgical and medical interventions. Pharmacological therapies to combat muscle wasting are currently limited but considered as an important unmet medical need. Muscle wasting has been attributed to increased muscle proteolysis, and in particular ubiquitin 26S-proteasome system (UPS)-dependent protein breakdown. However, rates of muscle protein synthesis are also subject to extensive (patho) physiological regulation, and the balance between synthesis and degradation ultimately determines net muscle protein turnover. As multinucleated muscle fibers accommodate threshold changes in muscle protein content by the accretion and loss of muscle nuclei, myonuclear turnover may additionally determine muscle mass. Current insights in the mechanisms dictating muscle mass plasticity not only reveal intricate interactions and crosstalk between these processes, but imply the existence of signaling molecules that act as molecular switchboards, which coordinate and integrate cellular responses upon conditions that evoke changes in muscle mass. These "master regulators" of skeletal muscle mass plasticity are preferred targets for pharmacological modulation of skeletal muscle wasting. In this review Glycogen synthase kinase-3β (GSK-3β) is highlighted as a master regulator of muscle mass plasticity since, in addition to its role in UPS-mediated muscle protein degradation, it also controls protein synthesis, and influences myonuclear accretion and cell death. Moreover, the regulation of GSK-3β activity as well as currently available pharmacological inhibitors are described and discussed in the context of multimodal treatment strategies aimed at the inhibition of GSK-3β, and optimal exploitation of its potential role as a central regulator of skeletal muscle mass plasticity for the treatment of muscle wasting.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2010-01-01

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

  2. Enhanced glycogen synthase kinase-3β activity mediates podocyte apoptosis under diabetic conditions.

    PubMed

    Paeng, Jisun; Chang, Jae Hyun; Lee, Sun Ha; Nam, Bo Young; Kang, Hye-Young; Kim, Seonghun; Oh, Hyung Jung; Park, Jung Tak; Han, Seung Hyeok; Yoo, Tae-Hyun; Kang, Shin-Wook

    2014-12-01

    Glycogen synthase kinase-3β (GSK-3β) is involved in the pathogenesis of various kidney diseases. This study was undertaken to examine the changes in GSK-3β activity in podocytes under diabetic conditions and to elucidate the functional role of GSK-3β in podocyte apoptosis. In vivo, 32 rats were injected with either diluent (n = 16, C) or with streptozotocin intraperitoneally (n = 16, DM), and 8 rats from each group were treated with 6-bromoindirubin-3'-oxime (BIO) for 3 months. In vitro, immortalized mouse podocytes were exposed to 5.6 mM glucose or 30 mM glucose (HG) with or without 10 μM BIO. Western blot analysis and TUNEL or Hoechst 33342 staining were performed to identify apoptosis. Urinary albumin excretion was significantly higher in DM rats, and this increase was significantly abrogated in DM rats by BIO treatment. The protein expression of Tyr216-phospho-GSK-3β was significantly increased in DM glomeruli and in cultured podocytes exposed to HG. Western blot analysis revealed that the protein expression of Bax and active fragments of caspase-3 were significantly increased, whereas phospho-Akt, β-catenin, and Bcl-2 protein expression were significantly decreased in DM glomeruli and HG-stimulated podocytes. Apoptosis, determined by TUNEL assay and Hoechst 33342 staining, was also significantly increased in podocytes under diabetic conditions. The changes in the expression of apoptosis-related molecules and the increase in the number of apoptotic cells in DM glomeruli as well as in HG-stimulated podocytes were significantly ameliorated by BIO. These findings suggest that enhanced GSK-3β activity within podocytes under diabetic conditions is associated with podocyte loss in diabetic nephropathy.

  3. Glycogen synthase kinase 3α regulates urine concentrating mechanism in mice.

    PubMed

    Nørregaard, Rikke; Tao, Shixin; Nilsson, Line; Woodgett, James R; Kakade, Vijayakumar; Yu, Alan S L; Howard, Christiana; Rao, Reena

    2015-03-15

    In mammals, glycogen synthase kinase (GSK)3 comprises GSK3α and GSK3β isoforms. GSK3β has been shown to play a role in the ability of kidneys to concentrate urine by regulating vasopressin-mediated water permeability of collecting ducts, whereas the role of GSK3α has yet to be discerned. To investigate the role of GSK3α in urine concentration, we compared GSK3α knockout (GSK3αKO) mice with wild-type (WT) littermates. Under normal conditions, GSK3αKO mice had higher water intake and urine output. GSK3αKO mice also showed reduced urine osmolality and aquaporin-2 levels but higher urinary vasopressin. When water deprived, they failed to concentrate their urine to the same level as WT littermates. The addition of 1-desamino-8-d-arginine vasopressin to isolated inner medullary collecting ducts increased the cAMP response in WT mice, but this response was reduced in GSK3αKO mice, suggesting reduced responsiveness to vasopressin. Gene silencing of GSK3α in mpkCCD cells also reduced forskolin-induced aquaporin-2 expression. When treated with LiCl, an isoform nonselective inhibitor of GSK3 and known inducer of polyuria, WT mice developed significant polyuria within 6 days. However, in GSK3αKO mice, the polyuric response was markedly reduced. This study demonstrates, for the first time, that GSK3α could play a crucial role in renal urine concentration and suggest that GSK3α might be one of the initial targets of Li(+) in LiCl-induced nephrogenic diabetes insipidus.

  4. 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.

  5. 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.

  6. Loss of glycogen synthase kinase 3 isoforms during murine oocyte growth induces offspring cardiac dysfunction.

    PubMed

    Monteiro da Rocha, André; Ding, Jun; Slawny, Nicole; Wolf, Amber M; Smith, Gary D

    2015-05-01

    Glycogen synthase kinase-3 (GSK3) is a constitutively active serine threonine kinase with 1) two isoforms (GSK3A and GSK3B) that have unique and overlapping functions, 2) multiple molecular intracellular mechanisms that involve phosphorylation of diverse substrates, and 3) implications in pathogenesis of many diseases. Insulin causes phosphorylation and inactivation of GSK3 and mammalian oocytes have a functional insulin-signaling pathway whereby prolonged elevated insulin during follicle/oocyte development causes GSK3 hyperphosphorylation, reduced GSK3 activity, and altered oocyte chromatin remodeling. Periconceptional diabetes and chronic hyperinsulinemia are associated with congenital malformations and onset of adult diseases of cardiovascular origin. Objectives were to produce transgenic mice with individual or concomitant loss of GSK3A and/or GSK3B and investigate the in vivo role of oocyte GSK3 on fertility, fetal development, and offspring health. Wild-type males bred to females with individual or concomitant loss of oocyte GSK3 isoforms did not have reduced fertility. However, concomitant loss of GSK3A and GSK3B in the oocyte significantly increased neonatal death rate due to congestive heart failure secondary to ventricular hyperplasia. Individual loss of oocyte GSK3A or GSK3B did not induce this lethal phenotype. In conclusion, absence of oocyte GSK3 in the periconceptional period does not alter fertility yet causes offspring cardiac hyperplasia, cardiovascular defects, and significant neonatal death. These results support a developmental mechanism by which periconceptional hyperinsulinemia associated with maternal metabolic syndrome, obesity, and/or diabetes can act on the oocyte and affect offspring cardiovascular development, function, and congenital heart malformation.

  7. Identification of a glycogen synthase kinase-3β inhibitor that attenuates hyperactivity in CLOCK mutant mice.

    PubMed

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

    2011-09-05

    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 mg kg⁻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⁻¹) and the antipsychotic haloperidol (1 mg kg⁻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.

  8. Efficacy of glycogen synthase kinase-3β targeting against osteosarcoma via activation of β-catenin

    PubMed Central

    Yamamoto, Norio; Nishida, Hideji; Hayashi, Katsuhiro; Kimura, Hiroaki; Takeuchi, Akihiko; Miwa, Shinji; Igarashi, Kentaro; Kato, Takashi; Aoki, Yu; Higuchi, Takashi; Hirose, Mayumi; Hoffman, Robert M; Minamoto, Toshinari; Tsuchiya, Hiroyuki

    2016-01-01

    Development of innovative more effective therapy is required for refractory osteosarcoma patients. We previously established that glycogen synthase kinase-3β (GSK- 3β) is a therapeutic target in various cancer types. In the present study, we explored the therapeutic efficacy of GSK-3β inhibition against osteosarcoma and the underlying molecular mechanisms in an orthotopic mouse model. Expression and phosphorylation of GSK-3β in osteosarcoma and normal osteoblast cell lines was examined, together with efficacy of GSK-3β inhibition on cell survival, proliferation and apoptosis and on the growth of orthotopically-transplanted human osteosarcoma in nude mice. We also investigated changes in expression, phosphorylation and co-transcriptional activity of β-catenin in osteosarcoma cells following GSK-3β inhibition. Expression of the active form of GSK- 3β (tyrosine 216-phosphorylated) was higher in osteosarcoma than osteoblast cells. Inhibition of GSK-3β activity by pharmacological inhibitors or of its expression by RNA interference suppressed proliferation of osteosarcoma cells and induced apoptosis. Treatment with GSK-3β-specific inhibitors attenuated the growth of orthotopic osteosaroma in mice. Inhibition of GSK-3β reduced phosphorylation at GSK- 3β-phospho-acceptor sites in β-catenin and increased β-catenin expression, nuclear localization and co-transcriptional activity. These results suggest the efficacy of GSK-3β inhibitors is associated with activation of β-catenin, a putative tumor suppressor in bone and soft tissue sarcoma and an important component of osteogenesis. Our study thereby demonstrates a critical role for GSK-3β in sustaining survival and proliferation of osteosarcoma cells, and identifies this kinase as a potential therapeutic target against osteosarcoma. PMID:27780915

  9. Glycogen synthase kinase 3 regulates photic signaling in the suprachiasmatic nucleus.

    PubMed

    Paul, Jodi R; McKeown, Alex S; Davis, Jennifer A; Totsch, Stacie K; Mintz, Eric M; Kraft, Timothy W; Cowell, Rita M; Gamble, Karen L

    2017-04-01

    Glycogen synthase kinase 3 (GSK3) is a serine-threonine kinase that regulates mammalian circadian rhythms at the behavioral, molecular and neurophysiological levels. In the central circadian pacemaker, the suprachiasmatic nucleus (SCN), inhibitory phosphorylation of GSK3 exhibits a rhythm across the 24 h day. We have recently shown that GSK3 is capable of influencing both the molecular clock and SCN neuronal activity rhythms. However, it is not known whether GSK3 regulates the response to environmental cues such as light. The goal of this study was to test the hypothesis that GSK3 activation mediates light-induced SCN excitability and photic entrainment. Immunofluorescence staining in the SCN of mice showed that late-night light exposure significantly increased GSK3 activity (decreased pGSK3β levels) 30-60 min after the light-pulse. In addition, pharmacological inhibition of GSK3 blocked the expected light-induced excitability in SCN neurons; however, this effect was not associated with changes in resting membrane potential or input resistance. Behaviorally, mice with constitutively active GSK3 (GSK3-KI) re-entrained to a 6-h phase advance in the light-dark cycle in significantly fewer days than WT control animals. Furthermore, the behavioral and SCN neuronal activity of GSK3-KI mice was phase-advanced compared to WT, in both normal and light-exposed conditions. Finally, GSK3-KI mice exhibited normal negative-masking behavior and electroretinographic responses to light, suggesting that the enhanced photic entrainment is not due to an overall increased sensitivity to light in these animals. Taken together, these results provide strong evidence that GSK3 activation contributes to light-induced phase-resetting at both the neurophysiological and behavioral levels. © 2017 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

  10. Modulation of glycogen synthase kinase-3β following TRAIL combinatorial treatment in cancer cells

    PubMed Central

    Santha, Sreevidya; Davaakhuu, Gantulga; Basu, Aninda; Ke, Rong; Das, Subhasis; Rana, Ajay; Rana, Basabi

    2016-01-01

    Glycogen Synthase Kinase-3β (GSK3β) is a serine/threonine kinase, known to regulate various cellular processes including proliferation, differentiation, survival, apoptosis as well as TRAIL-resistance. Thus pathways that can modulate GSK3β axis are important targets for cancer drug development. Our earlier studies have shown that combinatorial treatment with Troglitazone (TZD) and TRAIL can induce apoptosis in TRAIL-resistant cancer cells. The current studies were undertaken to investigate whether GSK3β pathway was modulated during this apoptosis. Our results indicated an increase in inhibitory GSK3βSer9 phosphorylation during apoptosis, mediated via AKT. At a later time, however, TZD alone and TRAIL-TZD combination produced a dramatic reduction of GSK3β expression, which was abolished by cycloheximide. Luciferase assays with GSK3β-luc promoter reporter showed that TZD can effectively antagonize GSK3β promoter activity. Since TZD is a ligand for transcription factor PPARγ and can activate AMPK, we determined their roles on antagonism of GSK3β. Knockdown of PPARγ was unable to restore GSK3β expression or antagonize GSK3βSer9 phosphorylation. Although pretreatment with Compound C (pharmacological inhibitor of AMPK) partially rescued GSK3β expression, knockdown of AMPKα1 or α2 alone or in combination were ineffective. These studies suggested a novel PPARγ-AMPK-independent mechanism of targeting GSK3β by TZD, elucidation of which might provide newer insights to improve our understanding of TRAIL-resistance. PMID:27602497

  11. Glycogen synthase kinase-3β is a pivotal mediator of cancer invasion and resistance to therapy.

    PubMed

    Domoto, Takahiro; Pyko, Ilya V; Furuta, Takuya; Miyashita, Katsuyoshi; Uehara, Masahiro; Shimasaki, Takeo; Nakada, Mitsutoshi; Minamoto, Toshinari

    2016-10-01

    Tumor cell invasion and resistance to therapy are the most intractable biological characteristics of cancer and, therefore, the most challenging for current cancer research and treatment paradigms. Refractory cancers, including pancreatic cancer and glioblastoma, show an inextricable association between the highly invasive behavior of tumor cells and their resistance to chemotherapy, radiotherapy and targeted therapies. These aggressive properties of cancer share distinct cellular pathways that are connected to each other by several molecular hubs. There is increasing evidence to show that glycogen synthase kinase (GSK)-3β is aberrantly activated in various cancer types and this has emerged as a potential therapeutic target. In many but not all cancer types, aberrant GSK3β sustains the survival, immortalization, proliferation and invasion of tumor cells, while also rendering them insensitive or resistant to chemotherapeutic agents and radiation. Here we review studies that describe associations between therapeutic stimuli/resistance and the induction of pro-invasive phenotypes in various cancer types. Such cancers are largely responsive to treatment that targets GSK3β. This review focuses on the role of GSK3β as a molecular hub that connects pathways responsible for tumor invasion and resistance to therapy, thus highlighting its potential as a major cancer therapeutic target. We also discuss the putative involvement of GSK3β in determining tumor cell stemness that underpins both tumor invasion and therapy resistance, leading to intractable and refractory cancer with dismal patient outcomes. © 2016 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

  12. Inhibition of glycogen synthase kinase 3 increased subventricular zone stem cells proliferation.

    PubMed

    Pachenari, Narges; Kiani, Sahar; Javan, Mohammad

    2017-09-01

    The effects of Wnt signaling modifiers on cell proliferation, seem to be cell specific. Enhancing the proliferation of subventricular zone (SVZ) progenitors has been in the focus of research in recent years. Here we investigate the effect of CHIR99021, a Glycogen Synthase Kinase 3 (GSk-3) inhibitor, on SVZ progenitor's proliferation both in vivo and in vitro. Neural stem cells were extracted from the adult C57bl/6 by mincing and trypsin treatment followed by culturing in specific medium. Sphere cells formed within about 7-10days and were characterized by immunostaining. Number of spheres and their size was assessed following exposure to different concentration of CHIR99021 or vehicle. For in vivo studies, animals received intracerebroventricular (i.c.v.) injection of CHIR99021 or vehicle for four days. A subgroup of animals, after 4days treatment with CHIR99021 received intranasal kainic acid to induce local neurodegeneration in CA3 area of hippocampus. Inhibition of GSk-3 by CHIR99021 increased neural progenitor proliferation and the effect of CHIR99021 was long lasting so that the treated cells showed higher proliferation even after CHIR99021 removal. In vivo administration of CHIR99021 increased the number of neural progenitors at the rims of lateral ventricles especially when the treatment was followed by kainic acid administration which induces neural insult. Results showed that direct administration of CHIR99021 into the culture medium or animal brain increased the number of SVZ progenitors, especially when a neural insult was induced in the hippocampus. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  13. 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

  14. Glycogen synthase kinase-3: a potential preventive target for prostate cancer management.

    PubMed

    Li, Benyi; Thrasher, James Brantley; Terranova, Paul

    2015-11-01

    Prostate cancers are the frequently diagnosed cancers in men, and patients with metastatic disease only have 28% chance for 5-year survival. Patients with low-risk tumors are subjected to active surveillance, whereas high-risk cases are actively treated. Unfortunately, there is no cure for patients with late-stage disease. Glycogen synthase kinase-3 (GSK-3, α and β) is a protein serine/threonine kinase and has diverse cellular functions and numerous substrates. We sought to summarize all the studies done with GSK-3 in prostate cancers and to provide a prospective direction for future work. A comprehensive search of the literature on the electronic databases PubMed was conducted for the subject terms of GSK-3 and prostate cancer. Gene mutation and expression information was extracted from Oncomine and COSMIC databases. Case reports were not included. Accumulating evidence indicates that GSK-3α is mainly expressed in low-risk prostate cancers and is related to hormone-dependent androgen receptor (AR)-mediated gene expression, whereas GSK-3β is mainly expressed in high-risk prostate cancers and is related to hormone-independent AR-mediated gene expression. GSK-3 has been demonstrated as a positive regulator in AR transactivation and prostate cancer growth independent of the Wnt/β-catenin pathway. Different types of GSK-3inhibitors including lithium show promising results in suppressing tumor growth in different animal models of prostate cancer. Importantly, clinical use of lithium is associated with reduced cancer incidence in psychiatric patients. Taken together, GSK-3 inhibition might be implicated in prostate cancer management as a preventive treatment. Copyright © 2015 Elsevier Inc. All rights reserved.

  15. Glycogen synthase kinase 3 is a potential drug target for African trypanosomiasis therapy.

    PubMed

    Ojo, Kayode K; Gillespie, J Robert; Riechers, Aaron J; Napuli, Alberto J; Verlinde, Christophe L M J; Buckner, Frederick S; Gelb, Michael H; Domostoj, Mathias M; Wells, Susan J; Scheer, Alexander; Wells, Timothy N C; Van Voorhis, Wesley C

    2008-10-01

    Development of a safe, effective, and inexpensive therapy for African trypanosomiasis is an urgent priority. In this study, we evaluated the validity of Trypanosoma brucei glycogen synthase kinase 3 (GSK-3) as a potential drug target. Interference with the RNA of either of two GSK-3 homologues in bloodstream-form T. brucei parasites led to growth arrest and altered parasite morphology, demonstrating their requirement for cell survival. Since the growth arrest after RNA interference appeared to be more profound for T. brucei GSK-3 "short" (Tb10.161.3140) than for T. brucei GSK-3 "long" (Tb927.7.2420), we focused on T. brucei GSK-3 short for further studies. T. brucei GSK-3 short with an N-terminal maltose-binding protein fusion was cloned, expressed, and purified in a functional form. The potency of a GSK-3-focused inhibitor library against the recombinant enzyme of T. brucei GSK-3 short, as well as bloodstream-form parasites, was evaluated with the aim of determining if compounds that inhibit enzyme activity could also block the parasites' growth and proliferation. Among the compounds active against the cell, there was an excellent correlation between activity inhibiting the T. brucei GSK-3 short enzyme and the inhibition of T. brucei growth. Thus, there is reasonable genetic and chemical validation of GSK-3 short as a drug target for T. brucei. Finally, selective inhibition may be required for therapy targeting the GSK-3 enzyme, and a molecular model of the T. brucei GSK-3 short enzyme suggests that compounds that selectively inhibit T. brucei GSK-3 short over the human GSK-3 enzymes can be found.

  16. Disruption of glycogen synthase kinase-3-beta activity leads to abnormalities in physiological measures in mice.

    PubMed

    Ahnaou, A; Drinkenburg, W H I M

    2011-08-01

    Dysregulation of glycogen synthase kinase-3-beta (GSK-3β) signaling pathways is thought to underlie the pathophysiology of mood disorders. In order to demonstrate that the loss of normal GSK-3β activity results in disturbances of physiological measures, we attempted to determine whether sleep-wake architecture, circadian rhythms of core body temperature and activity were altered in transgenic mice overexpressing GSK-3β activity specifically in the brain. Cortical electroencephalographic activity, body temperature (BT) and body locomotor activity (LMA) were continuously monitored using a biopotential telemetry probe. Normal circadian patterns were maintained for different measurements in both genotypes. No differences were found in total time spent asleep and waking over the 24-h recording session. However, transgenic animals overexpressing GSK-3β showed alteration in sleep continuity characterized by an increases in number of non rapid eye movement (NREM) sleep episodes (GSK-3β, 227.2 ± 1.7 vs. WT, 172.6 ± 1.4, p < 0.05) and decreases in mean episode duration (GSK-3β, 3.0 ± 0.1 vs. WT, 4.4 ± 0.2, p < 0.05). Additionally, transgenic animals exhibited marked enhancement of basal LMA and BT levels during the first part of the dark period, under both light-dark and free running dark-dark circadian cycles. Our findings indicate that transgenic mice overexpressing GSK-3β activity exhibit severe fragmentation of sleep-wake cycle during both the light and dark periods, without showing deviancy in total durations of vigilance states. The results strongly suggest that GSK-3β activity is elemental for the maintenance of circadian motor behavior levels required for proper regulation of BT and sleep-wake organization.

  17. The Antimalarial Effect of Curcumin Is Mediated by the Inhibition of Glycogen Synthase Kinase-3β.

    PubMed

    Ali, Amatul Hamizah; Sudi, Suhaini; Basir, Rusliza; Embi, Noor; Sidek, Hasidah Mohd

    2017-02-01

    Curcumin, a bioactive compound in Curcuma longa, exhibits various pharmacological activities, including antimalarial effects. In silico docking simulation studies suggest that curcumin possesses glycogen synthase kinase-3β (GSK3β)-inhibitory properties. The involvement of GSK3 in the antimalarial effects in vivo is yet to be demonstrated. In this study, we aimed to evaluate whether the antimalarial effects of curcumin involve phosphorylation of host GSK3β. Intraperitoneal administration of curcumin into Plasmodium berghei NK65-infected mice resulted in dose-dependent chemosuppression of parasitemia development. At the highest dose tested (30 mg/kg body weight), both therapeutic and prophylactic administrations of curcumin resulted in suppression exceeding 50% and improved median survival time of infected mice compared to control. Western analysis revealed a 5.5-fold (therapeutic group) and 1.8-fold (prophylactic group) increase in phosphorylation of Ser 9 GSK3β and 1.6-fold (therapeutic group) and 1.7-fold (prophylactic group) increase in Ser 473 Akt in liver of curcumin-treated infected animals. Following P. berghei infection, levels of pro- and anti-inflammatory cytokines, tumor necrosis factor (TNF)-α, interferon (IFN)-γ, interleukin (IL)-10, and IL-4 were elevated by 7.5-, 35.0-, 33.0-, and 2.2-fold, respectively. Curcumin treatment (therapeutic) caused a significant decrease (by 6.0- and 2.0-fold, respectively) in serum TNF-α and IFN-γ level, while IL-10 and IL-4 were elevated (by 1.4- and 1.8-fold). Findings from the present study demonstrate for the first time that the antimalarial action of curcumin involved inhibition of GSK3β.

  18. 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

  19. Small molecules targeting glycogen synthase kinase 3 as potential drug candidates for the treatment of retinitis pigmentosa.

    PubMed

    Marchena, Miguel; Villarejo-Zori, Beatriz; Zaldivar-Diez, Josefa; Palomo, Valle; Gil, Carmen; Hernández-Sánchez, Catalina; Martínez, Ana; de la Rosa, Enrique J

    2017-12-01

    Retinitis pigmentosa (RP) is an inherited retinal dystrophy that courses with progressive degeneration of retinal tissue and loss of vision. Currently, RP is an unpreventable, incurable condition. We propose glycogen synthase kinase 3 (GSK-3) inhibitors as potential leads for retinal cell neuroprotection, since the retina is also a part of the central nervous system and GSK-3 inhibitors are potent neuroprotectant agents. Using a chemical genetic approach, diverse small molecules with different potency and binding mode to GSK-3 have been used to validate and confirm GSK-3 as a pharmacological target for RP. Moreover, this medicinal chemistry approach has provided new leads for the future disease-modifying treatment of RP.

  20. Glycogen Synthase Kinase-3β, NF-κB Signaling, and Tumorigenesis of Human Osteosarcoma

    PubMed Central

    Tang, Qing-Lian; Xie, Xian-Biao; Wang, Jin; Chen, Qiong; Han, An-Jia; Zou, Chang-Ye; Yin, Jun-Qiang; Liu, Da-Wei; Liang, Yi; Zhao, Zhi-Qiang; Yong, Bi-Cheng; Zhang, Ru-Hua; Feng, Qi-Sheng; Deng, Wu-Guo; Zhu, Xiao-Feng; Zhou, Binhua P.; Zeng, Yi-Xin

    2012-01-01

    Background Glycogen synthase kinase-3β (GSK-3β), a serine/threonine protein kinase, may function as a tumor suppressor or an oncogene, depending on the tumor type. We sought to determine the biological function of GSK-3β in osteosarcoma, a rare pediatric cancer for which the identification of new therapeutic targets is urgent. Methods We used cell viability assays, colony formation assays, and apoptosis assays to analyze the effects of altered GSK-3β expression in U2OS, MG63, SAOS2, U2OS/MTX300, and ZOS osteosarcoma cell lines. Nude mice (n = 5–8 mice per group) were injected with U2OS/MTX300, and ZOS cells to assess the role of GSK-3β in osteosarcoma growth in vivo and to evaluate the effects of inhibitors and/or anticancer drugs on tumor growth. We used an antibody array, polymerase chain reaction, western blotting, and a luciferase reporter assay to establish the effect of GSK-3β inhibition on the nuclear factor-κB (NF-κB) pathway. Immunochemistry was performed on primary tumor specimens from osteosarcoma patients (n = 74) to determine the relationship of GSK-3β activity with overall survival. Results Osteosarcoma cells with low levels of inactive p-Ser9-GSK-3β formed colonies in vitro and tumors in vivo more readily than cells with higher levels and cells in which GSK-3β had been silenced formed fewer colonies and smaller tumors than parental cells. Silencing or pharmacological inhibition of GSK-3β resulted in apoptosis of osteosarcoma cells. Inhibition of GSK-3β resulted in inhibition of the NF-κB pathway and reduction of NF-κB-mediated transcription. Combination treatments with GSK-3β inhibitors, NF-κB inhibitors, and chemotherapy drugs increased the effectiveness of chemotherapy drugs in vitro and in vivo. Patients whose osteosarcoma specimens had hyperactive GSK-3β, and nuclear NF-κB had a shorter median overall survival time (49.2 months) compared with patients whose tumors had inactive GSK-3β and NF-κB (109.2 months). Conclusion GSK

  1. Identification and characterization of two wheat Glycogen Synthase Kinase 3/ SHAGGY-like kinases

    PubMed Central

    2013-01-01

    Background Plant Glycogen Synthase Kinase 3/ SHAGGY-like kinases (GSKs) have been implicated in numerous biological processes ranging from embryonic, flower, stomata development to stress and wound responses. They are key regulators of brassinosteroid signaling and are also involved in the cross-talk between auxin and brassinosteroid pathways. In contrast to the human genome that contains two genes, plant GSKs are encoded by a multigene family. Little is known about Liliopsida resp. Poaceae in comparison to Brassicaceae GSKs. Here, we report the identification and structural characterization of two GSK homologs named TaSK1 and TaSK2 in the hexaploid wheat genome as well as a widespread phylogenetic analysis of land plant GSKs. Results Genomic and cDNA sequence alignments as well as chromosome localization using nullisomic-tetrasomic lines provided strong evidence for three expressed gene copies located on homoeolog chromosomes for TaSK1 as well as for TaSK2. Predicted proteins displayed a clear GSK signature. In vitro kinase assays showed that TaSK1 and TaSK2 possessed kinase activity. A phylogenetic analysis of land plant GSKs indicated that TaSK1 and TaSK2 belong to clade II of plant GSKs, the Arabidopsis members of which are all involved in Brassinosteroid signaling. Based on a single ancestral gene in the last common ancestor of all land plants, paralogs were acquired and retained through paleopolyploidization events, resulting in six to eight genes in angiosperms. More recent duplication events have increased the number up to ten in some lineages. Conclusions To account for plant diversity in terms of functionality, morphology and development, attention has to be devoted to Liliopsida resp Poaceae GSKs in addition to Arabidopsis GSKs. In this study, molecular characterization, chromosome localization, kinase activity test and phylogenetic analysis (1) clarified the homologous/paralogous versus homoeologous status of TaSK sequences, (2) pointed out their

  2. Glycogen synthase kinase-3β in patients with bipolar I disorder: results from a prospective study.

    PubMed

    Jacoby, Anne S; Munkholm, Klaus; Vinberg, Maj; Joaquim, Helena Gp; Talib, Leda L; Gattaz, Wagner F; Kessing, Lars V

    2016-06-01

    The enzyme glycogen synthase kinase-3β (GSK3β) is involved in the mechanisms of action of lithium and may play a role in relation to affective states in bipolar disorder. The objectives of the present study were to compare the activity of GSK-3β (measured as levels of phosphorylated GSK-3β [p-GSK-3β]) between patients with bipolar disorder in the euthymic state and healthy control subjects, and to investigate whether GSK-3β activity varies with affective states in patients with bipolar I disorder. In a prospective 6-12-month follow-up study, we investigated state-specific, intraindividual alterations in the activity of GSK-3β in 60 patients with bipolar I disorder with an acute severe manic index episode and in subsequent euthymic, depressive and manic states and compared this with repeated measurements in healthy control subjects. Data were analyzed using linear mixed-effects models. From baseline to the end of follow-up, blood samples were drawn from the 60 patients during 181 affective states, comprising 60 manic, 11 mixed, 23 depressive, and 87 states of euthymia. A total of 69 blood samples were drawn from 35 healthy control subjects, with two samples from the same subject taken three months apart. In mixed-model analysis, p-GSK-3β was decreased in the euthymic state of subjects with bipolar disorder compared with healthy control subjects (b=0.63, 95% confidence interval [CI]: 0.42-0.96, P=.03). In addition, p-GSK-3β varied with affective states, being increased in depressive (b=1.68, 95% CI: 1.08-2.62, P=.02) and mixed (b=2.07, 95% CI: 1.12-3.84, P=.02) states but not in mania compared with euthymia. The activity of GSK-3β is altered in euthymic bipolar disorder compared with healthy control subjects and varies with affective states. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  3. An acute increase in fructose concentration increases hepatic glucose-6-phosphatase mRNA via mechanisms that are independent of glycogen synthase kinase-3 in rats.

    PubMed

    Wei, Yuren; Bizeau, Michael E; Pagliassotti, Michael J

    2004-03-01

    It appears that low amounts of fructose improve, whereas increased concentrations impair glucose tolerance and hepatic glucose metabolism. In this study, we compared directly the effects of low vs. high portal vein fructose concentrations on hepatic glucose metabolism in rats, using glucose-6-phosphatase gene expression as an endpoint. In the control group (C; n = 7), pancreatic clamps were performed using somatostatin and replacement of insulin such that basal glucose levels were maintained. In the experimental groups (n = 8/group), hyperglycemic, hyperinsulinemic pancreatic clamps were performed in which glucose (G) or glucose + fructose was infused into a jejunal vein. Fructose was infused to achieve either low (F1; <0.3 mmol/L) or high (F2; >1.0 mmol/L) portal vein concentrations. Total sugar load to the liver was equalized among the 3 experimental groups. Compared with C, liver glucose-6-phosphatase catalytic subunit mRNA was reduced by approximately 55% in G and F1, whereas it was increased approximately 180% in F2. F2 did not differentially affect glucose-6-phosphate translocase or phosphoenolpyruvate carboxykinase mRNA levels in liver, nor kidney glucose-6-phosphatase catalytic subunit mRNA. Livers from the F2 group were characterized by an accumulation of pentose phosphate intermediates and reduced phosphorylation of glycogen synthase kinase-3 (active form). However, in separate studies (n = 5/group), the infusion of a glycogen synthase kinase-3 inhibitor did not prevent the effects of F2 on glucose-6-phosphatase gene expression. We hypothesize that elevated fructose concentrations, similar to levels achieved after ingestion of sucrose- or fructose-enriched meals, initiate signals within the liver that elicit selective changes in hepatic gene expression.

  4. 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

    2012-02-03

    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.

  5. 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.

  6. 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.

  7. Impaired glycogen synthesis causes metabolic overflow reactions and affects stress responses in the cyanobacterium Synechocystis sp. PCC 6803.

    PubMed

    Gründel, Marianne; Scheunemann, Ramon; Lockau, Wolfgang; Zilliges, Yvonne

    2012-12-01

    The biosynthesis of glycogen or starch is one of the main strategies developed by living organisms for the intracellular storage of carbon and energy. In phototrophic organisms, such polyglucans accumulate due to carbon fixation during photosynthesis and are used to provide maintenance energy for cell integrity, function and viability in dark periods. Moreover, it is assumed that glycogen enables cyanobacteria to cope with transient starvation conditions, as observed in most micro-organisms. Here, glycogen accumulates when an appropriate carbon source is available in sufficient amounts but growth is inhibited by lack of other nutrients. In this study, the role of glycogen in energy and carbon metabolism of phototrophic cyanobacteria was first analysed via a comparative physiological and metabolic characterization of knockout mutants defective in glycogen synthesis. We first proved the role of glycogen as a respiratory substrate in periods of darkness, the role of glycogen as a reserve to survive starvation periods such as nitrogen depletion and the role of glycogen synthesis as an ameliorator of carbon excess conditions in the model organism Synechocystis sp. PCC 6803. We provide striking new insights into the complex carbon and nitrogen metabolism of non-diazotrophic cyanobacteria: a phenotype of sensitivity to photomixotrophic conditions and of reduced glucose uptake, a non-bleaching phenotype based on an impaired acclimation response to nitrogen depletion and furthermore a phenotype of energy spilling. This study shows that the analysis of deficiencies in glycogen metabolism is a valuable tool for the identification of metabolic regulatory principles and signals.

  8. Glycogen Metabolic Genes Are Involved in Trehalose-6-Phosphate Synthase-Mediated Regulation of Pathogenicity by the Rice Blast Fungus Magnaporthe oryzae

    PubMed Central

    Wilson, Richard A.; Wang, Zheng-Yi; Kershaw, Michael J.; Talbot, Nicholas J.

    2013-01-01

    The filamentous fungus Magnaporthe oryzae is the causal agent of rice blast disease. Here we show that glycogen metabolic genes play an important role in plant infection by M. oryzae. Targeted deletion of AGL1 and GPH1, which encode amyloglucosidase and glycogen phosphorylase, respectively, prevented mobilisation of glycogen stores during appressorium development and caused a significant reduction in the ability of M. oryzae to cause rice blast disease. By contrast, targeted mutation of GSN1, which encodes glycogen synthase, significantly reduced the synthesis of intracellular glycogen, but had no effect on fungal pathogenicity. We found that loss of AGL1 and GPH1 led to a reduction in expression of TPS1 and TPS3, which encode components of the trehalose-6-phosphate synthase complex, that acts as a genetic switch in M. oryzae. Tps1 responds to glucose-6-phosphate levels and the balance of NADP/NADPH to regulate virulence-associated gene expression, in association with Nmr transcriptional inhibitors. We show that deletion of the NMR3 transcriptional inhibitor gene partially restores virulence to a Δagl1Δgph1 mutant, suggesting that glycogen metabolic genes are necessary for operation of the NADPH-dependent genetic switch in M. oryzae. PMID:24098112

  9. Inhibition of hippocampal long-term potentiation by high-fat diets: is it related to an effect of palmitic acid involving glycogen synthase kinase-3?

    PubMed

    Contreras, Ana; Del Rio, Danila; Martínez, Ana; Gil, Carmen; Morales, Lidia; Ruiz-Gayo, Mariano; Del Olmo, Nuria

    2017-04-12

    High-fat diets (HFD) impair hippocampal-dependent learning and memory and produce important changes in synaptic transmission by enhancing glutamate uptake, decreasing synaptic efficacy, and inhibiting plasticity mechanisms such as N-methyl-D-aspartate-mediated long-term depression (LTD) within the hippocampus. Adolescent animals seem to be particularly susceptible to the detrimental effect of HFD as dietary treatments carried out between weaning and early adulthood are much more efficient in terms of hippocampal damage that those carried out during the adult period. As palmitic acid is the most abundant saturated fatty acid in HFD, its effect on hippocampal function needs to be studied. However, glycogen synthase kinase-3 (GSK-3), a pleiotropic enzyme highly expressed in the central nervous system, modulates both hippocampal long-term potentiation (LTP) and LTD, and has been implicated in neurological disorders including Alzheimer's disease. In this study, we have characterized in mice hippocampus the effect of (i) a 48 h HFD intervention and (ii) in-vitro palmitic acid, as well as the possible involvement of GSK-3 in the above-mentioned plasticity mechanisms. Our results show that both 48 h HFD and palmitic acid inhibit LTP in hippocampal slices, whereas no effect on LTD was observed. Moreover, tideglusib, an ATP-noncompetitive inhibitor of GSK-3, induced hippocampal LTP and partially reversed the impairment of LTP induced by palmitic acid.

  10. Probing the PI3K/Akt/mTor pathway using 31P-NMR spectroscopy: routes to glycogen synthase kinase 3

    PubMed Central

    Phyu, Su M.; Tseng, Chih-Chung; Fleming, Ian N.; Smith, Tim A. D.

    2016-01-01

    Akt is an intracellular signalling pathway that serves as an essential link between cell surface receptors and cellular processes including proliferation, development and survival. The pathway has many downstream targets including glycogen synthase kinase3 which is a major regulatory kinase for cell cycle transit as well as controlling glycogen synthase activity. The Akt pathway is frequently up-regulated in cancer due to overexpression of receptors such as the epidermal growth factor receptor, or mutation of signalling pathway kinases resulting in inappropriate survival and proliferation. Consequently anticancer drugs have been developed that target this pathway. MDA-MB-468 breast and HCT8 colorectal cancer cells were treated with inhibitors including LY294002, MK2206, rapamycin, AZD8055 targeting key kinases in/associated with Akt pathway and the consistency of changes in 31P-NMR-detecatable metabolite content of tumour cells was examined. Treatment with the Akt inhibitor MK2206 reduced phosphocholine levels in MDA-MB-468 cells. Treatment with either the phosphoinositide-3-kinase inhibitor, LY294002 and pan-mTOR inhibitor, AZD8055 but not pan-Akt inhibitor MK2206 increased uridine-5′-diphosphate-hexose cell content which was suppressed by co-treatment with glycogen synthase kinase 3 inhibitor SB216763. This suggests that there is an Akt-independent link between phosphoinositol-3-kinase and glycogen synthase kinase3 and demonstrates the potential of 31P-NMR to probe intracellular signalling pathways. PMID:27811956

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

    PubMed

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

    2006-06-01

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

  12. Glycogen synthase kinase-3β regulates leucine-309 demethylation of protein phosphatase-2A via PPMT1 and PME-1.

    PubMed

    Yao, Xiu-Qing; Li, Xia-Chun; Zhang, Xiao-Xue; Yin, Yang-Yang; Liu, Bin; Luo, Dan-Ju; Wang, Qun; Wang, Jian-Zhi; Liu, Gong-Ping

    2012-07-30

    Protein phosphatase-2A (PP2A) activity is significantly suppressed in Alzheimer's disease. We have reported that glycogen synthase kinase-3β (GSK-3β) inhibits PP2A via upregulating the phosphorylation of PP2A catalytic subunit (PP2A(C)). Here we studied the effects of GSK-3β on the inhibitory demethylation of PP2A at leucine-309 (dmL309-PP2A(C)). We found that GSK-3β regulates dmL309-PP2A(C) level by regulating PME-1 and PPMT1. Knockdown of PME-1 or PPMT1 eliminated the effects of GSK-3β on PP2A(C). GSK-3 could negatively regulate PP2A regulatory subunit protein level. We conclude that GSK-3β can inhibit PP2A by increasing the inhibitory L309-demethylation involving upregulation of PME-1 and inhibition of PPMT1.

  13. Marine Fungi as Producers of Benzocoumarins, a New Class of Inhibitors of Glycogen-Synthase-Kinase 3β

    PubMed Central

    Wiese, Jutta; Imhoff, Johannes F.; Gulder, Tobias A. M.; Labes, Antje; Schmaljohann, Rolf

    2016-01-01

    The glycogen-synthase-kinase 3 (GSK-3) is an important target in drug discovery. This enzyme is involved in the signaling pathways of type 2 diabetes, neurological disorders, cancer, and other diseases. Therefore, inhibitors of GSK-3 are promising drug candidates for the treatment of a broad range of diseases. Here we report pannorin (1), alternariol (2), and alternariol-9-methylether (3) to be promising inhibitors of the isoform GSK-3β showing sub-μM IC50 values. The in vitro inhibition is in the range of the known highly active GSK-3β inhibitor TDZD-8. Compounds 1–3 have a highly oxygenated benzocoumarin core structure in common, which suggests that this may be a new structural feature for efficient GSK-3β inhibition. PMID:27801816

  14. Cyclin-Dependent kinase 5 targeting prevents β-Amyloid aggregation involving glycogen synthase kinase 3β and phosphatases.

    PubMed

    Castro-Alvarez, John Fredy; Uribe-Arias, Alejandro; Cardona-Gómez, Gloria Patricia

    2015-08-01

    Inappropriate activation of cyclin-dependent kinase 5 (CDK5) resulting from proteolytic release of the activator fragment p25 from the membrane contributes to the formation of neurofibrillary tangles, β-amyloid (βA) aggregation, and chronic neurodegeneration. At 18 months of age, 3× Tg-AD mice were sacrificed after either 3 weeks (short term) or 1 year (long term) of CDK5 knockdown. In short-term-treated animals, CDK5 knockdown reversed βA aggregation in the hippocampi via inhibitory phosphorylation of glycogen synthase kinase 3β Ser9 and activation of phosphatase PP2A. In long-term-treated animals, CDK5 knockdown induced a persistent reduction in CDK5 and prevented βA aggregation, but the effect on amyloid precursor protein processing was reduced, suggesting that yearly booster therapy would be required. These findings further validate CDK5 as a target for preventing or blocking amyloidosis in older transgenic mice.

  15. Marine Fungi as Producers of Benzocoumarins, a New Class of Inhibitors of Glycogen-Synthase-Kinase 3β.

    PubMed

    Wiese, Jutta; Imhoff, Johannes F; Gulder, Tobias A M; Labes, Antje; Schmaljohann, Rolf

    2016-10-28

    The glycogen-synthase-kinase 3 (GSK-3) is an important target in drug discovery. This enzyme is involved in the signaling pathways of type 2 diabetes, neurological disorders, cancer, and other diseases. Therefore, inhibitors of GSK-3 are promising drug candidates for the treatment of a broad range of diseases. Here we report pannorin (1), alternariol (2), and alternariol-9-methylether (3) to be promising inhibitors of the isoform GSK-3β showing sub-μM IC50 values. The in vitro inhibition is in the range of the known highly active GSK-3β inhibitor TDZD-8. Compounds 1-3 have a highly oxygenated benzocoumarin core structure in common, which suggests that this may be a new structural feature for efficient GSK-3β inhibition.

  16. Identification of lead small molecule inhibitors of glycogen synthase kinase-3 beta using a fragment-linking strategy.

    PubMed

    Kim, Jinhee; Moon, Yonghoon; Hong, Sungwoo

    2016-12-01

    Glycogen synthase kinase-3 beta (GSK3β) kinase serves as a promising therapeutic target for the treatment of various human diseases, such as diabetes, obesity, and Alzheimer's disease. In this study, we report lead GSK3β inhibitors identified using a fragment-linking strategy. Through the systematic exploration, a six-atom chain unit bearing the rigid double bond was found to be a suitable linker connecting two fragments, which enables favorable contacts with backbone groups of residues in the pockets. As a consequence, potent GSK3β inhibitor 9i was found with IC50 values of 19nM. The binding mode analysis indicates that the activities of the inhibitors appear to be achieved by the establishment of multiple hydrogen bonds and hydrophobic interactions in the ATP-binding site of GSK3β. The good biochemical potencies and structural uniqueness of the inhibitors support consideration in the further study to optimize the biological activity.

  17. 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.

  18. 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.

  19. Glycogen Synthase Kinase-3β (GSK-3β) and Nuclear Factor Kappa-B (NFKB) in Childhood Acute Lymphoblastic Leukemia.

    PubMed

    Tovar, Cristian Fabian Layton; Zerón, Hugo Mendieta; Romero, Maria Del Socorro Camarillo; Sánchez, Yanko V Fabila; Romero, Isidoro Tejocote

    2016-01-01

    Acute lymphocytic leukemia (ALL) is the most common hematologic malignancy in early childhood. In children with acute lymphoblastic leukemia (ALL), the activity of glycogen synthase kinase (GSK-3β) has been associated with changes in the transcriptional activity and expression of nuclear factor kappa beta (NFKB) in the mononuclear cells of bone marrow. The aim of the study was to determine the possible role of glycogen synthase kinase 3beta (GSK-3β) and nuclear factor kappa beta (NFKB) as prognostic variables in pediatric patients with ALL. This was a descriptive, transversal, and observational study. Bone marrow and blood samples were obtained from 30 children with newly-diagnosed ALL, who were seen at the Hematology-Oncology Service, Hospital para el Niño (HPN), Toluca, Mexico, from 2014‒2015. Anthropometric variables, clinical lab results, immunophenotype and cytogenetic abnormalities were registered. GSK-3β was evaluated through immunohistochemistry, and NFKB messenger RNA (mRNA) with real-time polymerase chain reaction (qPCR). The cases of ALL were classified into two groups of risk: high and habitual. Thirty patients were included in this study, with a mean age of 7.1 years (range 2‒13 years). Twenty-one were male and 9 female. Employing the morphological classification, 26 patients had type L1 ALL and the remaining 4 patients had type L2 ALL. Abnormal genes were found in 7 (23.33%) patients, ETV-RUNX1 in 3, followed by TCF3-PBX1 (two), STL1-TAL1 (one), and BCR-ABL1 (one). NFKB relative expression levels, in comparison to the GSK-3β immunohistochemistry results of the bone marrow samples, showed significant differences between positive and negative cases (p = 0.001) and between weak-positive and negative cases (p = 0.002). These results suggest that GSK-3β may be a prognostic biomarker in childhood ALL.

  20. Anti-obesity effects of 3-hydroxychromone derivative, a novel small-molecule inhibitor of glycogen synthase kinase-3.

    PubMed

    Lee, Sooho; Yang, Woo Kyeom; Song, Ji Ho; Ra, Young Min; Jeong, Jin-Hyun; Choe, Wonchae; Kang, Insug; Kim, Sung-Soo; Ha, Joohun

    2013-04-01

    Glycogen synthase kinase 3 (GSK-3) plays a central role in cellular energy metabolism, and dysregulation of GSK-3 activity is implicated in a variety of metabolic disorders, including obesity, type 2 diabetes, and cancer. Hence, GSK-3 has emerged as an attractive target molecule for the treatment of metabolic disorders. Therefore, this research focused on identification and characterization of a novel small-molecule GSK-3 inhibitor. Compound 1a, a structure based on 3-hydroxychromone bearing isothiazolidine-1,1-dione, was identified from chemical library as a highly potent GSK-3 inhibitor. An in vitro kinase assay utilizing a panel of kinases demonstrated that compound 1a strongly inhibits GSK-3β. The potential effects of compound 1a on the inactivation of GSK-3 were confirmed in human liver HepG2 and human embryonic kidney HEK293 cells. Stabilization of glycogen synthase and β-catenin, which are direct targets of GSK-3, by compound 1a was assessed in comparison with two other GSK-3 inhibitors: LiCl and SB-415286. In mouse 3T3-L1 preadipocytes, compound 1a markedly blocked adipocyte differentiation. Consistently, intraperitoneal administration of compound 1a to diet-induced obese mice significantly ameliorated their key symptoms such as body weight gain, increased adiposity, dyslipidemia, and hepatic steatosis due to the marked reduction of whole-body lipid level. In vitro and in vivo effects were accompanied by upregulation of β-catenin stability and downregulation of the expression of several critical genes related to lipid metabolism. From these results, it can be concluded that compound 1a, a novel small-molecule inhibitor of GSK-3, has potential as a new class of therapeutic agent for obesity treatment. Copyright © 2013 Elsevier Inc. All rights reserved.

  1. A New Muscle Glycogen Storage Disease Associated with Glycogenin-1 Deficiency

    PubMed Central

    Malfatti, Edoardo; Nilsson, Johanna; Hedberg-Oldfors, Carola; Hernandez-Lain, Aurelio; Michel, Fabrice; Dominguez-Gonzalez, Cristina; Viennet, Gabriel; Akman, H. Orhan; Kornblum, Cornelia; den Bergh, Peter Van; Romero, Norma B.; Engel, Andrew G.; DiMauro, Salvatore; Oldfors, Anders

    2015-01-01

    We describe a slowly progressive myopathy in 7 unrelated adult patients with storage of polyglucosan in muscle fibers. Genetic investigation revealed homozygous or compound heterozygous deleterious variants in the glycogenin-1 gene (GYG1). Most patients showed depletion of glycogenin-1 in skeletal muscle, whereas 1 showed presence of glycogenin-1 lacking the C-terminal that normally binds glycogen synthase. Our results indicate that either depletion of glycogenin-1 or impaired interaction with glycogen synthase underlies this new form of glycogen storage disease that differs from a previously reported patient with GYG1 mutations who showed profound glycogen depletion in skeletal muscle and accumulation of glycogenin-1. PMID:25272951

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

    PubMed

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

    2015-01-15

    IL-33, a relatively new member of the IL-1 cytokine family, plays a crucial role in allergic inflammation and acute lung injury. Long form ST2 (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. In this study, we demonstrate that glycogen synthase kinase 3β (GSK3β) regulates ST2L internalization and IL-33 signaling. IL-33 treatment induced ST2L internalization, and 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 (ST2L(S446A)) and phosphorylation site mutant (ST2L(S442A)) 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. Furthermore, 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.

  3. Expression pattern of ataxia telangiectasia mutated (ATM), p53, Akt, and glycogen synthase kinase-3β in the striatum of rats treated with 3-nitropropionic acid.

    PubMed

    Duran-Vilaregut, Joaquim; Manich, Gemma; Del Valle, Jaume; Camins, Antoni; Pallàs, Mercè; Vilaplana, Jordi; Pelegrí, Carme

    2012-09-01

    3-Nitropropionic acid (3-NPA) is a mitochondrial toxin used in the laboratory to replicate neurodegenerative conditions that are accompanied by degeneration of the caudate-putamen. 3-NPA induces depletion in ATP production, reactive oxygen species production, and secondary excitotoxicity mediated by activation of N-methyl-D-aspartate receptors that culminates in the triggering of cell death mechanisms, including apoptosis. We here examined by immunohistochemical methods whether cellular expression of phospho(Ser1981) -ataxia telangiectasia mutated (ATM), phospho(Ser15) -p53, phospho(Ser473) -Akt, and phospho(Ser9) -glycogen synthase kinase-3β (GSK3β), which are key signal molecules that play a critical role in regulating cellular processes related to cell survival and demise, were involved in the striatal neurodegeneration in the brains of rats treated with 3-NPA. Our results indicate that the toxin induced the activation of ATM and p53 only in astrocytes, and a role for these proteins in neuronal degeneration was ruled out. On the other hand, striatal neurons lost the active form of Akt as soon as they began to appear pyknotic, indicating impairment of the PI3K/Akt/GSK3 pathway in their degenerative process. The inactive form of GSK3β was detected extensively, mainly in the rim of the striatal lesions around degenerating neurons, which could be attributed to a cell death or cell survival response.

  4. Downregulation of SIRT1 signaling underlies hepatic autophagy impairment in glycogen storage disease type Ia.

    PubMed

    Cho, Jun-Ho; Kim, Goo-Young; Pan, Chi-Jiunn; Anduaga, Javier; Choi, Eui-Ju; Mansfield, Brian C; Chou, Janice Y

    2017-05-01

    A deficiency in glucose-6-phosphatase-α (G6Pase-α) in glycogen storage disease type Ia (GSD-Ia) leads to impaired glucose homeostasis and metabolic manifestations including hepatomegaly caused by increased glycogen and neutral fat accumulation. A recent report showed that G6Pase-α deficiency causes impairment in autophagy, a recycling process important for cellular metabolism. However, the molecular mechanism underlying defective autophagy is unclear. Here we show that in mice, liver-specific knockout of G6Pase-α (L-G6pc-/-) leads to downregulation of sirtuin 1 (SIRT1) signaling that activates autophagy via deacetylation of autophagy-related (ATG) proteins and forkhead box O (FoxO) family of transcriptional factors which transactivate autophagy genes. Consistently, defective autophagy in G6Pase-α-deficient liver is characterized by attenuated expressions of autophagy components, increased acetylation of ATG5 and ATG7, decreased conjugation of ATG5 and ATG12, and reduced autophagic flux. We further show that hepatic G6Pase-α deficiency results in activation of carbohydrate response element-binding protein, a lipogenic transcription factor, increased expression of peroxisome proliferator-activated receptor-γ (PPAR-γ), a lipid regulator, and suppressed expression of PPAR-α, a master regulator of fatty acid β-oxidation, all contributing to hepatic steatosis and downregulation of SIRT1 expression. An adenovirus vector-mediated increase in hepatic SIRT1 expression corrects autophagy defects but does not rectify metabolic abnormalities associated with G6Pase-α deficiency. Importantly, a recombinant adeno-associated virus (rAAV) vector-mediated restoration of hepatic G6Pase-α expression corrects metabolic abnormalities, restores SIRT1-FoxO signaling, and normalizes defective autophagy. Taken together, these data show that hepatic G6Pase-α deficiency-mediated down-regulation of SIRT1 signaling underlies defective hepatic autophagy in GSD-Ia.

  5. Glycogen synthase kinase 3 inhibition promotes lysosomal biogenesis and autophagic degradation of the amyloid-β precursor protein.

    PubMed

    Parr, Callum; Carzaniga, Raffaela; Gentleman, Steve M; Van Leuven, Fred; Walter, Jochen; Sastre, Magdalena

    2012-11-01

    Alzheimer's disease (AD) has been associated with altered activity of glycogen synthase kinase 3 (GSK3) isozymes, which are proposed to contribute to both neurofibrillary tangles and amyloid plaque formation. However, the molecular basis by which GSK3 affects the formation of Aβ remains unknown. Our aim was to identify the underlying mechanisms of GSK3-dependent effects on the processing of amyloid precursor protein (APP). For this purpose, N2a cells stably expressing APP carrying the Swedish mutation were treated with specific GSK3 inhibitors or transfected with GSK3α/β short interfering RNA. We show that inhibition of GSK3 leads to decreased expression of APP by enhancing its degradation via an increase in the number of lysosomes. This induction of the lysosomal/autophagy pathway was associated with nuclear translocation of transcription factor EB (TFEB), a master regulator of lysosomal biogenesis. Our data indicate that GSK3 inhibition reduces Aβ through an increase of the degradation of APP and its carboxy-terminal fragment (CTF) by activation of the lysosomal/autophagy pathway. These results suggest that an increased propensity toward autophagic/lysosomal alterations in AD patients could have consequences for neuronal function.

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

    PubMed

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

    2013-09-06

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

  7. Association of cellular localization of glycogen synthase kinase 3beta in the digestive tract with cancer development.

    PubMed

    Hirakawa, Hiroshi; Nakayama, Toshiyuki; Shibata, Kenichiro; Sekine, Ichiro

    2009-09-01

    Glycogen synthase kinase 3beta (GSK-3beta) is a multifunctional serine/threonine kinase involved in several signaling pathways. Recently, we reported the polarized localization of GSK-3beta on the apical membrane of normal colon epithelium. To investigate the functions of this molecule, we studied stomach and colorectal cancer tissues. In normal simple columnar epithelium, GSK-3beta was localized with tight junction-associated protein ZO-1 in a single line at the apical cell border. GSK-3beta and ZO-1 were localized in the apical regions of tubular adenocarcinoma, similar to their localization in normal epithelium; however, their localization was different at the invasive front of the cancer and was found to be associated with lymphatic invasion. In signet-ring cell carcinoma of the stomach, the expression of these proteins was reduced and dot-like expression was observed in each cell of the signet-ring cell carcinoma. We speculated that GSK-3beta is involved in glandular structure formation and that the non-apical localization of membrane-localized GSK-3beta in tubular adenocarcinoma is associated with cancer development.

  8. 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

  9. Phosphatidylinositol 3-Kinase (PI3K) Signaling via Glycogen Synthase Kinase-3 (Gsk-3) Regulates DNA Methylation of Imprinted Loci*

    PubMed Central

    Popkie, Anthony P.; Zeidner, Leigh C.; Albrecht, Ashley M.; D'Ippolito, Anthony; Eckardt, Sigrid; Newsom, David E.; Groden, Joanna; Doble, Bradley W.; Aronow, Bruce; McLaughlin, K. John; White, Peter; Phiel, Christopher J.

    2010-01-01

    Glycogen synthase kinase-3 (Gsk-3) isoforms, Gsk-3α and Gsk-3β, are constitutively active, largely inhibitory kinases involved in signal transduction. Underscoring their biological significance, altered Gsk-3 activity has been implicated in diabetes, Alzheimer disease, schizophrenia, and bipolar disorder. Here, we demonstrate that deletion of both Gsk-3α and Gsk-3β in mouse embryonic stem cells results in reduced expression of the de novo DNA methyltransferase Dnmt3a2, causing misexpression of the imprinted genes Igf2, H19, and Igf2r and hypomethylation of their corresponding imprinted control regions. Treatment of wild-type embryonic stem cells and neural stem cells with the Gsk-3 inhibitor, lithium, phenocopies the DNA hypomethylation at these imprinted loci. We show that inhibition of Gsk-3 by phosphatidylinositol 3-kinase (PI3K)-mediated activation of Akt also results in reduced DNA methylation at these imprinted loci. Finally, we find that N-Myc is a potent Gsk-3-dependent regulator of Dnmt3a2 expression. In summary, we have identified a signal transduction pathway that is capable of altering the DNA methylation of imprinted loci. PMID:21047779

  10. Functional Interactions between Heterologously Expressed Starch-Branching Enzymes of Maize and the Glycogen Synthases of Brewer's Yeast1

    PubMed Central

    Seo, Beom-seok; Kim, Seungtaek; Scott, M. Paul; Singletary, George W.; Wong, Kit-sum; James, Martha G.; Myers, Alan M.

    2002-01-01

    Starch-branching enzymes (SBEs) catalyze the formation of α(1→6) glycoside bonds in glucan polymers, thus, affecting the structure of amylopectin and starch granules. Two distinct classes of SBE are generally conserved in higher plants, although the specific role(s) of each isoform in determination of starch structure is not clearly understood. This study used a heterologous in vivo system to isolate the function of each of the three known SBE isoforms of maize (Zea mays) away from the other plant enzymes involved in starch biosynthesis. The ascomycete Brewer's yeast (Saccharomyces cerevisiae) was employed as the host species. All possible combinations of maize SBEs were expressed in the absence of the endogenous glucan-branching enzyme. Each maize SBE was functional in yeast cells, although SBEI had a significant effect only if SBEIIa and SBEIIb also were present. SBEI by itself did not support glucan accumulation, whereas SBEIIa and SBEIIb both functioned along with the native glycogen synthases (GSs) to produce significant quantities of α-glucan polymers. SBEIIa was phenotypically dominant to SBEIIb in terms of glucan structure. The specific branching enzyme present had a significant effect on the molecular weight of the product. From these data we suggest that SBEs and GSs work in a cyclically interdependent fashion, such that SBE action is needed for optimal GS activity; and GS, in turn, influences the further effects of SBE. Also, SBEIIa and SBEIIb appear to act before SBEI during polymer assembly in this heterologous system. PMID:11950968

  11. Regulation of autophagic cell death by glycogen synthase kinase-3β in adult hippocampal neural stem cells following insulin withdrawal.

    PubMed

    Ha, Shinwon; Ryu, Hye Young; Chung, Kyung Min; Baek, Seung-Hoon; Kim, Eun-Kyoung; Yu, Seong-Woon

    2015-05-19

    Neural stem cells (NSCs) hold great potential for the treatment of neurodegenerative diseases. However, programmed cell death (PCD) provoked by the harsh conditions evident in the diseased brain greatly undermines the potential of NSCs. Currently, the mechanisms of PCD that effect NSCs remain largely unknown. We have previously reported that hippocampal neural stem (HCN) cells derived from the adult rat brain undergo autopahgic cell death (ACD) following insulin withdrawal without hallmarks of apoptosis despite their normal apoptotic capabilities. In this study, we demonstrate that glycogen synthase kinase 3β (GSK-3β) induces ACD in insulin-deprived HCN cells. Both pharmacological and genetic inactivation of GSK-3β significantly decreased ACD, while activation of GSK-3β increased autophagic flux and caused more cell death without inducing apoptosis following insulin withdrawal. In contrast, knockdown of GSK-3α barely affected ACD, lending further support to the critical role of GSK-3β. Collectively, these data demonstrate that GSK-3β is a key regulator of ACD in HCN cells following insulin withdrawal. The absence of apoptotic indices in GSK-3β-induced cell death in insulin-deprived HCN cells corroborates the notion that HCN cell death following insulin withdrawal represents the genuine model of ACD in apoptosis-intact mammalian cells and identifies GSK-3β as a key negative effector of NSC survival downstream of insulin signaling.

  12. 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.

  13. Biological basis and clinical study of glycogen synthase kinase- 3β-targeted therapy by drug repositioning for glioblastoma

    PubMed Central

    Furuta, Takuya; Sabit, Hemragul; Dong, Yu; Miyashita, Katsuyoshi; Kinoshita, Masashi; Uchiyama, Naoyuki; Hayashi, Yasuhiko; Hayashi, Yutaka; Minamoto, Toshinari; Nakada, Mitsutoshi

    2017-01-01

    Background Glycogen synthase kinase (GSK)-3β has emerged as an appealing therapeutic target for glioblastoma (GBM). Here, we investigated the therapeutic effect of the current approved drugs against GBM via inhibition of GSK3β activity both, in experimental setting and in a clinical study for recurrent GBM patients by repositioning existent drugs in combination with temozolomide (TMZ). Materials and Methods Progression-free and overall survival rates were compared between patients with low or high expression of active GSK3β in the primary tumor. GBM cells and a mouse model were examined for the effects of GSK3β-inhibitory drugs, cimetidine, lithium, olanzapine, and valproate. The safety and efficacy of the cocktail of these drugs (CLOVA cocktail) in combination with TMZ were tested in the mouse model and in a clinical study for recurrent GBM patients. Results Activation of GSK3β in the tumor inversely correlated with patient survival as an independent prognostic factor. CLOVA cocktail significantly inhibited cell invasion and proliferation. The patients treated with CLOVA cocktail in combination with TMZ showed increased survival compared to the control group treated with TMZ alone. Conclusions Repositioning of the GSK3β-inhibitory drugs improved the prognosis of refractory GBM patients with active GSK3β in tumors. Combination of CLOVA cocktail and TMZ is a promising approach for recurrent GBM. PMID:28423558

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

    PubMed Central

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

    2010-01-01

    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. PMID:20617560

  15. Glycogen Synthase Kinase 3 influences cell motility and chemotaxis by regulating PI3K membrane localization in Dictyostelium.

    PubMed

    Sun, Tong; Kim, Bohye; Kim, Lou W

    2013-10-01

    Glycogen Synthase Kinase 3 (GSK3) is a multifunctional kinase involved in diverse cellular activities such as metabolism, differentiation, and morphogenesis. Recent studies showed that GSK3 in Dictyostelium affects chemotaxis via TorC2 pathway and Daydreamer. Now we report that GSK3 affects PI3K membrane localization, of which the mechanism has remained to be fully understood in Dictyostelium. The membrane localization domain (LD) of Phosphatidylinositol-3-kinase 1 (PI3K1) is phosphorylated on serine residues in a GSK3 dependent mechanism and PI3K1-LD exhibited biased membrane localization in gsk3(-) cells compared to the wild type cells. Furthermore, multiple GSK3-phosphorylation consensus sites exist in PI3K1-LD, of which phosphomimetic substitutions restored cAMP induced transient membrane localization of PI3K1-LD in gsk3(-) cells. Serine to alanine substitution mutants of PI3K1-LD, in contrast, displayed constitutive membrane localization in wild type cells. Biochemical analysis revealed that GSK3 dependent serine phosphorylation of PI3K1-LD is constitutive during the course of cAMP stimulation. Together, these data suggest that GSK3 dependent serine phosphorylation is a prerequisite for chemoattractant cAMP induced PI3K membrane localization.

  16. Up-regulation of insulin-like growth factor 2 by ketamine requires glycogen synthase kinase-3 inhibition.

    PubMed

    Grieco, Steven F; Cheng, Yuyan; Eldar-Finkelman, Hagit; Jope, Richard S; Beurel, Eléonore

    2017-01-04

    An antidepressant dose of the rapidly-acting ketamine inhibits glycogen synthase kinase-3 (GSK3) in mouse hippocampus, and this inhibition is required for the antidepressant effect of ketamine in learned helplessness depression-like behavior. Here we report that treatment with an antidepressant dose of ketamine (10mg/kg) increased expression of insulin-like growth factor 2 (IGF2) in mouse hippocampus, an effect that required ketamine-induced inhibition of GSK3. Ketamine also inhibited hippocampal GSK3 and increased expression of hippocampal IGF2 in mice when administered after the induction of learned helplessness. Treatment with the specific GSK3 inhibitor L803-mts was sufficient to up-regulate hippocampal IGF2 expression. Administration of IGF2 siRNA reduced ketamine's antidepressant effect in the learned helplessness paradigm. Mice subjected to the learned helplessness paradigm were separated into two groups, those that were resilient (non-depressed) and those that were susceptible (depressed). Non-depressed resilient mice displayed higher expression of IGF2 than susceptible mice. These results indicate that IGF2 contributes to ketamine's antidepressant effect and that IGF2 may confer resilience to depression-like behavior. Copyright © 2016 Elsevier Inc. All rights reserved.

  17. 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.

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

    PubMed

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

    2011-07-01

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

  19. Glycogen Synthase Kinase 3 influences cell motility and chemotaxis by regulating PI3K membrane localization in Dictyostelium

    PubMed Central

    Sun, Tong; Kim, Bohye; Kim, Lou W.

    2013-01-01

    Glycogen Synthase Kinase 3 (GSK3) is a multifunctional kinase involved in diverse cellular activities such as metabolism, differentiation, and morphogenesis. Recent studies showed that GSK3 in Dictyostelium affects chemotaxis via TorC2 pathway and Daydreamer. Now we report that GSK3 affects PI3K membrane localization, of which mechanism has remained to be fully understood in Dictyostelium. The membrane localization domain (LD) of Phosphatidylinositol-3-kinase 1 (PI3K1) is phosphorylated on serine residues in a GSK3 dependent mechanism and PI3K1-LD exhibited biased membrane localization in gsk3− cells compared to the wild type cells. Furthermore, multiple GSK3-phosphorylation consensus sites exist in PI3K1-LD, of which phosphomimetic substitutions restored cAMP induced transient membrane localization of PI3K1-LD in gsk3− cells. Serine to alanine substitution mutants of PI3K1-LD, in contrast, displayed constitutive membrane localization in wild type cells. Biochemical analysis revealed that GSK3 dependent serine phosphorylation of PI3K1-LD is constitutive during the course of cAMP stimulation. Together, these data suggest that GSK3 dependent serine phosphorylation is a prerequisite for chemoattractant cAMP induced PI3K membrane localization. PMID:24102085

  20. A role for Akt and glycogen synthase kinase-3 as integrators of dopamine and serotonin neurotransmission in mental health

    PubMed Central

    Beaulieu, Jean-Martin

    2012-01-01

    Mental illnesses, such as bipolar disorder, attention-deficit/hyperactivity disorder, depression and schizophrenia are a major public health concern worldwide. Several pharmacologic agents acting on monoamine neurotransmission are used for the management of these disorders. However, there is still little understanding of the ultimate molecular mechanisms responsible for the therapeutic effects of these drugs or their relations with disease etiology. Here I provide an overview of recent advances on the involvement of the signalling molecules Akt and glycogen synthase kinase-3 (GSK3) in the regulation of behaviour by the monoamine neurotransmitters dopamine (DA) and serotonin (5-HT). I examine the possible participation of these signalling molecules to the effects of antidepressants, lithium and antipsychotics, as well as their possible contribution to mental disorders. Regulation of Akt and GSK3 may constitute an important signalling hub in the subcellular integration of 5-HT and DA neurotransmission. It may also provide a link between the action of these neurotransmitters and gene products, like disrupted in schizophrenia 1 (DISC1) and neuregulin (NRG), that are associated with increased risk for mental disorders. However, changes in Akt and GSK3 signalling are not restricted to a single disorder, and their contribution to specific behavioural symptoms or therapeutic effects may be modulated by broader changes in biologic contexts or signalling landscapes. Understanding these interactions may provide a better understanding of mental illnesses, leading to better efficacy of new therapeutic approaches. PMID:21711983

  1. Elevated glycogen synthase kinase-3 activity in Fragile X mice: key metabolic regulator with evidence for treatment potential.

    PubMed

    Min, Wenzhong William; Yuskaitis, Christopher J; Yan, Qijiang; Sikorski, Christopher; Chen, Shengqiang; Jope, Richard S; Bauchwitz, Robert P

    2009-02-01

    Significant advances have been made in understanding the underlying defects of and developing potential treatments for Fragile X syndrome (FXS), the most common heritable mental retardation. It has been shown that neuronal metabotropic glutamate receptor 5 (mGluR5)-mediated signaling is affected in FX animal models, with consequent alterations in activity-dependent protein translation and synaptic spine functionality. We demonstrate here that a central metabolic regulatory enzyme, glycogen synthase kinase-3 (GSK3) is present in a form indicating elevated activity in several regions of the FX mouse brain. Furthermore, we show that selective GSK3 inhibitors, as well as lithium, are able to revert mutant phenotypes of the FX mouse. Lithium, in particular, remained effective with chronic administration, although its effects were reversible even when given from birth. The combination of an mGluR5 antagonist and GSK3 inhibitors was not additive. Instead, it was discovered that mGluR5 signaling and GSK3 activation in the FX mouse are coordinately elevated, with inhibition of mGluR5 leading to inhibition of GSK3. These findings raise the possibility that GSK3 is a fundamental and central component of FXS pathology, with a substantial treatment potential.

  2. Elevated glycogen synthase kinase-3 activity in Fragile X mice: Key metabolic regulator with evidence for treatment potential

    PubMed Central

    Min, Wenzhong William; Yuskaitis, Christopher J.; Yan, Qijiang; Sikorski, Christopher; Chen, Shengqiang; Jope, Richard S.; Bauchwitz, Robert P.

    2009-01-01

    Significant advances have been made in understanding the underlying defects of and developing potential treatments for Fragile X syndrome (FXS), the most common heritable mental retardation. It has been shown that neuronal metabotropic glutamate receptor 5 (mGluR5)-mediated signaling is affected in FX animal models, with consequent alterations in activity-dependent protein translation and synaptic spine functionality. We demonstrate here that a central metabolic regulatory enzyme, glycogen synthase kinase-3 (GSK3) is present in a form indicating elevated activity in several regions of the FX mouse brain. Furthermore, we show that selective GSK3 inhibitors, as well as lithium, are able to revert mutant phenotypes of the FX mouse. Lithium, in particular, remained effective with chronic administration, although its effects were reversible even when given from birth. The combination of an mGluR5 antagonist and GSK3 inhibitors was not additive. Instead, it was discovered that mGluR5 signaling and GSK3 activation in the FX mouse are coordinately elevated, with inhibition of mGluR5 leading to inhibition of GSK3. These findings raise the possibility that GSK3 is a fundamental and central component of FXS pathology, with a substantial treatment potential. PMID:18952114

  3. Wound healing activity and docking of glycogen-synthase-kinase-3-beta-protein with isolated triterpenoid lupeol in rats.

    PubMed

    Harish, B G; Krishna, V; Santosh Kumar, H S; Khadeer Ahamed, B M; Sharath, R; Kumara Swamy, H M

    2008-09-01

    A triterpene compound lupeol isolated from petroleum ether extract of leaves of Celastrus paniculatus was screened for wound healing activity (8 mg/ml of 0.2% sodium alginate gel) by excision, incision and dead space wound models on Swiss Albino rats (175-225 g). In lupeol treated groups wound healing activity was more significant (17.83+/-0.48) than the standard skin ointment nitrofurazone (18.33+/-0.42). Epithelialization of the incision wound was faster with a high rate of wound contraction (571.50+/-5.07) as compared with the control group. In dead space wound model also the weight of the granulation tissue of the lupeol treated animal was increased indicating increase of collagenation and absence of monocytes. The comparative docking of isolated lupeol molecule and standard drug nitrofurazone to glycogen synthase kinase 3-beta protein by Wnt signaling pathway also supported the wound healing property of lupeol. The activation domain of GSK3-beta consisted of Tyr216, with residues Asn64, Gly65, Ser66, Phe67, Gly68, Val70, Lys85, Leu132, Val135, Asp181 in the active pocket docked with lupeol at the torsional degree of freedom 0.5 units with Lamarckian genetic algorithm showed the inhibition constant of 1.38 x 10(-7). The inhibition constant of nitrofurazone was only 1.35 x 10(-4).

  4. Lithium Blocks the c-Jun Stress Response and Protects Neurons via Its Action on Glycogen Synthase Kinase 3

    PubMed Central

    Hongisto, Vesa; Smeds, Nina; Brecht, Stephan; Herdegen, Thomas; Courtney, Michael J.; Coffey, Eleanor T.

    2003-01-01

    Lithium has been used as an effective mood-stabilizing drug for the treatment of manic episodes and depression for 50 years. More recently, lithium has been found to protect neurons from death induced by a wide array of neurotoxic insults. However, the molecular basis for the prophylactic effects of lithium have remained obscure. A target of lithium, glycogen synthase kinase 3 (GSK-3), is implicated in neuronal death after trophic deprivation. The mechanism whereby GSK-3 exerts its neurotoxic effects is also unknown. Here we show that lithium blocks the canonical c-Jun apoptotic pathway in cerebellar granule neurons deprived of trophic support. This effect is mimicked by the structurally independent inhibitors of GSK-3, FRAT1, and indirubin. Like lithium, these prevent the stress induced c-Jun protein increase and subsequent apoptosis. These events are downstream of c-Jun transactivation, since GSK-3 inhibitors block neuronal death induced by constitutively active c-Jun (Ser/Thr→Asp) and FRAT1 expression inhibits AP1 reporter activity. Consistent with this, AP1-dependent expression of proapoptotic Bim requires GSK-3-like activity. These data suggest that a GSK-3-like kinase acts in tandem with c-Jun N-terminal kinase to coordinate the full execution of the c-Jun stress response and neuronal death in response to trophic deprivation. PMID:12917327

  5. 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

  6. 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

  7. Lithium Chloride Promotes Apoptosis in Human Leukemia NB4 Cells by Inhibiting Glycogen Synthase Kinase-3 Beta.

    PubMed

    Li, Liu; Song, Hao; Zhong, Liang; Yang, Rong; Yang, Xiao-Qun; Jiang, Kai-Ling; Liu, Bei-Zhong

    2015-01-01

    Acute promyelocytic leukemia (APL) is a subtype of acute myeloid leukemia (AML). With the application of all-trans retinoic acid (ATRA) and arsenic trioxide (ATO), APL becomes one of best prognosis of leukemia. However, ATRA and ATO are not effective against all APLs. Therefore, a new strategy for APL treatment is necessary. Here, we investigated whether lithium chloride (LiCl), a drug used for the treatment of mental illness, could promote apoptosis in human leukemia NB4 cells. We observed that treatment with LiCl significantly accelerated apoptosis in NB4 cells and led to cell cycle arrest at G2/M phase. Moreover, LiCl significantly increased the level of Ser9-phosphorylated glycogen synthase kinase 3β(p-GSK-3β), and decreased the level of Akt1 protein in a dose-dependent manner. In addition, LiCl inhibition of c-Myc also enhanced cell death with a concomitant increase in β-catnin. Taken together, these findings demonstrated that LiCl promoted apoptosis in NB4 cells through the Akt signaling pathway and that G2/M phase arrest was induced by increase of p-GSK-3β(S9).

  8. Glycogen synthase kinase-3 inhibition by 3-anilino-4-phenylmaleimides: insights from 3D-QSAR and docking

    NASA Astrophysics Data System (ADS)

    Prasanna, Sivaprakasam; Daga, Pankaj R.; Xie, Aihua; Doerksen, Robert J.

    2009-02-01

    Glycogen synthase kinase-3, a serine/threonine kinase, has been implicated in a wide variety of pathological conditions such as diabetes, Alzheimer's disease, stroke, bipolar disorder, malaria and cancer. Herein we report 3D-QSAR analyses using CoMFA and CoMSIA and molecular docking studies on 3-anilino-4-phenylmaleimides as GSK-3α inhibitors, in order to better understand the mechanism of action and structure-activity relationship of these compounds. Comparison of the active site residues of GSK-3α and GSK-3β isoforms shows that all the key amino acids involved in polar interactions with the maleimides for the β isoform are the same in the α isoform, except that Asp133 in the β isoform is replaced by Glu196 in the α isoform. We prepared a homology model for GSK-3α, and showed that the change from Asp to Glu should not affect maleimide binding significantly. Docking studies revealed the binding poses of three subclasses of these ligands, namely anilino, N-methylanilino and indoline derivatives, within the active site of the β isoform, and helped to explain the difference in their inhibitory activity.

  9. 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.

  10. Glycogen Synthase Kinase-3β Is Involved in Ligand-Dependent Activation of Transcription and Cellular Localization of the Glucocorticoid Receptor

    PubMed Central

    Rubio-Patiño, Camila; Palmeri, Claudia M.; Pérez-Perarnau, Alba; Cosialls, Ana M.; Moncunill-Massaguer, Cristina; González-Gironès, Diana M.; Pons-Hernández, Lluís; López, José M.; Ventura, Francesc; Gil, Joan; Iglesias-Serret, Daniel

    2012-01-01

    Glucocorticoids (GC) induce cell cycle arrest and apoptosis in different cell types and therefore are widely used to treat a variety of diseases including autoimmune disorders and cancer. This effect is mediated by the GC receptor (GR), a ligand-activated transcription factor that translocates into the nucleus where it modulates transcription of target genes in a promoter-specific manner. Glycogen synthase kinase-3 (GSK3) regulates GR response by genomic and nongenomic mechanisms, although the specific role of each isoform is not well defined. We used GSK3 pharmacological inhibitors and isoform-specific small interfering RNA to evaluate the role of GSK3 in the genomic regulation induced by GC. GSK3 inhibition resulted in the reduction of GC-induced mRNA expression of GC-induced genes such as BIM, HIAP1, and GILZ. Knockdown of GSK3β but not GSK3α reduced endogenous GILZ induction in response to dexamethasone and GR-dependent reporter gene activity. Chromatin immunoprecipitation experiments revealed that GSK3 inhibition impaired the dexamethasone-mediated binding of GR and RNA polymerase II to endogenous GILZ promoter. These results indicate that GSK3β is important for GR transactivation activity and that GSK3β inhibition suppresses GC-stimulated gene expression. Furthermore, we show that genomic regulation by the GR is independent of known GSK3β phosphorylation sites. We propose that GC-dependent transcriptional activation requires functional GSK3β signaling and that altered GSK3β activity influences cell response to GC. PMID:22771494

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

    PubMed

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

    2012-05-15

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

  12. Glucocorticoids and hepatic glycogen metabolism.

    PubMed

    Stalmans, W; Laloux, M

    1979-01-01

    The steady accumulation of glycogen in fetal rat liver during the last fifth of gestation is elicited by a transient rise in the level of circulating corticosterone. One effect of glucocorticoids is to induce glycogen synthase. The actual deposition of glycogen, however, depends on the appearance of a small amount of glycogen synthase in the active, dephosphorylated form. Induction of glycogen synthase phosphatase by glucocorticoids may explain the latter crucial process. Insulin enhances further the rate of glycogen deposition. The effect of insulin requires a previous exposure of the fetal liver to glucocorticoids. It is exerted on the enzyme interconversion system and appears not to involve new protein synthesis. Administration of glucocorticoids to adult fed or fasted animals causes within 3 h an intensive deposition of glycogen in the liver. This phenomenon is ultimately explained by both an activation of glycogen synthase and an inactivation of glycogen phosphorylase. The latter process may be due to an enhanced activity of phosphorylase phosphatase, or possibly of phosphorylase kinase phosphatase. The activation of glycogen synthase is explained by an enhanced activity of glycogen synthase phosphatase. The latter enzyme is normally profoundly inhibited by phosphorylase a; glucocorticoids cause the appearance in the liver of a protein factor that decreases and eventually cancels this inhibitory effect of phosphorylase a. It remains to be established whether or not some part of the glucocorticoid effect on adult liver is mediated by insulin.

  13. Glycogen storage disease type Ib neutrophils exhibit impaired cell adhesion and migration.

    PubMed

    Kim, Goo-Young; Lee, Young Mok; Kwon, Joon Hyun; Jun, Hyun Sik; Chou, Janice

    2017-01-22

    Glycogen storage disease type Ib (GSD-Ib), characterized by impaired glucose homeostasis, neutropenia, and neutrophil dysfunction, is an inherited autosomal recessive disorder caused by a deficiency in the glucose-6-phosphate transporter (G6PT). Neutrophils play an essential role in the defense against invading pathogens. The recruitment of neutrophils towards the inflammation sites in response to inflammatory stimuli is a tightly regulated process involving rolling, adhesion, and transmigration. In this study, we investigated the role of G6PT in neutrophil adhesion and migration using in vivo and in vitro models. We showed that the GSD-Ib (G6pt(-/-)) mice manifested severe neutropenia in both blood and bone marrow, and treating G6pt(-/-) mice with granulocyte colony-stimulating factor (G-CSF) corrected neutropenia. However, upon thioglycolate challenge, neutrophils from both untreated and G-CSF-treated G6pt(-/-)mice exhibited decreased ability to migrate to the peritoneal cavity. In vitro migration and cell adhesion of G6PT-deficient neutrophils were also significantly impaired. Defects in cell migration were not due to enhanced apoptosis or altered fMLP receptor expression. Remarkably, the expression of the β2 integrins CD11a and CD11b, which are critical for cell adhesion, was greatly decreased in G6PT-deficient neutrophils. This study suggests that deficiencies in G6PT cause impairment in neutrophil adhesion and migration via aberrant expression of β2 integrins, and our finding should facilitate the development of novel therapies for GSD-Ib.

  14. 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.

  15. Utilizing glycogen synthase kinase-3β as a marker for the diagnosis of graft-versus-host disease.

    PubMed

    Orbach, A; Bassan-Levin, T; Dan, P; Hihinashvilli, B; Marx, S

    2013-06-01

    Graft-versus-host disease (GVHD) is a deadly complication of allogeneic hematopoietic stem cell transplantation. Timely diagnosis is critical, because mortality rates for GVHD are high, increasing with disease severity. A diagnostic tool to predict GVHD before the onset of clinical symptoms could save many lives. On the cellular level, GVHD occurs when T cells from the transplant attack the tissues of the host, after perceiving them to be foreign. T-cell proliferation occurs even before clinical symptoms appear. Glycogen synthase kinase (GSK)-3β is a protein which regulates proliferation in many cell types including T-cells. GSK-3β has never been directly connected with GVHD and we applied GSK-3β as a novel marker for GVHD prediction, seeking herein to determine whether GSK-3β can be utilized as a marker for the early diagnosis of GVHD. For the mouse model of acute GVHD, irradiated mice underwent allogeneic splenocyte transplantation and GSK-3β expression levels and phosphorylation states were monitored in harvested spleens by western blot. FACS analysis was used to measure the number of T cells within the harvested spleens. Mice developed observable GVHD symptoms by day 5 post-transplantation, with severe symptoms on day 6 requiring mice to be killed for humane reasons. A significantly increased number of T cells in the allogeneic mice correlated with GVHD development. GSK-3β protein expression levels and phosphorylation levels were significantly lower in allogeneic (GVHD) mice compared with negative (untreated) and positive (syngeneic transplant; non-GVHD) controls over time. GSK-3β was directly connected with the onset and progression of GVHD. Therefore, it can be utilized as a marker for GVHD diagnosis in animals and potentially in humans. Copyright © 2013 Elsevier Inc. All rights reserved.

  16. Glycogen synthase kinase-3 is essential for β-arrestin-2 complex formation and lithium-sensitive behaviors in mice

    PubMed Central

    O’Brien, W. Timothy; Huang, Jian; Buccafusca, Roberto; Garskof, Julie; Valvezan, Alexander J.; Berry, Gerard T.; Klein, Peter S.

    2011-01-01

    Lithium is the first-line therapy for bipolar disorder. However, its therapeutic target remains controversial. Candidates include inositol monophosphatases, glycogen synthase kinase-3 (GSK-3), and a β-arrestin-2/AKT/protein phosphatase 2A (β-arrestin-2/AKT/PP2A) complex that is known to be required for lithium-sensitive behaviors. Defining the direct target(s) is critical for the development of new therapies and for elucidating the molecular pathogenesis of this major psychiatric disorder. Here, we show what we believe to be a new link between GSK-3 and the β-arrestin-2 complex in mice and propose an integrated mechanism that accounts for the effects of lithium on multiple behaviors. GSK-3β (Gsk3b) overexpression reversed behavioral defects observed in lithium-treated mice and similar behaviors observed in Gsk3b+/– mice. Furthermore, immunoprecipitation of striatial tissue from WT mice revealed that lithium disrupted the β-arrestin-2/Akt/PP2A complex by directly inhibiting GSK-3. GSK-3 inhibitors or loss of one copy of the Gsk3b gene reduced β-arrestin-2/Akt/PP2A complex formation in mice, while overexpression of Gsk3b restored complex formation in lithium-treated mice. Thus, GSK-3 regulates the stability of the β-arrestin-2/Akt/PP2A complex, and lithium disrupts the complex through direct inhibition of GSK-3. We believe these findings reveal a new role for GSK-3 within the β-arrestin complex and demonstrate that GSK-3 is a critical target of lithium in mammalian behaviors. PMID:21821916

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

    PubMed

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

    2014-11-21

    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(+/-) 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. Copyright © 2014 Elsevier Inc. All rights reserved.

  18. Glycogen Synthase Kinase-3β: Variation over Time and the Possible Association with Mood and Cognition in Healthy Individuals.

    PubMed

    Munkholm, Klaus; Lenskjold, Toke; Jacoby, Anne Sophie; Miskowiak, Kamilla Woznica; Vinberg, Maj; Joaquim, Helena Giroud Passarelli; Talib, Leda Leme; Gattaz, Wagner Farid; Kessing, Lars Vedel

    2016-01-01

    Evidence indicates a role for glycogen synthase kinase-3β (GSK-3β) in the pathophysiology of mood disorders and in cognitive disturbances; however, the natural variation in GSK-3β activity over time is unknown. We aimed to investigate GSK-3β activity over time and its possible correlation with emotional lability, subjective mood fluctuations and cognitive function in healthy individuals. Thirty-seven healthy subjects were evaluated with neuropsychological tests and blood samples at baseline and 12-week follow-up. Total GSK-3β and serine-9-phosphorylated GSK-3β in peripheral blood mononuclear cells were quantitated using enzyme immunometric assays. The activity of GSK-3β (serine-9-phosphorylated GSK-3β/total GSK-3β) was lower at baseline compared with follow-up. No significant mean change over time was observed in levels of total GSK-3β and serine-9-phosphorylated GSK-3β. Exploratory analysis revealed lower activity of GSK-3β in spring and summer compared with the fall season. No correlation was observed between GSK-3β activity and emotional lability, subjective mood fluctuations or cognitive function. The results suggest that intra- and interindividual variation in GSK-3β activity over time could contribute to the heterogeneity of findings in clinical studies. The stability of GSK-3β activity and the role of potential moderators of GSK-3β activity warrant further investigation. Clinical studies of GSK-3β should consider including repeated measures of both cases and healthy individuals. © 2016 S. Karger AG, Basel.

  19. CB1 cannabinoid receptors increase neuronal precursor proliferation through AKT/glycogen synthase kinase-3beta/beta-catenin signaling.

    PubMed

    Trazzi, Stefania; Steger, Martin; Mitrugno, Valentina Maria; Bartesaghi, Renata; Ciani, Elisabetta

    2010-03-26

    The endocannabinoid system is involved in the regulation of many physiological effects in the central and peripheral nervous system. Recent findings have demonstrated the presence of a functional endocannabinoid system within neuronal progenitors located in the hippocampus and ventricular/subventricular zone that participates in the regulation of cell proliferation. It is presently unknown whether the endocannabinoid system exerts a widespread effect on neuronal precursors from different neurogenic regions, and very little is known about the signaling by which it regulates neuronal precursor proliferation. Herein, we demonstrate the presence of cannabinoid CB(1) receptors in granule cell precursors (GCPs) during early cerebellar development. Activation of CB(1) receptors by HU-210 promoted GCP proliferation in vitro, an effect that was prevented by a selective CB(1) antagonist. Accordingly, in vivo experiments showed that GCP proliferation was increased by chronic HU-210 treatment and that in CB(1)-deficient mice cell proliferation was significantly lower than in wild-type littermates, indicating that the endocannabinoid system is physiologically involved in regulation of GCP proliferation. The pro-proliferative effect of cannabinoids in GCPs was mediated through the CB(1)/AKT/glycogen synthase kinase-3beta/beta-catenin pathway. Involvement of this pathway was also observed in cultures of neuronal precursors from the subventricular zone, suggesting that this pathway may be a general mechanism by which endocannabinoids regulate proliferation of neuronal precursors. These observations suggest that endocannabinoids constitute a new family of lipid signaling cues that may exert a widespread effect on neuronal precursor proliferation during brain development.

  20. Tissue kallikrein promotes neovascularization and improves cardiac function by the Akt-glycogen synthase kinase-3β pathway

    PubMed Central

    Yao, Yu-Yu; Yin, Hang; Shen, Bo; Smith, Robert S.; Liu, Yuying; Gao, Lin; Chao, Lee; Chao, Julie

    2008-01-01

    Aims We investigated the role of the Akt-glycogen synthase kinase (GSK)-3β signalling pathway in mediating the protective effects of tissue kallikrein on myocardial injury by promoting angiogenesis and blood flow in rats after myocardial infarction (MI). Methods and results Human tissue kallikrein gene in an adenoviral vector, with or without co-administration of dominant-negative Akt (Ad.DN-Akt) or constitutively active GSK-3β (Ad.GSK-3βS9A), was injected into rat myocardium after MI. The expression of recombinant human kallikrein in rat heart significantly improved cardiac function and reduced infarct size 10 days after gene delivery. Kallikrein administration significantly increased myocardial blood flow as well as capillary and arteriole densities in the infarcted myocardium. Kallikrein increased cardiac Akt and GSK-3β phosphorylation in conjunction with decreased GSK-3β activity and the upregulation of vascular endothelial growth factor (VEGF) and VEGF receptor-2 (VEGFR-2). All of kallikrein’s effects on the myocardium were abrogated by Ad.DN-Akt and Ad.GSK-3βS9A. Moreover, in cultured human aortic endothelial cells, tissue kallikrein stimulated capillary tube formation and promoted cell migration; however, these effects were blocked by Ad.DN-Akt, Ad.GSK-3βS9A, icatibant (a kinin B2 receptor antagonist), Tki (a VEGF receptor tyrosine kinase inhibitor), and a neutralizing VEGF antibody. In addition, tissue kallikrein decreased GSK-3β activity via the phosphatidylinositol 3-kinase-Akt pathway and enhanced VEGF and VEGFR-2 expression in endothelial cells. Conclusion These data provide the first direct evidence that tissue kallikrein protects against acute-phase MI by promoting neovascularization, restoring regional blood flow and improving cardiac function through the kinin B2 receptor-Akt-GSK-3β and VEGF signalling pathways. PMID:18689794

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

    PubMed Central

    Gao, Mei; Yan, Xisheng; Weng, Han-Rong

    2013-01-01

    Paclitaxel (taxol) is a first-line chemotherapy-drug used to treat many types of cancers. Neuropathic pain and sensory dysfunction are the major toxicities, which are dose-limiting and significantly reduce the quality of life in patients. Two known critical spinal mechanisms underlying taxol-induced neuropathic pain are an increased production of pro-inflammatory cytokines including interleukin-1β (IL-1β) and suppressed glial glutamate transporter activities. In this study, we uncovered that increased activation of glycogen synthase kinase 3beta (GSK3β) in the spinal dorsal horn was concurrently associated with increased protein expressions of GFAP, IL-1β and a decreased protein expression of glial glutamate transporter 1 (GLT-1), as well as the development and maintenance of taxol-induced neuropathic pain. The enhanced GSK3β activities were supported by the concurrently decreased AKT and mTOR activities. The changes of all these biomarkers were basically prevented when animals received pre-emptive lithium (a GSK3β inhibitor) treatment, which also prevented the development of taxol-induced neuropathic pain. Further, chronic lithium treatment, which began on day 11 after the first taxol injection, reversed the existing mechanical and thermal allodynia induced by taxol. The taxol-induced increased GSK3β activities and decreased AKT and mTOR activities in the spinal dorsal horn were also reversed by lithium. Meanwhile, protein expressions of GLT-1, GFAP and IL-1β in the spinal dorsal horn were improved. Hence, suppression of spinal GSK3β activities is a key mechanism used by lithium to reduce taxol-induced neuropathic pain, and targeting spinal GSK3β is an effective approach to ameliorate GLT-1 expression and suppress activation of astrocytes and IL-1β over-production in the spinal dorsal horn. PMID:24070631

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

    PubMed

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

    2010-06-01

    Increased medial arterial thickness is a structural change in pulmonary arterial hypertension (PAH). The role of smooth muscle hypertrophy in this process has not been well studied. Bone morphogenetic proteins (BMPs), transforming growth factor (TGF)-beta1, serotonin (or 5-hydroxytryptamine; 5-HT), and endothelin (ET)-1 have been implicated in PAH pathogenesis. We examined the effect of these mediators on human pulmonary artery smooth muscle cell size, contractile protein expression, and contractile function, as well on the roles of glycogen synthase kinase (GSK)-3beta and p70 ribosomal S6 kinase (p70S6K), two proteins involved in translational control, in this process. Unlike epidermal growth factor, BMP-4, TGF-beta1, 5-HT, and ET-1 each increased smooth muscle cell size, contractile protein expression, fractional cell shortening, and GSK-3beta phosphorylation. GSK-3beta inhibition by lithium or SB-216763 increased cell size, protein synthesis, and contractile protein expression. Expression of a non-phosphorylatable GSK-3beta mutant blocked BMP-4-, TGF-beta1-, 5-HT-, and ET-1-induced cell size enlargement, suggesting that GSK-3beta phosphorylation is required and sufficient for cellular hypertrophy. However, BMP-4, TGF-beta1, 5-HT, and ET-1 stimulation was accompanied by an increase in serum response factor transcriptional activation but not eIF2 phosphorylation, suggesting that GSK-3beta-mediated hypertrophy occurs via transcriptional, not translational, control. Finally, BMP-4, TGF-beta1, 5-HT, and ET-1 treatment induced phosphorylation of p70S6K and ribosomal protein S6, and siRNAs against p70S6K and S6 blocked the hypertrophic response. We conclude that mediators implicated in the pathogenesis of PAH induce pulmonary arterial smooth muscle hypertrophy. Identification of the signaling pathways regulating vascular smooth muscle hypertrophy may define new therapeutic targets for PAH.

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

    PubMed Central

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

    2010-01-01

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

  4. Airway smooth muscle hyperplasia and hypertrophy correlate with glycogen synthase kinase-3(beta) phosphorylation in a mouse model of asthma.

    PubMed

    Bentley, J Kelley; Deng, Huan; Linn, Marisa J; Lei, Jing; Dokshin, Gregoriy A; Fingar, Diane C; Bitar, Khalil N; Henderson, William R; Hershenson, Marc B

    2009-02-01

    Increased airway smooth muscle (ASM) mass, a characteristic finding in asthma, may be caused by hyperplasia or hypertrophy. Cell growth requires increased translation of contractile apparatus mRNA, which is controlled, in part, by glycogen synthase kinase (GSK)-3beta, a constitutively active kinase that inhibits eukaryotic initiation factor-2 activity and binding of methionyl tRNA to the ribosome. Phosphorylation of GSK-3beta inactivates it, enhancing translation. We sought to quantify the contributions of hyperplasia and hypertrophy to increased ASM mass in ovalbumin (OVA)-sensitized and -challenged BALB/c mice and the role of GSK-3beta in this process. Immunofluorescent probes, confocal microscopy, and stereological methods were used to analyze the number and volume of cells expressing alpha-smooth muscle actin and phospho-Ser(9) GSK-3beta (pGSK). OVA treatment caused a 3-fold increase in ASM fractional unit volume or volume density (Vv) (PBS, 0.006 +/- 0.0003; OVA, 0.014 +/- 0.001), a 1.5-fold increase in ASM number per unit volume (Nv), and a 59% increase in volume per cell (Vv/Nv) (PBS, 824 +/- 76 microm(3); OVA, 1,310 +/- 183 mum(3)). In OVA-treated mice, there was a 12-fold increase in the Vv of pGSK (+) ASM, a 5-fold increase in the Nv of pGSK (+) ASM, and a 1.6-fold increase in Vv/Nv. Lung homogenates from OVA-treated mice showed increased GSK-3beta phosphorylation and lower GSK-3beta activity. Both hyperplasia and hypertrophy are responsible for increased ASM mass in OVA-treated mice. Phosphorylation and inactivation of GSK-3beta are associated with ASM hypertrophy, suggesting that this kinase may play a role in asthmatic airway remodeling.

  5. Lovastatin Modulates Glycogen Synthase Kinase-3β Pathway and Inhibits Mossy Fiber Sprouting after Pilocarpine-Induced Status Epilepticus

    PubMed Central

    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

  6. Glycogen synthase kinase 3β gene polymorphisms may be associated with bipolar I disorder and the therapeutic response to lithium.

    PubMed

    Lin, Yen-Feng; Huang, Ming-Chyi; Liu, Hsing-Cheng

    2013-05-01

    Glycogen Synthase Kinase 3β (GSK-3β) is thought to be a key feature in the therapeutic mechanism of mood stabilizers (e.g., lithium). Overexpression of GSK-3β might play a role in the pathogenesis of bipolar I disorder. Within the GSK-3β gene, a promoter single nucleotide polymorphism (SNP) rs334558 was identified associated with transcriptional strength, and an intronic SNP rs6438552 was found to regulate selection of splice acceptor sites. The aim of this study is to test the association between the two polymorphisms and bipolar I disorder. We genotyped the two SNPs in 138 Taiwanese bipolar I disorder patients and 131 controls. Lithium treatment efficacy was evaluated for 83 patients who had been treated with lithium carbonate for at least 24 months. We found no association between each of the two SNPs and the risk of bipolar I disorder. Following correction for multiple testing, CT genotype at rs6438552 was associated with an older age of onset than other genotypes (P=0.042) in female patients. Patients with genotype TT at rs334558 (P=0.044) had poorer response to lithium treatment. There was a trend that haplotype C-T increased the risk for bipolar I disorder (adjusted OR=4.22, corrected P=0.084), and patients with haplotype T-T had poorer treatment response to lithium than those with haplotype C-C. Limitations included small sample size, retrospective data collection, and a potential sampling bias. Despite the several limitations of the study, our results suggested GSK-3β genetic variants may be associated with the risk of bipolar I disorder, age of disease onset in females, and the therapeutic response to lithium. Copyright © 2012 Elsevier B.V. All rights reserved.

  7. Altered Wnt signalling in the teenage suicide brain: focus on glycogen synthase kinase-3β and β-catenin.

    PubMed

    Ren, Xinguo; Rizavi, Hooriyah S; Khan, Mansoor A; Dwivedi, Yogesh; Pandey, Ghanshyam N

    2013-06-01

    Glycogen synthase kinase (GSK)-3β and β-catenin are important components of the Wnt signalling pathway, which is involved in numerous physiological functions such as cognition, brain development and cell survival. Their abnormalities have been implicated in mood disorders and schizophrenia. Teenage suicide is a major public health concern; however, very little is known about its neurobiology. In order to examine if abnormalities of GSK-3β and β-catenin are associated with teenage suicide, we determined the gene and protein expression of GSK-3β and β-catenin in the prefrontal cortex (PFC) and hippocampus obtained from 24 teenage suicide victims and 24 normal control subjects. Protein expression was determined using Western blot with specific antibodies and gene expression (mRNA levels) was determined using the real-time polymerase chain reaction method. No significant change was observed in the GSK-3β protein levels either in the PFC or hippocampus of suicide victims compared to controls. However, protein levels of pGSK-3β-ser(9) were significantly decreased in the PFC and hippocampus of suicide victims compared to normal controls. We also found that GSK-3β mRNA levels were significantly decreased in the PFC but not in the hippocampus of teenage suicide victims compared to controls. Mean protein and mRNA levels of β-catenin were significantly decreased in both the PFC and hippocampus of teenage suicide group compared to controls. The observation that there is a decrease in β-catenin and pGSK-3β-ser(9) in the PFC and hippocampus of teenage suicide victims does indicate a disturbance in the Wnt signalling pathway in teenage suicide.

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

    PubMed

    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.

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

    PubMed

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

    2015-07-01

    Cardiac hypertrophy is a leading cause of serious heart diseases. Although many signaling molecules are involved in hypertrophy, the functions of some proteins in this process are still unknown. Calcineurin B homologous protein 3 (CHP3)/tescalcin is an EF-hand Ca(2+)-binding protein that is abundantly expressed in the heart; however, the function of CHP3 is unclear. Here, we aimed to identify the cardiac functions of CHP3. CHP3 was expressed in hearts at a wide range of developmental stages and was specifically detected in neonatal rat ventricular myocytes (NRVMs) but not in cardiac fibroblasts in culture. Moreover, knockdown of CHP3 expression using adenoviral-based RNA interference in NRVMs resulted in enlargement of cardiomyocyte size, concomitant with increased expression of a pathological hypertrophy marker ANP. This same treatment elevated glycogen synthase kinase (GSK3α/β) phosphorylation, which is known to inhibit GSK3 function. In contrast, CHP3 overexpression blocked the insulin-induced phosphorylation of GSK3α/β without affecting the phosphorylation of Akt, which is an upstream kinase of GSK3α/β, in HEK293 cells, and it inhibited both IGF-1-induced phosphorylation of GSK3β and cardiomyocyte hypertrophy in NRVMs. Co-immunoprecipitation experiments revealed that GSK3β interacted with CHP3. However, a Ca(2+)-binding-defective mutation of CHP3 (CHP3-D123A) also interacted with GSK3β and had the same inhibitory effect on GSK3α/β phosphorylation, suggesting that the action of CHP3 was independent of Ca(2+). These findings suggest that CHP3 functions as a novel negative regulator of cardiomyocyte hypertrophy via inhibition of GSK3α/β phosphorylation and subsequent enzymatic activation of GSK3α/β. Copyright © 2015 Elsevier Ltd. All rights reserved.

  10. Discovery of novel potent and highly selective glycogen synthase kinase-3β (GSK3β) inhibitors for Alzheimer's disease: design, synthesis, and characterization of pyrazines.

    PubMed

    Berg, Stefan; Bergh, Margareta; Hellberg, Sven; Högdin, Katharina; Lo-Alfredsson, Yvonne; Söderman, Peter; von Berg, Stefan; Weigelt, Tatjana; Ormö, Mats; Xue, Yafeng; Tucker, Julie; Neelissen, Jan; Jerning, Eva; Nilsson, Yvonne; Bhat, Ratan

    2012-11-08

    Glycogen synthase kinase-3β, also called tau phosphorylating kinase, is a proline-directed serine/threonine kinase which was originally identified due to its role in glycogen metabolism. Active forms of GSK3β localize to pretangle pathology including dystrophic neuritis and neurofibrillary tangles in Alzheimer's disease (AD) brain. By using a high throughput screening (HTS) approach to search for new chemical series and cocrystallization of key analogues to guide the optimization and synthesis of our pyrazine series, we have developed highly potent and selective inhibitors showing cellular efficacy and blood-brain barrier penetrance. The inhibitors are suitable for in vivo efficacy testing and may serve as a new treatment strategy for Alzheimer's disease.

  11. 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

  12. 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

  13. Glycogen Synthase Kinase-3α Limits Ischemic Injury, Cardiac Rupture, Post–Myocardial Infarction Remodeling and Death

    PubMed Central

    Lal, Hind; Zhou, Jibin; Ahmad, Firdos; Zaka, Raihana; Vagnozzi, Ronald J.; DeCaul, Morgan; Woodgett, James; Gao, Erhe; Force, Thomas

    2015-01-01

    Background The molecular pathways that regulate the extent of ischemic injury and post–myocardial infarction (MI) remodeling are not well understood. We recently demonstrated that glycogen synthase kinase-3α (GSK-3α) is critical to the heart’s response to pressure overload. However, the role, if any, of GSK-3α in regulating ischemic injury and its consequences is not known. Methods and Results MI was induced in wild-type (WT) versus GSK-3α(−/−) (KO) littermates by left anterior descending coronary artery ligation. Pre-MI, WT, and KO hearts had comparable chamber dimensions and ventricular function, but as early as 1 week post-MI, KO mice had significantly more left ventricular dilatation and dysfunction than WT mice. KO mice also had increased mortality during the first 10 days post-MI (43% versus 22%; P=0.04), and postmortem examination confirmed cardiac rupture as the cause of most of the deaths. In the mice that survived the first 10 days, left ventricular dilatation and dysfunction remained worse in the KO mice throughout the study (8 weeks). Hypertrophy, fibrosis, and heart failure were all increased in the KO mice. Given the early deaths due to rupture and the significant reduction in left ventricular function evident as early as 1 week post-MI, we examined infarct size following a 48-hour coronary artery ligation and found it to be increased in the KO mice. This was accompanied by increased apoptosis in the border zone of the MI. This increased susceptibility to ischemic injury–induced apoptosis was also seen in cardiomyocytes isolated from the KO mice that were exposed to hypoxia. Finally, Bax translocation to the mitochondria and cytochrome C release into the cytosol were increased in the KO mice. Conclusion GSK-3α confers resistance to ischemic injury, at least in part, via limiting apoptosis. Loss of GSK-3α promotes ischemic injury, increases risk of cardiac rupture, accentuates post-MI remodeling and left ventricular dysfunction, and

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2012-01-01

    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

  16. 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.

  17. Skeletal muscle metabolism is impaired during exercise in glycogen storage disease type III.

    PubMed

    Preisler, Nicolai; Laforêt, Pascal; Madsen, Karen Lindhardt; Prahm, Kira Philipsen; Hedermann, Gitte; Vissing, Christoffer Rasmus; Galbo, Henrik; Vissing, John

    2015-04-28

    Glycogen storage disease type IIIa (GSDIIIa) is classically regarded as a glycogenosis with fixed weakness, but we hypothesized that exercise intolerance in GSDIIIa is related to muscle energy failure and that oral fructose ingestion could improve exercise tolerance in this metabolic myopathy. We challenged metabolism with cycle-ergometer exercise and measured substrate turnover and oxidation rates using stable isotope methodology and indirect calorimetry in 3 patients and 6 age-matched controls on 1 day, and examined the effect of fructose ingestion on exercise tolerance in the patients on another day. Total fatty acid oxidation rates during exercise were higher in patients than controls, 32.1 (SE 1.2) vs 20.7 (SE 0.5; range 15.8-29.3) μmol/kg/min (p = 0.048), and oxidation of carbohydrates was lower in patients, 1.0 (SE 5.4) vs 38.4 (SE 8.0; range 23.0-77.1) μmol/kg/min (p = 0.024). Fructose ingestion improved exercise tolerance in the patients. Similar to patients with McArdle disease, in whom muscle glycogenolysis is also impaired, GSDIIIa is associated with a reduced skeletal muscle oxidation of carbohydrates and a compensatory increase in fatty acid oxidation, and fructose ingestion improves exercise tolerance. Our results indicate that GSDIIIa should not only be viewed as a glycogenosis with fixed skeletal muscle weakness, but should also be considered among the glycogenoses presenting with exercise-related dynamic symptoms caused by muscular energy deficiency. This study provides Class IV evidence that ingestion of fructose improves exercise tolerance in patients with GSDIIIa. © 2015 American Academy of Neurology.

  18. 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.

  19. 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

  20. 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.

  1. Characterization of Recombinant UDP- and ADP-Glucose Pyrophosphorylases and Glycogen Synthase To Elucidate Glucose-1-Phosphate Partitioning into Oligo- and Polysaccharides in Streptomyces coelicolor

    PubMed Central

    Asención Diez, Matías D.; Peirú, Salvador; Demonte, Ana M.; Gramajo, Hugo

    2012-01-01

    Streptomyces coelicolor exhibits a major secondary metabolism, deriving important amounts of glucose to synthesize pigmented antibiotics. Understanding the pathways occurring in the bacterium with respect to synthesis of oligo- and polysaccharides is of relevance to determine a plausible scenario for the partitioning of glucose-1-phosphate into different metabolic fates. We report the molecular cloning of the genes coding for UDP- and ADP-glucose pyrophosphorylases as well as for glycogen synthase from genomic DNA of S. coelicolor A3(2). Each gene was heterologously expressed in Escherichia coli cells to produce and purify to electrophoretic homogeneity the respective enzymes. UDP-glucose pyrophosphorylase (UDP-Glc PPase) was characterized as a dimer exhibiting a relatively high Vmax in catalyzing UDP-glucose synthesis (270 units/mg) and with respect to dTDP-glucose (94 units/mg). ADP-glucose pyrophosphorylase (ADP-Glc PPase) was found to be tetrameric in structure and specific in utilizing ATP as a substrate, reaching similar activities in the directions of ADP-glucose synthesis or pyrophosphorolysis (Vmax of 0.15 and 0.27 units/mg, respectively). Glycogen synthase was arranged as a dimer and exhibited specificity in the use of ADP-glucose to elongate α-1,4-glucan chains in the polysaccharide. ADP-Glc PPase was the only of the three enzymes exhibiting sensitivity to allosteric regulation by different metabolites. Mannose-6-phosphate, phosphoenolpyruvate, fructose-6-phosphate, and glucose-6-phosphate behaved as major activators, whereas NADPH was a main inhibitor of ADP-Glc PPase. The results support a metabolic picture where glycogen synthesis occurs via ADP-glucose in S. coelicolor, with the pathway being strictly regulated in connection with other routes involved with oligo- and polysaccharides, as well as with antibiotic synthesis in the bacterium. PMID:22210767

  2. Ventricular hypertrophy abrogates intralipid-induced cardioprotection by alteration of reperfusion injury salvage kinase/glycogen synthase kinase 3β signal.

    PubMed

    Ma, Lei-Lei; Ge, Hong-Wei; Kong, Fei-Juan; Qian, Ling-Bo; Hu, Bang-Chuan; Li, Qian; Xu, Liang; Liu, Jing-Quan; Xu, Yun-Xiang; Sun, Ren-Hua

    2014-05-01

    Recent studies have demonstrated that intralipid (ILP) conferred myocardial protection against ischemia-reperfusion (IR) injury through activation of reperfusion injury salvage kinase (RISK) pathway. As RISK signal has been shown to be impaired in hypertrophied myocardium, we investigated whether ILP-induced cardiac protection was maintained in hypertrophied rat hearts. Transverse aortic constriction was performed on male Sprague-Dawley rats to induce left ventricular hypertrophy, then sham-operated or hypertrophied rat hearts were isolated and perfused retrogradely by the Langendorff for 30 min (equilibration) followed by 40 min of ischemia and then 120 min of reperfusion. The isolated hearts received 15-min episode of 1% ILP separated by 15 min of washout or three episodes of 5-min ischemia followed by 5-min reperfusion before ischemia. The hemodynamics, infarct size, apoptosis, phosphorylated protein kinase B (p-Akt), phosphorylated extracellular regulated protein kinase 1/2 (ERK1/2), phosphorylated glycogen synthase kinase 3β (GSK3β), Bcl-2, phosphorylated Bad, and Bax were determined. We found that ILP significantly improved left ventricular hemodynamics and reduced infarct size and the number of TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling)-positive cells in the sham-operated rat hearts exposed to IR. However, such myocardial infarct-sparing effect of ILP was completely blocked by phosphatidylinositol-3-kinase inhibitor wortmannin, but only partially by mitogen-activated protein kinase kinase inhibitor PD98059 in sham-operated hearts. Intralipd upregulated the phosphorylation of Akt, extracellular regulated protein kinase 1/2 (ERK1/2), and their downstream target of GSK3β and antiapoptotic Bcl-2 expression in healthy rat hearts. Nonetheless, ILP failed to improve left ventricular hemodynamics and reduced infarct size and apoptosis and increase the phosphorylated Akt, ERK1/2, GSK3β, and antiapoptotic Bcl-2 in hypertrophied myocardium

  3. 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

  4. Effects of adrenaline on whole-body glucose metabolism and insulin-mediated regulation of glycogen synthase and PKB phosphorylation in human skeletal muscle.

    PubMed

    Jensen, Jørgen; Ruge, Toralph; Lai, Yu-Chiang; Svensson, Maria K; Eriksson, Jan W

    2011-02-01

    In the present study, we investigated the effect of adrenaline on insulin-mediated regulation of glucose and fat metabolism with focus on regulation of skeletal muscle PKB, GSK-3, and glycogen synthase (GS) phosphorylation. Ten healthy subjects (5 men and 5 women) received a 240-minute intravenous infusion of adrenaline (0.05 μg/[kg min]) or saline; after 120 minutes, a hyperinsulinemic-euglycemic clamp was added. Adrenaline infusion increased blood glucose concentration by approximately 50%, but the hyperinsulinemic clamp normalized blood glucose within 30 minutes. Glucose infusion rate during the last hour was approximately 60% lower during adrenaline infusion compared with saline (4.3 ± 0.5 vs 11.2 ± 0.6 mg/kg lean body mass per minute). Insulin increased PKB Ser⁴⁷³, PKB Thr³⁰⁸, and GSK-3β Ser⁹ phosphorylation in skeletal muscles; coinfusion of adrenaline did not influence insulin-stimulated PKB and GSK-3 phosphorylation. Adrenaline alone did not influence phosphorylation of PKB and GSK-3β. Insulin increased GS fractional activity and decreased GS Ser⁶⁴¹ and Ser⁶⁴⁵,⁶⁴⁹,⁶⁵³,⁶⁵⁷ phosphorylation. In the presence of adrenaline, insulin did neither activate GS nor dephosphorylate GS Ser⁶⁴¹. Surprisingly, GS Ser⁷ phosphorylation was not influenced by adrenaline. Adrenaline increased plasma lactate concentration; and muscle glycogen content was reduced in skeletal muscle the day after adrenaline infusion, supporting that insulin does not stimulate glycogen synthesis in skeletal muscles when adrenaline is present. In conclusion, adrenaline did not influence basal or insulin-stimulated PKB and GSK-3β phosphorylation in muscles, but completely blocked insulin-mediated GS activation and Ser⁶⁴¹ dephosphorylation. Still, insulin normalized adrenaline-mediated hyperglycemia.

  5. Discovery of a highly selective glycogen synthase kinase-3 inhibitor (PF-04802367) that modulates tau phosphorylation in brain: Translation for PET neuroimaging

    PubMed Central

    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; Patnaik, Debasis; Wagner, Florence F.; Perlis, Roy; Holson, Edward B.; Haggarty, Stephen J.; Fakhri, Georges El

    2016-01-01

    Glycogen synthase kinase-3 (GSK-3) regulates multiple cellular processes in diabetes, oncology and neurology. We have identified N-(3-(1H-1,2,4-triazol-1-yl)propyl)-5-(3-chloro-4-methoxyphenyl)oxazole-4-carboxamide (PF-04802367 or PF-367) as a highly potent inhibitor, which is among the most selective antagonists of GSK-3 to date. We demonstrated its efficacy 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 11C-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

  6. Novel, Starch-Like Polysaccharides Are Synthesized by an Unbound Form of Granule-Bound Starch Synthase in Glycogen-Accumulating Mutants of Chlamydomonas reinhardtii1

    PubMed Central

    Dauvillée, David; Colleoni, Christophe; Shaw, Eudean; Mouille, Gregory; D'Hulst, Christophe; Morell, Matthew; Samuel, Michael S.; Bouchet, Brigitte; Gallant, Daniel J.; Sinskey, Anthony; Ball, Steven

    1999-01-01

    In vascular plants, mutations leading to a defect in debranching enzyme lead to the simultaneous synthesis of glycogen-like material and normal starch. In Chlamydomonas reinhardtii comparable defects lead to the replacement of starch by phytoglycogen. Therefore, debranching was proposed to define a mandatory step for starch biosynthesis. We now report the characterization of small amounts of an insoluble, amylose-like material found in the mutant algae. This novel, starch-like material was shown to be entirely dependent on the presence of granule-bound starch synthase (GBSSI), the enzyme responsible for amylose synthesis in plants. However, enzyme activity assays, solubilization of proteins from the granule, and western blots all failed to detect GBSSI within the insoluble polysaccharide matrix. The glycogen-like polysaccharides produced in the absence of GBSSI were proved to be qualitatively and quantitatively identical to those produced in its presence. Therefore, we propose that GBSSI requires the presence of crystalline amylopectin for granule binding and that the synthesis of amylose-like material can proceed at low levels without the binding of GBSSI to the polysaccharide matrix. Our results confirm that amylopectin synthesis is completely blocked in debranching-enzyme-defective mutants of C. reinhardtii. PMID:9880375

  7. The marine natural-derived inhibitors of glycogen synthase kinase-3β phenylmethylene hydantoins: In vitro and in vivo activities and pharmacophore modeling

    PubMed Central

    Khanfar, Mohammad A.; Asal, Bilal Abu; Mudit, Mudit; Kaddoumi, Amal; El Sayed, Khalid A.

    2009-01-01

    The Red Sea sponge Hemimycale arabica afforded the known (Z)-5-(4-hydroxybenzylidene)-hydantoin (1). This natural phenylmethylene hydantoin (PMH) 1 and the synthetic (Z)-5-(4-(ethylthio)benzylidene)-hydantoin (2) showed potent in vitro and in vivo anti-growth and anti-invasive properties against PC-3M prostate cancer cells in MTT, spheroid disaggregation, and in mice models. To explore a possible molecular target of PMHs, the most potent synthetic analogue 2 has been virtually screened against various protein kinases. Molecular modeling study has shown that 2 can be successfully docked within the binding pocket of glycogen synthase kinase-3beta (GSK-3β) similar to the well-known GSK-3β inhibitor I-5. Several PMHs showed potent in vitro GSK-3β inhibitory activity with an IC50 range of 4–20 µM. The most potent analogue 3 showed a significant increase in liver glycogen level at the 5, 15, and 25 mg/kg dose levels, in vivo. Pharmacophore model was built and validated using in-house database of active and inactive GSK-3β inhibitors. The GSK-3β inhibitory activity of PMHs entitles them to be potential leads for the treatment of cancer, Alzheimer’s disease, bipolar disorders, stroke, different tau pathologies, and type-2 diabetes. PMID:19616957

  8. Treadmill exercise decreases incidence of Alzheimer's disease by suppressing glycogen synthase kinase-3β expression in streptozotocin-induced diabetic rats.

    PubMed

    Kim, Dae-Young; Jung, Sun-Young; Kim, Tae-Woon; Lee, Kwang-Sik; Kim, Kijeong

    2015-04-01

    Diabetes is a metabolic disorder, and it is considered as a major risk factor for Alzheimer's disease (AD). In the present study, we evaluated whether treadmill exercise ameliorates progression of AD in relation with glycogen synthase kinase-3β (GSK-3β) activity using streptozotocin (STZ)-induced diabetic rats. For this study, step-down avoidance task, immunohistochemistry for glycogen synthase kinase-3β (GSK-3β) and tau, and western blot for phosphor-phosphoinositide 3 kinase (p-PI3K)/PI3K and phosphor-Akt (p-Akt)/Akt were performed. Diabetes mellitus was induced by intraperitoneal injection of STZ. The rats in the exercise groups were made to run on the treadmill for 30 min per one day, five times a week, during 12 weeks. The present results showed that short-term and long-term latencies in the step-down avoidance task were decreased by induction of diabetes, and treadmill exercise inhibited these latencies in the diabetic rats. Induction of diabetes suppressed the ratio of p-PI3K to PI3K and the ratio of p-Akt to Akt, and treadmill exercise increased these ratios in the diabetic rats. The numbers of GSK-3β-positive and tau-positive cells in the hippocampal dentate gyrus was higher in the diabetes-induction group than that in the control group, and treadmill exercise inhibited these numbers in the diabetic rats. In the present study, treadmill exercise suppressed hyperphosphorylation of tau in the hippocampus by decreased GSK-3β activity through PI3K/Akt pathway activation in the diabetic rats. Based on the present results, treadmill exercise may helpful to prevent diabetes-associated AD occurrence.

  9. 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

  10. Arabidopsis Brassinosteroid-Insensitive dwarf12 Mutants Are Semidominant and Defective in a Glycogen Synthase Kinase 3β-Like Kinase1

    PubMed Central

    Choe, Sunghwa; Schmitz, Robert J.; Fujioka, Shozo; Takatsuto, Suguru; Lee, Mi-Ok; Yoshida, Shigeo; Feldmann, Kenneth A.; Tax, Frans E.

    2002-01-01

    Mutants defective in the biosynthesis or signaling of brassinosteroids (BRs), plant steroid hormones, display dwarfism. Loss-of-function mutants for the gene encoding the plasma membrane-located BR receptor BRI1 are resistant to exogenous application of BRs, and characterization of this protein has contributed significantly to the understanding of BR signaling. We have isolated two new BR-insensitive mutants (dwarf12-1D and dwf12-2D) after screening Arabidopsis ethyl methanesulfonate mutant populations. dwf12 mutants displayed the characteristic morphology of previously reported BR dwarfs including short stature, short round leaves, infertility, and abnormal de-etiolation. In addition, dwf12 mutants exhibited several unique phenotypes, including severe downward curling of the leaves. Genetic analysis indicates that the two mutations are semidominant in that heterozygous plants show a semidwarf phenotype whose height is intermediate between wild-type and homozygous mutant plants. Unlike BR biosynthetic mutants, dwf12 plants were not rescued by high doses of exogenously applied BRs. Like bri1 mutants, dwf12 plants accumulated castasterone and brassinolide, 43- and 15-fold higher, respectively, providing further evidence that DWF12 is a component of the BR signaling pathway that includes BRI1. Map-based cloning of the DWF12 gene revealed that DWF12 belongs to a member of the glycogen synthase kinase 3β family. Unlike human glycogen synthase kinase 3β, DWF12 lacks the conserved serine-9 residue in the auto-inhibitory N terminus. In addition, dwf12-1D and dwf12-2D encode changes in consecutive glutamate residues in a highly conserved TREE domain. Together with previous reports that both bin2 and ucu1 mutants contain mutations in this TREE domain, this provides evidence that the TREE domain is of critical importance for proper function of DWF12/BIN2/UCU1 in BR signal transduction pathways. PMID:12428015

  11. Impaired metabolic function of polymorphonuclear leukocytes in glycogen storage disease Ib.

    PubMed

    Gahr, M; Heyne, K

    1983-09-01

    To elucidate the basis for the recurrent infections in patients with glycogen storage disease (GSD) Ib we tested polymorphonuclear leukocyte (PMN) function in one patient. Bactericidal capacity and phagocytosis-induced O2 consumption were reduced. Also, phorbol myristate acetate-stimulated superoxide production and glucose oxidation through the hexose monophosphate shunt were diminished compared to control subjects. Therefore it could be speculated that in PMN of patients with GSD Ib, glucose-6-phosphate has no access to the enzymes of the hexose monophosphate shunt due to a transport-related defect as shown for glucogenesis in hepatocytes.

  12. Impaired autophagy contributes to muscle atrophy in glycogen storage disease type II patients.

    PubMed

    Nascimbeni, Anna Chiara; Fanin, Marina; Masiero, Eva; Angelini, Corrado; Sandri, Marco

    2012-11-01

    The autophagy-lysosome system is essential for muscle cell homeostasis and its dysfunction has been linked to muscle disorders that are typically distinguished by massive autophagic buildup. Among them, glycogen storage disease type II (GSDII) is characterized by the presence of large glycogen-filled lysosomes in the skeletal muscle, due to a defect in the lysosomal enzyme acid α-glucosidase (GAA). The accumulation of autophagosomes is believed to be detrimental for myofiber function. However, the role of autophagy in the pathogenesis of GSDII is still unclear. To address this issue we monitored autophagy in muscle biopsies and myotubes of early and late-onset GSDII patients at different time points of disease progression. Moreover we also analyzed muscles from patients treated with enzyme replacement therapy (ERT). Our data suggest that autophagy is a protective mechanism that is required for myofiber survival in late-onset forms of GSDII. Importantly, our findings suggest that a normal autophagy flux is important for a correct maturation of GAA and for the uptake of recombinant human GAA. In conclusion, autophagy failure plays an important role in GSDII disease progression, and the development of new drugs to restore the autophagic flux should be considered to improve ERT efficacy.

  13. Synthesis and evaluation of 8-amino-[1,2,4]triazolo[4,3-a]pyridin-3(2H)-one derivatives as glycogen synthase kinase-3 (GSK-3) inhibitors.

    PubMed

    Chun, Kwangwoo; Park, Ji-Seon; Lee, Han-Chang; Kim, Young-Ha; Ye, In-Hea; Kim, Kang-Jeon; Ku, Il-Whea; Noh, Min-Young; Cho, Goang-Won; Kim, Heejaung; Kim, Seung Hyun; Kim, Jeongmin

    2013-07-01

    New potent glycogen synthase kinase-3 (GSK-3) inhibitors, 8-amino-[1,2,4]triazolo[4,3-a]pyridin-3(2H)-one derivatives, were designed by modeling, synthesized and evaluated in vitro. Compound 17c showed good potency in enzyme and cell-based assays (IC50=111 nM, EC50=1.78 μM). Moreover, it has demonstrated desirable water solubility, PK profile, and moderate brain penetration.

  14. Mechanisms limiting glycogen storage in muscle during prolonged insulin stimulation

    SciTech Connect

    Richter, E.A.; Hansen, S.A.; Hansen, B.F. )

    1988-11-01

    The extent to which muscle glycogen concentrations can be increased during exposure to maximal insulin concentrations and abundant glucose was investigated in the isolated perfused rat hindquarter preparation. Perfusion for 7 h in the presence of 20,000 {mu}U/ml insulin and 11-13 mM glucose increased muscle glycogen concentrations to maximal values 2, 3, and 3.5 times above normal fed levels in fast-twitch white, slow-twitch red, and fast-twitch red fibers, respectively. Glucose uptake decreased from 34.9 {mu}mol{center dot}g{sup {minus}1}{center dot}h{sup {minus}1} at 0 h to 7.5 after 7 h of perfusion. During the perfusion muscle glycogen synthase activity decreased and free intracellular glucose and glucose 6-phosphate increased indicating that glucose disposal was impaired. However, glucose transport as measured by the uptake of 3-O-({sup 14}C)methyl-D-glucose was also markedly decreased after 5 and 7 h of perfusion compared with initial values. Total muscle water concentration decreased during glycogen loading of the muscles. Mechanisms limiting glycogen storage under maximal insulin stimulation include impaired insulin-stimulated membrane transport of glucose as well as impaired intracellular glucose disposal.

  15. Protein kinase A negatively modulates the nuclear accumulation of NF-ATc1 by priming for subsequent phosphorylation by glycogen synthase kinase-3.

    PubMed

    Sheridan, Colleen M; Heist, E Kevin; Beals, Chan R; Crabtree, Gerald R; Gardner, Phyllis

    2002-12-13

    The nuclear localization and transcriptional activity of the NF-ATc family of transcription factors, essential to many developmental, differentiation, and adaptation processes, are determined by the opposing activities of the phosphatase calcineurin, which promotes nuclear accumulation of NF-ATc, and several kinases, which promote cytoplasmic accumulation. Many reports suggest that protein kinase A (PKA) negatively modulates calcineurin-mediated NF-ATc activation. Here we show that overexpression of PKA causes phosphorylation and cytoplasmic accumulation of NF-ATc1 in direct opposition to calcineurin by phosphorylating Ser-245, Ser-269, and Ser-294 in the conserved serine-proline repeat domain, and that mutation of these serines blocks the effect of PKA. Activation of endogenous PKA is similarly able to promote phosphorylation of these sites on NF-ATc1 in two lymphoid cell lines. We further show that a complete block of NF-ATc1 nuclear localization by PKA requires a second kinase activity that can be supplied by glycogen synthase kinase-3 (GSK-3), and that mutation of either the PKA phosphorylation sites or the upstream GSK-3 sites prevents the effect of PKA. Thus, we propose that PKA functions cooperatively as a priming kinase for further phosphorylation by GSK-3 to oppose calcineurin-mediated nuclear accumulation and transcriptional activity of NF-ATc1 and that, through this mechanism, PKA may be an important modulator of many NF-ATc-dependent processes.

  16. 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.

  17. 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.

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

    PubMed Central

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

    2015-01-01

    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

  19. Trypanosoma brucei Glycogen Synthase Kinase-3, A Target for Anti-Trypanosomal Drug Development: A Public-Private Partnership to Identify Novel Leads

    PubMed Central

    Oduor, Richard O.; Ojo, Kayode K.; Williams, Gareth P.; Bertelli, Francois; Mills, James; Maes, Louis; Pryde, David C.; Parkinson, Tanya; Van Voorhis, Wesley C.; Holler, Tod P.

    2011-01-01

    Background Trypanosoma brucei, the causative agent of Human African Trypanosomiasis (HAT), expresses two proteins with homology to human glycogen synthase kinase 3β (HsGSK-3) designated TbruGSK-3 short and TbruGSK-3 long. TbruGSK-3 short has previously been validated as a potential drug target and since this enzyme has also been pursued as a human drug target, a large number of inhibitors are available for screening against the parasite enzyme. A collaborative industrial/academic partnership facilitated by the World Health Organisation Tropical Diseases Research division (WHO TDR) was initiated to stimulate research aimed at identifying new drugs for treating HAT. Methodology/Principal Findings A subset of over 16,000 inhibitors of HsGSK-3 β from the Pfizer compound collection was screened against the shorter of two orthologues of TbruGSK-3. The resulting active compounds were tested for selectivity versus HsGSK-3β and a panel of human kinases, as well as in vitro anti-trypanosomal activity. Structural analysis of the human and trypanosomal enzymes was also performed. Conclusions/Significance We identified potent and selective compounds representing potential attractive starting points for a drug discovery program. Structural analysis of the human and trypanosomal enzymes also revealed hypotheses for further improving selectivity of the compounds. PMID:21483717

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

    PubMed

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

    2016-03-22

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

  1. 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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

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

    2011-01-01

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

  4. Glycogen synthase kinase-3beta (GSK3beta) negatively regulates PTTG1/human securin protein stability, and GSK3beta inactivation correlates with securin accumulation in breast tumors.

    PubMed

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

    2011-08-26

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

  5. N-(4-bromophenethyl) Caffeamide Inhibits Melanogenesis by Regulating AKT/Glycogen Synthase Kinase 3 Beta/Microphthalmia-associated Transcription Factor and Tyrosinase-related Protein 1/Tyrosinase.

    PubMed

    Kuo, Yueh-Hsiung; Chen, Chien-Chia; Lin, Ping; You, Ya-Jhen; Chiang, Hsiu-Mei

    2015-01-01

    Skin color is primarily produced by melanin, which is a crucial pigment that protects the skin from UV-induced damage and prevents carcinogenesis. However, accumulated melanin in the skin may cause hyperpigmentation and related disorders. Melanin synthesis comprises consecutive oxidative reactions, and tyrosinase is the enzyme that catalyzes the rate-limiting process of melanogenesis. In this study, tyrosinase-related protein 1 (TRP-1) and TRP-2 contributed to melanin formation. N-(4-bromophenethyl) caffeamide ((E)-N-(4-bromophenethyl)-3-(3,4-dihydroxyphenyl)acrylamide; K36H), a caffeic acid phenyl amide derivative, inhibited α-melanocyte-stimulating hormone (α-MSH)-induced melanogenesis and tyrosinase activity in B16F0 cells. In addition, K36H reduced the protein expression of the phospho-cAMP response element binding protein (p-CREB), microphthalmia-associated transcription factor (MITF), tyrosinase, and TRP-1. Moreover, K36H promoted AKT and glycogen synthase kinase 3 beta (GSK3β) phosphorylation, thereby inhibiting MITF transcription activity. Thus, K36H attenuated α-MSH-induced cAMP pathways, contributing to hypopigmentation. The results of a safety assay revealed that K36H did not exhibit cytotoxicity or irritate the skin or eyes. According to these results, K36H may have the potential to be used as a whitening agent in the cosmetic and pharmaceutical industries.

  6. Measurement of effector protein injection by type III and type IV secretion systems by using a 13-residue phosphorylatable glycogen synthase kinase tag.

    PubMed

    Garcia, Julie Torruellas; Ferracci, Franco; Jackson, Michael W; Joseph, Sabrina S; Pattis, Isabelle; Plano, Lisa R W; Fischer, Wolfgang; Plano, Gregory V

    2006-10-01

    Numerous bacterial pathogens use type III secretion systems (T3SSs) or T4SSs to inject or translocate virulence proteins into eukaryotic cells. Several different reporter systems have been developed to measure the translocation of these proteins. In this study, a peptide tag-based reporter system was developed and used to monitor the injection of T3S and T4S substrates. The glycogen synthase kinase (GSK) tag is a 13-residue phosphorylatable peptide tag derived from the human GSK-3beta kinase. Translocation of a GSK-tagged protein into a eukaryotic cell results in host cell protein kinase-dependent phosphorylation of the tag, which can be detected with phosphospecific GSK-3beta antibodies. A series of expression plasmids encoding Yop-GSK fusion proteins were constructed to evaluate the ability of the GSK tag to measure the injection of Yops by the Yersinia pestis T3SS. GSK-tagged YopE, YopH, LcrQ, YopK, YopN, and YopJ were efficiently phosphorylated when translocated into HeLa cells. Similarly, the injection of GSK-CagA by the Helicobacter pylori T4SS into different cell types was measured via phosphorylation of the GSK tag. The GSK tag provides a simple method to monitor the translocation of T3S and T4S substrates.

  7. Glycogen synthase kinase-3β positively regulates protein synthesis and cell proliferation through the regulation of translation initiation factor 4E-binding protein 1.

    PubMed

    Shin, S; Wolgamott, L; Tcherkezian, J; Vallabhapurapu, S; Yu, Y; Roux, P P; Yoon, S-O

    2014-03-27

    Protein synthesis has a key role in the control of cell proliferation, and its deregulation is associated with pathological conditions, notably cancer. Rapamycin, an inhibitor of mammalian target of rapamycin complex 1 (mTORC1), was known to inhibit protein synthesis. However, it does not substantially inhibit protein synthesis and cell proliferation in many cancer types. We were interested in finding a novel target in rapamycin-resistant cancer. The rate-limiting factor for translation is eukaryotic translation initiation factor 4E (eIF4E), which is negatively regulated by eIF4E-binding protein 1 (4E-BP1). Here, we provide evidence that glycogen synthase kinase (GSK)-3β promotes cell proliferation through positive regulation of protein synthesis. We found that GSK-3β phosphorylates and inactivates 4E-BP1, thereby increasing eIF4E-dependent protein synthesis. Considering the clinical relevance of pathways regulating protein synthesis, our study provides a promising new strategy and target for cancer therapy.

  8. Neuropilin 2 Signaling Is Involved in Cell Positioning of Adult-born Neurons through Glycogen Synthase Kinase-3β (GSK3β).

    PubMed

    Ng, Teclise; Hor, Catherine H H; Chew, Benjamin; Zhao, Jing; Zhong, Zhong; Ryu, Jae Ryun; Goh, Eyleen L K

    2016-11-25

    Proper positioning of neurons is fundamental for brain functions. However, little is known on how adult-born neurons generated in the hilar side of hippocampal dentate gyrus migrate into the granular cell layer. Because class 3 Semaphorins (Sema3) are involved in dendritic growth of these newborn neurons, we examined whether they are essential for cell positioning. We disrupted Sema3 signaling by silencing neuropilin 1 (NRP1) or 2 (NRP2), the main receptors for Sema3A and Sema3F, in neural progenitors of adult mouse dentate gyrus. Silencing of NRP2, but not NRP1, affected cell positioning of adult newborn neurons. Glycogen synthase kinase-3β (GSK3β) knockdown phenocopied this NRP2 silencing-mediated cell positioning defect, but did not affect dendritic growth. Furthermore, GSK3β is activated upon stimulation with Sema3F, and GSK3β overexpression rescued the cell positioning phenotypes seen in NRP2-deficient neurons. These results point to a new role for NRP2 in the positioning of neurons during adult hippocampal neurogenesis, acting via the GSK3β signaling pathway. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. 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

  10. 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.

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

    PubMed

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

    2014-10-01

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

  12. Glycogen synthase kinase-3 inactivation and stabilization of beta-catenin induce nephron differentiation in isolated mouse and rat kidney mesenchymes.

    PubMed

    Kuure, Satu; Popsueva, Anna; Jakobson, Madis; Sainio, Kirsi; Sariola, Hannu

    2007-04-01

    Wnt proteins are required for induction of nephrons in mouse metanephric kidneys, but the downstream pathways that mediate tubule induction and epithelial differentiation have remained obscure. The intracellular mechanisms by which Wnt signaling mediates nephron induction in embryonic kidney mesenchymes were studied. First is shown that transient exposure of isolated kidney mesenchymes to structurally different glycogen synthase kinase-3 (GSK3) inhibitors lithium or 6-bromoindirubin-3'-oxime results in abundant epithelial differentiation and full segregation of nephrons. Shown further by mice with genetically disrupted ureteric bud or Wolffian duct development is that this nephrogenic competence arises independent of the influence of Wolffian duct-derived epithelia. Analysis of the intracellular signaling cascades downstream of GSK3 inhibition revealed stabilization of beta-catenin and upregulation of Lef1 and Tcf1, both events that are associated with the active canonical Wnt signaling. Last, genetic evidence that metanephric mesenchyme-specific stabilization of beta-catenin is sufficient to induce nephron differentiation in isolated kidney mesenchymes, similar to that induced by GSK3 inhibitors, is provided. These data show that activation of canonical Wnt pathway is sufficient to induce nephrogenesis and suggest that this pathway mediates the nephron induction in murine kidney mesenchymes.

  13. 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

  14. Glycogen synthase kinase-3 beta regulates Snail and β-catenin expression during Fas-induced epithelial-mesenchymal transition in gastrointestinal cancer.

    PubMed

    Zheng, Haoxuan; Li, Wenjing; Wang, Yadong; Liu, Zhizhong; Cai, Yidong; Xie, Tingting; Shi, Meng; Wang, Zhiqing; Jiang, Bo

    2013-08-01

    Fas signalling has been shown to induce the epithelial-mesenchymal transition (EMT) to promote gastrointestinal (GI) cancer metastasis, but its mechanism of action is still unknown. The effects of Fas-ligand (FasL) treatment and inhibition of Fas signalling on GI cancer cells were tested using invasion assay, immunofluorescence, immunoblot, Reverse Transcription Polymerase Chain Reaction (RT-PCR), quantitative Real-time PCR (qRT-PCR), immunoprecipitation and luciferase reporter assay. Immunohistochemistry was used to analyse the EMT-associated molecules in GI cancer specimens. FasL treatment inhibited E-cadherin transcription by upregulation of Snail in GI cancer cells. The nuclear expression and transcriptional activity of Snail and β-catenin were increased by inhibitory phosphorylation of glycogen synthase kinase-3 beta (GSK-3β) at Ser9 by FasL-induced extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) signalling. Snail associated with β-catenin in the nucleus and, thus, increased β-catenin transcriptional activity. Evaluation of human GI cancer specimens showed that the expression of FasL, phospho-GSK-3β, Snail and β-catenin increase during GI cancer progression. An EMT phenotype was shown to correlate with an advanced cancer stage, and a non-EMT phenotype significantly correlated with a better prognosis. Collectively, these data indicate that GSK-3β regulates Snail and β-catenin expression during Fas-induced EMT in gastrointestinal cancer.

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

    PubMed

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

    2015-09-01

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

  16. Valproic Acid Modifies Synaptic Structure and Accelerates Neurite Outgrowth Via the Glycogen Synthase Kinase-3β Signaling Pathway in an Alzheimer's Disease Model.

    PubMed

    Long, Zhi-Min; Zhao, Lei; Jiang, Rong; Wang, Ke-Jian; Luo, Shi-Fang; Zheng, Min; Li, Xiao-Feng; He, Gui-Qiong

    2015-11-01

    Tau hyperphosphorylation and amyloid β-peptide overproduction, caused by altered localization or abnormal activation of glycogen synthase kinase-3β (GSK-3β), is a pathogenic mechanism in Alzheimer's disease (AD). Valproic acid (VPA) attenuates senile plaques and neuronal loss. Here, we confirmed that VPA treatment improved spatial memory in amyloid precursor protein (APP)/presenilin 1 (PS 1) double-transgenic mice and investigated the effect of VPA on synaptic structure and neurite outgrowth. We used ultrastructural analysis, immunocytochemistry, immunofluorescence staining, and Western blot analysis to assess the effect of VPA treatment in mice. VPA treatment thickened the postsynaptic density, increased the number of presynaptic vesicles, and upregulated the expression of synaptic markers PSD-95 and GAP43. VPA increased neurite length of hippocampal neurons in vivo and in vitro. In VPA-treated AD mouse brain, inactivated GSK-3β (pSer9-GSK-3β) was markedly increased, while hyperphosphorylation of tau at Ser396 and Ser262 was decreased; total tau levels remained similar. VPA treatment notably improved pSer133-cAMP response element-binding protein (CREB) and brain-derived neurotrophic factor (BDNF) levels, which are associated with synaptic function and neurite outgrowth. VPA improves behavioral deficits in AD, modifies synaptic structure, and accelerates neurite outgrowth, by inhibiting the activity of GSK-3β, decreasing hyperphosphorylated tau, enhancing CREB and BDNF expression. © 2015 John Wiley & Sons Ltd.

  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.

  18. Intracellular domains of amyloid precursor-like protein 2 interact with CP2 transcription factor in the nucleus and induce glycogen synthase kinase-3beta expression.

    PubMed

    Xu, Y; Kim, H-S; Joo, Y; Choi, Y; Chang, K-A; Park, C H; Shin, K-Y; Kim, S; Cheon, Y-H; Baik, T-K; Kim, J-H; Suh, Y-H

    2007-01-01

    Amyloid precursor protein (APP) is a member of a gene family that includes two APP-like proteins, APLP1 and 2. Recently, it has been reported that APLP1 and 2 undergo presenilin-dependent gamma-secretase cleavage, as does APP, resulting in the release of an approximately 6 kDa intracellular C-terminal domain (ICD), which can translocate into the nucleus. In this study, we demonstrate that the APLP2-ICDs interact with CP2/LSF/LBP1 (CP2) transcription factor in the nucleus and induce the expression of glycogen synthase kinase 3beta (GSK-3beta), which has broad-ranged substrates such as tau- and beta-catenin. The significance of this finding is substantiated by the in vivo evidence of the increase in the immunoreactivities for the nuclear C-terminal fragments of APLP2, and for GSK-3beta in the AD patients' brain. Taken together, these results suggest that APLP2-ICDs contribute to the AD pathogenesis, by inducing GSK-3beta expression through the interaction with CP2 transcription factor in the nucleus.

  19. 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

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

    NASA Astrophysics Data System (ADS)

    Huang, Lei; Wu, Shengnan; Xing, Da

    2011-03-01

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

  1. 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

  2. Decreased Glycogen Content Might Contribute to Chronic Stress-Induced Atrophy of Hippocampal Astrocyte volume and Depression-like Behavior in Rats

    PubMed Central

    Zhao, Yunan; Zhang, Qiang; Shao, Xiao; Ouyang, Liufeng; Wang, Xin; Zhu, Kexuan; Chen, Lin

    2017-01-01

    The involvement of brain glycogen in the progress of chronic stress-induced impairment of hippocampal astrocyte structural plasticity and depression-like behavior is yet to be clarified. The present study designed three experiments to determine the role of brain glycogen in the plasticity and behavioral consequences of chronic stress. Time course studies on brain glycogen, astrocytes, and behavioral responses to stress were conducted in Experiment 1. Chronic stress decreased the hippocampal glycogen levels, reduced astrocytic size and protrusion length in the hippocampus, and induced depression-like behavior. Glycogen synthase 1 mRNA in the hippocampus was silenced by lentiviral vector-based RNA interference (RNAi) in Experiment 2. This RNAi produced a lack of glycogen in the hippocampus, decreased the hippocampal astrocyte size, and induced depressive behavior in rats. The mechanisms of chronic stress-induced brain glycogen decrease were investigated in Experiment 3. Chronic stress promoted hippocampal glycogen breakdown and increased hippocampal glycogen synthesis. Results suggest that decreased glycogen content was associated with chronic stress-induced atrophy of hippocampal astrocyte size and depression-like behavior. Furthermore, the decrease of glycogen content in the hippocampus might be due to the compensation of glycogen synthesis for breakdown in an insufficient manner. PMID:28233800

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

    PubMed Central

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

    2004-01-01

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

  4. Glycogen synthase kinase-3 (GSK3) regulates TNF production and haemocyte phagocytosis in the immune response of Chinese mitten crab Eriocheir sinensis.

    PubMed

    Li, Xiaowei; Jia, Zhihao; Wang, Weilin; Wang, Lingling; Liu, Zhaoqun; Yang, Bin; Jia, Yunke; Song, Xiaorui; Yi, Qilin; Qiu, Limei; Song, Linsheng

    2017-03-29

    Glycogen synthase kinase-3 (GSK3) is a serine/threonine protein kinase firstly identified as a regulator of glycogen synthesis. Recently, it has been proved to be a key regulator of the immune reaction. In the present study, a GSK3 homolog gene (designated as EsGSK3) was cloned from Chinese mitten crab, Eriocheir sinensis. The open reading frame (ORF) was 1824 bp, which encoded a predicted polypeptide of 607 amino acids. There was a conserved Serine/Threonine Kinase domain and a DNA binding domain found in EsGSK3. Phylogenetic analysis showed that EsGSK3 was firstly clustered with GSK3-β from oriental river prawn Macrobrachium nipponense in the invertebrate branch, while GSK3s from vertebrates formed the other distinct branch. EsGSK3 mRNA transcripts could be detected in all tested tissues of the crab including haepatopancreas, eyestalk, muscle, gonad, haemocytes and haematopoietic tissue with the highest expression level in haepatopancreas. And EsGSK3 protein was mostly detected in the cytoplasm of haemocyte by immunofluorescence analysis. The expression levels of EsGSK3 mRNA increased significantly at 6 h after Aeromonas hydrophila challenge (p < 0.05) in comparison with control group, and then gradually decreased to the initial level at 48 h (p > 0.05). The mRNA expression of lipopolysaccharide-induced tumor necrosis factor (TNF)-α factor (EsLITAF) was also induced by A. hydrophila challenge. However, the mRNA expression of EsLITAF and TNF-α production was significantly suppressed after EsGSK3 was blocked in vivo with specific inhibitor lithium, while the phagocytosis of crab haemocytes was significantly promoted. These results collectively demonstrated that EsGSK3 could regulate the innate immune responses of E. sinensis by promoting TNF-α production and inhibiting haemocyte phagocytosis.

  5. Impaired Nitric Oxide Synthase Signaling Dissociates Social Investigation and Aggression

    PubMed Central

    Trainor, Brian C.; Workman, Joanna L.; Jessen, Ruth; Nelson, Randy J.

    2007-01-01

    A combination of social withdrawal and increased aggression is characteristic of several mental disorders. Most previous studies have investigated the neurochemical bases of social behavior and aggression independently, as opposed to how these behaviors are regulated in concert. Neuronal nitric oxide synthase (nNOS) produces gaseous nitric oxide, which functions as a neurotransmitter and is known to affect several types of behavior including mating and aggression. Compared with wild-type mice, we observed that nNOS knockout mice showed reduced behavioral responses to an intruder behind a wire barrier. Similar results were observed in mice treated with the selective nNOS inhibitor 3-bromo-7-nitroindazole (3BrN). In habituation–dishabituation tests, treatment with 3BrN did not block recognition of male urine but did attenuate investigation time compared with oil-treated animals. Finally, nNOS knockout mice and 3BrN treated mice were significantly more aggressive than wild-type and oil-treated males, respectively. In general, these behavioral effects are less pronounced in pair-housed males compared with singly-housed males. Thus, nNOS inhibition results in a phenotype that displays reduced social investigation and increased aggression. These data suggest that further study of nNOS signaling is warranted in mental disorders characterized by social withdrawal and increased aggression. PMID:17469926

  6. Glycogen Phosphorylation and Lafora disease

    PubMed Central

    Roach, Peter J.

    2015-01-01

    Covalent phosphorylation of glycogen, first described 35 years ago, was put on firm ground through the work of the Whelan laboratory in the 1990s. But glycogen phosphorylation lay fallow until interest was rekindled in the mid 2000s by the finding that it could be removed by a glycogen-binding phosphatase, laforin, and that mutations in laforin cause a fatal teenage-onset epilepsy, called Lafora disease. Glycogen phosphorylation is due to phosphomonoesters at C2, C3 and C6 of glucose residues. Phosphate is rare, ranging from 1:500 - 1:5000 phosphates/glucose depending on the glycogen source. The mechanisms of glycogen phosphorylation remain under investigation but one hypothesis to explain C2 and perhaps C3 phosphate is that it results from a rare side reaction of the normal synthetic enzyme glycogen synthase. Lafora disease is likely caused by over-accumulation of abnormal glycogen in insoluble deposits termed Lafora bodies in neurons. The abnormality in the glycogen correlates with elevated phosphorylation (at C2, C3 and C6), reduced branching, insolubility and an enhanced tendency to aggregate and become insoluble. Hyperphosphorylation of glycogen is emerging as an important feature of this deadly childhood disease PMID:26278984

  7. Inhibition of Hepatic Glycogen Synthesis by Hyperhomocysteinemia Mediated by TRB3

    PubMed Central

    Liu, Wen-Jing; Ma, Lan-Qing; Liu, Wei-Hua; Zhou, Wei; Zhang, Ke-Qin; Zou, Cheng-Gang

    2011-01-01

    Recently, epidemiological and experimental studies have linked hyperhomocysteinemia (HHcy) to insulin resistance. However, whether HHcy impairs glucose homeostasis by affecting glycogenesis in the liver is not clear. In the present study, we investigated the effect of HHcy on hepatic glycogen synthesis. Hyperhomocysteinemia was induced in mice by drinking water containing two percent methionine. Mice with HHcy showed an increase in the phosphorylation of glycogen synthase and a significant decrease in hepatic glycogen content and the rate of glycogen synthesis. The expression of TRB3 (tribbles-related protein 3) was up-regulated in the liver of mice with HHcy, concomitantly with the dephosphorylation of glycogen synthase kinase-3β and Akt. The knockdown of TRB3 by short hairpin RNA suppressed the dephosphorylation of these two kinases. Homocysteine induced an increase in the levels of hepatic cAMP and cAMP response element-binding protein phosphorylation, which in turn up-regulated the expression of peroxisome proliferator–activated receptor (PPAR)-γ coactivator-1α and TRB3. The inhibition of PPAR-α by its inhibitor, MK886, or knockdown of PPAR-α by small interfering RNA significantly inhibited the expression of TRB3 induced by homocysteine. The current study demonstrates that HHcy impairs hepatic glycogen synthesis by inducing the expression of TRB3. These results provide a novel explanation for the development and progression of insulin resistance in HHcy. PMID:21435438

  8. Inhibition of Cyclin-Dependent Kinase 5 but Not of Glycogen Synthase Kinase 3-β Prevents Neurite Retraction and Tau Hyperphosphorylation Caused by Secretable Products of Human T-Cell Leukemia Virus Type I-Infected Lymphocytes

    PubMed Central

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

    2012-01-01

    Human T-cell leukemia virus type I (HTLV-I)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a neurodegenerative disease characterized by selective loss of axons and myelin in the corticospinal tracts. This central axonopathy may originate from the impairment of anterograde axoplasmic transport. Previous work showed tau hyperphosphorylation at T181 in cerebrospinal fluid of HAM/TSP patients. Similar hyperphosphorylation occurs in SH-SY5Y cells incubated with supernatant from MT-2 cells (HTLV-I-infected lymphocytes secreting viral proteins, including Tax) that produce neurite shortening. Tau phosphorylation at T181 is attributable to glycogen synthase kinase 3-β (GSK3-β) and cyclin-dependent kinase 5 (CDK5) activation. Here we investigate whether neurite retraction in the SH-SY5Y model associates with concurrent changes in other tau hyperphosphorylable residues. Threonine 181 turned out to be the only tau hyperphosphorylated residue. We also evaluate the role of GSK3-β and CDK5 in this process by using specific kinase inhibitors (LiCl, TDZD-8, and roscovitine). Changes in both GSK3-β active and inactive forms were followed by measuring the regulatory phosphorylable sites (S9 and Y216, inactivating and activating phosphorylation, respectively) together with changes in β-catenin protein levels. Our results showed that LiCl and TDZD-8 were unable to prevent MT-2 supernatant-mediated neurite retraction and also that neither Y216 nor S9 phosphorylations were changed in GSK3-β. Thus, GSK3-β seems not to play a role in T181 hyperphosphorylation. On the other hand, the CDK5 involvement in tau phosphorylation was confirmed by both the increase in its enzymatic activity and the absence of MT-2 neurite retraction in the presence of roscovitine or CDK5 siRNA transfection. PMID:21671254

  9. Glycogen Synthase Kinase-3β (GSK-3β) Inhibition Enhances Dendritic Cell-based Cancer Vaccine Potency via Suppression of Interferon-γ-induced Indoleamine 2,3-Dioxygenase Expression.

    PubMed

    Noh, Kyung Tae; Son, Kwang Hee; Jung, In Duk; Kang, Tae Heung; Choi, Chang Hun; Park, Yeong-Min

    2015-05-08

    Indoleamine 2,3-dioxygenase (IDO) functions as a crucial mediator of tumor-mediated immune tolerance by causing T-cell suppression via tryptophan starvation in a tumor environment. Glycogen synthase kinase-3β (GSK-3β) is also involved in immune and anti-tumor responses. However, the relativity of these proteins has not been as well defined. Here, we found that GSK-3β-dependent IDO expression in the dendritic cell (DC) plays a role in anti-tumor activity via the regulation of CD8(+) T-cell polarization and cytotoxic T lymphocyte activity. By the inhibition of GSK-3β, attenuated IDO expression and impaired JAK1/2-Stat signaling crucial for IDO expression were observed. Protein kinase Cδ (PKCδ) activity and the interaction between JAK1/2 and Stat3, which are important for IDO expression, were also reduced by GSK-3β inhibition. CD8(+) T-cell proliferation mediated by OVA-pulsed DC was blocked by interferon (IFN)-γ-induced IDO expression via GSK-3β activity. Specific cytotoxic T lymphocyte activity mediated by OVA-pulsed DC against OVA-expressing EG7 thymoma cells but not OVA-nonexpressing EL4 thymoma cells was also attenuated by the expressed IDO via IFN-γ-induced activation of GSK-3β. Furthermore, tumor growth that was suppressed with OVA-pulsed DC vaccination was restored by IDO-expressing DC via IFN-γ-induced activation of GSK-3β in an OVA-expressing murine EG7 thymoma model. Taken together, DC-based immune response mediated by interferon-γ-induced IDO expression via GSK-3β activity not only regulates CD8(+) T-cell proliferation and cytotoxic T lymphocyte activity but also modulates OVA-pulsed DC vaccination against EG7 thymoma.

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

    PubMed Central

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

    2016-01-01

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

  11. Glycogen Synthase Kinase-3 and Mammalian Target of Rapamycin Pathways Contribute to DNA Synthesis, Cell Cycle Progression, and Proliferation in Human Islets

    PubMed Central

    Liu, Hui; Remedi, Maria S.; Pappan, Kirk L.; Kwon, Guim; Rohatgi, Nidhi; Marshall, Connie A.; McDaniel, Michael L.

    2009-01-01

    OBJECTIVE—Our previous studies demonstrated that nutrient regulation of mammalian target of rapamycin (mTOR) signaling promotes regenerative processes in rodent islets but rarely in human islets. Our objective was to extend these findings by using therapeutic agents to determine whether the regulation of glycogen synthase kinase-3 (GSK-3)/β-catenin and mTOR signaling represent key components necessary for effecting a positive impact on human β-cell mass relevant to type 1 and 2 diabetes. RESEARCH DESIGN AND METHODS—Primary adult human and rat islets were treated with the GSK-3 inhibitors, LiCl and the highly potent 1-azakenpaullone (1-Akp), and with nutrients. DNA synthesis, cell cycle progression, and proliferation of β-cells were assessed. Measurement of insulin secretion and content and Western blot analysis of GSK-3 and mTOR signaling components were performed. RESULTS—Human islets treated for 4 days with LiCl or 1-Akp exhibited significant increases in DNA synthesis, cell cycle progression, and proliferation of β-cells that displayed varying degrees of sensitivity to rapamycin. Intermediate glucose (8 mmol/l) produced a striking degree of synergism in combination with GSK-3 inhibition to enhance bromodeoxyuridine (BrdU) incorporation and Ki-67 expression in human β-cells. Nuclear translocation of β-catenin responsible for cell proliferation was found to be particularly sensitive to rapamycin. CONCLUSIONS—A combination of GSK-3 inhibition and nutrient activation of mTOR contributes to enhanced DNA synthesis, cell cycle progression, and proliferation of human β-cells. Identification of therapeutic agents that appropriately regulate GSK-3 and mTOR signaling may provide a feasible and available approach to enhance human islet growth and proliferation. PMID:19073772

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

    PubMed Central

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

    2013-01-01

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

  13. Identification of an N-terminal glycogen synthase kinase 3 phosphorylation site which regulates the functional localisation of polycystin-2 in vivo and in vitro

    PubMed Central

    Streets, Andrew J; Moon, David J; Kane, Michelle E; Obara, Tomoko; Ong, Albert CM

    2008-01-01

    PKD2 is mutated in 15% of patients with autosomal dominant polycystic kidney disease (ADPKD). Polycystin-2 (PC2), the PKD2 protein, is a nonselective Ca2+-permeable cation channel which may function at the cell surface and ER. Nevertheless, the factors that regulate the dynamic translocation of PC2 between the ER and other compartments are not well understood. Constitutive phosphorylation of PC2 at a single C-terminal site (Ser812) has been previously reported. Since we were unable to abolish phospholabelling of PC2 in HEK293 cells by site-directed mutagenesis of Ser812 or all 5 predicted phosphorylation sites in the C-terminus, we hypothesised that PC2 could also be phosphorylated at the N-terminus. In this paper, we report the identification of a new phosphorylation site for PC2 within its N-terminal domain (Ser76) and demonstrate that this residue is phosphorylated by glycogen synthase kinase 3 (GSK-3). The consensus recognition sequence for GSK-3 (Ser76/Ser80) is evolutionarily conserved down to lower vertebrates. In the presence of specific GSK-3 inhibitors, the lateral plasma membrane pool of endogenous PC2 redistributes into an intracellular compartment in MDCK cells without a change in primary cilia localization. Finally, co-injection of wild-type but not a S76A/S80A mutant PKD2 capped mRNA could rescue the cystic phenotype induced by an antisense morpholino oligonucleotide to pkd2 in zebrafish pronephric kidney. We conclude that surface localization of PC2 is regulated by phosphorylation at a unique GSK-3 site in its N-terminal domain in vivo and in vitro. This site is functionally significant for the maintenance of normal glomerular and tubular morphology. PMID:16551655

  14. Synthesis and evaluation of [11C]PyrATP-1, a novel radiotracer for PET imaging of glycogen synthase kinase-3β (GSK-3β)

    PubMed Central

    Cole, Erin L.; Shao, Xia; Sherman, Phillip; Quesada, Carole; Fawaz, Maria V.; Desmond, Timothy J.; Scott, Peter J. H.

    2014-01-01

    Introduction The dysfunction of glycogen synthase kinase-3β(GSK-3β) has been implicated in a number of diseases, including Alzheimer’s disease. The ability to non-invasively quantify GSK-3βactivity in vivo is therefore of critical importance, and this work is focused upon development of inhibitors of GSK-3βradiolabeled with carbon-11 to examine quantification of the enzyme using positron emission tomography (PET) imaging. Methods [11C]PyrATP-1 was prepared from the corresponding desmethyl-piperazine precursor in an automated synthesis module. In vivo rodent and primate imaging studies were conducted on a Concorde MicroPET P4 scanner to evaluate imaging properties, and in vitro autoradiography studies with rat brain samples were carried out to examine specific binding. Results 2035 ± 518 MBq (55 ± 14 mCi) of [11C]PyrATP-1 were obtained (1–2% noncorrected radiochemical yield at end-of-synthesis based upon [11C]CO2) with high chemical (>95%) and radiochemical (>99%) purities, and good specific activities (143 ± 52 GBq/µmol (3874 ± 1424 Ci/mmol)), n = 5. In vivo microPET imaging studies revealed poor brain uptake in rodents and non-human primates. Pretreatment of rodents with cyclosporin A resulted in moderately increased brain uptake suggesting Pgp transporter involvement. Autoradiography demonstrated high levels of specific binding in areas of the rodent brain known to be rich in GSK-3β. Conclusion [11C]PyrATP-1 is readily synthesized using standard carbon-11 radiochemistry. However, the poor brain uptake in rodents and non-human primates indicates that the radiotracer is not suitable for the purposes of quantifying GSK-3βin neurological and psychiatric disorders. PMID:24768148

  15. Inhibiting glycogen synthase kinase-3 and transforming growth factor-β signaling to promote epithelial transition of human adipose mesenchymal stem cells.

    PubMed

    Setiawan, Melina; Tan, Xiao-Wei; Goh, Tze-Wei; Hin-Fai Yam, Gary; Mehta, Jodhbir S

    2017-09-02

    This study was aimed to investigate the epithelial differentiation of human adipose-derived mesenchymal stem cells (ADSCs) by inhibiting glycogen synthase kinase-3 (GSK3) and transforming growth factor β (TGFβ) signaling. STEMPRO human ADSCs at passage 2 were treated with CHIR99021 (GSK3 inhibitor), E-616452 (TGFβ1 receptor kinase inhibitor), A-83-01 (TGFβ type 1 receptor inhibitor), valproic acid (histone deacetylase inhibitor), tranylcypromine (monoamine oxidase inhibitor) and all-trans retinoic acid for 72 h. The mesenchymal-epithelial transition was shown by down-regulation of mesenchymal genes (Slug, Zinc Finger E-box Binding Homeobox 1 ZEB1, integrin α5 ITGA5 and vimentin VIM) and up-regulation of epithelial genes (E-cadherin, Epithelial Cell Adhesion Molecule EpCAM, Zonula Occludens-1 ZO-1, occludin, deltaN p63 δNp63, Transcription Factor 4 TCF4 and Twist Family bHLH Transcription Factor TWIST), compared to untreated ADSCs. Cell morphology and stress fiber pattern were examined and the treated cells became less migratory in scratch wound closure assay. The formation of cell junction complexes was observed under transmission electron microscopy. Global gene expression using GeneChip(®) Human Genome U133 Array (Affymetrix) showed that the treatment up-regulated 540 genes (containing genes for cell cycle, cytoskeleton reorganization, chemotaxis, epithelium development and regulation of cell migration) and down-regulated 483 genes. Human ADSCs were transited to epithelial lineage by inhibiting GSK3 and TGFβ signaling. It can be an adult stem cell source for epithelial cell-based therapy. Copyright © 2017 Elsevier Inc. All rights reserved.

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

    PubMed

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

    2015-01-01

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

  17. An essential role for the integrin-linked kinase-glycogen synthase kinase-3 beta pathway during dendrite initiation and growth.

    PubMed

    Naska, Sibel; Park, Katya J; Hannigan, Gregory E; Dedhar, Shoukat; Miller, Freda D; Kaplan, David R

    2006-12-20

    Multiple cues, including growth factors and circuit activity, signal to regulate the initiation and growth of mammalian dendrites. In this study, we have asked how these environmental cues regulate dendrite formation, and in particular, whether dendrite initiation and growth requires integrin-linked kinase (ILK) or its downstream effector, glycogen synthase kinase-3beta (GSK-3beta). In cultured sympathetic neurons, NGF and neuronal depolarization activated ILK and promoted dendrite initiation and growth, and inhibition of ILK (either pharmacologically, with a dominant-negative form of ILK, or by genetic knockdown) reduced depolarization-induced dendrite formation. In sympathetic neurons, ILK phosphorylated and inhibited GSK-3beta, and inhibition of GSK-3beta (either pharmacologically, with dominant-negative GSK-3beta, or by genetic knockdown) caused robust dendrite initiation. GSK-3beta inhibition also caused dendrite initiation in cultured cortical neurons and growth of hippocampal neurons in slice cultures. GSK-3beta functioned downstream of ILK to regulate dendrite formation, because inhibition of GSK-3beta promoted dendrite initiation even when ILK was simultaneously inhibited. Moreover, GSK-3beta promoted dendrite formation in sympathetic neurons by regulating the activity of a key dendrite formation effector, the MAP (microtubule-associated protein) kinase kinase (MEK)-extracellular signal-regulated protein kinase (ERK) pathway. Specifically, inhibition of GSK-3beta led to increased ERK phosphorylation, and inhibition of MEK completely blocked the effects of GSK-3beta inhibition on dendrite initiation and growth. Thus, the ILK-GSK-3beta pathway plays a key role in regulating dendrite formation in developing mammalian neurons.

  18. Glycogen Synthase Kinase 3β Is a Negative Regulator of Growth Factor-induced Activation of the c-Jun N-terminal Kinase*

    PubMed Central

    Liu, Shuying; Yu, Shuangxing; Hasegawa, Yutaka; LaPushin, Ruth; Xu, Hong-Ji; Woodgett, James R.; Mills, Gordon B.; Fang, Xianjun

    2016-01-01

    The c-Jun N-terminal kinase (JNK)/stress activated protein kinase is preferentially activated by stress stimuli. Growth factors, particularly ligands for G protein-coupled receptors, usually induce only modest JNK activation, although they may trigger marked activation of the related extracellular signal-regulated kinase. In the present study, we demonstrated that homozygous disruption of glycogen synthase kinase 3β (GSK-3β) dramatically sensitized mouse embryonic fibroblasts (MEFs) to JNK activation induced by lysophosphatidic acid (LPA) and sphingosine-1-phosphate, two prototype ligands for G protein-coupled receptors. To a lesser degree, a lack of GSK-3β also potentiated JNK activation in response to epidermal growth factor. In contrast, the absence of GSK-3β decreased UV light-induced JNK activation. The increased JNK activation induced by LPA in GSK-3β null MEFs was insufficient to trigger apoptotic cell death or growth inhibition. Instead, the increased JNK activation observed in GSK-3β−/− MEFs was associated with an increased proliferative response to LPA, which was reduced by the inhibition of JNK. Ectopic expression of GSK-3β in GSK-3β-negative MEFs restrained LPA-triggered JNK phosphorylation and induced a concomitant decrease in the mitogenic response to LPA compatible with GSK-3β through the inhibition of JNK activation, thus limiting LPA-induced cell proliferation. Mutation analysis indicated that GSK-3β kinase activity was required for GSK-3β to optimally inhibit LPA-stimulated JNK activation. Thus GSK-3β serves as a physiological switch to specifically repress JNK activation in response to LPA, sphingosine-1-phosphate, or the epidermal growth factor. These results reveal a novel role for GSK-3β in signal transduction and cellular responses to growth factors. PMID:15466414

  19. Kallikrein/kinin protects against myocardial apoptosis after ischemia/reperfusion via Akt-glycogen synthase kinase-3 and Akt-Bad.14-3-3 signaling pathways.

    PubMed

    Yin, Hang; Chao, Lee; Chao, Julie

    2005-03-04

    Our previous study has shown that human tissue kallikrein protected against ischemia/reperfusion-induced myocardial injury. In the present study, we investigated the protective role of local kallikrein gene delivery in ischemia/reperfusion-induced cardiomyocyte apoptosis and its signaling mechanisms in promoting cardiomyocyte survival. Adenovirus carrying the human tissue kallikrein gene was delivered locally into the heart using a catheter-based technique. Expression and localization of recombinant human kallikrein in rat myocardium after gene transfer were determined immunohistochemically. Kallikrein gene delivery markedly reduced reperfusion-induced cardiomyocyte apoptosis identified by both in situ nick end-labeling and DNA fragmentation. Delivery of the kallikrein gene increased phosphorylation of Src, Akt, glycogen synthase kinase (GSK)-3beta, and Bad(Ser-136) but reduced caspase-3 activation in rat myocardium after reperfusion. The protective effect of kallikrein on apoptosis and its signaling mediators was blocked by icatibant and dominant-negative Akt, indicating a kinin B2 receptor-Akt-mediated event. Similarly, kinin or transduction of kallikrein in cultured cardiomyocytes promoted cell viability and attenuated apoptosis induced by hypoxia/reoxygenation. The effect of kallikrein on cardiomyocyte survival was blocked by dominant-negative Akt and a constitutively active mutant of GSK-3beta, but it was facilitated by constitutively active Akt, catalytically inactive GSK-3beta, lithium, and caspase-3 inhibitor. Moreover, kallikrein promoted Bad.14-3-3 complex formation and inhibited Akt-GSK-3beta-dependent activation of caspase-3, whereas caspase-3 administration caused reduction of the Bad.14-3-3 complex, indicating an interaction between Akt-GSK-caspase-3 and Akt-Bad.14-3-3 signaling pathways. In conclusion, kallikrein/kinin protects against cardiomyocyte apoptosis in vivo and in vitro via Akt-Bad.14-3-3 and Akt-GSK-3beta-caspase-3 signaling pathways.

  20. Glycogen synthase kinase-3β in the ventral tegmental area mediates diurnal variations in cocaine-induced conditioned place preference in rats.

    PubMed

    Li, Su-Xia; Wei, Yi-Ming; Shi, Hai-Shui; Luo, Yi-Xiao; Ding, Zeng-Bo; Xue, Yan-Xue; Lu, Lin; Yu, Chang-Xi

    2014-11-01

    Cocaine sensitization and reward are reported to be under the influence of diurnal rhythm. However, no previous studies have reported brain areas that play a role as modulators and underlie the mechanism of diurnal variations in cocaine reward. We examined (1) the diurnal rhythm of glycogen synthase kinase-3β (GSK-3β) activity in the suprachiasmatic nucleus (SCN) and reward-related brain areas in naive rats; (2) the effect of day and night on the acquisition of cocaine-induced conditioned place preference (CPP); (3) the influence of cocaine-induced CPP on GSK-3β activity in the SCN and reward-related brain areas; and (4) the effect of the GSK-3β inhibitor SB216763 microinjected bilaterally into the ventral tegmental area (VTA) on cocaine-induced CPP. A significant diurnal rhythm of GSK-3β activity was found in the SCN and reward-related brain areas, with diurnal variations in cocaine-induced CPP. GSK-3β activity in the SCN and reward-related brain areas exhibited marked diurnal variations in rats treated with saline. GSK-3β activity in rats treated with cocaine exhibited distinct diurnal variations only in the prefrontal cortex and VTA. Cocaine decreased the expression of phosphorylated GSK-3β (i.e. increased GSK-3β activity) only in the VTA in rats trained and tested at ZT4 and ZT16. SB216763 microinjected into the VTA bilaterally eliminated the diurnal variations in cocaine-induced CPP, but did not affect the acquisition of cocaine-induced CPP. These findings suggest that the VTA may be a critical area involved in the diurnal variations in cocaine-induced CPP, and GSK-3β may be a regulator of diurnal variations in cocaine-induced CPP. © 2013 Society for the Study of Addiction.

  1. Disruption of circadian rhythmicity and suprachiasmatic action potential frequency in a mouse model with constitutive activation of glycogen synthase kinase 3.

    PubMed

    Paul, J R; Johnson, R L; Jope, R S; Gamble, K L

    2012-12-13

    Glycogen synthase kinase 3 (GSK3) is a serine/threonine kinase that has been implicated in psychiatric diseases, neurodevelopment, and circadian regulation. Both GSK3 isoforms, α and β, exhibit a 24-h variation of inhibitory phosphorylation within the suprachiasmatic nucleus (SCN), the primary circadian pacemaker. We examined the hypothesis that rhythmic GSK3 activity is critical for robust circadian rhythmicity using GSK3α(21A/21A)/β(9A/9A) knock-in mice with serine-alanine substitutions at the inhibitory phosphorylation sites, making both forms constitutively active. We monitored wheel-running locomotor activity of GSK3 knock-in mice and used loose-patch electrophysiology to examine the effect of chronic GSK3 activity on circadian behavior and SCN neuronal activity. Double transgenic GSK3α/β knock-in mice exhibit disrupted behavioral rhythmicity, including significantly decreased rhythmic amplitude, lengthened active period, and increased activity bouts per day. This behavioral disruption was dependent on chronic activation of both GSK3 isoforms and was not seen in single transgenic GSK3α or GSK3β knock-in mice. Underlying the behavioral changes, SCN neurons from double transgenic GSK3α/β knock-in mice exhibited significantly higher spike rates during the subjective night compared to those from wild-type controls, with no differences detected during the subjective day. These results suggest that constitutive activation of GSK3 results in the loss of the typical day/night variation of SCN neuronal activity. Together, these results implicate GSK3 activity as a critical regulator of circadian behavior and neurophysiological rhythms. Because GSK3 has been implicated in numerous pathologies, understanding how GSK3 modulates circadian rhythms and neurophysiological activity may lead to novel therapeutics for pathological disorders and circadian rhythm dysfunction. Copyright © 2012 IBRO. Published by Elsevier Ltd. All rights reserved.

  2. DISRUPTION OF CIRCADIAN RHYTHMICITY AND SUPRACHIASMATIC ACTION POTENTIAL FREQUENCY IN A MOUSE MODEL WITH CONSTITUTIVE ACTIVATION OF GLYCOGEN SYNTHASE KINASE-3

    PubMed Central

    Paul, Jodi R.; Johnson, Russell L.; Jope, Richard S.; Gamble, Karen L.

    2012-01-01

    Glycogen synthase kinase 3 (GSK3) is a serine/threonine kinase that has been implicated in psychiatric diseases, neurodevelopment, and circadian regulation. Both GSK3 isoforms, α and β, exhibit a 24-hour variation of inhibitory phosphorylation within the suprachiasmatic nucleus (SCN), the primary circadian pacemaker. We examined the hypothesis that rhythmic GSK3 activity is critical for robust circadian rhythmicity using GSK3α21A/21A/β9A/9A knock-in mice with serine-alanine substitutions at the inhibitory phosphorylation sites, making both forms constitutively active. We monitored wheel-running locomotor activity of GSK3 knock-in mice and used loose-patch electrophysiology to examine the effect of chronic GSK3 activity on circadian behavior and SCN neuronal activity. Double transgenic GSK3α/β knock-in mice exhibit disrupted behavioral rhythmicity, including significantly decreased rhythmic amplitude, lengthened active period, and increased activity bouts per day. This behavioral disruption was dependent on chronic activation of both GSK3 isoforms and was not seen in single transgenic GSK3α or GSK3β knock-in mice. Underlying the behavioral changes, SCN neurons from double transgenic GSK3α/β knock-in mice exhibited significantly higher spike rates during the subjective night compared to those from WT controls, with no differences detected during the subjective day. These results suggest that constitutive activation of GSK3 results in loss of the typical day/night variation of SCN neuronal activity. Together, these results implicate GSK3 activity as a critical regulator of circadian behavior and neurophysiological rhythms. Because GSK3 has been implicated in numerous pathologies, understanding how GSK3 modulates circadian rhythms and neurophysiological activity may lead to novel therapeutics for pathological disorders and circadian rhythm dysfunction. PMID:22986169

  3. Glycogen synthase kinase 3 activation is important for anthrax edema toxin-induced dendritic cell maturation and anthrax toxin receptor 2 expression in macrophages.

    PubMed

    Larabee, Jason L; Maldonado-Arocho, Francisco J; Pacheco, Sergio; France, Bryan; DeGiusti, Kevin; Shakir, Salika M; Bradley, Kenneth A; Ballard, Jimmy D

    2011-08-01

    Anthrax edema toxin (ET) is one of two binary toxins produced by Bacillus anthracis that contributes to the virulence of this pathogen. ET is an adenylate cyclase that generates high levels of cyclic AMP (cAMP), causing alterations in multiple host cell signaling pathways. We previously demonstrated that ET increases cell surface expression of the anthrax toxin receptors (ANTXR) in monocyte-derived cells and promotes dendritic cell (DC) migration toward the lymph node-homing chemokine MIP-3β. In this work, we sought to determine if glycogen synthase kinase 3 (GSK-3) is important for ET-induced modulation of macrophage and DC function. We demonstrate that inhibition of GSK-3 dampens ET-induced maturation and migration processes of monocyte-derived dendritic cells (MDDCs). Additional studies reveal that the ET-induced expression of ANTXR in macrophages was decreased when GSK-3 activity was disrupted with chemical inhibitors or with small interfering RNA (siRNA) targeting GSK-3. Further examination of the ET induction of ANTXR revealed that a dominant negative form of CREB could block the ET induction of ANTXR, suggesting that CREB or a related family member was involved in the upregulation of ANTXR. Because CREB and GSK-3 activity appeared to be important for ET-induced ANTXR expression, the impact of GSK-3 on ET-induced CREB activity was examined in RAW 264.7 cells possessing a CRE-luciferase reporter. As with ANTXR expression, the ET induction of the CRE reporter was decreased by reducing GSK-3 activity. These studies not only provide insight into host pathways targeted by ET but also shed light on interactions between GSK-3 and CREB pathways in host immune cells.

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

    PubMed Central

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

    1999-01-01

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

  5. 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.

  6. Inhibition of glycogen synthase kinase-3 by SB 216763 affects acquisition at lower doses than expression of amphetamine-conditioned place preference in rats.

    PubMed

    Wickens, Rebekah H; Quartarone, Susan E; Beninger, Richard J

    2016-12-14

    Dopamine (DA) drives incentive learning, whereby neutral stimuli acquire the ability to elicit responses. DA influences the signaling molecule glycogen synthase kinase-3 (GSK3). Inhibition of GSK3 attenuates the development of behavioral sensitization to stimulant drugs and conditioned place preference (CPP), a measure of incentive learning. We examined the role of GSK3 in the nucleus accumbens (NAc) of rats in CPP produced by amphetamine (1.5 mg/kg, i.p. or 20.0 μg/0.5 μl/side intra-NAc) by administering the inhibitor SB 216763 (1.0, 2.0, and 2.5 mg/kg, i.p. or 0.03, 0.30, 3.00, and 5.00 μg/0.5 μl/side intra-NAc) during acquisition or expression. We hypothesized a dose-dependent effect of SB 216763 and that acquisition would be affected by smaller doses than expression. For the systemic groups, 1.0 mg/kg of SB 216763 did not block CPP; 2.0 mg/kg administered in acquisition but not expression blocked CPP; and 2.5 mg/kg administered in either phase blocked CPP. For the central groups, 0.03 μg/0.5 μl/side of SB 216763 prevented acquisition but not expression, whereas larger doses administered in either phase blocked CPP. Thus, systemic or NAc inhibition of GSK3 by SB 216763 during acquisition or expression blocks amphetamine-produced CPP and acquisition is sensitive to lower doses than expression.

  7. 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.

  8. Inhibition of Glycogen Synthase Kinase 3β Ameliorates D-GalN/LPS-Induced Liver Injury by Reducing Endoplasmic Reticulum Stress-Triggered Apoptosis

    PubMed Central

    Zhang, Haiyan; Wen, Tao; Piao, Zhengfu; Zhou, Li; Zheng, Sujun; Zhang, Jing; Chen, Yu; Han, Yuanping; Duan, Zhongping; Ma, Yingji

    2012-01-01

    Background Glycogen synthase kinase 3β(GSK3β) is a ubiquitous serine-threonine protein kinase that participates in numerous cellular processes and disease pathophysiology. We aimed to determine therapeutic potential of GSK3β inhibition and its mechanism in a well-characterized model of lipopolysaccharide (LPS)-induced model of acute liver failure (ALF). Methodology In a murine ALF model induced by D-GalN(700 mg/kg)/LPS(10 µg/kg), we analyzed GSK3β mechanisms using a specific chemical inhibitor, SB216763, and detected the role of endoplasmic reticulum stress (ERS). Mice were administered SB216763 at 2 h before or after D-GalN/LPS injection, respectively, and then sacrificed 6 h after D-GalN/LPS treatment to evaluate its prophylactic and therapeutic function. The lethality rate, liver damage, ERS, cytokine expression, MAP kinase, hepatocyte apoptosis and expression of TLR 4 were evaluated, respectively. Whether the inhibition of GSK3β activation protected hepatocyte from ERS-induced apoptosis was investigated in vitro. Principal Findings GSK3β became quickly activated (dephosphorylated) upon D-GalN/LPS exposure. Administration of SB216763 not only ameliorated liver injury, as evidenced by reduced transaminase levels, and well-preserved liver architecture, but also decreased lethality. Moreover, GSK3β inhibition resulted in down-regulation of pro-apoptotic proteins C/EBP–homologous protein(CHOP) and caspase-12, which are related to ERS. To further demonstrate the role of ERS, we found that GSK3β inhibition protected hepatocyte from ERS-induced cell death. GSK3β inhibition down-regulated the MAPK pathways, reduced expression of inflammatory cytokines and decreased expression of TLR4. Conclusions Our findings demonstrate the key function of GSK3β signaling in the pathophysiology of ALF, especially in regulating the ERS, and provide a rationale for targeting GSK3β as a potential therapeutic strategy to ameliorate ALF. PMID:23028846

  9. Phosphorylation of glycogen synthase kinase-3 and stimulation of T-cell factor signaling following activation of EP2 and EP4 prostanoid receptors by prostaglandin E2.

    PubMed

    Fujino, Hiromichi; West, Kimberly A; Regan, John W

    2002-01-25

    Recently we have shown that the FP(B) prostanoid receptor, a G-protein-coupled receptor that couples to Galpha(q), activates T-cell factor (Tcf)/lymphoid enhancer factor (Lef)-mediated transcriptional activation (Fujino, H., and Regan, J. W. (2001) J. Biol. Chem. 276, 12489-12492). We now report that the EP(2) and EP(4) prostanoid receptors, which couple to Galpha(s), also activate Tcf/Lef signaling. By using a Tcf/Lef-responsive luciferase reporter gene, transcriptional activity was stimulated approximately 10-fold over basal by 1 h of treatment with prostaglandin E(2) (PGE(2)) in HEK cells that were stably transfected with the human EP(2) and EP(4) receptors. This stimulation of reporter gene activity was accompanied by a PGE(2)-dependent increase in the phosphorylation of both glycogen synthase kinase-3 (GSK-3) and Akt kinase. H-89, an inhibitor of protein kinase A (PKA), completely blocked the agonist-dependent phosphorylation of GSK-3 in both EP(2)- and EP(4)-expressing cells. However, H-89 pretreatment only blocked PGE(2)-stimulated Lef/Tcf reporter gene activity by 20% in EP(4)-expressing cells compared with 65% inhibition in EP(2)-expressing cells. On the other hand wortmannin, an inhibitor of phosphatidylinositol 3-kinase, had the opposite effect and inhibited PGE(2)-stimulated reporter gene activity to a much greater extent in EP(4)-expressing cells as compared with EP(2)-expressing cells. These findings indicate that the activation of Tcf/Lef signaling by EP(2) receptors occurs primarily through a PKA-dependent pathway, whereas EP(4) receptors activate Tcf/Lef signaling mainly through a phosphatidylinositol 3-kinase-dependent pathway. This is the first indication of a fundamental difference in the signaling potential of EP(2) and EP(4) prostanoid receptors.

  10. Mechanical Stretch Up-regulates MicroRNA-26a and Induces Human Airway Smooth Muscle Hypertrophy by Suppressing Glycogen Synthase Kinase-3β*

    PubMed Central

    Mohamed, Junaith S.; Lopez, Michael A.; Boriek, Aladin M.

    2010-01-01

    Airway smooth muscle hypertrophy is one of the hallmarks of airway remodeling in severe asthma. Several human diseases have been now associated with dysregulated microRNA (miRNA) expression. miRNAs are a class of small non-coding RNAs, which negatively regulate gene expression at the post-transcriptional level. Here, we identify miR-26a as a hypertrophic miRNA of human airway smooth muscle cells (HASMCs). We show that stretch selectively induces the transcription of miR-26a located in the locus 3p21.3 of human chromosome 3. The transcription factor CCAAT enhancer-binding protein α (C/EBPα) directly activates miR-26a expression through the transcriptional machinery upon stretch. Furthermore, stretch or enforced expression of miR-26a induces HASMC hypertrophy, and miR-26 knockdown reverses this effect, suggesting that miR-26a is a hypertrophic gene. We identify glycogen synthase kinase-3β (GSK-3β), an anti-hypertrophic protein, as a target gene of miR-26a. Luciferase reporter assays demonstrate that miR-26a directly interact with the 3′-untranslated repeat of the GSK-3β mRNA. Stretch or enforced expression of miR-26a attenuates the endogenous GSK-3β protein levels followed by the induction of HASMC hypertrophy. miR-26 knockdown reverses this effect, suggesting that miR-26a-induced hypertrophy occurs via its target gene GSK-3β. Overall, as a first time, our study unveils that miR-26a is a mechanosensitive gene, and it plays an important role in the regulation of HASMC hypertrophy. PMID:20525681

  11. Mechanical stretch up-regulates microRNA-26a and induces human airway smooth muscle hypertrophy by suppressing glycogen synthase kinase-3β.

    PubMed

    Mohamed, Junaith S; Lopez, Michael A; Boriek, Aladin M

    2010-09-17

    Airway smooth muscle hypertrophy is one of the hallmarks of airway remodeling in severe asthma. Several human diseases have been now associated with dysregulated microRNA (miRNA) expression. miRNAs are a class of small non-coding RNAs, which negatively regulate gene expression at the post-transcriptional level. Here, we identify miR-26a as a hypertrophic miRNA of human airway smooth muscle cells (HASMCs). We show that stretch selectively induces the transcription of miR-26a located in the locus 3p21.3 of human chromosome 3. The transcription factor CCAAT enhancer-binding protein α (C/EBPα) directly activates miR-26a expression through the transcriptional machinery upon stretch. Furthermore, stretch or enforced expression of miR-26a induces HASMC hypertrophy, and miR-26 knockdown reverses this effect, suggesting that miR-26a is a hypertrophic gene. We identify glycogen synthase kinase-3β (GSK-3β), an anti-hypertrophic protein, as a target gene of miR-26a. Luciferase reporter assays demonstrate that miR-26a directly interact with the 3'-untranslated repeat of the GSK-3β mRNA. Stretch or enforced expression of miR-26a attenuates the endogenous GSK-3β protein levels followed by the induction of HASMC hypertrophy. miR-26 knockdown reverses this effect, suggesting that miR-26a-induced hypertrophy occurs via its target gene GSK-3β. Overall, as a first time, our study unveils that miR-26a is a mechanosensitive gene, and it plays an important role in the regulation of HASMC hypertrophy.

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

    PubMed

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

    2014-09-01

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

  13. Activation of the Wnt pathway through use of AR79, a glycogen synthase kinase 3β inhibitor, promotes prostate cancer growth in soft tissue and bone

    PubMed Central

    Jiang, Yuan; Dai, Jinlu; Zhang, Honglai; Sottnik, Joe L.; Keller, Jill M.; Escott, Katherine J.; Sanganee, Hitesh J.; Yao, Zhi; McCauley, Laurie K.; Keller, Evan T.

    2013-01-01

    Due to its bone anabolic activity, methods to increase Wnt activity, such as inhibitors of dickkopf-1 and sclerostin, are being clinically explored. Glycogen synthase kinase (GSK3β) inhibits Wnt signaling through inducing β-catenin degradation. Therefore, AR79, an inhibitor of GSK3β, is being evaluated as a bone anabolic agent. However, Wnt activation has potential to promote tumor growth. The goal of this study was to determine if AR79 impacted progression of prostate cancer (PCa). PCa tumors were established in subcutaneous and bone sites of mice followed by AR79 administration. Tumor growth, β-catenin activation, proliferation (Ki67 expression) and apoptosis (caspase 3 activity) were measured. Additionally, PCa and osteoblast cell lines were treated with AR79 and β-catenin status, proliferation (with β-catenin knocked down in some cases) and proportion of the ALDH+CD133+ stem-like cells was determined. AR79 promoted PCa growth, decreased phospho-β-catenin expression and increased total and nuclear β-catenin expression in tumors and increased tumor-induced bone remodeling. Additionally, it decreased caspase 3 and increased Ki67 expression. In addition, AR79 increased bone formation in normal mouse tibiae. AR79 inhibited β-catenin phosphorylation, increased nuclear β-catenin accumulation in PCa and osteoblast cell lines and increased proliferation of PCa cells in vitro through β-catenin. Furthermore, AR79 increased the ALDH+CD133+ cancer stem cell-like proportion of the PCa cell lines. We conclude that AR79, while being bone anabolic, promotes PCa cell growth through Wnt pathway activation. PMID:24088787

  14. Constitutive activation of glycogen synthase kinase-3β correlates with better prognosis and cyclin-dependent kinase inhibitors in human gastric cancer

    PubMed Central

    2010-01-01

    Background Aberrant regulation of glycogen synthase kinase-3β (GSK-3β) has been implicated in several human cancers; however, it has not been reported in the gastric cancer tissues to date. The present study was performed to determine the expression status of active form of GSK-3β phosphorylated at Tyr216 (pGSK-3β) and its relationship with other tumor-associated proteins in human gastric cancers. Methods Immunohistochemistry was performed on tissue array slides containing 281 human gastric carcinoma specimens. In addition, gastric cancer cells were cultured and treated with a GSK-3β inhibitor lithium chloride (LiCl) for immunoblot analysis. Results We found that pGSK-3β was expressed in 129 (46%) of 281 cases examined, and was higher in the early-stages of pathologic tumor-node-metastasis (P < 0.001). The expression of pGSK-3β inversely correlated with lymphatic invasion (P < 0.001) and lymph node metastasis (P < 0.001) and correlated with a longer patient survival (P < 0.001). In addition, pGSK-3β expression positively correlated with that of p16, p21, p27, p53, APC, PTEN, MGMT, SMAD4, or KAI1 (P < 0.05), but not with that of cyclin D1. This was confirmed by immunoblot analysis using SNU-668 gastric cancer cells treated with LiCl. Conclusions GSK-3β activation was frequently observed in early-stage gastric carcinoma and was significantly correlated with better prognosis. Thus, these findings suggest that GSK-3β activation is a useful prognostic marker for the early-stage gastric cancer. PMID:20704706

  15. 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.

  16. Glycogen synthase kinase-3 inhibitor AR-A014418 suppresses pancreatic cancer cell growth via inhibition of GSK-3-mediated Notch1 expression

    PubMed Central

    Kunnimalaiyaan, Selvi; Gamblin, T Clark; Kunnimalaiyaan, Muthusamy

    2015-01-01

    Background Glycogen synthase kinase-3 (GSK-3) can act as either a tumour promoter or suppressor by its inactivation depending on the cell type. There are conflicting reports on the roles of GSK-3 isoforms and their interaction with Notch1 in pancreatic cancer. It was hypothesized that GSK-3α stabilized Notch1 in pancreatic cancer cells thereby promoting cellular proliferation. Methods The pancreatic cancer cell lines MiaPaCa2, PANC-1 and BxPC-3, were treated with 0–20 μM of AR-A014418 (AR), a known GSK-3 inhibitor. Cell viability was determined by the MTT assay and Live-Cell Imaging. The levels of Notch pathway members (Notch1, HES-1, survivin and cyclinD1), phosphorylated GSK-3 isoforms, and apoptotic markers were determined by Western blot. Immunoprecipitation was performed to identify the binding of GSK-3 specific isoform to Notch1. Results AR-A014418 treatment had a significant dose-dependent growth reduction (P < 0.001) in pancreatic cancer cells compared with the control and the cytotoxic effect is as a result of apoptosis. Importantly, a reduction in GSK-3 phosphorylation lead to a reduction in Notch pathway members. Overexpression of active Notch1 in AR-A014418-treated cells resulted in the negation of growth suppression. Immunoprecipitation analysis revealed that GSK-3α binds to Notch1. Conclusions This study demonstrates for the first time that the growth suppressive effect of AR-A014418 on pancreatic cancer cells is mainly mediated by a reduction in phosphorylation of GSK-3α with concomitant Notch1 reduction. GSK-3α appears to stabilize Notch1 by binding and may represent a target for therapeutic development. Furthermore, downregulation of GSK-3 and Notch1 may be a viable strategy for possible chemosensitization of pancreatic cancer cells to standard therapeutics. PMID:26147011

  17. Glycogen synthase kinase-3β is required for epithelial-mesenchymal transition and barrier dysfunction in mouse podocytes under high glucose conditions

    PubMed Central

    Guo, Jia; Yang, Lili; Qiao, Yingjin; Liu, Zhangsuo

    2016-01-01

    Epithelial-mesenchymal transition (EMT) is important for diabetic nephropathy (DN). Podocytes are specialized epithelial cells, which form a major component of the glomerular filtration barrier. Podocyte damage has been suggested to be the primary mechanism behind the albuminuria associated with DN. The present study aimed to determine the function of glycogen synthase kinase (GSK)-3β in EMT and barrier dysfunction of mouse podocytes exposed to high glucose (HG) conditions. Matured and differentiated podocytes were treated with normal glucose (NG), HG or NG + mannitol. Podocytes were also transfected with a small interfering RNA (siRNA) against GSK-3β or a scrambled siRNA, or were treated with lithium chloride (LiCl), a GSK-3β inhibitor, under NG or HG conditions. The expression levels of the epithelial cell markers, nephrin and podocin, and the myofibroblast cell markers, α-smooth muscle actin (SMA) and fibronectin, in podocytes by western blot analysis and immunofluorescence staining, respectively. The monolayer barrier function was assessed by albumin inflow. The phosphorylation and activity levels of GSK-3β were also quantified. It was observed that HG promotes EMT in podocytes, due to the increased levels of podocin and nephrin expression and the reduced α-SMA and fibronectin expression levels. HG also induced barrier dysfunction and increased the expression level of total GSK-3β, Try216-phosphorylated-GSK-3β and the GSK-3β activity in podocytes. Transfection of GSK-3β siRNA or treatment with LiCl reversed the HG-induced EMT and barrier dysfunction in podocytes. In conclusion, the present study determined that GSK-3β is required for EMT and barrier dysfunction in podocytes under HG conditions; therefore, GSK-3β may be a novel target for the treatment of DN. PMID:27748847

  18. Interactions between glycogen synthase kinase 3beta, protein kinase B, and protein phosphatase 2A in tau phosphorylation in mouse N2a neuroblastoma cells.

    PubMed

    Zhou, Xin-Wen; Winblad, Bengt; Guan, Zhizhong; Pei, Jin-Jing

    2009-01-01

    In this study, we investigated how tau phosphorylation is regulated by protein kinase glycogen synthase kinase 3beta (GSK3 beta), protein kinase B (PKB), and protein phosphatase 2A (PP2A) in mouse N2a neuroblastoma cells. Results showed that GSK3 beta overexpression significantly increased PKB phosphorylation at the S473 site but not the T308 site. Neither GSK3 beta nor PKB overexpression could reduce the PP2AC phosphorylation at the Y307 site. In contrast, either PKB or GSK3 beta knockdown could increase PP2A phosphorylation at the Y307 site. PP2AC knockdown increased GSK3 beta phosphorylation at the S9 site but not at the Y216 site, and PKB phosphorylation at the T308 site but not at the S473 site. Tau phosphorylation at the S396 site was increased by GSK3 beta or PKB overexpression. Tau phosphorylation at the S214 site was only induced by PKB overexpression in the study. While GSK3 beta knockdown decreased tau phosphorylation at the S396 site, PKB knockdown increased tau phosphorylation at both the S396 and S214 sites. PP2AC knockdown decreased tau phosphorylation at the S396 and S214 sites. These findings suggest that tau phosphorylation at the S396 and S214 sites is differentially regulated by GSK3 beta, PKB, and PP2A in N2a cells. The final phosphorylation state of tau is possibly caused by the synergic action of the three enzymes.

  19. Glycogen synthase kinase 3 beta alters anxiety-, depression-, and addiction-related behaviors and neuronal activity in the nucleus accumbens shell.

    PubMed

    Crofton, Elizabeth J; Nenov, Miroslav N; Zhang, Yafang; Scala, Federico; Page, Sean A; McCue, David L; Li, Dingge; Hommel, Jonathan D; Laezza, Fernanda; Green, Thomas A

    2017-05-01

    Psychiatric disorders such as anxiety, depression and addiction are often comorbid brain pathologies thought to share common mechanistic biology. As part of the cortico-limbic circuit, the nucleus accumbens shell (NAcSh) plays a fundamental role in integrating information in the circuit, such that modulation of NAcSh circuitry alters anxiety, depression, and addiction-related behaviors. Intracellular kinase cascades in the NAcSh have proven important mediators of behavior. To investigate glycogen-synthase kinase 3 (GSK3) beta signaling in the NAcSh in vivo we knocked down GSK3beta expression with a novel adeno-associated viral vector (AAV2) and assessed changes in anxiety- and depression-like behavior and cocaine self-administration in GSK3beta knockdown rats. GSK3beta knockdown reduced anxiety-like behavior while increasing depression-like behavior and cocaine self-administration. Correlative electrophysiological recordings in acute brain slices were used to assess the effect of AAV-shGSK3beta on spontaneous firing and intrinsic excitability of tonically active interneurons (TANs), cells required for input and output signal integration in the NAcSh and for processing reward-related behaviors. Loose-patch recordings showed that TANs transduced by AAV-shGSK3beta exhibited reduction in tonic firing and increased spike half width. When assessed by whole-cell patch clamp recordings these changes were mirrored by reduction in action potential firing and accompanied by decreased hyperpolarization-induced depolarizing sag potentials, increased action potential current threshold, and decreased maximum rise time. These results suggest that silencing of GSK3beta in the NAcSh increases depression- and addiction-related behavior, possibly by decreasing intrinsic excitability of TANs. However, this study does not rule out contributions from other neuronal sub-types. Copyright © 2017 Elsevier Ltd. All rights reserved.

  20. Convergence of 3',5'-cyclic adenosine 5'-monophosphate/protein kinase A and glycogen synthase kinase-3beta/beta-catenin signaling in corpus luteum progesterone synthesis.

    PubMed

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

    2009-11-01

    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)-3beta and beta-catenin contributes to LH-dependent steroidogenesis in luteal cells. We observed that LH via a cAMP/PKA-dependent mechanism stimulated the phosphorylation of GSK3beta at N-terminal Ser9 causing its inactivation and resulted in the accumulation of beta-catenin. Overexpression of N-terminal truncated beta-catenin (Delta90 beta-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 GSK3beta (GSK-S9A) reduced beta-catenin levels and inhibited LH-stimulated steroidogenesis. Chromatin immunoprecipitation assays demonstrated the association of beta-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 GSK3beta/beta-catenin signaling may contribute to the acute increase in progesterone production in response to LH.

  1. Activation of AMP-activated protein kinase and phosphorylation of glycogen synthase kinase3 β mediate ursolic acid induced apoptosis in HepG2 liver cancer cells.

    PubMed

    Son, Hyun-Soo; Kwon, Hee Young; Sohn, Eun Jung; Lee, Jang-Hoon; Woo, Hong-Jung; Yun, Miyong; Kim, Sung-Hoon; Kim, Young-Chul

    2013-11-01

    Despite the antitumour effect of ursolic acid observed in several cancers, the underlying mechanism remains unclear. Thus, in the present study, the roles of AMP-activated protein kinase (AMPK) and glycogen synthase kinase 3 beta (GSK3β) were examined in ursolic acid induced apoptosis in HepG2 hepatocellular carcinoma cells. Ursolic acid significantly exerted cytotoxicity, increased the sub-G1 population and the number of ethidium homodimer and terminal deoxynucleotidyl transferase(TdT) mediated dUTP nick end labeling positive cells in HepG2 cells. Also, ursolic acid enhanced the cleavages of poly-ADP-ribose polymerase (PARP) and caspase3, attenuated the expression of astrocyte elevated gene (AEG1) and survivin in HepG2 cells. Interestingly, ursolic acid increased the phosphorylation of AMPK and coenzyme A carboxylase and also enhanced phosphorylation of GSK3β at inactive form serine 9, whereas ursolic acid attenuated the phosphorylation of AKT and mTOR in HepG2 cells. Conversely, AMPK inhibitor compound C or GSK3β inhibitor SB216763 blocked the cleavages of PARP and caspase 3 induced by ursolic acid in HepG2 cells. Furthermore, proteosomal inhibitor MG132 suppressed AMPK activation, GSK3β phosphorylation, cleaved PARP and deceased AEG-1 induced by ursolic acid in HepG2 cells. Overall, our findings suggest that ursolic acid induced apoptosis in HepG2 cells via AMPK activation and GSK3β phosphorylation as a potent chemopreventive agent. Copyright © 2013 John Wiley & Sons, Ltd.

  2. Coupling of mammalian target of rapamycin with phosphoinositide 3-kinase signaling pathway regulates protein phosphatase 2A- and glycogen synthase kinase-3 -dependent phosphorylation of Tau.

    PubMed

    Meske, Volker; Albert, Frank; Ohm, Thomas Georg

    2008-01-04

    Tau is an important microtubule-stabilizing protein in neurons. In its hyperphosphorylated form, Tau protein loses its ability to bind to microtubules and then accumulates and is part of pathological lesions characterizing tauopathies, e.g. Alzheimer disease. Glycogen synthase kinase-3beta (GSK-3beta), antagonized by protein phosphatase 2A (PP2A), regulates Tau phosphorylation at many sites. Diabetes mellitus is linked to an increased risk of developing Alzheimer disease. This could be partially caused by dysregulated GSK-3beta. In a long term experiment (-16 h) using primary murine neuron cultures, we interfered in the insulin/phosphoinositide 3-kinase (PI3K) (LY294002 treatment and insulin boost) and mammalian target of rapamycin (mTor) (AICAR and rapamycin treatment) signaling pathways and examined consequent changes in the activities of PP2A, GSK-3beta, and Tau phosphorylation. We found that the coupling of PI3K with mTor signaling, in conjunction with a regulatory interaction between PP2A and GSK-3beta, changed activities of both enzymes always in the same direction. These balanced responses seem to ensure the steady Tau phosphorylation at GSK/PP2A-dependent sites observed over a long period of time (>/=6 h). This may help in preventing severe changes in Tau phosphorylation under conditions when neurons undergo transient fluctuations either in insulin or nutrient supply. On the other hand, the investigation of Tau protein at Ser-262 showed that interference in the insulin/PI3K and mTor signaling potentially influenced the Tau phosphorylation status at sites where only one of two enzymes (in this case PP2A) is involved in the regulation.

  3. Glycogen synthase kinase-3beta heterozygote knockout mice as a model of findings in postmortem schizophrenia brain or as a model of behaviors mimicking lithium action: negative results.

    PubMed

    Bersudsky, Yuly; Shaldubina, Alona; Kozlovsky, Nitzan; Woodgett, James R; Agam, Galila; Belmaker, R H

    2008-05-01

    In mice glycogen synthase kinase (GSK)-3beta heterozygote knockout status was reported to cause reduced immobility in the Porsolt forced swim test and reduced amphetamine-induced hyperactivity, behaviors that mimic the effects of lithium. GSK-3beta protein and mRNA level and activity have been reported to be reduced in the postmortem brain of schizophrenia patients and this could suggest the involvement of GSK-3beta in the etiology of schizophrenia. However, apomorphine-induced stereotyping was reported to be unchanged in GSK-3beta heterozygote (HZ) knockout (KO) mice. As such behaviors are not always robust, study in another laboratory seemed indicated. Motor activity and coordination were assessed in the rotarod test. Behavior was studied in the following tests: pilocarpine-induced seizures model for lithium action, Porsolt forced swim test, tail suspension test, elevated plus-maze, large open field, startle response and prepulse inhibition of acoustic startle response, amphetamine-induced hyperactivity, and apomorphine-induced stereotypic climbing. We could not confirm the report that GSK-3beta HZ KO mice exhibit reduced immobility in the Porsolt forced swim or reduced amphetamine-induced hyperactivity in a manner mimicking the behavioral effects of lithium. We did not find increased apomorphine-induced stereotypic climbing or disruption of prepulse inhibition, suggesting that human postmortem findings regarding GSK-3beta in schizophrenia are not mediated by changes in dopamine receptors and are not the cause of prepulse inhibition deficits in schizophrenia. These data do not support the role of GSK-3beta in schizophrenia or in the mechanism of therapeutic action of lithium. Although differences in the genetic background of the GSK-3beta HZ KOs used in the present study compared with that of the previous study could be responsible, such results could suggest that the previously reported effects of GSK-3beta knockout on behavior are not robust.

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

    PubMed

    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.

  5. 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

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

    PubMed Central

    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

    2015-01-01

    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

  7. 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.

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

    PubMed

    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; Jessen, N

    2012-01-15

    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.

  9. Regulation of glycogen resynthesis following exercise. Dietary considerations.

    PubMed

    Friedman, J E; Neufer, P D; Dohm, G L

    1991-04-01

    With the cessation of exercise, glycogen repletion begins to take place rapidly in skeletal muscle and can result in glycogen levels higher than those present before exercise. Understanding the rate-limiting steps that regulate glycogen synthesis will provide us with strategies to increase the resynthesis of glycogen during recovery from exercise, and thus improve performance. Given the importance of muscle glycogen to endurance performance, various factors which may optimise glycogen resynthesis rate and insure complete restoration have been of interest to both the scientist and athlete. The time required for complete muscle glycogen resynthesis after prolonged moderate intensity exercise is generally considered to be 24 hours provided approximately 500 to 700g of carbohydrate is ingested. Muscle glycogen synthesis rate is highest during the first 2 hours after exercise. Ingestion of 0.70g glucose/kg bodyweight every 2 hours appears to maximise glycogen resynthesis rate at approximately 5 to 6 mumol/g wet weight/h during the first 4 to 6 hours after exhaustive exercise. Further enhancement of glycogen resynthesis rate with ingestion of greater than 0.70g glucose/kg bodyweight appears to be limited by the constraints imposed by gastric emptying. Ingestion of glucose or sucrose results in similar muscle glycogen resynthesis rates while glycogen synthesis in liver is better served with the ingestion of fructose. Also, increases in muscle glycogen content during the first 4 to 6 hours after exercise are greater with ingestion of simple as compared with complex carbohydrate. Glycogen synthase activity is a key component in the regulation of glycogen resynthesis. Glycogen synthase enzyme exists in 2 states: the less active, more phosphorylated (D) form which is under allosteric control of glucose-6-phosphate, and the more active, less phosphorylated (I) form which is independent of glucose-6-phosphate. There is generally an inverse relationship between glycogen content

  10. Glycogen synthase kinase-3 inhibitors suppress the AR-V7-mediated transcription and selectively inhibit cell growth in AR-V7-positive prostate cancer cells.

    PubMed

    Nakata, Daisuke; Koyama, Ryokichi; Nakayama, Kazuhide; Kitazawa, Satoshi; Watanabe, Tatsuya; Hara, Takahito

    2017-06-01

    Recent evidence suggests that androgen receptor (AR) splice variants, including AR-V7, play a pivotal role in resistance to androgen blockade in prostate cancer treatment. The development of new therapeutic agents that can suppress the transcriptional activities of AR splice variants has been anticipated as the next generation treatment of castration-resistant prostate cancer. High-throughput screening of AR-V7 signaling inhibitors was performed using an AR-V7 reporter system. The effects of a glycogen synthase kinase-3 (GSK3) inhibitor, LY-2090314, on endogenous AR-V7 signaling were evaluated in an AR-V7-positive cell line, JDCaP-hr, by quantitative reverse transcription polymerase chain reaction. The relationship between AR-V7 signaling and β-catenin signaling was assessed using RNA interference. The effect of LY-2090314 on cell growth in various prostate cancer cell lines was also evaluated. We identified GSK3 inhibitors as transcriptional suppressors of AR-V7 using a high-throughput screen with an AR-V7 reporter system. LY-2090314 suppressed the reporter activity and endogenous AR-V7 activity in JDCaP-hr cells. Because silencing of β-catenin partly rescued the suppression, it was evident that the suppression was mediated, at least partially, via the activation of β-catenin signaling. AR-V7 signaling and β-catenin signaling reciprocally regulate each other in JDCaP-hr cells, and therefore, GSK3 inhibition can repress AR-V7 transcriptional activity by accumulating intracellular β-catenin. Notably, LY-2090314 selectively inhibited the growth of AR-V7-positive prostate cancer cells in vitro. Our findings demonstrate the potential of GSK3 inhibitors in treating advanced prostate cancer driven by AR splice variants. In vivo evaluation of AR splice variant-positive prostate cancer models will help illustrate the overall significance of GSK3 inhibitors in treating prostate cancer. © 2017 Wiley Periodicals, Inc.

  11. Kinase Inhibitor Screening Identifies Cyclin-Dependent Kinases and Glycogen Synthase Kinase 3 as Potential Modulators of TDP-43 Cytosolic Accumulation during Cell Stress.

    PubMed

    Moujalled, Diane; James, Janine L; Parker, Sarah J; Lidgerwood, Grace E; Duncan, Clare; Meyerowitz, Jodi; Nonaka, Takashi; Hasegawa, Masato; Kanninen, Katja M; Grubman, Alexandra; Liddell, Jeffrey R; Crouch, Peter J; White, Anthony R

    2013-01-01

    Abnormal processing of TAR DNA binding protein 43 (TDP-43) has been identified as a major factor in neuronal degeneration during amyotrophic lateral sclerosis (ALS) or frontotemporal lobar degeneration (FTLD). It is unclear how changes to TDP-43, including nuclear to cytosolic translocation and subsequent accumulation, are controlled in these diseases. TDP-43 is a member of the heterogeneous ribonucleoprotein (hnRNP) RNA binding protein family and is known to associate with cytosolic RNA stress granule proteins in ALS and FTLD. hnRNP trafficking and accumulation is controlled by the action of specific kinases including members of the mitogen-activated protein kinase (MAPK) pathway. However, little is known about how kinase pathways control TDP-43 movement and accumulation. In this study, we used an in vitro model of TDP-43-positve stress granule formation to screen for the effect of kinase inhibitors on TDP-43 accumulation. We found that while a number of kinase inhibitors, particularly of the MAPK pathways modulated both TDP-43 and the global stress granule marker, human antigen R (HuR), multiple inhibitors were more specific to TDP-43 accumulation, including inhibitors of cyclin-dependent kinases (CDKs) and glycogen synthase kinase 3 (GSK3). Close correlation was observed between effects of these inhibitors on TDP-43, hnRNP K and TIAR, but often with different effects on HuR accumulation. This may indicate a potential interaction between TDP-43, hnRNP K and TIAR. CDK inhibitors were also found to reverse pre-formed TDP-43-positive stress granules and both CDK and GSK3 inhibitors abrogated the accumulation of C-terminal TDP-43 (219-414) in transfected cells. Further studies are required to confirm the specific kinases involved and whether their action is through phosphorylation of the TDP-43 binding partner hnRNP K. This knowledge provides a valuable insight into the mechanisms controlling abnormal cytoplasmic TDP-43 accumulation and may herald new opportunities

  12. 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

  13. Inhibition of Glycogen Synthase Kinase or the Apoptotic Protein p53 Lowers the Threshold of Helium Cardioprotection In Vivo: The Role of Mitochondrial Permeability Transition

    PubMed Central

    Pagel, Paul S.; Krolikowski, John G.; Pratt, Phillip F.; Shim, Yon Hee; Amour, Julien; Warltier, David C.; Weihrauch, Dorothee

    2008-01-01

    BACKGROUND Prosurvival signaling kinases inhibit glycogen synthase kinase-3β (GSK-3β) activity and stimulate apoptotic protein p53 degradation. Helium produces cardioprotection by activating prosurvival kinases, but whether GSK and p53 inhibition mediate this process is unknown. We tested the hypothesis that inhibition of GSK or p53 lowers the threshold of helium cardioprotection via a mitochondrial permeability transition pore (mPTP)-dependent mechanism. METHODS Rabbits (n = 85) instrumented for hemodynamic measurement and subjected to a 30 min left anterior descending coronary artery (LAD) occlusion and 3 h reperfusion received 0.9% saline (control), or 1, 3, or 5 cycles of 70% helium-30% oxygen administered for 5 min interspersed with 5 min of an air-oxygen mixture (fraction of inspired oxygen concentration = 0.30) before LAD occlusion. Other rabbits received the GSK inhibitor SB 216763 (SB21; 0.2 or 0.6 mg/kg), the p53 inhibitor pifithrin-α (PIF; 1.5 or 3.0 mg/kg), or SB21 (0.2 mg/kg) or PIF (1.5 mg/kg) plus helium (1 cycle) before LAD occlusion in the presence or absence of the mPTP opener atractyloside (5 mg/kg). RESULTS Helium reduced (P < 0.05) myocardial infarct size (35 ± 6 [n = 7], 25 ± 4 [n = 7], and 20 ± 3% [n = 6] of area at risk, 1, 3, and 5 cycles, respectively) compared with control (44 ± 6% [n = 7]). SB21 (0.6 [n = 7] but not 0.2 mg/kg [n = 6]) and PIF (3.0 [n = 6] but not 1.5 mg/kg [n = 7]) also reduced necrosis. SB21 (0.2 mg/kg) or 1.5 mg/kg PIF (1.5 mg/kg) plus helium (1 cycle; n = 6 per group) decreased infarct size to an equivalent degree as three cycles of helium alone, and this cardioprotection was blocked by atractyloside (n = 7 per group). CONCLUSIONS Inhibition of GSK or p53 lowers the threshold of helium-induced preconditioning via a mPTP-dependent mechanism in vivo. PMID:18713881

  14. An inhibitor-driven study for enhancing the selectivity of indirubin derivatives towards leishmanial Glycogen Synthase Kinase-3 over leishmanial cdc2-related protein kinase 3.

    PubMed

    Efstathiou, Antonia; Gaboriaud-Kolar, Nicolas; Smirlis, Despina; Myrianthopoulos, Vassilios; Vougogiannopoulou, Konstantina; Alexandratos, Alexandros; Kritsanida, Marina; Mikros, Emmanuel; Soteriadou, Ketty; Skaltsounis, Alexios-Leandros

    2014-05-20

    In search of new antiparasitic agents for overcoming the limitations of current leishmaniasis chemotherapy, we have previously shown that 6-bromoindirubin-3'-oxime (6BIO) and several other 6-substituted analogues of indirubin, a naturally occurring bis-indole present in mollusks and plants, displayed reverse selectivity from the respective mammalian kinases, targeting more potently the leishmanial Cyclin-Dependent Kinase-1 (CDK1) homologue [cdc2-related protein kinase 3 (LCRK3)] over leishmanial Glycogen Synthase Kinase-3 (LGSK-3). This reversal of selectivity in Leishmania parasites compared to mammalian cells makes the design of specific indirubin-based LGSK-3 inhibitors difficult. In this context, the identification of compounds bearing specific substitutions that shift indirubin inhibition towards LGSK-3, previously found to be a potential drug target, over LCRK3 is imperative for antileishmanial targeted drug discovery. A new in-house indirubin library, composed of 35 compounds, initially designed to target mammalian kinases (CDKs, GSK-3), was tested against Leishmania donovani promastigotes and intracellular amastigotes using the Alamar blue assay. Indirubins with antileishmanial activity were tested against LGSK-3 and LCRK3 kinases, purified from homologous expression systems. Flow cytometry (FACS) was used to measure the DNA content for cell-cycle analysis and the mode of cell death. Comparative structural analysis of the involved kinases was then performed using the Szmap algorithm. We have identified 7 new indirubin analogues that are selective inhibitors of LGSK-3 over LCRK3. These new inhibitors were also found to display potent antileishmanial activity with GI50 values of <1.5 μΜ. Surprisingly, all the compounds that displayed enhanced selectivity towards LGSK-3, were 6BIO analogues bearing an additional 3'-bulky amino substitution, namely a piperazine or pyrrolidine ring. A comparative structural analysis of the two aforementioned leishmanial

  15. 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

  16. 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

  17. [Protective effect of glycogen synthase kinase 3β inhibition via peroxisome proliferator-activated receptor alpha activation in mice with acute liver failure].

    PubMed

    Shi, H B; Shi, H L; Zhang, X Y; Chen, D X; Duan, Z P; Ren, F

    2017-03-20

    Objective: To investigate the role of the glycogen synthase kinase 3β (GSK3β) and the peroxisome proliferator-activated receptor alpha (PPARα) signaling pathway in acute liver failure and related mechanisms in a mouse model of acute liver failure induced by D-galactosamine/lipopolysaccharide (D-GalN/LPS). Methods: C57BL/6 mice were given intraperitoneal injection of D-GalN/LPS to establish a mouse model of acute liver failure. SB216763 was used to inhibit the activity of GSK3β and PPARα siRNA was used to inhibit the expression of PPARα. Western blotting was used to measure the expression of PPARα protein. The changes in liver pathology were observed to evaluate liver injury, and the serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured to assess liver function. Quantitative real-time PCR was used to measure the mRNA expression of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-12p40 (IL-12p40), and PPARα. A one-way analysis of variance was used for comparison of means between multiple groups; the least significant difference test was used for data with homogeneity of variance, and the Games-Howell method was used for data with heterogeneity of variance. Results: In the mice with liver failure induced by D-GalN/LPS, GSK3β inhibition promoted the mRNA and protein expression of PPARα (F = 13.18 and 301.36, P = 0.00 and 0.00). In the mice with acute liver failure induced by D-GalN/LPS, GSK3β inhibition alleviated liver bleeding, inflammation, and necrosis and reduced the serum levels of ALT (F = 25.16, P = 0.000) and AST (F = 12.96, P = 0.001), as well as the mRNA expression of TNF-α (F = 32.17, P = 0.00), IL-1β (F = 11.57, P = 0.005), and IL-12p40 (F = 14.17, P = 0.015) in liver tissue. The inhibition of PPARα expression reversed the liver-protecting effect of GSK3β inhibition, which manifested as aggravation in liver bleeding, inflammation, and necrosis, increases in the serum levels

  18. Insufficient Astrocyte-Derived Brain-Derived Neurotrophic Factor Contributes to Propofol-Induced Neuron Death Through Akt/Glycogen Synthase Kinase 3β/Mitochondrial Fission Pathway.

    PubMed

    Liu, Yanan; Yan, Yasheng; Inagaki, Yasuyoshi; Logan, Sarah; Bosnjak, Zeljko J; Bai, Xiaowen

    2017-07-01

    Growing animal evidence demonstrates that prolonged exposure to propofol during brain development induces widespread neuronal cell death, but there is little information on the role of astrocytes. Astrocytes can release neurotrophic growth factors such as brain-derived neurotrophic factor (BDNF), which can exert the protective effect on neurons in paracrine fashion. We hypothesize that during propofol anesthesia, BDNF released from developing astrocytes may not be sufficient to prevent propofol-induced neurotoxicity. Hippocampal astrocytes and neurons isolated from neonatal Sprague Dawley rats were exposed to propofol at a clinically relevant dose of 30 μM or dimethyl sulfoxide as control for 6 hours. Propofol-induced cell death was determined by propidium iodide (PI) staining in astrocyte-alone cultures, neuron-alone cultures, or cocultures containing either low or high density of astrocytes (1:9 or 1:1 ratio of astrocytes to neurons ratio [ANR], respectively). The astrocyte-conditioned medium was collected 12 hours after propofol exposure and measured by protein array assay. BDNF concentration in astrocyte-conditioned medium was quantified using enzyme-linked immunosorbent assay. Neuron-alone cultures were treated with BDNF, tyrosine receptor kinase B inhibitor cyclotraxin-B, glycogen synthase kinase 3β (GSK3β) inhibitor CHIR99021, or mitochondrial fission inhibitor Mdivi-1 before propofol exposure. Western blot was performed for quantification of the level of protein kinase B and GSK3β. Mitochondrial shape was visualized through translocase of the outer membrane 20 staining. Propofol increased cell death in neurons by 1.8-fold (% of PI-positive cells [PI%] = 18.6; 95% confidence interval [CI], 15.2-21.9, P < .05) but did not influence astrocyte viability. The neuronal death was attenuated by a high ANR (1:1 cocultures; fold change [FC] = 1.17, 95% CI, 0.96-1.38, P < .05), but not with a low ANR [1:9 cocultures; FC = 1.87, 95% CI, 1.48-2.26, P > .05

  19. 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

  20. Early growth and development impairments in patients with ganglioside GM3 synthase deficiency.

    PubMed

    Wang, H; Wang, A; Wang, D; Bright, A; Sency, V; Zhou, A; Xin, B

    2016-05-01

    Ganglioside GM3 synthase is a key enzyme involved in the biosynthesis of gangliosides. GM3 synthase deficiency (GSD) causes a complete absence of GM3 and all downstream biosynthetic derivatives. The individuals affected by this disorder manifest severe irritability, intractable seizures and profound intellectual disability. However, we have found that most newborns seem symptom-free for a period of time after birth. In order to further understand the onset of the disease, we investigated the early growth and development of patients with this condition through this study. We compared 37 affected individuals with their normal siblings and revealed that all children with GSD had relatively normal intrauterine growth and development, as their weight, length and head circumference were similar to their normal siblings at birth. However, the disease progresses quickly after birth and causes significant constitutional impairments of growth and development by 6 months of age. Neither breastfeeding nor gastrostomy tube placement made significant difference on growth and development as all groups of patients showed the similar pattern. We conclude that GSD causes significant postnatal growth and developmental impairments and the amount of gangliosides in breast milk and general nutritional intervention do not seem to alter these outcomes. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  1. Anti-insulin effects of amylin and calcitonin-gene-related peptide on hepatic glycogen metabolism.

    PubMed Central

    Gómez-Foix, A M; Rodriguez-Gil, J E; Guinovart, J J

    1991-01-01

    To evaluate the effects of amylin and calcitonin-gene-related peptide (CGRP) as anti-insulin agents in hepatic tissue, we have studied whether these two agents counteracted the action of insulin on glycogen metabolism in isolated rat hepatocytes. In this system insulin stimulates [14C]glucose incorporation into glycogen and activates glycogen synthase. Incubation of the cells with insulin in the presence of amylin or CGRP markedly blocked the insulin stimulation of these two parameters, whereas amylin or CGRP acting alone did not induce any effect. We also examined the ability of amylin and CGRP to modify the anti-glucagon effects of insulin. In the presence of 100 nM-amylin or -CGRP, 10 nM-insulin was almost unable to counteract the inactivation of glycogen synthase and the activation of phosphorylase induced by glucagon. In contrast, neither amylin nor CGRP modified the effect of glucagon on these two enzymes. Our results indicate that amylin and CGRP are able to impair the action of insulin on hepatic glycogen metabolism. PMID:1905922

  2. Impaired cognitive performance in neuronal nitric oxide synthase knockout mice is associated with hippocampal protein derangements.

    PubMed

    Kirchner, Liselotte; Weitzdoerfer, Rachel; Hoeger, Harald; Url, Angelika; Schmidt, Peter; Engelmann, Mario; Villar, Santiago Rosell; Fountoulakis, Michael; Lubec, Gert; Lubec, Barbara

    2004-12-01

    Nitric oxide is implicated in modulation of memory and pharmacological as well as genetic inhibition of neuronal nitric oxide synthase (nNOS) leads to impaired cognitive function. We therefore decided to study learning and memory functions and cognitive flexibility in the Morris water maze (MWM) in 1-month-old male mice lacking nNOS (nNOS KO). Hippocampal protein profiling was carried out to possibly link protein derangement to impaired cognitive function. Two-dimensional gel electrophoresis with in-gel digestion of spots and subsequent MALDI-TOF identification of proteins and quantification of proteins using specific software was applied. In the memory as well as in the relearning task of the MWM, most of the nNOS KO failed to find the submerged platform within a given time. Proteomic evaluation of hippocampus, the main anatomical structure computing cognitive functions, revealed aberrant expression of a synaptosomal associated protein of the exocytotic machinery (NSF), glycolytic enzymes, chaperones 78 kDa glucose-regulated protein, T-complex protein 1; the signaling structure guanine nucleotide-binding protein G(I)/G(S)/G(T) and heterogeneous nuclear ribonucleoprotein H of the splicing machinery. We conclude that nNOS knockout mice show impaired spatial performance in the MWM, a finding that may be either linked to direct effects of nNOS/NO and/or to specific hippocampal protein derangements.

  3. Ganglioside synthase knockout in oncogene-transformed fibroblasts depletes gangliosides and impairs tumor growth.

    PubMed

    Liu, Y; Yan, S; Wondimu, A; Bob, D; Weiss, M; Sliwinski, K; Villar, J; Notario, V; Sutherland, M; Colberg-Poley, A M; Ladisch, S

    2010-06-03

    Biologically active membrane gangliosides, expressed and released by many human tumors, are hypothesized to significantly impact tumor progression. Lack of a model of complete and specific tumor ganglioside depletion in vivo, however, has hampered elucidation of their role. Here, we report the creation of a novel, stable, genetically induced tumor cell system resulting in specific and complete blockade of ganglioside synthesis. Wild-type (WT) and GM3 synthase/GM2 synthase double knockout (DKO) murine embryonic fibroblasts were transformed using amphotropic retrovirus-transduced oncogenes (pBABE-c-Myc(T58A)+H-RasG12V). The transformed cells, WT(t) and DKO(t) respectively, evidenced comparable integrated copy numbers and oncogene expression. Ganglioside synthesis was completely blocked in the DKO(t) cells, importantly without triggering an alternate pathway of ganglioside synthesis. Ganglioside depletion (to <0.5 nmol/10(7) cells from 9 to 11 nmol/10(7) WT(t) or untransfected normal fibroblasts) did not adversely affect cell proliferation kinetics but did reduce cell migration on fibronectin-coated wells, consistent with our previous observations in ganglioside-depleted normal human fibroblasts. Strikingly, despite similar oncogene expression and growth kinetics, DKO(t) cells evidenced significantly impaired tumor growth in syngeneic immunocompetent mice, underscoring the pivotal role of tumor cell gangliosides and providing an ideal system for probing their mechanisms of action in vivo.

  4. Ganglioside synthase knockout in oncogene-transformed fibroblasts depletes gangliosides and impairs tumor growth

    PubMed Central

    Liu, Yihui; Yan, Su; Wondimu, Assefa; Bob, Daniel; Weiss, Michael; Sliwinski, Konrad; Villar, Joaquín; Notario, Vicente; Sutherland, Margaret; Colberg-Poley, Anamaris M.; Ladisch, Stephan

    2010-01-01

    Biologically active membrane gangliosides, expressed and released by many human tumors, are hypothesized to significantly impact tumor progression. Lack of a model of complete and specific tumor ganglioside depletion in vivo, however, has hampered elucidation of their role. Here we report the creation of a novel, stable, genetically induced tumor cell system resulting in specific and complete blockade of ganglioside synthesis. Wild type (WT) and GM3 synthase/GM2 synthase double knockout (DKO) murine embryonic fibroblasts were transformed using amphotropic retrovirus-transduced oncogenes (pBABE-c-MycT58A+H-RasG12V). The transformed cells, WTt and DKOt respectively, evidenced comparable integrated copy numbers and oncogene expression. Ganglioside synthesis was completely blocked in the DKOt cells, importantly without triggering an alternate pathway of ganglioside synthesis. Ganglioside depletion (to <0.5 nmol/107 cells from 9-11 nmol/107 WTt or untransfected normal fibroblasts) did not adversely affect cell proliferation kinetics but did reduce cell migration on fibronectin-coated wells, consistent with our previous observations in ganglioside-depleted normal human fibroblasts. Strikingly, despite similar oncogene expression and growth kinetics, DKOt cells evidenced significantly impaired tumor growth in syngeneic immunocompetent mice, underscoring the pivotal role of tumor cell gangliosides and providing an ideal system for probing their mechanisms of action in vivo. PMID:20305696

  5. 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

  6. 25-Hydroxycholesterol impairs endothelial function and vasodilation by uncoupling and inhibiting endothelial nitric oxide synthase.

    PubMed

    Ou, Zhi-Jun; Chen, Jing; Dai, Wei-Ping; Liu, Xiang; Yang, Yin-Ke; Li, Yan; Lin, Ze-Bang; Wang, Tian-Tian; Wu, Ying-Ying; Su, Dan-Hong; Cheng, Tian-Pu; Wang, Zhi-Ping; Tao, Jun; Ou, Jing-Song

    2016-10-01

    Endothelial dysfunction is a key early step in atherosclerosis. 25-Hydroxycholesterol (25-OHC) is found in atherosclerotic lesions. However, whether 25-OHC promotes atherosclerosis is unclear. Here, we hypothesized that 25-OHC, a proinflammatory lipid, can impair endothelial function, which may play an important role in atherosclerosis. Bovine aortic endothelial cells were incubated with 25-OHC. Endothelial cell proliferation, migration, and tube formation were measured. Nitric oxide (NO) production and superoxide anion generation were determined. The expression and phosphorylation of endothelial NO synthase (eNOS) and Akt as well as the association of eNOS and heat shock protein (HSP)90 were detected by immunoblot analysis and immunoprecipitation. Endothelial cell apoptosis was monitored by TUNEL staining and caspase-3 activity, and expression of Bcl-2, Bax, cleaved caspase-9, and cleaved caspase-3 were detected by immunoblot analysis. Finally, aortic rings from Sprague-Dawley rats were isolated and treated with 25-OHC, and endothelium-dependent vasodilation was evaluated. 25-OHC significantly inhibited endothelial cell proliferation, migration, and tube formation. 25-OHC markedly decreased NO production and increased superoxide anion generation. 25-OHC reduced the phosphorylation of Akt and eNOS and the association of eNOS and HSP90. 25-OHC also enhanced endothelial cell apoptosis by decreasing Bcl-2 expression and increasing cleaved caspase-9 and cleaved caspase-3 expressions as well as caspase-3 activity. 25-OHC impaired endothelium-dependent vasodilation. These data demonstrated that 25-OHC could impair endothelial function by uncoupling and inhibiting eNOS activity as well as by inducing endothelial cell apoptosis. Our findings indicate that 25-OHC may play an important role in regulating atherosclerosis. Copyright © 2016 the American Physiological Society.

  7. Biomarker for Glycogen Storage Diseases

    ClinicalTrials.gov

    2017-07-03

    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

  8. Impaired neuronal nitric oxide synthase-mediated vasodilator responses to mental stress in essential hypertension.

    PubMed

    Khan, Sitara G; Geer, Amber; Fok, Henry W; Shabeeh, Husain; Brett, Sally E; Shah, Ajay M; Chowienczyk, Philip J

    2015-04-01

    Neuronal NO synthase (nNOS) regulates blood flow in resistance vasculature at rest and during mental stress. To investigate whether nNOS signaling is dysfunctional in essential hypertension, forearm blood flow responses to mental stress were examined in 88 subjects: 48 with essential hypertension (42±14 years; blood pressure, 141±17/85±15 mm Hg; mean±SD) and 40 normotensive controls (38±14 years; 117±13/74±9 mm Hg). A subsample of 34 subjects (17 hypertensive) participated in a single blind 2-phase crossover study, in which placebo or sildenafil 50 mg PO was administered before an intrabrachial artery infusion of the selective nNOS inhibitor S-methyl-l-thiocitrulline (SMTC, 0.05, 0.1, and 0.2 μmol/min) at rest and during mental stress. In a further subsample (n=21) with an impaired blood flow response to mental stress, responses were measured in the presence and absence of the α-adrenergic antagonist phentolamine. The blood flow response to mental stress was impaired in hypertensive compared with normotensive subjects (37±7% versus 70±8% increase over baseline; P<0.001). SMTC blunted responses to mental stress in normotensive but not in hypertensive subjects (reduction of 40±11% versus 3.0±14%, respectively, P=0.01, between groups). Sildenafil reduced the blood flow response to stress in normotensive subjects from 89±14% to 43±14% (P<0.03) but had no significant effect in hypertensive subjects. Phentolamine augmented impaired blood flow responses to mental stress from 39±8% to 67±13% (P<0.02). Essential hypertension is associated with impaired mental stress-induced nNOS-mediated vasodilator responses; this may relate to increased sympathetic outflow in hypertension. nNOS dysfunction may impair vascular homeostasis in essential hypertension and contribute to stress-induced cardiovascular events. © 2015 American Heart Association, Inc.

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

    PubMed

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

    2016-09-09

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

  10. Baicalin attenuates high fat diet-induced insulin resistance and ectopic fat storage in skeletal muscle, through modulating the protein kinase B/Glycogen synthase kinase 3 beta pathway.

    PubMed

    Xi, You-Li; Li, Hong-Xia; Chen, Chen; Liu, Ya-Qun; Lv, Hong-Mei; Dong, Shi-Qi; Luo, Er-Fei; Gu, Ming-Bo; Liu, Hua

    2016-01-01

    Insulin resistance is the pathophysiological basis of many diseases. Overcoming early insulin resistance highly significant in prevention diabetes, non-alcoholic fatty liver, and atherosclerosis. The present study aimed at evaluating the therapeutic effects of baicalin on insulin resistance and skeletal muscle ectopic fat storage in high fat diet-induced mice, and exploring the potential molecular mechanisms. Insulin resistance in mice was induced with a high fat diet for 16 weeks. Animals were then treated with three different doses of baicalin (100, 200, and 400 mg·kg(-1)·d(-1)) for 14 weeks. Fasting blood glucose, fasting serum insulin, glucose tolerance test (GTT), insulin tolerance test (ITT), and skeletal muscle lipid deposition were measured. Additionally, the AMP-activated protein kinase/acetyl-CoA carboxylase and protein kinase B/Glycogen synthase kinase 3 beta pathways in skeletal muscle were further evaluated. Baicalin significantly reduced the levels of fasting blood glucose and fasting serum insulin and attenuated high fat diet induced glucose tolerance and insulin tolerance. Moreover, insulin resistance was significantly reversed. Pathological analysis revealed baicalin dose-dependently decreased the degree of the ectopic fat storage in skeletal muscle. The properties of baicalin were mediated, at least in part, by inhibition of the AMPK/ACC pathway, a key regulator of de novo lipogenesis and activation of the Akt/GSK-3β pathway, a key regulator of Glycogen synthesis. These data suggest that baicalin, at dose up to 400 mg·kg(-1)·d(-1), is safe and able to attenuate insulin resistance and skeletal muscle ectopic fat storage, through modulating the skeletal muscle AMPK/ACC pathway and Akt/GSK-3β pathway.

  11. 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. Copyright © 2013 Elsevier Ltd. All rights reserved.

  12. Glycogen metabolism in brain and neurons - astrocytes metabolic cooperation can be altered by pre- and neonatal lead (Pb) exposure.

    PubMed

    Baranowska-Bosiacka, Irena; Falkowska, Anna; Gutowska, Izabela; Gąssowska, Magdalena; Kolasa-Wołosiuk, Agnieszka; Tarnowski, Maciej; Chibowska, Karina; Goschorska, Marta; Lubkowska, Anna; Chlubek, Dariusz

    2017-09-12

    Lead (Pb) is an environmental neurotoxin which particularly affects the developing brain but the molecular mechanism of its neurotoxicity still needs clarification. The aim of this paper was to examine whether pre- and neonatal exposure to Pb (concentration of Pb in rat offspring blood below the "threshold level") may affect the brain's energy metabolism in neurons and astrocytes via the amount of available glycogen. We investigated the glycogen concentration in the brain, as well as the expression of the key enzymes involved in glycogen metabolism in brain: glycogen synthase 1 (Gys1), glycogen phosphorylase (PYGM, an isoform active in astrocytes; and PYGB, an isoform active in neurons) and phosphorylase kinase β (PHKB). Moreover, the expression of connexin 43 (Cx43) was evaluated to analyze whether Pb poisoning during the early phase of life may affect the neuron-astrocytes' metabolic cooperation. This work shows for the first time that exposure to Pb in early life can impair brain energy metabolism by reducing the amount of glycogen and decreasing the rate of its metabolism. This reduction in brain glycogen level was accompanied by a decrease in Gys1 expression. We noted a reduction in the immunoreactivity and the gene expression of both PYGB and PYGM isoform, as well as an increase in the expression of PHKB in Pb-treated rats. Moreover, exposure to Pb induced decrease in connexin 43 immunoexpression in all the brain structures analyzed, both in astrocytes as well as in neurons. Our data suggests that exposure to Pb in the pre- and neonatal periods results in a decrease in the level of brain glycogen and a reduction in the rate of its metabolism, thereby reducing glucose availability, which as a further consequence may lead to the impairment of brain energy metabolism and the metabolic cooperation between neurons and astrocytes. Copyright © 2017 Elsevier B.V. All rights reserved.

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

    PubMed

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

    2015-01-01

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

  14. Brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein-1 cooperates with glycogen synthase kinase-3β to regulate osteogenesis of bone-marrow mesenchymal stem cells in type 2 diabetes.

    PubMed

    Li, Xiaoguang; Liu, Na; Wang, Yizhu; Liu, Jinglong; Shi, Haigang; Qu, Zhenzhen; Du, Tingting; Guo, Bin; Gu, Bin

    2017-01-15

    Type 2 diabetes mellitus (T2DM) is associated with inhibited osteogenesis of bone marrow mesenchymal stem cells (BMSCs). Brain and muscle ARNT-like protein 1 (BMAL1) has been linked to the T2DM-related bone remodeling, however, the specific mechanism is still unclear. Herein, we aimed to determine the role of BMAL1 in T2DM-induced suppression of BMSCs osteogenesis. Inhibited osteogenesis and BMAL1 expression were showed in diabetic BMSCs. And while β-catenin and T cell factor (TCF) expression were decreased, the glycogen synthase kinase-3β (GSK-3β) and nemo-like kinase (NLK) expression were increased in diabetic BMSCs. Moreover, over-expression of BMAL1 led to recovered osteogenesis ability and activation of Wnt/β-catenin pathway, which was partially due to inhibition of GSK-3β caused by over-expression of BMAL1. Taken together, our findings provide new insights into the role of BMAL1 in T2DM-induced suppression of BMSCs osteogenesis. Over-expressed BMAL1 could recover BMSCs osteogenesis in T2DM partially by decreasing GSK-3β expression to activate Wnt/β-catenin pathway. BMAL1 may have a potential use in repairing diabetic bone metabolic disorders. Copyright © 2016. Published by Elsevier Ireland Ltd.

  15. 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.

  16. 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

    Chen, Po C; Gaisina, Irina N; El-Khodor, Bassem F; Ramboz, Sylvie; Makhortova, Nina R; Rubin, Lee L; Kozikowski, Alan P

    2012-01-18

    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.

  17. 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

    2009-04-09

    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.

  18. [Experimental models of diabetes mellitus of the 1st and 2nd types in rats: regulation of activity of glycogen synthase by peptides of the insulin superfamily and by epidermal growth factor in skeletal muscles].

    PubMed

    Kuznetsova, L A; Chistiakova, O V

    2012-01-01

    The regulatory effect of peptides of the insulin hyperfamily--insulin, insulin-like growth factor (IGF-1), and relaxin, as well as of epidermal growth factor (EGF) on activity of glycogen synthase (GS) in rat skeletal muscles was studied in norm and in experimental diabetes mellitus of the 1st and 2nd types (DM1, DM2). In norm, peptides in vitro stimulated maximally the GS activity at a concentration of 10-8 M. The row of efficiency of the peptide action was as follows: insulin > IGF-1 > relaxin. In DM1 the basal GS activity did not change, while effect of insulin in vitro was decreased more sharply as compared with action of IGF-1 and relaxin at the 30th day of development of diabetes, i. e., the efficiency row was as follows: IGF-1 = relaxin > insulin. Administration of insulin in vivo did not restore sensitivity of the enzyme to the action of hormone in DM1. In DM2, the GS activity (both the total and active form) decreased. while the stimulatory effect ofpeptides and EGF on the enzyme was absent. Insulin introduced in vitro did not lead to restoration of the enzyme reaction. The conclusion has been made that the insulin resistance affects the basal GS activity in rat skeletal muscles as well as the regulation of the enzyme by peptides of the insulin nature and by EGF, which is more obvious in DM2, than in DM1.

  19. 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

  20. New insights concerning insulin synthesis and its secretion in rat hippocampus and cerebral cortex: amyloid-β1-42-induced reduction of proinsulin level via glycogen synthase kinase-3β.

    PubMed

    Nemoto, Takayuki; Toyoshima-Aoyama, Fumiyo; Yanagita, Toshihiko; Maruta, Toyoaki; Fujita, Hiroshi; Koshida, Tomohiro; Yonaha, Tetsu; Wada, Akihiko; Sawaguchi, Akira; Murakami, Manabu

    2014-02-01

    The reduction of insulin levels in hippocampal areas is associated with Alzheimer's disease. The present study using rat brain explores the mechanisms of insulin synthesis and secretion, as well as amyloid-β1-42 (Aβ(1-42))-induced reduction of proinsulin expression. After confirming the expression of insulin mRNA and proinsulin in rat brain, we visualized and analyzed the motion of insulin secretion in rat hippocampal neurons using pH-sensitive green fluorescent protein (pHluorin) fused to the insulin. In the rat hippocampal neurons expressing insulin-pHluorin, time-lapse confocal laser scanning microscopy revealed the appearance of fluorescent spots induced by depolarization after stimulation with 50 mM KCl. In these fluorescent spots, Ca(2+)-dependent activator protein for secretion 2 (CAPS2), which is the regulator of the dense-core vesicle involving neuronal peptides, was co-localized with insulin-pHluorin. However, Aβ(1-42)-induced reduction of proinsulin in rat hippocampal neurons was inhibited by treatment with lithium and transfection with glycogen synthase kinase-3β (GSK-3β) siRNA. These results demonstrate that synthesized insulin is secreted from rat hippocampal and cortical neuron's dense-core vesicles, and that activation of GSK-3β in Aβ(1-42)-induced Alzheimer's model hippocampal neurons decreases the insulin synthesis.

  1. 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

    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.

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

    PubMed

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

    2012-03-02

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

  3. Activation of Casein Kinase II and Inhibition of Phosphatase and Tensin Homologue Deleted on Chromosome 10 Phosphatase by Nerve Growth Factor/p75NTR Inhibit Glycogen Synthase Kinase-3β and Stimulate Axonal Growth

    PubMed Central

    Arevalo, María-Angeles

    2006-01-01

    Axonal elongation and guidance are controlled by extracellular factors such as the neurotrophins. Indeed, nerve growth factor (NGF) seems to promote axon growth through binding to its p75NTR receptor and inactivating RhoA. Furthermore, the local inhibition of glycogen synthase kinase (GSK)-3β by NGF also favors microtubule polymerization and axon extension. Inactivation of GSK-3β may be due to the NGF/TrkA-mediated activation of phosphatidylinositol-3 kinase (PI-3 kinase), which increases the levels of phosphatydilinositol 3-phosphate [PI(3)P]. However, we show here that NGF may inactivate GSK-3β through an alternative mechanism. In cultured hippocampal neurons, the capacity of NGF to promote axon elongation is mostly mediated by p75NTR, and the activation of this pathway leads to the inactivation of GSK-3β. However, the signaling pathway triggered by NGF/p75NTR acts through casein kinase II (CK2). NGF/p75NTR-activated CK2 phosphorylates the phosphatase and tensin homologue deleted on chromosome 10 (PTEN), thus rendering this phosphatase inactive. Like activation of the PI-3 kinase, PTEN inactivation allows PI(3)P levels to increase, thus favoring GSK-3β inactivation and axon outgrowth. This newly disclosed mechanism may help to extend the repertoire of pharmacological agents that activate CK2 or that inhibit PTEN to stimulate axon regeneration after trauma or disease. PMID:16723502

  4. Inactivation of fatty acid synthase impairs hepatocarcinogenesis driven by AKT in mice and humans

    PubMed Central

    Li, Lei; Pilo, Giulia M.; Li, Xiaolei; Cigliano, Antonio; Latte, Gavinella; Che, Li; Joseph, Christy; Mela, Marta; Wang, Chunmei; Jiang, Lijie; Ribback, Silvia; Simile, Maria M.; Pascale, Rosa M.; Dombrowski, Frank; Evert, Matthias; Semenkovich, Clay F.; Chen, Xin; Calvisi, Diego F.

    2015-01-01

    Background & Aims Cumulating evidence underlines the crucial role of aberrant lipogenesis in human hepatocellular carcinoma (HCC). Here, we investigated the oncogenic potential of fatty acid synthase (FASN), the master regulator of de novo lipogenesis, in the mouse liver. Methods FASN was overexpressed in the mouse liver, either alone or in combination with activated N-Ras, c-Met, or SCD1, via hydrodynamic injection. Activated AKT was overexpressed via hydrodynamic injection in livers of conditional FASN or Rictor knockout mice. FASN was suppressed in human hepatoma cell lines via specific small interfering RNA. Results Overexpression of FASN, either alone or in combination with other genes associated with hepatocarcinogenesis, did not induce histological liver alterations. In contrast, genetic ablation of FASN resulted in the complete inhibition of hepatocarcinogenesis in AKT-overexpressing mice. In human HCC cell lines, FASN inactivation led to a decline in cell proliferation and a rise in apoptosis, which were paralleled by a decrease in the levels of phosphorylated/activated AKT, an event controlled by the mammalian target of rapamycin complex 2 (mTORC2). Downregulation of AKT phosphorylation/activation following FASN inactivation was associated with strong inhibition of rapamycin-insensitive companion of mTOR (Rictor), the major component of mTORC2, at post-transcriptional level. Finally, genetic ablation of Rictor impaired AKT-driven hepatocarcinogenesis in mice. Conclusions FASN is not oncogenic per se in the mouse liver, but is necessary for AKT-driven hepatocarcinogenesis. Pharmacological blockade of FASN might be highly useful in the treatment of human HCC characterized by activation of the AKT pathway. PMID:26476289

  5. Lithium Suppresses Astrogliogenesis by Neural Stem and Progenitor Cells by Inhibiting STAT3 Pathway Independently of Glycogen Synthase Kinase 3 Beta

    PubMed Central

    Zhu, Zhenzhong; Kremer, Penny; Tadmori, Iman; Ren, Yi; Sun, Dongming; He, Xijing; Young, Wise

    2011-01-01

    Transplanted neural stem and progenitor cells (NSCs) produce mostly astrocytes in injured spinal cords. Lithium stimulates neurogenesis by inhibiting GSK3b (glycogen synthetase kinase 3-beta) and increasing WNT/beta catenin. Lithium suppresses astrogliogenesis but the mechanisms were unclear. We cultured NSCs from subventricular zone of neonatal rats and showed that lithium reduced NSC production of astrocytes as well as proliferation of glia restricted progenitor (GRP) cells. Lithium strongly inhibited STAT3 (signal transducer and activator of transcription 3) activation, a messenger system known to promote astrogliogenesis and cancer. Lithium abolished STAT3 activation and astrogliogenesis induced by a STAT3 agonist AICAR (5-aminoimidazole-4-carboxamide 1-beta-D-ribofuranoside), suggesting that lithium suppresses astrogliogenesis by inhibiting STAT3. GSK3β inhibition either by a specific GSK3β inhibitor SB216763 or overexpression of GID5-6 (GSK3β Interaction Domain aa380 to 404) did not suppress astrogliogenesis and GRP proliferation. GSK3β inhibition also did not suppress STAT3 activation. Together, these results indicate that lithium inhibits astrogliogenesis through non-GSK3β-mediated inhibition of STAT. Lithium may increase efficacy of NSC transplants by increasing neurogenesis and reducing astrogliogenesis. Our results also may explain the strong safety record of lithium treatment of manic depression. Millions of people take high-dose (>1 gram/day) lithium carbonate for a lifetime. GSK3b inhibition increases WNT/beta catenin, associated with colon and other cancers. STAT3 inhibition may reduce risk for cancer. PMID:21931595

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

    PubMed

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

    2013-02-01

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

  7. 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.

  8. Role of Maltose Enzymes in Glycogen Synthesis by Escherichia coli▿

    PubMed Central

    Park, Jong-Tae; Shim, Jae-Hoon; Tran, Phuong Lan; Hong, In-Hee; Yong, Hwan-Ung; Oktavina, Ershita Fitria; Nguyen, Hai Dang; Kim, Jung-Wan; Lee, Tae Soo; Park, Sung-Hoon; Boos, Winfried; Park, Kwan-Hwa

    2011-01-01

    Mutants with deletion mutations in the glg and mal gene clusters of Escherichia coli MC4100 were used to gain insight into glycogen and maltodextrin metabolism. Glycogen content, molecular mass, and branch chain distribution were analyzed in the wild type and in ΔmalP (encoding maltodextrin phosphorylase), ΔmalQ (encoding amylomaltase), ΔglgA (encoding glycogen synthase), and ΔglgA ΔmalP derivatives. The wild type showed increasing amounts of glycogen when grown on glucose, maltose, or maltodextrin. When strains were grown on maltose, the glycogen content was 20 times higher in the ΔmalP strain (0.97 mg/mg protein) than in the wild type (0.05 mg/mg protein). When strains were grown on glucose, the ΔmalP strain and the wild type had similar glycogen contents (0.04 mg/mg and 0.03 mg/mg protein, respectively). The ΔmalQ mutant did not grow on maltose but showed wild-type amounts of glycogen when grown on glucose, demonstrating the exclusive function of GlgA for glycogen synthesis in the absence of maltose metabolism. No glycogen was found in the ΔglgA and ΔglgA ΔmalP strains grown on glucose, but substantial amounts (0.18 and 1.0 mg/mg protein, respectively) were found when they were grown on maltodextrin. This demonstrates that the action of MalQ on maltose or maltodextrin can lead to the formation of glycogen and that MalP controls (inhibits) this pathway. In vitro, MalQ in the presence of GlgB (a branching enzyme) was able to form glycogen from maltose or linear maltodextrins. We propose a model of maltodextrin utilization for the formation of glycogen in the absence of glycogen synthase. PMID:21421758

  9. Role of maltose enzymes in glycogen synthesis by Escherichia coli.

    PubMed

    Park, Jong-Tae; Shim, Jae-Hoon; Tran, Phuong Lan; Hong, In-Hee; Yong, Hwan-Ung; Oktavina, Ershita Fitria; Nguyen, Hai Dang; Kim, Jung-Wan; Lee, Tae Soo; Park, Sung-Hoon; Boos, Winfried; Park, Kwan-Hwa

    2011-05-01

    Mutants with deletion mutations in the glg and mal gene clusters of Escherichia coli MC4100 were used to gain insight into glycogen and maltodextrin metabolism. Glycogen content, molecular mass, and branch chain distribution were analyzed in the wild type and in ΔmalP (encoding maltodextrin phosphorylase), ΔmalQ (encoding amylomaltase), ΔglgA (encoding glycogen synthase), and ΔglgA ΔmalP derivatives. The wild type showed increasing amounts of glycogen when grown on glucose, maltose, or maltodextrin. When strains were grown on maltose, the glycogen content was 20 times higher in the ΔmalP strain (0.97 mg/mg protein) than in the wild type (0.05 mg/mg protein). When strains were grown on glucose, the ΔmalP strain and the wild type had similar glycogen contents (0.04 mg/mg and 0.03 mg/mg protein, respectively). The ΔmalQ mutant did not grow on maltose but showed wild-type amounts of glycogen when grown on glucose, demonstrating the exclusive function of GlgA for glycogen synthesis in the absence of maltose metabolism. No glycogen was found in the ΔglgA and ΔglgA ΔmalP strains grown on glucose, but substantial amounts (0.18 and 1.0 mg/mg protein, respectively) were found when they were grown on maltodextrin. This demonstrates that the action of MalQ on maltose or maltodextrin can lead to the formation of glycogen and that MalP controls (inhibits) this pathway. In vitro, MalQ in the presence of GlgB (a branching enzyme) was able to form glycogen from maltose or linear maltodextrins. We propose a model of maltodextrin utilization for the formation of glycogen in the absence of glycogen synthase.

  10. Effects of Valproic Acid Derivatives on Inositol Trisphosphate Depletion, Teratogenicity, Glycogen Synthase Kinase-3β Inhibition, and Viral Replication: A Screening Approach for New Bipolar Disorder Drugs Derived from the Valproic Acid Core Structure

    PubMed Central

    Eickholt, B. J.; Towers, G. J.; Ryves, W. J.; Eikel, D.; Adley, K.; Ylinen, L. M. J.; Chadborn, N. H.; Harwood, A. J.; Nau, H.; Williams, R. S. B.

    2005-01-01

    Inositol-1,4,5-trisphosphate (InsP3) depletion has been implicated in the therapeutic action of bipolar disorder drugs, including valproic acid (VPA). It is not currently known whether the effect of VPA on InsP3 depletion is related to the deleterious effects of teratogenicity or elevated viral replication, or if it occurs via putative inhibitory effects on glycogen synthase kinase-3β(GSK-3β). In addition, the structural requirements of VPA-related compounds to cause InsP3 depletion are unknown. In the current study, we selected a set of 10 VPA congeners to examine their effects on InsP3 depletion, in vivo teratogenic potency, HIV replication, and GSK-3β activity in vitro. We found four compounds that function to deplete InsP3 in the model eukaryote Dictyostelium discoideum, and these drugs all cause growth-cone enlargement in mammalian primary neurons, consistent with the effect of InsP3 depletion. No relationship was found between InsP3 depletion and teratogenic or elevated viral replication effects, and none of the VPA congeners were found to affect GSK-3β activity. Structural requirements of VPA congers to maintain InsP3 depletion efficacy greater than that of lithium are a carboxylic-acid function without dependence on side-chain length, branching, or saturation. Noteworthy is the enantiomeric differentiation if a chiral center exists, suggesting that InsP3 depletion is mediated by a stereoselective mode of action. Thus, the effect of InsP3 depletion can be separated from that of teratogenic potency and elevated viral replication effect. We have used this to identify two VPA derivatives that share the common InsP3-depleting action of VPA, lithium and carbamazepine, but do not show the side effects of VPA, thus providing promising novel candidates for bipolar disorder treatment. PMID:15687223

  11. 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

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

    2015-09-01

    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.

  12. 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.

  13. Impaired inflammatory and pain responses in mice lacking an inducible prostaglandin E synthase

    PubMed Central

    Trebino, Catherine E.; Stock, Jeffrey L.; Gibbons, Colleen P.; Naiman, Brian M.; Wachtmann, Timothy S.; Umland, John P.; Pandher, Karamjeet; Lapointe, Jean-Martin; Saha, Sipra; Roach, Marsha L.; Carter, Demetrius; Thomas, Nathalie A.; Durtschi, Becky A.; McNeish, John D.; Hambor, John E.; Jakobsson, Per-Johan; Carty, Thomas J.; Perez, Jose R.; Audoly, Laurent P.

    2003-01-01

    Prostaglandin (PG)E2 is a potent mediator of pain and inflammation, and high levels of this lipid mediator are observed in numerous disease states. The inhibition of PGE2 production to control pain and to treat diseases such as rheumatoid arthritis to date has depended on nonsteroidal antiinflammatory agents such as aspirin. However, these agents inhibit the synthesis of all prostanoids. To produce biologically active PGE2, PGE synthases catalyze the isomerization of PGH2 into PGE2. Recently, several PGE synthases have been identified and cloned, but their role in inflammation is not clear. To study the physiological role of the individual PGE synthases, we have generated by targeted homologous recombination a mouse line deficient in microsomal PGE synthase 1 (mPGES1) on the inbred DBA/1lacJ background. mPGES1-deficient (mPGES1-/-) mice are viable and fertile and develop normally compared with wild-type controls. However, mPGES1-/- mice displayed a marked reduction in inflammatory responses compared with mPGES1+/+ mice in multiple assays. Here, we identify mPGES1 as the PGE synthase that contributes to the pathogenesis of collagen-induced arthritis, a disease model of human rheumatoid arthritis. We also show that mPGES1 is responsible for the production of PGE2 that mediates acute pain during an inflammatory response. These findings suggest that mPGES1 provides a target for the treatment of inflammatory diseases and pain associated with inflammatory states. PMID:12835414

  14. Glycogen and Glucose Metabolism Are Essential for Early Embryonic Development of the Red Flour Beetle Tribolium castaneum

    PubMed Central

    Fraga, Amanda; Ribeiro, Lupis; Lobato, Mariana; Santos, Vitória; Silva, José Roberto; Gomes, Helga; da Cunha Moraes, Jorge Luiz; de Souza Menezes, Jackson

    2013-01-01

    Control of energy metabolism is an essential process for life. In insects, egg formation (oogenesis) and embryogenesis is dependent on stored molecules deposited by the mother or transcribed later by the zygote. In oviparous insects the egg becomes an isolated system after egg laying with all energy conversion taking place during embryogenesis. Previous studies in a few vector species showed a strong correlation of key morphogenetic events and changes in glucose metabolism. Here, we investigate glycogen and glucose metabolism in the red flour beetle Tribolium castaneum, an insect amenable to functional genomic studies. To examine the role of the key enzymes on glycogen and glucose regulation we cloned and analyzed the function of glycogen synthase kinase 3 (GSK-3) and hexokinase (HexA) genes during T. castaneum embryogenesis. Expression analysis via in situ hybridization shows that both genes are expressed only in the embryonic tissue, suggesting that embryonic and extra-embryonic cells display different metabolic activities. dsRNA adult female injection (parental RNAi) of both genes lead a reduction in egg laying and to embryonic lethality. Morphological analysis via DAPI stainings indicates that early development is impaired in Tc-GSK-3 and Tc-HexA1 RNAi embryos. Importantly, glycogen levels are upregulated after Tc-GSK-3 RNAi and glucose levels are upregulated after Tc-HexA1 RNAi, indicating that both genes control metabolism during embryogenesis and oogenesis, respectively. Altogether our results show that T. castaneum embryogenesis depends on the proper control of glucose and glycogen. PMID:23750237

  15. Glycogen and glucose metabolism are essential for early embryonic development of the red flour beetle Tribolium castaneum.

    PubMed

    Fraga, Amanda; Ribeiro, Lupis; Lobato, Mariana; Santos, Vitória; Silva, José Roberto; Gomes, Helga; da Cunha Moraes, Jorge Luiz; de Souza Menezes, Jackson; de Oliveira, Carlos Jorge Logullo; Campos, Eldo; da Fonseca, Rodrigo Nunes

    2013-01-01

    Control of energy metabolism is an essential process for life. In insects, egg formation (oogenesis) and embryogenesis is dependent on stored molecules deposited by the mother or transcribed later by the zygote. In oviparous insects the egg becomes an isolated system after egg laying with all energy conversion taking place during embryogenesis. Previous studies in a few vector species showed a strong correlation of key morphogenetic events and changes in glucose metabolism. Here, we investigate glycogen and glucose metabolism in the red flour beetle Tribolium castaneum, an insect amenable to functional genomic studies. To examine the role of the key enzymes on glycogen and glucose regulation we cloned and analyzed the function of glycogen synthase kinase 3 (GSK-3) and hexokinase (HexA) genes during T. castaneum embryogenesis. Expression analysis via in situ hybridization shows that both genes are expressed only in the embryonic tissue, suggesting that embryonic and extra-embryonic cells display different metabolic activities. dsRNA adult female injection (parental RNAi) of both genes lead a reduction in egg laying and to embryonic lethality. Morphological analysis via DAPI stainings indicates that early development is impaired in Tc-GSK-3 and Tc-HexA1 RNAi embryos. Importantly, glycogen levels are upregulated after Tc-GSK-3 RNAi and glucose levels are upregulated after Tc-HexA1 RNAi, indicating that both genes control metabolism during embryogenesis and oogenesis, respectively. Altogether our results show that T. castaneum embryogenesis depends on the proper control of glucose and glycogen.

  16. Convergence of glycogen synthase kinase 3β and GR signaling in response to fluoxetine treatment in chronically stressed female and male rats.

    PubMed

    Mitic, Milos; Brkic, Zeljka; Lukic, Iva; Adzic, Miroslav

    2017-08-30

    Accumulating evidence strongly suggest that impaired glucocorticoid receptor (GR) signaling is involved in stress-related mood disorders, and nominate GR as a potential target for antidepressants (ADs). It is known that different classes of ADs affects the GR action via modifying its phosphorylation, while the mechanism through which ADs alter GR phosphorylation targeted by GSK3β, a kinase modulated via serotonin neurotransmission, are unclear. On this basis, we investigated whether GSK3β-GR signaling could be a convergence point of fluoxetine action on brain function and behavior, by examining its effect on GSK3β targeted-GR phosphorylation on threonine 171 (pGR171), and expression of GR-regulated genes in the hippocampus of female and male rats exposed to chronic isolation stress. Stress induced sex-specific GSK3β-targeted phosphorylation of pGR171 in the nucleus of the hippocampus of stressed animals. Namely, while in females stress triggered coupled action of GSK3β-pGR171 signaling, in males changes in pGR171 levels did not correspond to GSK3β activity. On the other hand, fluoxetine managed to up-regulate this pathway in sex-unbiased manner. Furthermore, fluoxetine reverted stress-induced changes in most of the analyzed genes in males, CRH, 5-HT1a and p11, while in females its effect was limited to CRH. These data further suggest that pGR171 signaling affects cellular localization of GR in response to chronic stress and fluoxetine in both sexes. Collectively, our results describe a novel convergence point between GR signaling and GSK3β pathway in rat hippocampus in response to stress and fluoxetine in both sexes and its involvement in fluoxetine-regulated brain function in males. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Neuronal glycogen synthesis contributes to physiological aging

    PubMed Central

    Sinadinos, Christopher; Valles-Ortega, Jordi; Boulan, Laura; Solsona, Estel; Tevy, Maria F; Marquez, Mercedes; Duran, Jordi; Lopez-Iglesias, Carmen; Calbó, Joaquim; Blasco, Ester; Pumarola, Marti; Milán, Marco; Guinovart, Joan J

    2014-01-01

    Glycogen is a branched polymer of glucose and the carbohydrate energy store for animal cells. In the brain, it is essentially found in glial cells, although it is also present in minute amounts in neurons. In humans, loss-of-function mutations in laforin and malin, proteins involved in suppressing glycogen synthesis, induce the presence of high numbers of insoluble polyglucosan bodies in neuronal cells. Known as Lafora bodies (LBs), these deposits result in the aggressive neurodegeneration seen in Lafora’s disease. Polysaccharide-based aggregates, called corpora amylacea (CA), are also present in the neurons of aged human brains. Despite the similarity of CA to LBs, the mechanisms and functional consequences of CA formation are yet unknown. Here, we show that wild-type laboratory mice also accumulate glycogen-based aggregates in the brain as they age. These structures are immunopositive for an array of metabolic and stress-response proteins, some of which were previously shown to aggregate in correlation with age in the human brain and are also present in LBs. Remarkably, these structures and their associated protein aggregates are not present in the aged mouse brain upon genetic ablation of glycogen synthase. Similar genetic intervention in Drosophila prevents the accumulation of glycogen clusters in the neuronal processes of aged flies. Most interestingly, targeted reduction of Drosophila glycogen synthase in neurons improves neurological function with age and extends lifespan. These results demonstrate that neuronal glycogen accumulation contributes to physiological aging and may therefore constitute a key factor regulating age-related neurological decline in humans. PMID:25059425

  18. Amyloid β oligomer-induced ERK1/2-dependent serine 636/639 phosphorylation of insulin receptor substrate-1 impairs insulin signaling and glycogen storage in human astrocytes.

    PubMed

    Zhang, Qinghua; Guo, Shougang; Zhang, Xiao; Tang, Shi; Wang, Lu; Han, Xiaojuan; Shao, Wen; Cong, Lin; Du, Yifeng

    2015-04-25

    This study is to investigate the effect of amyloid β1-42 oligomers on insulin signaling in astrocytes. Synthetic Aβ1-42 oligomers were prepared and the oligomeric form of Aβ1-42 was verified by an electron microscope. Normal human astrocytes were cultured in Dulbecco's Modified Eagle Medium. Western blotting was employed to measure the amount of lysate proteins. Immunofluorescence was performed to detect the distribution of phosphorylated insulin receptor substrate-1 and expression of P-GSK3β in astrocytes under confocal microscopy and fluorescent microscopy, respectively. Periodic Acid-Schiff staining was used to detect glycogen, the content of which was measured using glycogen assay. Our data showed that Aβ1-42 oligomers inhibited insulin-induced serine phosphorylation of Akt at 473 and GSK3β at serine 9, as well as glycogen storage. However, the levels of phosphorylated GSK3β at tyrosine 216 were significantly increased in the presence of Aβ1-42 oligomers. In addition, the levels of phosphorylated ERK1/2 and insulin receptor substrate-1 at serine 636/639 were significantly increased in response to treatment with Aβ1-42 oligomers. Of note, the responses and inhibitory effects of Aβ1-42 oligomers on insulin signaling were partially reversed by ERK1/2 upstream inhibitor PD98059. Our results demonstrated that Aβ1-42 oligomers impaired insulin signaling and suppressed insulin-induced glycogen storage in human astrocytes, probably due to ERK1/2-dependent serine phosphorylation of insulin receptor substrate-1 at 636/639 induced by Aβ1-42 oligomers. Copyright © 2015 Elsevier B.V. All rights reserved.

  19. Transcriptional regulation, stabilization, and subcellular redistribution of multidrug resistance-associated protein 1 (MRP1) by glycogen synthase kinase 3αβ: novel insights on modes of cadmium-induced cell death stimulated by MRP1.

    PubMed

    Kim, Hak-Ryul; Lee, Kang-Yo; Lee, Kang-Yoo; Ahn, Sang-Gun; Lee, Byung-Hoon; Jung, Ki-Tae; Yoon, Jung-Hoon; Yoon, Hyo-Eun; Oh, Seon-Hee

    2015-08-01

    Cadmium (Cd) resistance is associated with the suppression of autophagy in H460 lung cancer cells, which is regulated by phospho(p)serine-glycogen synthase kinase (GSK) 3αβ. However, the involvement of multidrug resistance (MDR) in this signaling pathway and its underlying mechanisms remain to be elucidated. In this study, we used Cd-resistant cells (RH460), developed from H460 lung cancer cells, to demonstrate that the induction of MDR-associated protein (MRP1) in response to Cd is enhanced in H460 cells compared to RH460. Treating RH460 cells with Cd induced large cytoplasmic vacuoles, which was inhibited by the autophagy inhibitor 3-methyladenine. MRP1 was detected in the nuclear-rich membrane fractions and redistributed from the perinuclear to the cytoplasmic compartment following exposure to Cd. Cd-induced MRP1, p-Ser/p-Tyr GSK3αβ, and LC3-II were all suppressed by the GSK3 inhibitor SB216763, but increased by lithium. Furthermore, MRP1 was upregulated by the Ser/Thr phosphatase inhibitor okadaic acid and downregulated by the tyrosine phosphatase inhibitor vanadate, suggesting that MRP1 protein was stabilized by p-Ser GSK3αβ. In addition, co-immunoprecipitation and co-localization analyzes revealed a physical interaction between MRP1 and p-Ser GSK3αβ. Genetic knockdown of GSK3β decreased Cd-induced MRP1 mRNA and protein levels, whereas its overexpression upregulated MRP1 protein expression. MRP1 also co-localized with lysosomal membrane protein-2, which may cause lysosomal membrane permeabilization and the subsequent release of cathepsins into the cytosol. In mice chronically injected with Cd, MRP1 localized to the perinuclear region of bronchial and alveolar epithelial cells. Collectively, these data suggest that Cd toxicity is regulated by the transcriptional regulation, stabilization, and subcellular redistribution of MRP1 via the posttranslational modification of GSK3αβ. Therefore, the serine phosphorylation of GSK3αβ plays a critical role in

  20. Nitric oxide synthase inhibitor, aminoguanidine reduces intracerebroventricular colchicine induced neurodegeneration, memory impairments and changes of systemic immune responses in rats.

    PubMed

    Sil, Susmita; Ghosh, Tusharkanti; Ghosh, Rupsa; Gupta, Pritha

    2017-02-15

    Intracerebroventricular (i.c.v.) injection of colchicine induces neurodegeneration, memory impairments and changes of some systemic immune responses in rats. Though the role of cox 2 in these colchicine induced changes have been evaluated, the influence of nitric oxide synthase (NOS) remains to be studied. The present study was designed to assess the role of NOS on the i.c.v. colchicine induced neurodegeneration, memory impairments and changes of some systemic immune responses by inhibiting its activity with aminoguanidine. In the present study the impairments of working and reference memories, neurodegeneration (chromatolysis and plaque formation) and changes of neuroinflammatory markers in the hippocampus (increased TNF α, IL 1β, ROS and nitrite) along with changes of serum inflammatory markers (TNF α, IL 1β, ROS and nitrite) and alteration of systemic immune responses (higher phagocytic activity of blood WBC and splenic PMN, higher cytotoxicity and lower leukocyte adhesion inhibition index of splenic MNC) were measured in the intracerebroventricular colchicine injected rats (ICIR). Administration of aminoguanidine (p.o. 30/50mg/kg body weight) to ICIR resulted in recovery of neuroinflammation and partial prevention of neurodegeneration which could be corroborated with the partial recovery of memory impairments in this model. The recovery of serum inflammatory markers and the systemic immune responses in ICIR was also observed after administration of aminoguanidine. Therefore, the present study shows that aminoguanidine can protect the colchicine induced neurodegeneration, memory impairments, and changes of systemic immune systemic responses in ICIR by inhibiting the iNOS. Copyright © 2016 Elsevier B.V. All rights reserved.

  1. 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.

  2. 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.

  3. 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.

  4. 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

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

    PubMed

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

    2015-06-01

    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. Copyright © 2015 the American Physiological Society.

  6. Isolation and characterization of a Saccharomyces cerevisiae mutant with impaired glutamate synthase activity.

    PubMed

    Folch, J L; Antaramián, A; Rodríguez, L; Bravo, A; Brunner, A; González, A

    1989-12-01

    A mutant of Saccharomyces cerevisiae that lacks glutamate synthase (GOGAT) activity has been isolated. This mutant was obtained after chemical mutagenesis of a NADP-glutamate dehydrogenase-less mutant strain. The gdh gus mutant is a glutamate auxotroph. The genetic analysis of the gus mutant showed that the GOGAT-less phenotype is due to the presence of two loosely linked mutations. Evidence is presented which suggests the possibility that S. cerevisiae has two GOGAT activities, designated GOGAT A and GOGAT B. These activities can be distinguished by their pH optima and by their regulation by glutamate. Furthermore, one of the mutations responsible for the GOGAT-less phenotype affected GOGAT A activity, while the other mutation affected GOGAT B activity.

  7. Isolation and characterization of a Saccharomyces cerevisiae mutant with impaired glutamate synthase activity.

    PubMed Central

    Folch, J L; Antaramián, A; Rodríguez, L; Bravo, A; Brunner, A; González, A

    1989-01-01

    A mutant of Saccharomyces cerevisiae that lacks glutamate synthase (GOGAT) activity has been isolated. This mutant was obtained after chemical mutagenesis of a NADP-glutamate dehydrogenase-less mutant strain. The gdh gus mutant is a glutamate auxotroph. The genetic analysis of the gus mutant showed that the GOGAT-less phenotype is due to the presence of two loosely linked mutations. Evidence is presented which suggests the possibility that S. cerevisiae has two GOGAT activities, designated GOGAT A and GOGAT B. These activities can be distinguished by their pH optima and by their regulation by glutamate. Furthermore, one of the mutations responsible for the GOGAT-less phenotype affected GOGAT A activity, while the other mutation affected GOGAT B activity. PMID:2687252

  8. The inhibition of glycogen synthase kinase-3 by insulin or insulin-like growth factor 1 in the rat skeletal muscle cell line L6 is blocked by wortmannin, but not by rapamycin: evidence that wortmannin blocks activation of the mitogen-activated protein kinase pathway in L6 cells between Ras and Raf.

    PubMed Central

    Cross, D A; Alessi, D R; Vandenheede, J R; McDowell, H E; Hundal, H S; Cohen, P

    1994-01-01

    Glycogen synthase kinase-3 (GSK3) is inactivated in vitro by p70 S6 kinase or MAP kinase-activated protein kinase-1 beta (MAPKAP kinase-1 beta; also known as Rsk-2). Here we show that GSK3 isoforms are inhibited by 40% within minutes after stimulation of the rat skeletal-muscle cell line L6 with insulin-like growth factor-1 (IGF-1) or insulin. GSK3 was similarly inhibited in rabbit skeletal muscle after an intravenous injection of insulin. Inhibition resulted from increased phosphorylation of GSK3, probably at a serine/threonine residue(s), because it was reversed by incubation with protein phosphatase-2A. Rapamycin blocked the activation of p70 S6 kinase by IGF-1 in L6 cells, but had no effect on the inhibition of GSK3 or the activation of MAPKAP kinase-1 beta. In contrast, wortmannin, a potent inhibitor of PtdIns 3-kinase, prevented the inactivation of GSK3 and the activation of MAPKAP kinase-1 beta and p70 S6 kinase by IGF-1 or insulin. Wortmannin also blocked the activation of p74raf-1. MAP kinase kinase and p42 MAP kinase, but not the formation of GTP-Ras by IGF-1. The results suggest that the stimulation of glycogen synthase by insulin/IGF-1 in skeletal muscle involves the MAP-KAP kinase-1-catalysed inhibition of GSK3, as well as the previously described activation of the glycogen-associated form of protein phosphatase-1. Images Figure 1 PMID:7945242

  9. Dysfunctional Muscle and Liver Glycogen Metabolism in mdx Dystrophic Mice

    PubMed Central

    Stapleton, David I.; Lau, Xianzhong; Flores, Marcelo; Trieu, Jennifer; Gehrig, Stefan M.; Chee, Annabel; Naim, Timur; Lynch, Gordon S.; Koopman, René

    2014-01-01

    Background Duchenne muscular dystrophy (DMD) is a severe, genetic muscle wasting disorder characterised by progressive muscle weakness. DMD is caused by mutations in the dystrophin (dmd) gene resulting in very low levels or a complete absence of the dystrophin protein, a key structural element of muscle fibres which is responsible for the proper transmission of force. In the absence of dystrophin, muscle fibres become damaged easily during contraction resulting in their degeneration. DMD patients and mdx mice (an animal model of DMD) exhibit altered metabolic disturbances that cannot be attributed to the loss of dystrophin directly. We tested the hypothesis that glycogen metabolism is defective in mdx dystrophic mice. Results Dystrophic mdx mice had increased skeletal muscle glycogen (79%, (P<0.01)). Skeletal muscle glycogen synthesis is initiated by glycogenin, the expression of which was increased by 50% in mdx mice (P<0.0001). Glycogen synthase activity was 12% higher (P<0.05) but glycogen branching enzyme activity was 70% lower (P<0.01) in mdx compared with wild-type mice. The rate-limiting enzyme for glycogen breakdown, glycogen phosphorylase, had 62% lower activity (P<0.01) in mdx mice resulting from a 24% reduction in PKA activity (P<0.01). In mdx mice glycogen debranching enzyme expression was 50% higher (P<0.001) together with starch-binding domain protein 1 (219% higher; P<0.01). In addition, mdx mice were glucose intolerant (P<0.01) and had 30% less liver glycogen (P<0.05) compared with control mice. Subsequent analysis of the enzymes dysregulated in skeletal muscle glycogen metabolism in mdx mice identified reduced glycogenin protein expression (46% less; P<0.05) as a possible cause of this phenotype. Conclusion We identified that mdx mice were glucose intolerant, and had increased skeletal muscle glycogen but reduced amounts of liver glycogen. PMID:24626262

  10. Novel method for detection of glycogen in cells.

    PubMed

    Skurat, Alexander V; Segvich, Dyann M; DePaoli-Roach, Anna A; Roach, Peter J

    2017-05-01

    Glycogen, a branched polymer of glucose, functions as an energy reserve in many living organisms. Abnormalities in glycogen metabolism, usually excessive accumulation, can be caused genetically, most often through mutation of the enzymes directly involved in synthesis and degradation of the polymer leading to a variety of glycogen storage diseases (GSDs). Microscopic visualization of glycogen deposits in cells and tissues is important for the study of normal glycogen metabolism as well as diagnosis of GSDs. Here, we describe a method for the detection of glycogen using a renewable, recombinant protein which contains the carbohydrate-binding module (CBM) from starch-binding domain containing protein 1 (Stbd1). We generated a fusion protein containing g lutathione S-transferase, a cM c eptitope and the tbd1 BM (GYSC) for use as a glycogen-binding probe, which can be detected with secondary antibodies against glutathione S-transferase or cMyc. By enzyme-linked immunosorbent assay, we demonstrate that GYSC binds glycogen and two other polymers of glucose, amylopectin and amylose. Immunofluorescence staining of cultured cells indicate a GYSC-specific signal that is co-localized with signals obtained with anti-glycogen or anti-glycogen synthase antibodies. GYSC-positive staining inside of lysosomes is observed in individual muscle fibers isolated from mice deficient in lysosomal enzyme acid alpha-glucosidase, a well-characterized model of GSD II (Pompe disease). Co-localized GYSC and glycogen signals are also found in muscle fibers isolated from mice deficient in malin, a model for Lafora disease. These data indicate that GYSC is a novel probe that can be used to study glycogen metabolism under normal and pathological conditions.

  11. 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. © 2016 Wiley Periodicals, Inc.

  12. Expression of glycogenes in differentiating human NT2N neurons. Downregulation of fucosyltransferase 9 leads to decreased Lewis(x) levels and impaired neurite outgrowth.

    PubMed

    Gouveia, Ricardo; Schaffer, Lana; Papp, Suzanne; Grammel, Nicolas; Kandzia, Sebastian; Head, Steven R; Kleene, Ralf; Schachner, Melitta; Conradt, Harald S; Costa, Júlia

    2012-12-01

    Several glycan structures are functionally relevant in biological events associated with differentiation and regeneration which occur in the central nervous system. Here we have analysed the glycogene expression and glycosylation patterns during human NT2N neuron differentiation. We have further studied the impact of downregulating fucosyltransferase 9 (FUT9) on neurite outgrowth. The expression of glycogenes in human NT2N neurons differentiating from teratocarcinoma NTERA-2/cl.D1 cells has been analysed using the GlycoV4 GeneChip expression microarray. Changes in glycosylation have been monitored by immunoblot, immunofluorescence microscopy, HPLC and MALDI-TOF MS. Peptide mass fingerprinting and immunoprecipitation have been used for protein identification. FUT9 was downregulated using silencing RNA. One hundred twelve mRNA transcripts showed statistically significant up-regulation, including the genes coding for proteins involved in the synthesis of the Lewis(x) motif (FUT9), polysialic acid (ST8SIA2 and ST8SIA4) and HNK-1 (B3GAT2). Accordingly, increased levels of the corresponding carbohydrate epitopes have been observed. The Lewis(x) structure was found in a carrier glycoprotein that was identified as the CRA-a isoform of human neural cell adhesion molecule 1. Downregulation of FUT9 caused significant decreases in the levels of Lewis(x), as well as GAP-43, a marker of neurite outgrowth. Concomitantly, a reduction in neurite formation and outgrowth has been observed that was reversed by FUT9 overexpression. These results provided information about the regulation of glycogenes during neuron differentiation and they showed that the Lewis(x) motif plays a functional role in neurite outgrowth from human neurons. Copyright © 2012 Elsevier B.V. All rights reserved.

  13. 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

  14. Impairment of Cellulose Synthases Required for Arabidopsis Secondary Cell Wall Formation Enhances Disease Resistance[W

    PubMed Central

    Hernández-Blanco, Camilo; Feng, Dong Xin; Hu, Jian; Sánchez-Vallet, Andrea; Deslandes, Laurent; Llorente, Francisco; Berrocal-Lobo, Marta; Keller, Harald; Barlet, Xavier; Sánchez-Rodríguez, Clara; Anderson, Lisa K.; Somerville, Shauna; Marco, Yves; Molina, Antonio

    2007-01-01

    Cellulose is synthesized by cellulose synthases (CESAs) contained in plasma membrane–localized complexes. In Arabidopsis thaliana, three types of CESA subunits (CESA4/IRREGULAR XYLEM5 [IRX5], CESA7/IRX3, and CESA8/IRX1) are required for secondary cell wall formation. We report that mutations in these proteins conferred enhanced resistance to the soil-borne bacterium Ralstonia solanacearum and the necrotrophic fungus Plectosphaerella cucumerina. By contrast, susceptibility to these pathogens was not altered in cell wall mutants of primary wall CESA subunits (CESA1, CESA3/ISOXABEN RESISTANT1 [IXR1], and CESA6/IXR2) or POWDERY MILDEW–RESISTANT5 (PMR5) and PMR6 genes. Double mutants indicated that irx-mediated resistance was independent of salicylic acid, ethylene, and jasmonate signaling. Comparative transcriptomic analyses identified a set of common irx upregulated genes, including a number of abscisic acid (ABA)–responsive, defense-related genes encoding antibiotic peptides and enzymes involved in the synthesis and activation of antimicrobial secondary metabolites. These data as well as the increased susceptibility of ABA mutants (abi1-1, abi2-1, and aba1-6) to R. solanacearum support a direct role of ABA in resistance to this pathogen. Our results also indicate that alteration of secondary cell wall integrity by inhibiting cellulose synthesis leads to specific activation of novel defense pathways that contribute to the generation of an antimicrobial-enriched environment hostile to pathogens. PMID:17351116

  15. 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. © 2013 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.

  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. Enhanced generation of human embryonic stem cells from single blastomeres of fair and poor-quality cleavage embryos via inhibition of glycogen synthase kinase β and Rho-associated kinase signaling.

    PubMed

    Taei, Adeleh; Hassani, Seyedeh-Nafiseh; Eftekhari-Yazdi, Poopak; Rezazadeh Valojerdi, Mojtaba; Nokhbatolfoghahai, Mohsen; Masoudi, Najmeh-Sadat; Pakzad, Mohammad; Gourabi, Hamid; Baharvand, Hossein

    2013-10-01

    Could selected pluripotency-enhancing small molecules (SMs) lead to efficient derivation of human embryonic stem cells (hESCs) from cleavage embryos-derived single blastomeres (SBs)? Inhibition of glycogen synthase kinase β (GSK3β) and Rho-associated kinase (ROCK) signaling can enhance the derivation of hESCs from cleavage embryo-derived SBs. Parameters involved in sustaining the pluripotency of biopsied blastomeres for generating hESCs without causing injury to a viable embryo have remained obscure. This research seeks to improve the culture conditions for increasing the efficiency of deriving hESCs from SBs from cleavage-stage embryos by using SMs. In order to identify SMs which may enhance hESC generation from SBs, 11 pluripotency-enhancing SMs were screened and CHIR99021 (CH), a GSK3β inhibitor, was selected. To optimize culture condition in hESC generation from SMs, we used ROCK inhibitor Y27632 (Y) and basic fibroblast growth factor in combination with CH or its alternative, Kenpaullone, in different time courses over 12 days. We also assessed a critical time point for CH + Y treatment of cleavage embryos from 4- to 8-cell embryo. In total, 224 embryos and 1607 SBs were used in the study. Blastomeres of fair and poor-quality from 6- to 8-cell stage human embryos were mechanically dispersed and individually seeded into a 96-well plate that was precoated with mitotically inactivated feeder cells. Derivation of hESC line from each SB was carried out in hESC defined medium supplemented with SMs. Randomly selected hESC lines were evaluated by immunostaining for pluripotency markers, karyotype analysis and differentiation potential into the three embryonic germ layer derivatives. We found that 3 μM CH was the only SM that was capable of directing SBs from fair and poor-quality 6-8-cell embryos into hESC lines. The application of hESC-conditioned medium had no additive effect on hESC establishment from SBs. Also, we indicated that CH combined with Y improved h

  18. Impaired Wound Induction of 3-Deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) Synthase and Altered Stem Development in Transgenic Potato Plants Expressing a DAHP Synthase Antisense Construct.

    PubMed Central

    Jones, J. D.; Henstrand, J. M.; Handa, A. K.; Herrmann, K. M.; Weller, S. C.

    1995-01-01

    Potato (Solanum tuberosum L.) cells were transformed with an antisense DNA construct encoding part of 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase (EC 4.1.2.15), the first enzyme of the shikimate pathway, to examine the role(s) of this protein in plant growth and development. Chimeric DNA constructs contained the transcript start site, the first exon, and part of the first intron of the shkA gene in antisense or sense orientations under the control of the cauliflower mosaic virus 35S promoter. Some, but not all, of the transgenic plants expressing antisense DAHP synthase RNA showed reduced levels of wound-induced DAHP synthase enzyme activity, polypeptide, and mRNA 12 and 24 h after wounding. No alteration in the wound induction of DAHP synthase gene expression was observed in transgenic potato tubers containing the chimeric sense construct. Reduced steady-state levels of DAHP synthase mRNA were observed in stem and shoot tip tissue. Some plants with the chimeric antisense construct had reduced stem length, stem diameter, and reduced stem lignification. PMID:12228551

  19. 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. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. Impaired ovulation in mice with targeted deletion of the neuronal isoform of nitric oxide synthase.

    PubMed Central

    Klein, S. L.; Carnovale, D.; Burnett, A. L.; Wallach, E. E.; Zacur, H. A.; Crone, J. K.; Dawson, V. L.; Nelson, R. J.; Dawson, T. M.

    1998-01-01

    BACKGROUND: Nitric oxide (NO) plays an important role in numerous reproductive processes. To date, most studies have assessed the role of NO by using nonspecific pharmacological inhibitors of the precursor to NO, nitric oxide synthase (NOS). These pharmacological NOS inhibitors suppress all isoforms of NOS; thus, the precise contribution of each isoform to female reproductive physiology is unknown. The purpose of this study was to determine the specific role of neuronal NOS (nNOS) in the regulation of ovulation in female mice lacking the gene that encodes for nNOS (nNOS-/-). MATERIALS AND METHODS: Ovulation was assessed in wild-type (WT) and nNOS-/- female mice by examining the number of ovarian rupture sites and number of oocytes recovered from the oviducts following mating or exposure to exogenous gonadotropins (i.e., 5 IU pregnant mares serum gonadotropin [PMSG] and 5 IU human chorionic gonadotropin [hCG]). Ovulatory efficiency was determined as the number of ovulated oocytes per number of ovarian rupture sites. To examine whether ovulatory deficits in nNOS-/- mice were due to alternations in central mechanisms, plasma luteinizing hormone (LH) concentrations were assessed in WT and nNOS-/- mice that were challenged with 25 ng of gonadotropin-releasing hormone (GnRH). To determine whether ovulatory deficits in nNOS-/- mice were due to local ovulation processes, nerves innervating the reproductive tract of WT and nNOS-/- females were examined for the presence of nNOS protein. RESULTS: There were substantial fertility deficits in nNOS-/- female mice; the nNOS-/- mice had fewer oocytes in their oviducts following spontaneous and gonadotropin-stimulated ovulation. Pituitary responsiveness to exogenous GnRH challenge was intact in nNOS-/- mice. Dense nNOS protein staining was observed in nerves innervating the reproductive tracts of WT mice. CONCLUSIONS: The reproductive deficits in nNOS-/- females are most likely due to alternations in the transfer of oocytes from

  1. Antisense inhibition of NADH glutamate synthase impairs carbon/nitrogen assimilation in nodules of alfalfa (Medicago sativa L.).

    PubMed

    Cordoba, Elizabeth; Shishkova, Svetlana; Vance, Carroll P; Hernández, Georgina

    2003-03-01

    Legumes acquire significant amounts of nitrogen for growth from symbiotic nitrogen fixation. The glutamine synthetase (GS)/NADH-dependent glutamate synthase (NADH-GOGAT) cycle catalyzes initial nitrogen assimilation. This report describes the impact of specifically reducing nodule NADH-GOGAT activity on symbiotic performance of alfalfa (Medicago sativa L.). Four independent transgenic alfalfa lines, designated GA89, GA87, GA88, and GA82 (for GOGATantisense), containing an antisense NADH-GOGAT cDNA fragment under the control of the soybean leghemoglobin (lbc3) promoter were evaluated. The GA plants were fertile and showed normal growth in non-symbiotic conditions. The NADH-GOGAT antisense transgene was heritable and the T1 plants showed phenotypic alterations - similar to primary transformants. Clonally propagated plants were inoculated with Sinorhizobium meliloti after rooting and the symbiotic phenotype was analyzed 21 days post-inoculation. Nodules of each GA line had reduced NADH-GOGAT activity, ranging from 33 to 87% of control plants, that was accompanied by comparable decreases in RNA and protein. Plants from the GA89 line, with the lowest NADH-GOGAT activity (c. 30%), presented a strikingly altered symbiotic phenotype: concomitantly activities of key enzyme for carbon and nitrogen assimilation decreased; nodule amino acids and amides were reduced while sucrose accumulated. Antisense GOGAT plants were chlorotic, reduced in fresh weight, and had a lower N content than control plants. Photosynthesis was also impaired in antisense plants. Specifically, reducing NADH-GOGAT in nodules resulted in plants having impaired nitrogen assimilation and altered carbon/nitrogen metabolic flux.

  2. Selective Inducible Nitric Oxide Synthase Inhibitor Reversed Zinc Chloride-Induced Spatial Memory Impairment via Increasing Cholinergic Marker Expression.

    PubMed

    Tabrizian, Kaveh; Azami, Kian; Belaran, Maryam; Soodi, Maliheh; Abdi, Khosrou; Fanoudi, Sahar; Sanati, Mehdi; Mottaghi Dastjerdi, Negar; Soltany Rezaee-Rad, Mohammad; Sharifzadeh, Mohammad

    2016-10-01

    Zinc, an essential micronutrient and biochemical element of the human body, plays structural, catalytic, and regulatory roles in numerous physiological functions. In the current study, the effects of a pretraining oral administration of zinc chloride (10, 25, and 50 mg/kg) for 14 consecutive days and post-training bilateral intra-hippocampal infusion of 1400W as a selective inducible nitric oxide synthase (iNOS) inhibitor (10, 50, and 100 μM/side), alone and in combination, on the spatial memory retention in Morris water maze (MWM) were investigated. Animals were trained for 4 days and tested 48 h after completion of training. Also, the molecular effects of these compounds on the expression of choline acetyltransferase (ChAT), as a cholinergic marker in the CA1 region of the hippocampus and medial septal area (MSA), were evaluated. Behavioral and molecular findings of this study showed that a 2-week oral administration of zinc chloride (50 mg/kg) impaired spatial memory retention in MWM and decreased ChAT expression. Immunohistochemical analysis of post-training bilateral intra-hippocampal infusion of 1400W revealed a significant increase in ChAT immunoreactivity. Furthermore, post-training bilateral intra-hippocampal infusion of 1400W into the CA1 region of the hippocampus reversed zinc chloride-induced spatial memory impairment in MWM and significantly increased ChAT expression in comparison with zinc chloride-treated animals. Taken together, these results emphasize the role of selective iNOS inhibitors in reversing zinc chloride-induced spatial memory deficits via modulation of cholinergic marker expression.

  3. 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.

  4. 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.

  5. Fractionation of particulate glycogen and bound enzymes using high-performance liquid chromatography.

    PubMed

    Vardanis, A

    1990-05-15

    During a study aimed at the specificity of binding of phosphorylase and glycogen synthase to glycogen particles we investigated the separation of these particles with the technique of high-performance liquid chromatography using several media. The technique allowed relatively rapid fractionations with little, if any, loss of enzyme activity. The basis of some separations was molecular weight of the particle. Of the molecular exclusion media used, Superose 6 had an exclusion limit large enough to accommodate most glycogens but excluded mouse liver and bovine liver glycogen particles. Toyopearl HW-75 F was a medium that could accommodate liver glycogen and gave us reasonably well-defined separation of the particles that carried phosphorylase and glycogen synthase. Separations on ion-exchange columns, that were obviously based on the overall charge of proteins associated with the glycogen particles, were not very effective in terms of allowing the separation of different types of particle. Of all the media used the hydrophobic interaction column was best in terms of separating what appeared to be distinct populations of particles that had definite preferences in terms of binding phosphorylase and glycogen synthase. As expected, these separations were based on the hydrophobic interaction of the proteins associated with the glycogen particles, since particles that had been deproteinized by a cold water extraction procedure did not bind to the column.

  6. Impaired Endothelial Nitric Oxide Synthase Homodimer Formation Triggers Development of Transplant Vasculopathy - Insights from a Murine Aortic Transplantation Model

    PubMed Central

    Oberhuber, Rupert; Riede, Gregor; Cardini, Benno; Bernhard, David; Messner, Barbara; Watschinger, Katrin; Steger, Christina; Brandacher, Gerald; Pratschke, Johann; Golderer, Georg; Werner, Ernst R.; Maglione, Manuel

    2016-01-01

    Transplant vasculopathy (TV) represents a major obstacle to long-term graft survival and correlates with severity of ischemia reperfusion injury (IRI). Donor administration of the nitric oxide synthases (NOS) co-factor tetrahydrobiopterin has been shown to prevent IRI. Herein, we analysed whether tetrahydrobiopterin is also involved in TV development. Using a fully allogeneic mismatched (BALB/c to C57BL/6) murine aortic transplantation model grafts subjected to long cold ischemia time developed severe TV with intimal hyperplasia (α-smooth muscle actin positive cells in the neointima) and endothelial activation (increased P-selectin expression). Donor pretreatment with tetrahydrobiopterin significantly minimised these changes resulting in only marginal TV development. Severe TV observed in the non-treated group was associated with increased protein oxidation and increased occurrence of endothelial NOS monomers in the aortic grafts already during graft procurement. Tetrahydrobiopterin supplementation of the donor prevented all these early oxidative changes in the graft. Non-treated allogeneic grafts without cold ischemia time and syngeneic grafts did not develop any TV. We identified early protein oxidation and impaired endothelial NOS homodimer formation as plausible mechanistic explanation for the crucial role of IRI in triggering TV in transplanted aortic grafts. Therefore, targeting endothelial NOS in the donor represents a promising strategy to minimise TV. PMID:27883078

  7. Mitochondrial impairment and melatonin protection in parkinsonian mice do not depend of inducible or neuronal nitric oxide synthases

    PubMed Central

    López, Ana; Ortiz, Francisco; Doerrier, Carolina; Venegas, Carmen; Fernández-Ortiz, Marisol; Aranda, Paula; Díaz-Casado, María E.; Fernández-Gil, Beatriz; Barriocanal-Casado, Eliana; Escames, Germaine; López, Luis C.

    2017-01-01

    MPTP-mouse model constitutes a well-known model of neuroinflammation and mitochondrial failure occurring in Parkinson’s disease (PD). Although it has been extensively reported that nitric oxide (NO●) plays a key role in the pathogenesis of PD, the relative roles of nitric oxide synthase isoforms iNOS and nNOS in the nigrostriatal pathway remains, however, unclear. Here, the participation of iNOS/nNOS isoforms in the mitochondrial dysfunction was analyzed in iNOS and nNOS deficient mice. Our results showed that MPTP increased iNOS activity in substantia nigra and striatum, whereas it sharply reduced complex I activity and mitochondrial bioenergetics in all strains. In the presence of MPTP, mice lacking iNOS showed similar restricted mitochondrial function than wild type or mice lacking nNOS. These results suggest that iNOS-dependent elevated nitric oxide, a major pathological hallmark of neuroinflammation in PD, does not contribute to mitochondrial impairment. Therefore, neuroinflammation and mitochondrial dysregulation seem to act in parallel in the MPTP model of PD. Melatonin administration, with well-reported neuroprotective properties, counteracted these effects, preventing from the drastic changes in mitochondrial oxygen consumption, increased NOS activity and prevented reduced locomotor activity induced by MPTP. The protective effects of melatonin on mitochondria are also independent of its anti-inflammatory properties, but both effects are required for an effective anti-parkinsonian activity of the indoleamine as reported in this study. PMID:28800639

  8. Mitochondrial impairment and melatonin protection in parkinsonian mice do not depend of inducible or neuronal nitric oxide synthases.

    PubMed

    López, Ana; Ortiz, Francisco; Doerrier, Carolina; Venegas, Carmen; Fernández-Ortiz, Marisol; Aranda, Paula; Díaz-Casado, María E; Fernández-Gil, Beatriz; Barriocanal-Casado, Eliana; Escames, Germaine; López, Luis C; Acuña-Castroviejo, Darío

    2017-01-01

    MPTP-mouse model constitutes a well-known model of neuroinflammation and mitochondrial failure occurring in Parkinson's disease (PD). Although it has been extensively reported that nitric oxide (NO●) plays a key role in the pathogenesis of PD, the relative roles of nitric oxide synthase isoforms iNOS and nNOS in the nigrostriatal pathway remains, however, unclear. Here, the participation of iNOS/nNOS isoforms in the mitochondrial dysfunction was analyzed in iNOS and nNOS deficient mice. Our results showed that MPTP increased iNOS activity in substantia nigra and striatum, whereas it sharply reduced complex I activity and mitochondrial bioenergetics in all strains. In the presence of MPTP, mice lacking iNOS showed similar restricted mitochondrial function than wild type or mice lacking nNOS. These results suggest that iNOS-dependent elevated nitric oxide, a major pathological hallmark of neuroinflammation in PD, does not contribute to mitochondrial impairment. Therefore, neuroinflammation and mitochondrial dysregulation seem to act in parallel in the MPTP model of PD. Melatonin administration, with well-reported neuroprotective properties, counteracted these effects, preventing from the drastic changes in mitochondrial oxygen consumption, increased NOS activity and prevented reduced locomotor activity induced by MPTP. The protective effects of melatonin on mitochondria are also independent of its anti-inflammatory properties, but both effects are required for an effective anti-parkinsonian activity of the indoleamine as reported in this study.

  9. Single fiber analyses of glycogen-related proteins reveal their differential association with glycogen in rat skeletal muscle.

    PubMed

    Murphy, Robyn M; Xu, Hongyang; Latchman, Heidy; Larkins, Noni T; Gooley, Paul R; Stapleton, David I

    2012-12-01

    To understand how glycogen affects skeletal muscle physiology, we examined enzymes essential for muscle glycogen synthesis and degradation using single fibers from quiescent and stimulated rat skeletal muscle. Presenting a shift in paradigm, we show these proteins are differentially associated with glycogen granules. Protein diffusibility and/or abundance of glycogenin, glycogen branching enzyme (GBE), debranching enzyme (GDE), phosphorylase (GP), and synthase (GS) were examined in fibers isolated from rat fast-twitch extensor digitorum longus (EDL) and slow-twitch soleus (SOL) muscle. GDE and GP proteins were more abundant (~10- to 100-fold) in fibers from EDL compared with SOL muscle. GS and glycogenin proteins were similar between muscles while GBE had an approximately fourfold greater abundance in SOL muscle. Mechanically skinned fibers exposed to physiological buffer for 10 min showed ~70% total pools of GBE and GP were diffusible (nonbound), whereas GDE and GS were considerably less diffusible. Intense in vitro stimulation, sufficient to elicit a ~50% decrease in intracellular glycogen, increased diffusibility of GDE, GP, and GS (~15-60%) and decreased GBE diffusibility (~20%). Amylase treatment, which breaks α-1,4 linkages of glycogen, indicated differential diffusibilities and hence glycogen associations of GDE and GS. Membrane solubilization (1% Triton-X-100) allowed a small additional amount of GDE and GS to diffuse from fibers, suggesting the majority of nonglycogen-associated GDE/GS is associated with myofibrillar/contractile network of muscle rather than membranes. Given differences in enzymes required for glycogen metabolism, the current findings suggest glycogen particles have fiber-type-dependent structures. The greater catabolic potential of glycogen breakdown in fast-twitch fibers may account for different contraction induced rates of glycogen utilization.

  10. 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

  11. Neuronal nitric-oxide synthase deficiency impairs the long-term memory of olfactory fear learning and increases odor generalization.

    PubMed

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

    2013-08-16

    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 lacking nNOS received six training trials, each consisting of an odor-CS co-terminating with a foot shock-US. Mice showed reduced freezing responses to the trained odor 24 h and 7 d after training, compared to wild-type mice. Pretraining systemic injections of the NO donor, molsidomine, rescued fear retention in nNOS knockout mice. In wild-type mice, pretraining systemic injections of L-NAME, a nonspecific nNOS blocker, disrupted odor-CS fear retention in a dose-dependent manner. To evaluate whether NO signaling is involved in generalization of fear memories, nNOS knockout mice and wild-type mice receiving L-NAME were trained to one odor and tested with a series of similar odors. In both cases, we found increased generalization, as measured by increased freezing to similar, unpaired odors. Despite the impairment in fear memory retention and generalization, neither mice receiving injections of L-NAME nor nNOS knockout mice showed any deficits in either novel odor investigation time or odor habituation, suggesting intact olfactory perception and short-term memory olfactory learning. These results support a necessary role for neuronal NO signaling in the normal expression and generalization of olfactory conditioned fear.

  12. Glycogen controls Caenorhabditis elegans lifespan and resistance to oxidative stress

    PubMed Central

    Gusarov, Ivan; Pani, Bibhusita; Gautier, Laurent; Smolentseva, Olga; Eremina, Svetlana; Shamovsky, Ilya; Katkova-Zhukotskaya, Olga; Mironov, Alexander; Nudler, Evgeny

    2017-01-01

    A high-sugar diet has been associated with reduced lifespan in organisms ranging from worms to mammals. However, the mechanisms underlying the harmful effects of glucose are poorly understood. Here we establish a causative relationship between endogenous glucose storage in the form of glycogen, resistance to oxidative stress and organismal aging in Caenorhabditis elegans. We find that glycogen accumulated on high dietary glucose limits C. elegans longevity. Glucose released from glycogen and used for NADPH/glutathione reduction renders nematodes and human hepatocytes more resistant against oxidative stress. Exposure to low levels of oxidants or genetic inhibition of glycogen synthase depletes glycogen stores and extends the lifespan of animals fed a high glucose diet in an AMPK-dependent manner. Moreover, glycogen interferes with low insulin signalling and accelerates aging of long-lived daf-2 worms fed a high glucose diet. Considering its extensive evolutionary conservation, our results suggest that glycogen metabolism might also have a role in mammalian aging. PMID:28627510

  13. Liver glycogen metabolism during short-term insulin-induced hypoglycemia in fed rats.

    PubMed

    Obici, Simoni; Lopes-Bertolini, Gisele; Curi, Rui; Bazotte, Roberto Barbosa

    2008-10-01

    The activities of glycogen phosphorylase and synthase during infusions of glucagon, isoproterenol, or cyanide in isolated liver of fed rats submitted to short-term insulin-induced hypoglycemia (IIH) was investigated. A condition of hyperinsulinemia/hypoglycemia was obtained with an intraperitoneal injection of regular insulin (1.0 U kg(-1)). The control group received ip saline. The experiments were carried out 60 min after insulin (IIH group) or saline (COG group) injection. The rats were anesthetized and after laparotomy, blood was collected from the vena cava for glucose and insulin measurements. The liver was then infused with glucagon (1 nM), isoproterenol (2 microM), or cyanide (0.5 mM) during 20 min and a sample of the organ was collected for determination of the activities of glycogen phosphorylase and synthase 5 min after starting and 10 min after stopping the infusions. The infusions of cyanide, glucagons, and isoproterenol did not change the activities of glycogen synthase and glycogen phosphorylase. However, glycogen catabolism was decreased during the infusions of glucagon and isoproterenol in IIH rats, being more intense with isoproterenol (p < 0.05), than glucagon. It was concluded that short-term IIH promoted changes in the liver responsiveness of glycogen degradation induced by glucagon and isoproterenol without a change in the activities of glycogen phosphorylase and synthase.

  14. Prevalent role of the insulin receptor isoform A in the regulation of hepatic glycogen metabolism in hepatocytes and in mice.

    PubMed

    Diaz-Castroverde, Sabela; Baos, Selene; Luque, María; Di Scala, Marianna; González-Aseguinolaza, Gloria; Gómez-Hernández, Almudena; Beneit, Nuria; Escribano, Oscar; Benito, Manuel

    2016-12-01

    In the postprandial state, the liver regulates glucose homeostasis by glucose uptake and conversion to glycogen and lipids. Glucose and insulin signalling finely regulate glycogen synthesis through several mechanisms. Glucose uptake in hepatocytes is favoured by the insulin receptor isoform A (IRA), rather than isoform B (IRB). Thus, we hypothesised that, in hepatocytes, IRA would increase glycogen synthesis by promoting glucose uptake and glycogen storage. We addressed the role of insulin receptor isoforms on glycogen metabolism in vitro in immortalised neonatal hepatocytes. In vivo, IRA or IRB were specifically expressed in the liver using adeno-associated virus vectors in inducible liver insulin receptor knockout (iLIRKO) mice, a model of type 2 diabetes. The role of IR isoforms in glycogen synthesis and storage in iLIRKO was subsequently investigated. In immortalised hepatocytes, IRA, but not IRB expression induced an increase in insulin signalling that was associated with elevated glycogen synthesis, glycogen synthase activity and glycogen storage. Similarly, elevated IRA, but not IRB expression in the livers of iLIRKO mice induced an increase in glycogen content. We provide new insight into the role of IRA in the regulation of glycogen metabolism in cultured hepatocytes and in the livers of a mouse model of type 2 diabetes. Our data strongly suggest that IRA is more efficient than IRB at promoting glycogen synthesis and storage. Therefore, we suggest that IRA expression in the liver could provide an interesting therapeutic approach for the regulation of hepatic glucose content and glycogen storage.

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

    PubMed

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

    2013-05-07

    Laforin or malin deficiency causes Lafora disease, characterized by altered glycogen metabolism and teenage-onset neurodegeneration with intractable and invariably fatal epilepsy. Plant starches possess small amounts of metabolically essential monophosphate esters. Glycogen contains similar phosphate amounts, which are thought to originate from a glycogen synthase error side reaction and therefore lack any specific function. Glycogen is also believed to lack monophosphates at glucosyl carbon C6, an essential phosphorylation site in plant starch metabolism. We now show that glycogen phosphorylation is not due to a glycogen synthase side reaction, that C6 is a major glycogen phosphorylation site, and that C6 monophosphates predominate near centers of glycogen molecules and positively correlate with glycogen chain lengths. Laforin or malin deficiency causes C6 hyperphosphorylation, which results in malformed long-chained glycogen that accumulates in many tissues, causing neurodegeneration in brain. Our work advances the understanding of Lafora disease pathogenesis and suggests that glycogen phosphorylation has important metabolic function. Copyright © 2013 Elsevier Inc. All rights reserved.

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

    PubMed Central

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

    2014-01-01

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

  17. Role of Autophagy in Glycogen Breakdown and Its Relevance to Chloroquine Myopathy

    PubMed Central

    Zirin, Jonathan; Nieuwenhuis, Joppe; Perrimon, Norbert

    2013-01-01

    Several myopathies are associated with defects in autophagic and lysosomal degradation of glycogen, but it remains unclear how glycogen is targeted to the lysosome and what significance this process has for muscle cells. We have established a Drosophila melanogaster model to study glycogen autophagy in skeletal muscles, using chloroquine (CQ) to simulate a vacuolar myopathy that is completely dependent on the core autophagy genes. We show that autophagy is required for the most efficient degradation of glycogen in response to starvation. Furthermore, we show that CQ-induced myopathy can be improved by reduction of either autophagy or glycogen synthesis, the latter possibly due to a direct role of Glycogen Synthase in regulating autophagy through its interaction with Atg8. PMID:24265594

  18. Impairment of Nitric Oxide Synthase but Not Heme Oxygenase Accounts for Baroreflex Dysfunction Caused by Chronic Nicotine in Female Rats

    PubMed Central

    Fouda, Mohamed A.; El-Gowelli, Hanan M.; El-Gowilly, Sahar M.; Rashed, Laila; El-Mas, Mahmoud M.

    2014-01-01

    We recently reported that chronic nicotine impairs reflex chronotropic activity in female rats. Here, we sought evidence to implicate nitric oxide synthase (NOS) and/or heme oxygenase (HO) in the nicotine-baroreflex interaction. Baroreflex curves relating changes in heart rate to increases (phenylephrine) or decreases (sodium nitroprusside) in blood pressure were generated in conscious female rats treated with nicotine or saline in absence and presence of pharmacological modulators of NOS or HO activity. Compared with saline-treated rats, nicotine (2 mg/kg/day i.p., for 14 days) significantly reduced the slopes of baroreflex curves, a measure of baroreflex sensitivity (BRS). Findings that favor the involvement of NOS inhibition in the nicotine effect were (i) NOS inhibition (Nω-Nitro-L-arginine methyl ester, L-NAME) reduced BRS in control rats but failed to do so in nicotine-treated rats, (ii) L-arginine, NO donor, reversed the BRS inhibitory effect of nicotine. Alternatively, HO inhibition (zinc protoporphyrin IX, ZnPP) had no effect on BRS in nicotine- or control rats and failed to reverse the beneficial effect of L-arginine on nicotine-BRS interaction. Similar to female rats, BRS was reduced by L-NAME, but not ZnPP, in male rats and the L-NAME effect was not accentuated after concomitant administration of nicotine. Baroreflex dysfunction caused by nicotine in female rats was blunted after supplementation with hemin (HO inducer) but not tricarbonyldichlororuthenium(II) dimer (CORM-2), a carbon monoxide (CO) releasing molecule, or bilirubin, the breakdown product of heme catabolism. The facilitatory effect of hemin was abolished upon simultaneous treatment with L-NAME or 1H-[1], [2], [4] oxadiazolo[4,3-a] quinoxalin-1-one (inhibitor of soluble guanylate cyclase, sGC). The activities of HO and NOS in brainstem tissues were also significantly increased by hemin. Thus, the inhibition of NOS, but not HO, accounts for the baroreflex depressant of chronic nicotine

  19. 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

  20. Postsynaptic α1-Adrenergic Vasoconstriction Is Impaired in Young Patients With Vasovagal Syncope and Is Corrected by Nitric Oxide Synthase Inhibition.

    PubMed

    Stewart, Julian M; Suggs, Melissa; Merchant, Sana; Sutton, Richard; Terilli, Courtney; Visintainer, Paul; Medow, Marvin S

    2016-08-01

    Syncope is a sudden transient loss of consciousness and postural tone with spontaneous recovery; the most common form is vasovagal syncope (VVS). During VVS, gravitational pooling excessively reduces central blood volume and cardiac output. In VVS, as in hemorrhage, impaired adrenergic vasoconstriction and venoconstriction result in hypotension. We hypothesized that impaired adrenergic responsiveness because of excess nitric oxide can be reversed by reducing nitric oxide. We recorded cardiopulmonary dynamics in supine syncope patients and healthy volunteers (aged 15-27 years) challenged with a dose-response using the α1-agonist phenylephrine (PE), with and without the nitric oxide synthase inhibitor N(G)-monomethyl-L-arginine, monoacetate salt (L-NMMA). Systolic and diastolic pressures among control and VVS were the same, although they increased after L-NMMA and saline+PE (volume and pressor control for L-NMMA). Heart rate was significantly reduced by L-NMMA (P<0.05) for control and VVS compared with baseline, but there was no significant difference in heart rate between L-NMMA and saline+PE. Cardiac output and splanchnic blood flow were reduced by L-NMMA for control and VVS (P<0.05) compared with baseline, while total peripheral resistance increased (P<0.05). PE dose-response for splanchnic flow and resistance were blunted for VVS compared with control after saline+PE, but enhanced after L-NMMA (P<0.001). Postsynaptic α1-adrenergic vasoconstrictive impairment was greatest in the splanchnic vasculature, and splanchnic blood flow was unaffected by PE. Forearm and calf α1-adrenergic vasoconstriction were unimpaired in VVS and unaffected by L-NMMA. Impaired postsynaptic α1-adrenergic vasoconstriction in young adults with VVS can be corrected by nitric oxide synthase inhibition, demonstrated with our use of L-NMMA. © 2016 American Heart Association, Inc.

  1. Last step in the conversion of trehalose to glycogen: a mycobacterial enzyme that transfers maltose from maltose 1-phosphate to glycogen.

    PubMed

    Elbein, Alan D; Pastuszak, Irena; Tackett, Alan J; Wilson, Tyler; Pan, Yuan T

    2010-03-26

    We show that Mycobacterium smegmatis has an enzyme catalyzing transfer of maltose from [(14)C]maltose 1-phosphate to glycogen. This enzyme was purified 90-fold from crude extracts and characterized. Maltose transfer required addition of an acceptor. Liver, oyster, or mycobacterial glycogens were the best acceptors, whereas amylopectin had good activity, but amylose was a poor acceptor. Maltosaccharides inhibited the transfer of maltose from [(14)C]maltose-1-P to glycogen because they were also acceptors of maltose, and they caused production of larger sized radioactive maltosaccharides. When maltotetraose was the acceptor, over 90% of the (14)C-labeled product was maltohexaose, and no radioactivity was in maltopentaose, demonstrating that maltose was transferred intact. Stoichiometry showed that 0.89 micromol of inorganic phosphate was produced for each micromole of maltose transferred to glycogen, and 56% of the added maltose-1-P was transferred to glycogen. This enzyme has been named alpha1,4-glucan:maltose-1-P maltosyltransferase (GMPMT). Transfer of maltose to glycogen was inhibited by micromolar amounts of inorganic phosphate or arsenate but was only slightly inhibited by millimolar concentrations of glucose-1-P, glucose-6-P, or inorganic pyrophosphate. GMPMT was compared with glycogen phosphorylase (GP). GMPMT catalyzed transfer of [(14)C]maltose-1-P, but not [(14)C]glucose-1-P, to glycogen, whereas GP transferred radioactivity from glucose-1-P but not maltose-1-P. GMPMT and GP were both inhibited by 1,4-dideoxy-1,4-imino-d-arabinitol, but only GP was inhibited by isofagomine. Because mycobacteria that contain trehalose synthase accumulate large amounts of glycogen when grown in high concentrations of trehalose, we propose that trehalose synthase, maltokinase, and GMPMT represent a new pathway of glycogen synthesis using trehalose as the source of glucose.

  2. 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…

  3. 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…

  4. Estradiol stimulates glycogen synthesis whereas progesterone promotes glycogen catabolism in the uterus of the American mink (Neovison vison).

    PubMed

    Bowman, Kole; Rose, Jack

    2017-01-01

    Glycogen synthesis by mink uterine glandular and luminal epithelia (GE and LE) is stimulated by estradiol (E2 ) during estrus. Subsequently, the glycogen deposits are mobilized to near completion to meet the energy requirements of pre-embryonic development and implantation by as yet undetermined mechanisms. We hypothesized that progesterone (P4 ) was responsible for catabolism of uterine glycogen reserves as one of its actions to ensure reproductive success. Mink were treated with E2 , P4 or vehicle (controls) for 3 days and uteri collected 24 h (E2 , P4 and vehicle) and 96 h (E2 ) later. To evaluate E2 priming, mink were treated with E2 for 3 days, then P4 for an additional 3 days (E2 →P4 ) and uteri collected 24 h later. Percent glycogen content of uterine epithelia was greater at E2 + 96 h (GE = 5.71 ± 0.55; LE = 11.54 ± 2.32) than E2 +24 h (GE = 3.63 ± 0.71; LE = 2.82 ± 1.03), and both were higher than controls (GE = 0.27 ± 0.15; LE = 0.54 ± 0.30; P < 0.05). Treatment as E2 →P4 reduced glycogen content (GE = 0.61 ± 0.16; LE = 0.51 ± 0.13), to levels not different from controls, while concomitantly increasing catabolic enzyme (glycogen phosphorylase m and glucose-6-phosphatase) gene expression and amount of phospho-glycogen synthase protein (inactive) in uterine homogenates. Interestingly, E2 →P4 increased glycogen synthase 1 messenger RNA (mRNA) and hexokinase 1mRNA and protein. Our findings suggest to us that while E2 promotes glycogen accumulation by the mink uterus during estrus and pregnancy, it is P4 that induces uterine glycogen catabolism, releasing the glucose that is essential to support pre-embryonic survival and implantation.

  5. Enzymatic regulation of seasonal glycogen cycling in the freeze-tolerant wood frog, Rana sylvatica.

    PubMed

    do Amaral, M Clara F; Lee, Richard E; Costanzo, Jon P

    2016-12-01

    Liver glycogen is an important energy store in vertebrates, and in the freeze-tolerant wood frog, Rana sylvatica, this carbohydrate also serves as a major source of the cryoprotectant glucose. We investigated how variation in the levels of the catalytic subunit of protein kinase A (PKAc), glycogen phosphorylase (GP), and glycogen synthase (GS) relates to seasonal glycogen cycling in a temperate (Ohioan) and subarctic (Alaskan) populations of this species. In spring, Ohioan frogs had reduced potential for glycogen synthesis, as evidenced by low GS activity and high PKAc protein levels. In addition, glycogen levels in spring were the lowest of four seasonal samples, as energy input was likely directed towards metabolism and somatic growth during this period. Near-maximal glycogen levels were reached by mid-summer, and remained unchanged in fall and winter, suggesting that glycogenesis was curtailed during this period. Ohioan frogs had a high potential for glycogenolysis and glycogenesis in winter, as evidenced by large glycogen reserves, high levels of GP and GS proteins, and high GS activity, which likely allows for rapid mobilization of cryoprotectant during freezing and replenishing of glycogen reserves during thawing. Alaskan frogs also achieved a near-maximal liver glycogen concentration by summer and displayed high glycogenic and glycogenolytic potential in winter, but, unlike Ohioan frogs, started replenishing their energy reserves early in spring. We conclude that variation in levels of both glycogenolytic and glycogenic enzymes likely happens in response to seasonal changes in energetic strategies and demands, with winter survival being a key component to understanding the regulation of glycogen cycling in this species.

  6. Multiple requirements for glycogen synthesis by hepatocytes isolated from fasted rats.

    PubMed

    Chen, K S; Lardy, H A

    1985-11-25

    Glycogen synthesis from various combinations of substrates by hepatocytes isolated from rats fasted 24 h was studied. As reported by Katz et al. (Katz, J., Golden, S., and Wals, P. A. (1976) Proc. Natl. Acad. Sci. U. S. A. 73, 3433-3437), appreciable rates of glycogen synthesis occurred only in the presence of gluconeogenic precursors and one of several amino acids, which includes L-glutamine. L-Leucine had negligible effects on glycogen synthesis from 20 mM dihydroxyacetone and/or 15 mM glucose when L-glutamine was not added to the medium. In the presence of 10 mM L-glutamine, L-leucine greatly increased glycogen synthesis from these substrates. alpha-Ketoisocaproate was ineffective, as was oleate. NH4Cl depressed glycogen synthesis from 10 mM glucose plus 20 mM dihydroxyacetone in the absence of added L-glutamine and enhanced that in its presence, but these effects were weak compared to those of L-leucine. The amino acid analogues L-norvaline and L-norleucine exerted effects that were similar to those exerted by L-leucine. Under all conditions studied, cycloheximide and puromycin inhibited net glycogen synthesis. Cycloheximide did not stimulate gluconeogenesis from dihydroxyacetone, or phosphorylase in hepatocytes from starved rats, or glycogenolysis in hepatocytes from fed rats. Puromycin, however, stimulated glycogenolysis in hepatocytes from fed rats. Glycogen synthesis from 20 mM dihydroxyacetone proceeds with a pronounced initial lag phase that can be shortened by incubation of cells with glutamine plus leucine before addition of dihydroxyacetone. Concurrent measurements of glycogen synthesis, glycogen synthase, and gluconeogenesis under different conditions reveal that in addition to protein synthesis, activation of glycogen synthase, which must occur to allow glycogen synthesis in hepatocytes, requires a second component which can be satisfied by addition of dihydroxyacetone or fructose to the cells.

  7. PER2 promotes glucose storage to liver glycogen during feeding and acute fasting by inducing Gys2 PTG and G L expression.

    PubMed

    Zani, Fabio; Breasson, Ludovic; Becattini, Barbara; Vukolic, Ana; Montani, Jean-Pierre; Albrecht, Urs; Provenzani, Alessandro; Ripperger, Juergen A; Solinas, Giovanni

    2013-01-01

    The interplay between hepatic glycogen metabolism and blood glucose levels is a paradigm of the rhythmic nature of metabolic homeostasis. Here we show that mice lacking a functional PER2 protein (Per2 (Brdm1) ) display reduced fasting glycemia, altered rhythms of hepatic glycogen accumulation, and altered rhythms of food intake. Per2 (Brdm1) mice show reduced hepatic glycogen content and altered circadian expression during controlled fasting and refeeding. Livers from Per2 (Brdm1) mice display reduced glycogen synthase protein levels during refeeding, and increased glycogen phosphorylase activity during fasting. The latter is explained by PER2 action on the expression of the adapter proteins PTG and GL, which target the protein phosphatase-1 to glycogen to decrease glycogen phosphorylase activity. Finally, PER2 interacts with genomic regions of Gys2, PTG, and G L . These results indicate an important role for PER2 in the hepatic transcriptional response to feeding and acute fasting that promotes glucose storage to liver glycogen.

  8. Cyclin Partners Determine Pho85 Protein Kinase Substrate Specificity In Vitro and In Vivo: Control of Glycogen Biosynthesis by Pcl8 and Pcl10

    PubMed Central

    Huang, Dongqing; Moffat, Jason; Wilson, Wayne A.; Moore, Lynda; Cheng, Christine; Roach, Peter J.; Andrews, Brenda

    1998-01-01

    In Saccharomyces cerevisiae, PHO85 encodes a cyclin-dependent protein kinase (Cdk) with multiple roles in cell cycle and metabolic controls. In association with the cyclin Pho80, Pho85 controls acid phosphatase gene expression through phosphorylation of the transcription factor Pho4. Pho85 has also been implicated as a kinase that phosphorylates and negatively regulates glycogen synthase (Gsy2), and deletion of PHO85 causes glycogen overaccumulation. We report that the Pcl8/Pcl10 subgroup of cyclins directs Pho85 to phosphorylate glycogen synthase both in vivo and in vitro. Disruption of PCL8 and PCL10 caused hyperaccumulation of glycogen, activation of glycogen synthase, and a reduction in glycogen synthase kinase activity in vivo. However, unlike pho85 mutants, pcl8 pcl10 cells had normal morphologies, grew on glycerol, and showed proper regulation of acid phosphatase gene expression. In vitro, Pho80-Pho85 complexes effectively phosphorylated Pho4 but had much lower activity toward Gsy2. In contrast, Pcl10-Pho85 complexes phosphorylated Gsy2 at Ser-654 and Thr-667, two physiologically relevant sites, but only poorly phosphorylated Pho4. Thus, both the in vitro and in vivo substrate specificity of Pho85 is determined by the cyclin partner. Mutation of PHO85 suppressed the glycogen storage deficiency of snf1 or glc7-1 mutants in which glycogen synthase is locked in an inactive state. Deletion of PCL8 and PCL10 corrected the deficit in glycogen synthase activity in both the snf1 and glc7-1 mutants, but glycogen synthesis was restored only in the glc7-1 mutant strain. This genetic result suggests an additional role for Pho85 in the negative regulation of glycogen accumulation that is independent of Pcl8 and Pcl10. PMID:9584169

  9. Dichloroacetate inhibits glycolysis and augments insulin-stimulated glycogen synthesis in rat muscle.

    PubMed Central

    Clark, A S; Mitch, W E; Goodman, M N; Fagan, J M; Goheer, M A; Curnow, R T

    1987-01-01

    The decrease in plasma lactate during dichloroacetate (DCA) treatment is attributed to stimulation of lactate oxidation. To determine whether DCA also inhibits lactate production, we measured glucose metabolism in muscles of fed and fasted rats incubated with DCA and insulin. DCA increased glucose-6-phosphate, an allosteric modifier of glycogen synthase, approximately 50% and increased muscle glycogen synthesis and glycogen content greater than 25%. Lactate release fell; inhibition of glycolysis accounted for greater than 80% of the decrease. This was associated with a decrease in intracellular AMP, but no change in citrate or ATP. When lactate oxidation was increased by raising extracellular lactate, glycolysis decreased (r = - 0.91), suggesting that lactate oxidation regulates glycolysis. When muscle lactate production was greatly stimulated by thermal injury, DCA increased glycogen synthesis, normalized glycogen content, and inhibited glycolysis, thereby reducing lactate release. The major effect of DCA on lactate metabolism in muscle is to inhibit glycolysis. PMID:3543056

  10. FGF19 as a postprandial, insulin-independent activator of hepatic protein and glycogen synthesis.

    PubMed

    Kir, Serkan; Beddow, Sara A; Samuel, Varman T; Miller, Paul; Previs, Stephen F; Suino-Powell, Kelly; Xu, H Eric; Shulman, Gerald I; Kliewer, Steven A; Mangelsdorf, David J

    2011-03-25

    Fibroblast growth factor (FGF) 19 is an enterokine synthesized and released when bile acids are taken up into the ileum. We show that FGF19 stimulates hepatic protein and glycogen synthesis but does not induce lipogenesis. The effects of FGF19 are independent of the activity of either insulin or the protein kinase Akt and, instead, are mediated through a mitogen-activated protein kinase signaling pathway that activates components of the protein translation machinery and stimulates glycogen synthase activity. Mice lacking FGF15 (the mouse FGF19 ortholog) fail to properly maintain blood concentrations of glucose and normal postprandial amounts of liver glycogen. FGF19 treatment restored the loss of glycogen in diabetic animals lacking insulin. Thus, FGF19 activates a physiologically important, insulin-independent endocrine pathway that regulates hepatic protein and glycogen metabolism.

  11. The effect of antenatal administration of solcoseryl on hepatic glycogen synthesis in rat fetuses with intrauterine growth retardation.

    PubMed

    Takahashi, H; Cheng, K M; Araki, T

    1993-06-01

    The effect of antenatal solcoseryl administration on hepatic glycogen synthesis and storage was studied in normal developing and intrauterine growth-retarded (IUGR) rat fetuses using biochemical analyses. The maximal effect of solcoseryl occurred 2 hours after administration. The glycogen content of the liver showed a significant increase in normal and IUGR fetuses with antenatal solcoseryl administration compared to their non-solcoseryl counterparts (p < 0.05). The activities of glycogen synthase enzymes, total and active forms, showed significant increases, at p < 0.05 and p < 0.005, respectively, in IUGR fetuses with antenatal solcoseryl administration. Active synthase also increased in normal fetuses with antenatal solcoseryl administration (p < 0.05). There were no significant changes in the activities of glycogen phosphorylase enzyme. These findings suggest that antenatal solcoseryl administration stimulates hepatic glycogen synthesis and storage in IUGR rat fetuses, and thus might favorably influence the development of neonatal hypoglycemia.

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

    PubMed

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

    2016-02-01

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

  13. Dysregulation of multiple facets of glycogen metabolism in a murine model of Pompe disease.

    PubMed

    Taylor, Kristin M; Meyers, Elizabeth; Phipps, Michael; Kishnani, Priya S; Cheng, Seng H; Scheule, Ronald K; Moreland, Rodney J

    2013-01-01

    Pompe disease, also known as glycogen storage disease (GSD) type II, is caused by deficiency of lysosomal acid α-glucosidase (GAA). The resulting glycogen accumulation causes a spectrum of disease severity ranging from a rapidly progressive course that is typically fatal by 1 to 2 years of age to a slower progressive course that causes significant morbidity and early mortality in children and adults. The aim of this study is to better understand the biochemical consequences of glycogen accumulation in the Pompe mouse. We evaluated glycogen metabolism in heart, triceps, quadriceps, and liver from wild type and several strains of GAA(-/-) mice. Unexpectedly, we observed that lysosomal glycogen storage correlated with a robust increase in factors that normally promote glycogen biosynthesis. The GAA(-/-) mouse strains were found to have elevated glycogen synthase (GS), glycogenin, hexokinase, and glucose-6-phosphate (G-6-P, the allosteric activator of GS). Treating GAA(-/-) mice with recombinant human GAA (rhGAA) led to a dramatic reduction in the levels of glycogen, GS, glycogenin, and G-6-P. Lysosomal glycogen storage also correlated with a dysregulation of phosphorylase, which normally breaks down cytoplasmic glycogen. Analysis of phosphorylase activity confirmed a previous report that, although phosphorylase protein levels are identical in muscle lysates from wild type and GAA(-/-) mice, phosphorylase activity is suppressed in the GAA(-/-) mice in the absence of AMP. This reduction in phosphorylase activity likely exacerbates lysosomal glycogen accumulation. If the dysregulation in glycogen metabolism observed in the mouse model of Pompe disease also occurs in Pompe patients, it may contribute to the observed broad spectrum of disease severity.

  14. Dysregulation of Multiple Facets of Glycogen Metabolism in a Murine Model of Pompe Disease

    PubMed Central

    Taylor, Kristin M.; Meyers, Elizabeth; Phipps, Michael; Kishnani, Priya S.; Cheng, Seng H.; Scheule, Ronald K.; Moreland, Rodney J.

    2013-01-01

    Pompe disease, also known as glycogen storage disease (GSD) type II, is caused by deficiency of lysosomal acid α-glucosidase (GAA). The resulting glycogen accumulation causes a spectrum of disease severity ranging from a rapidly progressive course that is typically fatal by 1 to 2 years of age to a slower progressive course that causes significant morbidity and early mortality in children and adults. The aim of this study is to better understand the biochemical consequences of glycogen accumulation in the Pompe mouse. We evaluated glycogen metabolism in heart, triceps, quadriceps, and liver from wild type and several strains of GAA−/− mice. Unexpectedly, we observed that lysosomal glycogen storage correlated with a robust increase in factors that normally promote glycogen biosynthesis. The GAA−/− mouse strains were found to have elevated glycogen synthase (GS), glycogenin, hexokinase, and glucose-6-phosphate (G-6-P, the allosteric activator of GS). Treating GAA−/− mice with recombinant human GAA (rhGAA) led to a dramatic reduction in the levels of glycogen, GS, glycogenin, and G-6-P. Lysosomal glycogen storage also correlated with a dysregulation of phosphorylase, which normally breaks down cytoplasmic glycogen. Analysis of phosphorylase activity confirmed a previous report that, although phosphorylase protein levels are identical in muscle lysates from wild type and GAA−/− mice, phosphorylase activity is suppressed in the GAA−/− mice in the absence of AMP. This reduction in phosphorylase activity likely exacerbates lysosomal glycogen accumulation. If the dysregulation in glycogen metabolism observed in the mouse model of Pompe disease also occurs in Pompe patients, it may contribute to the observed broad spectrum of disease severity. PMID:23457523

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

    PubMed Central

    DEAN, Matthew; HUNT, Jason; MCDOUGALL, Lisa; ROSE, Jack

    2014-01-01

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

  16. Insights into Brain Glycogen Metabolism

    PubMed Central

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

    2016-01-01

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

  17. Stable expression of lipocalin-type prostaglandin D synthase in cultured preadipocytes impairs adipogenesis program independently of endogenous prostanoids

    SciTech Connect

    Hossain, Mohammad Salim; Chowdhury, Abu Asad; Rahman, Mohammad Sharifur; Nishimura, Kohji; Jisaka, Mitsuo; Nagaya, Tsutomu; Shono, Fumiaki; Yokota, Kazushige

    2012-02-15

    Lipocalin-type prostaglandin D synthase (L-PGDS) expressed preferentially in adipocytes is responsible for the synthesis of PGD{sub 2} and its non-enzymatic dehydration products, PGJ{sub 2} series, serving as pro-adipogenic factors. However, the role of L-PGDS in the regulation of adipogenesis is complex because of the occurrence of several derivatives from PGD{sub 2} and their distinct receptor subtypes as well as other functions such as a transporter of lipophilic molecules. To manipulate the expression levels of L-PGDS in cultured adipocytes, cultured preadipogenic 3T3-L1 cells were transfected stably with a mammalian expression vector having cDNA encoding murine L-PGDS oriented in the sense direction. The isolated cloned stable transfectants with L-PGDS expressed higher levels of the transcript and protein levels of L-PGDS, and synthesized PGD{sub 2} from exogenous arachidonic acid at significantly higher levels. By contrast, the synthesis of PGE{sub 2} remained unchanged, indicating no influence on the reactions of cyclooxygenase (COX) and PGE synthase. Furthermore, the ability of those transfectants to synthesize {Delta}{sup 12}-PGJ{sub 2} increased more greatly during the maturation phase. The sustained expression of L-PGDS in cultured stable transfectants hampered the storage of fats during the maturation phase of adipocytes, which was accompanied by the reduced gene expression of adipocyte-specific markers reflecting the down-regulation of the adipogenesis program. The suppressed adipogenesis was not rescued by either exogenous aspirin or peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) agonists including troglitazone and {Delta}{sup 12}-PGJ{sub 2}. Taken together, the results indicate the negative regulation of the adipogenesis program by the enhanced expression of L-PGDS through a cellular mechanism involving the interference of the PPAR{gamma} signaling pathway without the contribution of endogenous pro-adipogenic prostanoids

  18. Acid Hydrolysis and Molecular Density of Phytoglycogen and Liver Glycogen Helps Understand the Bonding in Glycogen α (Composite) Particles

    PubMed Central

    Powell, Prudence O.; Sullivan, Mitchell A.; Sheehy, Joshua J.; Schulz, Benjamin L.; Warren, Frederick J.; Gilbert, Robert G.

    2015-01-01

    Phytoglycogen (from certain mutant plants) and animal glycogen are highly branched glucose polymers with similarities in structural features and molecular size range. Both appear to form composite α particles from smaller β particles. The molecular size distribution of liver glycogen is bimodal, with distinct α and β components, while that of phytoglycogen is monomodal. This study aims to enhance our understanding of the nature of the link between liver-glycogen β particles resulting in the formation of large α particles. It examines the time evolution of the size distribution of these molecules during acid hydrolysis, and the size dependence of the molecular density of both glucans. The monomodal distribution of phytoglycogen decreases uniformly in time with hydrolysis, while with glycogen, the large particles degrade significantly more quickly. The size dependence of the molecular density shows qualitatively different shapes for these two types of molecules. The data, combined with a quantitative model for the evolution of the distribution during degradation, suggest that the bonding between β into α particles is different between phytoglycogen and liver glycogen, with the formation of a glycosidic linkage for phytoglycogen and a covalent or strong non-covalent linkage, most probably involving a protein, for glycogen as most likely. This finding is of importance for diabetes, where α-particle structure is impaired. PMID:25799321

  19. Hormonal control of hepatic glycogen metabolism in food-deprived, continuously swimming coho salmon Oncorhynchus kisutch

    USGS Publications Warehouse

    Vijayan, M.M.; Maule, A.G.; Schreck, C.B.; Moon, T.W.

    1993-01-01

    The plasma cortisol concentration and liver cytosolic glucocorticoid receptor activities of continuously swimming, food-deprived coho salmon (Oncorhynchus kisutch) did not differ from those of resting, fed fish. Plasma glucose concentration was significantly higher in the exercising, starved fish, but there were no significant differences in either hepatic glycogen concentration or hepatic activities of glycogen phosphorylase, glycogen synthase, pyruvate kinase, or lactate dehydrogenase between the two groups. Total glucose production by hepatocytes did not differ significantly between the two groups; glycogen breakdown accounted for all the glucose produced in the resting, fed fish whereas it explained only 59% of the glucose production in the exercised animals. Epinephrine and glucagon stimulation of glucose production by hepatocytes was decreased in the exercised fish without significantly affecting hepatocyte glycogen breakdown in either group. Insulin prevented glycogen breakdown and enhanced glycogen deposition in exercised fish. The results indicate that food-deprived, continuously swimming coho salmon conserve glycogen by decreasing the responsiveness of hepatocytes to catabolic hormones and by increasing the responsiveness to insulin (anabolic hormone).

  20. Starch Binding Domain-containing Protein 1/Genethonin 1 Is a Novel Participant in Glycogen Metabolism*

    PubMed Central

    Jiang, Sixin; Heller, Brigitte; Tagliabracci, Vincent S.; Zhai, Lanmin; Irimia, Jose M.; DePaoli-Roach, Anna A.; Wells, Clark D.; Skurat, Alexander V.; Roach, Peter J.

    2010-01-01

    Stbd1 is a protein of previously unknown function that is most prevalent in liver and muscle, the major sites for storage of the energy reserve glycogen. The protein is predicted to contain a hydrophobic N terminus and a C-terminal CBM20 glycan binding domain. Here, we show that Stbd1 binds to glycogen in vitro and that endogenous Stbd1 locates to perinuclear compartments in cultured mouse FL83B or Rat1 cells. When overexpressed in COSM9 cells, Stbd1 concentrated at enlarged perinuclear structures, co-localized with glycogen, the late endosomal/lysosomal marker LAMP1 and the autophagy protein GABARAPL1. Mutant Stbd1 lacking the N-terminal hydrophobic segment had a diffuse distribution throughout the cell. Point mutations in the CBM20 domain did not change the perinuclear localization of Stbd1, but glycogen was no longer concentrated in this compartment. Stable overexpression of glycogen synthase in Rat1WT4 cells resulted in accumulation of glycogen as massive perinuclear deposits, where a large fraction of the detectable Stbd1 co-localized. Starvation of Rat1WT4 cells for glucose resulted in dissipation of the massive glycogen stores into numerous and much smaller glycogen deposits that retained Stbd1. In vitro, in cells, and in animal models, Stbd1 consistently tracked with glycogen. We conclude that Stbd1 is involved in glycogen metabolism by binding to glycogen and anchoring it to membranes, thereby affecting its cellular localization and its intracellular trafficking to lysosomes. PMID:20810658