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Sample records for muscle glycogen synthase

  1. 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

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

    PubMed

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

    2012-11-14

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

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

    PubMed

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

    1979-06-01

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

  4. 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

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

    PubMed Central

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

    2016-01-01

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

  6. 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.

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

    PubMed

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

    2014-10-01

    Glycogen synthase, which catalyzes the synthesis of glycogen, is especially important for Old World (Pteropodidae) and New World (Phyllostomidae) fruit bats that ingest high-carbohydrate diets. Glycogen synthase 1, encoded by the Gys1 gene, is the glycogen synthase isozyme that functions in muscles. To determine whether Gys1 has undergone adaptive evolution in bats with carbohydrate-rich diets, in comparison to insect-eating sister bat taxa, we sequenced the coding region of the Gys1 gene from 10 species of bats, including two Old World fruit bats (Pteropodidae) and a New World fruit bat (Phyllostomidae). Our results show no evidence for positive selection in the Gys1 coding sequence on the ancestral Old World and the New World Artibeus lituratus branches. Tests for convergent evolution indicated convergence of the sequences and one parallel amino acid substitution (T395A) was detected on these branches, which was likely driven by natural selection. PMID:25001420

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

    PubMed

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

    2014-01-01

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

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

    PubMed

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

    2016-02-01

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

  10. 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

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

    PubMed Central

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

    1997-01-01

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

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

    SciTech Connect

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

    1985-10-05

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

  13. 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.

  14. 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.

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed Central

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

    1993-01-01

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

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

    PubMed Central

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

    2012-01-01

    Chronic exercise training results in numerous skeletal muscle adaptations, including increases in insulin sensitivity and glycogen content. To understand the mechanism for increased muscle glycogen, we studied the effects of exercise training on glycogen regulatory proteins in rat skeletal muscle. Female Sprague Dawley rats performed voluntary wheel running for 1, 4, or 7 weeks. After 7 weeks of training, insulin-stimulated glucose uptake was increased in epitrochlearis muscle. Compared to sedentary control rats, muscle glycogen did not change after 1 week of training, but increased significantly after 4 and 7 weeks. The increases in muscle glycogen were accompanied by elevated glycogen synthase activity and protein expression. To assess the regulation of glycogen synthase, we examined its major activator, protein phosphatase 1 (PP1), and its major deactivator, glycogen synthase kinase 3 (GSK3). Consistent with glycogen synthase activity, PP1 activity was unchanged after 1 week of training but significantly increased after 4 and 7 weeks of training. Protein expression of RGL(GM), another regulatory PP1 subunit, significantly decreased after 4 and 7 weeks of training. Unlike PP1, GSK3 phosphorylation did not follow the pattern of glycogen synthase activity. The ~40% decrease in GSK-3α phosphorylation after 1 week of exercise training persisted until 7 weeks and may function as a negative feedback to elevated glycogen. Our findings suggest that exercise training-induced increases in muscle glycogen content could be regulated by multiple mechanisms including enhanced insulin sensitivity, glycogen synthase expression, allosteric activation of glycogen synthase and PP1activity. PMID:23206309

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

    PubMed

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

    1989-06-01

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

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

    SciTech Connect

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

    1993-02-01

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

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

    PubMed

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

    2003-08-01

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

  1. 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.

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

    PubMed

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

    2004-04-01

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

  3. 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.

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

    PubMed

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

    2002-10-10

    Electro-stimulation alters muscle metabolism and the extent of this change depends on application intensity and duration. The effect of 14 days of chronic electro-stimulation on glycogen turnover and on the regulation of glycogen synthase in fast-twitch muscle was studied. The results showed that macro- and proglycogen degrade simultaneously during the first hour of stimulation. After 3 h, the muscle showed net synthesis, with an increase in the proglycogen fraction. The glycogen content peaked after 4 days of stimulation, macroglycogen being the predominant fraction at that time. Glycogen synthase was determined during electro-stimulation. The activity of this enzyme was measured at low UDPG concentration with either high or low Glu-6-P content. Western blots were performed against glycogen synthase over a range of stimulation periods. Activation of this enzyme was maximum before the net synthesis of glycogen, partial during net synthesis, and low during late synthesis. These observations suggest that the more active, dephosphorylated and very low phosphorylated forms of glycogen synthase may participate in the first steps of glycogen resynthesis before net synthesis is observed, while partially phosphorylated forms are most active during glycogen elongation. PMID:12383944

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

    PubMed

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

    2013-01-01

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

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

    PubMed

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

    2013-05-01

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

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

    PubMed Central

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

    2011-01-01

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

  8. 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.

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

    PubMed Central

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

    2004-01-01

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

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

    PubMed Central

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

    2014-01-01

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

  11. Insulin-independent glycogen supercompensation in isolated mouse skeletal muscle: role of phosphorylase inactivation.

    PubMed

    Sandström, Marie E; Abbate, Fabio; Andersson, Daniel C; Zhang, Shi-Jin; Westerblad, Håkan; Katz, Abram

    2004-08-01

    Glycogen supercompensation (increase in muscle glycogen content above basal) is an established phenomenon induced by unknown mechanisms. It consists of both insulin-dependent and -independent components. Here, we investigate insulin-independent glycogen supercompensation in isolated, intact extensor digitorum longus muscles from mice. Muscles were stimulated electrically, incubated in vitro with 5.5 mM glucose for up to 16 h and then analysed for glycogen, glucose uptake and enzyme activities. Basal glycogen was 84+/-6 micro mol glucosyl units/g dry muscle and was depleted by 80% after 10 min contraction. Glycogen increased after contraction, reaching a peak value of 113+/-9 micro mol glucosyl units/g dry muscle ( P<0.05 vs. basal) by 6 h, and returned to basal values by 16 h (84+/-8). Maximal activities of glycogen synthase, phosphorylase and alpha-glucosidase were not significantly altered by contraction or during the 6-h recovery period. Glycogen synthase fractional activity (0.17/7.2 mM glucose-6-P; inversely related to phosphorylation state of the enzyme) was increased about twofold early after contraction but then decreased and was slightly lower than baseline during the period of supercompensation (4-6 h). Phosphorylase fractional activity (+/-adenosine monophosphate; directly related to phosphorylation state of the enzyme) decreased to 60% of basal after contraction and decreased further during the initial 4 h of recovery to 40% of basal ( P<0.01 vs. basal). After 4 h recovery, glucose uptake was slightly (50%) higher in the stimulated than in the non-stimulated muscle ( P<0.01). Thus, insulin-independent glycogen supercompensation involves inactivation of phosphorylase and hence an inhibition of glycogen breakdown. PMID:15085341

  12. Effect of endurance exercise training on muscle glycogen supercompensation in rats.

    PubMed

    Nakatani, A; Han, D H; Hansen, P A; Nolte, L A; Host, H H; Hickner, R C; Holloszy, J O

    1997-02-01

    The purpose of this study was to test the hypothesis that the rate and extent of glycogen supercompensation in skeletal muscle are increased by endurance exercise training. Rats were trained by using a 5-wk-long swimming program in which the duration of swimming was gradually increased to 6 h/day over 3 wk and then maintained at 6 h/day for an additional 2 wk. Glycogen repletion was measured in trained and untrained rats after a glycogen-depleting bout of exercise. The rats were given a rodent chow diet plus 5% sucrose in their drinking water and libitum during the recovery period. There were remarkable differences in both the rates of glycogen accumulation and the glycogen concentrations attained in the two groups. The concentration of glycogen in epitrochlearis muscle averaged 13.1 +/- 0.9 mg/g wet wt in the untrained group and 31.7 +/- 2.7 mg/g in the trained group (P < 0.001) 24 h after the exercise. This difference could not be explained by a training effect on glycogen synthase. The training induced approximately 50% increases in muscle GLUT-4 glucose transporter protein and in hexokinase activity in epitrochlearis muscles. We conclude that endurance exercise training results in increases in both the rate and magnitude of muscle glycogen supercompensation in rats. PMID:9049757

  13. Enhanced Symbiotic Performance by Rhizobium tropici Glycogen Synthase Mutants

    PubMed Central

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

    2001-01-01

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

  14. Postexercise muscle glycogen resynthesis in obese insulin-resistant Zucker rats.

    PubMed

    Bruce, C R; Lee, J S; Hawley, J A

    2001-10-01

    We determined the effect of an acute bout of swimming (8 x 30 min) followed by either carbohydrate administration (0.5 mg/g glucose ip and ad libitum access to chow; CHO) or fasting (Fast) on postexercise glycogen resynthesis in soleus muscle and liver from female lean (ZL) and obese insulin-resistant (ZO) Zucker rats. Resting soleus muscle glycogen concentration ([glycogen]) was similar between genotypes and was reduced by 73 (ZL) and 63% (ZO) after exercise (P < 0.05). Liver [glycogen] at rest was greater in ZO than ZL (334 +/- 31 vs. 247 +/- 16 micromol/g wet wt; P < 0.01) and fell by 44 and 94% after exercise (P < 0.05). The fractional activity of glycogen synthase (active/total) increased immediately after exercise (from 0.22 +/- 0.05 and 0.32 +/- 0.04 to 0.63 +/- 0.08 vs. 0.57 +/- 0.05; P < 0.01 for ZL and ZO rats, respectively) and remained elevated above resting values after 30 min of recovery. During this time, muscle [glycogen] in ZO increased 68% with CHO (P < 0.05) but did not change in Fast. Muscle [glycogen] was unchanged in ZL from postexercise values after both treatments. After 6 h recovery, GLUT-4 protein concentration was increased above resting levels by a similar extent for both genotypes in both fasted (approximately 45%) and CHO-supplemented (approximately 115%) rats. Accordingly, during this time CHO refeeding resulted in supercompensation in both genotypes (68% vs. 44% for ZL and ZO). With CHO, liver [glycogen] was restored to resting levels in ZL but remained at postexercise values for ZO after both treatments. We conclude that the increased glucose availability with carbohydrate refeeding after glycogen-depleting exercise resulted in glycogen supercompensation, even in the face of muscle insulin-resistance. PMID:11568131

  15. 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.

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

    PubMed

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

    2003-12-01

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

  17. Muscle glycogen and cell function--Location, location, location.

    PubMed

    Ørtenblad, N; Nielsen, J

    2015-12-01

    The importance of glycogen, as a fuel during exercise, is a fundamental concept in exercise physiology. The use of electron microscopy has revealed that glycogen is not evenly distributed in skeletal muscle fibers, but rather localized in distinct pools. In this review, we present the available evidence regarding the subcellular localization of glycogen in skeletal muscle and discuss this from the perspective of skeletal muscle fiber function. The distribution of glycogen in the defined pools within the skeletal muscle varies depending on exercise intensity, fiber phenotype, training status, and immobilization. Furthermore, these defined pools may serve specific functions in the cell. Specifically, reduced levels of these pools of glycogen are associated with reduced SR Ca(2+) release, muscle relaxation rate, and membrane excitability. Collectively, the available literature strongly demonstrates that the subcellular localization of glycogen has to be considered to fully understand the role of glycogen metabolism and signaling in skeletal muscle function. Here, we propose that the effect of low muscle glycogen on excitation-contraction coupling may serve as a built-in mechanism, which links the energetic state of the muscle fiber to energy utilization. PMID:26589115

  18. Muscle glycogen loading with a liquid carbohydrate supplement.

    PubMed

    Lamb, D R; Snyder, A C; Baur, T S

    1991-03-01

    This study compared two high carbohydrate (CHO) diets in 14 male runners for effects on muscle glycogen deposition, endurance, and sensations of gastrointestinal discomfort. Muscle glycogen was measured in the vastus lateralis at rest and run time to exhaustion at 75% VO2max was measured following 3-1/2 days on a 50% CHO diet. After 14 days the subjects consumed a 20% CHO diet and continued training to reduce glycogen. During the next 3-1/2 days, subjects ran less and consumed a 90% CHO diet emphasizing pasta and rice (Pasta, n = 7) or lesser amounts of pasta and rice supplemented by a maltodextrin beverage (Supplement, n = 7). Glycogen was again measured, followed by a second run to exhaustion. Compared to the 50% CHO diet, Pasta increased muscle glycogen by 27.1 +/- 12.2 mmoles/kg muscle (M +/- SE; P < 0.05) and run time by 15.7 +/- 5.9 min; Supplement increased glycogen by 43.2 +/- 13.5 mmoles/kg (P < 0.05) and run time by 29.0 +/- 7.4 min (P < 0.05). Total glycogen concentrations and run times were not significantly different for Pasta versus Supplement. Subjects reported less gastrointestinal discomfort and greater overall preference for Supplement than for Pasta. Thus, glycogen loading can be accomplished at least as effectively and more comfortably by substituting a maltodextrin drink for some of the pasta and rice in a glycogen loading diet. PMID:1844402

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

    PubMed

    Takezawa, Yosuke; Kohsaka, Shinichi; Nakajima, Kazuyuki

    2014-10-24

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

  20. 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

  1. Leptin promotes osteoblast differentiation and mineralization of primary cultures of vascular smooth muscle cells by inhibiting glycogen synthase kinase (GSK)-3{beta}

    SciTech Connect

    Zeadin, Melec G.; Butcher, Martin K.; Shaughnessy, Stephen G.; Werstuck, Geoff H.

    2012-09-07

    Highlights: Black-Right-Pointing-Pointer Leptin promotes osteoblast differentiation of primary smooth muscle cells. Black-Right-Pointing-Pointer Leptin regulates the expression of genes involved in osteoblast differentiation. Black-Right-Pointing-Pointer Constitutively active GSK-3{beta} attenuates leptin-induced osteoblast differentiation. Black-Right-Pointing-Pointer This suggests that leptin signals through GSK-3{beta} to promote osteoblast differentiation. -- Abstract: In this study, we begin to investigate the underlying mechanism of leptin-induced vascular calcification. We found that treatment of cultured bovine aortic smooth muscle cells (BASMCs) with leptin (0.5-4 {mu}g/ml) induced osteoblast differentiation in a dose-dependent manner. Furthermore, we found that leptin significantly increased the mRNA expression of osteopontin and bone sialoprotein, while down-regulating matrix gla protein (MGP) expression in BASMCs. Key factors implicated in osteoblast differentiation, including members of the Wnt signaling pathway, were examined. Exposure to leptin enhanced phosphorylation of GSK-3{beta} on serine-9 thereby inhibiting activity and promoting the nuclear accumulation of {beta}-catenin. Transfection of BASMCs with an adenovirus that expressed constitutively active GSK-3{beta} (Ad-GSK-3{beta} S9A) resulted in a >2-fold increase in GSK-3{beta} activity and a significant decrease in leptin-induced alkaline phosphatase (ALP) activity. In addition, qRT-PCR analysis showed that GSK-3{beta} activation resulted in a significant decrease in the expression of osteopontin and bone sialoprotein, but a marked increase in MGP mRNA expression. When taken together, our results suggest a mechanism by which leptin promotes osteoblast differentiation and vascular calcification in vivo.

  2. Glycogen Synthase Kinase-3 (GSK-3)-Targeted Therapy and Imaging.

    PubMed

    Pandey, Mukesh K; DeGrado, Timothy R

    2016-01-01

    Glycogen synthase kinase-3 (GSK-3) is associated with various key biological processes, including glucose regulation, apoptosis, protein synthesis, cell signaling, cellular transport, gene transcription, proliferation, and intracellular communication. Accordingly, GSK-3 has been implicated in a wide variety of diseases and specifically targeted for both therapeutic and imaging applications by a large number of academic laboratories and pharmaceutical companies. Here, we review the structure, function, expression levels, and ligand-binding properties of GSK-3 and its connection to various diseases. A selected list of highly potent GSK-3 inhibitors, with IC50 <20 nM for adenosine triphosphate (ATP)-competitive inhibitors and IC50 <5 μM for non-ATP-competitive inhibitors, were analyzed for structure activity relationships. Furthermore, ubiquitous expression of GSK-3 and its possible impact on therapy and imaging are also highlighted. Finally, a rational perspective and possible route to selective and effective GSK-3 inhibitors is discussed. PMID:26941849

  3. Glycogen Synthase Kinase-3 (GSK-3)-Targeted Therapy and Imaging

    PubMed Central

    Pandey, Mukesh K.; DeGrado, Timothy R.

    2016-01-01

    Glycogen synthase kinase-3 (GSK-3) is associated with various key biological processes, including glucose regulation, apoptosis, protein synthesis, cell signaling, cellular transport, gene transcription, proliferation, and intracellular communication. Accordingly, GSK-3 has been implicated in a wide variety of diseases and specifically targeted for both therapeutic and imaging applications by a large number of academic laboratories and pharmaceutical companies. Here, we review the structure, function, expression levels, and ligand-binding properties of GSK-3 and its connection to various diseases. A selected list of highly potent GSK-3 inhibitors, with IC50 <20 nM for adenosine triphosphate (ATP)-competitive inhibitors and IC50 <5 μM for non-ATP-competitive inhibitors, were analyzed for structure activity relationships. Furthermore, ubiquitous expression of GSK-3 and its possible impact on therapy and imaging are also highlighted. Finally, a rational perspective and possible route to selective and effective GSK-3 inhibitors is discussed. PMID:26941849

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

    PubMed

    Tejeda-Muñoz, Nydia; Robles-Flores, Martha

    2015-12-01

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

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

    PubMed

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

    1988-06-15

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

  6. Investigation of potential glycogen synthase kinase 3 inhibitors using pharmacophore mapping and virtual screening.

    PubMed

    Dessalew, Nigus; Bharatam, Prasad V

    2006-09-01

    Glycogen synthase kinase-3 is a serine/threonine kinase that has attracted significant drug discovery attention in recent years. To investigate the identification of new potential glycogen synthase kinase-3 inhibitors, a pharmacophore mapping study was carried out using a set of 21 structurally diverse glycogen synthase kinase-3 inhibitors. A hypothesis containing four features: two hydrophobic, one hydrogen bond donor and another hydrogen bond acceptor was found to be the best from the 10 common feature hypotheses produced by HipHop module of Catalyst. The best hypothesis has a high cost of 156.592 and higher best fit values were obtained for the 21 inhibitors using this best hypothesis than the other HipHop hypotheses. The best hypothesis was then used to screen electronically the NCI2000 database. The hits obtained were docked into glycogen synthase kinase-3beta active site. A total of five novel potential leads were proposed after: (i) visual examination of how well they dock into the glycogen synthase kinase-3beta-binding site, (ii) comparative analysis of their FlexX, G-Score, PMF-Score, ChemScore and D-Scores values, (iii) comparison of their best fit value with the known inhibitors and (iv) examination of the how the hits retain interactions with the important amino acid residues of glycogen synthase kinase-3beta-binding site. PMID:17062013

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

    PubMed Central

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

    2015-01-01

    We report the successful expression and purification of functional human muscle glycogen synthase (GYS1) in complex with human glycogenin-1 (GN1). Stoichiometric GYS1:GN1 complex was produced by co-expression of GYS1 and GN1 using a bicistronic pFastBac™-Dual expression vector, followed by affinity purification and subsequent size-exclusion chromatography. Mass spectrometry analysis identified that GYS1 is phosphorylated at several well-characterised and uncharacterised Ser/Thr residues. Biochemical analysis, including activity ratio (in the absence relative to that in the presence of glucose-6-phosphate) measurement, covalently attached phosphate estimation as well as phosphatase treatment, revealed that recombinant GYS1 is substantially more heavily phosphorylated than would be observed in intact human or rodent muscle tissues. A large quantity of highly-pure stoichiometric GYS1:GN1 complex will be useful to study its structural and biochemical properties in the future, which would reveal mechanistic insights into its functional role in glycogen biosynthesis. PMID:25527037

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

    PubMed Central

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

    2014-01-01

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

  9. 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

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

    PubMed

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

    2012-06-01

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

  11. 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.

  12. Role of glucose transport in glycogen supercompensation in reweighted rat skeletal muscle.

    PubMed

    Henriksen, E J; Stump, C S; Trinh, T H; Beaty, S D

    1996-05-01

    Hindlimb weight bearing after a 3-day period of hindlimb suspension (reweighting) of juvenile rats results in a marked transient elevation in soleus glycogen concentration that cannot be explained on the basis of the activities of glycogen synthase and phosphorylase. We have hypothesized that enhanced glucose transport activity could underlie this response. We directly tested this hypothesis by assessing the response of insulin-dependent and insulin-independent glucose transport activity (in vitro 2-[1,2-3H]deoxy-D-glucose uptake) as well as glucose transporter (GLUT-4) protein levels during a 48-h reweighting period. After a net glycogen loss (from 29 +/- 2 to 16 +/- 1 nmol/mg muscle; P < 0.05) during the first 2 h of reweighting, glycogen accumulated at an average rate of 1.4 nmol.mg-1.h-1 up to 18 h, reaching an apex of 38 +/- 1 nmol/mg. During this same reweighting period, insulin-independent, but not insulin-dependent, glucose transport activity was significantly enhanced (P < 0.05 vs. weight-bearing control values) and was associated with an elevated level of GLUT-4 protein and the specific activity of total hexokinase. The specific activity of citrate synthase was also increased. By 24 h of reweighting, although insulin-independent glucose transport activity and GLUT-4 protein remained elevated, glycogen accumulation had ceased, likely due to enhanced phosphorylase activity at this time point. These results are consistent with the interpretation that the glycogen supercompensation seen during reweighting of the rat soleus may be regulated in part by an enhanced glucose flux arising from an increase in insulin-independent glucose transport activity and hexokinase activity. PMID:8727537

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

    PubMed

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

    2004-03-01

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

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

    PubMed

    Maqbool, Mudasir; Mobashir, Mohammad; Hoda, Nasimul

    2016-01-01

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

  15. Muscle glycogen supercompensation: absence of a gender-related difference.

    PubMed

    James, A P; Lorraine, M; Cullen, D; Goodman, C; Dawson, B; Palmer, T N; Fournier, P A

    2001-10-01

    Recently it has been reported that women do not have the capacity to accumulate supranormal levels of muscle glycogen when subjected to a carbohydrate (CHO) loading regimen [Tarnopolsky et al. (1995) J Appl Physiol 78:1360-1368]. Since, in this study, CHO intake relative to body mass in the female subjects was much lower than that in males, our primary aim was to re-examine this issue using subjects fed comparable amounts of CHO. Endurance-trained female and male subjects ingested 12 g CHO x kg(-1) lean body mass day(-1) in conjunction with the cessation of their daily physical training. A 3-day exposure to this diet resulted in a marked rise in muscle glycogen levels from [mean (SD)] 108 (15) mmol x kg(-1) wet weight to 193 (14) mmol x kg(-1) wet weight and 111 (16) m mol x kg(-1) wet weight to 202 (20) mmol x kg(-1) wet weight in the female participants during the post-menstrual and pre-menstrual phases of their menstrual cycle, respectively, and from 109 (27) mmol x kg(-1) wet weight to 183 (25) mmol x kg(-1) wet weight in males. We conclude that (1) female athletes have the capacity to accumulate supranormal levels of muscle glycogen, and (2) when exercise-trained males and females are fed comparable amounts of CHO relative to lean body mass, there is no gender-related difference in their ability to accumulate supranormal levels of muscle glycogen. PMID:11718281

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

    PubMed

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

    2016-06-01

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

  17. Supercompensation of muscle glycogen in trained and untrained subjects.

    PubMed

    Roedde, S; MacDougall, J D; Sutton, J R; Green, H J

    1986-03-01

    The purpose of this study was to determine whether or not trained athletes have the same capacity for supercompensation of muscle glycogen as untrained subjects. Muscle glycogen was measured in 4 highly trained cyclists and 4 untrained controls over a 6 day period of exercise and dietary manipulation. During the week prior to the investigation the trained group tapered their training load but maintained a high carbohydrate intake as they would in preparation for a major competition. Needle biopsies were taken from the vastus lateralis before and after exhaustive cycle ergometry at 73% VO2 max followed by several sprint intervals, after 3 days on a carbohydrate-restricted diet and after 2 and 3 days on a high carbohydrate diet. All food intake was quantified and plasma insulin and glucose were monitored daily. The mean initial glycogen concentration for the trained group was 115 mmol X kg-1 wet muscle weight and 92 mmol X kg-1 for the untrained group. Both groups showed similar post exercise depletion and recovery patterns when expressed as a % of their initial values. Following 3 days of high carbohydrate diet, the glycogen concentration for the trained cyclists reached 174 mmol X kg-1 or 152% of its initial value while the untrained-group reached 143 mmol X kg-1 or 155% of its initial value. It was concluded that a regimen of exhaustive exercise, followed by a period of carbohydrate restriction and a period of high carbohydrate intake, results in substantially higher muscle glycogen storage than can be achieved by a reduction in training in combination with high carbohydrate intake.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:3698159

  18. Glycogen metabolism in humans.

    PubMed

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

    2016-06-01

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

  19. Carbohydrate supplementation spares muscle glycogen during variable-intensity exercise.

    PubMed

    Yaspelkis, B B; Patterson, J G; Anderla, P A; Ding, Z; Ivy, J L

    1993-10-01

    Effects of carbohydrate (CHO) supplementation on muscle glycogen utilization and endurance were evaluated in seven well-trained male cyclists during continuous cycling exercise that varied between low [45% maximal O2 uptake (VO2 max)] and moderate intensity (75% VO2 max). During each exercise bout the subjects received either artificially flavored placebo (P), 10% liquid CHO supplement (L; 3 x 18 g CHO/h), or solid CHO supplement (S; 2 x 25 g CHO/h). Muscle biopsies were taken from vastus lateralis during P and L trials immediately before exercise and after first (124 min) and second set (190 min) of intervals. Subjects then rode to fatigue at 80% VO2 max. Plasma glucose and insulin responses during L treatment reached levels of 6.7 +/- 0.7 mM and 70.6 +/- 17.2 microU/ml, respectively, and were significantly greater than those of P treatment (4.4 +/- 0.1 mM and 17.7 +/- 1.6 microU/ml) throughout the exercise bout. Plasma glucose and insulin responses of S treatment were intermediate to those of L and P treatments. Times to fatigue for S (223.9 +/- 3.5 min) and L (233.4 +/- 7.5 min) treatments did not differ but were significantly greater than that of P treatment (202.4 +/- 9.8 min). After the first 190 min of exercise, muscle glycogen was significantly greater during L (79 +/- 3.5 mumol/g wet wt) than during P treatment (58.5 +/- 7.2 mumol/g wet wt). Furthermore, differences in muscle glycogen concentrations between L and P treatments after 190 min of exercise and in time to fatigue for these treatments were positively related (r = 0.76, P < 0.05). These results suggest that CHO supplementation can enhance prolonged continuous variable-intensity exercise by reducing dependency on muscle glycogen as a fuel source. PMID:8282593

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

    PubMed

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

    2014-12-15

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

  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. PMID:27208262

  2. Effects of genetic and environmental factors on muscle glycogen content in Japanese Black cattle

    PubMed Central

    Komatsu, Tomohiko; Shoji, Noriaki; Saito, Kunihiko; Suzuki, Keiichi

    2014-01-01

    Monosaccharides such as glucose contribute to the development of meat flavor upon heating via the Maillard reaction; therefore, monosaccharide content is related to beef palatability. Here, we analyzed the effects of genetic and environmental factors on the content of glycogen, one of the precursors of monosaccharides, in the muscles of 958 fattened Japanese Black cattle from Yamagata Prefecture. Analysis of variance showed that muscle glycogen content was affected by the farm and postmortem periods, but not by sex, slaughter age, slaughter month or number of days detained at the slaughter yard. Additionally, consumption of digestible brown rice feed elevated muscle glycogen levels. Glycogen heritability was estimated to be 0.34, and genetic correlations between glycogen and carcass weight (CW) or beef marbling standard (BMS) were weak. The predicted breeding values varied among paternal lines. These results demonstrated that genetic factors might improve muscle glycogen content and therefore beef palatability, but do not influence CW or BMS. PMID:24716455

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

    PubMed

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

    2016-01-20

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

  4. Influence of circadian rhythms on rat muscle glycogen metabolism during and after exercise.

    PubMed

    Garetto, L P; Armstrong, R B

    1983-01-01

    Marked circadian fluctuations in skeletal muscle glycogen concentrations have previously been reported. The purpose of the present study was to estimate the influence of these rhythms on muscle glycogen metabolism during and after high-intensity treadmill exercise. Male Sprague-Dawley rats ran five 1-min sprints at 75 m min-1 interspersed by 1-3 min rest intervals either at 08.00 h (morning) or at 20.00 h (night). All muscles sampled lost significant amounts of glycogen during exercise at both time periods. There were no differences in rates of loss between morning and night, even though glycogen levels in several muscles (high-oxidative muscles) were significantly higher before exercise in the morning. Following exercise, glycogen restoration in muscle samples primarily composed of fast-twitch fibres was more rapid in the morning than at night. There was no difference in glycogen restoration rates between the two time periods in the muscle primarily composed of slow-twitch fibres. Although liver glycogen was lower after exercise at night than in the morning, there were no differences in post-exercise blood glucose levels between the two time periods. In conclusion, circadian rhythms do not appear to influence rates of glycogen loss during high-speed running. However, since glycogen loss is the same at all times of day, one would predict that circadian changes in pre-exercise muscle glycogen concentrations would affect muscular endurance. Muscle glycogen restoration after exercise does appear to be affected by circadian rhythms, although interpretation of these data is complicated by possible changes in patterns of muscle fibre contraction at different times of the day. These circadian influences should be considered in the design of exercise studies using laboratory rodents. PMID:6833943

  5. Possible mechanism for changes in glycogen metabolism in unloaded soleus muscle

    NASA Technical Reports Server (NTRS)

    Henriksen, E. J.; Tischler, M. E.

    1985-01-01

    Carbohydrate metabolism has been shown to be affected in a number of ways by different models of hypokinesia. In vivo glycogen levels in the soleus muscle are known to be increased by short-term denervation and harness suspension. In addition, exposure to 7 days of hypogravity also caused a dramatic increase in glycogen concentration in this muscle. The biochemical alterations caused by unloading that may bring about these increases in glycogen storage in the soleus were sought.

  6. Metabolic responses to adrenaline and muscle glycogen content in dogs treated with thyroxine.

    PubMed

    Brzezińska, Z; Kaciuba-Uściłko, H

    1978-01-01

    Lipolytic, hyperglycaemic and lactacidaemic responses to 1h adrenaline infusion (0.1 microgram/kg/min) were compared in resting dogs before (control) and after prolonged thyroxine (T4) treatment. Besides, the effect of 2-week thyroxine administration on muscle glycogen content, and its changes following adrenaline infusion were examined. Prolonged T4-treatment of dogs resulted in considerable alterations of the metabolic actions of adrenaline. A marked difference between the control and T4-treated dogs was also found in the muscle glycogen content, which was significantly lower in the latter. Both in the control and T4-injected dogs adrenaline infusion caused similar depletion of the muscle glycogen store. However, in all the control animals examined supercompensation of muscle glycogen was noted 1 h following termination of adrenaline infusion, whereas T4-treated dogs were unable of incurring any significant muscle glycogen deposition. PMID:742367

  7. Postexercise glucose uptake and glycogen synthesis in skeletal muscle from GLUT4-deficient mice.

    PubMed

    Ryder, J W; Kawano, Y; Galuska, D; Fahlman, R; Wallberg-Henriksson, H; Charron, M J; Zierath, J R

    1999-12-01

    To determine the role of GLUT4 on postexercise glucose transport and glycogen resynthesis in skeletal muscle, GLUT4-deficient and wild-type mice were studied after a 3 h swim exercise. In wild-type mice, insulin and swimming each increased 2-deoxyglucose uptake by twofold in extensor digitorum longus muscle. In contrast, insulin did not increase 2-deoxyglucose glucose uptake in muscle from GLUT4-null mice. Swimming increased glucose transport twofold in muscle from fed GLUT4-null mice, with no effect noted in fasted GLUT4-null mice. This exercise-associated 2-deoxyglucose glucose uptake was not accompanied by increased cell surface GLUT1 content. Glucose transport in GLUT4-null muscle was increased 1.6-fold over basal levels after electrical stimulation. Contraction-induced glucose transport activity was fourfold greater in wild-type vs. GLUT4-null muscle. Glycogen content in gastrocnemius muscle was similar between wild-type and GLUT4-null mice and was reduced approximately 50% after exercise. After 5 h carbohydrate refeeding, muscle glycogen content was fully restored in wild-type, with no change in GLUT4-null mice. After 24 h carbohydrate refeeding, muscle glycogen in GLUT4-null mice was restored to fed levels. In conclusion, GLUT4 is the major transporter responsible for exercise-induced glucose transport. Also, postexercise glycogen resynthesis in muscle was greatly delayed; unlike wild-type mice, glycogen supercompensation was not found. GLUT4 it is not essential for glycogen repletion since muscle glycogen levels in previously exercised GLUT4-null mice were totally restored after 24 h carbohydrate refeeding.-Ryder, J. W., Kawano, Y., Galuska, D., Fahlman, R., Wallberg-Henriksson, H., Charron, M. J., Zierath, J. R. Postexercise glucose uptake and glycogen synthesis in skeletal muscle from GLUT4-deficient mice. PMID:10593872

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

    PubMed

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

    2015-01-01

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

  9. Effects of depletion exercise and light training on muscle glycogen supercompensation in men

    PubMed Central

    Goforth, Harold W.; Laurent, Didier; Prusaczyk, William K.; Schneider, Kevin E.; Petersen, Kitt Falk; Shulman, Gerald I.

    2010-01-01

    Supercompensated muscle glycogen can be achieved by using several carbohydrate (CHO)-loading protocols. This study compared the effectiveness of two “modified” CHO-loading protocols. Additionally, we determined the effect of light cycle training on muscle glycogen. Subjects completed a depletion (D, n = 15) or nondepletion (ND, n = 10) CHO-loading protocol. After a 2-day adaptation period in a metabolic ward, the D group performed a 120-min cycle exercise at 65% peak oxygen uptake (V̇O2 peak) followed by 1-min sprints at 120% V̇O2 peak to exhaustion. The ND group performed only 20-min cycle exercise at 65% V̇O2 peak. For the next 6 days, both groups ate the same high-CHO diets and performed 20-min daily cycle exercise at 65% V̇O2 peak followed by a CHO beverage (105 g of CHO). Muscle glycogen concentrations of the vastus lateralis were measured daily with 13C magnetic resonance spectroscopy. On the morning of day 5, muscle glycogen concentrations had increased 1.45 (D) and 1.24 (ND) times baseline (P < 0.001) but did not differ significantly between groups. However, on day 7, muscle glycogen of the D group was significantly greater (p < 0.01) than that of the ND group (130 ± 7 vs. 104 ± 5 mmol/l). Daily cycle exercise decreased muscle glycogen by 10 ± 2 (D) and 14 ± 5 mmol/l (ND), but muscle glycogen was equal to or greater than preexercise values 24 h later. In conclusion, a CHO-loading protocol that begins with a glycogen-depleting exercise results in significantly greater muscle glycogen that persists longer than a CHO-loading protocol using only an exercise taper. Daily exercise at 65% V̇O2 peak for 20 min can be performed throughout the CHO-loading protocol without negatively affecting muscle glycogen supercompensation. PMID:12902321

  10. Effects of depletion exercise and light training on muscle glycogen supercompensation in men.

    PubMed

    Goforth, Harold W; Laurent, Didier; Prusaczyk, William K; Schneider, Kevin E; Petersen, Kitt Falk; Shulman, Gerald I

    2003-12-01

    Supercompensated muscle glycogen can be achieved by using several carbohydrate (CHO)-loading protocols. This study compared the effectiveness of two "modified" CHO-loading protocols. Additionally, we determined the effect of light cycle training on muscle glycogen. Subjects completed a depletion (D, n = 15) or nondepletion (ND, n = 10) CHO-loading protocol. After a 2-day adaptation period in a metabolic ward, the D group performed a 120-min cycle exercise at 65% peak oxygen uptake (VO2 peak) followed by 1-min sprints at 120% VO2 peak to exhaustion. The ND group performed only 20-min cycle exercise at 65% VO2 peak. For the next 6 days, both groups ate the same high-CHO diets and performed 20-min daily cycle exercise at 65% VO2 peak followed by a CHO beverage (105 g of CHO). Muscle glycogen concentrations of the vastus lateralis were measured daily with 13C magnetic resonance spectroscopy. On the morning of day 5, muscle glycogen concentrations had increased 1.45 (D) and 1.24 (ND) times baseline (P < 0.001) but did not differ significantly between groups. However, on day 7, muscle glycogen of the D group was significantly greater (p < 0.01) than that of the ND group (130 +/- 7 vs. 104 +/- 5 mmol/l). Daily cycle exercise decreased muscle glycogen by 10 +/- 2 (D) and 14 +/- 5 mmol/l (ND), but muscle glycogen was equal to or greater than preexercise values 24 h later. In conclusion, a CHO-loading protocol that begins with a glycogen-depleting exercise results in significantly greater muscle glycogen that persists longer than a CHO-loading protocol using only an exercise taper. Daily exercise at 65% VO2 peak for 20 min can be performed throughout the CHO-loading protocol without negatively affecting muscle glycogen supercompensation. PMID:12902321

  11. Differential response of rat cardiac and skeletal muscle glycogen to glucocorticoids.

    PubMed

    Poland, J L; Poland, J W; Honey, R N

    1982-05-01

    Though glucocorticoids were previously implicated in the support of myocardial glycogen supercompensation after exercise, it was unclear why skeletal muscle glycogen did not simultaneously supercompensate since it was also exposed to the exercise-induced glucocorticoid increases. The current study shows that glucocorticoids differentially affect cardiac and skeletal muscle glycogen. Following dexamethasone administration (400 micrograms i.p.) myocardial glycogen peaked at 6 h while glycogen in the soleus, red vastus lateralis, and white vastus lateralis increased more slowly and reached the highest values 17 h postinjection. Concurrently, blood glucose, insulin, and glucagon remained at control levels. Liver glycogen increased within 2 h and continued to rise with a peak value at 17 h. Plasma free fatty acid (FFA) levels increased and remained high throughout the 26-h experimental period. High FFA levels inhibit glycogenolysis and thus could be partially responsible for glucocorticoid-induced glycogen increases. It is postulated that glycogen supercompensation does not readily occur in skeletal muscles after exercise because of the brevity of the corticosterone and FFA increases and the slowness of the skeletal muscle glycogen response to glucocorticoids. PMID:7104851

  12. A fermented soy permeate improves the skeletal muscle glucose level without restoring the glycogen content in streptozotocin-induced diabetic rats.

    PubMed

    Malardé, Ludivine; Vincent, Sophie; Lefeuvre-Orfila, Luz; Efstathiou, Théo; Groussard, Carole; Gratas-Delamarche, Arlette

    2013-02-01

    Exercise is essential into the therapeutic management of diabetic patients, but their level of exercise tolerance is lowered due to alterations of glucose metabolism. As soy isoflavones have been shown to improve glucose metabolism, this study aimed to assess the effects of a dietary supplement containing soy isoflavones and alpha-galactooligosaccharides on muscular glucose, glycogen synthase (GSase), and glycogen content in a type 1 diabetic animal model. The dietary supplement tested was a patented compound, Fermented Soy Permeate (FSP), developed by the French Company Sojasun Technologies. Forty male Wistar rats were randomly assigned to control or diabetic groups (streptozotocin, 45 mg/kg). Each group was then divided into placebo or FSP-supplemented groups. Both groups received by oral gavage, respectively, water or diluted FSP (0.1 g/day), daily for a period of 3 weeks. At the end of the protocol, glycemia was noticed after a 24-h fasting period. Glucose, total GSase, and the glycogen content were determined in the skeletal muscle (gastrocnemius). Diabetic animals showed a higher blood glucose concentration, but a lower glucose and glycogen muscle content than controls. Three weeks of FSP consumption allowed to restore the muscle glucose concentration, but failed to reduce glycemia and to normalize the glycogen content in diabetic rats. Furthermore, the glycogen content was increased in FSP-supplemented controls compared to placebo controls. Our results demonstrated that diabetic rats exhibited a depleted muscle glycogen content (-25%). FSP-supplementation normalized the muscle glucose level without restoring the glycogen content in diabetic rats. However, it succeeded to increase it in the control group (+20%). PMID:23356441

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

    PubMed

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

    2016-06-01

    Chronic activation of AMP-activated protein kinase (AMPK) increases glycogen content in skeletal muscle. Previously, we demonstrated that a mutation in the ryanodine receptor (RyR1(R615C)) blunts AMPK phosphorylation in longissimus muscle of pigs with a gain of function mutation in the AMPKγ3 subunit (AMPKγ3(R200Q)); this may decrease the glycogen storage capacity of AMPKγ3(R200Q) + RyR1(R615C) muscle. Therefore, our aim in this study was to utilize our pig model to understand how AMPKγ3(R200Q) and AMPK activation contribute to glycogen storage and metabolism in muscle. We selected and bred pigs in order to generate offspring with naturally occurring AMPKγ3(R200Q), RyR1(R615C), and AMPKγ3(R200Q) + RyR1(R615C) mutations, and also retained wild-type littermates (control). We assessed glycogen content and parameters of glycogen metabolism in longissimus muscle. Regardless of RyR1(R615C), AMPKγ3(R200Q) increased the glycogen content by approximately 70%. Activity of glycogen synthase (GS) without the allosteric activator glucose 6-phosphate (G6P) was decreased in AMPKγ3(R200Q) relative to all other genotypes, whereas both AMPKγ3(R200Q) and AMPKγ3(R200Q) + RyR1(R615C) muscle exhibited increased GS activity with G6P. Increased activity of GS with G6P was not associated with increased abundance of GS or hexokinase 2. However, AMPKγ3(R200Q) enhanced UDP-glucose pyrophosphorylase 2 (UGP2) expression approximately threefold. Although UGP2 is not generally considered a rate-limiting enzyme for glycogen synthesis, our model suggests that UGP2 plays an important role in increasing flux to glycogen synthase. Moreover, we have shown that the capacity for glycogen storage is more closely related to the AMPKγ3(R200Q) mutation than activity. PMID:27302990

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed

    Jensen, Thomas E; Richter, Erik A

    2012-03-01

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

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

    PubMed Central

    Jensen, Thomas E; Richter, Erik A

    2012-01-01

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

  17. Role of glycogen availability in sarcoplasmic reticulum Ca2+ kinetics in human skeletal muscle

    PubMed Central

    Ørtenblad, Niels; Nielsen, Joachim; Saltin, Bengt; Holmberg, Hans-Christer

    2011-01-01

    Little is known about the precise mechanism that relates skeletal muscle glycogen to muscle fatigue. The aim of the present study was to examine the effect of glycogen on sarcoplasmic reticulum (SR) function in the arm and leg muscles of elite cross-country skiers (n= 10, 72 ± 2 ml kg−1 min−1) before, immediately after, and 4 h and 22 h after a fatiguing 1 h ski race. During the first 4 h recovery, skiers received either water or carbohydrate (CHO) and thereafter all received CHO-enriched food. Immediately after the race, arm glycogen was reduced to 31 ± 4% and SR Ca2+ release rate decreased to 85 ± 2% of initial levels. Glycogen noticeably recovered after 4 h recovery with CHO (59 ± 5% initial) and the SR Ca2+ release rate returned to pre-exercise levels. However, in the absence of CHO during the first 4 h recovery, glycogen and the SR Ca2+ release rate remained unchanged (29 ± 2% and 77 ± 8%, respectively), with both parameters becoming normal after the remaining 18 h recovery with CHO. Leg muscle glycogen decreased to a lesser extent (71 ± 10% initial), with no effects on the SR Ca2+ release rate. Interestingly, transmission electron microscopy (TEM) analysis revealed that the specific pool of intramyofibrillar glycogen, representing 10–15% of total glycogen, was highly significantly correlated with the SR Ca2+ release rate. These observations strongly indicate that low glycogen and especially intramyofibrillar glycogen, as suggested by TEM, modulate the SR Ca2+ release rate in highly trained subjects. Thus, low glycogen during exercise may contribute to fatigue by causing a decreased SR Ca2+ release rate. PMID:21135051

  18. 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

  19. 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

  20. Carbohydrate supercompensation and muscle glycogen utilization during exhaustive running in highly trained athletes.

    PubMed

    Madsen, K; Pedersen, P K; Rose, P; Richter, E A

    1990-01-01

    Three female and three male highly trained endurance runners with mean maximal oxygen uptake (VO2max) values of 60.5 and 71.5 ml.kg-1.min-1, respectively, ran to exhaustion at 75%-80% of VO2max on two occasions after an overnight fast. One experiment was performed after a normal diet and training regimen (Norm), the other after a diet and training programme intended to increase muscle glycogen levels (Carb). Muscle glycogen concentration in the gastrocnemius muscle increased by 25% (P less than 0.05) from 581 mmol.kg-1 dry weight, SEM 50 to 722 mmol.kg-1 dry weight, SEM 34 after Carb. Running time to exhaustion, however, was not significantly different in Carb and Norm, 77 min, SEM 13 vs 70 min, SEM 8, respectively. The average glycogen concentration following exhaustive running was 553 mmol.kg-1 dry weight, SEM 70 in Carb and 434 mmol.kg-1 dry weight, SEM 57 in Norm, indicating that in both tests muscle glycogen stores were decreased by about 25%. Periodic acid-Schiff staining for semi-quantitative glycogen determination in individual fibres confirmed that none of the fibres appeared to be glycogen-empty after exhaustive running. The steady-state respiratory exchange ratio was higher in Carb than in Norm (0.92, SEM 0.01 vs 0.89, SEM 0.01; P less than 0.05). Since muscle glycogen utilization was identical in the two tests, the indication of higher utilization of total carbohydrate appears to be related to a higher utilization of liver glycogen. We have concluded that glycogen depletion of the gastrocnemius muscle is unlikely to be the cause of fatigue during exhaustive running at 75%-80% of VO2max in highly trained endurance runners. Furthermore, diet- and training-induced carbohydrate super-compensation does not appear to improve endurance capacity in such individuals. PMID:2079068

  1. Prevention of glycogen supercompensation prolongs the increase in muscle GLUT4 after exercise.

    PubMed

    Garcia-Roves, Pablo M; Han, Dong-Ho; Song, Zheng; Jones, Terry E; Hucker, Kathleen A; Holloszy, John O

    2003-10-01

    Exercise induces an increase in GLUT4 in skeletal muscle with a proportional increase in glucose transport capacity. This adaptation results in enhanced glycogen accumulation, i.e., "supercompensation," in response to carbohydrate feeding after glycogen-depleting exercise. The increase in GLUT4 reverses within 40 h after exercise in carbohydrate-fed rats. The purpose of this study was to determine whether prevention of skeletal muscle glycogen supercompensation after exercise results in maintenance of the increases in GLUT4 and the capacity for glycogen supercompensation. Rats were exercised by means of three daily bouts of swimming. GLUT4 mRNA was increased approximately 3-fold and GLUT4 protein was increased approximately 2-fold 18 h in epitrochlearis muscle after exercise. These increases in GLUT4 mRNA and protein reversed completely within 42 h after exercise in rats fed a high-carbohydrate diet. In contrast, the increases in GLUT4 protein, insulin-stimulated glucose transport, and increased capacity for glycogen supercompensation persisted unchanged for 66 h in rats fed a carbohydrate-free diet that prevented glycogen supercompensation after exercise. GLUT4 mRNA was still elevated at 42 h but had returned to baseline by 66 h after exercise in rats fed the carbohydrate-free diet. Glycogen-depleted rats fed carbohydrate 66 h after exercise underwent muscle glycogen supercompensation with concomitant reversal of the increase in GLUT4. These findings provide evidence that prevention of glycogen supercompensation after exercise results in persistence of exercise-induced increases in GLUT4 protein and enhanced capacity for glycogen supercompensation. PMID:12799316

  2. Persistence of supercompensated muscle glycogen in trained subjects after carbohydrate loading.

    PubMed

    Goforth, H W; Arnall, D A; Bennett, B L; Law, P G

    1997-01-01

    Several carbohydrate (CHO)-loading protocols have been used to achieve muscle glycogen supercompensation and prolong endurance performance. This study assessed the persistence of muscle glycogen supercompensation over the 3 days after the supercompensation protocol. Trained male athletes completed a 6-day CHO-loading protocol that included cycle ergometer exercise and dietary manipulations. The 3-day depletion phase began with 115 min of cycling at 75% peak oxygen uptake followed by 3 x 60-s sprints and included the subjects consuming a low-CHO/high-protein/high-fat (10:41:49%) diet. Subjects cycled 40 min at the same intensity for the next 2 days. During the 3-day repletion phase, subjects rested and consumed a high-CHO/low-protein/low-fat (85:08:07%) diet, including a glucose-polymer beverage. A 3-day postloading phase followed, which involved a moderately high CHO diet (60%) and no exercise. Glycogen values for vastus lateralis biopsies at baseline and postloading days 1-3 were 408 +/- 168 (SD), 729 +/- 222, 648 +/- 186, and 714 +/- 196 mmol/kg dry wt, respectively. The CHO-loading protocol increased muscle glycogen by 1.79 times baseline, and muscle glycogen remained near this level during the 3-day postloading period. Results indicate that supercompensated muscle glycogen levels can be maintained for at least 3 days in a resting athlete when a moderate-CHO diet is consumed. PMID:9029236

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  4. 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…

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

    PubMed Central

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

    2012-01-01

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

  6. Glycogen metabolism in humans☆☆☆

    PubMed Central

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

    2016-01-01

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

  7. 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

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

    PubMed

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

    2014-01-01

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

  9. 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

  10. 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.

  11. Subcellular distribution of glycogen and decreased tetanic Ca2+ in fatigued single intact mouse muscle fibres

    PubMed Central

    Nielsen, Joachim; Cheng, Arthur J; Ørtenblad, Niels; Westerblad, Håkan

    2014-01-01

    In skeletal muscle fibres, glycogen has been shown to be stored at different subcellular locations: (i) between the myofibrils (intermyofibrillar); (ii) within the myofibrils (intramyofibrillar); and (iii) subsarcolemmal. Of these, intramyofibrillar glycogen has been implied as a critical regulator of sarcoplasmic reticulum Ca2+ release. The aim of the present study was to test directly how the decrease in cytoplasmic free Ca2+ ([Ca2+]i) during repeated tetanic contractions relates to the subcellular glycogen distribution. Single fibres of mouse flexor digitorum brevis muscles were fatigued with 70 Hz, 350 ms tetani given at 2 s (high-intensity fatigue, HIF) or 10 s (low-intensity fatigue, LIF) intervals, while force and [Ca2+]i were measured. Stimulation continued until force decreased to 30% of its initial value. Fibres were then prepared for analyses of subcellular glycogen distribution by transmission electron microscopy. At fatigue, tetanic [Ca2+]i was reduced to 70 ± 4% and 54 ± 4% of the initial in HIF (P < 0.01, n = 9) and LIF (P < 0.01, n = 5) fibres, respectively. At fatigue, the mean inter- and intramyofibrillar glycogen content was 60–75% lower than in rested control fibres (P < 0.05), whereas subsarcolemmal glycogen was similar to control. Individual fibres showed a good correlation between the fatigue-induced decrease in tetanic [Ca2+]i and the reduction in intermyofibrillar (P = 0.051) and intramyofibrillar (P = 0.0008) glycogen. In conclusion, the fatigue-induced decrease in tetanic [Ca2+]i, and hence force, is accompanied by major reductions in inter- and intramyofibrillar glycogen. The stronger correlation between decreased tetanic [Ca2+]i and reduced intramyofibrillar glycogen implies that sarcoplasmic reticulum Ca2+ release critically depends on energy supply from the intramyofibrillar glycogen pool. PMID:24591577

  12. Glycogen supercompensation masks the effect of a traininginduced increase in GLUT-4 on muscle glucose transport.

    PubMed

    Host, H H; Hansen, P A; Nolte, L A; Chen, M M; Holloszy, J O

    1998-07-01

    Endurance exercise training induces a rapid increase in the GLUT-4 isoform of the glucose transporter in muscle. In fasted rats, insulin-stimulated muscle glucose transport is increased in proportion to the increase in GLUT-4. There is evidence that high muscle glycogen may decrease insulin-stimulated glucose transport. This study was undertaken to determine whether glycogen supercompensation interferes with the increase in glucose transport associated with an exercise-induced increase in GLUT-4. Rats were trained by means of swimming for 6 h/day for 2 days. Rats fasted overnight after the last exercise bout had an approximately twofold increase in epitrochlearis muscle GLUT-4 and an associated approximately twofold increase in maximally insulin-stimulated glucose transport activity. Epitrochlearis muscles of rats fed rodent chow after exercise were glycogen supercompensated (86.4 +/- 4.8 micromol/g wet wt) and showed no significant increase in maximally insulin-stimulated glucose transport above the sedentary control value despite an approximately twofold increase in GLUT-4. Fasting resulted in higher basal muscle glucose transport rates in both sedentary and trained rats but did not significantly increase maximally insulin-stimulated transport in the sedentary group. We conclude that carbohydrate feeding that results in muscle glycogen supercompensation prevents the increase in maximally insulin-stimulated glucose transport associated with an exercise training-induced increase in muscle GLUT-4. PMID:9655766

  13. Adenovirus-mediated delivery into myocytes of muscle glycogen phosphorylase, the enzyme deficient in patients with glycogen-storage disease type V.

    PubMed Central

    Baqué, S; Newgard, C B; Gerard, R D; Guinovart, J J; Gómez-Foix, A M

    1994-01-01

    The feasibility of using adenovirus as a vector for the introduction of glycogen phosphorylase activity into myocytes has been examined. We used the C2C12 myoblast cell line to assay the impact of phosphorylase gene transfer on myocyte glycogen metabolism and to reproduce in vitro the two strategies proposed for the treatment of muscle genetic diseases, myoblast transplantation and direct DNA delivery. In this study, a recombinant adenovirus containing the muscle glycogen phosphorylase cDNA transcribed from the cytomegalovirus promoter (AdCMV-MGP) was used to transduce both differentiating myoblasts and nondividing mature myotube cells. Muscle glycogen phosphorylase mRNA levels and total phosphorylase activity were increased in both cell types after viral treatment although more efficiently in the differentiated myotubes. The increase in phosphorylase activity was transient (15 days) in myoblasts whereas in myotubes higher levels of phosphorylase gene expression and activity were reached, which remained above control levels for the duration of the study (20 days). The introduction of muscle phosphorylase into myotubes enhanced their glycogenolytic capacity. AdCMV MGP-transduced myotubes had lower glycogen levels under basal conditions. In addition, these engineered cells showed more extensive glycogenolysis in response to both adrenaline, which stimulates glycogen phosphorylase phosphorylation, and carbonyl cyanide m-chlorophenylhydrazone, a metabolic uncoupler. In conclusion, transfer of the muscle glycogen phosphorylase cDNA into myotubes confers an enhanced and regulatable glycogenolytic capacity. Thus this system might be useful for delivery of muscle glycogen phosphorylase and restoration of glycogenolysis in muscle cells from patients with muscle phosphorylase deficiency (McArdle's disease). Images Figure 1 Figure 2 Figure 5 PMID:7818463

  14. Role of submaximal exercise in promoting creatine and glycogen accumulation in human skeletal muscle.

    PubMed

    Robinson, T M; Sewell, D A; Hultman, E; Greenhaff, P L

    1999-08-01

    We examined the effect of glycogen-depleting exercise on subsequent muscle total creatine (TCr) accumulation and glycogen resynthesis during postexercise periods when the diet was supplemented with carbohydrate (CHO) or creatine (Cr) + CHO. Fourteen subjects performed one-legged cycling exercise to exhaustion. Muscle biopsies were taken from the exhausted (Ex) and nonexhausted (Nex) limbs after exercise and after 6 h and 5 days of recovery, during which CHO (CHO group, n = 7) or Cr + CHO (Cr+CHO group, n = 7) supplements were ingested. Muscle TCr concentration ([TCr]) was unchanged in both groups 6 h after supplementation commenced but had increased in the Ex (P < 0.001) and Nex limbs (P < 0.05) of the Cr+CHO group after 5 days. Greater TCr accumulation was achieved in the Ex limbs (P < 0.01) of this group. Glycogen was increased above nonexercised concentrations in the Ex limbs of both groups after 5 days, with the concentration being greater in the Cr+CHO group (P = 0.06). Thus a single bout of exercise enhanced muscle Cr accumulation, and this effect was restricted to the exercised muscle. However, exercise also diminished CHO-mediated insulin release, which may have attenuated insulin-mediated muscle Cr accumulation. Ingesting Cr with CHO also augmented glycogen supercompensation in the exercised muscle. PMID:10444618

  15. Creatine supplementation spares muscle glycogen during high intensity intermittent exercise in rats

    PubMed Central

    2010-01-01

    Background The effects of creatine (CR) supplementation on glycogen content are still debatable. Thus, due to the current lack of clarity, we investigated the effects of CR supplementation on muscle glycogen content after high intensity intermittent exercise in rats. Methods First, the animals were submitted to a high intensity intermittent maximal swimming exercise protocol to ensure that CR-supplementation was able to delay fatigue (experiment 1). Then, the CR-mediated glycogen sparing effect was examined using a high intensity intermittent sub-maximal exercise test (fixed number of bouts; six bouts of 30-second duration interspersed by two-minute rest interval) (experiment 2). For both experiments, male Wistar rats were given either CR supplementation or placebo (Pl) for 5 days. Results As expected, CR-supplemented animals were able to exercise for a significant higher number of bouts than Pl. Experiment 2 revealed a higher gastrocnemius glycogen content for the CR vs. the Pl group (33.59%). Additionally, CR animals presented lower blood lactate concentrations throughout the intermittent exercise bouts compared to Pl. No difference was found between groups in soleus glycogen content. Conclusion The major finding of this study is that CR supplementation was able to spare muscle glycogen during a high intensity intermittent exercise in rats. PMID:20205834

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

    PubMed

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

    2014-01-01

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

  17. Carbohydrate supplementation during prolonged cycling exercise spares muscle glycogen but does not affect intramyocellular lipid use

    PubMed Central

    Boon, Hanneke; Gijsen, Annemie P.; Stegen, Jos H. C. H.; Kuipers, Harm; van Loon, Luc J. C.

    2007-01-01

    Using contemporary stable-isotope methodology and fluorescence microscopy, we assessed the impact of carbohydrate supplementation on whole-body and fiber-type-specific intramyocellular triacylglycerol (IMTG) and glycogen use during prolonged endurance exercise. Ten endurance-trained male subjects were studied twice during 3 h of cycling at 63 ± 4% of maximal O2 uptake with either glucose ingestion (CHO trial; 0.7 g CHO kg−1 h−1) or without (CON placebo trial; water only). Continuous infusions with [U-13C] palmitate and [6,6-2H2] glucose were applied to quantify plasma free fatty acids (FFA) and glucose oxidation rates and to estimate intramyocellular lipid and glycogen use. Before and after exercise, muscle biopsy samples were taken to quantify fiber-type-specific IMTG and glycogen content. Plasma glucose rate of appearance (Ra) and carbohydrate oxidation rates were substantially greater in the CHO vs CON trial. Carbohydrate supplementation resulted in a lower muscle glycogen use during the first hour of exercise in the CHO vs CON trial, resulting in a 38 ± 19 and 57 ± 22% decreased utilization in type I and II muscle-fiber glycogen content, respectively. In the CHO trial, both plasma FFA Ra and subsequent plasma FFA concentrations were lower, resulting in a 34 ± 12% reduction in plasma FFA oxidation rates during exercise (P < 0.05). Carbohydrate intake did not augment IMTG utilization, as fluorescence microscopy revealed a 76 ± 21 and 78 ± 22% reduction in type I muscle-fiber lipid content in the CHO and CON trial, respectively. We conclude that carbohydrate supplementation during prolonged cycling exercise does not modulate IMTG use but spares muscle glycogen use during the initial stages of exercise in endurance-trained men. PMID:17333244

  18. Effect of exercise-diet manipulation on muscle glycogen and its subsequent utilization during performance.

    PubMed

    Sherman, W M; Costill, D L; Fink, W J; Miller, J M

    1981-05-01

    This study examined the effect of three exercise-diet regimens on muscle glycogen supercompensation and subsequent performance during a 20.9-km run. A diet containing 15% carbohydrate (CHO,L), 50% CHO (M), or 70% (CHO (H) was arranged in three trials as follows: trial A = 3 days L, 3 days H; trial B = 3 days M, 3 days H; trial C = 6 days M. For each trial a 5-day depletion-taper exercise sequence was conducted on the treadmill at 73% VO2 max. The runs were 90, 40, 40, 20, and 20 min, respectively. A day of rest preceded the 20.9-km performance run. Muscle biopsies were obtained from the gastrocnemius on days 4 and 7 (both prior to and after the performance run). Trials A, B, and C elevated muscle glycogen to 207, 203, and 159 mmol glucosyl units/kg wet tissue (mmG), respectively. The performance run in both trials A and B utilized significantly more glycogen than in trial C: 5.0 and 5.1 mmG/km vs. 3.1 mmG/km. There were, however, no differences in either performance run times or post-performance run glycogen levels between the trials. These data demonstrate that (1) muscle glycogen can be elevated to high levels with a moderate exercise-diet regimen; (2) initial muscle glycogen levels influence the amount subsequently utilized during exercise; (3) carbohydrate loading is of no benefit to performance for trained runners during a 20.9-km run. PMID:7333741

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

    PubMed

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

    2000-12-01

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

  20. Effects of caffeine on muscle glycogen utilization and the neuroendocrine axis during exercise.

    PubMed

    Laurent, D; Schneider, K E; Prusaczyk, W K; Franklin, C; Vogel, S M; Krssak, M; Petersen, K F; Goforth, H W; Shulman, G I

    2000-06-01

    To examine the effect of caffeine ingestion on muscle glycogen utilization and the neuroendocrine axis during exercise, we studied 20 muscle glycogen-loaded subjects who were given placebo or caffeine (6 mg/kg) in a double blinded fashion 90 min before cycling for 2 h at 65% of their maximal oxygen consumption. Exercise-induced glycogen depletion in the thigh muscle was noninvasively measured by means of 13C nuclear magnetic resonance spectroscopy (NMR) spectroscopy, and plasma concentrations of substrates and neuroendocrine hormones, including beta-endorphins, were also assessed. Muscle glycogen content was increased 140% above normal values on the caffeine trial day (P < 0.001). After cycling for 2 h, caffeine ingestion was associated with a greater increase in plasma lactate (caffeine: +1.0 +/- 0.2 mmol/L; placebo, +0.1 +/- 0.2 mmol/L; P < 0.005), epinephrine (caffeine, +223 +/- 82 pg/mL; placebo, +56 +/- 26 pg/mL; P < 0.05), and cortisol (caffeine, +12 +/- 3 mg/mL; placebo, +2 +/- 2 mg/mL; P < 0.001) levels. However, plasma free fatty acid concentrations increased (caffeine, +814 +/- 133 mmol/L; placebo, +785 +/- 85 mmol/L; P = NS), and muscle glycogen content decreased (caffeine, -57 +/- 6 mmol/L muscle; placebo, -53 +/- 5 mmol/L muscle; P = NS) to the same extent in both groups. At the same time, plasma beta-endorphin levels almost doubled (from 30 +/- 5 to 53 +/- 13 pg/mL; P < 0.05) in the caffeine-treated group, whereas no change occurred in the placebo group. We conclude that caffeine ingestion 90 min before prolonged exercise does not exert a muscle glycogen-sparing effect in athletes with high muscle glycogen content. However, these data suggest that caffeine lowers the threshold for exercise-induced beta-endorphin and cortisol release, which may contribute to the reported benefits of caffeine on exercise endurance. PMID:10852448

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

    PubMed Central

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

    2008-01-01

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

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

    PubMed

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

    2016-07-15

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

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

    Maixner, Dylan Warren; Weng, Han-Rong

    2013-01-01

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

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

    PubMed

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

    2004-10-01

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

  6. 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

  7. 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

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  13. Effect of muscle glycogen on glucose, lactate and amino acid metabolism during exercise and recovery in human subjects

    PubMed Central

    Blomstrand, Eva; Saltin, Bengt

    1999-01-01

    Eight subjects performed two-legged exercise, one leg with low and the other with normal muscle glycogen content. The purpose was to study the effect of low initial muscle glycogen content on the metabolic response during 1 h of exercise and 2 h of recovery. This model allows direct comparison of net fluxes of substrates and metabolites over the exercising legs receiving the same arterial inflow.Muscle glycogen breakdown during exercise was 60% lower in the leg with a reduced pre-exercise glycogen concentration and the rate of glucose uptake during exercise was 30% higher.The amount of pyruvate that was oxidized during exercise was calculated to be approximately 450 mmol in the low-glycogen leg and 750 mmol in the normal-glycogen leg, which suggests more fat and amino acid oxidation in the low-glycogen leg.During exercise, there was a significant release of amino acids not metabolized in the muscle, e.g. tyrosine and phenylalanine, only from the low-glycogen leg, suggesting an increased rate of net protein degradation in this leg.The release of tyrosine and phenylalanine from the low-glycogen leg during the exercise period and the change in their muscle concentrations yield a net tyrosine and phenylalanine production rate of 1.4 and 1.5 mmol h−1, respectively. The net rate of protein degradation was then calculated to be 7–12 g h−1.The results suggest that the observed differences in metabolism between the low-glycogen and the normal-glycogen leg are induced by the glycogen level per se, since the legs received the same arterial supply of hormones and substrates. PMID:9831734

  14. Muscle glycogen levels and blood metabolites in reindeer (Rangifer tarandus tarandus L.) after transport and lairage.

    PubMed

    Wiklund, E; Andersson, A; Malmfors, G; Lundström, K

    1996-01-01

    A total of 66 reindeer cows and calves were included in a study on the effects of supplementary feeding, transport and lairage on muscle glycogen content, ultimate pH and blood metabolite values. Thirty reindeer (10 not transported, 20 transported 800 km) received no supplementary feed (groups A-C), another 30 animals (10 not transported, 20 transported 1000 km) were given a supplementary reindeer feed mixture 2 months prior to slaughter (groups D-F) and six animals, which had been part of a feeding experiment, were fed for 5 months and slaughtered at the research unit. Glycogen determinations and pH measurements were made in m. longissimus, m. biceps femoris and m. triceps brachii. Blood samples were collected at slaughter and muscle samples were taken 30 min after slaughter. Ultimate pH was measured 30 hr post mortem. The glycogen content in the muscles of groups A-C was very low (100-200 mmol/kg). In groups D-G, the glycogen content was equivalent to normal beef muscle values (300-500 mmol/kg). The values of the blood metabolites urea and creatinine, both of which could indicate protein catabolism caused by stress, were significantly (p < 0.05) higher in animals not having received supplemental feed (groups A-C). Alkaline phosphatase values were significantly (p < 0.05) higher in supplemental fed animals (groups D-G), indicating that their nutritional status was good. Total protein values were significantly (p < 0.05) higher in groups A, D, E and F compared to the other groups. Lorry transport did not significantly (p > 0.05) reduce the muscle glycogen content. Lairage (groups C and F) showed no negative effect on the parameters examined. These results suggest that the animals' physical condition and nutritional status have a considerable effect on their ability to tolerate various stress factors, such as lorry transport and lairage. PMID:22060679

  15. The effect of high-intensity intermittent swimming on post-exercise glycogen supercompensation in rat skeletal muscle.

    PubMed

    Sano, Akiko; Koshinaka, Keiichi; Abe, Natsuki; Morifuji, Masashi; Koga, Jinichiro; Kawasaki, Emi; Kawanaka, Kentaro

    2012-01-01

    A single bout of prolonged endurance exercise stimulates glucose transport in skeletal muscles, leading to post-exercise muscle glycogen supercompensation if sufficient carbohydrate is provided after the cessation of exercise. Although we recently found that short-term sprint interval exercise also stimulates muscle glucose transport, the effect of this type of exercise on glycogen supercompensation is uncertain. Therefore, we compared the extent of muscle glycogen accumulation in response to carbohydrate feeding following sprint interval exercise with that following endurance exercise. In this study, 16-h-fasted rats underwent a bout of high-intensity intermittent swimming (HIS) as a model of sprint interval exercise or low-intensity prolonged swimming (LIS) as a model of endurance exercise. During HIS, the rats swam for eight 20-s sessions while burdened with a weight equal to 18% of their body weight. The LIS rats swam with no load for 3 h. The exercised rats were then refed for 4, 8, 12, or 16 h. Glycogen levels were almost depleted in the epitrochlearis muscles of HIS- or LIS-exercised rats immediately after the cessation of exercise. A rapid increase in muscle glycogen levels occurred during 4 h of refeeding, and glycogen levels had peaked at the end of 8 h of refeeding in each group of exercised refed rats. The peak glycogen levels during refeeding were not different between HIS- and LIS-exercised refed rats. Furthermore, although a large accumulation of muscle glycogen in response to carbohydrate refeeding is known to be associated with decreased insulin responsiveness of glucose transport, and despite the fact that muscle glycogen supercompensation was observed in the muscles of our exercised rats at the end of 4 h of refeeding, insulin responsiveness was not decreased in the muscles of either HIS- or LIS-exercised refed rats compared with non-exercised fasted control rats at this time point. These results suggest that sprint interval exercise

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

    PubMed

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

    2013-10-01

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

  17. Enhanced Glycogen Storage of a Subcellular Hot Spot in Human Skeletal Muscle during Early Recovery from Eccentric Contractions.

    PubMed

    Nielsen, Joachim; Farup, Jean; Rahbek, Stine Klejs; de Paoli, Frank Vincenzo; Vissing, Kristian

    2015-01-01

    Unaccustomed eccentric exercise is accompanied by muscle damage and impaired glucose uptake and glycogen synthesis during subsequent recovery. Recently, it was shown that the role and regulation of glycogen in skeletal muscle are dependent on its subcellular localization, and that glycogen synthesis, as described by the product of glycogen particle size and number, is dependent on the time course of recovery after exercise and carbohydrate availability. In the present study, we investigated the subcellular distribution of glycogen in fibers with high (type I) and low (type II) mitochondrial content during post-exercise recovery from eccentric contractions. Analysis was completed on five male subjects performing an exercise bout consisting of 15 x 10 maximal eccentric contractions. Carbohydrate-rich drinks were subsequently ingested throughout a 48 h recovery period and muscle biopsies for analysis included time points 3, 24 and 48 h post exercise from the exercising leg, whereas biopsies corresponding to prior to and at 48 h after the exercise bout were collected from the non-exercising, control leg. Quantitative imaging by transmission electron microscopy revealed an early (post 3 and 24 h) enhanced storage of intramyofibrillar glycogen (defined as glycogen particles located within the myofibrils) of type I fibers, which was associated with an increase in the number of particles. In contrast, late in recovery (post 48 h), intermyofibrillar, intramyofibrillar and subsarcolemmal glycogen in both type I and II fibers were lower in the exercise leg compared with the control leg, and this was associated with a smaller size of the glycogen particles. We conclude that in the carbohydrate-supplemented state, the effect of eccentric contractions on glycogen metabolism depends on the subcellular localization, muscle fiber's oxidative capacity, and the time course of recovery. The early enhanced storage of intramyofibrillar glycogen after the eccentric contractions may

  18. Enhanced Glycogen Storage of a Subcellular Hot Spot in Human Skeletal Muscle during Early Recovery from Eccentric Contractions

    PubMed Central

    Nielsen, Joachim; Farup, Jean; Rahbek, Stine Klejs; de Paoli, Frank Vincenzo; Vissing, Kristian

    2015-01-01

    Unaccustomed eccentric exercise is accompanied by muscle damage and impaired glucose uptake and glycogen synthesis during subsequent recovery. Recently, it was shown that the role and regulation of glycogen in skeletal muscle are dependent on its subcellular localization, and that glycogen synthesis, as described by the product of glycogen particle size and number, is dependent on the time course of recovery after exercise and carbohydrate availability. In the present study, we investigated the subcellular distribution of glycogen in fibers with high (type I) and low (type II) mitochondrial content during post-exercise recovery from eccentric contractions. Analysis was completed on five male subjects performing an exercise bout consisting of 15 x 10 maximal eccentric contractions. Carbohydrate-rich drinks were subsequently ingested throughout a 48 h recovery period and muscle biopsies for analysis included time points 3, 24 and 48 h post exercise from the exercising leg, whereas biopsies corresponding to prior to and at 48 h after the exercise bout were collected from the non-exercising, control leg. Quantitative imaging by transmission electron microscopy revealed an early (post 3 and 24 h) enhanced storage of intramyofibrillar glycogen (defined as glycogen particles located within the myofibrils) of type I fibers, which was associated with an increase in the number of particles. In contrast, late in recovery (post 48 h), intermyofibrillar, intramyofibrillar and subsarcolemmal glycogen in both type I and II fibers were lower in the exercise leg compared with the control leg, and this was associated with a smaller size of the glycogen particles. We conclude that in the carbohydrate-supplemented state, the effect of eccentric contractions on glycogen metabolism depends on the subcellular localization, muscle fiber’s oxidative capacity, and the time course of recovery. The early enhanced storage of intramyofibrillar glycogen after the eccentric contractions may

  19. Oral conjugated linoleic acid supplementation enhanced glycogen resynthesis in exercised human skeletal muscle.

    PubMed

    Tsao, Jung-Piao; Liao, Su-Fen; Korivi, Mallikarjuna; Hou, Chien-Wen; Kuo, Chia-Hua; Wang, Hsueh-Fang; Cheng, I-Shiung

    2015-01-01

    Present study examined the effects of conjugated linoleic acid (CLA) supplementation on glycogen resynthesis in exercised human skeletal muscle. Twelve male participants completed a cross-over trial with CLA (3.8 g/day for 8 week) or placebo supplements by separation of 8 weeks. CLA is a mixture of trans-10 cis-12 and cis-9 trans-11 isomers (50:50). On experiment day, all participants performed 60-min cycling exercise at 75% VO2 max, then consumed a carbohydrate meal immediately after exercise and recovered for 3 h. Biopsied muscle samples from vastus lateralis were obtained immediately (0 h) and 3 h following exercise. Simultaneously, blood and gaseous samples were collected for every 30 min during 3-h recovery. Results showed significantly increased muscle glycogen content with CLA after a single bout of exercise (P < 0.05). Muscle glucose transporter type 4 expression was significantly elevated immediately after exercise, and this elevation was continued until 3 h after exercise in CLA trial. However, P-Akt/Akt ratio was not significantly altered, while glucose tolerance was impaired with CLA. Gaseous exchange data showed no beneficial effect of CLA on fat oxidation, instead lower non-esterified fatty acid and glycerol levels were found at 0 h. Our findings conclude that CLA supplementation can enhance the glycogen resynthesis rate in exercised human skeletal muscle. PMID:25385360

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

    PubMed

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

    2003-12-01

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

  1. 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

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

    PubMed

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

    2011-06-30

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

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

    PubMed

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

    2010-07-01

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

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

    PubMed Central

    Jope, Richard Scott

    2011-01-01

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

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

    PubMed

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

    2015-11-15

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

  7. 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

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

    PubMed

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

    2015-09-01

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

  9. Carbohydrate ingestion and glycogen utilization in different muscle fibre types in man.

    PubMed Central

    Tsintzas, O K; Williams, C; Boobis, L; Greenhaff, P

    1995-01-01

    1. The effect of carbohydrate (CHO) ingestion on muscle glycogen utilization during exercise was examined on seven male subjects completing two 60 min treadmill runs at 70% maximum oxygen uptake (VO2,max), 1 week apart. On each occasion the subjects consumed either water or a 5.5% CHO-electrolyte solution immediately before and during exercise. Muscle samples were obtained from the vastus lateralis by needle biopsy before and immediately after exercise. Venous blood samples were also collected from an ante-cubital vein at rest and at 10, 20, 40 and 60 min into the run. 2. Higher blood glucose concentrations (P < 0.01) were observed throughout the run during the CHO trial compared with the water trial. Serum insulin concentration was only higher after 20 min of exercise (P < 0.01). 3. A 28% reduction in mixed glycogen utilization was observed as a result of CHO ingestion when compared with water ingestion (108.7 +/- 16.3 vs. 150.9 +/- 19.9 mmol (kg dry matter)-1, respectively; P < 0.01). 4. The ingestion of the CHO solution resulted in sparing of glycogen in type I (slow twitch) fibres only (38 +/- 7% degradation of glycogen as opposed to 66 +/- 3% during the water trial; P = 0.01). PMID:8583408

  10. Glucosidic pathways of glycogen breakdown and glucose production by muscle from postexercised frogs.

    PubMed

    Fournier, P A; Guderley, H

    1993-11-01

    Muscle and body glucose in frogs increases markedly during the initial hour of recovery after strenuous exercise. The liver is not the major source responsible for this accumulation. This is indicated by the stability of liver glycogen levels after exercise and by the observation that hepatectomized and normal frogs accumulate similar amounts of glucose in their muscles and body during recovery. The renal contribution cannot account for this increase in body glucose. Most of the glucose that accumulates in the body after exercise has a muscular origin, as indicated by the facts that two-thirds of the body glucose is found in muscle and that the intracellular levels of muscle glucose are much higher than those of the plasma. The glucose that accumulates outside muscle may also have a muscular origin. The glucosidic pathways of glycogen breakdown are the only metabolic avenue with sufficient capacity to account for the amount of glucose accumulated in muscle during the first hour of recovery. These results indicate that the ability of an isolated preparation of frog muscle to liberate glucose during recovery from exercise (Fournier et al. J. Biol. Chem. 267: 8234-8238, 1992) is not an artifactual metabolic curiosity but rather a metabolic reality that takes place in vivo. Glucose accumulation during recovery is thought to facilitate the metabolic transition of frog carbohydrate metabolism from a catabolic state, characteristic of exercise, to an anabolic one. PMID:8238616

  11. Correction of glycogen storage disease type II by an adeno-associated virus vector containing a muscle-specific promoter.

    PubMed

    Sun, Baodong; Zhang, Haoyue; Franco, Luis M; Brown, Talmage; Bird, Andrew; Schneider, Ayn; Koeberl, Dwight D

    2005-06-01

    Glycogen storage disease type II (Pompe disease) causes death in infancy from cardiorespiratory failure due to acid alpha-glucosidase (GAA; acid maltase) deficiency. An AAV2 vector pseudotyped as AAV6 (AAV2/6 vector) transiently expressed high-level human GAA in GAA-knockout (GAA-KO) mice without reducing glycogen storage; however, in immunodeficient GAA-KO/SCID mice the AAV2/6 vector expressed high-level GAA and reduced the glycogen content of the injected muscle for 24 weeks. A CD4+/CD8+ lymphocytic infiltrate was observed in response to the AAV2/6 vector in immunocompetent GAA-KO mice. When a muscle-specific creatine kinase promoter was substituted for the CB promoter (AAV-MCKhGAApA), that AAV2/6 vector expressed high-level GAA and reduced glycogen content in immunocompetent GAA-KO mice. Muscle-restricted expression of hGAA provoked only a humoral (not cellular) immune response. Intravenous administration of a high number of particles of AAV-MCKhGAApA as AAV2/7 reduced the glycogen content of the heart and skeletal muscle and corrected individual myofibers in immunocompetent GAA-KO mice 24 weeks postinjection. In summary, persistent correction of muscle glycogen content was achieved with an AAV vector containing a muscle-specific promoter in GAA-KO mice, and this approach should be considered for muscle-targeted gene therapy in Pompe disease. PMID:15922959

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

    PubMed

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

    2004-07-01

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

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed

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

    2012-08-01

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

  16. Muscle protein and glycogen responses to recovery from hypogravity and unloading by tail-cast suspension

    NASA Technical Reports Server (NTRS)

    Henriksen, E. J.; Tischler, M. E.; Jacob, S.; Cook, P. H.

    1985-01-01

    Previous studies in this laboratory using the tail-bast hindlimb suspension model have shown that there are specific changes in protein and carbohydrate metabolism in the soleus muscle due to unloading. For example, 6 days of unloading caused a 27% decrease in mass and a 60% increase in glycogen content in the soleus muscle, while the extensor digitorum longus muscle was unaffected. Also, fresh tissue tyrosine and its in vitro release from the muscle are increased in the unloaded soleus, indicating that this condition causes a more negative protein balance. With these results in mind, studies to investigate the effect of hypogravity on protein and carbohydrate metabolism in a number of rat hindlimb muscles were carried out.

  17. Muscle protein and glycogen responses to recovery from hypogravity and unloading by tail-cast suspension

    NASA Technical Reports Server (NTRS)

    Henriksen, E. J.; Tischler, M. E.; Jacob, S.; Cook, P. H.

    1985-01-01

    Previous studies in this laboratory using the tail-bast hindlimb suspension model have shown that there are specific changes in protein and carbohydrate metabolism in the soleus muscle due to unloading. For example, 6 days of unloading caused a 27 percent decrease in mass and a 60 percent increse in glycogen content in the soleus muscle, while the extensor digitorum longus muscle was unaffected. Also, fresh tissue tyrosine and its in vitro release from the muscle are increased in the unloaded soleus, indicating that this condition causes a more negative protein balance. With these results in mind, studies to investigate the effect of hypogravity on protein and carbohydrate metabolism in a number of rat hindlimb muscles were carried out.

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

    PubMed

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

    2012-02-01

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

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

    PubMed Central

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

    2009-01-01

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

  20. Hyperthermia, but not muscle water deficit, increases glycogen use during intense exercise.

    PubMed

    Fernández-Elías, V E; Hamouti, N; Ortega, J F; Mora-Rodríguez, R

    2015-06-01

    We determined if dehydration alone or in combination with hyperthermia accelerates muscle glycogen use during intense exercise. Seven endurance-trained cyclists (VO2max  = 54.4 ± 1.05 mL/kg/min) dehydrated 4.6% of body mass (BM) during exercise in the heat (150 min at 33 ± 1 °C, 25 ± 2% humidity). During recovery (4 h), subjects remained dehydrated (HYPO trial) or recovered all fluid losses (REH trials). Finally, subjects exercised intensely (75% VO2max ) for 40 min in a neutral (25 ± 1 °C; HYPO and REH trials) or in a hot environment (36 ± 1 °C; REHHOT ). Before the final exercise bout vastus lateralis glycogen concentration was similar in all three trials (434 ± 57 mmol/kg of dry muscle (dm)) but muscle water content was lower in the HYPO (357 ± 14 mL/100 g dm) than in REH trials (389 ± 25 and 386 ± 25 mL/100 g dm; P < 0.05). After 40 min of intense exercise, intestinal temperature was similar between the HYPO and REHHOT trials (39.2 ± 0.5 and 39.2 ± 0.4 °C, respectively) and glycogen use was similar (172 ± 86 and 185 ± 97 mmol/kg dm, respectively) despite large differences in muscle water content. In contrast, during REH, intestinal temperature (38.5 ± 0.4 °C) and glycogen use (117 ± 52 mmol/kg dm) were significantly lower than during HYPO and REHHOT . Our data suggest that hyperthermia stimulates glycogen use during intense exercise while muscle water deficit has a minor role. PMID:25943663

  1. Beta-endorphin infusion during exercise in rats does not alter hepatic or muscle glycogen.

    PubMed

    Jamurtas, A Z; Goldfarb, A H; Chung, S C; Hegde, S; Marino, C; Fatouros, I G

    2001-12-01

    The aim of this study was to determine whether beta-endorphin infusion influences liver or muscle glycogen concentration during exercise. Thirty-two rats (Harlan Co., IN, USA) with a body mass of 265-290 g were assigned at random to four groups, each of eight rats: (1) beta-endorphin infusion for 90 min at rest; (2) beta-endorphin infusion for 90 min while running on a rodent treadmill at 22 m x min(-1) and 0% grade; (3) saline infusion (0.9% NaCl) for 90 min at rest; and (4) saline infusion for 90 min while running on a rodent treadmill at 22 m x min(-1) and 0% grade. Beta-endorphin infusion elevated plasma beta-endorphin concentration by 2.5-fold at rest compared with saline infusion at rest, and by two-fold after exercise compared with saline infusion after exercise. Beta-endorphin infusion attenuated exercise-induced glucose concentration but did not alter the fasting hepatic glycogen concentration at rest or after exercise compared with saline infusion. Fasting hepatic glycogen decreased significantly as a result of 90 min of exercise independent of treatment. Deep intermedius muscle glycogen concentration at rest was similar after 90 min of both beta-endorphin and saline infusion and decreased significantly as a result of 90 min of exercise independent of treatment. Our results suggest that liver and muscle glycogenolysis is not responsible for the differences in plasma glucose with beta-endorphin infusion during exercise. PMID:11820687

  2. 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.

  3. Effects of glucose on contractile function, [Ca2+]i, and glycogen in isolated mouse skeletal muscle.

    PubMed

    Helander, Ingrid; Westerblad, Håkan; Katz, Abram

    2002-06-01

    Extensor digitorum longus muscles were stimulated to contract to fatigue and allowed to recover for 2 h in the absence or presence of 5.5 or 11 mM extracellular glucose. This was followed by a second fatigue run, which ended when the absolute force was the same as at the end of the first run. During the first fatigue run, the fluorescence ratio for indo 1 increased [reflecting an increase in myoplasmic free Ca2+ concentration ([Ca2+]i)] during the initial tetani, peaking at approximately 115% of the first tetanic value, followed by a continuous decrease to approximately 90% at fatigue. During the first fatigue run, myofibrillar Ca2+ sensitivity was significantly decreased. During the second run, the number of tetani was 57 +/- 6% of initial force in muscles that recovered in the absence of glucose and 110 +/- 6 and 119 +/- 2% of initial force in muscles that recovered in 5.5 and 11 mM glucose, respectively. Fluorescence ratios during the first, peak, and last tetani did not differ significantly between the first and second fatigue runs during any of the three conditions. Glycogen decreased by almost 50% during the first fatigue run and did not change further after recovery in the absence of glucose. After recovery in the presence of 5.5 and 11 mM glucose, glycogen increased 32 and 42% above the nonstimulated control value (P < 0.01). These data demonstrate that extracellular glucose delays the decrease of tetanic force and [Ca2+]i during fatiguing stimulation and that glycogen supercompensation following contraction can occur in the absence of insulin. PMID:11997245

  4. TFEB overexpression promotes glycogen clearance of Pompe disease iPSC-derived skeletal muscle.

    PubMed

    Sato, Yohei; Kobayashi, Hiroshi; Higuchi, Takashi; Shimada, Yohta; Ida, Hiroyuki; Ohashi, Toya

    2016-01-01

    Pompe disease (PD) is a lysosomal disorder caused by acid α-glucosidase (GAA) deficiency. Progressive muscular weakness is the major symptom of PD, and enzyme replacement therapy can improve the clinical outcome. However, to achieve a better clinical outcome, alternative therapeutic strategies are being investigated, including gene therapy and pharmacological chaperones. We previously used lentiviral vector-mediated GAA gene transfer in PD patient-specific induced pluripotent stem cells. Some therapeutic efficacy was observed, although glycogen accumulation was not normalized. Transcription factor EB is a master regulator of lysosomal biogenesis and autophagy that has recently been associated with muscular pathology, and is now a potential therapeutic target in PD model mice. Here, we differentiated skeletal muscle from PD patient-specific induced pluripotent stem cells by forced MyoD expression. Lentiviral vector-mediated GAA and transcription factor EB gene transfer independently improved GAA enzyme activity and reduced glycogen content in skeletal muscle derived from PD-induced pluripotent stem cells. Interestingly, GAA and transcription factor EB cooperatively improved skeletal muscle pathology, both biochemically and morphologically. Thus, our findings show that abnormal lysosomal biogenesis is associated with the muscular pathology of PD, and transcription factor EB gene transfer is effective as an add-on strategy to GAA gene transfer. PMID:27556060

  5. TFEB overexpression promotes glycogen clearance of Pompe disease iPSC-derived skeletal muscle

    PubMed Central

    Sato, Yohei; Kobayashi, Hiroshi; Higuchi, Takashi; Shimada, Yohta; Ida, Hiroyuki; Ohashi, Toya

    2016-01-01

    Pompe disease (PD) is a lysosomal disorder caused by acid α-glucosidase (GAA) deficiency. Progressive muscular weakness is the major symptom of PD, and enzyme replacement therapy can improve the clinical outcome. However, to achieve a better clinical outcome, alternative therapeutic strategies are being investigated, including gene therapy and pharmacological chaperones. We previously used lentiviral vector-mediated GAA gene transfer in PD patient-specific induced pluripotent stem cells. Some therapeutic efficacy was observed, although glycogen accumulation was not normalized. Transcription factor EB is a master regulator of lysosomal biogenesis and autophagy that has recently been associated with muscular pathology, and is now a potential therapeutic target in PD model mice. Here, we differentiated skeletal muscle from PD patient-specific induced pluripotent stem cells by forced MyoD expression. Lentiviral vector-mediated GAA and transcription factor EB gene transfer independently improved GAA enzyme activity and reduced glycogen content in skeletal muscle derived from PD-induced pluripotent stem cells. Interestingly, GAA and transcription factor EB cooperatively improved skeletal muscle pathology, both biochemically and morphologically. Thus, our findings show that abnormal lysosomal biogenesis is associated with the muscular pathology of PD, and transcription factor EB gene transfer is effective as an add-on strategy to GAA gene transfer. PMID:27556060

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

    PubMed

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

    2016-08-15

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

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

    PubMed

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

    2014-01-01

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

  8. 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.

  9. Role of NADH/NAD+ transport activity and glycogen store on skeletal muscle energy metabolism during exercise: in silico studies

    PubMed Central

    Li, Yanjun; Dash, Ranjan K.; Kim, Jaeyeon; Saidel, Gerald M.; Cabrera, Marco E.

    2009-01-01

    Skeletal muscle can maintain ATP concentration constant during the transition from rest to exercise, whereas metabolic reaction rates may increase substantially. Among the key regulatory factors of skeletal muscle energy metabolism during exercise, the dynamics of cytosolic and mitochondrial NADH and NAD+ have not been characterized. To quantify these regulatory factors, we have developed a physiologically based computational model of skeletal muscle energy metabolism. This model integrates transport and reaction fluxes in distinct capillary, cytosolic, and mitochondrial domains and investigates the roles of mitochondrial NADH/NAD+ transport (shuttling) activity and muscle glycogen concentration (stores) during moderate intensity exercise (60% maximal O2 consumption). The underlying hypothesis is that the cytosolic redox state (NADH/NAD+) is much more sensitive to a metabolic disturbance in contracting skeletal muscle than the mitochondrial redox state. This hypothesis was tested by simulating the dynamic metabolic responses of skeletal muscle to exercise while altering the transport rate of reducing equivalents (NADH and NAD+) between cytosol and mitochondria and muscle glycogen stores. Simulations with optimal parameter estimates showed good agreement with the available experimental data from muscle biopsies in human subjects. Compared with these simulations, a 20% increase (or ∼20% decrease) in mitochondrial NADH/NAD+ shuttling activity led to an ∼70% decrease (or ∼3-fold increase) in cytosolic redox state and an ∼35% decrease (or ∼25% increase) in muscle lactate level. Doubling (or halving) muscle glycogen concentration resulted in an ∼50% increase (or ∼35% decrease) in cytosolic redox state and an ∼30% increase (or ∼25% decrease) in muscle lactate concentration. In both cases, changes in mitochondrial redox state were minimal. In conclusion, the model simulations of exercise response are consistent with the hypothesis that mitochondrial NADH

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

    PubMed Central

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

    2014-01-01

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

  11. Immunological detection of degradation intermediates of skeletal-muscle glycogen phosphorylase in vitro and in vivo.

    PubMed Central

    Cookson, E J; Flannery, A V; Cidlowski, J A; Beynon, R J

    1992-01-01

    Over 95% of the pyridoxal phosphate (PLP) in skeletal is bound to one protein, glycogen phosphorylase. This, and the fact that phosphorylase constitutes approx. 5% of the soluble protein in skeletal muscle, introduce the possibility that PLP might be used as a specific label to identify degradation intermediates of the enzyme. In this investigation, we have developed immunological methods, using a monoclonal antibody to PLP and polyclonal antibodies to phosphorylase, to detect degradation intermediates in vitro and in vivo. We have identified a family of degradation intermediates of glycogen phosphorylase in the high-speed-supernatant fraction of mouse skeletal muscle. These peptides react with both types of antibodies and are in the size and concentration range expected for degradation intermediates in a model in which the committed step is followed by rapid clearance of the products. Changes in amounts of degradation intermediates are examined in physiological or pathological conditions in which the rate of degradation of phosphorylase is altered. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 PMID:1445274

  12. Hindlimb muscle fibre size and glycogen stores in bank voles with increased aerobic exercise metabolism.

    PubMed

    Jaromin, Ewa; Wyszkowska, Julia; Labecka, Anna Maria; Sadowska, Edyta Teresa; Koteja, Paweł

    2016-02-01

    To test hypotheses concerning physiological factors limiting the rate of aerobic exercise metabolism, we used a unique experimental evolution model: lines of bank voles selected for high swim-induced aerobic metabolism (A) and unselected, control lines (C). We investigated putative adaptations that result in the increased performance of the hindlimb muscle (gastrocnemius joined with plantaris). The body mass-adjusted muscle mass was higher in A-lines (0.093 g) than in C-lines (0.083 g; P=0.01). However, selection did not affect mean muscle fibre cross-sectional area (P=0.34) or glycogen content assessed with a histochemical periodic acid-Schiff reaction (PAS; P=0.82). The results suggest that the increased aerobic performance is achieved by an increase of total muscle mass, without major qualitative changes in the muscle fibre architecture. However, such a conclusion should be treated with caution, because other modifications, such as increased density of capillaries or mitochondria, could occur. PMID:26685167

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

    PubMed

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

    2016-07-01

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

  14. The impact of beef cattle temperament assessed using flight speed on muscle glycogen, muscle lactate and plasma lactate concentrations at slaughter.

    PubMed

    Coombes, S V; Gardner, G E; Pethick, D W; McGilchrist, P

    2014-12-01

    This study evaluated the effect of animal temperament measured using flight speed (FS) on plasma lactate, muscle glycogen and lactate concentrations at slaughter plus ultimate pH in 648 lot finished cattle of mixed breed and sex. Muscle samples were collected at slaughter from the m. semimembranosus, m. semitendinosus and m. longissimus thoracis (LT) for analysis of glycogen and lactate concentration. Blood was collected after exsanguination and analysed for plasma lactate concentration and ultimate pH of the LT was measured. FS had no effect on muscle glycogen concentration in any muscle or ultimate pH of the LT (P>0.05). As FS increased from 1 to 5m/s, plasma and muscle lactate concentration increased by 54% and 11.4% (P<0.01). The mechanisms through which temperament contributes to variation in glycogen metabolism remain unclear. The risk of dark cutting was not impacted by temperament, indicating that other production and genetic factors have a greater impact on the incidence of dark cutting. PMID:25170817

  15. Mastoparan binds to glycogen phosphorylase to regulate sarcoplasmic reticular Ca2+ release in skeletal muscle.

    PubMed Central

    Hirata, Yutaka; Atsumi, Masanori; Ohizumi, Yasushi; Nakahata, Norimichi

    2003-01-01

    The ryanodine receptor, a Ca(2+)-releasing channel in sarcoplasmic reticulum (SR), plays an important role in the excitation-contraction coupling of skeletal muscle. In a previous study [Hirata, Nakahata and Ohizumi (2000) Mol. Pharmacol. 57, 1235-1242], we reported that mastoparan caused Ca(2+) release through ryanodine receptor from the heavy fraction of SR (HSR) isolated from rabbit skeletal muscle, and that it specifically bound to a 97 kDa protein which was distinct from Ca(2+)-pump or triadin. The present study was undertaken to identify and characterize the 97 kDa mastoparan-binding protein. The 97 kDa protein was purified from solubilized HSR by DEAE-Sepharose column chromatography and preparative SDS/PAGE. The partial amino acid sequence of the purified 97 kDa protein was matched with that of glycogen phosphorylase (GP). The proteolytic cleavage pattern of the 97 kDa protein was identical with that of GP. Furthermore, [(125)I-Tyr(3)]mastoparan specifically bound to GP. Interestingly, mastoparan-induced Ca(2+) release was inhibited by exogenous addition of GP-a, and mastoparan dissociated GP from HSR. These results indicate that the 97 kDa mastoparan-binding protein is GP, which negatively regulates Ca(2+) release from HSR. There may be a functional cross-talk between Ca(2+) release from HSR and glycogenolysis for energy supply mediated through GP in skeletal muscles. PMID:12519071

  16. 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

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

    SciTech Connect

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

    1986-05-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    SciTech Connect

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

    2010-01-01

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

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

    PubMed

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

    2010-01-01

    Patients with alveolar rhabdomyosarcoma (ARMS) have poorer response to conventional chemotherapy and lower survival rates than those with embryonal RMS (ERMS). To identify compounds that preferentially block the growth of ARMS, we conducted a small-scale screen of 160 kinase inhibitors against the ARMS cell line Rh30 and ERMS cell line RD and identified inhibitors of glycogen synthase kinase 3 (GSK3), including TWS119 as ARMS-selective inhibitors. GSK3 inhibitors inhibited cell proliferation and induced apoptosis more effectively in Rh30 than RD cells. Ectopic expression of fusion protein PAX3-FKHR in RD cells significantly increased their sensitivity to TWS119. Down-regulation of GSK3 by GSK3 inhibitors or siRNA significantly reduced the transcriptional activity of PAX3-FKHR. These results suggest that GSK3 is directly involved in regulating the transcriptional activity of PAX3-FKHR. Also, GSK3 phosphorylated PAX3-FKHR in vitro, suggesting that GSK3 might regulate PAX3-FKHR activity via phosphorylation. These findings support a novel mechanism of PAX3-FKHR regulation by GSK3 and provide a novel strategy to develop GSK inhibitors as anti-ARMS therapies. PMID:19995556

  1. 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.

  2. 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

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

    PubMed

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

    2014-08-01

    Glycogen synthase kinase 3 (GSK-3) is associated with several cellular systems, including immune response. Lithium, a widely used pharmacological treatment for bipolar disorder, is a GSK-3 inhibitor. GSK-3α is the predominant isoform in human neutrophils. In this study, we examined the effect of GSK-3 inhibition on the production of TNF-α by neutrophils. In the murine air pouch model of inflammation, lithium chloride (LiCl) amplified TNF-α release. In lipopolysaccharide-stimulated human neutrophils, GSK-3 inhibitors mimicked the effect of LiCl, each potentiating TNF-α release after 4 h, in a concentration-dependent fashion, by up to a 3-fold increase (ED50 of 1 mM for lithium). LiCl had no significant effect on cell viability. A positive association was revealed between GSK-3 inhibition and prolonged activation of the p38/MNK1/eIF4E pathway of mRNA translation. Using lysine and arginine labeled with stable heavy isotopes followed by quantitative mass spectrometry, we determined that GSK-3 inhibition markedly increases (by more than 3-fold) de novo TNF-α protein synthesis. Our findings shed light on a novel mechanism of control of TNF-α expression in neutrophils with GSK-3 regulating mRNA translation and raise the possibility that lithium could be having a hitherto unforeseen effect on inflammatory diseases. PMID:24803542

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

    PubMed Central

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

    1997-01-01

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

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

    PubMed Central

    Sun, Ye

    2014-01-01

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

  6. 5-imino-1,2,4-thiadiazoles: first small molecules as substrate competitive inhibitors of glycogen synthase kinase 3.

    PubMed

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

    2012-02-23

    Cumulative evidence strongly supports that glycogen synthase kinase-3 (GSK-3) is a pathogenic molecule when it is up-dysregulated, emerging as an important therapeutic target in severe unmet human diseases. GSK-3 specific inhibitors might be promising effective drugs for the treatment of devastating pathologies such as neurodegenerative diseases, stroke, and mood disorders. As GSK-3 has the ability to phosphorylate primed substrates, small molecules able to bind to this site should be perfect drug candidates, able to partially block the activity of the enzyme over some specific substrates. Here, we report substituted 5-imino-1,2,4-thiadiazoles as the first small molecules able to inhibit GSK-3 in a substrate competitive manner. These compounds are cell permeable, able to decrease inflammatory activation and to selectively differentiate neural stem cells. Overall, 5-imino-1,2,4-thiadiazoles are presented here as new molecules able to decrease neuronal cell death and to increase endogenous neurogenesis blocking the GSK-3 substrate site. PMID:22257026

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

    PubMed Central

    SA, Perrine; JS, Miller; EM, Unterwald

    2008-01-01

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

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

    PubMed Central

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

    2005-01-01

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

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

    PubMed

    Eldar-Finkelman, Hagit

    2006-04-01

    Dr Hagit Eldar-Finkelman (Sackler School of Medicine, Israel) was interviewed by Emma Quigley (Commissioning Editor, Expert Opinion on Therapeutic Targets) on 16th February 2006. Born in Jerusalem, Dr Eldar-Finkelman received her BSc in Chemistry in 1984 and both her MSc in Physical Chemistry (1986) and PhD in Life Science (1993) from the Weizmann Institute of Science. She was a recipient of the British Council Award, which allowed her to conduct research in biological nuclear magnetic resonance at the University of Oxford in the laboratory of Professor George K Radda. Following postdoctoral work at the School of Medicine of the University of Washington with Nobel Laureate Professor Edwin G Krebs, she became an Assistant Professor in the Department of Medicine at Harvard Medical School. Dr Eldar-Finkelman joined the Sackler School of Medicine at Tel Aviv University in 1999. Dr Eldar-Finkelman's research focuses on the molecular mechanisms regulating the protein kinase glycogen synthase kinase-3 (GSK-3), and their implications in negative regulation of signalling pathways. In particular, her work aims to develop specific inhibitors for GSK-3 and to test their functions in vitro and in vivo, considering the concept that such inhibitors may be useful in insulin resistance and Type 2 diabetes. These studies provide a conceptual basis for development of GSK-3 inhibitors and may lead to design of small molecules for treatment of diabetes and or neurodegenerative disorders. PMID:16548769

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

    PubMed Central

    Landis, Justine; Shaw, Leslie M.

    2014-01-01

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

  11. 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.

  12. 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

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed

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

    2016-03-01

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

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

    PubMed Central

    Yoshino, Yuki; Ishioka, Chikashi

    2015-01-01

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

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

    PubMed

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

    2016-06-30

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

  17. Role of insulin on exercise-induced GLUT-4 protein expression and glycogen supercompensation in rat skeletal muscle.

    PubMed

    Kuo, Chia-Hua; Hwang, Hyonson; Lee, Man-Cheong; Castle, Arthur L; Ivy, John L

    2004-02-01

    The purpose of this study was to investigate the role of insulin on skeletal muscle GLUT-4 protein expression and glycogen storage after postexercise carbohydrate supplementation. Male Sprague-Dawley rats were randomly assigned to one of six treatment groups: sedentary control (Con), Con with streptozocin (Stz/C), immediately postexercise (Ex0), Ex0 with Stz (Stz/Ex0), 5-h postexercise (Ex5), and Ex5 with Stz (Stz/Ex5). Rats were exercised by swimming (2 bouts of 3 h) and carbohydrate supplemented immediately after each exercise session by glucose intubation (1 ml of a 50% wt/vol). Stz was administered 72-h before exercise, which resulted in hyperglycemia and elimination of the insulin response to the carbohydrate supplement. GLUT-4 protein of Ex0 rats was 30% above Con in fast-twitch (FT) red and 21% above Con in FT white muscle. In Ex5, GLUT-4 protein was 52% above Con in FT red and 47% above Con in FT white muscle. Muscle glycogen in FT red and white muscle was also increased above Con in Ex5 rats. Neither GLUT-4 protein nor muscle glycogen was increased above Con in Stz/Ex0 or Stz/Ex5 rats. GLUT-4 mRNA in FT red muscle of Ex0 rats was 61% above Con but only 33% above Con in Ex5 rats. GLUT-4 mRNA in FT red muscle of Stz/C and Stz/Ex0 rats was similar but significantly elevated in Ex5/Stz rats. These results suggest that insulin is essential for the increase in GLUT-4 protein expression following postexercise carbohydrate supplementation. PMID:14555686

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

    PubMed Central

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

    2007-01-01

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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    1997-01-01

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

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

    PubMed

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

    2009-03-01

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

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

    PubMed

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

    2003-08-01

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

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

    PubMed

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

    2016-08-01

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

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

    PubMed

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

    2016-02-01

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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2012-01-01

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

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

    PubMed

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

    2016-09-01

    To explore the effect of glycogen synthase kinase 3β (GSK-3β) silencing on Tau-5 phosphorylation in mice suffering Alzheimer disease (AD). GSK-3β was firstly silenced in human neuroblastoma SH-SY5Y cells using special lentivirus (LV) and the content of Tau (A-12), p-Tau (Ser396) and p-Tau (PHF-6) proteins. GSK-3β was also silenced in APP/PS1 mouse model of AD mice, which were divided into three groups (n = 10): AD, vehicle, and LV group. Ten C57 mice were used as control. The memory ability of mice was tested by square water maze, and the morphological changes of hippocampus and neuron death were analyzed by haematoxylin-eosin staining. Moreover, the levels of Tau and phosphorylated Tau (p-Tau) were detected by western blotting and immunohistochemistry, respectively. The lentivirus-mediated GSK-3β silencing system was successfully developed and silencing GSK-3β at the cellular level reduced Tau phosphorylation obviously. Moreover, GSK-3β silence significantly improved the memory ability of AD mice in LV group compared with AD group (P < 0.05) according to the latency periods and error numbers. As for the hippocampus morphology and neuron death, no significant change was observed between LV group and normal control. Immunohistochemical detection and western blotting revealed that the levels of Tau and p-Tau were significantly down-regulated after GSK-3β silence. Silencing GSK-3β may have a positive effect on inhibiting the pathologic progression of AD through down-regulating the level of p-Tau. PMID:27255602

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    2006-01-01

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

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

    PubMed Central

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

    2014-01-01

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

  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. 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

  14. Investigation and management of the hepatic glycogen storage diseases.

    PubMed

    Bhattacharya, Kaustuv

    2015-07-01

    The glycogen storage diseases (GSD) comprise a group of disorders that involve the disruption of metabolism of glycogen. Glycogen is stored in various organs including skeletal muscle, the kidneys and liver. The liver stores glycogen to supply the rest of the body with glucose when required. Therefore, disruption of this process can lead to hypoglycaemia. If glycogen is not broken down effectively, this can lead to hepatomegaly. Glycogen synthase deficiency leads to impaired glycogen synthesis and consequently the liver is small. Glycogen brancher deficiency can lead to abnormal glycogen being stored in the liver leading to a quite different disorder of progressive liver dysfunction. Understanding the physiology of GSD I, III, VI and IX guides dietary treatments and the provision of appropriate amounts and types of carbohydrates. There has been recent re-emergence in the literature of the use of ketones in therapy, either in the form of the salt D,L-3-hydroxybutyrate or medium chain triglyceride (MCT). High protein diets have also been advocated. Alternative waxy maize based starches seem to show promising early data of efficacy. There are many complications of each of these disorders and they need to be prospectively surveyed and managed. Liver and kidney transplantation is still indicated in severe refractory disease. PMID:26835382

  15. The role of blood glucose in the restoration of muscle glycogen during recovery from exhaustive exercise in rainbow trout (Oncorhynchus mykiss) and winter flounder (Pseudopleuronectes americanus).

    PubMed

    Pagnotta, A; Milligan, C L

    1991-11-01

    The role of blood-borne glucose in the restoration of white muscle glycogen following exhaustive exercise in the active, pelagic rainbow trout (Oncorhynchus mykiss) and the more sluggish, benthic winter flounder (Pseudopleuronectes americanus) were examined. During recovery from exhaustive exercise, the animals were injected with a bolus of universally labelled [14C]glucose via dorsal aortic (trout) or caudal artery (flounder) catheters. The bulk of the injected label (50-70%) remained as glucose in the extracellular fluid in both species. The major metabolic fates of the injected glucose were oxidation to CO2 (6-8%) and production of lactate (6-8%), the latter indicative of continued anaerobic metabolism post-exercise. Oxidation of labelled glucose could account for up to 40% and 15% of the post-exercise MO2 in trout and flounder, respectively. Exhaustive exercise resulted in a reduction of muscle glycogen stores and accumulation of muscle lactate. Glycogen restoration in trout began 2-4h after exercise, whereas in flounder, glycogen restoration began within 2h. Despite a significant labelling of the intramuscular glucose pool, less than 1% of the infused labelled glucose was incorporated into muscle glycogen. This suggests that blood-borne glucose does not contribute significantly to the restoration of muscle glycogen following exhaustive exercise in either trout or flounder and provides further evidence against a prominent role for the Cori cycle in these species. PMID:1757776

  16. Nitric oxide synthase inhibition reduces muscle inflammation and necrosis in modified muscle use

    NASA Technical Reports Server (NTRS)

    Pizza, F. X.; Hernandez, I. J.; Tidball, J. G.

    1998-01-01

    The objective of this study was to determine the role of nitric oxide in muscle inflammation, fiber necrosis, and apoptosis of inflammatory cells in vivo. The effects of nitric oxide synthase (NOS) inhibition on the concentrations of neutrophils, ED1+ and ED2+ macrophages, apoptotic inflammatory cells, and necrotic muscle fibers in rats subjected to 10 days of hindlimb unloading and 2 days of reloading were determined. Administration of NOS inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME) significantly reduced the concentrations of neutrophils, ED1+ and ED2+ macrophages, and necrotic fibers in soleus muscle relative to water-treated controls. The concentration of apoptotic inflammatory cells was also significantly lower for L-NAME-treated animals compared with water-treated controls. However, the proportion of the inflammatory cell population that was apoptotic did not differ between L-NAME-treated and control animals, suggesting that L-NAME treatment did not decrease inflammatory cell populations by increasing the frequency of apoptosis. Thus, nitric oxide or one of its intermediates promotes muscle inflammation and fiber necrosis during modified muscle use and plays no more than a minor role in the resolution of muscle inflammation by inducing apoptosis of inflammatory cells.

  17. 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.

  18. Creatine ingestion augments dietary carbohydrate mediated muscle glycogen supercompensation during the initial 24 h of recovery following prolonged exhaustive exercise in humans.

    PubMed

    Roberts, Paul A; Fox, John; Peirce, Nicholas; Jones, Simon W; Casey, Anna; Greenhaff, Paul L

    2016-08-01

    Muscle glycogen availability can limit endurance exercise performance. We previously demonstrated 5 days of creatine (Cr) and carbohydrate (CHO) ingestion augmented post-exercise muscle glycogen storage compared to CHO feeding alone in healthy volunteers. Here, we aimed to characterise the time-course of this Cr-induced response under more stringent and controlled experimental conditions and identify potential mechanisms underpinning this phenomenon. Fourteen healthy, male volunteers cycled to exhaustion at 70 % VO2peak. Muscle biopsies were obtained at rest immediately post-exercise and after 1, 3 and 6 days of recovery, during which Cr or placebo supplements (20 g day(-1)) were ingested along with a prescribed high CHO diet (37.5 kcal kg body mass(-1) day(-1), >80 % calories CHO). Oral-glucose tolerance tests (oral-GTT) were performed pre-exercise and after 1, 3 and 6 days of Cr and placebo supplementation. Exercise depleted muscle glycogen content to the same extent in both treatment groups. Creatine supplementation increased muscle total-Cr, free-Cr and phosphocreatine (PCr) content above placebo following 1, 3 and 6 days of supplementation (all P < 0.05). Creatine supplementation also increased muscle glycogen content noticeably above placebo after 1 day of supplementation (P < 0.05), which was sustained thereafter. This study confirmed dietary Cr augments post-exercise muscle glycogen super-compensation, and demonstrates this occurred during the initial 24 h of post-exercise recovery (when muscle total-Cr had increased by <10 %). This marked response ensued without apparent treatment differences in muscle insulin sensitivity (oral-GTT, muscle GLUT4 mRNA), osmotic stress (muscle c-fos and HSP72 mRNA) or muscle cell volume (muscle water content) responses, such that another mechanism must be causative. PMID:27193231

  19. Segmental extracellular and intracellular water distribution and muscle glycogen after 72-h carbohydrate loading using spectroscopic techniques.

    PubMed

    Shiose, Keisuke; Yamada, Yosuke; Motonaga, Keiko; Sagayama, Hiroyuki; Higaki, Yasuki; Tanaka, Hiroaki; Takahashi, Hideyuki

    2016-07-01

    Body water content increases during carbohydrate loading because 2.7-4-g water binds each 1 g of glycogen. Bioelectrical impedance spectroscopy (BIS) allows separate assessment of extracellular and intracellular water (ECW and ICW, respectively) in the whole body and each body segment. However, BIS has not been shown to detect changes in body water induced by carbohydrate loading. Here, we aimed to investigate whether BIS had sufficient sensitivity to detect changes in body water content and to determine segmental water distribution after carbohydrate loading. Eight subjects consumed a high-carbohydrate diet containing 12 g carbohydrates·kg body mass(-1)·day(-1) for 72 h after glycogen depletion cycling exercise. Changes in muscle glycogen concentration were measured by (13)C-magnetic resonance spectroscopy, and total body water (TBW) was measured by the deuterium dilution technique (TBWD2O). ICW and ECW in the whole body (wrist-to-ankle) and in each body segment (arm, trunk, and leg) were assessed by BIS. Muscle glycogen concentration [72.7 ± 10.0 (SD) to 169.4 ± 55.9 mmol/kg wet wt, P < 0.001] and TBWD2O (39.3 ± 3.2 to 40.2 ± 3.0 kg, P < 0.05) increased significantly 72 h after exercise compared with baseline, respectively. Whole-body BIS showed significant increases in ICW (P < 0.05), but not in ECW. Segmental BIS showed significant increases in ICW in the legs (P < 0.05), but not in the arms or trunk. Our results suggest that increase in body water after carbohydrate loading can be detected by BIS and is caused by segment-specific increases in ICW. PMID:27231310

  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. PMID:19458083

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

    PubMed Central

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

    2009-01-01

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

  2. When phosphorylated at Thr148, the β2-subunit of AMP-activated kinase does not associate with glycogen in skeletal muscle.

    PubMed

    Xu, Hongyang; Frankenberg, Noni T; Lamb, Graham D; Gooley, Paul R; Stapleton, David I; Murphy, Robyn M

    2016-07-01

    The 5'-AMP-activated protein kinase (AMPK), a heterotrimeric complex that functions as an intracellular fuel sensor that affects metabolism, is activated in skeletal muscle in response to exercise and utilization of stored energy. The diffusibility properties of α- and β-AMPK were examined in isolated skeletal muscle fiber segments dissected from rat fast-twitch extensor digitorum longus and oxidative soleus muscles from which the surface membranes were removed by mechanical dissection. After the muscle segments were washed for 1 and 10 min, ∼60% and 75%, respectively, of the total AMPK pools were found in the diffusible fraction. After in vitro stimulation of the muscle, which resulted in an ∼80% decline in maximal force, 20% of the diffusible pool became bound in the fiber. This bound pool was not associated with glycogen, as determined by addition of a wash step containing amylase. Stimulation of extensor digitorum longus muscles resulted in 28% glycogen utilization and a 40% increase in phosphorylation of the downstream AMPK target acetyl carboxylase-CoA. This, however, had no effect on the proportion of total β2-AMPK that was phosphorylated in whole muscle homogenates measured by immunoprecipitation. These findings suggest that, in rat skeletal muscle, β2-AMPK is not associated with glycogen and that activation of AMPK by muscle contraction does not dephosphorylate β2-AMPK. These findings question the physiological relevance of the carbohydrate-binding function of β2-AMPK in skeletal muscle. PMID:27099349

  3. An Optimized Histochemical Method to Assess Skeletal Muscle Glycogen and Lipid Stores Reveals Two Metabolically Distinct Populations of Type I Muscle Fibers

    PubMed Central

    Prats, Clara; Gomez-Cabello, Alba; Nordby, Pernille; Andersen, Jesper L.; Helge, Jørn W.; Dela, Flemming; Baba, Otto; Ploug, Thorkil

    2013-01-01

    Skeletal muscle energy metabolism has been a research focus of physiologists for more than a century. Yet, how the use of intramuscular carbohydrate and lipid energy stores are coordinated during different types of exercise remains a subject of debate. Controversy arises from contradicting data from numerous studies, which used different methodological approaches. Here we review the “pros and cons” of previously used histochemical methods and describe an optimized method to ensure the preservation and specificity of detection of both intramyocellular carbohydrate and lipid stores. For optimal preservation of muscle energy stores, air drying cryosections or cycles of freezing-thawing need to be avoided. Furthermore, optimization of the imaging settings in order to specifically image intracellular lipid droplets stained with oil red O or Bodipy-493/503 is shown. When co-staining lipid droplets with associated proteins, Bodipy-493/503 should be the dye of choice, since oil red O creates precipitates on the lipid droplets blocking the light. In order to increase the specificity of glycogen stain, an antibody against glycogen is used. The resulting method reveals the existence of two metabolically distinct myosin heavy chain I expressing fibers: I-1 fibers have a smaller crossectional area, a higher density of lipid droplets, and a tendency to lower glycogen content compared to I-2 fibers. Type I-2 fibers have similar lipid content than IIA. Exhaustive exercise lead to glycogen depletion in type IIA and IIX fibers, a reduction in lipid droplets density in both type I-1 and I-2 fibers, and a decrease in the size of lipid droplets exclusively in type I-1 fibers. PMID:24204959

  4. Effects of PCB on the adrenergic response in perfused gills and on levels of muscle glycogen in rainbow trout (Salmo gairdneri Rich. )

    SciTech Connect

    Kiessling, A; Paert, P.; Ring, O; Lindahl-Keesling, K.

    1983-12-01

    Alterations in behavior have been observed in PCB exposed fish. The response to external disturbance seems to be less evident in the PCB exposed fish than in unexposed ones. Furthermore, the levels of muscle glycogen are reported to be higher in fish exposed to PCB. The question arose if these PCB related effects on fish were the result of an interaction between PCB and the stress system. Response to stress in fish is either mediated by neurons direct to the target organ or by catecholamines released to the blood circulation. The gills are sensitive to circulating adrenaline, increasing the oxygen uptake. Adrenaline also participates in mobilization of muscle glycogen. The purpose of this study was to find out whether PCB influences the adrenaline response in gills and/or glycogen storage in muscle.

  5. Ingestion of glucose or sucrose prevents liver but not muscle glycogen depletion during prolonged endurance-type exercise in trained cyclists.

    PubMed

    Gonzalez, Javier T; Fuchs, Cas J; Smith, Fiona E; Thelwall, Pete E; Taylor, Roy; Stevenson, Emma J; Trenell, Michael I; Cermak, Naomi M; van Loon, Luc J C

    2015-12-15

    The purpose of this study was to define the effect of glucose ingestion compared with sucrose ingestion on liver and muscle glycogen depletion during prolonged endurance-type exercise. Fourteen cyclists completed two 3-h bouts of cycling at 50% of peak power output while ingesting either glucose or sucrose at a rate of 1.7 g/min (102 g/h). Four cyclists performed an additional third test for reference in which only water was consumed. We employed (13)C magnetic resonance spectroscopy to determine liver and muscle glycogen concentrations before and after exercise. Expired breath was sampled during exercise to estimate whole body substrate use. After glucose and sucrose ingestion, liver glycogen levels did not show a significant decline after exercise (from 325 ± 168 to 345 ± 205 and 321 ± 177 to 348 ± 170 mmol/l, respectively; P > 0.05), with no differences between treatments. Muscle glycogen concentrations declined (from 101 ± 49 to 60 ± 34 and 114 ± 48 to 67 ± 34 mmol/l, respectively; P < 0.05), with no differences between treatments. Whole body carbohydrate utilization was greater with sucrose (2.03 ± 0.43 g/min) vs. glucose (1.66 ± 0.36 g/min; P < 0.05) ingestion. Both liver (from 454 ± 33 to 283 ± 82 mmol/l; P < 0.05) and muscle (from 111 ± 46 to 67 ± 31 mmol/l; P < 0.01) glycogen concentrations declined during exercise when only water was ingested. Both glucose and sucrose ingestion prevent liver glycogen depletion during prolonged endurance-type exercise. Sucrose ingestion does not preserve liver glycogen concentrations more than glucose ingestion. However, sucrose ingestion does increase whole body carbohydrate utilization compared with glucose ingestion. This trial was registered at https://www.clinicaltrials.gov as NCT02110836. PMID:26487008

  6. [Dependence of creatine kinase and glycogen synthetase activities of skeletal muscles on state of adenine nucleotide phosphorylation and cAMP metabolism].

    PubMed

    Iakovlev, N N; Chagovets, N R; Maksimova, L V

    1980-01-01

    Changes in the contents of adenine nucleotides, creatine phosphate, inorganic phosphate, creatine, glucose-6-phosphate and glycogen and the activity of adenylate cyclase, creatine kinase, glycogen phosphorylase 31:51-AMP-phosphodiesterase and glycogen synthetase in muscles and of blood catecholamines were studied in adult rats before loading, immediately after the cessation of the muscular activity, and at rest. Adenine nucleotides are established to play a regulatory role in catabolic and anabolic processes nucleotides are established to play a regulatory role in catabolic and anabolic processes related to the muscular activity. It is established that compensation and supercompensation of the working losses of muscular creatine phosphate and glycogen are due to activation of anabolic processes under conditions of higher phosphorylation of the adenylic system. PMID:6247797

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

    PubMed

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

    2015-08-14

    Cancer cell invasion is a critical phenomenon in cancer pathogenesis. Glycogen synthase kinase-3β (GSK-3β) has been reported to regulate cancer cell invasion both negatively and positively. Thus, the net effect of GSK-3β on invasion is unclear. In this report, we showed that GSK-3β inhibitors induced dysregulation of the actin cytoskeleton and functional insufficiency of focal adhesion, which resulted in suppressed invasion. In addition, WAVE2, an essential molecule for actin fibre branching, was down-regulated after GSK-3β inhibition. Collectively, we propose that the WAVE2-actin cytoskeleton axis is an important target of GSK-3β inhibitors in cancer cell invasion. PMID:26116771

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

    PubMed

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

    2016-08-01

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

  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. Effects in skeletal muscle.

    PubMed

    Young, Andrew

    2005-01-01

    The first biological action of amylin to be described was the inhibition of insulin-stimulated incorporation of radiolabeled glucose into glycogen in the isolated soleus muscle of the rat. This antagonism of insulin action in muscle was non-competitive, occurring with equal potency and efficacy at all insulin concentrations. Amylin inhibited activation of glycogen synthase, partially accounting for the inhibition of radiolabeled glucose incorporation. However, this did not account for a low rate of labeling at higher amylin concentrations, wherein the radioglycogen accumulation was even less than in incubations where insulin was absent. The principal action of amylin accounting for reduction of insulin-stimulated accumulation of glycogen was activation of glycogen phosphorylase via a cyclic AMP-, protein kinase C-dependent signaling pathway to cause glycogenolysis (glycogen breakdown). At physiological concentrations, amylin activated glycogen phosphorylase at its ED50, but because glycogen phosphorylase is present in such high activity, the resulting flux out of glycogen was estimated to be similar to insulin-mediated flux of glucosyl moieties into glycogen. Thus, in the rat, endogenous amylin secreted in response to meals appeared to mobilize carbon from skeletal muscle. Amylin-induced glycogenolysis resulted in intramuscular accumulation of glucose-6-phosphate and release of lactate from tissue beds that included muscle. When muscle glycogen was pre-labeled with tritium in the three position, amylin could be shown to evoke the release of free glucose. This is made possible by glucosyl moieties cleaved at the branch points in glycogen being released as free glucose, rather than being phosphorylated, as occurs with the bulk of the glycogen glucosyls. Free glucose is free to exit cells via facilitated transport, down a concentration gradient that might exist under such circumstances. When measured by a sensitive technique utilizing efflux of labeled glucose, amylin

  11. Increased dietary carbohydrate and endurance during single-leg cycling using a limb with normal muscle glycogen concentration.

    PubMed

    Hardman, A E; Williams, C

    1989-01-01

    The purpose of this study was to test the hypothesis that increased availability of blood-borne glucose would improve endurance after carbohydrate loading. A single-leg exercise model was employed, taking advantage of the fact that supercompensation of muscle glycogen occurs only in a previously exercised limb. Endurance time to exhaustion at 70% of maximal oxygen uptake (VO2 max) was determined for 11 males and three females who were then allocated to a control group or a high-carbohydrate (CHO) group. For 3 days following Test 1 the control group maintained a prescribed normal diet whilst the CHO group increased the proportion of energy derived from carbohydrate (62.1 +/- 4.3% cf. 43.9 +/- 2.0%, P less than 0.01). The endurance test was then repeated using the leg that was inactive during Test 1. Endurance time was increased on Test 2 (123.7 +/- 43.2 min cf. 98.5 +/- 21.9 min, P less than 0.05 one-tailed test) for the CHO group but not for the control group (101.8 +/- 21.7 min cf. 107.5 +/- 9.1 min, NS). There was no indication of enhanced carbohydrate metabolism during Test 2 for the CHO group but mean heart rate was lower during Test 2 than during Test 1 (145 +/- 14 beat min-1 cf. 152 +/- 12 beat min-1, P less than 0.05). These results suggest that the prior consumption of a high-carbohydrate diet improves endurance during high-intensity cycling with a limb with normal muscle glycogen concentration. PMID:2681820

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

    PubMed

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

    2015-04-01

    Glycogen synthase kinase 3 (GSK-3) is a serine-threonine kinase discovered decades ago to have an important role in glycogen metabolism. Today, we know that this kinase is involved in the regulation of many cell functions, including insulin signaling, specification of cell fate during embryonic development, and the control of cell division and apoptosis. Insulin and TDZD-8 (4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione) are inhibitors of GSK-3β that have been shown to possess organ-protective effects in inflammatory-mediated organ injury models. We aimed to evaluate the cytoprotective effect of GSK-3β inhibition on rat models of liver ischemia-reperfusion and thermal injury. In the liver ischemia-reperfusion model, TDZD-8 and insulin were administered at 5 mg/kg (i.v.) and 1.4 IU/kg (i.v.), respectively, 30 min before induction of ischemia and led to the significant reduction of the serum concentration of aspartate aminotransferase, alanine aminotransferase, γ-glutamyltransferase, and lactate dehydrogenase. Beneficial effects were found to be independent from blood glucose levels. In the thermal injury model, TDZD-8 was administered at 5 mg/kg (i.v.) 5 min before induction of injury and significantly reduced multiple organ dysfunction markers (liver, neuromuscular, and lung). In the lung, TDZD-8 reduced the histological signs of tissue injury, inflammatory markers (cytokines), and neutrophil chemotaxis/infiltration; reduced GSK-3β, nuclear factor-κB, and Akt activation; reduced caspase-3 and metalloproteinase-9 activation. Our study provides a new insight on the beneficial effects of GSK-3β inhibition on systemic inflammation and further elucidates the mechanism and pathway crosstalks by which TDZD-8 reduces the multiple organ injury elicited by thermal injury. PMID:25394244

  13. Adjunctive β2-agonist treatment reduces glycogen independently of receptor-mediated acid α-glucosidase uptake in the limb muscles of mice with Pompe disease

    PubMed Central

    Farah, Benjamin L.; Madden, Lauran; Li, Songtao; Nance, Sierra; Bird, Andrew; Bursac, Nenad; Yen, Paul M.; Young, Sarah P.; Koeberl, Dwight D.

    2014-01-01

    Enzyme or gene replacement therapy with acid α-glucosidase (GAA) has achieved only partial efficacy in Pompe disease. We evaluated the effect of adjunctive clenbuterol treatment on cation-independent mannose-6-phosphate receptor (CI-MPR)-mediated uptake and intracellular trafficking of GAA during muscle-specific GAA expression with an adeno-associated virus (AAV) vector in GAA-knockout (KO) mice. Clenbuterol, which increases expression of CI-MPR in muscle, was administered with the AAV vector. This combination therapy increased latency during rotarod and wirehang testing at 12 wk, in comparison with vector alone. The mean urinary glucose tetrasaccharide (Glc4), a urinary biomarker, was lower in GAA-KO mice following combination therapy, compared with vector alone. Similarly, glycogen content was lower in cardiac and skeletal muscle following 12 wk of combination therapy in heart, quadriceps, diaphragm, and soleus, compared with vector alone. These data suggested that clenbuterol treatment enhanced trafficking of GAA to lysosomes, given that GAA was expressed within myofibers. The integral role of CI-MPR was demonstrated by the lack of effectiveness from clenbuterol in GAA-KO mice that lacked CI-MPR in muscle, where it failed to reverse the high glycogen content of the heart and diaphragm or impaired wirehang performance. However, the glycogen content of skeletal muscle was reduced by the addition of clenbuterol in the absence of CI-MPR, as was lysosomal vacuolation, which correlated with increased AKT signaling. In summary, β2-agonist treatment enhanced CI-MPR-mediated uptake and trafficking of GAA in mice with Pompe disease, and a similarly enhanced benefit might be expected in other lysosomal storage disorders.—Farah, B. L., Madden, L., Li, S., Nance, S., Bird, A., Bursac, N., Yen, P. M., Young, S. P., Koeberl, D. D. Adjunctive β2-agonist treatment reduces glycogen independently of receptor-mediated acid α-glucosidase uptake in the limb muscles of mice with

  14. Endothelial nitric oxide synthase (NOS) deficiency affects energy metabolism pattern in murine oxidative skeletal muscle.

    PubMed Central

    Momken, Iman; Fortin, Dominique; Serrurier, Bernard; Bigard, Xavier; Ventura-Clapier, Renée; Veksler, Vladimir

    2002-01-01

    Oxidative capacity of muscles correlates with capillary density and with microcirculation, which in turn depend on various regulatory factors, including NO generated by endothelial nitric oxide synthase (eNOS). To determine the role of eNOS in patterns of regulation of energy metabolism in various muscles, we studied mitochondrial respiration in situ in saponin-permeabilized fibres as well as the energy metabolism enzyme profile in the cardiac, soleus (oxidative) and gastrocnemius (glycolytic) muscles isolated from mice lacking eNOS (eNOS(-/-)). In soleus muscle, the absence of eNOS induced a marked decrease in both basal mitochondrial respiration without ADP (-32%; P <0.05) and maximal respiration in the presence of ADP (-29%; P <0.05). Furthermore, the eNOS(-/-) soleus muscle showed a decrease in total creatine kinase (-29%; P <0.05), citrate synthase (-31%; P <0.01), adenylate kinase (-27%; P <0.05), glyceraldehyde-3-phosphate dehydrogenase (-43%; P <0.01) and pyruvate kinase (-26%; P <0.05) activities. The percentage of myosin heavy chains I (slow isoform) was significantly increased from 24.3+/-1.5% in control to 30.1+/-1.1% in eNOS(-/-) soleus muscle ( P <0.05) at the expense of a slight non-significant decrease in the three other (fast) isoforms. Besides, eNOS(-/-) soleus showed a 28% loss of weight. Interestingly, we did not find differences in any parameters in cardiac and gastrocnemius muscles compared with respective controls. These results show that eNOS knockout has an important effect on muscle oxidative capacity as well on the activities of energy metabolism enzymes in oxidative (soleus) muscle. The absence of such effects in cardiac and glycolytic (gastrocnemius) muscle suggests a specific role for eNOS-produced NO in oxidative skeletal muscle. PMID:12123418

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

    PubMed Central

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

    2012-01-01

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

  16. 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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-12-18

    Glycogen synthase kinase-3β (GSK-3β) was immobilized on magnetic beads (MBs) by affinity method for the first time. The enzyme-immobilized MBs were coupled with high-performance liquid chromatography-ultraviolet (HPLC-UV) technique to establish a cost-effective and reliable method for screening of inhibitors of GSK-3β. A peptide substrate of GSK-3β containing a tyrosine residue was employed since it can be sensitively detected by UV detector at 214nm. The substrate and its phosphorylated product were separated by baseline within 10min. The enzyme activity was determined by the quantification of peak area of the product. Parameters including enzyme immobilization, enzyme reaction and the performance of immobilized-enzyme were investigated. The immobilized enzyme can be reused for 10 times and remain stable for 4 days at 4°C. The inhibitory activities of extracts of 15 traditional Chinese medicines (TCMs) were screened. As a result, three of them including Euonymus fortunei, Amygdalus communis and Garcinia xanthochymus were found possessing high inhibitory activities (inhibition rate >90%). From G. xanthochymus, a new inhibitor of GSK-3β, fukugetin, was discovered with an IC50 value of 3.18±0.07μM. Enzyme kinetics and molecular docking experiments further revealed the inhibitory mechanism, indicating fukugetin was a non-ATP competitive inhibitor interacting with the phosphate recognizing substrate binding site of GSK-3β. PMID:26610618

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

    PubMed Central

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

    2016-01-01

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

  1. 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.

  2. 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

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

    PubMed

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

    2015-08-01

    The proximity of an enzyme to its substrate can influence rate and magnitude of catalysis. A-kinase anchoring protein 220 (AKAP220) is a multivalent anchoring protein that can sequester a variety of signal transduction enzymes. These include protein kinase A (PKA) and glycogen synthase kinase 3β (GSK3β). Using a combination of molecular and cellular approaches we show that GSK3β phosphorylation of Thr-1132 on AKAP220 initiates recruitment of this kinase into the enzyme scaffold. We also find that AKAP220 anchors GSK3β and its substrate β-catenin in membrane ruffles. Interestingly, GSK3β can be released from the multienzyme complex in response to PKA phosphorylation on serine 9, which suppresses GSK3β activity. The signaling scaffold may enhance this regulatory mechanism, as AKAP220 has the capacity to anchor two PKA holoenzymes. Site 1 on AKAP220 (residues 610-623) preferentially interacts with RII, whereas site 2 (residues 1633-1646) exhibits a dual specificity for RI and RII. In vitro affinity measurements revealed that site 2 on AKAP220 binds RII with ∼10-fold higher affinity than site 1. Occupancy of both R subunit binding sites on AKAP220 could provide a mechanism to amplify local cAMP responses and enable cross-talk between PKA and GSK3β. PMID:26088133

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

    PubMed

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

    2016-09-01

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

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

    PubMed

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

    2016-08-01

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

  6. 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

  7. 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.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

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

    PubMed

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

    2016-02-16

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-07-01

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

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

    PubMed Central

    McAlpine, Cameron S.; Werstuck, Geoff H.

    2014-01-01

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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2016-01-01

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

  17. 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

  18. 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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2012-01-01

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

  2. Effect of acetic acid feeding on the circadian changes in glycogen and metabolites of glucose and lipid in liver and skeletal muscle of rats.

    PubMed

    Fushimi, Takashi; Sato, Yuzo

    2005-11-01

    The aim of the present study is to investigate the effect of acetic acid feeding on the circadian changes in glycogen concentration in liver and skeletal muscle. Rats were provided meal once daily (09.00-13.00 hours) for 10 d. On the 11th day, they were either killed immediately or given 9 g diet containing either 0 (control) or 0.7 g/kg-diet acetic acid beginning at 09.00 hours for 4 h, as in the previous regimen. Rats in the fed group were killed at 4, 8 or 24 h after the start of feeding. At 4 h after the start of feeding, the acetic acid group had significantly greater liver and gastrocnemius muscle glycogen concentrations (P<0.05). Also, at this same point, liver xylulose-5-phosphate, a key stimulator of glycolysis, the ratio of fructose-1,6-bisphosphate to fructose-6-phosphate in skeletal muscle, which reflects phosphofructokinase-1 activity, and liver malonyl-CoA, an allosteric inhibitor of carnitine palmitoyl-transferase, were significantly lower in the acetic acid group than in the control group (P<0.05). In addition, the acetic acid group had a significantly lower serum lactate concentration and lower ratio of insulin to glucagon than the control group at the same point (P<0.05). We conclude that a diet containing acetic acid may enhance glycogen repletion but not induce supercompensation, a large increase in the glycogen level that is beneficial in improving performance, in liver and skeletal muscle by transitory inhibition of glycolysis. Further, we indicate the possibility of a transient enhancement of fatty acid oxidation in liver by acetic acid feeding. PMID:16277773

  3. Effect of chronic supplementation with branched-chain amino acids on the performance and hepatic and muscle glycogen content in trained rats.

    PubMed

    de Araujo, Jonas A; Falavigna, Gina; Rogero, Marcelo M; Pires, Ivanir S O; Pedrosa, Rogerio G; Castro, Inar A; Donato, Jose; Tirapegui, Julio

    2006-08-29

    The objective of this study was to evaluate the effects of a diet supplemented with branched-chain amino acids (BCAA; 3.57% and 4.76%) on the performance and glycogen metabolism of trained rats. Thirty-six adult male Wistar rats received the control diet (AIN-93M) (n=12) and two diets supplemented with BCAA (S1: AIN-93M+3.57% BCAA, n=12, and S2: AIN-93M+4.76% BCAA, n=12) for 6 weeks. The training protocol consisted of bouts of swimming exercise (60 min day(-1)) for 6 weeks at intensities close to the lactate threshold. On the last day of the experiment, all groups were trained for 1 h (1H) or were submitted to the exhaustion test (EX). The time to exhaustion did not differ between groups. The groups submitted to the exhaustion test presented a reduction in plasma glucose and an increase in plasma ammonia and blood lactate concentrations compared to the 1H condition. In the 1H condition, hepatic glycogen concentration was significantly higher in group S2 compared to the control diet and S1 groups (132% and 44%, respectively). Group S2 in the 1H condition presented a higher muscle glycogen concentration (45%) compared to the control diet group. In the EX condition, a significantly higher hepatic glycogen concentration was observed for group S2 compared to the control diet and S1 groups (262% and 222%, respectively). Chronic supplementation with BCAA promoted a higher hepatic and muscle glycogen concentration in trained animals, with this effect being dose dependent. PMID:16698042

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

    PubMed Central

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

    2015-01-01

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

  5. A Splice Mutation in the PHKG1 Gene Causes High Glycogen Content and Low Meat Quality in Pig Skeletal Muscle

    PubMed Central

    Zhou, Lisheng; Ren, Jun; Liu, Xianxian; Zhang, Hui; Yang, Bin; Zhang, Zhiyan; Ma, Huanban; Xie, Xianhua; Xing, Yuyun; Guo, Yuanmei; Huang, Lusheng

    2014-01-01

    Glycolytic potential (GP) in skeletal muscle is economically important in the pig industry because of its effect on pork processing yield. We have previously mapped a major quantitative trait loci (QTL) for GP on chromosome 3 in a White Duroc × Erhualian F2 intercross. We herein performed a systems genetic analysis to identify the causal variant underlying the phenotype QTL (pQTL). We first conducted genome-wide association analyses in the F2 intercross and an F19 Sutai pig population. The QTL was then refined to an 180-kb interval based on the 2-LOD drop method. We then performed expression QTL (eQTL) mapping using muscle transcriptome data from 497 F2 animals. Within the QTL interval, only one gene (PHKG1) has a cis-eQTL that was colocolizated with pQTL peaked at the same SNP. The PHKG1 gene encodes a catalytic subunit of the phosphorylase kinase (PhK), which functions in the cascade activation of glycogen breakdown. Deep sequencing of PHKG1 revealed a point mutation (C>A) in a splice acceptor site of intron 9, resulting in a 32-bp deletion in the open reading frame and generating a premature stop codon. The aberrant transcript induces nonsense-mediated decay, leading to lower protein level and weaker enzymatic activity in affected animals. The mutation causes an increase of 43% in GP and a decrease of>20% in water-holding capacity of pork. These effects were consistent across the F2 and Sutai populations, as well as Duroc × (Landrace × Yorkshire) hybrid pigs. The unfavorable allele exists predominantly in Duroc-derived pigs. The findings provide new insights into understanding risk factors affecting glucose metabolism, and would greatly contribute to the genetic improvement of meat quality in Duroc related pigs. PMID:25340394

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

    PubMed

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

    2015-02-01

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

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

    PubMed

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

    1999-10-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2016-08-01

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