This study examined the effects of a sports drink on the performance of young soccer players. Ten competitive young male soccer players, ages 10 and 11, performed two experimental trials while consuming 32 ounces of either a sports drink (G)- Gatorade or a placebo (P)- Crystal Light in a double-blind, crossover design. Both trials consisted of a 15-minute warm-up period, a pre and post exercise test protocol and a 40-minute indoor scrimmage with a five-minute half time. The assigned fluid was consumed just prior to the warm-up, pre-test protocol, scrimmage and post-test protocol as well as during the half time of the scrimmage. The exercise tests included six activities such as shooting velocity, dribbling, passing, jumping, backward running, and sprinting in order to measure skill, agility, power, and speed. The results showed that due to the interaction of the 40-minute scrimmage and the consumption of Gatorade, the post-test shot velocity measurement was significantly (p<0.01) lower for P while G remained similar to the pre-test measurement. Also, there was a significant (p<0.05) decrease in the number of jumps completed for both P and G during the post-test jumping exercise when compared to the pre-test measurement. However, there were no significant difference of treatment, time and/or their interaction for the dribbling, passing, backward running, and sprinting. Many possible reasons may account for this lack of effect. 1) Muscle glycogen may not have been substantially depleted, possibly because the prescribed exercise during the trial was not intense or long enough. 2) Prior to the experimental trials, muscle glycogen stores were sufficient where no additional CHO was necessary (due to the subject's diet on the day of the trial or the short fast prior to the experimental trials). 3) Alternative mechanism, such as increased lactate production or dehydration and not muscle glycogen depletion, may be the cause of impaired skills. 4) A child's increased fat utilization allows for less of a need for manipulation of glycogen stores. / Master of Science

The existence of a glycogen-sarcoplasmic reticulum has been demonstrated by a number of researchers. This complex is suspected to participate in the calcium uptake activities of the sarcoplasmic reticulum (SR). Removal of glycogen particles associated with this complex may alter the calcium handling abilities of the SR. This experiment sought to determine what effect exercise and treatment with a-amylase had on the abilities of the SR to regulate intracellular calcium ([Ca2+]i). Rats were either run on a treadmill for 60 min at a speed of 21 m/min and a 10% grade or were not exercised. Animals were then killed by decapitation after inhalation of CO2. Left and right gastrocnemius muscles were excised from both groups and underwent SR vesicle preparation to separate the heavy and light SR fractions (HSR and LSR respectively). Left hindlimb muscle homogenate also underwent 60 min incubation with a-amylase to digest glycogen before differential centrifugation. Treatment with a-amylase significantly depressed rate of calcium uptake by LSR and HSR fractions by 22.89% and 25.22% respectively (p<0.05). alpha-Amylase had no effect on SR's rate of calcium release. There was no effect of exercise on calcium uptake or release rates. Glycogen concentration associated with the SR was unaffected by either alpha-amylase treatment or exercise. These results indicate that treatment with alpha-amylase decreases the ability of the SR to sequester calcium ions. / Master of Science

Glycogen synthase kinase-3beta (GSK-3beta) has been demonstrated to play a critical role in a diverse range of cellular functions from cell fate determination to cancer development. It is also implicated to be involved in the pathogenesis of neurodegenerative diseases (e.g. Alzheimer's disease), cancers and endocrine disorders (e.g. Type II diabetes). To gain further insight into the cellular mechanisms mediated by GSK-3beta, proteomic approach to identify novel cellular targets has become popular in recent years. GSK-3beta was inhibited by treating with lithium and kenpaullone in SH-SY5Y cell model, and over-expressed in Chinese Hamster Ovary (CHO) Tet-Off cell model. To getting more reliable results, we have used both conventional 2-D approach and Difference Gel Electrophoresis (DIGE) approach for this proteomic study. In 2-D electrophoresis, samples were resolved by two-dimensional polyacrylamide gel-electrophoresis (2D-PAGE). Protein spots were excised from the gels for in-gel trypsin digestion and further subjected to protein identification using mass spectrometry (MALDI-TOF-MS or MS/MS). In the GSK-3beta inhibition approach, cofilin was found to be down-regulated and Pin1 was found to be up-regulated, these consequence events demonstrated inhibition of GSK-3beta would protect the cells from structure alteration and tau hyperphosphorylation. In the GSK-3beta activation approach, cyclin-dependent kinase 5 (CDK-5) was found to significantly up-regulated in tau and GSK-3beta/Tau over-expressed cells, confirmed by Western blotting and RT-PCR. This finding indicates there is a new pathway between GSK-3beta, tau and CDK-5. Pin1 is also identified to be up-regulated after GSK-3beta activation. This result indicated a protection mechanism in response to the accumulation of hyperphosphorylated tau proteins. Our study help to get a better understanding of the GSK-3beta mediated substrates and pathways that help us to identify novel targets for the treatment of neurodegenerative and other diseases mediated by GSK-3beta. / Mak, Ying Cheong. / "September 2007." / Source: Dissertation Abstracts International, Volume: 69-08, Section: B, page: 4588. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (p. 157-181). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Dendritic cells (DCs) are the most potent professional antigen presenting cells (pAPCs) of the immune system and play a fundamental role in coordinating innate and adaptive immune responses. Through the expression of a wide array of pattern recognition receptors (PRRs), such as toll-like receptors (TLRs), DCs recognize a variety of microbial pathogens and infectious stimuli. Stimulation of DCs through TLR ligation results in a rapid series of activation-associated events, termed "maturation," which include the upregulation of surface co-stimulatory molecule expression, inflammatory cytokine secretion, and stimulation of naïve T cells via antigen presentation by MHC molecules. Activation of DCs through TLRs is coupled with an increased metabolic demand fulfilled by a rapid change in DC glucose metabolism and characterized by increased aerobic glycolysis rates. TLR-driven glycolytic reprogramming plays an essential role in generating building blocks required for high level protein synthesis associated with maturation. Although glucose imported from extracellular environments has been broadly considered as the major driver of glycolytic metabolism in immune cells, the contributions of intracellular glucose stores to these processes are not well-defined. The role of intracellular stores of glucose, in the form of glycogen, is widely appreciated in non-immune systems. However, very little is known about the implication of glycogen metabolism in DC immune responses. This work unveils the role and potential regulatory mechanisms of glycogen metabolism in support of DC effector function. The first part of this work primarily focuses on our characterization of the role of glycogen metabolism in early DC activation responses; while in the last chapter, we describe a potential regulatory mechanism of DC glycogen metabolism by activation-associated nitric oxide (NO) production. In this work, we tested the overarching hypothesis that DC-intrinsic glycogen metabolism supports the early glycolytic reprogramming required for effector responses and that nitric oxide can regulate this metabolism. We demonstrate that DCs possess the enzymes required for glycogen metabolic machinery and that glycogen metabolism supports DC immune effector response, particularly during early activation and in nutrient-limited environments. More importantly, we uncover a very intriguing metabolic phenomenon, in which DCs engage in the differential metabolic pathways driven by carbons derived distinctively from glycogen and free glucose. Our studies present the fundamental role and regulatory mechanisms of DC-intrinsic glycogen metabolism and underline the differential utilization of glycogen and glucose metabolism to support their effector responses. Overall, this work adds to a growing field of immuno-metabolism an improved understanding of an intricate layer of metabolic mechanisms that immune cells undertake in response to immune stimuli.

Thesis (Ph. D.)--University of Missouri--Columbia, 2000. / Typescript. Vita. Includes bibliographical references (leaves 199-207). Also available on the Internet.

Indiana University-Purdue University Indianapolis (IUPUI) / Glycogen, a branched polymer of glucose, serves as an osmotically neutral means of storing glucose. Covalent phosphate is a trace component of mammalian glycogen and has been a point of interest with respect to Lafora disease, a fatal form of juvenile myoclonus epilepsy. Mutations in either the EPM2A or EPM2B genes, which encode laforin and malin respectively, account for ~90% of disease cases. A characteristic of Lafora disease is the formation of Lafora bodies, which are mainly composed of an excess amount of abnormal glycogen that is poorly branched and insoluble. Laforin-/- and malin-/- knockout mice share several characteristics of the human disease, formation of Lafora bodies in various tissues, increased glycogen phosphorylation and development of neurological symptoms. The source of phosphate in glycogen has been an area of interest and here we provide evidence that glycogen synthase is capable of incorporating phosphate into glycogen. Mice lacking the glycogen targeting subunit PTG of the PP1 protein phosphatase have decreased glycogen stores in a number of tissues. When crossed with mice lacking either laforin or malin, the double knockout mice no longer over-accumulate glycogen, Lafora body formation is almost absent and the neurological disorders are normalized. Another question has been whether the abnormal glycogen in the Lafora disease mouse models can be metabolized. Using exercise to provoke glycogen degradation, we show that in laforin-/- and malin-/- mice the insoluble, abnormal glycogen appears to be metabolically inactive. These studies suggest that a therapeutic approach to Lafora disease may be to reduce the overall glycogen levels in cells so that insoluble, metabolically inert pools of the polysaccharide do not accumulate.

This document only includes an excerpt of the corresponding thesis or dissertation. To request a digital scan of the full text, please contact the Ruth Lilly Medical Library's Interlibrary Loan Department (rlmlill@iu.edu).

Indiana University-Purdue University Indianapolis (IUPUI) / Glycogen is a major energy reserve in most eukaryotes and its rate of synthesis is controlled by glycogen synthase. The activity of eukaryotic glycogen synthase is regulated by the allosteric activator glucose-6-phosphate, which can overcome the inhibitory effects of phosphorylation. The effects of phosphorylation and glucose-6-phosphate on glycogen synthase are mediated by a cluster of six arginines located within a stretch of 12 amino acids near the C-terminus of the enzyme’s polypeptide chain. We studied isoform-2 of yeast glycogen synthase as a model to study the structural and molecular mechanisms that underlie the regulation of the eukaryotic enzymes and our primary tools of investigation were macromolecular X-ray crystallography, site-directed mutagenesis, intein-mediated peptide ligation and enzyme assays. We have solved the tetrameric structure of the yeast enzyme in two different activity states; the resting enzyme and the activated state when complexed with glucose-6-phosphate. Binding of glucose-6-phosphate to glycogen synthase induces large conformational changes that free the active site of the subunits to undergo conformational changes necessary to catalyze the reaction. Further, using site directed mutagenesis and intein-mediated peptide ligation to create specific phosphorylation states of the enzyme we were able to define specific roles for the arginine residues that mediate the regulatory effects of phosphorylation and glucose-6-phosphate activation. Based on these studies, we propose a three state structural model for the regulation of the enzyme, which relate the observed conformational states to specific activity levels. In addition to these regulatory studies, we have also solved the structure of the enzyme complexed with UDP and with substrate analogs, which provide detailed insight into the catalytic mechanism of the enzyme and the ability of glycogen synthase to remain tightly bound to its substrate glycogen.

BE [(1,4)-a-D-glucan:(I,4)-a-D-glucan 6-glucosyltransferase, EC 2.4.1.18] catalyses a transglycosylation reaction where a branch-point is created by the cleavage of an a-l,4 glycosidic bond to form an a-l,6 glycosidic bond. Branching enzyme (BE) from baker's yeast was purified to near homogeneity by chromatography on DEAE-cellulose, Sephacryl S-200 and Protein Pak Q. Electrophoresis on SDS-PAGE revealed one major band of molecular weight 74 kDa. Three distinct methods for determining BE activity (Phosphorylase Stimulation, Iodine- Binding and Branch-Linkage Assays) were used to characterise the purified protein. The enzyme displayed a temperature optimum between 15-25°C and a broad pH optimum of 6.5-7.5 with maximum activity occurring in phosphate buffer. The enzyme was fully stable after incubation at 20°C for 5 hours. A Km value of 1474 Jlg/ ml for amylose was obtained. Primary structural analysis involving N-terminal sequencing and amino acid composition suggested that yeast BE may share some homology with BEs isolated from other sources. Immunological comparisons between yeast, maize (BEll) and Escherichia coli BE using yeast polyclonal antiserum indicated that the enzymes may share antigenic determinants. However, similar comparisons between yeast BE and E.coli antiserum revealed that the antibody only recognised yeast BE in its denatured conformation. Yeast BE was used to modify potato amylose and amylopectin and wheat starch. The enzyme was capable of introducing additional branch points to these substrates resulting in a displacement of the iodine Amax from 629 nm to 568 nm, from 543 nm to 411 nm and from 632 nm to 568 nm for amylose, amylopectin and wheat starch, respectively. HPAEC-PAD analysis of the branched products produced by yeast BE revealed that predominantly short chains of dp 2 to I? were transferred. At least three BE fractions of higher specific activities were isolated from brewer's yeast hatyested at the late exponential phase, suggesting the expression of more than one BE in Saccharomyces cerevisiae.

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